R3: Reviews, Responses & Reflections in Neuroscience (2019 Edition) by utneuro

REVIEWS RESPONSES REFLECTIONS IN NEUROSCIENCE 1

Compiled By: Andrea Pinto Cover Image from Pixabay

Table of Contents Reaching new lights: A promising alternative to treating depression and anxiety symptoms using transcranial photobiomodulation

A Medial Prefrontal Cortex Receptor May be Involved in Mediating Stress Resilience by Reducing Inflammation

Gut Microbiome Disturbance Mediated Depression Induces GABA-A Receptor Subunit Changes in the Hippocampus.

Alzheimer’s Disease: Targeting Neuroinflammation with Minocycline

Examining the use of rapid eye movement sleep deprivation as antidepressant therapy in OBX rats

Multiple Sclerosis through an animal model: oral antibiotic treatment in Dark Agouti rat neonate exasperates central nervous system autoimmunity

Vagus Nerve Stimulation: Enhancing Outcomes in Ischemic-Stroke Motor Rehabilitation

Countering the Effects of Plasticity and Deficits in Long Term Depression Induced by Cocaine with the Use of ZetaInhibitory Peptide

Anhedonia: Examination of Future Biological Targets Involving Galanin Receptors and Galanin N-Terminal (1-15) for Depression

Inducing and Quantifying Lucid Dreams Using Galantamine and the Mild Technique

Liquiritigenin as an Antidepressant in Chronic Mild Unpredictable Stress Induced Mouse Models of Depression

Cannabis’ Effects on Mental Health and Cognition When Used as a Therapy for Chronic Pain in Lieu of Opioids

The Neuro-Immune Axis: Vagal Neuromodulation of Anti-Inflammatory Pathways in the Gastrointestinal Tract.

MS: Modulation of immunity and microbiome via probiotic supplementation

Human Amniotic Epithelial Stem Cells Aa A Therapy For Alzheimer’s Disease

Habitual Tea Drinking as a Protective Measure for Cognitive Decline in Older Adults

Genome-edited Skin Transplants Offer a Safe and Enduring Gene Therapy Approach for Treating Drug Addiction

An Appreciation for the Gut Microbiota: Antibiotic-Mediated Treatment from Early Adolescence Alters Brain Development and Behaviour

101

Investigating the role of neutrophils with respect to cortical blood flow and cognitive function in Alzheimer’s disease mouse models

106

A Novel Player in Alzheimer’s Disease Pathogenesis: SFRP1

111

Phantom Limb Pain: risk and protective factors.

116

Elucidating the Link Between Hyperinsulinemia and Dementia

122

Assessment of the Link between Plasma Cytokines and Rapid Eye Movement Sleep Behavior Disorder.

128

The Neurobiological Role of Mifepristone as a Potential Treatment for Anorexia Nervosa

134

Usage of Botulinum Toxin in Understanding Facial Feedback

139

What Happens to the Brain Following Sleep Loss?

144

Examination of Flammulina velutipes polysaccharides: A Gut Microbiota Modulation Resulting in Learning and Memory 149 Improvements 3

Investigating the Mechanisms of REM Sleep Control: Connecting the Hypothalamus and Brainstem REM Switch.

154

Binge eating disorder (BED) in both humans and rodents includes a change in emotional profile following binge eating, which can be assessed via behavioral and neurophysiological investigation.

159

Psychoplastogens: a growing class of compounds that promote long lasting structural, functional, and behavioral changes after only a single dose

166

The Effect of Zeta Inhibitory Peptide on Behavioral Patterns and Synaptic Plasticity Caused by Cocaine Use

175

Examination of the Effects of Acetaminophen on Empathic Behaviour and the Neuropeptides involved in Empathy

180

Alzheimer’s Disease Mouse Models Show Scanning Ultrasound Treatment Removes Amyloid-β Peptides and Restores 186 Performance on Memory Tasks The transcription factor Zfp189 orchestrates a gene network in the prefrontal cortex that mediates stress resilience and harbors antidepressant effects

192

A New Technique to Examine the Importance of Infralimbic Cortex in Retrieval of Extinction Memory: Optogenetics.

201

Exercise and Neuroplasticity in Brain Injury

209

The Highs and Lows of Pregnancy: Effects of Prenatal Cannabis Exposure on Dopaminergic Neurons in the Ventral Tegmental Area

215

Electrochemical and Structural Assessment of Neuron-Glioma Interactions

223

Rifampicin: A Light in the Search for Therapeutics Against Alzheimer’s Disease

230

Myopathic changes and motor neuron degeneration observed in a Matr3 overexpression transgenic mouse model of ALS

235

Characterization of Newborn Dentate Granule Cell Morphology and Loss of Innervation in Frontotemporal Dementia and Activity-Dependent Rescue

241

Alzheimer’s disease: Consequences of neurofilament light protein gene deletion in APP/PS1 mice model

247

The gut microbiome and the brain: how Akkermansia muciniphila can alleviate hippocampal-associated deficits that result from high fat diets

252

Exploring metformin potential as treatment for stroke recovery in different ages and sexes by examining neuronal pro- 257 genitor cell pool expansion and cognitive recovery Review article: Potential contribution of gut dysbiosis to Alzheimer’s Disease through the gut-brain axis

262

Elucidating the Role of mTORC2 Signalling in Syndromic Autism Spectrum Disorder.

269

Fecal Microbiota Transplantation as a Therapeutic Strategy for Alzheimer’s Disease

273

Axon Demyelination and Degeneration in White Matter As Biomarker for Traumatic Brain Injury.

279

Gut Microbiota Dysbiosis Impact On Memory in Mice

284

Understanding the Mechanism through which Type 2 Diabetes Mellitus Causes Alzheimer’s Pathophysiology

289

Potential Biomarkers for Alzheimer’s Disease in Human Retina

296

Evaluating Probiotic Supplementation as a Potential Treatment for Age-Related Long-Term Potentiation Loss and Increased Neuroinflammation

303

Overexpression of RGS8 Protein Correlation to Elongation of Primary Cilia Induces Antidepressant-like Phenotype

310

Apelin-36: An Overlooked Peptide with Promising Effects on Parkinson’s Disease

315

Bilateral Lesioned Hippocampus Inhibits Retrieval of Previously Learned Memories but Not New Postoperative Memo- 321 ries Determining optimal rTMS protocols for treatment of PTSD

326

Elevated levels of plasma neurofilament light chain concentration in patients with anorexia nervosa.MDD: Investigating 335 Microglial Polarization and Endoplasmic Reticulum Stress Using a Chronic Social Defeat Stress Mouse Model Overcoming Low-motivated Arousal after Early Life Stress using Chemogenetic Activation of the Lateral Hypothalamus 341 The Role of Drp1 Mediated Mitochondrial Fission in Glial Activation and Neuronal Injury, in Mice Models of Neurodegenerative Disease.

347

Examination of Gut Microbial Dysbiosis Induced by ASD-Associated Environmental Risk Factors.

353

Evaluating the efficacy of dance interventions on motor symptoms and quality of life aspects in Parkinson’s patients

358

Rifaximin Modulates Gut Microbiota in Rats with Induced Visceral Hyperalgesia, Leading to Reductions in Intestinal Permeability and Inflammation

363

Depression induced mice show increased interleukin-1β and NLRP3 inflammasome

369

Is hyperserotonemia due to gut dysbiosis the driving force for autism spectrum disorder!

374

The Lung-Brain Axis: Mechanisms of Ozone Exposure and Association with Alzheimer’s Disease

380

The Exploration of GUO as a Potential Therapeutic Treatment for Focal Ischemic Stroke

387

Modeling Traumatic Brain Injury in Adult Zebrafish Shows Relevant Mammalian Injury Characteristics and Treatment Outcomes

393

Social Deficits in SHANK3 mutant mice caused by Anterior Cingulate Cortex Impairments

399

Deciphering the role of the gut microbiome in the complementary relationship between stress and the development of depressive-type behaviors

404

Autism Spectrum Disorder: Improvements Post Microbiota Transplant correlating with gastrointestinal symptoms

413

The Neuroprotective Potential of Physical Exercise

418

Rapid eye movement sleep modulated by melanin-concentrating hormone neurons via the ventrolateral periaqueductal 423 gray The Use of Gold Nanoparticles to Treat Alzheimer’s Disease

429

The role of gut microbiome in contributing to the development of schizophrenia via kynurenine metabolism?

433

Antioxidant-like protein 1 loss of function in streptozotocin-induced diabetes rat model, indicates a mechanism for chronic hyperglycemia and cognitive impairment.

437

Running to the Rescue – How Exercise Induces Hippocampal Neurogenesis through Platelet Activation

442

Brain-Selective Estrogen improves α-Synuclein homeostasis, neuropathology, and motor phenotype in Parkinson’s Disease model

447

Estrogen Matters: The Impact of Neuron-Derived Estradiol

452

Bisphenol A Upregulates the Expression of Neuropeptide Y Through Disruptions of the Regulation of Circadian Rhythm

457

Physical Exercise as an Effective Therapy for Delaying Alzheimer’s Disease Progression

261

Linking the Hypotheses of Depression: Dopamine D3 Receptor Knockout Mice Exhibit Both Depressive Symptoms and Microglia Activation

467

Reaching new lights: A promising alternative to treating depression and anxiety symptoms using transcranial photobiomodulation Bushra Ahmed

The pathophysiology of depressive and anxiety disorders is strongly connected with serotonin and nitrous oxide (NO) levels. Transcranial photobiomodulation or TPBM is an effective tool that has been established as a promising method to reduce depression and anxiety related symptoms, by directly stimulating the cortical surface of the brain, specifically regions associated with depression and anxiety, i.e. the hippocampus, prefrontal cortex (PFC) and amygdala. However, the correct dosage to treat these symptoms is still unknown, which is why the Eshaghi et al demonstrated this method on a Chronic Restraint Stress (CRS) rodent model with three different dosage levels to identify the ideal dosage needed to maximize improvement of these symptoms. The four experimental groups received no dosage, 4, 8 and 16 J/cm2 with a near-infrared radiation or NIR diode laser, for 3 hours daily lasting three weeks and compared against the control group. The mice also underwent behavioural tests such as Open Field Test, Tail Suspension and Elevated plus Maze. Following the tests, the PFC and Hippocampus of all groups wereassessed for serotonin, serum cortisol and NO levels. The results from the study showed that the CRS+8NIR group received the ideal dosage and provided maximal effects of reduced depressive and anxiety symptoms, via maximal time spent exploring in the behavioural tests, reduced serum cortisol and NO levels and increased serotonin levels. The CRS Sham group, which received no dosage, exhibited depression and anxiety symptoms such as lowest time spent in exploration and high NO and serum cortisol levels with reduced serotonin expression. The CRS+4NIR and CRS+16NIR groups showed decline in these aspects compared to the 8NIR group, thus rendering 8J/ cm2 as the ideal dosage for treating these disorder symptoms.. Key words: Transcranial photobiomodulation, near infrared, depression, anxiety, serotonin, nitrous oxide, laser therapy

INTRODUCTION

(TST). Post mortem assessment of levels NO, serum cortisol and serotonin were measured in the prefrontal cortex (PFC) and Hippocampus, which are the regions primarily associated with depression and anxiety disorders. MAJOR RESULTS Among the five groups of mice that were tested upon in the study, the CRS+8NIR mice improved the most. They exceeded expectations in the behavioural tests, had the lowest time spent in attenuating immobilization and increased serotonin in the hippocampus and prefrontal cortex, and maximal reduction in serum cortisol and NO levels. Particularly, they showed most improvement in the open field test, tail suspension, elevated, and maze test. However, the CRS Sham group performed the least in the behavioural test, which is they gave up the fastest in the tail suspension test, spent the least amount of time exploring the open field and elevated and maze. This is significant because the CRS Sham model represents a typical depression model, and clearly exhibits the symptoms that depression mice models express, if they go without treatment. Whereas the CRS+8NIR mice showed the most improvement, thus enabling the correct dosage of TPBM required treating depressive symptoms. From a pathobiological perspective, the CRS Sham mice showed increased serum cortisol and NO and maximal reduction in serotonin/5-HT levels among all the other groups (Fig 1, 2 and 3), again reflecting the pathobiology of depression brain. Such results confirm the hypothesis of depression effects on NO, serum cortisol and serotonin levels, and highlights their role in treating depressive symptoms.

Depression and anxiety are two of the most common mental conditions that affect millions of people across the globe. The onset of depression and anxiety symptoms, which include anxious symptoms, anhedonia, continuous low mood etc. (Hennessey and Hamblin, 2017). 5-HT a.k.a Serotonin and Nitrous Oxide (NO) are two of the major components that underlies the pathobiology of anxiety and depression disorders. The monoamine-deficiency of hypothesis states that serotonin; dopamine and noradrenaline imbalance in the central nervous system can lead to express depressive symptoms (Jesulola et al, 2018). Moreover, NO, which plays an important role in neurogenesis, neuroinflammation, synaptic plasticity and neurotransmission, also underlies anxiety and depression pathology (Dhir et al, 2011). While medication and various forms of psychotherapy, various forms of low-level laser therapy primarily treat, these disorders are being tested in rodent models and clinical populations as an alternative form of treatment. Photobiomodulation is a method by which biological mechanisms are stimulated with the use of photons or light. Transcranial photobiomodulation (TPBM) is a cell regeneration, preservation and stimulation method that uses red or near infrared (NIR) light to activate signalling pathways on cellular and tissue level on the brain (Hennessey and Hamblin, 2017). This method allows stimulating the brain without causing any major side effects and it relatively safe to use as established by small clinical trials. TPBM has recently become a major player in treating various mood disorders, such as depression and anxiety. In a study conducted by Schiffer et al, treatment resistant patients with depression (who also had other mental disorders such as anxiety and post traumatic stress disorder), received TPBM exposure for four minutes at two sites of the forehead, reported significantly lower scores on the Hamilton Depression Rating scale, at 2-week and 4- week post treatment check in (2009). Moreover, Cassano et al produced similar results in their study, where two out of the four patients achieve remission upon TPBM treatment (2015). Significant improvement in depression scores have also been reported in patients with elevated depression symptoms (Morries et al, 2015; Disner et al, 2016). TPBM is an emerging method and a promising alternative to traditional treatment of depressive disorders, since 30-40% of patients do not respond to antidepressants (Salehpour and Rasta, 2017). The article chosen for the literature review (Eshaghi et al, 2019), builds upon the solid evidence of TPBM treatment of anxiety and depression disorder. The objective of the study was to investigate the effects of three different doses of NIR TPM on Chronic Restraint Stress (CRS) mice models via three behavioural tests consisting of Open Field Test (OFT), Elevated plus Maze (EPM) and Tail Suspension Test

Fig 1: The graph signifies Nitrous Oxide (NO) levels across the five groups. CRS+8NIR group had the least amount of NO expressed and the sham group had the most. All groups that received TPBM reported overall reduction in NO levels. (Eshaghi et. al, 2019)

DISCUSSION/ CONCLUSIONS As shown in the Results section, the CRS+8NIR group provided maximal benefits by most reduction in NO and serum cortisol levels, which have elevated levels in depression and anxiety disorders, and maximal increase in serotonin levels, which is significantly low in these mood disorders. The authors compare the various aspects of their results to the existing literature and reemphasize the previous existing hypotheses surrounding anxiety, depression and their underlying pathobiology with the results of the current study. They also report a negative correlation of NO levels with 5-HT, and its association with depression (Fig 4). The authors conclude the paper by emphasizing how anti anxiety and anti depressive symptoms can be mitigated by the TPBM procedure; however, this pathway is highly depended on the Fig 2: The graph illustrates Serotonin or 5-HT levels across the correct dosage being delivered to the cortical surface of the five groups. CRS+8NIR group had the most 5- HT expressed and brain. The correct amount of dosage was calculated in this the sham group had the least. All groups that received TPBM study, which is significant since TPBM requires varied dosage reported overall increase in 5-HT levels. (Eshaghi et. al, 2019) level depending on the area it is administered on. CRITICAL ANALYSIS The results obtained from the current study re-establishes the hypotheses around the efficacy of TPBM in treating mood disorders. However, the study needs to be performed among clinical populations. Human populations with mood disorders often have comorbidities associated, which is not the case in mice models. Therefore, it will be difficult to understand whether varying levels of neurotransmitters and other components are due to the target disorder itself or the comorbidity associated with it. Furthermore, the correct dosage may vary due to different physiological proportions in human beings, and skull size, presence of cerebral fluid etc. matter when TPBM is performed, since 100% of the light cannot pass through the barriers to get to the desired region and create optimal effects (Salehpour et. al, 2018). Another issue is the use of laser TPBM, since increased concentration on a small treatment area can lead to tissue damage, but provided increase stimulation compared to LED light (Passarella and Karu, 2014). Lastly, the research field for TPBM is still in its infancy, in regards to treating depressive disorders. Caldieraro and Cassano argue accurately that all the studies that were conducted, use different models of depression, thus making it difficult to replicate the studies. This is also true for article in review, since it does not specify what type of depression models the authors are investigating. However, the author of this literature review does believe the study is extremely well designed and acts as a guide for future researchers to continue improving upon TPBM treatment for mood disorders.

Fig 3: The graph illustrates serum cortisol levels across the five groups. CRS+4NIR group had the least serum cortisol expressed and the sham group had the most. All groups that received TPBM reported overall decrease in serum levels. (Eshaghi et. al, 2019)

FUTURE DIRECTIONS As described in the Critical Analysis section, it is important to test out the TPBM method with the same dosage in clinical populations, as well as replication in larger sample sizes of mice models. It is necessary to test it out in clinical populations because the dosage level may not translate the same to human population compared to mice. This method may be an alterna-

Fig 4: The graph illustrates the correlation between Nitrous Oxide (NO) and Serotonin (5-HT) levels in the prefrontal cortex (PFC) and the hippocampus. Increased NO levels tended to associate with lower 5-HT levels and vice versa, which suggested a connection between the neurotransmitter and chemical in depression models. (Eshaghi et. al, 2019) 8

tive to pharmaceuticals or perhaps an added enhancement to pharmaceuticals to treat depression and anxiety disorders. In most cases, there were no significant side effects reported for use of TPBM in both rodent and human populations, therefore it is increasingly becoming a better non-pharmaceutical alternative to treating mood disorders, and may lead to significant reduction of symptoms if combined with traditional treatment methods such as pharmaceuticals, therapy and other light therapy methods. (Salehpour and Rasta, 2017). It is also important to reiterate that mood disorders often have comorbidities, therefore it can be difficult to extricate the effects that are also associated with sleep, cognition, suicidal ideation etc., and these areas must be highlighted in future studies (Cassano et. al, 2016). More sham control studies must be conducted, as device procedures can be associated with increased placebo effects (Cassano et al, 2016; Kaptchuk et. al, 2000). Cassano et. al also argue that this method must be encouraged for greater dissemination for its cost-efficiency and safe use, among the wider community of psychiatric professionals (2016). Due to the diverse needs TPBM caters to, such as treating not only mood disorders, but also Parkinsonâ&#x20AC;&#x2122;s Disease, Alzheimerâ&#x20AC;&#x2122;s, other traumatic brain injuries, Hamblin also identifies the need for TPBM to be circulated for wider use, but only after replicating the already existing studies among clinical populations (2016). Therefore, it can be identified that TPBM is a promising tool that can treat various brain disorders; however, studies that are more rigorous must be conducted in larger samples for its widespread use.

REFRENCES 1.

Caldieraro, M. A., & Cassano, P. (2019). Transcranial and systemic photobiomodulation for major depressive disorder: A systematic review of efficacy, tolerability and biological mechanisms. Journal of Affective Disorders, 243, 262–273. doi: 10.1016/j.jad.2018.09.048

Cassano, P., Cusin, C., Mischoulon, D., Hamblin, M. R., De Taboada, L., Pisoni, A., … Iosifescu, D. V. (2015). Near-Infrared Transcranial Radiation for Major Depressive Disorder: Proof of Concept Study. Psychiatry journal, 2015, 352979. doi:10.1155/2015/352979

Cassano, P., Petrie, S. R., Hamblin, M. R., Henderson, T. A., & Iosifescu, D. V. (2016). Review of transcranial photobiomodulation for major depressive disorder: targeting brain metabolism, inflammation, oxidative stress, and neurogenesis. Neurophotonics, 3(3), 031404. doi:10.1117/1.NPh.3.3.031404

Dhir, A., & Kulkarni, S. K. (2011, April 30). Nitric oxide and major depression. Retrieved from https://www.ncbi.nlm.nih.gov/ pubmed/21335097.

Disner, S. G., Beevers, C. G., & Gonzalez-Lima, F. (2016). Transcranial Laser Stimulation as Neuroenhancement for Attention Bias Modification in Adults with Elevated Depression Symptoms. Brain Stimulation, 9(5), 780–787. doi: 10.1016/ j.brs.2016.05.009

Eshaghi, E., Sadigh‐Eteghad, S., Mohaddes, G., & Rasta, S. H. (2019). Transcranial photobiomodulation prevents anxiety and depression via changing serotonin and nitric oxide levels in brain of depression model mice: A study of three different doses of 810 nm laser. Lasers in Surgery and Medicine, 51(7), 634–642. doi: 10.1002/lsm.23082

Hamblin, M. R. (2016). Shining light on the head: Photobiomodulation for brain disorders. BBA Clinical, 6, 113–124. doi: 10.1016/j.bbacli.2016.09.002

Henderson, T. A., Morries, L., & Cassano, P. (2015). Treatments for traumatic brain injury with emphasis on transcranial near-infrared laser phototherapy. Neuropsychiatric Disease and Treatment, 2159. doi: 10.2147/ndt.s65809

Hennessy, M., & Hamblin, M. R. (2017). Photobiomodulation and the brain: a new paradigm. Journal of optics (2010), 19(1), 013003. doi:10.1088/2040- 8986/19/1/013003

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Jesulola, E., Micalos, P., & Baguley, I. J. (2018, April 2). Understanding the pathophysiology of depression: From monoamines to the neurogenesis hypothesis model - are we there yet? Retrieved from https://www.ncbi.nlm.nih.gov/ pubmed/29284108.

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Kaptchuk, T. J., Goldman, P., Stone, D. A., & Stason, W. B. (2000). Do medical devices have enhanced placebo effects? Journal of Clinical Epidemiology, 53(8), 786–792. doi: 10.1016/s0895-4356(00)00206-7

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Passarella, S., & Karu, T. (2014). Absorption of monochromatic and narrow band radiation in the visible and near IR by both mitochondrial and non-mitochondrial photoacceptors results in photobiomodulation. Journal of Photochemistry and Photobiology B: Biology, 140, 344–358. doi: 10.1016/j.jphotobiol.2014.07.021

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Salehpour, F., & Rasta, S. H. (2017, May 24). The potential of transcranial photobiomodulation therapy for treatment of major depressive disorder. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/28231069.

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Salehpour, F., Mahmoudi, J., Kamari, F., Sadigh-Eteghad, S., Rasta, S. H., & Hamblin, M. R. (2018). Brain Photobiomodulation Therapy: a Narrative Review. Molecular Neurobiology, 55(8), 6601–6636. doi: 10.1007/s12035-017-0852-4

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Schiffer, F., Johnston, A. L., Ravichandran, C., Polcari, A., Teicher, M. H., Webb, R. H., & Hamblin, M. R. (2009). Psychological benefits 2 and 4 weeks after a single treatment with near infrared light to the forehead: a pilot study of 10 patients with major depression and anxiety. Behavioral and brain functions: BBF, 5, 46. doi:10.1186/1744-9081-5-46 10

A Medial Prefrontal Cortex Receptor May be Involved in Mediating Stress Resilience by Reducing Inflammation. Sadia Ahmed

Individuals either express more resilience or vulnerability after exposure to a stressor. Having a stress vulnerable phenotype increases the susceptibility to various mental health illnesses, such as depression and posttraumatic stress disorder (PTSD), predisposing an individual to a lower quality of life (Wood et al., 2015). This paper will discuss and critique the research of Corbett et al. (2019), a group that studied rats for the potential neurobiological mechanisms underlying stress resilience. Rats that displayed resilience after stress exposure showed increased expression of sphingosine-1phosphate receptor 3 (S1PR3) in the medial prefrontal cortex (mPFC) compared to vulnerable and control, non-defeated rats (Corbett et al., 2019). Through viral-mediated techniques, the knockdown of S1PR3 in the mPFC increases vulnerability to stress, whereas S1PR3 overexpression increases resilience. The authors further observed that decreased expression of S1PR3 led to an increase in activated microglia and proinflammatory cytokines, such as tumor necrosis factor alpha (TNFÎą), after stress was induced. This suggests that having more S1PR3 in the mPFC reduces vulnerability to stress by attenuating stress-induced proinflammatory processes. Moreover, Corbett el al. demonstrated that the increased expression of S1PR3 in resilient rats was mediated by stress-induced glucocorticoid (GC) receptor pathways. To translate some of their findings related to S1PR3 to humans, combat-exposed individuals with and without PTSD were blood tested. The results showed that veterans with PTSD had relatively lower levels of S1PR3 mRNA in their blood compared to veterans without PTSD. Overall, the study suggests that having more S1PR3 in the mPFC reduces inflammation in the brain and promotes stress resilience. Key words: Stress, resilience, vulnerability, depression, post-traumatic stress disorder (PTSD), medial prefrontal cortex (mPFC), sphingosine-1-receptor protein 3 (S1PR3), microglial cells, proinflammatory processes, glucocorticoid (GC) receptor.

BACKGROUND Humans and even animals exhibit differences when exposed to stressful conditions (Franklin et al., 2012). For instance, certain populations succumb to stress more easily, while others are more resilient in strenuous situations. Stressvulnerable individuals have a higher chance of expressing mental health illnesses, such as depression and post-traumatic stress disorder (PTSD), predisposing the individual to a lower quality of life (Wood et al., 2015). Therefore, it is of interest in understanding what neurobiological mechanisms are at play in determining how we respond to stress and moreover, how those mechanisms relate to the expression of various kinds of mental health disorders.

ating resilience against stressful situations (Corbett et al., 2019). The researchers utilized white, male Sprague Dawley rats, and separated them into groups based on whether they expressed resilience or vulnerability after a social-defeat protocol, which involved a stressful encounter of an aggressive Long Evans rat. Resilient rats express proactive coping to stress, which includes behaviourally adopting upright postures, whereas vulnerable rats express passive coping and usually adopt supine postures (Wood et al., 2015). Through PCR analysis, the authors were able to distinguish that S1PR3 levels were elevated in resilient rats compared to the vulnerable group. Resilient rats also exhibited lower levels of inflammation within the mPFC compared to vulnerable rats. Overall, the authors were able to demonstrate that S1PR3 in the mPFC promotes stress resilience by reducing proinflammatory cytokines and activated microglial cells. The authors also established that war veterans with PTSD had less S1PR3 mRNA in their blood compared to veterans without PTSD, which is congruent with the results observed in rats.

A particular region of the brain that has been implicated to be involved in stress regulation is the medial prefrontal cortex (mPFC) (Diorio et al., 1993; Franklin et al., 2012; McKlveen et al., 2015). Its structural regions include the anterior cingulate (ACC), prelimbic (PLC) and infralimbic (ILC) cortex, which are involved in controlling and inhibiting neurons that are a part of the hypothaThis paper is relevant because it takes into consideration lamic-pituitaryadrenocortical (HPA) axis (Franklin et al., 2012). A the possible mechanisms underlying the expression of stressstudy has further demonstrated that lesions in the mPFC cause coping strategies. Individuals who display a more stressan increased response to stressors by the HPA axis (Diorio et al., vulnerable phenotype are also more prone to developing stress1993). related disorders, therefore understanding the underlying mechA stress activated HPA axis results in increased glucocorti- anisms could potentially be beneficial for the prognosis, diagnocoid (GC) secretion from the adrenal cortex (Leonard, 2001). sis and treatment of certain mental health illnesses. Individuals with depression and other stressrelated disorders MAJOR RESULTS tend to have high GC levels for an extended amount of time, which decreases the sensitivity of GC receptors in the central S1PR3 Expression in the mPFC Corbett and his colleagues nervous system (CNS), including the ones on microglial cells (2019) performed repeated social-defeat experiments on Spra(Leonard, 2001). As a result, this may account for the increased gue Dawley rats for seven days to differentiate them into groups inflammation within the CNS of individuals with stress-related that are stressvulnerable or resilient. Vulnerable rats submitted disorders (Leonard, 2001). For example, one study showed that to defeat more quickly, hence displayed shorter defeat latencies individuals with depression had increased levels of the proin- (Figure 1a). On the other hand, resilient rats exhibited longer flammatory cytokine tumor necrosis factor alpha (TNFÎą) in their defeat latencies. After performing the experiments, vulnerable, brain compared to healthy controls (Tuglu et al., 2003). Never- resilient and non-defeated rats were euthanized and their mPFC theless, treatment of antidepressant medication within these tissues were obtained for PCR array analysis. The mPFC tissue individuals reduces TNFÎą levels closer to the concentrations of was specifically chosen due to its well-known role in mediating healthy subjects. stress-related responses. Through PCR analysis, the authors found that S1PR3 mRNA was differentially expressed in the This paper will specifically analyze the results of a study mPFC between the vulnerable and resilient rats. Rats that exhibconducted by Corbett et al. (2019), whom have discovered a ited resilience after repeated stress exposure showed increased protein in the mPFC, which may be involved in promoting stress levels of S1PR3 in their mPFC, compared to vulnerable and nonresilience. The protein in question is sphingosine-1-phosphate defeated rats (Figure 1b). This suggests a role for S1PR3 in stress receptor 3 (S1PR3). S1PR3 is a Gprotein coupled receptor that is resilience. typically involved in cellular mechanisms within the periphery, such as inflammation, proliferation and angiogenesis (MahajanThakur et al., 2015). However, its exact mechanisms within the CNS are not clear. This particular study is one of the first to consider the role of S1PR3 within the brain, and its effects on medi12

iAAV-S1PR3 (red) rats. B. Represents the time (seconds) engaged in immobile, swim, climb behaviours in iAAV-scramble and iAAV-S1PR3 rats. C. Represents the time spent by iAAV-scramble and iAAV-S1PR3 rats interacting with a stimulus rat in the social interaction test.

To substantiate that S1PR3 attenuates stress-induced vulnerability, the authors performed experiments to overexpress S1PR3 in the mPFC of rats. They bilaterally injected AAV-S1PR3-GFP in the mPFC, leading to increased expression of S1PR3 and green fluorescent protein (GFP). On the other hand, the control group were injected AAV-GFP, which did not alter S1PR3 expression. All rats were then subjected to the social-defeat paradigm. Rats that Figure adapted from Corbett et al. (2019). Nature communi- overexpressed S1PR3 in their mPFC showed more resilience comcations, 10(1), 1-13. pared to controls. In particular, they had longer defeat latencies. Figure 1. A. Represents the mean defeat latencies in vulnerable and resilience Furthermore, S1PR3-overexpressing rats had decreased immobility rats. B. Represents the S1PR3 mRNA levels in the mPFC in non-defeated (ND), in the FST, and increased interactions with the stimulus rat in the vulnerable (VUL), and resilient (RES) rats. social interaction test, indicating reduced depression and anxietylike behaviour respectively. Overall, these results demonstrate that S1PR3 in the mPFC is important for producing a resilient phenotype Knockout & Overexpression of S1PR3 in the mPFC when exposed to stressful situations. To demonstrate that S1PR3 is necessary for a resilient phenotype, Corbett et al. (2019) performed knock-out and knock-in experiments with the use of viral-mediated techniques. Experi- S1PR3 Functions & Inflammation mental rats were bilaterally injected with iAAV-S1PR3 in the mPFC, The mechanisms by which S1PR3 in the mPFC promotes stresswhich reduced S1PR3 cell numbers by more than 70%. Control rats resilience is elucidated by the authors. In stress-related disorders, were injected with iAAV-scramble, which did not affect S1PR3 cell such as depression, individuals cope with stress passively and exnumbers. All rats were then subjected to the social-defeat parapress a vulnerable phenotype (Franklin et al., 2012). In the literadigm. The results demonstrated that S1PR3-knockout rats exhibited ture, humans and rodents with symptoms of depression generally a more vulnerable phenotype with quicker defeat latencies, comexhibit increased inflammation in their brain (Audet et al., 2011; pared to the control group (Figure 2a). Moreover, reduced S1PR3 Tuglu et al., 2003; Wood et al., 2015). Furthermore, S1PR3 in the levels in the mPFC was associated with depression-like behaviour as periphery has been implicated to be involved in regulating immunoobserved by the increased immobility in the Porsolt forced swim logical responses (Mahajan-Thakur et al., 2015). Hence, S1PR3 in test (FST) (Figure 2b). S1PR3-knockout rats also displayed more the mPFC may potentially exert its resilient-promoting effects by anxiety, as the rats spent less time interacting with a stimulus rat of reducing stress-induced inflammation. the same strain and size (Figure 2c). Therefore, knocking out S1PR3 Corbett et al. (2019) observed S1PR3-knockout rats (iAAVproduces a more vulnerable phenotype in rats when exposed to S1PR3) and compared the expression of proinflammatory markers stress. in the mPFC to the control groups, after social defeat experiments. They specifically looked for changes in microglial densities, which are the primary mediators of inflammation in the brain (Allan & Rothwell, 2003). The authors quantified the microglial cells with the marker, ionized calcium-binding adapter molecule 1 (IBA1), since IBA1 expression tends to be upregulated in activated microglial cells (Sasaki et al., 2001). Furthermore, the authors also looked for changes in the levels of tumor necrosis factor alpha (TNFÎą), which is one of the proinflammatory cytokines that are released in response to microglial activation. Overall, Corbett et al. (2019) observed that S1PR3-knockout rats, who were exposed to repeated stress, exhibited increased TNFÎą and IBA-1 cell densities within regions of the mPFC, compared to non-defeated S1PR3-knockout rats and defeated iAAV-scramble controls (Figure 3). These results were in line with previous studies that reported an increase in proinflammatory responses associated with passive coping strategies (Wood et al., 2015). Overall, Corbett et al. (2019) demonstrates that reduced S1PR3 levels in the mPFC increase proinflammation in rats, which may contribute to a stress-vulnerable phenotype. Figure adapted from Corbett et al. (2019). Nature communications, 10(1), 113. Figure 2. A. Represents the mean defeat latencies in iAAV-scramble (white) and

mPFC after stress is induced. The authors further observed that resilient rats generally express greater levels of GC receptors in the ILC and PLC structures of the mPFC compared to vulnerable rats (Figure 4a and 4b), corresponding to the higher levels of S1PR3 in resilient rodents.

Figure adapted from Corbett et al. (2019). Nature communications, 10(1), 113. Figure 3. Represents the IBA1 cell densities in the IL of the mPFC of iAAVscramble (white) and iAAV-S1PR3 (red) rats, in non-defeated and defeated conditions.

These results were authenticated by performing S1PR3 knock-in experiments (AAV-S1PR3-GFP) in the mPFC of rats. Defeated S1PR3-overexpressing rats showed decreased IBA-1 and TNFα cell densities in their mPFC compared to defeated AAV-GFP controls (Corbett et al., 2019). Therefore, the results suggest that having increased levels of S1PR3 promotes resilience by decreasing inflammation induced by repeated stress exposure. If the increase in brain inflammation causes a more stressvulnerable phenotype, then attenuating the inflammation with pharmacological inhibitors should promote a more resilient phenotype. The authors injected TNFα inhibitor infliximab or the control substance, saline, via intracerebral cannula into the mPFC of iAAVS1PR3 and iAAV-scramble control rats (Corbett et al., 2019). S1PR3knockout rats injected with infliximab, showed more resilience than S1PR3-knockout rats injected with saline after social defeat experiments. Furthermore, iAAV-S1PR3 rats injected with infliximab also exhibited a reduction in depression and anxiety-like behaviour, as observed by the decreased time spent immobile in the FST and increased time spent interacting with the stimulus rat. Therefore, a reduction in mPFC S1PR3 levels increases inflammation within the brain, leading to a stress vulnerable phenotype. Nevertheless, inhibiting inflammation caused by the reduced S1PR3 in the mPFC rescues the animal to become more resilient against stressful stimuli.

Figure adapted from Corbett et al. (2019). Nature communications, 10(1), 113. Figure 4. A. Represents the abundance of GC receptors in the PLC of nondefeated, vulnerable (VUL) and resilient (RES) rats. B. Represents the abundance of GC receptors in the ILC of non-defeated, VUL and RES rats.

In summary, stress activation of GC receptors in the mPFC upregulates the expression of S1PR3, leading to the attenuation of inflammatory markers such as TNFα. Because GC receptor levels are higher in resilient rats, they have a greater expression of S1PR3 in the mPFC, which consequently increases resilience against stress.

S1PR3 & PTSD in Humans

To translate their findings to humans, Corbett et al. (2019) observed S1PR3 serum levels in combat-exposed veterans with and without PTSD. S1PR3 mRNA in the blood were utilized as a proxy for its levels in the mPFC, since there are no current in-vivo methods for S1PR3 quantification in the brain. In general, PTSD symptom severity was inversely correlated with S1PR3 serum levels. War veterans with PTSD had much lower levels of S1PR3 in their blood compared to veterans without the stress disorder. These results are in parallel with the results observed in resilient and vulnerable rats. GC Receptors in the mPFC Therefore, there is a potential for S1PR3 to be used as a diagnostic Stress exposure activates c-Fos genes within the mPFC, marker for individuals with PTSD. indicating early activation of the brain region (Ostrander et al., 2003). In addition, immunohistochemical studies demonstrated that mPFC cells expressing c-Fos genes after stress was induced, also co- CONCLUSIONS expressed GC receptors (Ostrander et al., 2013). Previous research Corbett and his colleagues (2019) were able to demonhas further demonstrated that the prefrontal cortex expresses high strate that an mPFC protein is involved in the expression of a stress levels of GC receptors (Meaney & Aitken, 1985; Ostrander et al., resilient phenotype. In particular, having increased levels of S1PR3 2013). Moreover, GC receptors have also been implicated as tran- in the mPFC, reduces stress-induced inflammation thereby proscription factors and are close in proximity to the S1PR3 gene in the moting a resilient phenotype. On the other hand, having decreased mPFC of rodents (Corbett et al., 2019; Herman et al., 2012). S1PR3 levels leads to an increase in inflammation, which induces a Corbett et al. (2019) demonstrated that knocking out GC receptors in the mPFC, with the virus iAAV-GR, led to a reduction in S1PR3 expression after stress exposure, compared to control rats that did not have GC receptor knockdown (iAAV-scramble). Therefore, GC receptors seem to regulate the expression of S1PR3 in the

more stress-vulnerable phenotype. The mechanisms by which S1PR3 is increased in the brain of resilient rats compared to vulnerable rats is by virtue of increased GC receptors. Moreover, the authors were also able to show that human veterans with PTSD exhibited less S1PR3 mRNA in their serum compared to veterans without

PTSD, indicating a potential for S1PR3 markers in the diagnosis of Moreover, the authors only observed male Sprague DawPTSD in humans. ley rats, hence, the results can only be generalized in male rat populations. Similarly, the veterans in the authors’ research only includPrevious studies in the literature have also shown that the ed one female, while the rest were males (Corbett et al., 2019). To mPFC is involved in stress regulation (Diorio et al., 1993; Franklin et understand if the same effects of the social-defeat paradigm apply al., 2012; McKlveen et al., 2015). In particular, the ACC, ILC and PLC to female populations, more females should be implemented in the regions of the mPFC tends to reduce the activation of stress-related study next time. pathways, such as the HPA axis (Franklin et al., 2012). Furthermore, it has been implicated that GC receptors are greatly expressed in the prefrontal cortices, which also partakes in stress-response regu- FUTURE DIRECTIONS – lation (Herman et al., 2012; Leonard, 2001; Ostrander et al., 2003). In extension to the research conducted by Corbett et al., Individuals with depression tends to exhibit augmented activation future studies should implement female populations, in order to of the HPA-axis activation due to increased vulnerability to stressful translate the findings of the current study to female animals. Males stimuli (Leonard, 2001). Furthermore, individuals with depression and females exhibit differences in hormones (Wood & Bhatnagar, show protracted GC levels causing desensitization of GC receptors 2015), therefore there may also be differences in their responses to in the CNS, possibly resulting in increased inflammation in the brain a stressful situation. (Leonard, 2001). Generally, studies on depression and anxiety Furthermore, rats should be placed in cages that are enshowed that individuals express greater proinflammatory cytokines, such as TNFα (Audet et al., 2011; Leonard, 2001; Tuglu et al., 2003). riched with playful and less monotonous material to occupy the The current study by Corbett et al. (2016) fits together the results animal in isolated conditions and to reduce stress-related responses from past studies to show that a protein in the mPFC is upregulated prior to the actual experiments. This may result in reduced stressby GC receptors and mediates stress resilience by reducing inflam- defeat responses in the vulnerable rat group. Furthermore, there may be an observation of decreased immobility in the FST and more mation in the brain. interaction with the stimulus rat in the social interaction test. NevTo conclude, the study conducted by Corbett et al. was ertheless, EE may not be a solution if it prevents the animals from one of the first to look at the potential role of S1PR3 within the sleeping, since cage enrichment has previously been used for sleep brain. S1PR3 in the mPFC and blood seems to increase resilience in deprivation experiments (Zucconi et al., 2006). A lack of sleep may rats and veterans without PTSD respectively. Overall, the results end up impacting the results of subsequent experiments. from this study will help to elucidate the neurobiological mechanisms involved in exhibiting resilience or vulnerability to stressful situations. Having a stress-vulnerable phenotype predisposes an individual to stress-related disorders, such as depression and PTSD. Therefore, understanding the underlying mechanisms could potentially aid in the prognosis, diagnosis and treatment of certain mental health conditions.

CRITICAL ANALYSIS There were a few redundancies with regards to the experiments that were conducted by Corbett et al. For instance, it was unknown if the rats that were subjected to viral-mediated S1PR3 knockdown or overexpression were stress-vulnerable or resilient to begin with. Moreover, the controls that were utilized to compare the experimental models might have naturally been more resilient or vulnerable to stress, which could have had an influence on the results when comparing them with the experimental groups. Depicting if the animal was originally resilient or vulnerable may a bit too tedious, nevertheless. Despite these limitations, the authors were still able to show statistical significance with most of their results. The authors also mentioned that the rats were singly housed before they were subjected to social-defeat paradigms, which might have exacerbated the social-defeat results (Corbett et al., 2019). The conditions of where a rat is placed in before experimentation can greatly influence a study’s results (Belz et al., 2013). Rats are social animals and they prefer to not be isolated. Hence, a monotonous, isolated cage may increase stress hormone levels before the experiments (Belz et al., 2013). This issue can be potentially resolved with environmental enrichment (EE); having a full cage can distract the animal from the fact that they are isolated, thereby reducing stress (Belz et al., 2013). Thus, the authors should use EE to minimize any extraneous effects to their experimental results.

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Gut Microbiome Disturbance Mediated Depression Induces GABA-A Receptor Subunit Changes in the Hippocampus. Mrinal Anagal

Many stress related disorders have been seen to be linked with intestinal dysbiosis through the gutbrain axis. Additionally, it has been shown that the inhibitory neurotransmitter known as gammaaminobutyric acid (GABA) may influence emotional behaviours and be related to depression. How early gut microbiome disturbance (GMD) influences the GABAergic system in regards to emotional disorders has not yet been elucidated. Experimenters Liang et al. (2017) altered the gut microbiome of newborn rats and through the use of a tail suspension test (TST), forced swim test (FST) and the Morris water maze (MWM), they observed depression like behaviour compared to the controls. Not only were their emotional changes observed, the effects on the GABAergic system were seen in the hippocampus, specifically the reduction of GABAsubunits. GABA-A a5 and delta subunits are said to be involved in hippocampus-dependent associative memory. In addition, GABA-A a5 and delta subunits are thought to be mediated by stress hormones, thus changes in the receptor may be related to depression. Results also showed that the administration of probiotics produced less depressive-like symptoms in rats compared to treatment with a drug called clonazepam used to treat depression. These findings help us develop a better understanding of the origins of emotional disorders in humans. In addition, it leads us step closer in the direction towards nonâ&#x20AC;&#x201C; pharmaceutical alternatives for alleviation of gut microbial disturbance induced depression. Key words: GABAome disturbance(GMD), probiotics, antidepressants, clonazepam, hippocampus

BACKGROUND Stress plays a big role in the pathogenesis of many mental disorders (Belleau, Treadway, & Pizzagalli, 2019; López, Akil, & Watson, 1999). Depression is a complex and heterogeneous disorder with a wide variety of symptoms such as fatigue, anhedonia, sleeping problems, and unhappy thoughts (Belleau et al., 2019; de Groot et al., 2000; Korczyn & Halperin, 2009). Scientists have gained an interest in this mood disorder because it currently has no known mechanisms, unclear biological markers and an unknown definitive cause (Belleau et al., 2019; Korczyn & Halperin, 2009). Catecholaminergic mechanisms to understand mood disorders was the focus of neurobiological research for many years (Bhagwagar et al., 2004). Gamma-amino butyric acid (GABA) is an inhibitory neurotransmitter in the central nervous system (CNS) that interacts with its ionotropic GABA-A receptor and its metabotropic GABA-B receptor which play an important role in regulating emotional behaviours (Bravo et al., 2011; Kalueff & Nutt, 2007). There have been numerous animal models and human studies that found low levels of GABA in the brain in depressed subjects (Bhagwagar et al., 2004; Kalueff & Nutt, 2007; Sanacora et al., 1999). Additionally, it was found that blocking the GABA-A receptor in the hippocampus produced depressionlike symptoms in mice (Kalueff & Nutt, 2007). Over the last decade there has been a rise in research regarding the gut microbiome and how it may impact health and disease (Cresci & Bawden, 2015). The millions of intestinal micro -organisms are shaped by factors such as diet, exercise and the consumption of antibiotics (Mohajeri, La Fata, Steinert, & Weber, 2018). Significant interest has been placed in understanding the gut microbiota’s link with the brain --- this is known as the gut-brain axis (Kelly et al., 2016). Alterations in the connectivity of the brain and gut have been linked to neurodevelopmental disorders such as anxiety, depression, autism, schizophrenia, and dementia (Liang, Zhou, Zhang, Yuan, & Wu, 2017). In the research study by Liang et al., published in 2017, the experimenters examined neuronal samples from the hippocampus of juvenile and adult rats (Liang et al., 2017). They detected GABA-A receptor positive cells and examined their a5 and d subunits (Liang et al., 2017). Using 5 groups of rats with varying gut microbiota, they were able to visualize GABA-A receptor subunit changes via q-PCR and immunohistochemistry (Liang et al., 2017). Depression in the rats with an altered gut microbiota was evident and was tested using the tail suspension test (TST), forced swim test (FST) and the Morris water maze (MWM) (Liang et al., 2017). Results showed that adult rats with an affected gut microbial composition had increased depressive symptoms ac-

companied by a decrease in the a5 and d subunits in their hippocampus (Liang et al., 2017). Links between the intestinal flora and the GABA receptors have been explored through other studies which showed that patients that were given Lactobacillus rhamnosus bacteria reduced GABA-A receptor subunits in the prefrontal cortex and amygdala, but increased its expression in the hippocampus (Bravo et al., 2011). In addition, Liang et al.’s (2017) findings showed that when comparing 2 rats with gut microbial disturbance, the rat that was administered with probiotics showed reduced depressive symptoms than the rat given clonazepam, a drug used to treat depression (Liang et al., 2017). Similar to these results, animal and human studies have shown that the administration of different strains of Lactobacillus into depressed individuals improved mood and decreased the amount of serotonin and dopamine being metabolized in different areas of the brain suggesting that probiotics may carry antidepressant properties (Benton, Williams, & Brown, 2007; Rao et al., 2009; Tabouy et al., 2018). The current research published by Liang and his colleagues in 2017 is relevant because provides a possible nonpharmaceutical depression treatment and also provides greater insight to the field of the gut-brain axis and mood disorders (Liang et al., 2017).

MAJOR RESULTS Although it is known through previous studies that the hippocampus is an area of the brain affected by emotional destress, little is known how gut microbiome mediated depression plays a role in GABA-A receptor subunits in the hippocampus (Liang et al., 2017). In Liang et. al.’s research (2017), newborn Sprague Dawley rats were divided into 5 groups that each received varied treatments to alter their gut microbiome (Liang et al., 2017). Rats were fed ampicillin (antibiotic) to alter their gut microbiome composition (GMD) (Liang et al., 2017). Some of the rats undergoing gut microbiome disturbance were fed Lactobacillus rhamnosus, a probiotic (GMD+P), whilst others were given clonazepam, an antidepressant (GMD+D) (Liang et al., 2017). The other two groups were fed saline as a negative control (NC) or were given no treatment serving as a blank control (BC) (Liang et al., 2017). Through behavioural tests including TST, FST and MWM, the rats with an altered gut microbiome were seen to exhibit depressive-like behaviours (Liang et al., 2017). One of the major findings of this research was that rats with gut microbial disturbance saw a decrease in their a5 and d subunit of the GABA-A receptor in the hippocampus (Liang et al., 2017). Another major finding showed that the probiotic reduced depressive symptoms more than the clonazepam drug in rats with

altered gut bacteria compositions (Liang et al., 2017). These findings suggest that probiotics may be a valid candidate for mood disorder therapies (Liang et al., 2017). In addition, this may open doors to exploring subunit-specific GABAergic drugs as a cure for neurobiological mental illnesses (Kalueff & Nutt, 2007). Gut Microbiome and Depression: Behavioural Tests

tered levels of bacterial composition in their gut and the Shank3 gene is involved in neurodevelopmental disorders (Tabouy et al., 2018). One bacterial species they saw an abundance of in the gut microbiome of these KO mice was Lactobacillus reuteri (L.reuteri) (Tabouy et al., 2018). The authors were able to show that as L.reuteri increased in numbers, the expression of hippocampal GABA subunits also increased (Figure 2.2) (Tabouy et al., 2018). They also saw a reduction in the abnormal behaviours in the mice that were treated with L.reuteri (Tabouy et al., 2018). The results of this study coincides with the major findings of Liang et al.’s (2017) research as Tabouy et al. (2018) shows an association between altered gut microbiota and the GABAergic system in the hippocampus (Tabouy et al., 2018). However, the key difference between both experiments is that Liang et al. (2017) did not examine specific bacterial genera and species in the gut microbiota that may be contributing to the GABA-A receptor subunit changes (Liang et al., 2017; Tabouy et al., 2018). Additionally, studies have shown that genes encoding the a5 subunit in the GABA-A receptor is linked with cognitive functions (Kalueff & Nutt, 2007). Moreover, other subunits in the GABA-A receptor have also exhibited changes as seen through genetic studies in mice showing an association between a1, a6 and d2 subunits (Kalueff & Nutt, 2007).

To test if the rats in each group had depression, 3 behavioural tests were administered: TST, FST and MWM. Results showed that in the TST and FST, the immobility time in rats with altered gut microbiota was much longer than the NC and BC groups (Liang et al., 2017). An increase in immobility time in these tests represent depressive-like states (Zheng et al., 2016). Similar results were seen in a study published in 2016 by Zheng et al. (2016). where after showing a difference in bacterial compositions between the microbiota between major depressive disorder (MDD) patients and healthy patients, his team transferred the gut microbiome from human patients with (MDD) into germ-free (GF) rats (Zheng et al., 2016). They were able to show that mice with the “depression microbiota” exhibited longer immobility times in the TST and FST than mice with the “healthy microbiota” (Figure 1) (Zheng et al., 2016). These results align with the results of Liang et al.’s (2017) behavioural tests suggesting that the TST and FST may be reliable tests of depression in animal models if altered gut microbiome is present (Liang et al., 2017; Zheng et al., 2016). Additionally, in Liang et al.’s (2017) study, the MWM test was also used to further assess depressive symptoms in the rats (Liang et al., 2017). Results showed that overall, rats with altered gut microbiota show reduced spatial memory than NC and BC rats which is also associated with depression (Liang et al., 2017). These results suggest that GMD in juvenile rats is associated with depression (Liang Fig. 2.1. Overall expression of GABA-A receptor subunits in the hipet al., 2017). pocampus of adult rats. A) d subunit expression in the dentate gyrus (DG) of the hippocampus seen lowest in rats with gut microbial disturbances. B) a5 subunit expression in the commissure (CA3) region of the hippocampus seen lowest in rats with altered gut microbiome. Figure adapted from Liang et al. 2017.

Fig. 1. Behavioural test comparison in a) Forced swim test (FST). FST shows significant increase in immobility time (seconds) in mice containing “depression microbiota”. b) Tail suspension test (TST). Increase of duration of immobility in TST seen in mice containing Fig. 2.2. GABA receptor gene expression positively correlated with “depression microbiota”. Figure adapted from Zheng et al. 2016. levels of L.reuteri bacteria in Shank3 KO mice. B-D) As L.reuteri levels rise, there is a significant increase in the GABA receptor subuGut Microbiome and the GABAergic System nit expression. Figure adapted from Tabouy et al. 2018. Studies have shown that a dysfunctional GABAergic system is associated with mood disorders (Kalueff & Nutt, 2007). Liang et al.’s (2017) experimenters were able to show a change in the GABA-A receptor subunits in the hippocampus in adult rats exhibiting depressive symptoms and an altered gut microbiome as explained in figure 2.1 (Liang et al., 2017). A study published by Tabouy et al. (2018) explored a connection between an impairment in gut bacteria genera and GABA receptor subunits located in the brain (Tabouy et al., 2018). The authors used Shank3 knockout (KO) rats because these rats exhibited abnormal behaviours, al-

Probiotics: Potential Antidepressant Therapy for Gut Microbiome Mediated Depression? Probiotics are live, beneficial microbes to the host that are able to occupy the gut (Rao et al., 2009). Scientists are always looking for alternative theories to treat depression and there has been recent interest in the potential of probiotics on improving brain health and function (Benton et al., 2007; Rao et al., 2009) . Neufeld et al. (2018) did an experiment on two different strains of mice

symptoms assessed by the tail suspension test (McVey Neufeld, Kay, & Bienenstock, 2018). They compared the effects of the probiotic Lactobacillus rhamnosus (L.rhamnosus) and the antidepressant fluoxetine on depressive symptoms in these different strains to examine whether probiotics result in the same beneficial effects as antidepressants do (McVey Neufeld et al., 2018). The tail suspension test showed a reduction in depressive symptoms (decrease in immobility time) only in the BALB/c strain after the use of both the probiotic and the antidepressant suggesting that antidepressant properties may be strain specific (Figure 3.1) (McVey Neufeld et al., 2018). However, a significant difference in the effectiveness of the probiotic over the antidepressant or viceversa was not observed (McVey Neufeld et al., 2018). This differs from Liang et al.â&#x20AC;&#x2122;s (2017) study because in their behavioural tests, the GMD rats that had the antidepressant clonazepam administered in them showed more depressive symptoms than the probiotic-fed GMD rats suggesting that the probiotic was more effective in reducing depressive symptoms than the antidepressant (Figure 3.2) (Liang et al., 2017). However, both studies demonstrate that the L.rhamnosus probiotic carries antidepressant properties (Liang et al., 2017; McVey Neufeld et al., 2018). Although both the probiotic and clonazepam reduce depressive symptoms, the probiotic deems significantly more effective at reducing depressive symptoms in GMD rats than clonazepam (Figure 3.2) (Liang et al., 2017; McVey Neufeld et al., 2018).

CONCLUSIONS and DISCUSSION The current study by Liang et al. (2017) aimed to examine the alterations in GABA-A receptor subunits involved in the pathogenicity of altered gut microbiome in emotionally distressed juvenile rats (Liang et al., 2017). Evidence was in favour of their first hypothesis that changes would occur in the a5 and d subunits of the GABA-A receptor in the hippocampus as a reduction in both these subunits was observed in adult rats with gut microbiome induced depression (Liang et al., 2017). Additionally, results were in favour for their second hypothesis that GABA-A subunit densities would be increased by probiotics and clonazepam. (Liang et al., 2017). Significant improvements were seen in depressive symptoms in GMD rats treated with probiotics and clonazepam compared to GMD rats alone (Liang et al., 2017). In fact, probiotics showed more improvement in depressive symptoms than the clonazepam did (Liang et al., 2017). Moreover, the possibility of creating drugs targeted towards GABA-A receptor subunits could be explored through these new found results. The authors mention that their findings correspond with many studies in the field relating to probiotics and mood disorders as well as the GABAergic system and emotional behaviours (Liang et al., 2017). This study takes it a step further by linking these topics together to find an association not only between the gut microbiome and the GABAergic system, but with probiotics and GABA receptors (Liang et al., 2017). Unlike other pieces of literature in the field, Liang et al.â&#x20AC;&#x2122;s (2017) research has observed probiotics being a more successful treatment for depression than antidepressants, thus providing probiotics as a possible alternative non-pharmaceutical therapy for depression (Liang et al., 2017). CRITICAL ANALYSIS

Figure 3.1. Effects of feeding Lactobacillus rhamnosus (JB-1) probiotic or fluoxetine antidepressant to BALB/c and Swiss Webster strains of mice on immobility time in tail suspension test. Both JB-1 and fluoxetine significantly reduced immobility time, thus reducing depressive symptoms in BALB/c mice, but a significant effect was not seen in Swiss Webster mice. Figure adapted from Neufeld et al. 2018.

Figure 3.2. Morris water maze represents spatial memory. Probiotic administered GMD rats (GMD+P) have improved A) latency and B) distance to arrive at the target platform, have C) increased duration spent in the quadrant and D) they cross the platform more frequently compared to clonazepam administered GMD rats. Although both Figure adapted from Liang et al. 2017.

Liang et al. (2017) presented data showing changes in GABA-A receptor subunits as a result of gut microbial disturbance mediated depression (Liang et al., 2017). These findings are consistent with other studies regarding the role of the gut microbiome on neurobiological changes in the brain (Cresci & Bawden, 2015; Kaelberer et al., 2018; Kelly et al., 2016; Liang et al., 2017; Mohajeri et al., 2018; Zheng et al., 2016). Similar to depression, the gut microbiome has been associated with other disorders related to behaviour such as anxiety, autism, schizophrenia and bipolar disorder (Liang et al., 2017). However, since mood disorders involve more than just one neuronal system, the authors should have observed changes in other receptor types such as the glucocorticoid and glutamate receptors as they are present at high levels in the hippocampus (Liu et al., 2017). Though the hippocampus is known to be involved in mood disorders, depression is not localized to one brain region, thus they should have examined the effects in GABA receptors in different brain regions affected by depression such as the prefrontal cortex and amygdala (Liu et al., 2017). Through this, it would be interesting to see if similar subunit changes would be observed in these areas (Liu et al., 2017). Research by Neufeld et al. (2018) found that probiotics and antidepressants only produced positive effects in one out of the two strains of mice (McVey Neufeld et al., 2018). Due to this, Liang et alâ&#x20AC;&#x2122;s (2017) study should have used more than one strain of rats to explore the possibility that GABA-A receptor subunit changes could be strain specific. A greater emphasis should have been put on the role of probiotics and antidepressants on the GABA-A receptor subunits through a separate experiment comparing the effects of

the probiotics and antidepressants in GMD rats and healthy rats. Further research should be conducted to observe long term effects of the probiotic and clonazepam as it is currently unclear if the effectiveness of probiotics over clonazepam is maintained for longer periods of time.

difference in bacterial genera and species compositions, thus raising questions about if the type of bacterial species would elicit similar responses in the GABAergic system. If an abundance or reduction in a certain species associated with depressive or antidepressant symptoms in the brain is seen in humans and not in rats, it would mean that more research would need to be conducted to The focus of many depression studies involve the use of find probiotic treatments more relevant and beneficial to the hudepression-induced animal models which poses many issues since man gut microbiome. many aspects of human behavioural and cognitive functions cannot be reproduced in animals (Kalueff & Nutt, 2007). For example, Lastly, experiments could be done to see if environmental studies have shown that antidepressant effects seen in animal factors other than consuming probiotics could be possible nonmodels do not match the action of these drugs in humans (Kalueff pharmaceutical methods to increase a5 and d subunits in the & Nutt, 2007). GABA-A receptor. This could be done through building on the association between exercise and the gut microbiome. Research has This study simply altered the gut microbiome without shown that exercise alters the gut microbiome and leads to an observing the changes in the levels of the types of bacteria after increase in synaptic plasticity (Allen et al., 2018). Using GMD rats, gut microbial disturbance. The authors should have analyzed the adding the factor of exercise in a separate group of rats along with species present in control rats as well as GMD rats before and after the probiotic and clonazepam administered group, it would be administration of the probiotic and clonazepam to better under- interesting to see the effectiveness of exercise on the changes in stand the role the gut microbiome plays in their results. the GABA-A receptor subunits as compared to the probiotic and antidepressant. The authors did not express the extent of how long subunit downregulation was maintained. This is important because if the downregulation of subunits is not maintained longer in those with GMD alone as compared to those than those with GMD+P or GMD+D, it could contradict all of the results seen in the paper. FUTURE DIRECTIONS A major gap this paper and accompanying literature have is the failure to show a mechanism of action of how the gut bacteria causes changes to the neurobiological systems of the brain. Many studies have shown that the vagus nerve mediates the gut-brain axis (Kaelberer et al., 2018). An ideal experiment that could be done to test if gut microbial disturbances directly influences the GABA-A receptor subunits could be to vagomatize the GMD rats. If the a5 and d subunit mRNA and density reductions are still observed, it would suggest strong evidence for an association between gut microbiome and brain function. On the contrary, if the subunit expression is maintained at the level despite the rats having an altered gut microbiome, it would show that the gut microbiome may have indirect effects on the GABAergic system, thus requiring further research. Depression is found to affect multiple areas of the brain (Jesulola, Sharpley, & Agnew, 2017). This current study focused on the changes elicited in the hippocampus of adult rats (Liang et al., 2017). It would be interesting to see if other areas of the brain such as a limbic system and prefrontal cortex exhibit the same GABA receptor subunit changes there thus providing a deeper understanding of the relationship between the gut microbiome and the GABAergic system. As mentioned previously, rats and humans differ in their behaviour and cognition (Kalueff & Nutt, 2007). An experiment to better understand the human microbial effects on the brain can be done via transplanting fecal microbiota from human patients with major depressive disorder (MDD) into germ-free (GF) rats. Then, researchers can obtain fecal pellets from the rats with â&#x20AC;&#x153;humanized microbiotaâ&#x20AC;? and analyze it through 16S ribosomal RNA sequencing. Comparisons between rat and human microbiota can show a

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Alzheimer’s Disease: Targeting Neuroinflammation with Minocycline Leena Attyani

Alzheimer’s Disease (AD) is a progressive neurodegenerative disease affecting cognition and visuospatial orientation. Since its discovery in the early 20th century, several pathologies have been characterized including cholinergic neuron dysfunction, extracellular amyloid-beta (Aβ) plaques, and neuroinflammation. Currently, the only drugs approved by the US Food and Drug Administration (FDA) include, but are not limited to, acetylcholinesterase inhibitors such as Donepezil, Galantamine, and Rivastigmine – these treatments, however, solely act to alleviate the patients’ symptoms, and decelerate the disease-progression without achieving a definite cure. Thus, there is an imperative need to investigate other AD targets, such as neuroinflammation, in hopes of generating more effective therapeutic approaches. As a result, some research studies proposed minocycline, a tetracycline antibiotic that also exerts anti-inflammatory and neuroprotective factors, as a potential AD therapeutic. In the study conducted by Garcez et. al. (2017), minocycline is used on AD-like mice models (induced by Aβ (1-42) oligomers) in order to assess its effect on memory performance and cytokine levels (in serum, cortex, and hippocampus). The main results of minocycline treatment in AD mice include memory improvements; reversal of increases of inter-leukin 1β (IL-1β), IL-10, and tumor necrosis factor α (TNF-α) in the hippocampus; reversal of increases of IL-1β, IL-4, and TNFα in the cortex; and reversal of increases of IL-1β and IL-4 in serum. These results are critical as they show minocycline-induced reversals of AD memory impairments and inflammation, affirming its efficacy as an AD drug. The study also demonstrates direct associations between cognitive decline and cytokine production in the hippocampus – further emphasizing the neurodegenerative effects of neuroinflammation. Key words: Alzheimer’s disease, neurodegenerative, amyloid-beta plaques, neuroinflammation, minocycline, inter-leukin (IL-1β, IL-4), and tumor necrosis factor α (TNF-α), hippocampus, cortex, serum.

has targeted neuroinflammation in hopes to generate novel and effective therapeutic approaches against AD. Minocycline is a tetracycline antibiotic with neuroprotective effects that acts to inhibit M1 cells, apoptosis, and reactive oxidative species (Garrido-Mesa et al., 2013). The effectiveness of minocycline as a therapeutic agent for neurodegenerative disease – including AD – is actively being studied amongst researchers. In the study conducted by Garcez et. al. (2017), minocycline is used on AD-like mice models (induced by Aβ (142) oligomers) to evaluate its effectiveness in repairing memory impairments and neuroinflammation. Minocycline treatment was observed to stimulate the reversal of memory deficits; reversals of elevated IL-1β, IL-10, TNF-α in the hippocampus; reversals of elevated IL-1β, IL-4, and TNF- α in the cortex, and IL-1β and IL-4 in serum. Garcez et. al (2017) additionally displayed BACKGROUND or INTRODUCTION direct associations between neuroinflammation and cognitive decline. Collectively, these findings demonstrate the effectiveness of minocycline therapy on AD by reducing neuroinflammaAlzheimer’s Disease (AD) is a progressive neurodegenerative disorder that causes severe cognitive deficits and tion, and subsequently reducing memory deficits. This provides visuospatial disorientation. Since its discovery in the early 20th further evidence towards the efficacy of minocycline, or other century, several pathologies have been characterized including neuroinflammatory targets, as an AD drug. cortical and hippocampal atrophy, interrupted-cholinergic neurotransmission, intracellular neurofibrillary tangles (NFT), extra- MAJOR RESULTS cellular amyloid-beta (Aβ) senile plaques, and neuroinflammaIn Garcez et al’s research (2017), male BALB/c tion (Ryan et al., 2015). Currently, some of the only available mice were randomized into four groups. The first two groups treatments of AD approved by the US Food and Drug Admin- received water or minocycline through the oral gavage after 24 istration (FDA) include three different acetylcholinesterase inhib- hours from i.c.v. injections of Artificial Cerebrospinal Fluid itors – Donepezil, Galantamine and Rivastigmine (Mehta et al., (ACSF); whereas the latter two received water or minocycline 2011). These drugs are based on one of the earliest hypotheses through the oral gavage after 24 hours from i.c.v. injections of of AD, the Cholinergic hypothesis, that focuses on the role of Aβ (1-42) oligomers. Minocycline (or water) treatment where acetylcholine in memory and cognition (Hampel et al., 2018). given once a day for 17 days. To assess their memory perforThese drugs, however, show moderate involvement in deceler- mance, mice were subjected to a radial-arm maze task (food ating the disease progression and alleviating the patients’ symp- were placed on 4 of 8 arms) on 4 consecutive days starting from toms without attaining a definitive cure. As such, there is a wide- the 14th day of minocycline treatment. Compared to ACSF conspread interest, and an urgent need amongst researchers to trols, the results of this test demonstrate that AD (with the addifurther investigate differing AD targets to understand the under- tion of water) had greater memory impairments in finding food lying basis of this disease pathology, and thereby generate more (on the basis of the ‘latency to find food’ measurement). Minoeffective therapeutic approaches. Some studies have explored cycline, however, was observed to revert that impairment. AD the role of Aβ plaques (from Aβ (1-42) oligomers) in neuroin- (water) mice also showed greater ‘total errors to find food’ comflammation induction. Aβ (1-42) oligomers – produced by inter- pared to ACSF mice; minocycline was also able to revert that ADplays among neurons and astrocytes – have been observed to induced effect. Minocycline also restored working memory promote NFT, and neurotoxicity (Heppner et al., 2015). As the (amount of times a mouse entered an ‘arm’ loaded with food disease progresses, Aβ plaques appear to activate microglial that has been previously entered) and reference memory cells (M1) which induce the secretion of pro-inflammatory cyto- (amount of time a mouse entered an arm that did not contain kines including inter-leukin 1β (IL-1β), and tumor necrosis factor food) in AD mice (Figure 1). After the radial-arm maze task, seα (TNF-α). These cytokines, in turn, promote further synthesis rum was collected, and both the hippocampus and cortex were and secretion of Aβ (1-42) oligomers (Kumar et al., 2015). Addi- dissected to measure differences in cytokine levels. AD (water) tionally, anti-inflammatory cytokines (IL-10 and IL-4) have also mice – compared to ACSF – had elevated levels of Il-1β in hippobeen shown to elevate in response to Aβ plaques in AD patients; campus, cortex, and serum (Figure 2); elevated TNF-α in hippohowever, their role in AD is controversial as some studies that campus, and cortex (Figure 3); elevated Il-4 in cortex and serum have explored the anti-inflammatory effects of IL-4 or Il-10 as (Figure 4); and elevated Il-10 in in hippocampus and serum potential AD drugs show conflicting results in which they may (Figure 5). Minocycline treatment was successful in reverting exacerbate disease conditions (Zheng et al., 2016). Nevertheless, most of these AD-induce upregulated cytokines, except for IL-10 experimental studies involving intracerebroventricular (i.c.v.) in the serum (it remained elevated). administration of Aβ (1-42) oligomers (in mice) have shown to In Garcez et. al’s (2017) study, increased task-errors and produce these similar outcomes of cytokine production and cognitive deficits (Kumar et al., 2015). As a result, recent research memory-impairments of AD (water) mice in comparision to the 25

ACSF controls shows that Aβ (1-42) oligomers induction was successful in introducing the general cognitive and visuospatial deficits characteristic of AD patients. Additionally, authors showed that – compared to ACSF controls – Aβ (1-42) oligomers increased levels of key cytokines similar to that observed in AD patients. These results, therefore, reaffirm the induction of Aβ (1-42) oligomers as a suitable model of AD. Minocycline treatment on these established AD models not only shows expected neuroinflammation reductions, but also shows visuospatial-orientation improvements – suggesting a direct association between neuroinflammation and cognitive deficits as reducing neuroinflammation helps to ameliorate memory deteriorations. Collectively, these results help reinforce the efficacy of minocycline as a therapeutic for AD patients.

CONCLUSIONS/DISCUSSION Neuroinflammation has been targeted in many research studies as a key contributor to the pathogenesis in AD (Hepner, 2015). As such, several studies have focused on the utilization of anti-inflammatory agents as potential AD therapeutics. Notably, minocycline, a tetracycline antibiotic, has been considered a suitable contender for anti-inflammatory directed AD-treatment for its well characterized neuroprotective factors (Garrido-Mesa, 2013). In Gracez et al’s (2017) study, minocycline was observed to exert anti-inflammatory effects, and reverse spatial memory deficits in Aβ (1-42) oligomer, ADinduced mice. A direct association was also observed between elevated cytokine levels (in the hippocampus) to cognitive decline (in radial-arm maze test) – reinforcing the neurodegenerative role of neuroinflammation. Authors of this study concluded that their results largely aligned with other literature studies involving minocycline administration. For instance, rodent AD models demonstrate similar reversals of spatial-memory deficit administration. However, the authors also revealed few studies that show contrary findings – A study using ‘water maze tests’ to measure spatial memory show no improvements with minocycline treatment. Other studies also illustrate similar reversal effects of minocycline on pro and anti-inflammatory cytokines; however, Garcez et al (2017) revealed that this was the first study to demonstrate these effects on Aβ (1-42) oligomer induced AD-like models in mice, as other studies have utilized other AD-models including Tau-transgenic mice. Overall, these results establish the role of minocycline or other antiinflammatory agents as potential AD therapeutic agents by their ability to reverse Aβ-plaque induced cytokine increases and memory deficits. Critical Analysis In Garcez et al’s (2017) study, minocycline treatment largely reversed cytokine elevations and spatial impairments of AD-induced mice models. This generally aligned with metaanalytic studies exploring minocycline’s impact on neuroinflammation and memory-impairments (Li et al., 2013). One study, however, shows conflicting results as they purport relatively no benefits of minocycline on memory-deficits in AD-like models

(Seabrook, 2006). Nonetheless, Garcez et al’s (2017) findings largely complement current literature and reinforce minocycline’s efficacy as a potential AD therapeutic. Still, these results do not accomplish the goal of identifying a ‘definitive’ cure to AD as the authors of that study do not investigate potential pathways by which minocycline restores cytokine levels and enhances cognitive performance. Thus, the study lacks substantial evidence to the stability and durability of minocycline’s effectiveness as an AD-drug. Garcez et. al (2017) examined only the effect of minocycline on cytokine levels, and their subsequent influence on memory – however, no examination of microglial cells morphology or activity levels were performed. Thus, it is not clear whether minocycline directly suppresses secreted cytokines by destroying those soluble molecules, or indirectly inhibits cytokine secretion by targeting and inactivating their source – microglial cells. Targeting cytokines directly, rather that microglial cells, creates a relatively temporary property to minocycline-induced AD therapeutic, making it none the better than that of the current approved drugs. Additionally, Garcez et al’s (2017) study did not consider the effects of minocycline on Aβ plaques – the trigger to microglial activation, and therefore cytokine release. If minocycline does not affect (or rather exacerbates) Aβ plaques, this would also reduce the durability and value of minocycline-induced AD therapeutic. As a result, further work is needed to elucidate minocycline’s anti-inflammatory pathways, and their influence on Aβ plaques and microglial activities in order to verify minocycline’s potential in AD therapy.

Future Directions In Garcez et al’s (2017) study, hippocampal and cortical dissections of the four groups were examined for relative differences in cytokine levels. For future researches, it would be advised to take that investigation a step further and examine microglial differences in both activity and morphology within those brain-dissections. This may be performed through immunohistochemical (IA) and morphological (MA) assays. For IA, antibodies against upregulated molecules in M1 cells (relative to M2 cells) may be utilized. These molecules may include cluster of differentiation (CD) 11b and 45, ionizing calcium-binding adaptor molecule 1 (Iba1), Major histocompatibility molecule (MHC) class II and its associated accessory molecules (B7.1 and B7.2) (Biscaro et. al., 2012). If the AD-mice (water) show increased expression of these molecules, this would be indictive of an increase in activated microglia induced by Aβ (1-42) oligomers. Additionally, if AD-minocycline treated mice show decreased expression in this IA, this would be indictive of minocycline’s induced inhibition of microglial cells which reduces cytokine levels. If this were true, this would enhance minocycline’s effectiveness as an AD therapeutic. However, if expression levels are not decreased in AD-minocycline treated mice, this would indicate that minocycline works by directly suppressing cytokine molecules – diminishing minocycline’s effectiveness in AD-therapy as microglial cells remain activated, and therefore persist in their neuroinflammatory capacity. IA results may be

the dissected brain components by injecting fluores- water mice group. Figure Adapted from Garcez et al. (2017). Prog cent dyes (e.g. Lucifer Yellow) to aid in cells’ morphological vis- Neuropsychopharmacol Biol Psychiatry, 3;77:23-31 ualization (Kettenman et al., 2011). Smaller number and shorter lengths of microglial branches are reported in M1 cells. If AD (water) mice report with these characteristics, this would also provide indication to M1 activity, and cytokine release. Additionally, if AD-minocycline treated mice show contrary features (longer and increased branches, M2 cells), this would be indictive of minocycline’s direct inhibition of microglial cells, reducing cytokine levels. However, persistence of M1 features in ADminocycline mice would be indictive of minocycline’s direct suppression of cytokines – diminishing its role in AD-therapy. Finally, it would also be advised to examine the effect of minocycline on Aβ plaques as they trigger M1 cells, and cytokine release. IAs using antibodies against Aβ plaques markers (e.g. DW14 and R1282) may demonstrate relative plaque depositions (Seabrook et. al., 2006). Declined expression of these markers in AD-minocycline mice would be indictive of minocycline’s role plaque degradation, which in turn lessen M1 cells and their associated cytokines. However, plaque markers are not reduced in AD-minocycline mice, this would diminish minocycline’s effectiveness as an AD-therapeutic as Aβ plaques remain capable in inducing M1 formation, and cytokine release. By examining minocycline’s effect on microglial cells and Aβ plaques, experimenters would be able to dissect minocycline’s anti-inflammatory pathway. Thus, providing more information of the stability or durability of minocycline in AD therapy and whether it is capable of providing more than symptomatic relief to the disease pathogenesis.

Figure 1. Radial-Arm Maze Results for Four Mice-Groups. The circle signifies ASCF and water treated mice. The square signifies ASCF and minocycline treated mice. The upward triangle signifies Aβ (1-42) and water treated mice. The downward triangle signifies Aβ (1-42) and minocycline treated mice. A. Measures the Total errors to find food; there is a significant increase in the Aβ (1-42) and water mice group. B. Measures the latency to find food; there is a significant increase in the Aβ (142) and water mice group. C. Measures the Reference memory errors (amount of time a mouse entered an arm that did not contain food); there is a significant increase in Aβ (1-42) and water mice group. D. Measures the working memory errors (amount of times a mouse entered an ‘arm’ loaded with food that has been previously entered); there is a significant increase in Aβ (1-42) and

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Examining the use of rapid eye movement sleep deprivation as antidepressant therapy in OBX rats Jihyeun Angela Baek

In patients with depression, an already-known side-effect of some prescribed antidepressant medications is the reduction of the Rapid Eye Movement (REM) sleep stage. In the paper “REM Sleep Deprivation Reverses Neurochemical and Other Depressive-Like Alterations Induced by Olfactory Bulbectomy” by Maturana et al. (2015), the authors wanted to find if reducing the REM sleep stage first would have any tangible neurochemical effect on the depressed brain, and on the observable behaviour of a depressed animal. In order to test their hypothesis, they utilized the OBX rat model (rodent artificially induced for depression via olfactory bulbectomy) to represent depression, along with other animal controls. Some of the subjects, including some OBX and some controls, underwent Rapid Eye Movement Sleep Deprivation (REMSD). Later, all subjects underwent a series of behaviour tests, as well as a rebound period (REB), and then further testing. Immunoassay was performed on the rats’ brain tissue. After analyses, it was found that the OBX rats that had undergone REMSD showed behavioural signs of reduced depression, and had higher levels of hippocampal serotonin (5-HT) and serotonin metabolite products, as well as increased brain-derived neurotrophic factor (BDNF) levels in the substantia nigra pars compacta (SNpc). These results led the authors to conclude REMSD does indeed have an effect both neurochemically and behaviourally in the animal model of depression OBX. Thus, REMSD has some potential to become a useful method in depression therapy procedures in the future, whether utilized in treatment solely or in conjunction with other therapeutic methods. Key words: depression therapy, antidepressant mechanism, sleep deprivation , rapid eye movement sleep deprivation, Rebound, hippocampus, serotonin , brain-derived neurotrophic factor, substantia nigra pars compacta, olfactory bulbectomy

The researchers were aware of a paper which concluded that REMSD caused by some depression medications as a “side effect” is actually the functional aspect of antidepressants and the reason why mood can be improved and depression is reduced in depressed patients (Vogel, 1983). Additionally, more recent papers like Clark et al., 2000, McNamara et al., 2010, and McCarthy et al., 2016 continued to show that pharmacological antidepressants reduced or inhibited REM sleep in individuals taking these kinds of medications. Clark et al. found that antidepressants were more effective in patients who happened to have reduced density in their REM sleep. The authors thought however that the area of looking into sleep deprivation in general as a treatment for depression was still very much unexplored. They thought not enough research had been completed, especially regarding the neurochemistry behind those kinds of claims. So, using reverse logic, Maturana et al. creatively asked themselves whether a depressed animal’s mood and depressed state could be improved by depriving it of the REM stage in natural sleep first, mimicking some antidepressants’ side effects and testing Vogel’s results. Thus, they undertook the task to further explore REMSD’s potential behavioural and neurochemical effects on the rat brain and against mental health depression. To answer this question, they used OBX rats – rats artificially induced to have the signs and symptoms of depression via stereotactic surgical removal of their olfactory bulbs. The scientists particularly wanted to discover what neurochemical changes might occur if REMSD was implemented in such animals. Rats were divided into four groups. Two groups underwent REMSD (treatment) while two groups acted as controls and did not. One of the treatment groups were OBX rats (the animal model for depression in this study) and the other treatment group were “Sham”, or non-OBX rats. The first control group that did not undergo REMSD was comprised of a different group of OBX rats, and the second control group that also did not undergo REMSD was comprised of another group of “Sham” rats. The method used to inflict REMSD on the treatment groups was through the “single platform method” – utilization of a circular, disk-shaped platform that is 6.5 cm in diameter, a size known to fully make REM sleep impossible for rats without them falling into the water. After the treatment time, all groups of rats were subjected to various performance tests to observe their level of behaviour, and each group’s scores for the various tests were compared with each other. The two tests the rats underwent were the open-field test and the forced swimming test, with these tests measuring points such as distance travelled and mean velocity in the open-field test, and swimming time, immobility time, and climbing time in the forced swimming test. Rats were considered “less depressed” if they had lower distance

travelled and mean velocity in the open-field test, and if they had higher swimming and climbing times, and lower immobility time in the forced swimming test. Aside from these performance tests, some of the rats in each group were sacrificed (with scientists abiding by ethical lab practices) and immunoassays were performed on their brain tissue to quantify the levels of dopamine (DA), noradrenaline (NA), 5-HT, and these neurochemicals’ metabolites present in the striatum, and also in the hippocampus. Also, BDNF levels from the rats’ SNpc were quantified. Lastly, there is a “part two” to this research. In order to test if the potential antidepressant implications of REMSD could be long-lasting and not temporary, all measurements were taken again after a certain REB period had passed. The researchers took into consideration the existence of a rebound period for their rats – a time when possibly the REMSD previously implemented no longer has impact on the brain, or the possibility that such an effect would wear off. They wanted to test if REMSD could continue to have long-term effects on the rats. MAJOR RESULTS The key results they found for the OBX rat treatment group were an increase in BDNF levels in the SNpc area of the brain when compared to the control OBX rat group. Additional results arising from this fact was that this increase in BDNF levels within the SNpc of the OBX rats that underwent REMSD was found to be, statistically, strongly correlated with rats’ swimming time/willingness to keep swimming, and also with their level of serotonin/5-HT, the “happy chemical/neurotransmitter”, in their hippocampi – i.e., serotonin levels in the hippocampus were higher for OBX rats that were REM sleep=deprived. Another additional strong statistical relationship they found was between serotonin levels and swimming time, further confirming the importance and role of serotonin for brain and mental mood health. As for the “lasting power” of REMSD beyond the REB period, their results made them believe that yes, REMSD’s changes to the brain had a prolonged and positive effect on the OBX treatment group. Neurochemically, the BDNF levels in the SNpc remained as they were compared to before the REB period, and the serotonin levels in the hippocampus also remained at the same level as before the REB period. Also, the rats’ observed activity through a further swim test continued to show similar results after the rebound period had passed, and made the researchers notice the effects of REMSD showed a more permanent effect. All these significant observations they made, as well as the statistical correlations and tests they calculated made Maturana et al. ultimately conclude REMSD does indeed have an

antidepressant function on their depressed animal model, OBX, inducing events. Lastly, Nielsen et al. in 2010 found that lower and one that is lasting at that, and could be one more mode of amounts of REM sleep as well as disrupting it actually increased fighting depression in the future and potentially be used as ther- the frequency of nightmares in people. apy. However, on an excellent note, Maturana et al.’s findings on the neurochemical effects of REMSD bring valuable supDISCUSSION porting evidence to this area of neuroscience. The researchers’ There are some questionable aspects to this paper’s findings of increased hippocampal 5-HT and 5-HT metabolite research design which make its findings ultimately limited and levels, as well as increased nigral BDNF levels concur with other thus debatable, at least regarding the antidepressant properties teams’ research. Maturana et al.’s findings on increased nigral of REMSD argued by the researchers. For instance, the study BDNF levels are somewhat supported by Gorgulu and Caliyurt’s design is done in a “group comparison” model, comparing differ- 2009 paper on Total Sleep Deprivation (TSD), which found that ent groups of individuals, rather than focusing more on the TSD increased BDNF levels as well and reduced symptoms of changes over time occurring on the same individuals due to the depression and depressed mood. Giese et al. also found evitreatment. They only compared and contrasted between vari- dence of increased BDNF levels in their research on SD and Parous groups of healthy rats and their animal depression models. tial Sleep Deprivation (PSD) (Giese et al., 2014). Thus, Maturana Since they did not perform their research on a “before/after” et al.’s findings further illustrate to the scientific community that model, the researchers could only gauge correlations but not for REMSD too, increased BDNF levels are seen. causation or show any therapeutic benefit evidence. Thus, this Currently, extra supportive evidence of solely REMSD’s paper is inconclusive in regards to showing REMSD’s antidepres- direct, potential antidepressant effect is lacking in the field. sant benefits on the depressed brain model. However, there are other findings that highlight some possible In fact, in 2011, Landsness et al. rather observed antide- benefits of REMSD for mood and the brain. For example, Corsipressant effects through depriving the Non-REM (NREM) stage Cabrera et al. in 2015 found REMSD improved executive cogniof Slow Wave Sleep (SWS; Slow Wave Sleep Deprivation (SWD)) tive function as well as attention level. Further, Lau et al. in instead of the REM sleep stage and REMSD. Siddique et al. in 2020 (*research conducted in 2019) found people who had low2018 also subjected rats to REMSD and conducted swimming er levels of REM sleep during daytime napping were less likely to tests like Maturana et al. However, Siddique et al. found the be more emotionally perceptive, sensitive, and negativelyrats’ depressive behaviours worsened after REMSD. Another affected in their mood by looking at angry-looking faces. paper also found that “fragmenting” and altering with the REM sleep stage actually worsened the symptoms of depression CRITICAL ANALYSIS (Pesonen et al., 2019). However, Asakura et al. in 1993 obWhat’s remaining in the field to be asked is thus whethserved REMSD to increase swimming activity in mice subjected er REMSD, and SD kinds of therapies for that matter, really do to a forced swimming test, much like Maturana et al. have antidepressant effects on the brain of depressed individuAside from these papers, other research finds using SD als, and whether or not the potential side effects of the various and REMSD as potential antidepressant therapy too “costly” kinds of sleep deprivations themselves are not so harmful compared to the actual potential benefits of such therapies. enough to the brain and body to warrant further consideration Velazquez-Moctezuma et al. in 1996 found that although REMSD of utilizing such treatment methods in depression and mood could interestingly elevate sexual performance and increase therapy. sexual and mounting behaviour in male rats, REMSD could also A possibly very interesting research avenue is looking cause death in cases where the rats were deprived of REM sleep into conjunct therapies. Combining REMSD or SD with other for 12 days straight, highlighting the potential risk and dangers methods of treatment in depression and mood therapy is alassociated with REMSD. Davis et al. found in 2003 that REMSD ready continuously being done and tested by multiple teams impaired Long-Term Potentiation (LTP) and the brain’s ability to around the world. For example, chronotherapy (mood therapy form memories. Straus et al. in 2018 also echo Davis et al.’s using light) methods such as Bright Light Therapy (BLT) and Sleep findings in their research on memory recall in patients suffering Phase Advance (SPA) have improved symptoms of depression in from fear and Post-Traumatic Stress Disorder (PTSD); those pa- individuals with drug-resistant depression when these therapies tients with more REM sleep were better able to learn PTSD- were combined with TSD as part of the treatment plan overcoming strategies than patients who had lower REM sleep. (Echizenya et al., 2013; Kurczewska et al., 2019). Sikkens et al. Menz et al. (2013) found higher amounts of REM sleep led peo- additionally found the same positive effect with even treatmentple to better learn fear conditioning and remember fear- resistant depression that was co-morbid (more than one mood 31

disorder existing simultaneously together in an individual) with other mood disorders (Sikkens et al., 2019). Thus, exploring combination therapy plans that incorporate REMSD might shed more light on potential antidepressant effects REMSD itself may have. As well, looking at the observable changes in individual rats over time before and after treatment in a more “longitudinal” kind of study may be more helpful in discovering more direct evidence on REMSD’s antidepressant effects, if such effects truly exist. CONCLUSION & FUTURE DIRECTIONS Further research is still urgently needed to repeat, retest, and strengthen parts of the results of this paper, especially as the antidepressant efficacy of REMSD is still widely under scrutiny, highly debated in the science community, and there is still no conclusive evidence regarding that matter. However, continuing to create and test out new combinations of therapies for efficacy could add SD, REMSD, and other offshoots and combinations of these kinds of treatments as new treatment modes to the arsenal of alternate available therapy methods against depression, or even other neurological areas of interest or disorders. For example, REMSD was found to reduce negative side effects of medications for Parkinson’s Disease (PD) like raclopride, rotenone, and ibotenic acid (Targa et al., 2018). Another group of researchers found REMSD and REM REB (rebound) were able to manipulate and have a controlling impact on the cerebrospinal hypocretin system (Pedrazzoli et al., 2004), while Basheer et al. (1998) found REMSD to impact the norepinephrine (NE) system. Some researchers are exploring REMSD’s effect on the human body and brain at the cell/ molecular level; Mallick and Amar (2019) found REMSD caused more sodium-potassium pump activity due to causing noradrenaline (NA)-mediated promotion of sodium-potassium pumps’ individual subunit proteins’ mRNA production. Governments and the public health sector may be interested in REMSD, SD, or “combined therapies” research since this kind of research could have the ability to lessen the dependence on solely pharmacological antidepressants and medication for patients and the general public, as antidepressants sometimes come with their own negative or debilitating side effects, while some patients’ mood disorders are drug-resistant. Increasingly, depression and poor mental health is a social phenomenon and sadly an all-too-common problematic reality for the current times. Honestly, any research in finding new effective treatment methods and learning more about the brain and mood disorders in all directions would continually be incredibly invaluable and admirable to society.

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Multiple Sclerosis through an animal model: oral antibiotic treatment in Dark Agouti rat neonate exasperates central nervous system autoimmunity Sang Hee Baek

Gut microbiome and its potential function in the neuro-immune system have been extensively researched. Particularly, alteration in gut microbiome composition has been identified with the Central Nervous System (CNS) diseases such as Parkinson's disease, Alzheimer's disease, and Multiple Sclerosis (MS). Recent findings also suggest that this gut microbiome dysbiosis can occur due to changes in routinely activities involving diet, physical activity, and prolonged use of medication. Stanisavljevic et al. looked at how antibiotics administration affects neonatal gut microbiome in Dark Agouti (DA) rats, and how these changes exacerbate symptoms of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. DA rats in experimental group had oral administration of antibiotic starting two weeks before their delivery and were injected with encephalitogenic emulsion for EAE induction at 8th week post-delivery. Four weeks after the injection, DA rats with neonatal antibiotic administration experienced worsened symptoms of EAE, with significant changes in their gut microbiome profile and diversity, decreased the production of short chain fatty acids (SCFA), and finally changes in immune cell production and regulation. These findings using DA rats suggest the possibility of further investigation on human gut microbiome, and its relationship to CNS autoimmune diseases. Keywords: Gut microbiota, Central Nervous System, Multiple Sclerosis (MS), Experimental Autoimmune Encephalomyelitis (EAE), Short Chain Fatty Acids (SCFAs), Butyrate, Antibiotics, Autoimmunity, Neonatal

response in EAE, an animal model of Multiple Sclerosis, and observed an association between dysbiosis and EAE induced rats6. Therefore, gut microbiota has a strong association with CNS, while gut microbiota dysbiosis exhibits a causal effect on CNS immunity disorders. While its mechanism stays unclear, gut microbiota is also responsible for producing a critical immunomodulatory compound, short chain fatty acids (SCFAs). These carboxylic acid compounds are imperative in immune system equilibrium and prevention against neurological disease7. In the gut, three phyla â&#x20AC;&#x201C; Firmicutes, Bacteroidetes, and Actinobacteria â&#x20AC;&#x201C; are responsible for producing three major SCFAs, which are acetate, proportionate, and butyrate. They not only function as sources for an epithelial immune barrier but also regulate brain immunity 8. For example, butyrate is responsible for the differentiation of T lymphocytes into T regulatory cells, which is responsible for neuroinflammaBACKGROUND AND INTRODUCTION tion response3. These SCFAs are produced and modulated by gut microbiota, thus altogether participate in the "microbiota-gutMicrobiota refers to all of bacteria, viruses, archaea, and eukary- brain-axis," which is a downstream mechanism for CNS immune otes that are growing and residing in human body, and this di- regulatory function8. verse colonization is settled in mucosal tissues of respiratory, urogenital, and particularly, of gut1. This microbiota is coded This microbiota-gut-brain-axis can be modified through many from its genetic profile, microbiome. According to many findings, day- to-day activities, including food consumption, exercise, and gut microbiota formation begins neonatally, and there are differmedication such as antibiotics. There is increased attention on ences in microbiome if the infants are born vaginally or through antibiotics especially, as it is the most frequently used medica2 a cesarean section . As the infants are growing, the diversity and tion around the world, and many epidemiological studies have complexity of gut microbiota increases, and eventually, a human shown the association between antibiotic-related gut microbiota gut microbiome stabilizes with major enterotypes including Bacdysbiosis and diseases such as asthma, inflammatory bowel disteroidetes, Prevotella, and Ruminococcus genera, with individual ease, and obesity9. To examine further relationship of antibiotics 2 variabilities . This mature, healthy gut microbiota is required to use and gut microbiota, Stanisavlejic et al. conducted an original regulate and maintain the immune system, including CNS autoresearch to test the relationship between neonatal gut microbioimmunity. Therefore, maintaining a healthy gut microbiota is ta dysbiosis and CNS autoimmunity. The gut microbiota was disimperative for various immuno-regulatory responses and prevenrupted using antibiotics, and when the dysbiosis was no longer tion of disorders involving alteration in the immune system. present in their adult stage, rats were induced with EAE 4. Compared to the control group, antibiotic-treated rats exhibited agMany recent researches have appreciated the close relationship gravated symptoms of EAE, suggesting that increased EAE sympbetween gut microbiota and CNS. As mentioned, the gut micro- toms are associated with altered immune response created by biota is believed to play an essential role in immune response gut microbial dysbiosis4. regulation. For example, Gorecki et al. in 2019 conducted a study where Parkinson's disease patients and their gut microbiota MAJOR RESULTS were compared to mice that over-expresses alpha-Synuclein, a pathogenic compound associated with Parkinson's disease. The From two weeks before delivery, pregnant DA rats were admingut microbiome from Parkinson's disease patients exhibited sig- istered with antibiotics in drinking water. Antibiotic administranificantly reduced complexity and diversity. Furthermore, the gut tion on offspring rats was continued by using the same method, microbial diversity was inversely proportional to the severity of and feeding milk provided from dams during nursing. Fecal samParkinson's symptom3. Especially within CNS immunity, the ples of the antibiotic-treated offspring were collected for gut healthy gut microbiota is responsible for regulating the produc- microbiota analysis in the fourth week, and offspring were sepation and the delivery of immunomodulatory biochemical SCFAs rated from their dems. In the fourth week, antibiotic administrasuch as acetate, propionate, and butyrate. These SCFAs are es- tion halted as well. When analyzed, antibiotics and its strong sential for effector T helper cells and T regulatory cells in second- influence on gut microbiota were shown, evidenced by a signifiary lymphoid tissues such as gut-associated-lymphoid-tissues cant decrease in complexity and diversity of gut microbiota in (GALT)4. A study in 2015 had compared healthy individuals and the analysis. After four weeks from the separation, rats were MS patients to examine the gut microbial differences. When op- immunized using encephalitogenic emulsion, and again, their gut erational taxonomic units were compared, MS patients exhibited microbiota was assessed. There was no significant difference in not only the abundance of specific units but also decreased di- gut microbiota composition between experimental rats and conversity5. Previous study by the authors has also examined the trol rats, indicating that gut microbiota had restored to its origicorrelation between gut microbiota composition and immune 36

nal profile following the completion of antibiotics administration. Both groups of rats were at the peak of the disease at 12th week, with antibiotic-treated rats exhibiting exacerbated EAE symptoms by scoring worse in clinical parameters compared to control rats. Another fecal sample analyses were done on antibiotic-treated group and control group, and this time, the results were different.

munity in Dark Agouti rats. Scientific Reports, 9(1). doi: 10.1038/s41598-01837505-7

Antibiotics and SCFAs

The total production of SCFAs in both groups of offsprings was analyzed. Overall, antibiotic-treated offspring had a much lower concentration of SCFAs produced compared to the control while the composition ratio of butyrate increased at all times after EAE Antibiotics and Gut Microbiota immunization, shown in Figure 2-C. This finding is consistent with many other findings where the association between increased When fecal samples of antibiotic-treated offspring and controls butyrate production with increased Th17 response as well as IL-23 were compared at the time of separation, immunization, and at production were associated with results in the exasperated inflamthe peak of EAE, the most different composition of gut microbi- matory response11.

ota was detected at the separation (the fourth week), indicating antibiotics use in neonatal has a causal effect on gut microbiome dysbiosis. In the 12th week, which was the peak of EAE, antibiotic-treated offsprings showed decreased complexity and diversity of gut microbiota, suggesting the gut microbiota dysbiosis in an earlier age is implicated with gut microbiota composition and therefore, aggravated EAE. Gut microbial composition changes at the phylum level after antibiotic treatment during the fourth week was the most prominent and consistent across all antibiotic-treated offspring. Relative abundance analysis at the phylum level between the two groups of offspring revealed that, at the time of separation where gut microbial dysbiosis is present, antibiotic-treated offspring were seen with complete replacement of Firmicutes and Actinobacteria by Bacteroidetes and gamma- Proteobacteria. Notably, the gut microbial replacement to gammaProteobacteria and a decreased production in Turicibacter, which is a genus in the Firmicutes, are an essential finding as it was previously revealed that they may play an important role in EAE prevention and reduction in the severity of EAE symptoms10. Figure 1. Different EAE parameters and clinical scores were measured among offsprings in both the control group and antibiotictreated group shown in C-H, J. Overall, antibiotic-treated offsprings scored higher compared to the control group. Particularly in C, antibiotic-treated offsprings have shown the longer duration of EAE. Graph I depict a cross-section of the spinal cord in Control and Antibiotic treated offsprings. Antibiotics treated offsprings exhibit increased production of IFN-gamma and IL-17

Figure 2-C. Production of Butyrate (in percentage) compared to the total production of SCFA after EAE induction in antibiotic treated offsprings (grey) and control offsprings (black). Image received from: Stanisavljević, S., Čepić, A., Bojić, S., Veljović, K., Mihajlović, S., Đedović, N., … Golić, N. (2019). Oral neonatal antibiotic treatment perturbs gut microbiota and aggravates central nervous system autoimmunity in Dark Agouti rats. Scientific Reports, 9(1). doi: 10.1038/s41598-018-37505-7

Antibiotics and Immune Cells

Lymph nodes of both antibiotic-treated offspring and control were extracted and compared. Overall, Interferon-gamma (IFNgamma) and Th-17 productions significantly increased in antibiotic-treated offsprings. This is due to increased concentration of butyrate in antibiotic-treated offsprings, as butyrate is responsible for inducing Th17 response and TFN-gamma production. This key finding also aligns with previous study conducted by Donohoe et al., where they found altered cytokine production and intensified colitis when butyrate was orally administered in a murine model12. CONCLUSIONS/DISCUSSION

The present study suggested that antibiotics use in the neonatal stage of Dark Agouti rats significantly impact CNS autoimmunity and their subsequent adulthood 4. Despite the restoration of their gut microbiota after the administration of the antibiotics halted, when injected with encephalitogenic emulsion, antibiotic-treated offspring displayed worsened symptoms of EAE, strongly disrupted production and maintenance of SCFA including butyrate production, as well as prolonged inflammation due to an imbalance of immune cells such as Th17 4. It is Image received from: Stanisavljević, S., Čepić, A., Bojić, S., Veljović, K., important to acknowledge that disruptive changes occurring Mihajlović, S., Đedović, N., … Golić, N. (2019). Oral neonatal antibiotic treat- earlier in life is what resulted exaggerated EAE symptom in anment perturbs gut microbiota and aggravates central nervous system autoim-

tibiotic-treated DA rats, as the antibiotic-treated DA rats were not administered with antibiotics in their adulthood nor at the time of injection. With recent hypotheses, infants are born entirely gut microbiome free, but soon starts to establish gut microbial production, which takes 3-4 years to stabilize13. The present finding implies the significance of antibiotic usage in the neonatal stage, as antibiotics can disrupt the stabilization process of gut microbiome in newborns and leaves a life-long impact. When the antibiotic-treated offspring had fecal matter analyses to examine their gut microbial composition, Firmicutes and Actinobacteria were completely replaced with Proteobacteria and Bacteroidetes. Actinobacteria is the most important phyla in neonate gut microbiome, providing a nourishing environment for newborn hosts to stabilize their gut microbiome, and continues to be the most important phyla in adult stage for microbial diversity4,13. On the other hand, Proteobacteria initially exists in neonate gut microbiome but phases out in adulthood, therefore strongly associated with prolonged bowel inflammation when Proteobacteria are found in adult intestine14. Microbial diversity is also responsible for production in SCFA 15. SCFAs are microbial metabolites that are responsible for immune responses to mediate inflammation throughout the bodily system16. Many previous findings have associated changes in microbial diversity with altered SCFA production and shown to have implications with neuroinflammation such as Parkinson's diseases17. By aligning with these findings, the present study explains decreased microbial composition and diversity, and differential production in SCFA. An increased concentration of butyrate in fecal matter was also discovered in the present study. Butyrate, a main SCFA that is produced by gut microbiota, is strongly associated with immune-regulatory properties, such as amelioration of bowel inflammation through absorption at mucosal level4. A transport protein monocarboxylate transporter 1 (MTC 1) is found to be responsible for butyric acid transport from the gut to gut cells, and MTC 1 expression is negatively regulated by the presence of lymph node cells such as IFN-gamma18. The authors have suggested that the increased production of IFN-gamma due to gut dysbiosis and disruptive SCFA production downregulated the expression of MTC 1, therefore caused inappropriate transportation of butyrate to the intestinal level and ultimately, resulted in prolonged inflammation4. However, the finding seems only consistent with EAE model and is specific to the type of antibiotics used in the present study, as other researches have shown opposing results in different clinical conditions. CRITICAL ANALYSIS The present study is a very recent finding that shed light on the importance of gut microbiota regulation. Although transient gut microbiome dysbiosis may occur throughout an individual's lifetime, prolonged changes in the gut microbiota due to dysfunctional metabolism, altered diet, or even hygienic issues have been frequently reported with CNS autoimmune disease patients4. Extensive use of antibiotics has also been frequently

associated with a strong disruption in gut microbiome; however, these findings were not consistent as many of these studies have shown different consequences for their results. For example, the authors mentioned a study where ampicillin was administered in lupus patients and resulted in increased SCFA production was observed 4. There are more than hundreds of antibiotics that exist for different diseases, and even within the same disease, the types of antibiotics used may differ, as well as the dosage. For antibiotics administration, the authors have combined two antibiotics - Neosulfox (neomycin sulfate) and Pentrexyl (ampicillin). Neomycin sulfate is antibiotics that is prescribed for attenuating intestinal inflammation, and ampicillin is antibiotics for bacterial infection in general 20,21; therefore, the pharmacological effect of these two antibiotics must have differed when administered. As mentioned, different antibiotics bring different effects on gut microbiota and SCFA production; because the authors administered multiple antibiotics collectively, the causal relationship of each result - gut microbiota dysbiosis, altered SCFA production, and immune cell regulation - and each antibiotics used remained obscure. Stanisavljevic et al. also acknowledged that their findings are consistent with their previous research where altered gut microbial composition led to increased up-regulation of IFNgamma and IL-17 production, thus prolonged and increased inflammation response in EAE rats4. Previously, the authors have successfully demonstrated the rectification of EAE in EAEprone DA rat by transplanting healthy gut microbiota from Albino Oxford (AO) rats19. AO rats are known to be extremely resistant to EAE induction; therefore gut microbiota transplant in DA rats supports the importance of healthy gut microbiome production and CNS autoimmunity19. This consistency provides a critical future implication as DA rat gut microbiota shows the most similarity with human gut microbiota. Especially, the successful transplantation of healthy gut microbiome to EAE prone DA rats can suggest the future direction for Multiple Sclerosis and its treatment. While the exact mechanism on how transplanted gut microbiota stabilizes and reverses the EAE symptoms through immune cell regulation remains unknown, the authors have yet to perform genetic level profiling between the two rats. On genetic level, AO rats and DA rats may differ significantly; thus, the mechanism that gut microbiota act on SCFA production and immune cell regulation may differ as well. Performing analysis on In Vitro at genetic and histological level may strengthen the causal relationship proposed by the authors, collectively. FUTURE DIRECTIONS To distinguish how different antibiotics affect on gut microbiome dysbiosys and consequences, a future study should include multiple experimental groups that are administered with separate base-type antibiotics. As mentioned, authors used Neosulfox containing neomycin sulfate, an antibiotic for intestinal inflammation, and Pentrexyl containing ampicillin, an antibiotic for general bacterial infection20,21. The subsequent design of further research should divide the experimental group into two and should only administer either of neomycin sulfate or

ampicillin. Alternatively, the authors can include multiple subject groups. By having three experimental groups, authors can administer three different treatment to each group: all of neomycin sulfate + ampicillin group, neomycin sulfate only, and ampicillin only group. This will allow the authors to perform fecal matter analyses among the three groups, and establish the causal relationship by examining the association between three groups and independent variables such as SCFA production, and/or immune cell regulation. For example, if it is only neomycin sulfate, an antibiotic used to attenuate intestinal inflammation, resulted in profoundly increased production of IFN-gamma, the result should imply that antibiotics for intestinal inflammation are responsible for down-regulation of MTC 1, thus inhibiting butyrate transport to intestinal cells. The authors can also combine AO rat study and DA rat study to experiment with the mechanism behind gut microbiota transplant further. Genetic profiles of AO rat and DA rat should be performed first, especially on the Gastro-intestinal cellular level and CNS, to confirm if AO rat and DA rat have a similar mechanism in antibiotic digestion. If AO rat and DA rat have a similar genetic profile, the authors can integrate the differential antibiotic model, as mentioned above. For example, the authors can divide the antibiotic-treated DA rat groups into neomycin sulfate + ampicillin, neomycin only, and ampicillin only group. When gut microbial transplant from healthy AO rats to DA rats from a different group is completed, and DA rats start to show recovery signs, the authors can monitor the difference in amelioration of EAE symptoms. EAE-related clinical measurements such as tail agony, hind limb paresis, or hind limb paralysis can be used throughout the recovery4, and statistical analysis such as multiple regression can be performed to see if each group exhibits differential recovery depicted by different clinical scores. If different antibiotics are associated with different EAE symptoms in DA rats, the process of recovery must differ as well.

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Vagus Nerve Stimulation: Enhancing Outcomes in IschemicStroke Motor Rehabilitation

Sinamys Bagh

Although life after a stroke may be impacted by many obstacles ranging from cognitive deficits to emotional burdens, the loss in functional arm mobility is one of the most debilitating consequences. Treatments based on occupational therapy and physiotherapy are commonly implemented to recover arm mobility. Despite evidence supporting their efficacy, they have not been able to provide complete functional recovery to many stroke patients. Research investigating the mechanisms underlying the efficacy of the implemented therapies revealed neuroplasticity to be the key player in recovery. This has opened the door to the idea of utilizing methods that specifically target neuroplasticity in motor networks, one of which being Vagus Nerve Stimulation (VNS). Recently, it has been demonstrated that supplementing traditional motor rehabilitation with VNS allowed for rat stroke models to regain complete forelimb functionality, recovering twice as much as the rats that received motor rehabilitation alone. Although this was a big step forward in developing better treatment options for stroke patients, further investigation was needed. A more recent study by Meyers et al. (2018) aimed to expand on the previous research by investigating the generalizability, lasting effects, and potential mechanisms underlying the enhanced performance with the delivery of VNS. Stroke-induced rats that received VNS-paired rehabilitation performed better on a novel untrained task than rats that received motor rehabilitation alone; displaying generalizability. The enhanced performance in VNS treated rats persisted for at least 7 weeks after the end of rehabilitation; displaying lasting effects. In addition, neuroplasticity was revealed to also be the mechanism mediating the enhanced recovery in VNS treated rats. This evidence, expanding on the efficacy of VNS, provides more confidence in further investigations for developing better rehabilitation treatments for stroke patients. Key words: vagus nerve stimulation (VNS), ischemic stroke, rehabilitation, plasticity, neuroplasticity, motor networks, motor function, forelimb impairment, rats

BACKGROUND Among the many outcomes of ischemic strokes ranging from cognitive impairment to speech impairment, functional impairment related to arm motor deficits is often the most impactful stroke remnant in the day-to-day lives of the patients (Krakauer, 2005). In fact, this upper-limb motor impairment is the leading cause of acquired disability in adults worldwide (Donnan, Fisher, Macleod, & Davis, 2008). This drastic and sudden change in one’s functionality can negatively impact various aspects of life often as emotional, financial, and psychosocial strains. Motor rehabilitation approaches through physiotherapy and occupational therapy have been the most common treatment regimen provided to stroke patients (Claflin, Krishnan, & Khot, 2015). However, despite the functional benefits associated with this type of rehabilitation (Nudo, Wise, SiFuentes, & Milliken, 1996), a randomized control trial by Kwakkel, Kollen, and Wagenaar (2002) revealed that even with intensive motor rehabilitation, only about 60% of patients were able to regain useful function, not to be confused with normal function. This general outcome was also supported by multiple other studies (Lai, Studenski, Duncan, & Perera, 2002; Claflin et al., 2015). It has long been understood that neuronal plasticity is the key player underlying the improvements in arm functionality using motor rehabilitation approaches (Nudo et al.1996; Buonomano, & Merzenvich, 1998). This is in line with the more recent research reporting neuronal plasticity to be the only observable spontaneous stroke-recovery mechanism (Johansso, 2007; Cheatwood, Emerick, & Kartje, 2010) both in rodent models (Emerick, & Kartje, 2004) and in humans (Rossini et al., 2007; Tecchio et al., 2006). The way in which plasticity is thought to recover arm functionality is through its ability to reorganize the uncontrolled widespread neuronal activation that is observed in the motor and premotor cortices of stroke patients (Feydy et al., 2002; O’Shea, Johansen-Berg, Trief, Göbel, & Rushworth, 2007; Grefkes et al., 2008). This link between plasticity and arm motor recovery has put forth the notion that combining the wellestablished motor rehabilitation therapies with more direct techniques that enhance neuroplasticity in specific areas of the brain, namely the motor cortices, may result in a significant leap in post-stroke care and recovery. Following this, Porter et al. (2011), used healthy individuals to shine the spotlight on a technique that was able to enhance the plasticity within the primary motor cortex that is generally observable with forelimb training. The technique was the repeated delivery of vagus nerve stimulation (VNS). VNS utilizes time-controlled engagement of systems that modulate neuroplasticity, driving their function to increase plasticity in motor regions of the brain (Hulsey et al., 2017). Khodaparast et al. (2013) took the discovery by Porter et al. (2011) a step further by testing the effects of pairing motor rehabilitation with VNS in rat stroke models and the findings were remarkable. Rats that received VNS-paired motor rehabilitation were able to return to their pre-lesion mobility levels (Khodaparast et al. 2013). This is contrasted to the rats that received motor rehabilitation only and their failure to return to their pre-lesion state (Khodaparast et al. 2013). This was considered a milestone in post-stroke rehabilitation research as this study was the first to observe com-

plete upper-limb recovery in rodent models. The findings by Khodaparast et al. (2013) were then supported by subsequent research. (Hays, 2016; Khodaparast et al., 2016; Hays et al., 2014). This new accumulation of research in support of the benefit of introducing VNS to post-stroke motor rehabilitation seems promising but the benefits of VNS in stroke recovery need to be evaluated more extensively to look into its long-term performance compared to the traditional rehabilitation methods. Looking into the recovery’s generalizability is also crucial as regaining functional mobility in a specific task or movement without the transfer of treatment benefits to other movements does not adequately represent recovery of functionality. The paper reviewed here by Meyers et al. (2018) set out to fill these gaps by investigating the benefits of VNS-paired rehabilitation, their generalizability, and their stability over time in lesion-induced rats. Rats that received VNS-paired motor rehabilitation (VNS+Rehab) were compared to rats that received motor rehabilitation alone (Rehab) on various tests. Post-stroke performance on a previously trained and familiar upper-limb motor task was significantly better in the VNS+Rehab rats compared to the Rehab rats (Meyers et al., 2018). Post-stroke performance on a novel task was also better in the VNS+Rehab rats compared to the Rehab rats; increased generalizability (Meyers et al., 2018). The enhanced performances of the VNS+Rehab group lasted for at least 7 weeks. Additionally, increased plasticity was observed in the VNS+Rehab rats in motor networks through the injection of a fluorescent trans-synaptic tracer (Meyers et al., 2018). Their findings further consolidate the benefits associated with VNS-paired rehabilitation on a multitude of factors not just focused on the immediate and movement-specific benefits. This provides more confidence in the possibility of effectively introducing VNS-paired rehabilitation in stroke patients. MAJOR RESULTS

Forelimb Motor Impairment After Stroke In the study by Meyers et al. (2018), unilateral ischemic lesions were introduced into the primary motor cortices and the dorsolateral striata of female rats by injecting endothelin-1, similar to previous research (Khodaparast et al., 2013; Fang et al.,2010). One week after undergoing ischemic lesions, all rats displayed decreased performance on the trained supination task. This task allowed for the assessment of rotational forelimb strength, defined by turn angle and success rate; trials in which turn angle exceeded 60° (Meyers et al., 2018). All rats had been highly proficient at the supination tasks prior to the induced ischemic strokes. No significant performance differences were observed between the groups before and after introducing the lesions indicating that any subsequent difference in performance would be likely be attributed to the difference in treatment protocol; whether they received VNS-paired motor rehabilitation (VNS+Rehab) or just motor rehabilitation alone (Rehab).

Rehabilitation and VNS Pairing Enhanced Performance on persistence of motor benefits when rehabilitation is accompanied by VNS. These observations were also illustrated in Figure 2. Trained Task Vagus nerve cuffs were surgically implanted per the procedure previously described by Khodaparast et al. (2013). After a week of rest, rehabilitation training on the supination task, consisting of freely performing the behavioural task, took place over 6 weeks. During which, the performance of the rats in the VNS+Rehab condition was consistently higher than the Rehab condition. This was measured using peak turn angle and success rate. In addition, all VNS+Rehab rats (9 out of 9) achieved at least 50% recovery, compared to only 3 out of 10 Rehab rats as depicted in Figure 1.

Figure 2. VNS-paired training improves forelimb function on both trained (supination) and untrained (isometric pull) tasks when defined by peak turn angle and peak pull force. Similar trends were observed when performance was defined by success rate (not shown here). Improved performance was maintained at weeks 11 and 12, indicating benefits lasting at least 7 weeks after the cessation of VNS delivery. Figure adapted from Meyers et al. (2018) Stroke. 49(3), 710-717.

VNS Enhanced Recovery by Stimulating Plasticity; Increased Connectivity to Musculature Figure 1. Based on peak turn angle and success rate, all VNS+Rehab rats obtained at least 50% recovery, and only 3 out of 10 Rehab rats surThe rats were sacrificed after the last phase of supination passed the 50% recovery mark. In addition, trial numbers did not differ between the two groups during the training period, with the data here task training and lesion size was measured revealing no differences being from week 6. Figure adapted from Meyers et al. (2018) Stroke. 49 between the two groups (Meyers et al., 2018). This indicates that (3), 710-717. the benefits of VNS-paired rehabilitation may not mediated through

Enhanced Performance Generalized to Untrained Tasks Both groups were also tested on the isometric pull task to assess generalizability of the benefits of the VNS-paired rehabilitation. This task required similar mobility as in the supination task but it also, more specifically, assessed forelimb pulling strength. Rats in the VNS+Rehab group achieved significantly higher peak pull force compared to the rats in the Rehab group; increased forelimb strength in the VNS+Rehab rats (Meyers et al., 2018). Thus, as with the supination task, performance on the isometric pull task was significantly better in the VNS+Rehab group compared to the Rehab group, indicating that the enhanced performance of VNS-paired rehabilitation was generalizable to equivalent novel tasks. Figure 2 depicts a visual comparison of performance on the trained and untrained tasks over the course of the testing.

the reduction of lesion size but perhaps by alternative mechanisms possibly involving enhanced plasticity. This suggestion was supported when the injection of the retrograde trans-synaptic tracer pseudorabies virus: PRV-152 into the musculature of the trained forelimb of the VNS-Rehab group displayed 6 times the number of labelled cortical neurons of the Rehab group within the motor networks (Meyers et al., 2018). These findings, combined, indicate that the benefits of VNS-paired rehabilitation over rehabilitation alone are likely due to the enhanced synaptic connectivity in the motor circuits, mainly in the trained forelimb.

Enhanced Performance Was Long-Lasting After the assessment on the isometric pull task, rats were retested on the previously trained supination task to investigate the lasting effects of the benefits associated with VNS-paired rehabilitation compared to rehabilitation alone. The VNS+Rehab group performed significantly better than the Rehab group signifying that enhanced performance persisted for at least 7 weeks after the cessation of VNS (Meyers et al., 2018). In addition, no decrease in peak turn angle or success rate was observed in the VNS+Rehab condition. This is contrasted to the slight observed decline in performance of the Rehab rats. These findings together, indicate higher

Figure 3. VNS-paired increases plasticity and connectivity in motor networks. A, Location of induced lesion depicted with locations of PRV-152 positive neurons in Rehab rats shown via green triangles. B, Location of PRV-152 positive neurons in VNS+Rehab rats. C, Number of PRV-152 positive neurons was significantly higher in both cortices of the VNS+Rehab rats. Figure adapted from Meyers et al. (2018) Stroke. 49(3), 710-717.

CONCLUSIONS & DISCUSSION Meyers et al. (2018) concluded that pairing motor rehabilitation training with VNS results in enhanced upper forelimb functional recovery compared to rehabilitation training alone. This is in line with the initial study investigating the benefits of VNS-paired rehabilitation (Khodaparast et al., 2013). What Meyers et al. (2018) expanded on, however, was that VNS-paired rehabilitation was not task or movement specific but rather its benefits can generalize to similar untrained tasks. Another extension provided was the investigation of the longterm benefits of VNS-paired rehabilitation over rehabilitation alone. Meyers et al. (2018) found that the enhanced recover of VNS-paired rehabilitation lasted at least 7 weeks after the termination of VNS delivery. Meyers et al. (2018) were the first to join two links have previously been established: the link between VNS and increased neuroplasticity in brain motor region was previously established (Hulsey et al., 2017), along with the link between increasing plasticity and enhancing upper forelimb recovery in strokes (Feydy et al., 2002; Oâ&#x20AC;&#x2122;Shea et al., 2007; Grefkes et al., 2008). Meyers et al., (2018) were the first to demonstrate that enhanced upper forelimb functionality from VNS delivery was linked to the ability of VNS to drive plasticity in the motor networks of stroke models. This supports the idea that plasticity is an underlying mechanism of stroke recovery (Cheatwood, Emerick, & Kartje, 2010). The observed maintenance of lesion size post VNS-paired rehabilitation by Meyers et al. (2018) was consistent with previous findings (Khodaparast et al., 2016), further weakening the possibility that functional stroke recovery is associated with lesion-size reduction. In summation, the findings by Meyers et al. (2018) contribute to the field of poststroke recovery by demonstrating the efficacy, generalizability, and stability of VNS-paired rehabilitation and supporting the notion that plasticity is the driving mechanism in stroke functional recovery. All to justify further research on VNS delivery and other possible plasticity-inducing techniques as treatment options for stroke patients. With the research by Meyers et al. (2018), this next step can be taken more confidently; knowing that there is a real potential. CRITICAL ANALYSIS Overall, the present study provided valuable insight into the efficacy and the enhanced upper forelimb functionality outcomes of VNS-paired motor rehabilitation, building on the initial study by Khodaparast et al. (2013). Although the findings by Meyers et al. (2018) were mostly in line with the study by Khodaparast et al. (2013), one discrepancy remains; Khodaparast et al. (2013) reported overall performance recovery of post -stroke rats to pre-stroke levels which was not reported in the present study (Meyers et al., 2018). Although, both studies did report doubled recovery in the VNS-paired rehabilitation group compared to the rehabilitation group. This slight discrepancy, however, does not detract from the overall findings and may be a result of the small sample sizes used in both studies. Larger sample sizes in future studies may provide more accurate average recovery values.

Looking into the methodology of the paper by Meyers and et al. (2018), a few things stand out as improvements on previous research investigating VNS efficacy in stroke rehabilitation. For one, the use of two tasks to assess upper forelimb motor function, was a change to the previous research that all used only one task throughout the rehabilitation (Khodaparast et al., 2013; Hays, 2016; Khodaparast et al., 2016; Hays et al., 2014). This, as discussed throughout, allowed for the assessment of the generalizability of the VNS-associated outcomes. In addition, the choice of the supination task and the isometric task, specifically, allowed for the findings to contribute to the understanding of enhanced functionality and not just enhanced mobility. This is because both tasks mentioned assess the combination of fine motor movements involving the fingers for grasping and overall fore-limb strength. This is valuable when considering that fine motor functionality deficits is what attributes to post-stroke disability (Lambercy et al. 2011), so assessing such skills is crucial when investigating the efficacy of functional therapies. However, despite these improvements, it is important to keep in mind that the supination and the isometric pull tasks are still very similar in their mobility requirements; they both assess functionality in a rather stationary way. Both tasks require only slight movement; an emphasis on strength over mobility. Future studies could benefit from using tasks that differed more and assessed the need for greater mobility to further investigate generalizability. Including the current study, research conducted looking into the efficacy of VNS-paired rehabilitation have all utilized female rat models (Khodaparast et al. 2013; Hays, 2016; Khodaparast et al., 2016; Hays et al., 2014). This may have implication on the generalizability of the findings due to the reported differences in stroke susceptibility and outcomes across sexes, both in rats (Renolleau et al., 2007) and in humans (Murphy, McCullough & Smith, 2004). Commonly, this research indicated less susceptibility and less detrimental stroke outcomes in females due to the neuroprotective nature of estrogen. Utilizing male models would fill that gap in research and help uncover if the sex-related differences affect the efficacy of VNS-paired rehabilitation. In vain of the reported sex-related differences in stroke susceptibility and outcomes, it is important to be reminded of the multifaceted nature of strokes. Not all strokes are the same, not even are all ischemic strokes the same (Sommer, 2017). Many factors like age, severity of ischemia, localization, and genetic background play into the severity of stoke outcomes and the patientsâ&#x20AC;&#x2122; potential for recovery. So, although the findings obtained from the simplified rodent models are crucial in providing a basis for future research, it is still important to zoom out and look at the bigger picture of the disease when assessing potential treatments or therapies. FUTURE DIRECTIONS Building on my comments on the methodology of the paper by Meyers et al. (2018), it would be possible for future research to fill the gap associated with the limited generalizability assessed by the current study. Because of the similar mo-

mobility requirements of the supination task and the isometric pull task used by Meyers et al. (2018), the extent of the generalization of the benefits of VNS are not examined adequately. To remedy this, future research could use a task that involves movements not directly assessed by Meyers et al. (2018). A suggestion for a task would be the skilled reaching task devised by Whishaw, Oâ&#x20AC;&#x2122;Connor, and Dunnett (1986). This task can be easily customized to target dexterity involving the grabbing of objects or the requirement for a wider forelimb range of movement. This would provide a better sense of generalizability of VNS-paired rehabilitation. In addition, the issue with sample size could easily be circumvented if larger starting samples were used. This is because in the studies by Meyers et al. (2018) and Khodaparast et al. (2013), the number of rats decreased after the surgical implantation of the VNS cuffs, when complications were encountered resulting the death of some rats. By starting with a larger sample, the authors could account for a possible decrease in size without it being detrimental to the statistics of the study. The complications encountered during the surgical procedure raises some issues regarding the safety of the treatment on stroke patients. However, a first-in-human study by Dawson et al. (2016) demonstrated the safety of VNS-paired rehabilitation in stroke patients. Although the study by Dawson et al. (2016) preceded the current study, future research exploring the efficacy of VNS-paired rehabilitation on humans should follow more readily with the contributions of Meyers et al. (2018) expanding on the understanding of VNS-based treatment. Moreover, current research on VNS-paired rehabilitation lacks consideration of rehabilitation timing. It has been shown that, in stroke patients, recovery in arm mobility is predicted best based on the performance level one month after the stroke (Krakauer, 2005). So traditional treatments have focused on administering therapy within the first month after the stroke to maximize recovery. However, the possibility of an optimum time window for recovery has not been explored in VNS-paired rehabilitation research. Future research could compare the recovery levels among of rats that start VNS-paired rehabilitation at varying times after lesion-induction. Results could show a similar trend where therapy soon after the stroke would yield better results (Krakauer, 2005). On the other hand, therapy administration time could be revealed to have little to no effect on the recovery of the rats, which would be a great leap in the field of stroke recovery. This potential, along with accumulating research showing the efficacy, safety, and stability of VNS treatment could result in a major long-due shift in enhancing life post-stroke.

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Countering the Effects of Plasticity and Deficits in Long Term Depression Induced by Cocaine with the Use of ZetaInhibitory Peptide Yeasmin Sultana Begum

Zeta-Inhibitory Peptide (ZIP) is compound that was created to inhibit a protein involved in learning and memory—tenets of plasticity and Long Term Potentiation/Depression (LTP/D)—called atypical PKC (PKMζ). Evidence suggests that ZIP is capable of eliminating the preservation of memories in experience-dependent behaviors. Such behaviours are an integral component of drug-induced plasticity. Despite this role of ZIP in experience-dependent behavioral mechanisms, it is unknown whether this functionality actually translates to drug-induced plasticity. In order to determine the potential effects of ZIP on drug-induced plasticity and the reinstatement (RI) of cocaine-seeking behaviours, Deutschmann et. al (2019), conducted a study by administering ZIP into the nucleus accumbens (NAc) of rodent models. This study revealed that when ZIP was administered to these rats 24 hours or 1 week before cocaine priming, cocaine-seeking RI was blocked. The specificity of this effect was such that ZIP administration had no effect on other food-seeking behaviours. To further study the effects of ZIP on LTD, the ability of ZIP to counter LTD deficits caused by cocaine were also assessed. It was determined that not only did ZIP administration rescue NMDA-dependent and mGluR5 -dependent LTD in mice after cocaine administration and a period of withdrawal, but this effect is also independent of PKMζ inhibition. This suggests that ZIP is capable of countering the plasticityblocking effects of cocaine. These studies on ZIP exhibit that it is persistently able to counter the effects of cocaine-induced plasticity and deficits in LTD and may lead to future therapeutic mediations for cocaine addiction. Key words: atypical PKC; cocaine; LTD; mGluR; NMDAR; nucleus accumbens; nucleus accumbens core; plasticity; reinstatement, zeta-inhibitory peptide

INTRODUCTION

istration to the NAc block the RI of cocaine seeking, but this Experience-dependent as well as cue-driven behaviours are effect also did not generalize to block other feeding behaviours. a central component of drug association and addiction. This is ZIP was also administered to both PKMζ wild type (WT) and especially considered in the case of cocaine-induced plasticity PKMζ-KO mice to establish whether the previously determined which creates a great addiction problem with its high rates of effects of ZIP functioned independently of ZIP’s PKMζ-inhibition relapse and reinstatement (RI). Drug dependent behaviour is function. The results between the WT and KO mice were the immensely maladaptive yet extraordinarily resilient to change, same, suggesting independent function. posing a great challenge to find therapeutic preventions and Lastly, the ability of ZIP to remedy the deficits in plasticity remedies. within the NAc were tested in mice via self-administration of The underlying culprit to relapse is cue-driven withdrawal (Conrad et al., 2010). Cue-driven behaviours when paired with the experience of drug association induces the RI of cocaineseeking behaviour even after extinction. (Conrad et al., 2010). It has been suggested that these cocaine-seeking behaviours may be inhibited via pathways involving the nucleus accumbens core (NAcc) (Théberge et al., 2010). Kalivas and McFarland (2003), identified certain brain circuitries in the basolateral amygdala and the ventral tegmental area (VTA) that are involved in cue-primed RI and drug-primed RI respectively. These circuits have a common output in the NAcc. The ultimate underlying cause of relapse and addiction is then thought to be the inability to block the association of drug paired cues in these pathways. Many inhibitors of these pathways have been produced, but their effects on cocaine-seeking behaviours have been temporary and lack persistent results (Mahler et al., 2013). Zeta-Inhibitory Peptide (ZIP) is a compound created to inhibit atypical PKC (PKMζ), which is involved in learning and memory maintenance through Long Term Potentiation (LTP) (Pastalkova et al., 2006). Hence, ZIP is capable of eliminating the consolidation of memories created by experiencedependent behaviours. However, there is some controversy around the target effect of ZIP. Recent evidence using PKMζknock-out (KO) mice suggests that ZIP actually has off-target effects that mimic PKMζ in its absence (Sacktor and Hell, 2017). It is unclear whether PKMζ is necessary for ZIP to work on disrupting drug-associations and related plasticity. In the case of cocaine, self-administration of the drug results in the dampening of a type of plasticity known as Long Term Depression (LTD) (Martin et al., 2006). It is thought that drug self-administration causes the inability to stimulate LTD, resulting in memory maintenance of drug associations (Kelley, 2004). Despite all this, ZIP is able to impair many different types of memory pathways associated with LTP, including memories of drug associations. Such memories are important in the maintenance of drug-induced plasticity. However, it is unclear if the ability of ZIP to impair drug-association memories translates to drug-induced plasticity as well. Deutschmann et al. (2019) conducted a series of experiments to study and consolidate the unknown effects of ZIP on cocaine-induced behaviours and how it may be able to reverse the aforementioned plasticity in cocaine-primed associations. One of the first aspects of ZIP that was examined was its ability to interrupt RI of cocaine seeking. Since cocaine-seeking is derived from cue-driven behaviour which in turn is associated with NAc synaptic plasticity, ZIP was administered into the NAc of rat models. It was concluded that not only did ZIP admin-

cocaine. These results suggested that ZIP administration was able to rescue two forms of LTD, NMDA-dependent LTD and mGluR5-dependent LTD; an approach which proved to have more persistent effects than previously tested therapies. MAJOR RESULTS Administration of ZIP and the Interruption of Cocaine-Seeking RI To test the ability of ZIP to disrupt the RI of cocaine seeking, Deutschmann et al. (2019) used male Sprague Dawley rats. These rats had guide cannulas implanted to administer ZIP into the NAc. The rats were either assigned to cocaine selfadministering and sucrose self-administering. Within operant chambers, these cohorts of rats either self-administered cocaine or sucrose while being simultaneously classically conditioned to a cue-light which lit with each infusion. Each group then went under a course of extinction after addiction had been established. After two hours of extinction, the rats were given intra-accumbal microinjections of either ZIP or the inactive scrambled form of ZIP, SCR-ZIP (Hardt et al., 2010; Serrano et al., 2008). The rats then received a substance-primed RI session of either cocaine or sucrose as per their experimental group either 24 hours or 1 week after the compound administration.

Figure 1. Study Cohorts: Eight study cohorts were created to exam-

For the cocaine-seeking cohorts of rats, those that received SCR-ZIP prior the cocaine-primed RI session exhibited significant RI as opposed to those that that had ZIP administered (Fig. 2A,B). This shows that ZIP administration to the NAc was indeed able to block drug-induced plasticity stimulated by cocaine. Therefore, ZIP is able to translate its memory disrupting effects on drug-associations to drug-induced plasticity. To exclude other types of substance seeking behaviours from the functional repertoire of ZIP, the sucrose cohorts were

examined. Indiscriminate of whether the rats received ZIP or SCR-ZIP, they all displayed significant RI of sucrose (Fig. 2C). These results therefore depict that the function of ZIP in blocking RI of substanceseeking does not generalize when administered to the NAc. This may be an ideal factor when considering using ZIP as a therapeutic intervention to cocaine addiction because there would not be a side of effect lacking appetite (Rogers, 2017). The use of ZIP administration in both the 24-hrspost (Fig. 2A) and 1-week-post (Fig. 2B) groups show a consistency in results despite the difference in time passed for reintroduction to cocaine. There has largely been a predicament in finding therapeutic interventions to drug addiction that are persistent (Mahler et al., 2013), but Detschmann et al. (2019) show that ZIP promisingly has consistent effects at least up to a week after administration. Dependency of ZIP Function on PKMζ There is much controversy around whether or not these drug-association and drug-induced plasticity blocking effects of ZIP are dependent on its function of inhibiting PKMζ, or even if PKMζ has any function in plasticity and memory. A study by von Kraus et al. (2010) successfully attempted to disrupt sensorimotor memories in rats via the use of ZIP. However, a study conducted by Lee et al. (2013) on synaptic plasticity and memory in the hippocampus using PKMζ-KO mice demonstrated normal LTP and maintenance of memory. To further investigate the role of ZIP and its possible dependency on PKMζ, cocaine seeking was examined in PKMζ-WT vs PKMζ-KO mice. These mice underwent the same protocol as the rats in the previous study, save for a few adjustments.

Figure 2. Administration of ZIP and the Interruption of CocaineSeeking RI vs Sucrose Seeking RI 24 hrs vs 1 week Post Administration: A, In cocaine-seeking rats that were administered ZIP or SCR-ZIP 24 hrs before cocaine-primed RI, there was a significant exhibition of RI in the rats that did not receive the active compound (SCR-ZIP posttest vs ZIP posttest, p<0.0001; n=6/group). B, Rats that underwent cocaineprimed RI 1 week after being administered ZIP or SCR-ZIP showed similar results to those in A (SCR-ZIP posttest vs ZIP posttest, p=0.0025; n=6/group). C, RI of sucrose seeking was not affected in sucrose-seeking rats (SCR-ZIP and ZIP extinction vs posttest, p=0.0001; n=7-8/group). Figures adapted from Deutschmann AU, et al. (2019). J. Neurosci. 39 (39):7801-7809.

The first is that there were only two cohorts in this study, the WT and the KO mice. It had already been established via the previous study that intra-accumbal administration of ZIP disrupts drug-induced plasticity, hence, both these groups of mice were only classically conditioned with the light cue and cocaine. In mice, the most vigorous inducers of RI are cue driven (Sondheimer and Knackstedt, 2011). Hence, instead of implementing substance-primed RI sessions, these mice underwent cue-driven cocaine-seeking RI via the light they were classically conditioned to associate with cocaine. After a cue-driven TI session, regardless of whether the mice were WTs or KOs, the mice that received SCR-ZIP displayed significant cocaine RI whereas the mice that received ZIP did not (Fig. 3).

Figure 3. ZIP Function in PKMζ-WT vs PKMζ-KO Mice: Mice that received ZIP did not exhibit cue-driven cocaine-seeking RI but mice that received SCR-ZIP exhibited significant RI. (SCR-ZIP vs ZIP RI test, p=0.0009 (adjusted); n=5-7/group). Figure adapted from Deutschmann AU, et al. (2019). J. Neurosci. 39(39):7801-7809.

addictions and a major problem in the pharmacological industry in off target effects. Hence, the specificity of ZIP is very important to note.

McGrath et al. (2018), in their 2018 study on the effect of PKMζ in the NAc, found that the absence of PKMζ is correlated to an increase of cue-driven RI of cocaine-seeking behaviours. However, they also found that PKMζ expression increases after drug exposure. These mixed results expose the controversy around this protein. The study conducted by Deutschmann et al. (2019) has demonstrated that ZIP in fact functions independent of PKMζ in pathways regarding the blocking of drugassociated memories and drug-induced plasticity. Lee et al. (2013), who found that plasticity and memory did not require PKMζ also concluded from the same study that the effects of ZIP are independent of PKMζ as it counters the LTP deficits in PKMζ-KO mice. Countering Deficits in Plasticity Induce by Cocaine

Figure 4. Rescuing LTD Using ZIP: A, Bath application of ZIP to co-

The ability of ZIP to remedy the deficits in plasticity induced caine-inexperienced mice exhibits no significant change to their already by cocaine were also examined by Deutschmann et al. (2019). robust NMDAR-dependent LTD in their NAcc. B, Bath application of ZIP Dampened LTD is observed in the NAc after exposure to coIt was also established that using ZIP as an intervention recaine (Martin et al., 2006) as opposed to inexperienced mice sults in long lasting and persistent effects in blocking RI. Anoththat exhibit robust LTD in their nucleus accumbens core (NAcc). er issue with the pharmacological industry is that intervention A bath application to administer ZIP was used on both co- may not be long lasting (Mahler et al., 2013). Deutschmann et caine experienced and inexperienced C57BL/6J mice. NMDAR- al. (2019) have demonstrated that ZIP indeed has a long-lasting, dependent LTD is one form of plasticity that is disrupted by persistent effect on interrupting drug-induced plasticity. There cocaine use. This study examined whether ZIP would be able to is no current study that has been able to block such a vigorous reverse the dampened plasticity that was induced by cocaine. substance RI such as cocaine RI for such a long period of time. Mice that were cocaine inexperienced, the controls, demon- ZIP does not even remain in the brain for 24 hours (Kwapis et strated no changes in their NAcc after bath application of ZIP al., 2012), hence, in order for it to have had such long lasting (Fig. 4A). Bath application of ZIP did however produce strong effects, its function is clearly independent of being continuously LTD in the NAcc of cocaine-experienced mice (Fig. 4B). bound to its binding site. Though NMDAR-dependent LTD is able to be remedied by Even more so pertinent than the interventional aspects of ZIP administration, Deutschmann et al. (2019) wanted to exam- ZIP administration to the NAc is its ability to counter the conseine whether the effects of ZIP would generalize to other forms quences of two forms of dampened LTD in the NAcc. Both of dampened plasticity. mGluR5-dependent LTD in the NAcc is MNDAR-dependent and mGluR5-depeendent plasticity were also disrupted by cocaine. Results similar to that in the NMDAR- able to be rescued by ZIP infusion. This demonstrated that ZIP dependent LTD rescuing study were obtained in the studies is capable of restoring function where it was destroyed by coinvolved in rescuing mGluR5-dependent LTD (Fig. 4C,D). The caine. results demonstrated that ZIP administration was able to rescue two forms of LTD, NMDA-dependent LTD and mGluR5- CRITICAL ANALYSIS dependent LTD. Though Deutschmann et al. (2019) demonstrated that a single administration of ZIP into the NAc is enough to disrupt cocaine-seeking RI for as long as a week, there was no indicaCONCLUSIONS/DISCUSSION tion of what might happen with prolonged exposure. As with The use of ZIP administration to the NAc is not only able any pharmaceutical, prolonged exposure may present with adblock cocaine-seeking RI but its effects are specific enough that verse effects. In terms of drug addiction intervention, one week sucrose-seeking behaviours are not affected. The effects are is very little time for therapy. And one dose of an anti-addiction not generalized to other food-seeking behaviours. This distinc- compound is hardly enough for addictions as vigorous as cotion is an important one to make because many anti-addiction caine addiction. The authors should have examined the effects drugs have off target effects that may affect appetite (Rogers over a longer period of time as well as report the effects that 2017). The purpose of studying ZIP in this light is to assess were observed with ZIP administration. whether it can be a potential therapeutic intervention to drug 52

Secondly, the authors only administered ZIP to the NAc or its core. This method resulted in a very specific blocking of cocaine RI but not to other food-seeking behaviours such as sucrose. Even in PKMζ-KO models, ZIP was only infused to the NAc. Sucrose seeking, it seems, is not affected by intraaccumbal ZIP. However, what are the effects of ZIP in other brain regions? The authors did not discuss whether this specificity of ZIP to drug-induced plasticity is only so because of administration to the NAc. Also, ZIP was able to rescue two forms of dampened LTD localized in the NAcc. These were deficits in plasticity that were rescued by ZIP. However, Lee et al. (2013) found no changes in several different hippocampal-dependent plasticities after ZIP administration. Deutschmann et al. (2019) demonstrated that the druginduced plasticity blocking effects of ZIP worked independently of PKMζ by using PKMζ-KO mice. However, what if in the absence of PKMζ, another molecule is used as compensation for the same function as Sacktor and Hell (2017) have hypothesized? These various new findings on ZIP are indeed very important for understanding what therapeutic interventions can be devised in order. To tread drug addictions are dangerous and cocaine addiction. However, there are many areas that require further studies. FUTURE DIRECTIONS – An area that requires further study regarding the functions and effects of ZIP are how long the effects actually last. It has been established that effects last at least a week and that this is largely due to the fact the ZIP does not need to be continuously bound (Kwapis et al., 2012). One study. That can be conducted to assess the longevity of the effects is to have a dose of ZIP administered and then to examine how long until cocaineseeking RI resurfaces. The model may be similar to the first study conducted by Deutchmann et al. (2019). However, there should be more cohorts that are distinguished by timer intervals. Instead of just a post 1-hour group and a post 1 week, there should be more increments and more frequent as well. A study of this manner will not only show how long the effects last, but by what quantity does the effect change over time. Another study that should be considered is recreating Deutschmann et al. (2019) but in other brain regions also affected by drug-induced plasticity. These would include areas such as the hippocampus and the amygdala. One other direction would be to reconsider the independence of ZIP functioning to PKMζ expression. As Sacktor and Hell (2017) have hypothesized, there may be another molecule that compensates for PKMζ in its absence. This should be identified first before making any conclusions as it may be confounding factor.

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Anhedonia: Examination of Future Biological Targets Involving Galanin Receptors and Galanin N-Terminal (1-15) for Depression Tina Bohin

Anhedonia is characterized by the decreased ability to feel or experience pleasure. It is a multifaceted symptom understood as a result of disturbances in the reward system. Although not all diagnosed with depression experience anhedonia, it is considered to be a core feature in depression and other mood-related disorders. Galanin has been of particular interest in recent studies on depression and other mood-related disorders and is considered to be a potential target for the development of novel therapeutic treatments for depression. Previous studies have proposed a role of the galanin receptors and galanin n-terminal (1-15) (GAL-15) in reward-systems within depression and other moodrelated disorders. In the study conducted by Malton et al. (2019) GAL (1-15) is proposed as a possible therapeutic target for anhedonia-like symptoms. In this study, the behavioral effects of GAL (115) administration were explored using saccharin self-administration tests, sucrose preference tests, novelty-suppressed feeding tests and female urine sniffing tests. The neurochemical effects of GAL (1-15) were also studied using in-vivo topography, revealing significant alterations in the dopaminergic mesolimbic system. This study is the first of its kind of show a connection between GAL (1-15) and anhedonia-like behavior Millon et alâ&#x20AC;&#x2122;s (2019) study on the role of GAL (1-5) in anhedonia-like phenotype presents a possible novel therapeutic target specific for anhedonia symptoms experienced in depression, which has not been previously explored: allowing for a more personalized individualneed focused treatment for depression. Key words: anhedonia, depression, dopamine, galanin, galanin receptor

BACKGROUND/INTRODUCTION.

Anhedonia is a multi-facetted symptom commonly defined as the lack of pleasure in previously rewarding activities (et al. Hoflich, 2019). This may range from social disturbances to lack of pleasure while consuming food (et al. Hoflich, 2019). Anhedonia is considered to be one of the core symptoms of depression outlined within the Diagnostic and Statistical Manual of Mental Disorders, ( DSM-V; American Psychiatric Association, 2013). Anhedonia is also one of the two core diagnostic criteria in the melancholic subtype of depression and has shown to predict possible antidepressant response outcomes (et al. Hoflich, 2019). There presently are no known approved therapeutic treatments to specifically treat anhedonia in depression or other reward-related diseases. Anhedonia is treated primarily alongside conditions, where more general therapeutics are administered to individuals with depression (Lally et al., 2014). There has only been one proposed alternative specific to anhedonia symptoms. A study performed by Lally et al. (2014) presented that ketamine rapidly decreased anhedonia-like symptoms in patients, implicating its potential use as therapeutic alternative for the treatment of anhedonia in depression (Lally et al., 2014). It has become increasingly evident that standard depression treatments fail to address, or worsen symptoms specific to anhedonia, leading to further problems related to sexual and social anhedonia as well as emotional blunting (Lally et al., 2014). It has been identified that over 37% of those diagnosed with depression also have major symptoms of anhedonia (Ho et al., 2013). As anhedonia is defined as a core diagnostic tool for depression (Rizvi et al., 2016), its specific acknowledgement within research indicates it is a prevalent part of the depression experience and still remains to be explored. Although the entirety of underlying mechanisms of depression are still relatively unknown. The role of galanin has been explored within literature as playing a potential role in depression -like behavior and other mood-related disorders in animal models (Barde et al. 2016). There has been a recent interest in the role of galanin as a modulator for mood-related disorders as its receptors are primarily present within the limbic brain structure of rodents (Barde et al., 2016). A study performed by Kuteeya et. al (2005) has shown that the overexpression of galanin in mice lead to depression-like behavior (Kuteeva et al. 2005, Weiss et al., 1998). The role of galanin is primarily mediated by three receptor subtypes, GALR1, GALR2 and GALR3 (Millon et al., 2019). GALR1 and GALR3 serve to mainly activate inhibitory proteins while GALR2 primarily mediates excitatory signaling (Branchek et al., 2000). The involvement of GALR receptors have been linked to depression and anxiety-related behaviors (Kuteeva et al., 2008 Juhasz et al., 2014; Kuteeva et al., 2007; Mellon et al., 2017; Weiss et al., 1998; Ogren et al., 2006). In a study performed by Kuteeva et. al (2018) galanin receptor subtypes were explored within depression-like behavior. The study revealed that the stimulation of GALR1, GALR2 and GALR3 presented different roles in depression-like phenotype; where the activation of GALR1 and GALR3 receptors lead to depression-like behavior and the stimulation of GALR2 less-

ened depression-like behavior (Kuteeva et al., 2008; Bartfai et al., 2004). Results such as these further emphasize the potential role of galanin in depression and mood-related disorders. The specific role of galanin N-terminal GAL (1-15) has also been explored in depression-like behaviors in rodent models. A study performed by Millon et. al (2015) demonstrated the effects of on the galanin n-terminal (1 -15) on anxiety and depression behaviors in rats. Researchers Mellon et al.’s (2019) article published on the role of GAL (1-15) in anhedonia-like symptoms provides a potential direction for therapeutic depression alternatives, and provides evidence on the effect of GAL (1-15) in depression-like behavior. The primary focuses of the article were the behavioral and neurochemical effects of GAL (1-15) in rats (Millon et al., 2019). In the first section of the study involved an analysis of behavioral responses to GAL (1-15) using saccharin self-administration tests, sucrose preference tests and novelty-suppressed feeding tests (Millon et al., 2019). The second segment of the study involved exploring the neurochemical changes induced by GAL (1-15) using invivo imaging (Millon et al., 2019). The paper’s findings revealed that GAL (1-15) induces strong anhedonia-like phenotype in behavior, and causes neurochemical changes to the mesolimbic system (Millon et al., 2019). Furthermore, the study’s findings reinforce the importance of galanin, and particularly GAL receptors and specifically galanin N-terminal (1-15) in the role of depression-like behaviors and mood-related disorders. The study is the first of its kind creating a direct link between the neuropeptide GAL (1-15) and the anhedonia phenotype These concerted findings indicate that GAL (1-115) may potentially serve as a therapeutic target for anhedonia-like symptoms in depression as well as other reward-related diseases.

MAJOR RESULTS In Millón et. al’s research (2019) the effect of Galanin Nterminal fragment (1-15) was explored using saccharin selfadministration tests, sucrose preference tests, noveltysuppressed feeding tests and female urine sniffing tests. The effect of GAL (1-15) on the ventral tegmental area and the nucleus accumbens were also studied using in-vivo topography. One of the major findings of Millón et al’s research (2019) was that at a concentration of 3nmol, GAL (1-15) induced strong anhedonia-like phenotype in rats; this was apparent in all tests performed. GAL (1-15) at a concentration of 3nmol significantly decreased the amount of reinforcements during saccharin selfadministration while the administration of GAL had nonsignificant effects on the number of reinforcements and the administration of GALR2 antagonist M871 caused a block of the effects of GAL (1-15) (Millon et al., 2019). GAL (1-15) at a concentration of 3nmol led to a reduction of sucrose intake (Millon et al., 2019). The administration of GAL (1-15) 3nmol led to a significant change in duration of female urine cotton in the female urine sniffing test (FUST), as it caused an overall reduction in the sniffing duration compared to control group (Millon et al., 2019). In the novel suppressed feeding test (NSF) GAL (1-15) 3nmol significantly increased the amount of latency to eat

(Millon et al., 2019). Other major findings involve the neurochemical effects of GAL (1-15). GAL (1-15) was found to increase the mRNA expression of both GALR1 and GALR2, indicating their involvement in the GAL (1-15) anhedonia-like behavior inducing mechanism. It was also found that the mRNA expression of dopamine receptors in the ventral tegmental area (VTA) and the nucleus accumbens (NAc) were altered (Millon et al., 2019). Another neurochemical finding was that TH immunoreactivity was significantly decreased after 3nmol GAL (1-15) administration (Millon et al., 2019). These findings specific to galanin N-terminal (1-15) serve as a possible basis for the further exploration of alternative therapeutic treatments to address anhedonia-like symptoms.

tests, tail suspension tests, open field tests, and light/dark tests performed (Millon et al., 2015). The effects of GAL2 receptor antagonist M871 and GAL2 receptor knockdown were also implemented, and indicated that GAL2 receptor plays a role as GAL1 and GAL2 knockdown lead to changes in the effect of GAL (1-15) (Millon et al., 2015). This study further demonstrates that GAL (1-15) potentially induces depression and anxiogeniclike effects.

Figure Adapted from Millón et. Al (2015). Int J Neuropsychopharmacol 18:1–13.

Figure Adapted from Millón et. Al (2019). Journal of Psycho- Figure 4. Analysis of galanin (1-15) behavioral effects on forced swimming test (FST), tail suspension test and open field test and pharmacology, 33(6), 737–747 light vs. dark test. The duration of immobility time (a), duration of Figure 4. Analysis of galanin (1-15) behavioral effects on su- climbing time (b), duration of swimming time (c) and duration of crose intake test, novelty suppressed feeding test, saccharin time in center (d) are demonstrated with a control, GALR2 adminself-administration test, female urine sniffing test (a) Sucrose istration or GALR2 and GAL (1-15) administration. intake test 2-24hrs after administration of 3nmol GAL (1-15) (b) Novelty suppressed feeding test (NSF) after 3nmol administraThe upregulation of GALR1 and GALR2 mRNA, as well as the inhibition of GAL (1-15) and cerebrospinal fluid-injected rats. Vertical tion of the effects of GAL (1-15) by GALR2 antagonist found in the bars indicate the mean- standard error of the period of latency study performed by Millon et al. (2019) further implicates the role following the first feeding (c) Saccharin self-administration test of GAL receptors, and particulary the GALR1-GALR2 heteromer in after administration of CSFa for control group, GAL (1-15) depression-like behavior in rodent models. A study done by Fuxe 3nmol, GALR2 receptor antagonist M871 or M871 with GAL (1- et. al (2012) has identified that the GalR1-GalR2 heteromer is n15). (d) Female urine sniffing test after exposure to water, terminal fragment preferring receptor (1-15) and identified Urine-CSFa or Urine-GAL (1-15) at 3nmol. The vertical bars indi- GALR2’s important role in the mechanism. Both of these studies cate mean-standard error of the mean duration of sniffing in presenting evidence that anhedonia-like behavior may possibly be water. mediate by GAL (1-15) through GALR2 mechanisms within the heterodimer GALR1/GALR2. GAL Receptors and GAL (1-15) N-terminal in Mood-related Dis- Changes to the Mesolimbic system and mRNA transcription orders Research Dopamine signaling has shown to play an important role in several GAL (1-15) and its function in relation to anhedonia was the disorders, including depression (Xiang et al., 2008). In the study primary focus of Mellon et. al’s (2019) study. In a study pub- done by Millón et al. (2019) neurochemical changes were observed lished in 2015, Mellón identified a potential role for galanin N- after the administration of GAL (1-15). Increased dopamine recepterminal fragment in anxiety-and depression related behaviors tors in the VTA and NAc have previously been observed within in rats. This study indicated that GAL (1-15) caused significant subjects with depression (Xiang et al., 2008). In Millon et. al’s study anxiogenic-like and depression-like effects in forced swimming (2019) there was a significant change in dopamine receptors D1, 57

D2 and D3 in the VTA. There was also an increase in D3 receptor in the NAc in the depression-like phenotype (Millon et. al 2019; Xiang et. al 2008). Neurochemical changes in Vmat2 and Dat were also observed in depression-like phenotype (Millon et. al 2019; German et al., 2015; Lohr et al., 2014). Tyrosine immunoreactivity has been shown to be reduced in depressed patients (Braumann et al., 1999). Neurochemical changes in tyrosine immunoreactivity were also observed in Millon et al’s (2019) study. The neurochemical results observed in Millon et. al’s study are prevalent, as they have been previously demonstrated in literature, allowing for a further understanding of the mechanisms of GAL (1-15) and its potential role in depression and other mood-related disorders. CONCLUSIONS/DISCUSSION The main conclusions taken from Millon et al.’s (2019) article are that GAL (1-15) n-terminal causes anhedonia-like phenotype in rats, and causes prominent neurochemical changes to the dopaminergic system. Unlike other research papers regarding galanin’s role in depression, Millon et. al’s article focuses particularly on anhedonia-like symptoms, which is a core diagnostic feature of major depressive disorder as well as other mood-related diseases. The novelty of this focus on anhedonialike symptoms would allow for a potentially more personalized approach for the treatment of depression through the targeting of mechanisms involved with GAL (1-15), particularly the GALR1 -GALR2 heterodimer. A significant amount of previous literature has focused mainly on galanin receptor subtypes, while Millon et. al’s (2019) study has made a meaningful connection between the particular galanin n-terminal (1-15) and anhedonia -like symptoms often observed in depression.

administration of GAL (1-15) led to anxiety-like and depressionlike behavior in rats. In a study performed by Burgess et al. (2017), the effects of galanin (1-15) combined with fluoxetine was shown to have antidepressant-like effects (Burgess et al. 2017; Escuela et al. 2018). The study by Millón et. al could also have implemented the use of FLX in their study to analyse its interaction with GAL (1-15) particularly in anhedonia-like behavior. The role of GALR2 was identified in Millon et al.’s study using a GALR2 antagonist, which consequently blocked the effects of GAL (1-15). A study performed by Fuxe et. al (2012) is in agreement with this finding, showing the role GAL2 and its preference for the galanin n-terminal (1-15). The study also confirmed the presence of GAL1R/GAL2R heterodimers (Fuxe et al. 2012). The authors could have also implemented a GALR1 receptor antagonist, as previous literature has shown that both GALR1 and GALR2 receptor subtypes may be involved with depressionlike behavior (Bartfai et al., 2004). The addition of a GALR1 antagonist would allow for further confirmation of GALR1’s involvement with GAL (1-15) and the GALR1/GALR2 heterodimer, specifically within anhedonia-like behavior.

Previous studies have identified that the administration of GAL2 results in antidepressant-like behaviors, while the study performed by Millón et. al showed that the use of a GALR2 antagonist M871 blocked the depression-like phenotype caused by GAL (1-15) (Millon et. al 2019). This may indicate the different role of GALR2 within the heterodimer GALR1/GALR2; implying that the administration of GALR2 without the presence of GAL (1-15) may reveal anti-depressant-like phenotype (Kuteeva et. al 2008), while the administration of a GALR2 antagonist when GAL (1-15) is present blocks the anhedonia-like phenoPrevious literature on the specific galanin n-terminal (1-15) has type (Millon et al., 2019). primarily focused on the broader depression-like behaviors in rodents, while this study allows for a more specific look into A study done by Burgess et al. (2017) is in agreeance with the one of depressions and other mood-related diseases’ core fea- neurochemical changes found in Millon et al.’s findings, as a tures, anhedonia. There are no known approved therapeutic decrease in immunoreactivity was observed in depression. The treatments for the symptoms of anhedonia associated with neurochemical changes observed in the study involving Vmat major depressive disorders, and most anti-depressants availa- and Dat mRNA expression in depression-like phenotype are ble have shown to potentially worsen anhedonia symptoms. also in accordance with previous studies performed by Lohr et. The study performed by Millon et al. serves as a potential basis al (2014) as well as German et. al (2015), which both found for novel pharmaceutical targets for anhedonia involving disturbances in DAT and VMAT2 expression in depression-like behavior. C-fos mRNA expression was also found to be altered galanin receptors and galanin N-terminal (1-15). in Millon et. al’s study (2019), which is in accordance with Kung et. al’s study on c-fos activity and depressive-like phenotype; a study done by Slarkisova et. al (2002) also observed changes in CRITICAL ANALYSIS c-fos expression within dopamine structures in depression . Millon et. al’s findings revealed changes in mRNA expression of D1, D2 In their study exploring the role of galanin N-terminal (1-15), and D3 in the ventral tegmental area and an increase in D3 expresthe authors Millon et al. offer a novel mechanism involving GAL sion in the nucleus accumbens (NAc) following the administration (1-15) in anhedonia-like behavior. The study by Millón et. al of GAL (1-15). These results are in accordance with a study done by (2019) is in agreeance with previous literature regarding the Xiang et al. (2008), which demonstrated increased DA receptor role of GAL (1-15) in various mood-related disorders. The study mRNA expression in subjects with depression. offers a potential therapeutic target for specific anhedonia-like symptoms experienced in depression. A previous publication by The findings of Millón et al.’s article (2019) reveal a meaningful Millón et. al (2015) investigated the role of GAL (1-15) in anxie- connection between the roles of GAL (1-15) and GALR1/GALR2 ty and depression in a rodent model, which revealed that the receptors in anhedonia, an often-overlooked symptom of depres58

depression. These findings allow for the basis of further research shining a light on anhedonia-like symptoms in depression, and the potential role of galanin N-terminal (1-15) as a possible therapeutic target. FUTURE DIRECTIONS â&#x20AC;&#x201C; Since it is the first experiment of its kind introducing a direct link between GAL (1-5) and anhedonia-like phenotype in mice, future researchers should re-perform the experiment using other methods related to the measurement of anhedonia like-symptoms including social interaction tests, intracranial stimulation tests and chamber preference tests (Scheggi et al., 2018). A prominent symptom of anhedonia is connected to social interaction. The social aspect of anhedonia symptoms could be explored through a social interaction test performed on rodents. The test would measure the amount of time the rodent spends in close proximity to an inanimate object compared to a social target (Scheiggi et al., 2018). Other ways to explore anhedonia-like behavior could be tested such as intracranial self-stimulation, which allows rodents to stimulate particular regions of the brain within the reward circuit by pushing a lever (Scheiggi et al., 2018). Another test that could be implemented would be a test related to chamber preference, where a rewarding stimulus is placed in one chamber and a neutral stimulus in another chamber, and the time spent in each chamber could be measured (Scheiggi et al., 2018). If during the social interaction test the rodent spends more time in proximity to the inanimate target compared to the social target, this would show anhedonia-like behavior. During the intracranial self-stimulation test, if the rodent presents fewer self-administered stimulation, this would imply a disturbance in reward-circuits often observed in anhedonia, indication anhedonia-like behavior. Similarly, if the rodent spends more time in the neutral stimulus chamber compared to the positive chamber, this would indicate alterations in the reward symptom, as the positive stimulus may seem or feel less rewarding. This test could further solidify the role of GAL (1-15) in anhedonia-like behavior. GAL (1-15) has been shown to have specific affinity for GALR1GALR2 heterodimers. As the administration of GAL (1-15) lead to the appearance of depressive-like behavior in rodent models (Millon et al., 2019; Millon et al., 2015), the development of possible antagonists for this heterodimer may serve as a potential therapeutic target to mediate the effects depression-like effects of galanin n-terminal (1-15). The development of this antagonist could be administered in addition to GAL (1-15) and the same tests performed by Millon et al. (2019) could be used to analyze anhedonia-like behavior. If anhedonia-like phenotype is reduced through a GALR1-GALR2 hetorodimer selective antagonist, it could potentially serve as a basis for the further exploration of alternative therapeutic targets for anhedonia involving galanin N-terminal (1-15).

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Inducing and Quantifying Lucid Dreams Using Galantamine and the Mild Technique Hala Bucheeri

. Lucid dreaming is when an individual is consciously aware that he/she is dreaming. Previous studies have found that lucid dreaming occurs during REM sleep, where cortical activation is at its peak, especially in the frontal brain regions. To be able to lucidly dream, an individual can undergo training to develop this skill via the MILD technique (setting an intention to remember that one is dreaming). Another way to induce lucid dreams is via the use of acetylcholinesterase inhibitors such as galantamine. Galantamine increases the amount of acetylcholine, activating the cholinergic system; this system is found to be active during REM sleep. This review of LaBerge, LaMarcal, and Bairdâ&#x20AC;&#x2122;s (2018) study tackles the question of quantifying the amount of galantamine needed to induce lucid dreams. Their study uses an integrative approach: administering different doses of galantamine (0, 4, and 8 mg) along with rehearsing the MILD technique. The results of this study showed that the higher the doses of galantamine, the more lucid dreams reported. The experiment was only done by integrating both galantamine ingestion and the MILD technique; and this method resulted in more than half of the participants experiencing lucid dreaming. This protocol also increased lucid dream vividness, selfreflection, complexity, positive emotions, recall, and clarity for participants. The results provide an opportunity for future studies, such as testing the effect of galantamine on patients with altered states of consciousness and whether their quality of life and sleep can be improved by activating certain neurotransmitters, such as acetylcholine and dopamine. Key words: lucid dreams, sleep, REM sleep, MILD technique, acetylcholine, acetylcholinesterase inhibitor, galantamine, consciousness

BACKGROUND Lucid dreaming (LD) is the conscious awareness that one is dreaming (Baird, Mota-Rolim, & Dresler, 2019). Evidence from various studies show that an individual can attempt and control various behaviors and dream content in full awareness while being in a state of physiological sleep (Baird et al., 2019; MotaRolim et al., 2013). Using different variables to measure sleep, LaBerge in the late 1900s was able to prove that lucid dreaming is a real and verified phenomenon (LaBerge, Nagel, Dement, & Zarcone, 1981). Upon studying LD, it was found to occur during Rapid-Eye Movement Sleep (REM sleep) (Baird et al., 2019). To prove this, a previous study used the fact that eye muscles remain activated during REM sleep, Hearne (1978) asked sleepers prior to the experiment to gaze their eyes in a particular sequence once they begin to lucidly dream; the study found that eye gazing and the individual’s ability to lucidly dream occurred during REM sleep (Baird et al., 2019; Hearne, 1978). Ever since the discovery of LD, it is assumed that lucid dreaming is learnable and all individuals have the ability to experience that (Kern, Appel, Schredl, & Pipa, 2017). The functions of a lucid dream are localized to specific brain areas. For example, during an EEG study, beta frequency was found throughout the parietal lobe of the brain, an area that is responsible for episodic memory and self-reflection, both of which are functions of LDs (Baird et al., 2019). High cortical activity in frontal regions also seem to play a role in LD, specifically the prefrontal cortex; this region’s functions are also selfreflection, planning, as well as top-down processing such as regulating attention (Baird, Castelnovo, Gosseries, & Tononi, 2018; Stumbrys, Erlacher, Schädlich, & Schredl, 2012). Due to that, Mota-Rolim and Araujo (2013) hypothesized that LD is linked with high frontal cortex activity during REM sleep.

to induce LD can be done by inhibiting the enzyme that breaks down acetylcholine, acetylcholinesterase, using acetylcholinesterase inhibitors (AChEI) (Sparrow, Hurd, Carlson, & Molina, 2018). The mechanism by which these inhibitors work in LDs is poorly understood, but it is suggested to work by increasing the time the dreamer is in REM sleep, strengthening connections between brain areas that are highly linked with LD, or by affecting higher cognitive processes that are specific to LDs (Baird et al., 2019). Amatruda and colleagues (1975) found that agonists of ACh and antagonists of AChEI all increase REM sleep phases. AChEIs also alleviate some cognitive symptoms of Alzheimer’s Disease or vascular dementia due to their memoryimproving effect (Birks & Craig, 2006; Koontz and Baskys, 2005). Consequently, with LDs, these inhibitors can increase dream recall (Koontz and Baskys, 2005). Galantamine is a type of acetylcholinesterase inhibitor, this drug is known to increase the number of lucid dreams (Sparrow et al., 2018). In a study by Sparrow and colleagues (2016), lucid dreamers that took galantamine had much longer and more vivid dreams than subjects that did not. Galantamine is also used as a treatment in patients with severe sleep disorders; galantamine improved overall sleep quality and decreased awakening during sleep (Litvinenko, Krasakov, & Tikhomirova, 2013). Additional ways to induce LD include the usage of external and internal cues administered during REM sleep. External cues are for the dreamer to be aware that he/she is dreaming, such as a cue to be heard or a cue by touch, while internal cues are normally spontaneous phases that occur in the dream to notify the dreamer that he/she is dreaming (Gackenbach, 1985). LaBerge and colleagues (2018) also introduced the Mnemonic Induction of Lucid Dreams (MILD) technique: a subject makes an intention that he/she will lucidly dream, thus using prospective memory to remember to be conscious when dreaming. This technique is perhaps a better strategy to induce LD, more so than external cues alone; a combination of the two might even be better (Stumbrys et al., 2012). To maximize the MILD technique, LaBerge and coworkers (2018) suggest that the participant wake up a few hours into his/her sleep, remain awake for a range of 30-120 minutes, apply the MILD technique, and then go back to sleep. It is important for research on lucid dreams to continue and progress, this provides a gateway into studying consciousness, and various altered states that can be achieved during REM sleep, as well as a method for a physically impaired subject to be able to control his/her own motor abilities in a state of lucid dreaming (Mota-Rolim & Araujo, 2013).

Interestingly, LDs are experienced in different ways in different individuals, some people experience a more vivid dream, some have a stronger recall for the dream than others, and the degree of consciousness during the LD also varies among individuals (Baird et al., 2019). According to McLaughlin and coworkers (2015), different psychiatric disorders may be correlated with LDs. Such conditions include Post-Traumatic Stress Disorder (PTSD) and Reward Deficiency Syndrome-associated conditions. In their study, a dopamine agonist increased the occurrence of happy dreams. However, when a patient suffering from PTSD was also experiencing lucid nightmares, the same dopamine agonist was administered and in turn happy dreams were reported (McLaughlin et al., 2015). However, luThis review will be focusing on the paper by LaBerge and cid dreams and their association with the dopamine circuit are colleagues (2018). This study presumed that AChEIs induce LDs, not well understood. however the purpose of the experiment was to quantify the Many studies have proved that LDs are inducible (LaBerge, effect of galantamine on LD induction and whether or not this LaMarca, & Baird, 2018; Stumbrys et al., 2012). As mentioned method is reliable, as well as its dynamic relationship with the earlier, it is assumed that LD is a skill that can be learned and MILD technique. Perhaps a combination of both galantamine developed (Baird et al., 2019). One of the main ways to induce and the MILD technique will shift lucid dream induction in a LD is via the build-up of acetylcholine at synapses (Baird et al., favorable direction (Stumbrys et al., 2012). The reviewed study 2019). Acetylcholine plays a great role in REM sleep, and eviinvolved sleep interruption, galantamine ingestion, and then dence shows that REM sleep is controlled by brainstem cholinthe application of the MILD technique. The major result from ergic neurons which results in increased cortical activity this study is that the higher the dose of galantamine, the higher (Amatruda, Black, McKenna, McCarley, & Hobson, 1975). A the number of LD reports. Overall, the study shows that the proven more effective way of affecting the cholinergic system 62

method of integrating galantamine after sleep interruption as well as using prospective memory (via the MILD technique) is very effective for LD induction. MAJOR RESULTS A detailed schematic of the study’s methods is outlined in Figure 1. For three nights in a row, subjects wake up from their sleep 4.5 hours later, which is approximately at the third REM cycle. The participants recollected the dream they had up to that moment, took a galantamine capsule (either a placebo pill with 0 mg galantamine, 4 mg, or 8 mg), they then preoccupied themselves for at least the next half hour (sleep interruption), and then returned to bed, applied the MILD technique, and Figure 1. Detailed schematic of method used. Adapted from finally went back to sleep. The participants woke up after the Figure 1 by “Pre-sleep treatment with galantamine stimulates lucid dreaming: A double-blind placebo-controlled, crossover following REM cycle and reported their dream once again. study,” by S. LaBerge, K. LaMarca, & B. Baird, 2018, PLoS ONE, The major results of this study were that highest reports for 13(8), 4. Copyright 2018 by LaBerge et al. LDs were from participants who ingested 8 mg galantamine capsules (42%), then 4 mg (27%), and both were higher than the placebo pill (14%). Upon looking at the overall integration results (galantamine + MILD), the higher the galantamine dosage, the frequency of LD was way higher than expected. For example, lucid dream frequency was six times more frequent for 4 mg doses, while 8 mg resulted in a nine times greater frequency. The combined protocol increased LDs for approximate- Figure 2. Graph showing increase of lucid dream reports with an increase in galantamine dosage. Adapted from Figure 2 by ly 57% of the participants. “Pre-sleep treatment with galantamine stimulates lucid dreamFigure 2 shows the number of LD reports of lucid dreamers. ing: A double-blind placebo-controlled, crossover study,” by S. BASE is the frequency of LDs the lucid dreamers experienced LaBerge, K. LaMarca, & B. Baird, 2018, PLoS ONE, 13(8), 7. Copthe past six months prior to the experiment. G0 denotes the yright 2018 by LaBerge et al. placebo capsule (zero mg of galantamine), G4 is 4 mg, and G8 is 8 mg of Galantamine. Primarily, Figure 2 shows that LD reports DISCUSSION increased as the dose of galantamine increased. This study’s main findings are that higher doses of galantaParticipants that had their sleep interrupted for at least half an hour and then practicing the MILD technique for a minimum mine in integration with the MILD technique can effectively of ten minutes were also more likely to experience a lucid induce LDs in healthy individuals. This integration method can dream. This shows that the length of sleep interruption is cru- also improve many aspects of consciousness including selfcial for inducing a lucid dream as well as the time spent engag- reflection during LDs, vividness, and control. Researchers LaBarge, LaMarca, and Baird (2018) conclude that increasing the ing in the MILD technique. neurotransmitter acetylcholine can increase the frequency of As for other variables that were measured throughout the LDs, and the combined method increased LDs for more than experiment, LDs in the study resulted in improved conscioushalf of the participants (57%). ness in different dimensions. The aspects measured were recall, Researchers of this study were not very sure about the mechvividness, clarity, positive emotion, negative emotion, control, anism behind galantamine as an AChEI. What is understood, self-reflection, complexity, and bizarreness. All dimensions though, is that acetylcholine agonists and AChEI inhibitors are were increased in LD reports except for negative emotions that associated with REM sleep, the phase of sleep when LD occurs. decreased. As for the side-effects of the galantamine drug, Notable conclusions by the researchers include: injecting other most participants tolerated its effects well, with only 14 subACh agonists were able to prolong REM sleep, and inhibitors of jects (about 12% of the sample) experiencing an upset stomAChE also being able to manage cognitive and memory deficits, ach, fatigue, and insomnia. which is why they are used on patients with Alzheimer’s DisThese results are significant and important in acknowledging ease. Enhancing cholinergic effects may reduce the latency of that galantamine has a great effect in inducing LDs as well as REM sleep, and memory enhancements may be due to proimproving different aspects of consciousness. Additionally, the spective memory strategies (such as the MILD technique), beMILD technique shows the importance of setting an intention cause the individual is increasing his/her own propensity to as an internal cue when lucidly dreaming as well as the imlucidly dream. portance of time/sleep interruption throughout the study. Inhibitors of AChE may not completely act on the cholinergic However, the combination of galantamine and the MILD techsystem, but may also act on other neurotransmitters, such as nique is the most ideal protocol to undertake when inducing dopamine, serotonin, and noradrenaline; dopaminergic neuLDs. 63

rons being activated and latter two systems being completely shut-off during REM sleep (Hobson, 2009). As mentioned earlier in the Backgrounds section, dopamine agonists improve the quality of dreams, from lucid nightmares to happy dreams (McLaughlin et al., 2015). Researchers of the reviewed study acknowledge that the dopaminergic system may also increase LD frequency because by activating it, metacognition is improved, as well as self-reflection. An important note about this study is that the researchers predict similar results for individuals who generally have strong memory for their dreams and who have an interest in dreaming lucidly. However, the conclusions of this study regarding the reliability and effectiveness of galantamine is referring to, however not restricted to, Hawaii’s lucid dreaming training program where the subjects are able to learn how to lucidly dream.

participants, to have a set of findings specific to individuals with altered states of consciousness would be worth analyzing. FUTURE DIRECTIONS – Building on the first two limitations in the Critical Analysis section, these can be improved in future studies to conduct the same experiment in a sleep laboratory with an EEG monitor in order to confirm that REM sleep is the phase in which participants claim to lucidly dream, rather than relying on assumption and estimation of sleep phases (and instead based on EEG brain waves). Having this experiment done in a controlled lab setting is also useful to control the time at which sleep interruption occurs. For example, all participants will sleep at 10:00 p.m. and their sleep will be interrupted at 2:00 a.m.. This can be controlled and regulated by the researchers alone, instead of relying on subject reports and having the participants themselves interrupt their sleep 4.5 hours later. Additionally, a controlled setting can give researchers the opportunity to monitor the different ways each participant prepares his/her prospective memory during the MILD technique. The reason for this is that the MILD technique may be a technique that is unique to every individual, which would be worth looking into how different MILD preparatory methods can affect LD reports.

The primary aim of the study was to quantify galantamine’s effect and develop a set methodology for lucid dream induction. This aim was fulfilled and the current results give evidence for an established LD protocol. Since galantamine and the MILD technique were only studied in integration with each other, it is poorly understood how the results would look like if these variables were studied independently of one another. The significant findings of this study make way for further studies focusing on consciousness, and an individual’s capabilities when in a Having said that, the third limitation in the Critical Analysis state of LD. section can be improved by conducting the study using different galantamine doses and the MILD technique first in isolation CRITICAL ANALYSIS from one another, and then follow it with an integrated apThere were limitations to this study. Firstly, the data of the proach due to the possibility of the stronger method merely study relied heavily on subject report and inferring which stage masking the weaker one and being unable to distinguish the of sleep the LDs occurred in. The study was not executed in a effects between them. sleep laboratory nor used an EEG wave monitor, therefore, there was no way to confirm that the LDs actually occurred during the REM sleep phase. The researchers also specifically relied on a series of steps to authenticate the participant’s report, such as by firstly having the subjects understand what LDs are, by using participant reports of whether or not they achieved LDs after each night, by having them indicate the factors that make the dream lucid, and lastly by having participants recall the dream.

It would also be important to carry out the same study using participants with varying severities of mental disorders such as PTSD or sleep disorders. This would be a good way to assess whether the effects of galantamine, its different doses, as well as the MILD technique work the same way as they do in neurotypicals. Since dopamine agonists have proven to convert lucid nightmares into happy dreams, researchers should experiment this phenomenon on the cholinergic system; perhaps there is an association between the two systems which in turn can imAnother limitation, based on the first one, is the fact that the prove sleep quality in such individuals (McLaughlin et al., 2015). time at which sleep interruption occurred was estimated and In addition, given prior research, the mechanism through not regulated. This inferred whether the dreamer was in a peri- which acetylcholinesterase inhibitors work in relation with conod of REM sleep or not. For example, all participants were ex- sciousness and improving cognitive impairments is poorly unpected to wake up approximately 4.5 hours after initially falling derstood. Similar to the previous point, perhaps future reasleep, which is after about three cycles of REM. However, search ideas may include experimenting how galantamine or though research supports this, it is a major limitation to make other AChEIs, in a dose-related manner, affect the conscious or assumptions in a study. unconscious states of individuals with psychosis (keeping in It is also important to have studied the effects of both galan- mind that psychosis patients suffer from different positive tamine and the MILD technique independent of each other. symptoms for example hallucinations, as well as experience This is crucial to know if the statistically significant results are a ‘dream-like’ states during wakefulness) (Garety, Kuipers, result of an interaction between the two, or whether one has a Fowler, Freeman, & Bebbington, 2001). For an experiment like larger effect than the other and is merely masking the effects of this, the authors of this review would expect galantamine to instill happy lucid dreams in psychosis patients, given that the other, ‘weaker’ method. galantamine could work in association with the dopamine sysLastly, the study did not use participants with any sleep or tem and administering dopamine agonists in past studies have mental disorders. Though it is very important to have healthy alleviated nightmares (McLaughlin et al., 2015). Galantamine may also work to balance out their ‘awake’ life and produce a 64

more ‘dream-like’ dream states, and a more ‘awake-like’ awake state. For example, hallucinations may be reduced, and selfawareness may be enhanced. In terms of doses, the authors would expect that a higher dose of galantamine to indicate better improved sleep quality. However, this experiment should also consider other acetylcholinesterase inhibitors such as Donepezil. Donepezil has been used in the past on patients with Alzheimer’s Disease, however not particularly on patients with psychosis (Lilienfeld, 2002). It would be interesting to know how the different strengths of different AChEIs may have an impact on the quality of consciousness and self-awareness. Lastly, induction of LDs has potential to evolve into a therapeutic technique, especially for individuals with motor and physical impairments (Mota-Rolim & Araujo, 2013). Future studies can assess the degree to which physically impaired individuals are able to control their dreams and whether or not their motor impairments play a role in setting an intention to ‘move’ or ‘act’ in their dreams. To be able to induce LDs can be a method of motor rehabilitation for such individuals (MotaRolim & Araujo, 2013).

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Liquiritigenin as an Antidepressant in Chronic Mild Unpredictable Stress Induced Mouse Models of Depression Isabelle Carvalho

Major depressive disorder (MDD) is a mental illness marked by depressed mood, increased cognitive impairments, and diminished pleasure. MDD has had a greater economic and social impact than any other mental illness, affecting 4.4% of the global population and costing the Canadian economy 32.3 billion dollars a year. Stress has repeatedly been linked to the development of MDD symptoms and seems to affect similar regions of the body, as addressed in the paper by Tao et al. (2016). In unpredictable chronic mild stress induced (UCMS) mice, there was greater pro-inflammatory cytokine production and decreased antioxidant production. With regards to common behavioural tests of MDD symptomatology in mice, UCMS mice in the Tao et al. study also had decreased motivation in the forced swimming and tail suspension tests, alongside increased sucrose consumption. Known antidepressants such as fluoxetine hydrochloride (Flu) have been shown to markedly decrease the aforementioned effects. The study by Tao et al. found that 15mg/kg of Liquiritigenin (Liq) reduced behavioural and physiological symptoms of MDD and UCMS nearly as well as UCMS+Flu and significantly better than UCMS alone or UCMS+7.5mg/kg Liq. These findings suggest that Liq may serve as an alternative to common antidepressants in stress induced MDD and provides further evidence for a direct connection between UCMS and MDD. Key words: major depressive disorder, unpredictable chronic mild stress, treatment, Liquiritigenin, BDNF pathway, immobility mice tests of depression, antidepressants, fluoxetine hydrochloride

BACKGROUND or INTRODUCTION. In Canada alone, depression costs the economy 32.3 billion dollars a year (WHO, 2017) and affects 1 in every 4 Canadians over their lifetime (Ontario MoH, n.d.). Depression manifests both physically and psychologically. It lowers the individual’s motivation and memory capacity. It increases the individual’s pleasureseeking behaviours and bodily inflammation. It even increases the risk of neuronal death and has been known to permanently lower neuronal communication throughout the brain (Kubera et al., 2011). As such, major depressive disorder (MDD) is disabling to those with its most severe and/or long-lasting forms and, as such, is disabling to the greater Canadian economy for as long as it remains untreated.

Trauma and stress were found to be major contributors to MDD. 62.5% of individuals with MDD have faced two or more traumatic events in childhood, most of whom faced long-term traumas (parental neglect or repeated abuse) (Williams et al., 2016). Individuals with chronically high-stress jobs are 1.8 times more likely to struggle with MDD than their non-stressed counterparts (Siegrist, 2008). Chronically stressed animals had higher release of neuro-inflammatory markers such as interleukin-1beta (IL1beta), tumour necrosis factor alpha (TNF-alpha), and interleukin-6 (IL-6). With regards to depression-like behaviour, chronically stressed animals scored higher in tests of learned helplessness such as the tail suspension test (TST) and forced swim test (FST) (Kubera, 2011). In both animal models and human studies, stress has been directly correlated with MDD and depressionlike symptoms.

exposed to unpredictable chronic mild stress (UCMS) and was not provided any pharmaceutical intervention. Another control group was exposed to UCMS and not provided any pharmaceutical intervention. The last three groups were exposed to UCMS and a pharmaceutical intervention of either fluoxetine, a lower dose of liquiritigenin (7.5mg/kg), or a higher dose of liquiritigenin (15mg/kg). UCMS involved a multitude of mouse-specific distressing actions, including but not limited to inversion of the day -night light cycles, water or food deprivation, and tail pinching. Both physiological and behavioural tests were performed to test for depression-like responses to the stressful experiences and/or pharmaceutical treatments. While liquiritigenin did not limit depression-like responses to UCMS as well as the known antidepressant, fluoxetine, it did substantially lower depression-like responses when compared to the UCMS/non-pharmaceutical control condition (Tao et al., 2016). As liquiritigenin is known to have very few side effects and no serious side effects (Grady et al., 2009), these results do indicate that liquiritigenin may serve as an alternative for patients with a history of chronic stress who do not tolerate common SSRIs.

MAJOR RESULTS In the research of Tao et al. (2016), mice were exposed to UCMS and given either fluoxetine, a lower dose of liquiritigenin (7.5mg/kg), or a higher dose of liquiritigenin (15mg/kg). The flavonoid liquiritigenin has been known to aid in the reduction of beta amyloid induced neurotoxicity (Liu and Lu, 2010) and in the reduction of neuroinflammation (Kim et al., 2009). Neuroinflammation and neurotoxicity have been well accepted as potential causes of depression-like symptoms (Hurley and Tizabi, 2012) and psychological stress has been associated with increased neuroinflammation (Barnum et al., 2012). Tao et al. argue that the anti-inflammatory properties of liquiritigenin make the compound a potential alternative to common antidepressants. This is further supported by three major findings from their experiment. Figure 1 indicates that UCMS was found to increase depression-like behavioural symptoms in mice and that liquiritigenin was found to reverse this effect. Figures 2 and 3 indicate that UCMS-induced inflammatory and antioxidative-marker symptoms in mice were reduced in mice exposed to liquiritigenin.

While common SSRIs are touted as the first line of defense against depression, their lower-than-expected efficacy coupled with their high risk of side effects has left prescribers searching for better options. While 50% of those taking SSRIs responded positively, 32% of those in the placebo condition of the same study also did so, implying a minimal antidepressant effect (Moncrieff and Kirsch, 2005). Individuals with a childhood trauma history respond even less to antidepressants than the average depressed individual (Williams et al., 2016). As a majority of MDD patients are primed for stress-induced non-response to SSRIs, the search for a truly effective SSRI is as pressing as ever. This raises one major question: is there a medication that will reduce symptoms in individuals with stress-induced depression Behavioural Effect of Liquiritigenin on UCMS Exposed-Mice without serious side effects?

In the research performed by Tao et al. (2016), 10 mice were exposed to UCMS without pharmaceutical intervention and 10 mice consisted of a true control (no UCMS, no pharmaceutical intervention). After 40-42 days of UCMS exposure, behavioural tests of depression were performed. These tests include the forced swim test (FST), sucrose preference test (SPT), and tail suspension test (TST).

In the study of focus for this review, Tao et al proposed the use of liquiritigenin as a potential antidepressant for patients facing chronic stress (2016). Liquiritigenin is a naturally derived selective estrogen receptor beta antagonist (Grady et al., 2009). It has been used in Chinese medicine for centuries as a treatment option for the symptoms of menopause, antioxidation, inflammation, and diabetes (Alrushaid, 2016). Liquiritigenin’s attraction lies in its minimal side effects, of which the most common is mild According to Yankelevitch-Yahav et al. (2015), greater immobility loose stools (Grady et al.). In the study by Tao et al, fifty mice time indicated greater despair, as the mouse lost hope in its own were divided into five categories. One control group was not 69

escape. This despair and loss of hope serves as a model for depression in mice. UCMS-exposed mice were immobile during the TST and FST for substantially longer periods of time than nonUCMS-exposed mice as per figure 1a. This indicates that UCMSexposed mice gave up on survival more quickly and therefore had more severe depression-like behaviour.

Figure Adapted from Liquiritigenin Reverses Depression-Like Behavior in Unpredictable Chronic Mild Stress-Induced Mice by Regulating PI3K/Akt/Mtor Mediated BDNF/Trkb Pathway, by Tao et al. (2016).

Figure 1b. Effect of UCMS and/or Pharmaceutical interventions on Despair Models of Depression in Mice Measurements were taken after 40 days of UCMS and/or pharmaceutical intervention. The percent consumption of sucrose Anhedonia is a symptom required for a diagnosis of depression, water versus regular water determined the percent sucrose preferaccording to the DSM-5 (2013). In mouse models of anhedonia, ence. Scheggi et al. (2018) indicate that depressed mice seek out less sucrose than non-depressed mice. As sucrose consumption triggers reward centers in the brain, decreased sucrose consump- Physiological Effect of Liquiritigenin on UCMS-Exposed Mice tion indicates a lack of pleasure on a physiological level (Scheggi et al.). UCMS-exposed mice sought out sucrose significantly less often than their control counterparts, according to figure 1b. 10 mice were exposed to 7.5mg/kg of liquiritigenin, 10 to 15mg/ Therefore, it can be stated that highly stressed mice were more kg of liquiritigenin, and 10 to 20mg/kg of fluoxetine. After 42 anhedonic, indicating the presence of more severe depression- days of UCMS exposure, tests were performed to determine the concentration of inflammatory markers and markers of oxidative like symptoms. stress. 30 mice were also divided into three categories: UCMS with fluoxetine, UCMS with 7.5mg/kg of liquiritigenin, and UCMS with 15mg/kg of liquiritigenin. Figure 1a shows that exposure to liquiritigenin did successfully decreased immobility time in both the FST and TST, with 15mg/kg of liquiritigenin being nearly as effective as fluoxetine in both. Figure 1b shows that exposure to liquiritigenin successfully increased sucrose preference to nearly the same level as fluoxetine exposure.

In a study performed by Miller et al. (2002) it was found that in serum concentrations of inflammatory markers such as IL-6 and IL-beta were raised in depressed individuals. Similarly, UCMS exposed mice had greater concentrations of IL-6, IL-beta, and TNF-alpha in both the hippocampus and in serum, implying system-level inflammation as well as hippocampal-level inflammation. Inflammation in the brain is known to increase the risk of apoptosis of neurons. The presence of inflammation in the hippocampus is likely the cause of known decreases in hippocampal volume in depressed patients (Sheline, Mittler, and Mintun, 2002). Tao et al.â&#x20AC;&#x2122;s study indicates that this inflammation may be reversible. In figure 2, liquiritigenin significantly reduced the concentration of all three inflammatory markers, in both serum and the hippocampus. In figure 2d, 15mg/kg of liquiritigenin was equally effective to fluoxetine in decreasing IL-6 concentrations in the hippocampus. In figure 2e, 15mg/kg of liquiritigenin was more effective than fluoxetine in decreasing IL-1beta concentrations in the hippocampus. Therefore, it can be said that liquiritiFigure Adapted from Liquiritigenin Reverses Depression-Like Be- genin is an effective anti-inflammatory on both a system and havior in Unpredictable Chronic Mild Stress-Induced Mice by Reg- hippocampal level. ulating PI3K/Akt/Mtor Mediated BDNF/Trkb Pathway, by Tao et al. (2016). Figure 1a. Effect of UCMS and/or Pharmaceutical Interventions on Despair Models of Depression in Mice Measurements were taken after 42 days of UCMS and/or pharmaceutical intervention. Immobility time in both the forced swim test (FST) and tail suspension test (TST) was measured in seconds.

Antioxidative molecules such as GSH, CAT, and SOD are critical in the protection of the brain from oxidative species (Jiang et al., 2016). The concentration of these molecules decreases in stressed individuals (Olsvik et al., 2005) and depressed individuals (Bilici et al., 2001). The Tao et al. study indicates that this effect can be reversed. Liquiritigenin significantly increased the concentration of GSH, CAT, and SOD in serum according to figure 3b-3d. Furthermore, figures 2b and 3d show that 15mg/kg liquiritigenin increased the concentration of SOD and CAT as effectively as the known antidepressant, fluoxetine. It can be concluded that liquiritigenin was effective in raising concentrations of antioxidants.

MDA is a product of lipid oxidation and is therefore used as a marker to indicate the presence of oxidative stress (Islam et al., 2018). Higher concentrations of MDA are associated with higher oxidation and, therefore, greater symptoms of depression (Islam et al.). In figure 3a it can be seen that MDA levels were substantially higher in the UCMS category. While liquiritigenin did significantly lower the concentration of MDA in serum, it did not do so as well as fluoxetine did. This provides evidence for the claim that liquiritigenin successfully decreases lipid oxidation in serum.

of Oxidative Stress in Serum Measurements taken after 42 days of UCMS and/ or pharmaceutical intervention. Measurements for MDA in μmol/ml. Measurements for SOD and CAT in U/ml. Measurements for GSH in U.

CONCLUSIONS/DISCUSSION The study performed by Tao et al. (2016) contained two major discoveries. Firstly, it demonstrated that 15mg/kg of liquiritigenin is sufficient to decrease the UCMS-induced, depression-like behaviours in mice with nearly the same effectiveness as fluoxetine, a known antidepressant. Secondly, it demonstrated that 15mg/kg of liquiritigenin is sufficient to decrease the concentration of inflammatory molecules and MDA to nearly the same degree as fluoxetine. Finally, it demonstrated that 15mg/kg of liquiritigenin is sufficient to increase the concentration of antioxidants to nearly the same degree as fluoxetine. As such, it was concluded by the authors that liquiritigenin is an effective antidepressant as it is capable of reversing the depression-like effects of UCMS. As individuals with stress-induced depression are more likely to be unresponsive to common antidepressants (Siegrist, 2008; Williams et al., 2016), the discovery of a low-side effect antidepressant that functions for individuals with stress induced depression could be life saving.

The effectiveness of liquiritigenin in stress-induced depression-like symptoms specifically had not been tested prior to the paper by Tao et al. (2016). However, liquiritigenin has been found to reduce behavioural and inflammatory symptoms of depression in mice with non-UCMS induced depression (Su et al., 2016). Therefore, the antidepressant effect of liquiritigenin on behavioural symptoms that was found in figure 1 is supported by the literature. Figure Adapted from Liquiritigenin Reverses Depression-Like Behavior in UnpreWhen focusing on stress-induced depression, support for Tao et dictable Chronic Mild Stress-Induced Mice by Regulating PI3K/Akt/Mtor Mediatal.’s findings can also be found. Stress is known to trigger proed BDNF/Trkb Pathway, by Tao et al. (2016). Figure 2a-2f. Effect of UCMS and/or Pharmaceutical Interventions on Inflammatory Markers in Serum and the Hippocampus Measurements taken after 42 days of UCMS and/or pharmaceutical intervention. Measurements for TNFalpha, IL1beta, and IL-6 measured in pg/mg.

inflammatory cytokine release, depression-like symptoms are known to be triggered by pro-inflammatory cytokine release (Kubera et al., 2011), and liquiritigenin is known to reduce the concentration of pro-inflammatory cytokines in the blood (Kim et al., 2009). Therefore, the inflammatory marker results in figure 2 are also supported by the literature.

Finally, the antioxidant properties of liquiritigenin have also been supported by the literature. Antioxidant concentration is decreased and oxidative products are increased in both depressed (Bilici et al., 2000) and stressed (Olsvik et al., 2005) individuals. As liquiritigenin is known to have antioxidant properties (Alrushaid et al., 2016), the antioxidant-specific findings in Tao et al.’s paper (figure 3) are supported by the literature.

CRITICAL ANALYSIS The focus of this paper was on liquiritigenin and its potential use as an antidepressant (Tao et al., 2016). Liquiritigenin is a natural compound derived from liquorice, and has been used in traditional Chinese medicine for centuries (Tao et al.). It is known for its analFigure Adapted from Liquiritigenin Reverses Depression-Like Behavior in Unpre- gesic and anti-inflammatory properties, while also being used by dictable Chronic Mild Stress-Induced Mice by Regulating PI3K/Akt/Mtor Mediat- diabetics (Alrushaid, 2016). The use of so-called “traditional” remeed BDNF/Trkb Pathway, by Tao et al. (2016). dies has historically been frowned upon by doctors, although papers Figure 3a-d. Effect of UCMS and/or Pharmaceutical Interventions on Markers like the one written by Tao et al. demonstrate the importance of not

overlooking these traditions.

Focusing on the methods themselves, the use of three controltype conditions increases the significance of this paper. A true control (no UCMS, no pharmaceutical intervention) with a UCMS control allows the researchers to further confirm the presence of depression-like symptoms with UCMS exposure for all potential tests of depression, confirming the validity of UCMS in mouse models of depression. The use of a fluoxetine control allows researchers to determine the value of any effects liquiritigenin may have on depression-like symptoms. Were liquiritigenin not comparable in effect to a first-line drug such as fluoxetine, the benefit of this discovery to the psychiatric community would have been negligible at best.

A central issue with this paper was the definition of UCMS. By surveying multiple studies, it can be determined that there is no widely-accepted protocol for the frequency at which researchers should perform UCMS-inducing activities (Forbes et al., 1996; Nollet, Guisquet, and Belzung, 2013; Detanico et al., 2009). Researchers such as Tao et al. utilized a random selection model, which increases the risk of some mice being exposed for longer to more stressful activities than other mice (2016). While the use of random selection should result in relatively even exposure across a cohort, the small sample sizes per cohort (only 10 mice) decreases the chances of relatively even distribution and therefore decreases the viability of the research (Bean, Stafford, and Brashares, 2012). It is recommended that in the future, further studies either increase their sample sizes or regulate the amount of hours each mouse is exposed to a given stressful stimulus in order to ensure the validity of any future results.

the effectiveness of the drug on varying intensities of stressinduced depression, which would permit the determination of ideal doses in humans. Furthermore, the use of perceived rather than actual stress may lead to greater accuracy in results. In a paper by Aneshensel and Stone (1982), it was determined that perceived strain was a better predictor of depression symptom severity than actual life events. As perceived stress is impossible to test in mice, there is even greater incentive to begin human trials instead. As perceived stress is known to have a greater effect on human depression symptoms than the frequency of stress-inducing events, it is likely that individuals with a higher rating of perceived stress will also have more severe depression symptoms (Aneshensel and Stone). As individuals with higher reported stress levels also have a tendency to be minimally responsive to antidepressant treatments, it can be assumed that these individuals will respond less effectively to liquiritigenin treatment (Williams et al., 2016). If liquiritigenin works equally well between all levels of stress, it can be assumed that the level of stress is a minimally significant a predictor of liquiritigenin success. As the higher liquiritigenin dose was more effective according to Tao et al.â&#x20AC;&#x2122;s study, it can also be assumed that a higher dose will be more effective in any human trials. If at one point a higher dose is deemed to be just as effective as a lower dose, it can be assumed that maximal efficacy has been reached.

FUTURE DIRECTIONS Researchers should perform human trials with larger sample sizes in the future. Liquiritigenin is a compound already commonly used in China. It has well understood side effects, of which the worst is soft stools (Mersereau et al., 2008). This is comparatively mild when considering the most common side effects of SSRIs tend to be sexual dysfunction, exhaustion, and weight gain (Cascade et al., 2009). While mice are often used in research in order to prevent the ethical dilemmas of human testing, most ethical concerns are nulled by the fact that liquiritigenin has been safely used for centuries (Mersereau et al.). Human trials would also allow researchers to circumvent issues regarding UCMS and its definitions. While humans do have more complex lives with more complex stressors, a plethora of well vetted pencil-and-paper tests of stress are known to exist which can formally categorize the human stress experience. The Perceived Stress Scale is a well understood scale of human stress that is further supported by physiological and self-rating tests of stress (Lee, 2012). Such a scale could be used in future human trials in order to reduce the risk of variability. Individuals could also be divided into categories, with cohorts determined by the level of perceived stress. This would allow researchers to test 72

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Cannabisâ&#x20AC;&#x2122; Effects on Mental Health and Cognition When Used as a Therapy for Chronic Pain in Lieu of Opioids Xuan Chen

Due to its highly addictive nature, many patients who are prescribed opioids for chronic pain management turn to the usage of cannabis instead. Despite the wide usage of cannabis both recreationally and medically, the effects of its interactions with mood and cognition are not well understood. 187 individuals who currently use opioid medication were recruited from three outpatient pain clinics in Washington state and screened. The aim was to see whether they have ever used cannabis for their pain, and how this can be attributed to their mental state. 150 participants were determined as eligible based on pre-established inclusion and exclusion criterion. They were then asked to complete a cross-sectional survey where they self-reported cognition levels, depression and anxiety symptoms, as well as the potency and frequency of cannabis that they consume on average. Increased frequency and potency in the last 30 days typically led to worsened conditions of depression, anxiety, and cognition. This significantly worsened effects of already poor cognitive function. Interesting to note was that individuals who were already at increased risk for cognitive impairment were more likely to use cannabis in assistance to manage pain and other stressors. As the information was collected through surveys, cause and effect cannot be concluded, however it does set up for future studies as more research needs to be done on the exact effects of cannabidiol (CBD) and mood interactions, as well as how CBD and tetrahydrocannabinol (THC) differ. Keywords: cannabis, cannabidiol, tetrahydrocannabinol, opioid, chronic pain, pain management, depression, anxiety, cognition

Introduction: A significant portion of the adults in the United States live with chronic pain, putting them at increased risk for opioid dependence, mental health disorders such anxiety and depression as well as an overall decrease in quality of life 1. Typically, pain symptoms are managed by the effective use of opioids however with the epidemic and over-prescription of narcotics2 many patients are turning to the usage of cannabis instead11. Despite the constant controversies regarding cannabis in the media, it has long been used as a method for pain management. Cannabis describes a family of plants, including marijuana, and produces hundreds of both cannabinoids and terpenes. Of these, the most commonly used are cannabidiol (CBD) and delta9-tetrahydrocannabinol (THC) which act on different cannabinoid receptors in the human endocannabinoid system3. Between THC and CBD, the entire body can be affected as THC mainly targets receptors in the brain which controls memory, fear and motor responses; CBD targets the peripheral body such as various organs and muscle3. They can also be consumed via various methods such as inhalation or ingestion, although some routes act faster as they are closer to the bloodstream or target organ12. Currently, chronic pain is the most common reason that individuals inquire about medical cannabis4. However, despite the widespread use of cannabis both medically and recreationally, there are very mixed results regarding whether it truly has benefits and whether they outweigh possible consequences14.

tional Support Short Form allowed participants to rate how often in the past week they felt that they had social support. The General Anxiety Disorder Scale consisted of a 7-item scale that allowed individuals to indicate how often they have been bothered by symptoms of anxiety over the past two weeks. Lastly, the Patient Health Questionnaire was an eight-item questionnaire used to assess depression and its symptoms. Overall it was noted that the negative effects of depression and anxiety on cognition were worsened as the usage of cannabis increased, particularly if the products were high in CBD content.

Results: Participants were recruited from waiting areas of clinics, approximately 200 individuals were approached however not all qualified as there were a number of refusals as well as ineligible participants. The largest social group that data was collected from in this study were Caucasian females that were either unemployed, disabled or retired; a large portion of participants were also not educated past the secondary school level. There was an approximately even split for those who have or have not ever used any form of cannabis for their pain management. Within the cannabis user subpopulation, a majority reported usage of edible cannabis, another significant portion have used some form of cannabis in the past thirty days. Of all the participants sampled, 16% reported usage of products that were greater than 10% in CBD concentration, while 27% reported usage of THC concentration greater than 10%. A small portion One of the most important factors to consider is cognition, as it also did not know the average amount of THC and CBD content is negatively impacted by both chronic pain as well as drug us- consumed per use. age. Especially since patients are typically also at risk of poor mental health, it is important to investigate how this can impact cognition altogether. Overall the paper Cannabis Use and Cogni- More than two thirds of participants surveyed reported modertion in Adults Prescribed Opioids for Persistent Pain aims to un- ately severe pain (7 out of 10), similar proportion of results were derstand if the frequency of either THC or CBD usage can affect found for those experiencing moderate to severe anxiety howthe relationship between mental health and disorders, specifi- ever almost half of all participants were experiencing moderate cally between poor cognition with anxiety and depression. Addi- to severe depression symptoms. tionally, the study also wanted to determine whether having a strong social support network can alleviate any negative effects. As for the specific aims of this study, it was found that poor cognition levels were able to be attenuated with increased social The authors set out to investigate its two points of inquiry support, this was found to be significant at p=0.01. Unfortunatethrough the usage of surveys and questionnaires, completed by ly, social support was not enough to ameliorate anxiety and departicipants recruited from three outpatient pain clinics. The pression symptoms. However, the symptoms of these two illparticipants were recruited from an American state where recre- nesses largely contributed to the decreased cognition, also sigational and medical use of cannabis are legal, in order to bypass nificant at p < 0.01. The frequency of cannabis usage within the barriers of participants being afraid to share their activities. The past month were positively correlated with depression and anxisurveys were completed on paper by eligible participants and ety symptoms. All of this is summarized in the table below: included multiple sections: The Demographics and Participant (Figure 1) Characteristics Questionnaire collected demographic information that would allow participants to be stratified into social groups based on age, gender, ethnicity and overall background. The Cannabis Use Questionnaire was modeled off of national health surveys, with the first question asking whether participants have ever used marijuana. Those who have, would then indicate the frequency and reason of usage, such as registered medical usage for pain management. The Cognition Short Form asked participants to rate whether they felt like their cognition was as sharp or intact as usual for the past seven days. An Emo76

Symptom Poor cognition Anxiety

Depression

Attenuated By Social Support Yes, p=0.01

No, some positive but nonsignificant No

Effect of Anxiety Worsened

Effect of Depression Worsened

p < 0.01

N/A

Effect of Cannabis Indirectly worsened Worsened Worsened

fact that these two illnesses cause similar neurobiological changes, specifically loss of white matter9. Effects of the interaction between anxiety and cognition are even more elusive than that of depression, as anxiety is usually studied as a specific construct in laboratories. However, there are some research which suggests that there is decreased cognitive flexibility and decision making with increased anxiety. Mainly the flexibility pertains to the ability to shift between tasks and to focus despite the presence of distractors10. The main region responsible for this flexibility is in the prefrontal cortex, which is also responsible for higher level thinking and making logical decisions10.

Figure 1. This figure was created to clearly summarize the results from the 2 main aims of the study, effect of social support on Based on evidence from past research, the findings from this mental health, as well as how cannabis can affect cognition. study appear to be rather novel. There has not been much research done showcasing the exact connections between cannabis, cognition, and mental health the way that this paper did. Conclusion and Discussion: As iterated in the results section, it Moving forward, should the results from this paper hold true, was found that social support can be used as a means to attenu- there are major implications to be made regarding the usage ate cognition levels, should it be negatively affected by other and allowance of cannabis. Many states and countries are movfactors such as poor mental health induced by cannabis usage. ing towards legalizing it, however, based on the consequences Factors that can cause deterioration in cognition include wors- that it can cause, this may require further review. ened depression and anxiety symptoms due to frequency of cannabis consumption. These findings match with previous research which suggested that the most prominent predictor of problems Critical Analyses: A limitation to this study is that all factors induced by cannabis is based on its frequency of use 5. Its usage measured were done through self-reporting by patients, which has also been associated with numerous mental health prob- may hold biases13. There is always the risk of misunderstanding a lems, where increased frequency can lead to increased psychotic question, leading to inaccurate responses, or that participants symptoms such as manic and depressive symptoms5. Most re- want to appear positive even if their identities are anonymous 17. search regarding cannabis usage and mental health issues in the This unfortunate phenomenon of response bias is a widely dispast have been linked to schizophrenia, it is only in recent years cussed topic in healthcare research studies17. The researchers that more research has been done showcasing evidence towards attempted to minimize bias as much as possible through the an increase in depression as well6. One study conducted in Balti- usage of standardized tests and rating scales which are used in more showed that those with a baseline diagnosis of cannabis multiple other studies, and national surveys. The results were abuse were four times more likely to have depressive symptoms also stratified by social groups such as age, culture, gender than those without a baseline by the end of a 2-year study peri- which make the results more generalizable to specific groups 19. od7. While cannabis is thought to indirectly cause decreases in Another limitation was that the study was only done at a crosscognition, a critique is such that other research has shown that sectional level, and had participants recall their past week. Rethe long-term usage of cannabis on cognition is unknown. Espe- calling past experiences can cause a lot of inaccuracies to the cially as the effects of cannabis wears off with time and can be results due to participants not being able to remember precisely, reversed with abstinence8. Neuroimaging studies have shown or if the participants did not pay attention to how their cognition very minimal effects between chronic cannabis users compared and mental state changed in the past week 16. A study looking at to healthy controls in terms of their cognitive abilities8. recall bias in chronic pain patients showed that having increased social support can actually contribute to a false recall, of less pain16. This is highly applicable here as social support was seen Measuring human cognition has been proven to be difficult. Eve- to increase cognition and could have potentially also caused a ryone has different upbringings and areas of specialty, and false, positive, recall in cognition levels. Additionally, it would therefore cannot be subjected to standardized mazes the same have been interesting to see how the cognition and mental way that mice are, so the measurements of cognition are rather health of those who did not use cannabis compared to those indirect15. Furthermore, cognition levels were self-reported and who did. The overarching result is that the increased frequency although the majority of participants rated themselves to have of cannabis causes worsening in mental capabilities however decreased cognition, this was not confirmed through any kind of does that mean that those who do not use cannabis are the best testing. Cognition levels measured in this way is also relative to off? their baseline state and other participants rather than being an absolute value. Previous research has shown that there are associations between depression and dementia, particularly the cog- Future Directions: The authors or other interested labs can replinition deterioration aspect, such that early life depression led to cate the study in a prospective manner. Patients can be recruitan increased risk of developing dementia9. This is based on the ed and be prospectively followed for a short period of time. In 77

that time period, they can record each time that they used cannabis, the approximate dosage and how they would evaluate their cognitive ability each day. Although this requires more work from participants, it may be a more accurate capture18 of the usage of cannabis and how it affects their mental health. This can then also be compared with those who use opioids and cannabis to those who only use opioids, as well as those who only use cannabis. As these proposed studies are prospective and do not manipulate any factors, it will still not allow for any causations to be drawn however it will allow for a better comparison between different groups of chronic pain patients and their methods of management.

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The Neuro-Immune Axis: Vagal Neuromodulation of AntiInflammatory Pathways in the Gastrointestinal Tract. Andrew Cheon

The vagus nerve is regarded as a crucial modulator in the neuro-immune axis of inflammation. Vagal efferent fibers have been demonstrated to modulate the spleen-mediated systemic anti-inflammatory pathway. In cases of local inflammation, such as in gastrointestinal postoperative ileus (POI), circulating levels of cytokines are not high enough to elicit the systemic immune response. Intriguingly, vagus nerve stimulation (VNS) has been demonstrated to exert local anti-inflammatory effects in mouse models of POI. Matteoli et al. (2014) therefore proposed a local gastrointestinal cholinergic anti-inflammatory pathway (CAIP) independent of the spleen. Results indicated that vagal efferent fibers directly innervate the myenteric neurons, which leads to reduced proinflammatory cytokine release by intestinal macrophages. VNS applied to mice with POI resulted in reduced recruitment of myeloperoxidase (MPO)-positive immune cells and reduced release of proinflammatory cytokines such as interleukin (IL)-1, IL-6, and chemokine ligand 2 (CCL2). This suggests that the vagus nerve modulates a gastrointestinal CAIP independently of the spleen, ultimately inhibiting intestinal immune cells that express α7 nicotinic acetylcholine receptors (α7nAChR). Through a series of elegant experiments, Matteoli et al. (2014) provide novel anatomical and functional insights into the mechanisms underlying VNS-mediated CAIP, highlighting the potential for therapeutic developments in the treatment of POI and chronic inflammatory bowel diseases (IBDs). Key words: gastrointestinal tract, myenteric plexus, neuro-immune axis, vagus nerve, vagus nerve stimulation (VNS), cholinergic anti-inflammatory pathway (CAIP), spleen, α7 nicotinic acetylcholine receptor (α7nAChR), myeloperoxidase (MPO)-positive immune cells, enteric nervous system (ENS), postoperative ileus (POI), proinflammatory cytokines

INTRODUCTION

Quantifying the recruitment of immune cells and expression of proinflammatory cytokines

The vagus nerve is a cholinergic cranial nerve, extending from its origins in the brainstem to the visceral organs (Rosas-Ballina et al., 2011). Over the past two decades, researchers have highlighted the anti-inflammatory role of the vagus nerve in the systemic immune response (Andersson & Tracey, 2012; Borovikova et al., 2000). This neuromodulatory effect is referred to as the “cholinergic antiinflammatory pathway” (CAIP). In mouse models of systemic inflammation, electrical stimulation of the vagus nerve has been shown to inhibit splenic macrophages from releasing pro-inflammatory cytokines such as interleukin (IL)-1 and tumor necrosis factor alpha (TNFα) (Borovikova et al., 2000; Gallowitsch-Puerta & Pavlov, 2007). Researchers have also demonstrated that splenic nerve denervation reliably eliminates the systemic CAIP (Rosas-Ballina et al., 2011), suggesting that vagus nerve-mediated anti-inflammatory effects are dependent on the spleen (Vida, Peña, Deitch, & Ulloa, 2011). However, in cases of local inflammation, circulating levels of cytokines are not high enough to elicit the systemic immune response. For example, postoperative ileus (POI) is a condition characterized by local inflammation of the intestines due to surgery-induced macrophage activation (de Jonge et al., 2005). This gut-localized inflammatory response does not trigger systemic inflammation that activates splenic immune cells (Meregnani et al., 2011). Strikingly, vagus nerve stimulation (VNS) in mouse models of POI was still effective in attenuating surgery-induced inflammation (de Jonge et al., 2005). This suggests that the vagus nerve might directly modulate intestinal resident macrophages in a pathway independent of the spleen. Thus, in the study of interest conducted by Matteoli et al. (2014), the effects of VNS on mouse models of localized POI were investigated. Splenic denervated mice were used to investigate the involvement of the spleen in the gastrointestinal CAIP. Next, anterograde tracing and immunofluorescence techniques were used in wild-type mice to elucidate the anatomical pathway of vagal efferent fibers. Finally, bone marrow chimaera mice deficient in α7 nicotinic acetylcholine receptors (α7nAChR) were used to identify the final target cells of vagal efferent fibers in this CAIP. Researchers found that VNS was able to exert anti-inflammatory effects in splenic denervated mice with POI, demonstrating a local gastrointestinal CAIP independent of the spleen. Furthermore, researchers identified cholinergic myenteric neurons as the targets of vagal efferents in the intestines, while α7nAChR expression on bone marrowderived cells was necessary for anti-inflammatory effects of VNS.

The anti-inflammatory effects of VNS in mouse models of POI were quantified by measuring the recruitment of myeloperoxidase (MPO) -positive cells and expression of proinflammatory cytokines in the intestines. Since bone marrow-derived leukocytes can be stained by MPO, researchers used the Hanker-Yates reagent immunostaining technique to quantify the recruitment of proinflammatory immune cells (Wehner et al., 2007). Similarly, researchers quantified the expression of proinflammatory cytokines IL-1β, IL-1α, IL-6, and chemokine ligand 2 (CCL2) because these cytokines are reliable markers of acute inflammation found in POI (Wang et al., 2009). Quantification of proinflammatory cytokines was accomplished by real time quantitative polymerase chain reaction (RT-qPCR) analysis using primer sequences for il1β, il1α, il6, and ccl2 on homogenized intestinal muscularis tissue. Protocols for measuring the antiinflammatory effects of VNS by quantifying MPO-positive cell recruitment and proinflammatory cytokine expression have been well documented by previous research using murine models of POI (Baeur et al., 2004; de Jonge et al., 2005; Kalff et al., 2003). The anti-inflammatory effect of VNS on the gut is independent of the spleen Splenic denervated wild-type mice were used to investigate the role of the spleen in VNS-mediated local anti-inflammatory effects. Splenic denervated wild-type mice were subject to (a) laparotomy, (b) laparotomy and intestinal manipulation (IM) to induce POI, or (c) laparotomy and IM followed by VNS to model the anti-inflammatory effects of VNS on mice with POI. VNS in splenic denervated mice failed to lower LPS-induced TNFα serum levels, which indicated that splenic denervation was successful. Recruitment of MPO-positive immune cells was significantly reduced by VNS in splenic denervated mice with POI. Expression of proinflammatory cytokines (il6, il1β, il1α, and ccl2) was also significantly reduced by VNS in splenic denervated mice with POI.

Altogether, this study demonstrates that the vagus nerve exerts a local anti-inflammatory effect in the gut, independent of the spleen. The significance of these findings lies in the implication of the enteric nervous system (ENS) as a possible target for anti-inflammatory control in immune-related intestinal disorders such as inflammatory bowel disorders (IBDs) and POI (Bonaz et al., 2017; Goverse, Stakenborg, & Matteoli, 2016; Matteoli et al., 2014). Correspondingly, VNS may potentially be adapted as a therapeutic intervention for IBDs, POI, and other chronic inflammatory diseases (Majoie et al., 2011). Matteoli et al. (2014) have thus provided novel insight into the poEdited from Figure 1 by Matteoli,et al.(2014). Gut, 63(6), 938–948. tential pathways by which pharmacological, complementary mediciFigure 1. Vagus nerve stimulation decreases inflammation in nal, and bioelectrical methods may target the vagus nerve to treat splenic denervated mice with postoperative ileus. (A) Vagus nerve chronic intestinal inflammatory diseases. stimulation (VNS) significantly reduced recruitment of immune cells. (B) VNS significantly reduced expression of proinflammatory cytoMAJOR RESULTS

kines. The decreases in both MPO-positive cell recruitment and proinflammatory cytokine expression suggest that VNS is able to reduce the inflammatory symptoms of POI in splenic denervated mice. Thus, the gastrointestinal CAIP has been shown to be independent of the systemic spleen-mediated CAIP. The existence of a direct gastrointestinal CAIP is further corroborated by previous studies demonstrating the effectiveness of α7nAChR stimulation in ameliorating inflammatory symptoms in the intestines (Browning & Travagli, 2014; Costantini et al., 2012). Vagal efferent fibers terminate within the myenteric plexus In a separate phase of the experiment, researchers used anterograde tracing methods and immunofluorescence in wild-type mice to elucidate the circuitry and targets of vagal efferents. Biotinstreptaviding immunofluorescence was used in the dorsal motor nucleus of the vagus (DMV) followed by confocal microscopy at 19 days post-injection.

To identify the targets of cholinergic transmission as neural or immune cells, researchers produced α7nAChR-/- bone marrow (BM) chimaera mice. Mice deficient for α7nAChR on BM-derived immune cells were subject to (a) laparotomy, (b) laparotomy and intestinal manipulation (IM) to induce POI, or (c) laparotomy and IM followed by VNS to model the anti-inflammatory effects of VNS on mice with POI.

Edited from Figure 5 by Matteoli,et al.(2014). Gut, 63(6), 938–948. Figure 3. α7nAChR expression on immune cells is required for vagus nerve-mediated anti-inflammatory effects in mice with postoperative ileus. (A) Vagus nerve stimulation (VNS) significantly reduced recruitment of immune cells and expression of proinflammatory cytokines. (B) VNS did not reduce recruitment of immune cells or expression of proinflammatory cytokines.

Edited from Figure 3 by Matteoli,et al.(2014). Gut, 63(6), 938–948. Figure 2. Vagal efferent fibers directly contact cholinergic myenteric neurons associated with intestinal resident macrophages. The vagus nerve (green/yellow) terminates near the cholinergic enteric neurons (red). Intestinal resident macrophages (blue) are closely associated with neurons in the myenteric plexus. Vagal efferents were shown to target the myenteric neurons innervating the intestinal muscularis, rather than terminating directly on resident macrophages. The ENS, indicated as the anatomical interface between the vagus nerve and resident macrophages, may be implicated in neuromodulation of the gastrointestinal CAIP (Bratton et al., 2012). Previous research conducted by Walter et al. (2009) also used anterograde tracing methods to identify the terminal site of vagal efferents at the myenteric plexus. Furthermore, various studies have associated myenteric plexitis with Crohn’s disease pathogenesis, functionally corroborating the results of anatomical analysis in this model of POI (Ferrante et al., 2006). By including choline acetyltransferase staining (ChAT) to characterize the cholinergic nature of cells proximal to vagal efferent terminals, Matteoli et al. (2014) provided substantial evidence for a gastrointestinal CAIP modulated by the vagus nerve.

The reduction in both MPO-positive immune cell recruitment and proinflammatory cytokine expression suggest that VNS is able to reduce the inflammatory symptoms of POI in mice that express α7nAChR on immune cells. However, VNS was not found to affect the MPO-positive immune cell recruitment or proinflammatory cytokine expression in mice deficient for α7nAChR on immune cells. Immune cells were demonstrated to be the final target of the VNSmediated gastrointestinal CAIP. Accordingly, previous research has also demonstrated the necessity of α7nAChR expression on immune cells in regulating systemic inflammation (Olofsson et al., 2012; Vida et al., 2011; Wang et al, 2003). CONCLUSIONS/DISCUSSION Through a series of elegant experiments, Matteoli et al. (2014) provided novel evidence indicating that the vagus nerve-mediated gastrointestinal CAIP occurs independently of the systemic immune response. Furthermore, researchers determined that this mechanism is dependent on the expression of α7nAChR on resident macrophages associated with myenteric neurons, suggesting involvement at the level of the ENS as well. Correspondingly, Matteoli et al. (2014) interpreted the results of α7nAChR-/- bone marrow chimaera mice as evidence for resident macrophages being the final targets of cholinergic transmission, which ultimately results in the inhibition of pro-inflammatory cytokine release.

The gastrointestinal CAIP requires α7nAChR expression on immune Previously, it was thought that vagal efferents provided direct input cells to the immune cells involved in both the local and systemic inflammatory responses (Borovikova et al., 2000). Further research pro-

vided substantial evidence against this hypothesis, revealing an indirect pathway by which the vagus nerve signals the splenic nerve to inhibit splenic macrophages from releasing proinflammatory cytokines (Baeur et al., 2004; de Jonge et al., 2005; Rosas-Ballina et al., 2011). Accordingly, Matteoli et al. (2014) reasoned that local inflammation of the GI tract would activate a distinct, indirect pathway to inhibit non-splenic immune cells. Splenic denervated mice were responsive to the anti-inflammatory effects of VNS, leading to widespread acceptance that the vagus nerve does indeed neuromodulate the local gastrointestinal CAIP in tandem with, and independently from, the systemic immune response (Kimura et al., 2019). Matteoli et al. (2014) revealed the potential ENS-associated targets of pharmacological or bioelectrical interventions involved in this CAIP, providing valuable anatomical and biochemical avenues by which therapeutic treatments may be developed for POI or chronic inflammatory gastrointestinal diseases with no known cure to date (Browning & Travagli, 2014).

Non-invasive tVNS has been used to treat some cases of refractive depression and epilepsy as well, though its applications for antiinflammatory neuromodulation of GI conditions are still in early stages of development (Marshall et al., 2015). Methods for invasive VNS, on the other hand, have been thoroughly established for the treatment of neurological disorders ranging from epilepsy to mood disorders (Daban et al., 2008; Huston et al., 2018). Due to the welldefined therapeutic parameters for invasive VNS, this technique has been widely applied to the study of inflammatory bowel diseases (IBDs) (Lubbers et al., 2010; Stakenborg et al., 2017). For example, proinflammatory cytokines such as TNFα and IL-1 have long been known to characterize and exacerbate colitis and inflammatory bowel diseases (Ghia et al, 2007), and invasive VNS has been shown to reduce the expression of these cytokines in the intestines (Matteoli et al., 2014). This anti-inflammatory effect of VNS has even been evaluated in recent pilot studies using human IBD patients (Bonaz et al., 2017). Five out of seven patients were found to be in deep remission after three months of VNS based on clinical (Crohn’s disease activity index) and endoscopic analyses (Crohn’s disease endoscopic index of severity) (Bonaz et al., 2017). By providing novel insights into the direct gastrointestinal CAIP, findings of this study by Matteoli et al. (2014) have impacted the bioelectrical approach to treating chronic inflammatory disorders like IBDs.

expressing immune cells such as leukocytes, monocytes, and neutrophils expressing CD45, Cd11b–, or F4/80− have not been mentioned. Neutrophils, which express α7nAChR to a lesser extent than macrophages, have been identified as a potent source of IL-1β in vagotomized mice (Cunha & Verri, 2006). The inflammatory effects of BM-derived immune cells should be investigated as a whole and not simply by the presence of α7nAChR, as interactions between immune cells are complex and may provide further insight into the detailed mechanisms underlying neuromodulation of inflammatory cytokine release in the GI tract. Using anterograde tracing, Matteoli et al. (2014) provided further insight into the indirect nature of vagal neuromodulation at the myenteric plexus. Researchers speculated that, at this level, the ENS may amplify vagal cholinergic signals that result in modulation of local inflammatory pathways. Indeed, myenteric plexitis has been associated with chronic inflammation in patients with Crohn’s disease, which independently corroborates this speculation (Ferrante et al., 2006). However, the authors did not address the involvement of mytenteric neurons and other cells of the enteric nervous system. Further research must be conducted to characterize the involvement of myenteric neurons and associated cells of the ENS in the CAIP, beyond its ability to engage in cholinergic transmission at the interface between vagal efferents and resident macrophages. There are several reasons to believe that, although crucial to the antiinflammatory effects of VNS, cholinergic transmission alone may not fully explain the pathway from vagal efferent activation to reduced cytokine release. For example, once released, acetylcholine is rapidly broken down by acetylcholinesterase into choline, which is indeed a selective α7nAChR agonist. However, cholinergic neurons are known to release various other neurotransmitters such as glutamate and nitric oxide, which may affect the response of ENSassociated cells in the myenteric plexus (Volpi et al., 2012). This is an unanswered area of interest for future research because the myenteric plexus contains enteric glial cells (EGCs) that, as demonstrated by anatomical tracing results of this study, may be involved in modulating this anti-inflammatory pathway through indirect mechanisms downstream of macrophage activity (Olofsson et al., 2012; Walter et al., 2009). In fact, EGCs outnumber neurons 7 to 1 in the GI tract, which suggests a more prominent role of glia than myenteric neurons in this possible pathway between the vagus nerve and inflammation (Hoff et al., 2008). Research has implicated glia in gut motility disorders like POI and in inflammatory bowel diseases (Liñán-Rico et al., 2016). Inflammation of the GI tract has been shown to induce significant changes in EGCs, converting EGCs to the rEGC phenotype and initiating the pro-inflammatory NF-κB signalling pathway (Liñán-Rico et al., 2013). This NF-κB pathway is the pro-inflammatory mechanism that happens to be deactivated in response to α7nAChR activation in immune cells (Matteoli et al., 2014). Reactive EGCs (rEGCs) have been shown to upregulate the expression of specific pro-inflammatory cytokines, including the markers IL-1α and IL-1β measured by Matteoli et al. (2014), in the intestinal muscularis tissue of mice (Fujikawa et al., 2015). Future research should investigate the involvement of these enteric glial cell phenotypes in the gastrointestinal CAIP mediated by the vagus nerve.

CRITICAL ANALYSIS Matteoli et al. (2014) used α7nAChR–/– bone marrow chimaera mice to identify resident macrophages as the final targets of cholinergic transmission, but the functions of other α7nAChR-

Matteoli et al. (2014) did not further investigate the role of nonneural cells of the myenteric plexus, possibly because bone marrow chimaera mouse models revealed that α7nAChR expression was only necessary on immune cells for the gastrointestinal CAIP. De-

The clinical significance of experimental results demonstrating the efficacy of VNS for treating POI lies in the fact that this condition affects at least one in eight postoperative GI patients in the USA (Chapman et al., 2018). Even so, a majority of the evidence for current treatments of POI has been contentious, including intravenous administration of potentially addictive opioids, unspecific analgesics such as lidocaine, or even chewing gum to stimulate vagal activity (Kranke et al., 2015; Short et al., 2015). Upon the empirical foundations provided by Matteoli et al. (2014), various bioelectronic therapeutic approaches have been further developed. For example, one team has since targeted the gastrointestinal CAIP via non-invasive, low-risk transcutaneous VNS (tVNS) and successfully demonstrated a reduction in proinflammatory cytokine release in mouse models of POI (Hong et al., 2019).

spite this, it is important to note that EGCs do not communicate by action potentials as they are not excitable like myenteric neurons are (Ochoa-Cortes et al., 2016). Furthermore, apart from cholinergic receptors, EGCs express adrenergic (sympathetic) receptors as well as a variety of well-known receptors of the innate immune system, which suggest that its role in vagus nerve-mediated intestinal CAIP cannot be excluded (Gershon, 2011). Instead, through a complex repertoire of Ca2+ signalling, they communicate with neurons and other cells in the GI tract (Kabouridis et al., 2015). The reactive EGC contributes to proinflammatory cytokine production and compromised integrity of the intestinal epithelial barrier, which can also exacerbate the systemic proinflammatory effects of gut microbiota in response to intestinal barrier dysfunction (Brzozowski et al., 2016; Ochoa-Cortes et al., 2016). However, it has not yet been established whether EGC alterations from healthy to reactive phenotypes occurs before or after the inflammatory reactions. As such, future research should incorporate immunostaining methods for EGCs such as the glia-specific S100β. This can easily be adopted for experiments using murine models of POI to investigate the involvement of EGCs in the gastrointestinal CAIP. The response of glial cells should be further characterized to define the downstream effects induced by VNS, as this may potentially lead to the identification of effective targets for POI and IBD-related interventions (Bauer & Boeckxstaens, 2004). FUTURE DIRECTIONS Another interesting avenue for future research involves the effects of VNS on the gut microbiome. First of all, the gut microbiome is heavily implicated in local inflammatory signalling (Zuo & Ng, 2018). Gut dysbiosis has been associated with chronic GI inflammation, due to reductions in microbial biodiversity and decreases in bacteria that enhance immune tolerance or attenuate the inflammatory response (Atarashi et al., 2013; Henson & Phalak, 2017). Furthermore, gut microbiota have been demonstrated to regulate the development of ECGs, which suggests a potentially significant role of bacterial modulation in downstream VNS-induced processes (Kabouridis et al., 2015). The need for further research characterizing this interaction is highlighted by the fact that even today, the roles of innate lymphoid cells and muscularis macrophages have not been elucidated in detail, despite the well-known relationship between gut microbiota, muscularis macrophages, and the ENS in the maintaining the integrity of the intestinal wall (Wynn et al., 2013). Furthermore, gut microbiota have been demonstrated to educate the immune cells, including muscularis macrophages, which in turn regulate the microfloral ecology (Clemente et al., 2012). While VNS has been implicated in the preservation of the intestinal epithelial barrier via local release of acetylcholine (Krzyzaniak et al., 2011), researchers have not yet been able to define the sequence and interactions between gut microbiota, vagal neurotransmission, and EGC of the myenteric plexus. Cross-sectional compositional analyses of the gut microbiome in response to VNS, using metagenomic sequencing techniques such as RT-qPCR, may provide further insight into the complex interactions that underlie the gastrointestinal CAIP in humans. Novel pharmacological interventions may be developed to mimic the effects of VNS on the gut microbiome and cells of the myenteric plexus, in an effort to devise accessible, non-invasive alternatives to VNS in the treatment of IBDs. Future research efforts to adapt the use of VNS in mice with POI to human patients with IBDs should focus on performing robust randomized controlled trials to identify both the ideal duration of VNS as well as the systems-wide mechanisms of anti-inflammatory effects. In epilepsy and depression treatments, VNS is applied 30

seconds on and 5 minutes off (Nemeroff et al., 2006), whereas in rheumatoid arthritis treatments, VNS is applied for 60 seconds up to 4 times daily to reduce cytokine release and improve inflammatory symptoms (Koopman et al., 2016). Theoretically, these applications of VNS are thought to gradually restore the vagal tone, which may actually ameliorate the hyporeactivity of the hypothalamic-pituitary -adrenal (HPA) axis in chronic inflammatory diseases (Mogilevski et al., 2019). This may occur in tandem with VNS-induced amplification of cholinergic signaling in the ENS, further reducing the circulation of pro-inflammatory cytokines (Schütz et al., 2015). The interactions between these sympathetic and enteric nervous system responses to parasympathetic activation may provide more holistic insights into how the immune system is neuromodulated specifically in the gut. Further mechanistic understandings of bioelectrical vagus nerve modulation may be relevant to complementary medicinal approaches alongside existing anti-inflammatory treatments, such as anti-TNFα treatments used to treat IBDs (Bonaz et al., 2017).

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MS: Modulation of immunity and microbiome via probiotic supplementation Amy Iman Aicha Coulibaly

Multiple sclerosis, ordinarily abbreviated as MS, is an autoimmune disease characterized by demyelination and neurodegeneration in the central nervous system (CNS). Its cause is still unknown, however, experimental autoimmune encephalomyelitis (EAE) mouse model studies have shown that the gut microbiome may be implicated in MS progression. Crucial to our understanding is the link between microbiota and immunity in MS. In the five-month study conducted by Tankou et al. (2018), probiotic supplementation containing Lactobacillus, Bifidobacterium and Streptococcus (LBS) were administered orally to 9 MS patients and 13 healthy controls (HC). Blood and stool collection were performed at baseline, 2 months after probiotic administration and 3 months after discontinuation. Feces samples were used for metabolomics assessment and 16S rRNA profiling. Frozen peripheral blood mononuclear cells (PBMCs) were utilized for immune cell profiling. The main results of this research showed that probiotic supplementation enriches microbiome taxa discerned to be depleted in MS for example Lactobacillus. Moreover, those shown to contribute to dysbioses such as Akkermansia and Blautia had a reduction in abundance. Probiotic supplementation was shown to reduce methane metabolism as well as numerous KEGG (Kyoto Encyclopaedia of Genes and Genomes) pathways in MS patients. With regards to immunology, probiotic supplementation induced a shift to an anti-inflammatory state. There was a reduction in the frequency of inflammatory monocytes, mean fluorescence intensity (MFI) of CD80 ligands and HLA-DR on dendritic cells. Probiotic administration was also shown to decrease the expression of HLA-DQA1, an MS risk allele in controls Furthermore, a correlation between the enhanced abundance of Lactobacillus and Bifidobacterium and reduced expression of the MS risk allele HLA.DPB1 was shown in controls. These findings suggest that probiotics may serve as complementary treatments with existing MS therapies.

Keywords: multiple sclerosis, autoimmune, probiotics, microbiome, anti-inflammation, monocyte, MS risk allele, 16S rRNA, peripheral blood mononuclear cells (PBMC), Kyoto Encyclopaedia of Genes and Genomes (KEGG),

INTRODUCTION

Multiple Sclerosis (MS) is an autoimmune disease of which etiology is still unknown. It is characterized by chronic inflammation culminating in neurodegeneration and demyelination of the CNS (Mielcarz & Kasper, 2015). The experimental autoimmune encephalomyelitis (EAE) is a mice model used in various studies to examine the pathophysiology since it mimics some of the symptoms commonly seen in the disease. Studies have established T-cells as the major effectors of inflammation in MS (Fletcher, Lalor, Sweeney, Tubridy, & Mills, 2010). The Ephrin ligands B1 and B2 are implicated in the chemotaxis of CD4+ Th cells, Th17 & Th1 in MS and EAE models (Luo, et al., 2016). Moreover, diverse Th-polarizing proteins such as the cytokines IL-12 and IL-23 have been critical in driving demyelination in EAE models. Current MS medication using disease-modifying drugs (DMD) are preventive and for attenuation rather than curative. Chemical analogs of Beta interferon which are naturally occurring immune cell cytokines have been developed to modulate and regulate the immune system by preventing viral replication and promoting anti-inflammation (Goldenberg, 2012). Other medications include immune modulators, immunosuppressants, myelin basic protein (MBP) competitive inhibitors and monoclonal humanized antibodies that act on a variety of immune cells (Goldenberg, 2012). New findings suggest that the gut microbiome may be involved in MS, as it is with various other autoimmune diseases such as rheumatoid arthritis and inflammatory bowel disease ( Picchianti-Diamanti , et al., 2018) (Cammarota, et al., 2015). The gut microbiome consists of all microbial species as well as the genetic components encoded by them. Alteration in specific taxa found within the gut microbiome of MS individuals has been associated with the promotion of inflammatory cytokines and inflammation overall. In Regulatory T (Treg) cell deficiency, absence of the transcription factor: Forkhead box protein 3 (Foxp3) important in development and function leads to dysbiosis and autoimmunity (He, et al., 2017). In this study He et al., demonstrated that oral administration of Lactobacillus reuteri to reset the gut microbiome inhibited autoimmunity due to Treg deficiency via metabolic interaction with adenosine (A2A) receptors. Later on, it was demonstrated that oral administration of Lactobacillus reuteri suppresses the progression of EAE in mice by altering the gut microbiota and reducing Th1 & Th17 cells and their associated cytokines IFN-γ/IL-17 presence (He, et al., 2019). It is evident that the administration of probiotics to alter gut microbiota may help control autoimmune disorders. Growing evidence from animal and human studies propose an intrinsic interaction between gut flora and immunity (He, et al., 2007) (Spencer & Belkaid, 2012). Germ-free mice studies have recognized the importance of the gut microbiome in developing a healthy immune system (Round & Mazmanian, 2009). Finding a probiotic which can work in synergy with MS medication, and attenuate chronic inflammation would be of value to MS management.

The study conducted by Tankou et al. (2018) provides future directions on the treatment of patients with relapsingremitting MS who are taking glatiramer acetate as medication. Fecal samples and Frozen PBMC were used to perform microbiome and immune profiling. The effect of probiotics supplementation containing Lactobacillus, Bifidobacterium and Streptococcus (LBS) in MS and HC patients was measured. Overall, probiotics supplementation of LBS promoted anti-inflammation and enhanced the taxonomic abundance of Lactobacillus, Bifidobacterium and Streptococcus. Tankou et al. (2018) suggest that oral administration of probiotics to alter the gut microbiome, increasing ‘beneficial’ bacteria taxa may reduce inflammation in MS patients caused by T-cell release of inflammatory cytokines and have a synergistic purpose with MS therapies. MAJOR RESULTS LBS supplementation is associated with alterations in the structure and composition of the gut flora and KEGG pathways Tankou et al. (2018) used fecal samples obtained from HC and MS patients and performed 16S RNA sequencing to assess alpha diversity (species diversity in the microbiota) as well as beta diversity (species diversity between samples) within their gut microbiome. UniFrac analysis of beta diversity at different time points revealed that after LBS discontinuation, the microbial community shifted back to the baseline level. Calculated alpha diversity was measured using the Shannon diversity index. LBS administration had no significant effect on alpha diversity in MS patients, however, a reduction in diversity was shown in HCs (Tankou, et al., 2018); as shown in figure 1.

Figure 1 Effect of LBS on alpha diversity in HC and MS patients. Shannon diversity index calculated at a depth of 10,000 reads. Tankou, S. K., Regev, K., Healy, B. C., Tjon, E., Laghi, L., Cox, L. M., . . . Weiner, H. L. (2018). A probiotic modulates the microbiome and immunity in multiple sclerosis. Annals of Neurology, 83(6), 1147-1161.

Tankou et al. (2018) reported that LBS supplementation initiated a decrease in the KEGG pathway specifically those involved in regulating cellular processes, metabolism, organismal systems as well as the processing of environmental cues. Zhang et al. (2016) reported dysregulation in differentially expressed long non-coding RNA (lncRNAs) genes controlling molecules involved in various KEGG pathways in MS. In this study, KEGG pathways were significantly enriched in the HC compared to MS individuals (Zhang, et al., 2016). Indicating that they may be important in MS progression and that

they may be important in MS progression and that LBS administration may cause the upregulation of such lncRNAs, providing a mechanism of action to reduce MS advancement. LBS supplementation is associated with alterations in peripheral immune cell frequency and a reduction in MS risk alleles Tankou et al. (2018) demonstrated that the frequency of intermediate monocytes (CD14highCD16low), involved in proinflammatory response as reported by Chimen et al., (2017) decreases following probiotic administration in MS patients as well Figure 3 Correlation between immune markers of peripheral as the MFI of CD80 ligands; HLA-DR on dendritic cells was recells and microbiome species after LBS administration in MS duced after LBS administration in MS patients. patients. Spearmanâ&#x20AC;&#x2122;s correlation assessment between operaIn a previous study conducted by Lincoln. et al., an epistatic tional taxonomic units (OTUs) relative abundances and immune interaction among HLA-DQA1 and two other HLA class II loci markers Tankou, S. K., Regev, K., Healy, B. C., Tjon, E., Laghi, L., were identified as genetic factors contributing to MS risk Cox, L. M., . . . Weiner, H. L. (2018). A probiotic modulates the (Lincoln, et al., 2009). In the study conducted by Tankou et al. microbiome and immunity in multiple sclerosis. Annals of Neu(2018), Nanostring immunology panel was used to determine rology, 83(6), 1147-1161. the impact of LBS administration on gene expression of HLA class CONCLUSIONS/DISCUSSION II allele on monocytes retrieved from PBMC. Probiotic use was The results of the research conducted by Tankou et al. (2018) associated with a reduction in pro-inflammatory HLA genes and showed that LBS administration enriched microbiome taxa disan increase in anti-inflammatory genes linked to MS risk (Fig. 2); cerned to be depleted in MS such as Lactobacillus in both HC and however, these effects were reversed with probiotic discontinuHS patients. Furthermore, microbial taxa such as Akkermansia ation. Moreover, LBS supplementation caused an increase and and Blautia, associated with dysbiosis were depleted after LBS decrease in gene expression of proteins associated with antiadministration. Additionally, microbiome and metagenomics inflammation and pro-inflammation respectively. profiling further showed a reduction in the abundance of various KEGG associated with MS with the inclusion of methane metabolism and carbon fixation pathways. Immunological profiling revealed an induction of anti-inflammatory peripheral immunity evidenced by the decrease in intermediate monocytes as well as a reduction in inflammatory monocytes and MFI of CD80 markers on monocytes in MS patients and HC respectively following probiotics administration. The correlational assessment demonstrated that probiotics Figure 2 Effect of LBS on peripheral monocyte gene expression administration promotes anti-inflammation and increases the HC and MS patients; Tankou, S. K., Regev, K., Healy, B. C., Tjon, abundance of bacterial genera such as Lactobacillus; this interE., Laghi, L., Cox, L. M., . . . Weiner, H. L. (2018). A probiotic mod- pretation is concordant with the finding of Tamtaji, et al., (2017) ulates the microbiome and immunity in multiple sclerosis. An- whereby probiotics supplementation stimulated the downregulation of pro-inflammatory cytokines interleukin-8 and Tunals of Neurology, 83(6), 1147-1161. mour necrosis factor (Tamtaji, et al., 2017). Preference for antiImmune markers in peripheral blood are correlated with microbiinflammatory immune markers could aid in identifying biological ota changes targets for future MS treatment. The importance and impact of The authors examined a hypothetical link between gut bacteri- the microbial community have been studied extensively in the al species abundances with a collection of immune markers literature and shown to play a role in the pathogenesis of various whose MFI had significant changes following probiotics treat- diseases, not just autoimmune disorders (Sampson, et al., 2016). ment. The bacterial genera Bifidobacterium, Lactobacillus and This study conducted by Tankou et al. (2018) is relevant to the Streptococcus were shown to be negatively correlated with pro- field of clinical neuroscience as it provides insight into the possiinflammatory immune markers and positively correlated with ble impact probiotics supplementation could have on MS management. anti-inflammatory markers in MS individuals. To conclude, Tankou et al. (2018) provided evidence establishOTUs of Lactobacillus had a negative correlation with HLA-DR ing the safety of high dose probiotics administration for MS paMFI on dendritic cells, Streptococcus had a negative correlation tients under glatiramer acetate medication. Furthermore, their with CYBB and a positive correlation with LILRB2 (Leukocyte Imresults suggested a reversal in microbiome composition and munoglobulin Like Receptor B2); whereas Bifidobacterium was function after LBS supplementation. Finally, the changes in gut negatively correlated with CYBB (Fig. 3). microbiome were associated with an anti-inflammatory response by peripheral immune cells in both HC and MS patients; thus, indicating that LBS administration may act in synergy with DMD used by MS patients. 90

CRITICAL ANALYSIS The study conducted by Tankou et al. (2018) establishes an alternate route and perspective on MS therapy and treatment. They provide evidence on the structural and compositional changes occurring during and after LBS supplementation. Moreover, the immunologic response after LBS administration as well as the correlates between the gut microbiome and peripheral immune cells and their associated cytokines and proteins. The author and associates conclude that LBS administration may act in synergy with DMD by modulating the gut microbiome as well as immunity to control inflammation. The study conducted by Tankou et al. (2018) is significant and appropriate for other researchers or clinician to interpret and apply to the general MS population, as contrarily to mouse models like EAE the benefit of having humans as test participant is the ability of the results to be generalized and be predictive of human subjects with the disease/disorder (Shanks, Greek, & Greek, 2009); despite their sample size consisting of just 22 participants. The authors should instead look at examining the impact of probiotics supplementation for MS management with a greater sample group to provide more accurate and reliable interpretations and determine whether modulation of the gut microbiome may be the next step in MS therapy. Multiple variables have not been taken into consideration when assessing the impact of LBS administration on microbiome and immunity. Firstly, the impact of LBS supplementation on glatiramer acetate function. Tankou et al. (2018) excluded MS patients taking other medications other than glatiramer acetate; this is a strength as it maintains reproducibility and limits confounding results, therefore, all MS participants used the same medication with equivalent mechanisms of action. In-vitro and mice studies have suggested that glatiramer acetate administration activates specific Tregs which induce and activate peripheral immune cells to inhibit inflammatory responses against MBP (Arnon & Aharoni, 2004). Interestingly, mice studies examining the interaction between probiotics and the immune system have shown that probiotic administration upregulates the production of CD4+Foxp3+ Tregs increasing their suppressor activity (Kwon, et al., 2010). It would be of interest to the authors to assess whether probioticinduced microbiota changes alter the number of Treg activated by glatiramer acetate; thus, determining whether probiotic supplementation may indeed work in synergy with MS DMDs. Secondly, the functionality of multispecies probiotic. Multistrain probiotics have been reported to be more effective than singlestrain probiotics (Timmerman , Koning, Mulder, Rombouts, & Beynen, 2004) providing a strong rationale for the use of probiotics containing LBS. However, Inter-strain interaction between the three bacterial species has not been examined. It is reported that adhesive properties of bacteria are required for colonization of the gut microbiome (Bermudez-Brito, Plaza-Díaz, MuñozQuezada, Gómez-Llorente, & Gil, 2012) and in-vitro adhesion assays have shown that Lactobacillus GG doubles the adhesion properties of Bifidobacterium lactis Bb12 (Ouwehand, Isolauri , Kirjavainen, ölkkö, & Salminen, 2000). Thus, indicating that cooperation between bacterial species synergistically or antagonistically may be required for the increased colonization and enrichment of the gut microbiome and enhancement of anti-

inflammation. Understanding the interaction of probiotics containing more than one taxon can help researchers and later on clinician understand the appropriate supplementation combinations to use to assist with MS management. Lastly, diet. Although participants were asked to perform dietary surveys, diet remains a confounding variable on the clear effect of LBS administration on the pathogenesis of MS. Longterm consumption of high-fat food is positively associated with increased abundance of Bacteroides within the gut flora, conversely a high carbohydrate diet is associated with increased concentration of Prevotella and decreased abundance of lactic acid bacteria (Streptocococus and Lactobacillus) (Wu, et al., 2011) (Singh, et al., 2017). A diet limited in animal products leads to a significant reduction in Bifidobacteria, Bacteriodetes, and Enterobacteriaceae (Salonen & de Vos, 2014). Salonen & De Vos (2014) reviewed the impact of a high-fat diet on the gut flora and have established that overall, microbiota diversity and complexity is simplified and reduced. This is important to consider because disturbances in abundances due to diet may mask the clear effect of LBS supplementation, in terms of the extent to which LBS increased the abundances of Bifidobacterium, Lactobacillus and Streptococcus. The authors should aim at determining whether the respective increases and decreases in microbial abundances are largely due to probiotic supplementation or diet. FUTURE DIRECTIONS Although RRMS is the most common type of MS, the authors should consider assessing the impact of probiotics treatment on the other three subtypes of MS: primary progressive, secondary progressive MS and progressive relapsing MS. This is important because the 8 FDA approved MS drugs are used to treat the relapsing/remitting symptoms which are not consistent in all MS subtypes (Goldenberg, 2012).

This would allow for the results to be comprehensive of all the population affected by MS. For example, the authors should recruit participants exhibiting primary progressive MS and measure the effect of LBS supplementation on immunity and microbiome diversity. Probiotic supplementation may be beneficial in synergistically slowing down demyelination and neurodegeneration in primary progressive patients who are typically more resistant to DMD (Goldenberg, 2012). There is little evidence in literature demonstrating that probiotic treatment may be beneficial to other MS subtypes, however, given the results of the current study it would be advantageous for our understanding to know whether an anti-inflammation response is also favoured in other forms of MS. Establishing a possible field of study for future MS adjunct treatment. The lack of knowledge and understanding about the natural progression as well as curative medication available of MS patients makes it crucial for researcher to keep proposing and examining strategies to discover successful and empirical solutions for all MS treatment. Glatiramer acetate is commonly used to treat RRMS as it promotes oligodendrogenesis and remyelination via mechanisms involving growth factors that are beneficial for repair (Skihar, et al., 2009). It is administered subcutaneously and used as a firstline medication for patients who are intolerant to beta-interferon

medication (Goldenberg, 2012). Tankou et al. (2018) state that probiotic administration may have a synergistic function with DMD specifically glatiramer acetate; as the next step, the authors should investigate the impact of LBS administration on taking other DMD. This would be of pressing issue because MS patients taking other medication may not exhibit the similar changes in microbiome complexity and immune response; therefore, before recommending probiotics as adjunct treatment for MS the outcomes must be consistent for all medications. For example, by performing similar research, however, including MS patients who are taking only beta interferons as medication. Though, it would also be important to consider the impact of LBS administration on the efficacy and effectiveness of DMD significantly those such as beta interferons which are administered orally because probiotic administration may lead to fluctuations in the absorption of such drugs (Kim, et al., 2018). They should use pharmacological measurements of efficacy such as pharmacokinetic drug interaction (Boster, Ford, Neudorfer, & Gilgun-Sherki, 2015) to determine whether probiotics facilitate the mechanism by which orally administered drugs function. This would also allow them to examine the most effective LBS-DMD combination and delivery for MS management.

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Habitual Tea Drinking as a Protective Measure for Cognitive Decline in Older Adults Brittany Demircan

Cognitive decline is a natural sign of aging that spares no human. Cognitive decline can begin as early as age 40 and precede to higher levels with older age. It is often characterized by impairments in memory and reasoning, as well as slower processing speeds. The effects of aging have prominent effects on the brain structure and connectivity making the brain less efficient. There have been vigorous studies conducted to further understand and delay the cognitive decline process. One of these studies by Li et al. (2019) investigated the effects of habitual tea drinking on the global and regional brain connectivity in older adults using a between-subject design. The participants were put into two groups â&#x20AC;&#x201C; tea drinking group and non-tea drinking group â&#x20AC;&#x201C; in accordance to their average tea consumption prior to the study. The brains of both groups were studied using structural and functional imaging to evaluate the signs associated with cognitive decline in both groups. The main results of their study show that tea positively contributes to brain organization giving rise to greater efficiency in structural connectivity. Furthermore, they indicate tea has positive effects on hemispheric asymmetries, showing it leads to less leftward asymmetry in structural connectivity. These findings suggest lifestyle choices as simple as drinking tea can play a role in delaying age related cognitive decline. Key words: cognitive decline, brain health, cognitive aging, tea, L-theanine, caffeine, catechin, antioxidants

INTRODUCTION. Other than water, tea has been the most popular beverage around the world for centuries (Brody, 2019). It is estimated that two billion cups of tea are drunk each day all over the world (Brody, 2019). Tea has been known for its relaxing and uplifting properties for centuries but only recently has been an interest to the western scientific community. As the ancient wisdom surrounding tea became an area of interest to researchers around the world, the scientific literature concerning the benefits of tea rapidly grew (Gilbert, 2019). Some significant benefits include mood improvements, cardiovascular disease prevention, weight loss, as well as reduction in cognitive decline (Li et al., 2019). These benefits are believed to result from certain components found in tea such as catechin, L-theanine and caffeine. All three of these components individually have shown promising findings related to brain health. Catechin has shown improvements in memory recognition and working memory compared to placebo in human subjects (Torre et al., 2013). As such, the same results were replicated using Down syndrome mouse models which showed significant improvements in cognitive memory tasks. (Torre et al., 2013). Further benefits were found in L-theanine which was shown to cause a significant reduction in stress-response both physiologically and psychologically during a cognitive demanding task (Kimura et al., 2007). Other studies suggest that L-theanine crosses the brain blood barrier and can directly affect the plasticity of the brain, allowing it to regenerate itself (Gilbert, 2019). Moreover, a study by Owen et al. (2008), studied the synergistic effects of both L-theanine and caffeine and found it improved cognitive performance (accuracy and speed on cognitively demanding tasks), as well as mood. Along with the cognitive enhancements, the components of tea were also found to also have neuroprotective effects on the brain. A study on L-theanine showed neuroprotective effects on neuronal cell death after transient cerebral ischemia (Kakuda, 2011). Other studies found catechins played a role in decreasing amyloid‐β and amyloid precursor protein (APP) while increasing soluble amyloid precursor protein‐alpha (sAPPα) (Mandel et al., 2008). This suggests a possible neuroprotective strategy for Alzheimer’s and Parkinson’s Diseases (Mandel et al., 2008). All considered, the components of tea possess beneficial effects on brain health. Fortunately, the cognitive enhancements and neuroprotective effects were able to be replicated with tea alone. The Nakajima Project was a large study conducted on older adults (>60 years old) that found that tea consumption reduced the incidence of mild cognitive impairment (MCI) and dementia (Noguchi-Shinohara et al., 2014). Although these findings are promising, they lack justification of the physical changes that undergo in the brain as a result of tea consumption. In order to address this problem, researchers Li et al. (2019) studied the brain connectivity using global and regional metrics with structural and functional imaging to investigate the differences between the tea drinking group and the non-tea drinking group. In both groups they reviewed the leftward hemispheric asymmetry, which is associated with cognitive aging (Li et al., 2019). They found the tea drinking group had less leftward hemispheric asymmetry showing evidence of slower cognitive aging (Li et al., 2019). Furthermore, they found that the tea

drinking group had less reduction in interregional connectivity, also associated with aging induced cognitive decline (Li et al., 2019). Taken altogether, these findings could benefit individuals to delay the cognitive decline associated with aging, as well as other cognitive diseases. MAJOR RESULTS Demographics and Neuropsychological Measures Li et al.’s research (2019) included 36 participants divided amongst 15 participants in the tea drinking group and 21 participants in the non-tea drinking group. There were no significant differences between the two groups in age, gender, years of education, coffee consumption or ratio of left handedness to right handedness. Furthermore, only 1 out of 12 neuropsychological measures were significantly different between the two groups. The tea drinking group showed better performance in the Block design test compared to the non-tea drinking group. Leftward Hemispheric Asymmetry The hemispheric asymmetries in global parameters, Cw and Eloc, were significantly different in structural network between the tea drinking and non-tea drinking group. The tea drinking group displayed less leftward asymmetry between the hemispheres than the non-tea drinking group. The Lw and Eglob were not significant between the two groups in structural or functional network. Default Mode Network The interactions and strength of connections in the default mode network exhibited increased strength in the functional connectivity in the tea drinking group compared to the non-tea drinking group. There were 11 functional connections in the tea drinking group that displayed significantly increased strength compared to the non-tea drinking group. These 11 functional connections were dominantly part of the posterior cingulate gyrus, parahippocampal gyrus or angular gyrus.

Figure 1. The brain regions that show differences in structural efficiency between the tea drinking group and the non-tea drinking group.

chological measures. The novelty of these results allow for further research and development on how to maximize these properties in tea. Furthermore, it provides valuable information that implementing something as simple and accessible as tea can act to improve brain health. In their study, the tea drinking group exhibited significantly less leftward hemispheric asymmetry than the non-tea drinking group showing reduced signs of cognitive decline. Previous studies have proposed a U-shaped developmental trajectory in hemispheric asymmetry from leftward asymmetry to rightward asymmetry back to leftward asymmetry throughout adolescence, to middle age, to old age (Li et al., 2019). Thus, the Li et al. (2019) study suggests that habitual tea drinking may suppress leftward asymmetry associated with old age and conserve a structural connectivity similar to middle age in older adults. Additionally, they report a higher functional connectivity within the default mode network in the tea drinking group, suggesting tea has a role in connective strength alterations. Previous studies have proposed that reduction of interregional connectivity is associated with aging induced cognitive decline (Li et al., 2019) This suggests that tea consumption leads to retainment of the strength of connections and leads to a delay in cognitive decline.

Figure 2. The differences between the hemispheric asymmetries of global metrics between the tea drinking group (T) and non-tea drinking group (NT). Negative values represent a rightward hemispheric asymmetry while the positive values represent leftward hemispheric symmetry. A. Hemispheric asymmetries for functional connectivity network. B. Hemispheric asymmetries for structural connectivity network.

Figure 3. Strength of connections of the tea drinking group and non -tea drinking group in the default mode network. A. Differences in connective strengths between groups for functional network. B. Differences in connective strengths between groups for structural network.

CONCLUSIONS/DISCUSSION Li et al. (2019) conclude that tea consumption has beneficial effects on delaying cognitive decline associated with aging by studying the functional and structural networks in the human brain. They reveal that tea drinkers have less leftward hemispheric asymmetry and stronger connections within the default mode network. Their results are supported by previous studies that show individual constituents in tea produce cognitive enhancements and neuroprotective effects (Li et al., 2019). In regard to studies on tea, Li et al. (2019) was the first to show the effects of tea on a system-level brain network, while other studies focused primarily on neuropsy-

CRITICAL ANALYSIS It is essential to investigate the specific brain regions that showed significant difference in functional connectivity between the two groups. In the study, tea drinkers displayed higher strength connections that were dominantly part of the posterior cingulate gyrus (PCG), parahippocampal gyrus (PHG) or angular gyrus (ANG) (Li et al., 2019). These areas are particularly vulnerable to aging and have significant roles for everyday cognitive function. The posterior cingulate gyrus is a highly metabolically active brain region that is involved in attention, information processing and working memory (Leech & Sharp, 2013). Aging as well as Alzheimer’s disease is known to display dysfunction in the posterior cingulate cortex (Leech & Sharp, 2013), implying that tea may have neuroprotective effects. Next, the parahippocampal gyrus is associated with visuospatial processing and episodic memory (Aminoff et al., 2013). Previous studies have found that atrophy in this region are early biomarkers of mild cognitive impairments (MCI), dementia and Alzheimer’s disease (Burgmans et al., 2011; Hackert et al., 2002; Pennanen et al., 2004). This may suggest that tea drinkers are less susceptible to aging induced cognitive decline as well as other cognitive disorders. Furthermore, the angular gyrus plays a role in memory retrieval, language and attention (Sephier, 2012). A study by Scheff et al. (2016) has reported that synaptic change in this area is associated with cognitive aging and Alzheimer’s disease. Taken altogether, the findings of the Li et al. (2019) study that show non-tea drinkers have lower functional connectivity in PCG, PHG or ANG areas conform with current literature that show their involvement in cognitive decline and cognitive disorders. More research is required in order to understand the optimal type of tea that supports brain health. In the Li et al. (2019) study, the researchers only used the frequency of tea drinking to form the two groups (tea drinkers and non-tea drinkers). They included three types of tea (oolong, black and green tea) but did not analyze them individually. One of the major setbacks of this condition is all three teas contain different types of antioxidants (Leung et al., 2001). Antioxidants aid in the prevention of damage to biomolecules caused by free radicals (Meydani et al., 2009). The current literature indicates that oxida-

tive stress is one of the main contributors of free radicals that cause a decline in cognitive function (Meydani et al., 2009). It is currently unknown if certain types of antioxidants are more beneficial for brain health. While green tea has higher levels of antioxidant catechin, oolong and black tea contain higher levels of antioxidant theaflavin (Leung et al., 2001). Catechins, as previously mentioned, have numerous studies that claim they support cognitive function (Torre et al., 2013). As such, numerous studies on black tea have found no effect on cognitive decline (Feng et al., 2018). Additionally, a study on black tea found it suppresses long term memory (Zhang et al., 2018). The same negative effects were not found for green tea or oolong tea. Thus, the effects of tea should be examined individually by the type of tea to find the optimal one that supports brain health.

FUTURE DIRECTIONS Future researchers should try to isolate the optimal type of tea for brain health using tea instead of its constituents. As previously mentioned, individual teas may have different levels and types of antioxidants that may act differently on the brain and cognition (Meydani et al., 2009). Additionally, some studies suggest the constituents in tea have synergistic effects thus studying only the individual constituents may hinder the results supporting the benefits (Owen et al., 2008). Furthermore, long-term studies and follow up is necessary to ensure the benefits of tea persists. These gaps in the current literature can be addressed by conducting experiments in rural regions where certain types of teas are the most popular. To minimize the effects of different lifestyles, rural communities should be used since they possess more similarities within the communities (Pateman, 2011). As such, black tea is the most popular in England while China is the largest consumer of green tea (Brody, 2019). It would be valuable to conduct studies within rural communities in these countries to investigate the effects of green tea and black tea on the brain. Current literature suggests that green tea may be more beneficial for cognitive health, but the comparative studies are limited to only scientific review papers. If green tea shows greater reduction in cognitive decline it can be concluded that catechins in green tea are more beneficial than the theaflavins found in black tea. Otherwise, it can be concluded that all antioxidants found in tea are equally beneficial in reducing cognitive decline. In order to assess if a reduction in cognitive decline occurs, the specific functional connections must be studied. If there exists a stronger connection in the PCG, PHG or ANG in the tea drinking group compared to the non-tea drinking group, it can be suggested that a reduction in cognitive decline has occurred. The loss of connections in these brain regions have been demonstrated to be associated with cognitive decline and other cognitive disorders such as Alzheimerâ&#x20AC;&#x2122;s and dementia. These findings on tea as a neuroprotective measure for cognitive decline could aid in the implementation of tea programs in seniorsâ&#x20AC;&#x2122; homes and serve as a preventive measure for middle age individuals. With further research, it could potentially serve as a treatment for individuals with Alzheimerâ&#x20AC;&#x2122;s, dementia and other cognitive disorders.

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An Appreciation for the Gut Microbiota: Antibiotic-Mediated Treatment from Early Adolescence Alters Brain Development and Behaviour Mary Dominics

Perturbations of the gut microbiota may affect the communication between the brain and the microbiome. The importance of bacteria in the developmental processes in the brain behaviour has been reported. The alteration of the gut microbiome has been demonstrated to increase the vulnerability to psychiatric disorders. Researchers have identified ways to study the effects of microbiome perturbations through the use of germ-free (GF) mice. More recently, there has been growing appreciation for antibiotic-mediated microbiome depletion in mice to develop an understanding for the importance of the gut microbiota in the body. Desbonnet et. al. addressed the effects of antibiotic treatment in early adolescence on brain development and behaviour in adulthood through the measurement of neuromodulators, anxiety, cognition and memory. Mice were treated with an antibiotic mixture administered in drinking water and subjected to behavioural tests throughout the span of the treatment. Microbiota diversity and richness significantly decreased with antibiotic treatment as measured from caecal contents. It was discovered that depletion of the gut microbiota from weaning onwards reduced the expression of BDNF, vasopressin and oxytocin in the brain, demonstrating neuromodulation from chronic treatment of antibiotics. This paralleled with the induction of cognitive deficits and reduction in anxiety levels, indicating that key neuromodulators, altered by the microbiome composition, may induce behavioural changes as well. This dysregulation of the gut-brain axis suggests that environmental factors, such as antibiotic treatment, in the early adolescent period can alter both brain development and behaviour in adulthood. This may further suggest antibotic-mediated depletion as a more useful model for assessing the communication between the gut and the brain, and may potentially provide treatment for neurodevelopmental disorders through further studies. Key words: antibiotic treatment, gut microbiota depletion, brain development, bacterial diversity, early adolescence, anxiety, cognitive deficits, microbiota composition, gut-brain axis

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BACKGROUND The gut microbiota has an evident role in the regulation of behaviour (Ignatova 2019). Although the standard use of germfree (GF) mice to examine the importance of the microbiome has been consistently employed, the use of antibiotics has been commonly known to induce changes in the composition of the microbiome (Kennedy, King, & Baldridge, 2018; Jernberg, Lofmark, Edlund, & Jansson, 2010). This has sparked interest in the area of antibiotic treatment to formulate discoveries surrounding the effects of microbiome perturbations on the body, specifically in the brain and behaviour (Lavelle, et al., 2019). It has been well documented that the alteration of the microbiome thorugh antibiotics creates lasting effects on metabolism, immune responses, and gene expression (Cox, et al., 2014; Willing, Russell, & Finlay, 2011). Particularly, it has also been shown to hinder the development of the brain and behaviour such as social learning and memory (Desbonnet, Clarke, Shanahan, Dinan, & Cryan, 2013; Gareau, et al., 2010). Adolescence is defined by an important critical period for brain development and behaviour, highlighting the vulnerability of this period to environmental changes (Sturman & Moghaddam, 2011). Various environmental factors

observed, an observation that has been previously reported in antibiotic-treated mice (Sun, et al., 2019). Prior to sacrifice, the treated and non-treated mice were subject to an acute stressor, resulting in a decrease in the number of bacterial species and diversity in antibiotic-treated mice (Figure 1). A decrease in Bacteroidetes was also observed, which had been previously demonstrated from exposure to chronic psychosocial stress (Bailey, et al., 2011). In contrast, the untreated group indicated an increase in these measures, particularly in the number of bacterial species and richness (Figure 1).

Figure 1. Effect of antibiotic-mediated depletion on bacterial diversity and species. Antibiotic treatment initiated from weaning onwards through ingestion and an acute restraint stress was exposed to mice prior to sacrifice. High-throughput DNA sequencing measured bacterial species (A), bacterial diversity (B), and Chao1 species richness (C). Data is expressed as ÂąSEM with n=7-8 mice per group. GLM analysis with Least Significant Difference post hoc test was used; *p<0.05, ***p<0.0001 (antibiotic effect), #p<0.05 (stress effect) (Desbonnet et. al., 2015)

can contribute to this growth period by shaping neuronal architecture in the brain (Spear, 2011). Intuitively, antibiotics would be thought to induce such changes, as was observed in recent studies on the dangerous effects of antibiotic exposure Neuromodulation Results from Antibiotic Treatment during the early periods of life (Cho, et al., 2012). Serum samples collected from mice treated with antibiotics preDesbonnet et. al. (2015) aimed to further discover the sented an increase in tryptophan levels and a decrease in role of antibiotic treatment on the microbiota through investikynurenine, in comparison with the non-treated mice (Figure 2), gating the effects of treatment during the early adolescent peria finding that is inconsistent with a previous finding (Fujisaka, et od on adulthood. It was found that antibiotic treatment from al., 2018). Although acute stress resulted a decrease in tryptoweaning to adulthood significantly altered the gut microbiota by phan measures, antibiotic treatment did not seem to have a decreasing bacterial diversity and species richness. These changstatistically significant stress effect. However, tryptophan levels es were sought to induce alterations in neuromodulator concenafter this stress occurrence did differ between the treated and tration, such as tryptophan and kynurenine as well as BDNF in non-treated mice, demonstrating a lower concentration in the the hippocampus and oxytocin and vasopressin in the hypothallatter group (Figure 2). amus. Furthermore, antibiotic treatment contributed to a decrease in anxiety levels, non-spatial cognition, and social memory through a novel object recognition test, light-dark box test, and the social transmission of food preference test, respectively. This suggests a role that bacteria may play in the development of the brain and behaviour, further supporting the idea of a bidirectional communication between the gut and the brain. MAJOR RESULTS

Figure 2. Effect of antibiotic-mediated depletion from weaning onwards on tryptophan (A) and kynurenine (B) concentrations post-sacrifice. Data is expressed as ÂąSEM with n=7-8 mice per Following treatment of antibiotics from weaning onwards, group. GLM analysis with Least Significant Difference post hoc Desbonnet et. al. (2015) measured microbiota composition of test was used; *p<0.05, **p<0.005, ***p<0.0001 (antibiotic mice caecal samples using high-throughput DNA sequencing and effect), ###p<0.001 (stress effect) (Desbonnet et. al., 2015) yielded lower levels of bacterial species, phylogenetic diversity, and species richness (Chao1 index) compared to mice undergo- To assess anxiety levels, mice were placed in a light box with an ing no antibiotic treatment (Figure 1). A decrease in Firmicutes adjacent dark box to measure the produced amount of excreted and Bacteroidetes and an increase in Proteobacteria was also fecal pellets as well as the amount of time the animals spent in Antibiotic Treatment Induces Changes in the Gut Microbiome

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the light. This test is based on the mouse’s tendency to avoid bright, open spaces, which could trigger anxiety (Bourin & Hascoët, 2003; Campos, Fogaca, Aguiar, & Guimaraes, 2013). It was observed that antibiotic treated mice produced significantly more fecal pellets and spent more time in the light compared to non-treated mice, suggesting lower anxiety levels (Figure 4). Anxiety behaviours have been reported in GF mice (Heijtz, et al., 2011) Desbonnet et. al. (2015) also investigated the role of antibiotic treatment on social memory using the social transmission of food preference test. This involved the introduction of a novel food source to a demonstrator mouse which had then been exposed to an observer mouse in the absence of food, to facilitate social interaction. This observer mouse was then placed in a new environment containing the food source previously exposed to the demonstrator mouse and another food source one day after the social interaction. Antibiotic-treated mice performed poorly in this test as there was less preference for the cued food source compared with the non-treated mice (Figure 4). Therefore, changes in microbiota composition could have an effect on social memory, as well.

cognitive deficits that are also observed with antibioticmediated depletion of the microbiome. Anxiety levels, as demonstrated with the light/dark box test, are reduced in antibiotic-treated mice which have been previously observed in germ-free mice, providing even more evidence that these behavioural changes to derive from changes in the microbiome. Lastly, the capacity to retain social cues is diminished in antibiotic mice as observed through the social transmission of food test. However, Fröhlich, et al., 2016 contradicts this finding as they found that there were no changes in spatial learning and memory. Furthermore, this paper provides two new major findings including the effect of antibiotic treatment in early adolescence on brain development and behaviour, as well as the effect of a stress response on these changes.

CRITICAL ANALYSIS

Desbonnet et. al. (2015) formulated a strong suggestion that adolescence represents a critical period during which changes in the microbiota can have an impact on the development of the brain and behaviour in adulthood. However, it seems like a very forward conclusion to make about the cruciality of developmental periods in early adolescence. Exposure to antibiotics continued into adulthood so changes could have been initiated DISCUSSION & CONCLUSIONS elsewhere in the life cycle, rather than strictly from early adoDesbonnet et. al. (2015) reported that antibiotic treatment lescence. alters microbiome composition resulting in neuromodulation and behavioural changes. A decrease in bacterial diversity sug- Another consideration of this paper is the multiple time points gested significant impacts on oxytocin, vasopressin, and BDNF used to perform behavioural tests. For instance, the novel obin brain regions which can be paralleled with the behavioural ject recognition test was performed approximately 4 weeks changes involving reduced anxiety, social memory, and non- following the initial antibiotic treatment but the social transspatial memory. These results signify the importance of the mission of food test was conducted in adulthood. The beginmicrobiome by highlighting the significance of the gut-brain ning of the antibiotic treatment period could have yielded axis. different results compared to a test taken later on in the lifespan. Particularly, anxiety levels may have been either highChanges in the microbiome induced by antibiotic treatment er or lower immediately after the initial treatment of antibihas consistently been reported in previous studies, indicating a otics compared to when it was measured in the 9th week, decrease in bacterial species and diversity. This paper aimed to which is considered the beginning of adulthood (Foster, Small, test these findings in a particular point in development. The & Fox, 1982). Therefore, the behavioural tests should not only authors also tested with and without an acute stressor prior to have been done in the first few weeks of life, but they also sacrifice and demonstrated a decrease in these measures with should have been consistently measured in the same period in antibiotic treatment. Although this has not been previously order to correctly assume that adolescence is the period in studied, these measures have been observed in antibiotic- which these changes occur. treated mice exposed to chronic psychosocial stress, indicating that stress response can be altered with changes in microbiota. Another limitation of this study is the testing of the microbiota composition after sacrifice. Changes observed, such as a deTryptophan and 5-IAA levels were elevated in antibiotic- crease in bacterial diversity, may have resulted from any point treated mice, suggesting a role of serotonin in the communica- of the mouse’s life cycle, and should not be directly associated tion between the gut and the brain. The relationship between with the occurrence during adolescence. To gain further underneuromodulators and the microbiome has consistently been standing of the microbiota changes induced by antibiotic treatobserved in many senses including (Kannan, Krishnamoorthy, ment in adolescence, mice should have been sacrificed at the Palanichamy, & Marudhamuthu, 2017). The decrease in BDNF beginning of adulthood. with antibiotic treatment has been previously shown which further suggests a bidirectional communication between the This paper successfully addressed the usefulness of antibiotic gut and brain because BDNF is released from enterocytes and treatment to induce changes in the microbiota to effectively acts as a neurotransmitter within the enteric neuronal circuit measure the effects on the brain and behaviour. The authors (Bathina & Das, 2015). This decrease can be associated with argued that germ-free mice are disadvantageous as they re102

quire housing in sterile conditions which may become costly. It was suggested that antibiotic treatment may provide a costeffective way to investigate these microbiota effects on brain and behaviour. The results presented in this paper parallel with results obtained previously from germ-free mice models, demonstrating that antibiotics may be just as an effective model (Kennedy, King, & Baldridge, 2018).

FUTURE DIRECTIONS To further examine the significance of microbiota in brain development in early adolescence, it would be essential to look at the effects induced by treatment with antibiotics strictly during the adolescent period. This might include treating the mice with antibiotics in the first 6-9 weeks of their life and examining the effects in the adulthood period rather than continuing to treat with antibiotics into adulthood. To do this, antibiotics would be consumed by approximately 10 healthy mice during the early life stages up until the 8-9th week of life and sacrificed at around the 12th week to examine this antibiotic effect from adolescence on adulthood. It is expected that the microbiota consumption would be similar to the one observed in this study as GF mice have previously shown this result as well. However, in order to accurately assess the role of antibiotic-mediated depletion in adolescents on adult brain development, behavioural tests should be performed with a larger sample size of mice at a consistent time period. Experimenters might divide the mice into groups to conduct either a novel object recognition test, light-dark test, or a social transmission of food preference test to observe any cognitive deficits and changes in anxiety levels. This is suggested because conducting the behavioural tests in the same mouse at the same time may cause stress (Desbonnet et. al., 2015). Studies have also suggested a link between depression and the gut-brain axis, so it would be interesting to assess depression levels in mice treated with antibiotics using the forced swimming test (Peirce & AlviĂąa, 2019).

otic treatment in mice should be investigated to potentially develop a treatment for psychiatric disorders. It would also be interesting to examine the effects of an acute stressor earlier on in the lifespan. This study exposed the mice to an acute stressor just prior to sacrifice but what would happen if mice were exposed to this during adolescence? It would be interesting to look at anxiety levels using the light-dark box performed in adulthood to compare the effects of stress on mice between the antibiotic-treated group and the control group. It would be expected that antibiotic-treated mice would have higher anxiety levels as compared to without the presence of a stressor, but would still have lower levels than the non-treated group. This is hypothesized because Desbonnet et. al. (2015) study showed that there was a decline in anxiety levels already from antibiotictreated mice. Furthermore, antibiotics has been observed to induce dramatic effects on the brain and behaviour, thereby promoting the existence of the gut-brain axis and its capacity to serve as a treatment for psychological disorders in the near future.

It would be expected that these behavioural results would be more pronounced than what was observed in this study because it would be a better indicator of the impact of perturbations of the gut microbiota in adolescence on brain development and behaviour in adulthood. Anxiety and depression levels would be reduced accompanied with deficits in cognitive abilities (Desbonnet et. al., 2015). Although it would be expected to produce more telling results, there is also the possibility that no changes would be observed with antibiotic treatment. This may be because the gut could possibly recolonize and therefore, have a phenotype similar to a non-treated mouse in adulthood. This may provide contrasting results to Desbonnet et. al.â&#x20AC;&#x2122;s experiment as it would provide an even better estimate of how antibiotic treatment in adolescence affects adulthood. In addition, it would also be beneficial to observe these same measures in mice with antibioticdepletion only during the weaning period to investigate the importance of the microbiota during the juvenile period. This paper has also suggested a role of serotonin in the gut-brain axis. Previous literature has suggested that this key neurotransmitter is involved in both the central nervous system and the microbiome (Stasi, Sadalla, & Milani, 2019) and has been associated with depression (Neumeister, 2003). Therefore, experiments involving the examination of serotonin in this pathway using antibi-

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Investigating the role of neutrophils with respect to cortical blood flow and cognitive function in Alzheimer’s disease mouse models Alina Dormann

Alzheimer’s disease (AD) is a serious neurodegenerative condition, affecting over 35 million people worldwide. The disease is characterized by dementia and gradual declines in cognitive function, and has severe impacts on caretakers as well as the directly affected individual, thereby making this an important disease to understand. It is a continuous and progressive disorder, so any advances in the field that may contribute to a reversal of symptoms or slowing of its development will prove to be extremely valuable. In addition to the distinctive neuropathological features of amyloid-beta plaque accumulation and tau protein hyperphosphorylation, reductions in cerebral blood flow (CBF) have also been implicated in the development of AD. However, the method by which this vascular dysfunction impacts disease progression remains unclear. In this study, the decreased CBF seen in transgenic mouse models of AD (APP/PS1 mice) was attributed to an increased percentage of blocked capillary segments in the transgenic mice as compared to the wild-types. Neutrophils were demonstrated to be the source of the blockage, and removal of these leukocytes following specific antibody administration had immediate positive impacts on CBF as well as cognitive function. This indicates that the prevention or reduction of neutrophil adhesion in cortical capillaries may play an important role in improving the short-term memory function of those affected by AD. Key words: Alzheimer’s disease, cerebral blood flow, neutrophil adhesion, neurodegeneration, cognitive function , amyloid-beta

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INTRODUCTION Alzheimer’s disease is the world’s most common form of dementia1. Affected individuals suffer from progressive declines in cognitive function, and the impacts of this are so extensive that the G8 has identified the development of a cure or potential therapy as a global priority, with the aim to have a solution by 20252. A number of theories have been proposed for the origin of AD, however there have been no conclusive results and its cause is often attributed to a combination of different factors 3. While amyloid-beta plaque formation and tau protein hyperphosphorylation are two of the most commonly studied neuropathological features of the disease and often the targets of proposed treatments, vascular dysfunction has also been associated with AD development1. Efficient blood supply to the brain is crucial for proper functioning, and issues in the systems responsible for blood delivery are seen in a number of different neurological or psychiatric disorders, including AD4. There are several proposed vascular pathologies that may play a role in altered blood delivery to the brain, the majority of which result in sustained brain hypoperfusion5. Therefore, it is no surprise that significant reductions in cerebral blood flow have been observed in AD patients as compared to healthy controls6. Additionally, these decreases in CBF often precede the aforementioned neuropathologies that are more classically associated with AD, indicating that lower levels of blood delivery to the brain may play an important role in AD development, or in influencing the speed with which it progresses7. It is also worth mentioning that improvements in CBF have been linked to enhanced memory performance in individuals with mild cognitive impairment (a precursor to AD)8, and that reduced CBF is an accurate predictor of cognitive decline later in life for healthy individuals9.

neutrophil extracellular traps, both of which play a role in the body’s inflammatory response and could alter blood flow through the cerebral vasculature12. In this particular study, the experimenters chose to investigate physical blockages within the capillaries, rather than factors involved in the vessel’s constriction. It was determined that the obstructed capillaries contained neutrophils. This suggests the involvement of neutrophils in yet another mechanism that may contribute to reduced CBF in AD patients. Reducing the number of plugged capillaries by administrating antibodies against a specific neutrophil marker resulted in improved short-term memory function – an expected result given the previously established link between increased CBF and improved cognitive performance. The findings of this study provide valuable contributions to the body of knowledge aimed at understanding reduced CBF in Alzheimer’s. Since this decreased cerebral perfusion has important impacts on disease progression and on cognitive symptoms, better understanding of its cause can lead to potential treatments, or at least better management of the disease. MAJOR RESULTS Cortical vasculature imaging In this study, two-photon excited fluorescence microscopy was

It is now well-established that reduced CBF is a strong indicator for AD development and plays a role in impairing cognitive performance. However, the causes of this hypoperfusion remain to be elucidated. Studies have varied in their focus, indicating that the underlying mechanisms may be multi-faceted or vary between different brain regions. There is general consensus that changes in blood flow are due to changes in cerebral capillaries, as opposed to other vessel types10. However, what occurs within the capillaries to ultimately result in reduced CBF is the subject of many studies.

used to view the cortical vasculature in transgenic mouse models of AD (over-expressers of amyloid precursor protein). Capillaries were the only vessel type observed to have blockages, and four times more capillaries were obstructed in the transgenic mice as opposed to the wild type controls. As seen in Figure 1, about 1.8% of capillaries were occluded in the AD mouse models, a number that may seem small but is actually capable of producing the reductions in CBF observed in AD due to the cumulative impact of each occluded vessel. These results are consistent with previous research indicating that capillaries are the vessels responsible for producing the reduced CBF that is observed in AD10. In contrast to previous studies, this is the first one to postulate that neutrophil plugging plays a role in generating reduced blood flow through the capillaries. Additionally, stalled capillaries did not increase with amyloid-beta plaque frequency, further differentiating this mechanism from previous research in which correlations between plaque density/ location and decreases in blood flow were seen11.

A number of different cell types, including pericytes and neutrophils have been suggested to play a role in changing the structure or function of the capillaries, ultimately resulting in reduced blood flow to the brain. Pericytes are capable of regulating CBF via their ability to change contractile tone and thereby promote either vasoconstriction or vasodilation within capillaries. One study found that in the presence of amyloid beta plaques, pericytes were induced to cause capillary constriction11. This was attributed to the production of reactive oxygen species by amyloid beta, which then prompted release of an endothelin that acted on pericytes to generate vasocon- Figure 1. Indicating a significant increase in the fraction of stalled striction. Other studies have implicated neutrophils as having capillaries in APP/PS1 vs WT mice. an important role. There are a number of potential mechanisms Figure adapted from Hernández et al. (2019). Nature Neuroscience, 22 through which neutrophils may act to reduce CBF. When acti(3), 413-420 vated, they can produce reactive oxygen species and release 106

Role of neutrophils in capillary stalling

placement tests and Y-maze completion. Despite these gains in one aspect of cognitive functioning, no improvements were observed in depression- and anxiety-behavior or in sensory motor function, two other important elements of AD development. Finally, it is known that the vasculature plays an important role in the clearance of amyloid-beta peptides14. Therefore, the researchers investigated whether the improved blood flow as a result of depleted neutrophils and decreased capillary stalling would impact the density and number of amyloid-beta plaques. While injection of a-Ly6G antibodies led to slight reductions in the concentration of amyloid-beta1-40, there was no impact on amyloid-beta1-42 aggregations. Overall, despite the antibody’s effects on improving cognitive performance, there were no substantial impacts on amyloid-beta plaques.

Labeling strategies were used to identify the cell types located within the stalled capillaries. The most consistent result was the presence of a leukocyte. A specific subset of leukocytes, known as neutrophils, have previously been linked to AD development and its associated cognitive impairment. Earlier studies have observed correlations between neutrophil extravasation and regions with increased amyloid-beta plaque density. Activated LFA-1 integrin, triggered by a specific form of amyloidbeta acted on neutrophils to induce the release of neutrophil extracellular traps, eventually leading to impaired cognitive function13. However, this process was not examined in the research being reviewed. The neutrophil plugging hypothesis presented by Hernández et al. (2019) acts on capillaries, independently of proximity to amyloid-beta deposits. Following injection of fluorescently labeled antibodies against a specific neutrophil surface marker (Ly6G), it was determined that the majority of stalled capillaries included a labeled cell, implicating neutrophils as the key cell in the process and thereby forming the back bone of their “neutrophil plugging” hypothesis. Administering higher doses of the specific antibodies resulted in a decreased number of stalled capillaries, and subsequent flow cytometry indicated massive depletions in the numbers of cir- Figure 3. Improved scores on a test of short-term memory observed culating neutrophils. To see if these changes would have an in the APP/PS1 mice, following administration of a-Ly6G antibodies. impact on the fundamental issues of CBF in AD, arterial spinFigure adapted from Hernández et al. (2019). Nature Neuroscience, labeled MRI was used to measure cortical CBF. APP/PS1 mice 22(3), 413-420 that had been injected with the a-Ly6G recovered the majority of the CBF difference that had previously been observed beDISCUSSION tween them and WT animals. Reduced CBF is a well-established feature of AD15. However, the mechanisms leading to this reduction remain unclear. One of the most important results of this paper is the neutrophil plugging hypothesis, suggesting that neutrophils are the key cells forming blockages in individual capillaries, and thereby majorly contributing to decreases in CBF. Prior to this study, there was no data in support of neutrophils playing this role. Despite this, the hypothesis is consistent with previous research on CBF as it implicates capillaries as the primarily affected vessels10. Because administration of a-Ly6G antibodies led to rapid depleFigure 2. Neutrophil concentrations 24 h after injection of a-Ly6G antibodies. tion of neutrophils and decreases in the number of stalled capillaries, the researchers postulated that the neutrophil adheFigure adapted from Hernández et al. (2019). Nature Neuroscience, sion was a result of receptor-mediated interactions with the 22(3), 413-420 capillary endothelium. Since Ly6G is a marker specific to neutrophils, interfering with its signaling should act to alter neutroImproving AD symptoms via increased CBF phil migration and adhesion. This aspect of the study is purely Having established a connection between neutrophil levels, an interpretation rather than a conclusive finding, but previous stalled capillaries and overall CBF, the researchers chose to studies have suggested that this is a potential mechanism by 16 examine whether the antibody-mediated increases in CBF which the neutrophil binding occurs . would impact cognitive functioning. Improvements were ob- The authors reported no differences in capillary stalling in relaserved in tests of short-term and working memory. Continual tion to amyloid-beta plaque concentration, as well as no signifiantibody administration over the period of a month resulted in cant reductions in concentration following treatment with ashort-term memory performance at identical levels to wild-type Ly6G. This is in contrast to the aforementioned studies which animals. For example, in figure 3 it can be examined the role that pericytes play in generating CBF. These observed that APP/PS1 mice performed similarly to WT mice on cells were more likely to induce vasoconstriction in regions with 11 tests of novel object recognition, and these results were con- high levels of amyloid-beta aggregations . Although it is possisistent across a number of different tests including object re- ble that the differences between pericytes and neutrophils 107

would result in only one cell type being affected by the presence of amyloid-beta, other studies focusing solely on the role of neutrophils, have also obtained results impacted by amyloidbeta concentration. Interestingly, a study investigating LFA-1 integrinâ&#x20AC;&#x2122;s role in regulating neutrophil extravasation and neutrophil extracellular trap release reported LFA-1 activation by amyloid-beta42 peptide13. This is the same form of amyloid-beta that was unaffected by treatment in the paper focused on neutrophil plugging, emphasizing a need for a better understanding of different amyloid-beta peptides and their properties. An extremely important finding of this paper has to do with the improved memory performance and cognitive function that was observed following improvements in CBF. The ability to interfere with neutrophil adhesion and subsequently enhance cognitive function provides a new window of opportunity for potential therapeutic approaches. CRITICAL ANALYSIS The authors made an important contribution to the body of knowledge surrounding AD by determining a potential mechanism for improving CBF in affected individuals and thereby improving their cognitive performance. Despite this, there are some discrepancies between the literature and the results presented in the article, and areas where further experimentation is required. While decreased CBF is a hallmark of AD, the extent to which different brain regions are affected by blood flow alterations tends to vary15. Some regions, such as the right anterior cingulate gyrus actually experience increases in CBF when an individual is affected by AD15. Therefore, the authors should conduct a more specific examination of capillary location, which could lead to better targeting of treatment and potentially even greater impacts on cognitive function. Previous studies have found major roles for amyloid-beta with respect to neutrophil activation, pericyte activity and CBF regulation. For example, generation of reactive oxygen species triggered by amyloid-beta has been observed to increase pericyte contractile activity11 as well as induce neutrophil activation and adhesion13. While the authors of the reviewed paper mention that amyloid-beta may play a role in generating inflammation in this way, their results do not indicate any changes in blood flow based on proximity to or concentration of amyloid-beta plaques. Acknowledging this relationship is important, but the authors should aim to obtain a better understanding of the mechanisms underlying the neutrophil plugging. A more indepth investigation of reactive oxygen species levels or of neutrophil-binding receptor expression would be beneficial in determining why certain capillaries are becoming plugged. It has been previously established that human neutrophils respond more strongly to these reactive oxygen species when compared to other human or rodent cells that may come into contact with them17. Therefore, these promising results obtained from mouse models may be partially due to the fact that rodent neutrophils may be activated to a lesser extent than in their human counterparts, making treatment more effective. Further investigation into this area and the mechanism occurring within humans will be required.

Finally, the authors themselves mention that their study focused solely on capillaries that had been completely occluded. If this neutrophil adhesion is truly playing a role in decreased CBF, capillaries that still have some blood flow may be experiencing reductions in the speed with which it occurs. A much greater than anticipated increase in CBF was reported following treatment with a-Ly6G antibodies, but this may have been less of a surprise if slowed flow had also been investigated. Finally, mouse models of AD typically experience dramatic increases in the level of amyloid-beta plaques between the ages of 12 and 15 months18. However, mice aged 6 and 11 months were used in the tests of memory and cognition in this study. The rapid and substantial increases observed in cognitive function could potentially be due to the use of mouse models with lower levels of amyloid-beta plaques than would typically be seen in an individual affected by AD. FUTURE DIRECTIONS While this study provides a basis for a potential new AD therapy, a lot of research remains to be done in this area. First of all, the study will eventually need to be replicated in human brain tissue to determine whether treatment by a-Ly6G antibodies has the same results on decreased capillary stalling, improved CBF and greater cognitive function in humans. While we may be a long way from trying the therapy on a living human, studies using brain biopsies can be conducted to determine whether a-Ly6G antibody administration acts the same way on human neutrophils. As mentioned previously, it has been established that human neutrophils respond differently to reactive oxygen species compared to other human and rodent cells17, so it is important to investigate whether this promising therapy will be just as capable of restoring CBF and cognitive function in humans. While the authors considered amyloid-beta plaque density and concentration as a factor potentially impacting stalled capillaries, they did not examine reactive oxygen species levels or other markers of inflammation. Amyloid-beta has been implicated in many other studies as playing an important role in affecting cells (including neutrophils) which ultimately result in decreased CBF. While no association was found in this study, previous research indicates that the amyloid-beta plaques likely play a role in neutrophil activity13, and this relationship is worthy of further investigation. Levels of reactive oxygen species could be measured in various brain regions and used to asses whether there is any correlation between this measure and stalled capillaries. The location of stalled capillaries is another area requiring further research. As mentioned previously, cerebral regions vary in how their blood flow changes in humans affected by AD 13. Certain regions experience greater decreases than others, and in some areas, even increases have been observed. The cortex is the only region in which capillaries were assessed in this study, however there are a number of additional brain regions involved in the cognitive functions that were examined. It would be worth specifically investigating the blood supply in other

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regions playing important roles in learning and memory, such as the hypothalamus. Lastly, based on the relatively young age of the mouse models used in this study, the researchers should examine if their promising findings with respect to cognitive function are reproducible in older mice with higher concentrations of amyloidbeta plaques. Interestingly enough, upon further review of the literature it was found that the same research group has actually begun to produce results in this area, and the optimism with respect to decreasing neutrophil adhesion in order to improve cognition continues to grow.

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REFRENCES 1. Hernández J, Bracko O, Kersbergen C, Muse V, Haft-Javaherian M, Berg M, ... Schaffer C. (2019) Neutrophil adhesion in brain capillaries reduces cortical blood flow and impairs memory function in Alzheimer’s disease mouse models. Nature Neuroscience 22(3): 413-420. 2. Scheltens P, Blennow K, Breteler M, de Strooper B, Frisoni G, Salloway S, Van der Flier W. (2016) Alzheimer’s disease. Lancet 388(10043): 505-517. 3. Bathula S, Dustakar V. (2011) Alzheimer’s disease: Recent advances in diagnosis and overview of clinical research in newer treatments. Clinical Research and Regulatory Affairs 28(2): 34-37. 4. Shabir O, Berwick J, Francis S. (2018) Neurovascular dysfunction in vascular dementia, Alzheimer’s and atherosclerosis. BMC Neuroscience 19(62). 5. Ferraz Alves T, Busatto G. (2006) Regional cerebral blood flow reductions, heart failure and Alzheimer’s disease. Neurological Research 28(6): 579-587. 6. Maalikjy Akkawi N, Borroni B, Agosti C, Pezzini A, Magoni M, Rozzini L, ... Padovani A. (2003) Volume reduction in cerebral blood flow in patients with Alzheimer’s Disease: a sonographic study. Dementia and Geriatric Cognitive Disorders 16: 163-169. 7. Dickstein D, Walsh J, Brautigam H, Stockton S, Gandy S, Hof P. (2010) Role of vascular risk factors and vascular dysfunction in Alzheimer’s Disease. Mount Sinai Journal of Medicine 77(1): 82-102. 8. Bracko O, Njiru B, Swallow M, Ali M, Haft-Javaherian , Schaffer C. (2019) Increasing cerebral blood flow improves cognition into late stages in Alzheimer’s disease mice. Journal of Cerebral Blood Flow and Metabolism 39: 1-12. 9. Xekardaki A, Rodriguez C, Montandon M, Toma S, Tombeur E, Herrmann F ... Haller S. (2015) Arterial spin labeling may contribute to the prediction of cognitive deterioration in healthy elderly individuals. Radiology 274(2): 490-499. 10. Kimura T, Hashimuira T, Miyakawa T. (1991) Observations of microvessels in the brain with Alzheimer’s disease by the scanning electron microscopy. Japanese Journal of Psychiatry and Neurology 45(3): 671-676. 11. Nortley R, Korte N, Izquierdo P, Hirunpattarasilp C, Mishra A, Jaunmuktane Z ... Attwell D. (2019) Amyloid β oligomers constrict human capillaries in Alzheimer’s disease via signaling to pericytes. Science 365(6450): 250-261. 12. Dong Y, Lagarde J, Xicota L, Corne H, Chantran Y, Chaigneau T ... Elbim C. (2018) Neutrophil hyperactivation correlates with Alzheimer’s disease progression. Annals of Neurology 83(2):387-405. 13. Zenaro E, Pietronigro E, Della Bianca V, Piacentino G, Marongiu L, Budui S ... Constantin G. (2015) Neutrophils promote Alzheimer’s disease-like pathology and cognitive decline via LFA-1 integrin. Nature Medicine 21(8): 880-886. 14. Deane R, Bell R, Sagare A, Zlokovic B. (2009) Clearance of amyloid-β peptide across the blood-brain barrier: Implication for therapies in Alzheimer’s disease. CNS and Neurological Disorders – Drug Targets 8(1): 16-30. 15. Dai W, Lopez O, Carmicheal A, Becker J, Kuller L, Gach H. (2009) Mild cognitive impairment and Alzheimer Disease: Patterns of altered cerebral blood flow at MR Imaging. Radiology 250(3): 856-866. 16. Wang J, Bair A, King S, Shnayder R, Huang Ya, Shieh C ... Nigrovic P. (2012) Ly6G ligation blocks recruitment of neutrophils via a ß2-integrin-dependent mechanism. Blood 120(7): 1489-1498. 17. Della Bianca V, Dusi S, Bianchini E, Dal Prà I, Rossi F. (1999) ß-amyloid activates the O2 forming NADPH oxidase in microglia, monocytes and neutrophils. Journal of Biological Chemistry 274(22): 15493-15499. 18. Elder G, Sosa M, Gasperi R. (2010) Transgenic mouse models of Alzheimer’s Disease. Mount Sinai Journal of Medicine. 77(1): 69-81. 110

A Novel Player in Alzheimer’s Disease Pathogenesis: SFRP1 Maida Duncan

Alzheimer’s disease (AD) is currently the primary cause of dementia worldwide1. No drug has been approved to treat AD in the U.S. since 2003 2, making the development of new treatments a high priority. Most prospective therapies aim to interfere with the formation of amyloid plaques (APs), which form when the peptide amyloid-β (Aβ) aggregates. Efforts to target these plaques stem from the amyloid cascade hypothesis, in which Aβ aggregation is considered to play a causal role in AD progression3. Research has shown that the formation of Aβ, from the processing of amyloid precursor protein (APP) by γ-secretases, can be decreased by alternative APP cleavage by α-secretases4. A recent study by Esteve et al. indicates that Secreted Frizzled-Related Protein 1 (SFRP1), an inhibitor of α-secretase activity, is elevated in the brains of patients with AD, and likely contributes to AD pathology5. Investigation into this Aβ-promoting pathway has indicated that inhibiting SFRP1 decreases Aβ accumulation and AD symptoms in an AD mouse model, indicating that SFRP1 inhibition may be a novel route for treating AD progression in humans5. Key words: Alzheimer’s Disease (AD), Secreted Frizzled-Related Protein 1 (SFRP1), amyloid precursor protein (APP), amyloid-β (Aβ), amyloid plaques (APs), amyloid cascade hypothesis, A Disintegrin and Metalloproteinase 10 (ADAM10), soluble APPα (sAPPα)

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Introduction

SFRP1 in AD Patients

Alzheimer's disease (AD) is the world’s leading cause of dementia1. It is characterized by a gradual worsening of cognitive impairment and neuronal damage1. No single cause of AD has been identified, and it is likely that several factors contribute to its presentation. Numerous studies have noted the appearance of cellular aggregates of the peptide amyloid-β (Aβ) in AD patient’s brains4. From this observation, researchers hypothesize that Aβ aggregates are causal in AD pathology, a proposal known as the amyloid cascade hypothesis3. Research in animal models suggests that interfering with the formation of these aggregates may be a way to decrease the symptoms of AD4.

Using anti-SFRP1 monoclonal antibodies and an enzyme-linked immunosorbent assay (ELISA), researchers found that SFRP1 levels were significantly higher in the brains of humans with AD compared to non-AD, age-matched controls, specifically in the frontal and entorhinal brain regions.

Aβ is a product of the amyloidogenic processing pathway of amyloid precursor protein (APP), in which APP is cleaved by β- and γ-secretase proteins4. Aβ has the ability to aggregate and form amyloid plaques (APs), which are a hallmark of AD. However, APP can also be processed via the non-amyloidogenic pathway, in which α-secretase proteins cleave within the Aβ domain of APP and produces a larger peptide called soluble APPα (sAPPα), that has been shown to have neuroprotective effects4.

Figure 1.) Immunofluorescent staining of human AD patient amyloid plaques. Arrow heads in i and j show AP cores, small arrows in j show AP “halo”. Immunostaining of the frontal cortices of AD patients showed that SFRP1 levels were high in the core of APs and near the outer edge “halo” of APs. This finding significant because “halos” are the site of concentrated accumulation of the oligomeric form of Aβ5 (Figure 1). The oligomeric form of Aβ has been indicated as the potential root of the cognitive decline seen in AD11.

Much research has aimed at inhibiting the amyloidogenic pathway as a therapy for AD4. Early research pinpointing the protein BACE1 as the main β-secretase causing Aβ production and accumulation in AD seemed to offer an excellent target for AD treatment6. Initial experiments in animal models suggested inhibiting this protein led to improved symptoms, however, further research showed that side effects counteract- SFRP1-Aβ interaction in vivo ed the symptom relief BACE1 inhibitors offered 7. Finding treatIn order to investigate possible interactions between ments with observable benefits that also outweigh negative Aβ and SFRP1, these compounds were incubated apart, and side effects has proven difficult in numerous additional AD then together. In vitro, Aβ alone formed both oligomers and studies2, 3, 4. fibrils. SFRP1 alone formed spherical oligomers of varying size. Some research has thus taken a different approach by When incubated together, however, fibrillar Aβ and SFRP1 olitrying to enhance the processing of APP by α-secretases, which gomers significantly declined and Aβ oligomers dominated the cleave within the Aβ domain of APP, to reduce the formation of culture. These data suggest that SFRP1 and Aβ interact in vitro Aβ. One recent study has investigated how the interaction be- to affect each other’s aggregation. tween the Secreted Frizzled-Related Protein 1 (SFRP1) and the constitutive α-secretase, ADAM10 (A Disintegrin and Metalloproteinase 10), contributes to AD pathology5. Previous research SFRP1 in mouse models of AD showed that SFRP1 inhibits ADAM108, making it a novel target SFRP1 was also found at higher levels in the AD mouse in manipulating Aβ formation. In the current study by Esteve, et model APP/PS1 (with mutations in amyloid precursor protein al., it is shown that inhibition of SFRP1 results in increased αand presenilin 1) than in wild type mice. Using cosecretase activity, reduced formation of APs, and a significant immunoprecipitation techniques, researchers were able to reduction in AD symptoms5. show that SFRP1 and Aβ bind one another and that SFRP1 interacts with the α-secretase ADAM10 in vivo. Major Results Esteve et al. show that both human and mouse AD populations express higher levels of SFRP1 than do non-AD individuals5. Further analysis suggests that SFRP1 expression has a causal effect on increasing the rate of amyloid plaque formation, and on the worsening of the AD phenotype, making it a promising target for AD therapies5.

Modifications to SFRP1 levels and associated AD-related changes When SFRP1 and green fluorescent protein (GFP) were overexpressed in heterozygous APP/PS1 mice via a modified lentivirus, immunostaining of neural tissue showed a significant increase in SFRP1 levels and the rate of AP formation compared to mice infected with GFP only, or uninfected mice. These data

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are quite significant in that they indicate a causal relationship Esteve, et al. present strong evidence of a causal relabetween SFRP1 expression and Aβ aggregation. tionship between SFRP1 levels and the development of AD in a mouse model. It was determined that SFRP1 levels are higher in Researchers tested the effects of decreased SFRP1 the brains of human and mouse individuals with AD, and that levels on AD in Sfrp1 null mutant mice and in mice treated with SFRP1 binds to and inhibits the α-secretase ADAM10. They also an anti-SFRP1 monoclonal antibody. In APP/PS1 Sfrp1 null mushowed that AD symptoms are alleviated by a reduction in tant mice, lower levels of the amyloidogenic pathway products, SFRP1 levels, likely due to increased activity of the α-secretase including Aβ, were detected compared to APP/PS1 mice (Figure ADAM10. These findings suggest that the inhibition of SFRP1 is 2). a promising avenue to explore in future AD treatment research. With the high failure rate of recent AD drug trials2, it is critical that novel pathways be explored. SFRP1 has yet to be targeted in other AD studies and may finally lead to a successful human AD treatment. Many studies have targeted other proteins involved in Aβ formation, and have even targeted Aβ itself, but have failed to see improvements, or have seen negative side effects that outweigh AD improvements2, 3, 4. Esteve, et al. conclude that inhibiting SFRP1 may be more effective than other approaches because the use of antiSFRP1 antibodies resulted in significant AD improvement, while also leading to fewer of the negative side effects seen in other AD immunotherapies5. Despite these findings, however, there Figure 2.) a-c show mouse brain slices stained for Aβ in APP/ are still many details to investigate further. The potential to see PS1 mice. Fewer Aβ “hotspots” can be seen in APP/PS1 Sfrp1 side effects in humans may be quite high, especially because knock-out mice (d-f). Graphical representation of Aβ levels in g. the α-secretase ADAM10 interacts with additional targets beyond APP3. In addition, higher levels of α-secretase (ADAM10) and non-amyloidogenic products were detected. APP/PS1 Sfrp1-/mice performed similarly to wild type in Morris water maze and Critical Analysis novel object recognition tests, while APP/PS1 mice performed The research conducted by Esteve, et al. appears to poorly. In addition, APP/PS1 mice treated with a monoclonal elucidate a novel and promising route for potential new treatantibody against SFRP1 showed significantly less AP formation ments, however, there are aspects of this study that need imcompared to APP/PS1 mice treated with a nonspecific antiprovement. Firstly, this study manipulated SFRP1 levels in mice body. who were only a few months old. In order to increase the exResearchers electrophysiologically investigated the ternal validity of this study, researchers should experiment with effects of the anti-SFRP1 monoclonal antibody on long term SFRP1 levels in older mice with more progressed symptoms. By potentiation (LTP) and found that APP/PS1 mice treated with doing so, the experimental conditions will more closely resemanti-SFRP1 antibody had similar LTP responses to wild type ble the circumstances under which SFRP1 inhibitor treatments after 5 months of treatment while APP/PS1 mice treated with are generally administered to humans with AD. Furthermore, nonspecific antibody had significantly worse LTP responses this study does not control for the complete involvement that (Figure 3). SFRP1 has in Wnt signaling pathways. Esteve, et al. conclude that SFRP1’s binding to ADAM10 causes AD symptom progression, however, SFRP1 is known to have roles in Wnt signaling, which is important in numerous pathways12. In order to increase internal validity, future research should ensure that only ADAM10 - SFRP1 interactions are being manipulated by only inhibiting the ADAM10-binding domain of SFRP1.

Figure 3.) l shows long term potentiation responses in wild type, nonspecific antibody (IgG)-treated APP/PS1, and antiSfrp1 antibody-treated APP/PS1 mice. Those treated with antiSfrp1 antibody show similar response at wild type. Averaged data from time points 50-60 min shown in m. Conclusion

Beyond the experimental design of this study, there are reasons to be cautious about using SFRP1 inhibitors to increase ADAM10 α-secretase activity as a treatment for AD in humans. Specifically, ADAM10 acts on substrates other than APP, raising the possibility that unrelated processes could be affected by its elevation3. Of particular concern is the finding that elevated ADAM10 is associated with certain cancers13. This means that long term elevation of ADAM10 activity in humans may cause cancerous side effects that outweigh improvement in AD symptoms. Furthermore, research has shown that over-

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overaccumulation of sAPPα, which is normally considered to be neuroprotective, may cause mental deficits, as seen in Fragile X syndrome14. Elevation of sAPPα via the inhibition of SFRP1 may thus exacerbate the existing cognitive impairments seen in AD patients which may diminish the efficacy of SFRP1 inhibitors as an AD treatment. These are all factors that should be taken into account in future research. Future Directions Future research should begin by studying the effects of inhibiting the ADAM10-binding domain of SFRP1 on AD pathology. This would be done by introducing a ligand that selectively binds this domain into an AD mouse model and in wild type mice, both young and old. If not done selectively, this could lead to inhibition of numerous metalloproteinase inhibitors with similar domains. If selective inhibition of the ADAM10-binding SFRP1 domain improves AD pathology in an AD mouse model, the conclusions of Esteve, et al. will be validated and further investigation can take place. If inhibiting one domain of SFRP1 has no effect on AD progression trajectory, this would contradict the conclusions of Esteve, et al. and prompt reevaluation of their findings. In the case that inhibiting one domain of SFRP1 slows the progression of AD in mice, the potential of these domain-selective SFRP1 inhibitors to enter human drug trials would increase, as SFRP1 would still be able to function in its other roles in AD patients. In order to reach human drug trials, future research must also delve into the possibilities of severe side effects such as further cognitive decline induced by sAPPα, and cancer caused by ADAM10 elevation. Experiments addressing these issues should involve testing the long-term changes associated with elevated ADAM10 and sAPPα in an AD mouse model and in wild type mice. If the appearance of any negative side effects is dose-dependent, researchers must investigate whether the level of SFRP1 inhibition needed to treat AD would be high enough to cause severe side effects. Beyond these experiments, researchers would also need to test for unexpected consequences of SFRP1 inhibition. There are still many years of research before SFRP1 inhibition might be considered for treatment of AD in humans. However, the research conducted by Esteve, et al. highlights a promising target for future therapies.

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REFRENCES 1.

Alzheimer's Association: 2019 Alzheimer's disease facts and figures. 2019; 1-23.

Cummings J, Lee G, Ritter A, Sabbagh M, Zhong K. Alzheimer's disease drug development pipeline: 2019. Alzheimer's & Dementia: Translational Research & Clinical Interventions. 2019 Jan 1;5:272-93.

Wetzel S, Seipold L, Saftig P. The metalloproteinase ADAM10: A useful therapeutic target?. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research. 2017 Nov 1;1864(11):2071-81.

Zhang YW, Thompson R, Zhang H, Xu H. APP processing in Alzheimer's disease. Molecular brain. 2011 Dec 1;4(1):3.

Esteve P, Rueda-Carrasco J, Mateo MI, Martin-Bermejo MJ, Draffin J, Pereyra G, Sandonís Á, Crespo I, Moreno I, Aso E, Garcia-Esparcia P. Elevated levels of Secreted-Frizzled-Related-Protein 1 contribute to Alzheimer’s disease pathogenesis. Nature neuroscience. 2019 Aug;22(8):1258-68.

Luo Y, Bolon B, Kahn S, Bennett BD, Babu-Khan S, Denis P, Fan W, Kha H, Zhang J, Gong Y, Martin L. Mice deficient in BACE1, the Alzheimer's β-secretase, have normal phenotype and abolished β-amyloid generation. Nature neuroscience. 2001 Mar;4(3):231.

Dominguez D, Tournoy J, Hartmann D, Huth T, Cryns K, Deforce S, Serneels L, Camacho IE, Marjaux E, Craessaerts K, Roebroek AJ. Phenotypic and biochemical analyses of BACE1-and BACE2-deficient mice. Journal of Biological Chemistry. 2005 Sep 2;280(35):30797-806.

Esteve P, Sandonìs A, Cardozo M, Malapeira J, Ibañez C, Crespo I, Marcos S, Gonzalez-Garcia S, Toribio ML, Arribas J, Shimono A. SFRPs act as negative modulators of ADAM10 to regulate retinal neurogenesis. Nature neuroscience. 2011 May;14(5):562.

Alzheimer’s Association. FDA-approved treatments for Alzheimer’s. 2019.

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Mehta D, Jackson R, Paul G, Shi J, Sabbagh M. Why do trials for Alzheimer’s disease drugs keep failing? A discontinued drug perspective for 2010-2015. Expert opinion on investigational drugs. 2017 Jun 3;26(6):735-9.

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Selkoe DJ. Soluble oligomers of the amyloid β-protein: Impair synaptic plasticity and behavior. InSynaptic Plasticity and the Mechanism of Alzheimer's Disease 2008 (pp. 89-102). Springer, Berlin, Heidelberg.

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Doberstein K, Pfeilschifter J, Gutwein P. The transcription factor PAX2 regulates ADAM10 expression in renal cell carcinoma. Carcinogenesis. 2011 Aug 30;32(11):1713-23.

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Pasciuto E, Ahmed T, Wahle T, Gardoni F, D’Andrea L, Pacini L, Jacquemont S, Tassone F, Balschun D, Dotti CG, CallaertsVegh Z. Dysregulated ADAM10-mediated processing of APP during a critical time window leads to synaptic deficits in fragile X syndrome. Neuron. 2015 Jul 15;87(2):382-98.

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Phantom Limb Pain: risk and protective factors. Shahd Fares

Amputation resulting from war incidents, peripheral vascular disease, neoplasms and diabetes, among other reasons can lead to phantom limb pain (PLP).1 PLP is the severe painful sensation in an absent limb following amputation.1 Several theories have been proposed to attempt and explain this, however a lack of clear consensus on the etiology and pathophysiology of PLP has resulted in a lack of available treatment to such long term pain.2 Several studies have also attempted to discover potential predictors (risk and protective factors) of PLP, one of which is this paper. Questionnaires, pain diaries, interviews and pain measures were used to evaluate predictors in relation to PLP incidence and severity. Pre-operative depression, anxiety and chronic pain were risk factors of PLP, while pre-operative life control was a protective factor. Post-operative sub-acute pain was also a risk factor of PLP. Such findings although not completely new to the field were significant as this paper was one of the first and few that used a prospective research design and thereby evaluated factors preoperatively in direct relation to the expected PLP. Despite the lack of knowledge on the etiology of PLP, understanding its predictors and their respective pathways can be a gateway into uncovering the underlying mechanisms of the disease. Key words: Phantom limb pain, Phantom limb sensation, Residual limb pain, depression, anxiety, chronic pain, acute pain, subacute pain, preoperative, postoperative, predictive factors, 1 week, 3 months, 12 months, etiology.

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Background and Introduction:

sality) and confounding variables. Is it PLP that causes psychological factors such as depression (potentially due to the new disability experienced or the traumatic event encountered) or does pre-operative depression contribute to the intensity of pain in PLP? Is social support during the post-operative stage effective even if it was absent prior to the amputation? Or, does it need to build on established relationships with the amputee through friends and family? Such questions remain unanswered in a pre-operative viewpoint (due to minimal preoperative research), creating a gap in the literature.

Amputation resulting from war incidents, peripheral vascular disease, neoplasms and diabetes, among other reasons, is not only a traumatic experience for many amputees, but one that may also result in phantom limb sensation (PLS) and phantom limb pain (PLP).1-3 PLP is the severe painful sensation in an absent limb following amputation, while PLS is any sensation in the missing limb, other than pain.1 PLS is rarely a clinical problem. PLP on the other hand, challenges the medical field as it places amputees in a state of long-term pain that can be particularly difficult to treat (figure 1).4 Larbig et al. attempt to fill such a gap through the exploration of such predictive factors in a prospective study design. 1 They then extend this through a longitudinal study to observe how With an incidence rate of 60-80% of amputees, the occurrence these factors change between the pre- and post-operative stagof PLP is independent of age in adults, gender, or side of ampu- es with regards to PLP.1 Depression, anxiety, social support, life tation.2 This intermittent pain, usually experienced in the distal control, as well as chronic and subacute pain of 52 participants parts of the missing limb, is described as a shooting, stabbing, were measured using questionnaires, pain diaries, pain squeezing, or burning sensation by amputees.2 PLP may arise measures/index and interviews. several months or years after amputation, and is usually seen in regions with large cortical representation such as the hands and feet.4 Major Results:

Although a series of potential biological mechanisms behind PLP have been discussed in the literature in relation to the periphery and central nervous system2,3,5-7, the etiology and pathophysiology of PLP remain disputed in the field.3

The 52 patients (majority of which underwent lower limb amputation) who were eligible for the study and completed the one-year follow up were contacted a week prior to the amputation, a week after the amputation, 3 months after the amputation, and 12 months after the amputation. The measures taken at each of these intervals enabled the authors to identify risk This lack of consensus therefore limits the treatment options of factors and protective factors of PLP based on the relationship PLP (as it is unclear what aspects/regions should be targeted in observed between the measure at a particular time and the the course of therapy). This is where the exploration of PLP presence of PLP at 12 months after the operation (chronic PLP). predictors is thereby potentially very valuable. Studying PLP predictors not only aids the medical field in potential preventative measures, but it also aids in the discovery of potential biological pathways and mechanisms behind PLP (as understanding a predictive factor such as depression may allow researchers to use the existing knowledge in the field about depression in terms of its pathway and genes, in relation to the theories behind PLP in the literature).

Predictive factors of PLP are generally categorized into risk or protective factors.1 Risk factors (factors that increase likelihood of PLP incidence) have been identified to include psychological predictors such as depression, anxiety and stress, as well as immediate pre-amputation pain (measured by participant recall post amputation) and subacute post-operative pain.1,8,9 Protective factors (factors that reduce incidence of PLP) include social support and activity.1,8

Figure 1: Intensity and incidence of PLP, RLP, PLS over 1 week, 3 months, and 1 year. A. mean intensity of PLP, RLP, and PLS measured on a numerical rating score (NRS) between 0 and 6 where 0 is “no pain” and 6 is “most painful”, B. mean incidence of PLP, RLP and PLS. PLP and PLS show stable incidence and intensity over the three time periods while RLP decreases with It should be considered however, that such findings in the liter- time. Data obtained from primary paper reviewed. ature were all completed post-operatively.1,8,9 This is significant as it highlights the potential of recall bias (as participants had to recall pre-operative information), directionality (reverse cau117

Table 1: Predictors of PLP Phantom Pain Preoperative period Chronic Pain 76.84 before amputation (CPI) Pain Severity 4.02 1 week before amputation (MPI) Perioperative period Pain on day 3.74 before amputation (NRS 010) Postoperative 2.66 pain (NRS 0 10) Psychological variables before amputation BDI 7.83 Anxiety Trait 42.44 MPI life con2.86 trol MPI social 4.95 support

No phantom pain

Statistical Significance

31.56

1.74

***

Stronger acute pain severity one day before the operation was not observed to strongly predict PLP at 12 months (table 1). 1 Average postoperative pain (measured 1 week after the amputation using a numerical rating scale ranging from “no pain” at 0 to “worst pain possible” at 10) on the other hand, was four times higher in those with chronic PLP than those without (table 1).1

Psychological predictors and PLP: depression and anxiety acting as risk factors, life control acting as a protective factor

2.44

0.57

***

4.25 27.55 4.13

* ** *

5.13

The preoperative measures and the psychological tests were conducted a week prior to amputation. The post-operative pain during the perioperative period was measured a week postoperatively. Presence of pain refers to individuals having PLP at 12 months (chronic PLP) and those who do not have PLP at 12 months (non-chronic PLP). All values indicated in the table are the mean value of the 52 participants involved. CPI = Chronic Pain Index (measured as frequency x duration x intensity), MPI = West Haven-Yale Pain Inventory, NRS = Numerical rating scale (where participants rate their pain from 0-10, 10 being the worst possible pain), BDI = Beck Depression Inventory, Anxiety trait = State-Trait Anxiety Questionnaire. *= p<0.05; **=p<0.01, ***=p<0.005, ns= not significant. Data obtained from primary paper reviewed.1

Psychological factors such as depression and anxiety were measured a week prior to the operation. Beck Depression Inventory (BDI), a measure used to assess depression was scored at 7.83 in amputees experiencing chronic PLP. Those without chronic PLP had a BDI score of 4.25 pre-operatively (table 1).1 Further, those with PLP after amputation by 12 months had higher anxiety pre-operatively. This was evident as those with PLP had a state-trait anxiety questionnaire score of 42.44, while those without had a score of 27.55 (table 1).1

Life control on the other hand had the opposite results, where higher pre-operative life control was observed significantly in non-chronic PLP patients. (4.13 West Haven-Yale Multidimensional Pain Inventory (MPI) score while in those experiencing chronic PLP, MPI life control was 2.86) (table 1).1 Although pre-operative social support MPI also differed, where it was found to be 5.13 in participants without PLP at 12 months, and 4.95 in those with PLP at 12 months, this difference was very small and was not found to be statistically significant (table 1).1

Conclusion/Discussion section:

As a result of such findings this paper concludes that preoperative anxiety and depression, as well as pre-operative chronic pain are risk factors for PLP. Pre-operative life control Pre-amputation chronic pain is a risk factor of PLP on the other hand is a protective factor. Sub-acute postoperative pain is also a risk factor for PLP. No clear relationship Chronic Pain Index (CPI) – measured as: frequency x duration x was established between pre-operative acute pain and PLP or intensity (of pain) – was used as a measure of chronic pain pre- pre-operative social support and PLP. operatively. Amputees with chronic PLP had more than twice the mean CPI value of those without PLP (table 1).1 Additionally, patients with chronic PLP had reported higher pain severity The findings that prolonged pain before the amputation was a to the pain measured a day beat 1 week prior to the operation. This is evident as chronic PLP strong predictor in comparison 1 fore the amputation confirms that chronic pain before the patients had a higher mean MPI value (West Haven-Yale Pain surgery is a risk factor while acute pain is not. This suggests Inventory measure) in comparison to non-chronic PLP partici1 that pathways involved in PLP are built over the long term, popants (table 1). tentially signifying a re-organization or sensitization in existing pathways arising from the pain experienced in the limb before its amputation.10 These pathways may then be exacerbated Postoperative-perioperative pain is a risk factor of PLP post-operatively through changes in different parts of the nervous system at different times.10 This may also explain why pa118

tients with chronic PLP reported quadruple the amount of pain as those without chronic PLP a week after the operation (as they may already have established sensitized pathways in comparison to the others).

The psychological findings with regards to PLP are consistent with the literature and therefore validate previous postoperative research on depression, anxiety, and life control. 11,12 This is also evident in relation to the findings in the field that anti-depressant medication such as amitriptyline, mirtazapine, and tricyclic medications seem to present great, stable relief for PLP pain.2 This correspondence further implies that psychological pathways are involved in the etiology of PLP and therefore highlights the significance of further understanding them in future research.

study (since patients recalling chronic pain that was present long before they were questioned a week prior to the operation). An implication of such bias could be the overreporting or underreporting of pain which may have skewed the data. Furthermore, another potential limitation to this study is the small sample size as well as the significant loss of participants. Starting with 74 eligible patients, only 52 of those were evaluated throughout the whole longitudinal study as a result of multiple factors such as participant withdrawal and death.1This decline in patient numbers therefore not only reduced the sample size of the study, lowering its statistical power (a common issue in neuroscience)13, but it may have also resulted in some attrition bias affecting the findings.1

Additionally, this paper outlines the predictive factors of PLP and by doing so highlights potential pathways and treatments that can be used (in relation to the predictive factors). This does not however alleviate the need for further research into the etiology of PLP. Future in depth research of each factor and its functional and molecular mechanisms needs to build on the findings of this paper to fill such a gap in the literature.

Such findings are significant as to the authors knowledge, this is one of the first papers to have assessed pre-operative pain before amputation (pain measures administered before amputation) rather than asking patients to re-call their pain after the operation. Further, it is one of the few papers that assesses psychological factors before the amputation in the literature. This therefore had reduced recall bias and increased the confidence in the results obtained and the directionality between Furthermore, although the prospective nature of this study the variables (as one event occurred before another and therereduces the likelihood of reverse causality and places it in a by the predictor most likely causes the PLP). higher rank of internal validity in comparison to the literature, it remains a correlational study limiting the confidence in the results. Therefore, even though the conclusions of this paper are by no means new to the field, they contribute greatly towards con- Such critical analysis is significant as the factors explored by the firming previous studies and supporting results in the litera- study only explained a third of the variance,1 resulting in unexture. This is beneficial to the field as discussed earlier, since it plained variation waiting to be explored. This unresolved variahighlights the potential pathways further research should focus tion may also suggest the involvement of other risk and protecon to identify the etiology and pathophysiology of PLP in ampu- tive factors or genetic components that are yet to be discovtees to thereby aid in treatment development. ered.1

Critical analysis section:

Future directions section:

It should be considered however that this paper does not align with the literature in terms of social support. Although it finds no relationship between social support and PLP, the literature generally proposes it as a protective factor. 12 On the other hand, the literature usually looks at post-operative social support and so this may explain the difference (as this study looks at pre-operative support). Another potential reasoning behind such discrepancy is that social support measures may have differed across the studies. Further studies evaluating this are required to generate greater confidence in such findings.

Based on the limitations discussed above, the overall study should be replicated to reduce potential errors and gain greater confidence in the findings. This is especially important considering the small sample size discussed earlier.

It is also worth noting that even though this paper attempts to fill the gaps in the literature through the pre-operative study of factors reducing recall bias, it does this only a week prior to amputation. As a result, there could still be recall bias in the

The authors should also aim to explore more factors with regards to the incidence and severity of PLP. These may include pain catastrophizing (exaggerated and negative cognitive and emotional representation of pain by an individual). 14 Even though it has been examined in the literature14, like other factors this has been done in a post-operative perspective. It could be hypothesized that pre-amputation pain catastrophizing is a risk factor for PLP as patients may pay more attention to stimuli and thereby experience and report greater pain. This potential enhanced response to stimuli coupled with the possible sensiti-

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zation of relative pathways due to pre-amputation chronic pain (which has been established as a risk factor) 1 could help better explain PLP, highlighting its potential mechanism and thereby aiding in the unveiling of its etiology. As a result, completing more pre-operative studies involving factors such as pain catastrophizing may provide valuable findings that build off of the paper in review. Such a hypothesis can be explored using similar means to that of the paper reviewed1 with the addition of pain catastrophizing measures such as the Danish version of the pain catastrophizing scale.14

individual increasing the internal validity of potential findings.

Brain imaging studies could also be conducted to evaluate central nervous system (CNS) changes in patients from preoperative stages to post-operative stages with time (also evaluating the chronic and acute pain pathways triggered during such times in accordance with the finding of this study of them being risk factors).

In review of the literature, it can be hypothesized that cortical re-organization/re-mapping to neighboring regions may be observed in such comparisons.5 This may be identified through smaller activation sites observed in the area representing the limb of interest in fMRI imaging post-operatively in relation to the same movement/activation conducted pre-operatively (with larger area of activation in neighboring areas with their respective movements/tasks). Such smaller areas may include the somatosensory association cortex/ superior parietal lobe in Brodmann areas 5 and 7 for toe flexions for example.18

Such studies may enable better understanding of CNS pathways that could be involved in PLP. Functional magnetic resonance (fMRI) and diffusion tensor imaging (DTI) could be used to determine functional, metabolic, and white matter tractography changes in the brain following amputations. 16,17

Such comparisons will also be carried out alongside measures of risk factors and protective factors of individuals (using the same methods defined in the paper reviewed) so that CNS variations could be matched to such characteristics. This could help establish the relationship between a particular change observed and the predictive factors presented in the paper reviewed.

Furthermore, DTI results may indicate changes in white matter pathways in the corticospinal or cerebellar tract (in relation to coordination of fine motor tasks and timing coordination).19

Two groups may be recruited. An experimental group which includes individuals expecting an amputation in the future with Such findings could be especially significant since the literature varying reports of pain duration, frequency and intensity, as well as a control group which is not experiencing any pain and indicates a potential relationship between neuroplasticity/ structural re-organization, and pain.20 Furthermore, even if no is not expected to have an amputation (healthy group). significant differences were observed this may suggest that PLP involves more peripheral changes rather than central, again Both groups can then be asked to carry out specific tasks (such potentially providing significant findings to the field. as toe flexion or leg bending/extension) in the limb prior to and post amputation (imagining movements in that limb after it has been amputated) as well as in the healthy limb (in both healthy limbs for healthy individuals). This could be done at 1 month and 1-week pre-operatively as well as 1 week, 3 months, and 12 months post-operatively (as likelihood of amputation cannot be determined very early pre-operatively).

The fMRI and DTI results can then be compared between the control group and the experimental group (pre-and postoperatively) to identify differences in functional and anatomical regions in the brain, as well as white matter tracts. Such comparison may prove beneficial as general changes across all patients could be analyzed, identifying potential generalizable CNS re-organization in amputees.

These comparisons could also be drawn in the same individual in the experimental group (through contralateral brain activation evaluations between the movements in the healthy limb versus those in the amputated/painful limb). Such comparison may prove beneficial as other variables remain constant in that 120

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Nikolajsen L, Christensen KF. Chapter 2 - phantom limb pain. Nerves and Nerve Injuries 2015:23-34. ISBN 978-0-12-802653 -3

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Flor H, Nikolajsen L, Staehelin JT. Phantom Limb Pain: A case of maladaptive CNS plasticity? Nat. Rev. Neuroscie. 2006 Nov; 7(11): 873- 881.

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Valentini E, Hu L, Chakrabarti B, Hu Y, Aglioti SM, Lannetti GD. The primary somatosensory cortex largely contributes to the early part of the cortical response elicited by nociceptive stimuli. Neuroimage. Jan 2012; 59(2): 1571-1581.

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Antonio C, Gisela EH, Antonella P, Marta B, Cecillia G, Alberto C, Alessandro CS, Carlo C, Umberto S. Functional changes in the activity of cerebellum and frontostiatal regions during externally and internally timed movement in Parkinson’s disease. Brain Research Bulletin. 2006; 71: 259-269.

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Elucidating the Link Between Hyperinsulinemia and Dementia Aisha Faruqui

Hyperinsulinemia is a known risk factor for Alzheimer’s disease, but the pathways underlying this connection are poorly understood. In the study conducted by Chow et al. (2019), chronic insulin exposure was found to trigger insulin resistance in neurons, resulting in impaired glycolysis and ultimate accumulation of p25. p25 leads to build-up of β-catenin and subsequent neuronal senescence, while activation of neurotoxic CDK5-p25 causes cell death. These findings elucidate a previously unknown mechanism that has significance regarding the pathology of Alzheimer’s disease, and can explain cognitive decline and neurodegeneration in diabetics. Key words: Diabetes mellitus, hyperinsulinemia, insulin resistance, CDK5, p25, Alzheimer’s disease, neurodegeneration, neuron senescence

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INTRODUCTION

of dementia, and makes the first clear connection between Type 2 diabetes mellitus (T2DM) is among the top ten hyperinsulinemia, cognitive decline, and neurodegeneration. global causes of death and continues to be a major health concern in both developed and developing countries (“The top 10”, 2018). T2DM comorbidities include cardiovascular disease, hyMAJOR RESULTS pertension, obesity, and depression – all of which result in decreased quality of life (Goldney et al., 2004; Sowers et al., Chronic insulin exposure results in impaired glycolysis and sub2001). Aside from the body’s failure to properly regulate blood sequent accumulation of p25 glucose levels, diabetics often also have deficits in memory, Chow et al. (2019) used mouse neuronal cell lines to attention, and processing speed (Roriz-Filho et al., 2009). Inobserve the effects of chronic insulin exposure on the brain. deed, diabetics are found to have an increased risk of demenAfter 48 hours of exposure, they found a reduction in the trantia, as well as neurological symptoms described as ‘accelerated scription of genes involved in glycolysis. Notably, there was a brain ageing’ (Biessels, 2002). These symptoms include brain substantial decline in genes that encode glucose transporters, abnormalities, cerebral atrophy, and altered glutamatergic sigHK2, and pyruvate kinase. These changes were corroborated by naling (Biessels, 2002). similar in vivo findings in cortices from the IR mouse model. In The prediabetic stage is often associated with excess the neuronal cell culture, chronic insulin exposure resulted in insulin in the blood—a condition called hyperinsulinemia (Chow impaired glycolysis and reduced availability of intracellular gluet al., 2019). Hyperinsulinemia can induce insulin resistance (IR) cose, which was associated with increased levels of p35—the in multiple tissues before the onset of full-blown diabetes uncleaved form of cyclin-dependent kinase 5 (CDK5) activator. (Shanik et al., 2008). Insulin from the blood enters the central Chow et al. then observed cleavage of CDK5-p35 to the p25 nervous system (CNS) through active transporters (Steffens et form, leading to p25 accumulation (Fig. 1a). In order to deteral., 1988), which allows neurons to process glucose through mine the sequence of events among these protein changes, glycolysis and the tricarboxylic acid cycle (Gjedde et al., 2001; Chow et al. performed small interfering RNA (siRNA) knockNehlig et al., 2004). Prolonged hyperinsulinemia increases the down on the aforementioned glycolysis-related genes. Silencing flux of insulin into the CNS, leading to saturation of the cere- of the Hk2 gene (which encodes hexokinase) resulted in accubrospinal fluid (Begg et al., 2015) and subsequent development mulation of p25 (Fig. 1b), suggesting that hexokinase 2 is an of neuronal IR. This prediabetic stage was shown to quadruple upstream regulator of p25. the risk of Alzheimer’s disease (AD) (Ott et al., 1999), and many previous studies have associated brain hyperinsulinemia with cognitive decline and dementia (Lutski et al., 2017; Young et al., 2006). However, the mechanistic link between brain hyperinsulinemia and neurodegeneration remains elusive.

Neurodegeneration and brain ageing as a result of cellular senescence has been a topic of considerable interest in the past several years (Chinta et al., 2016). The senescence phenotype is defined by cellular growth arrest and includes changes in metabolism and protein expression (Tan et al., 2014). These alterations contribute to neurodegeneration and age-related pathologies (Chinta et al., 2016; Tan et al., 2014). While studies on diabetic patients have revealed associations between insulin resistance and cellular senescence (Burton, 2009; Morocutti et al., 1996), a direct link has yet to be made.

Figure adapted from Chow et al. (2019). Nature neuroscience, 22(11), 1806-1819. Figure 1. a, Western blots of HK2, p35, and p25 following chronic insulin exposure (100 nM insulin + 16 nM glucose) of neuronal cell cultures. Cells were exposed for various time intervals before collection, as indicated. b, Western blots of enzymes HK2, p35, and p25 following siRNA knockdown of HK2. All in vitro treatments were performed on mouse neu-

In the study conducted by Chow et al. (2019), the authors expose a neuronal cell culture to chronic insulin to observe cellular changes. Chronic insulin exposure results in impaired glycolysis due to decreased transcription of hexokinase 2 (HK2). This leads to downstream effects, including accumula- Accumulation of p25 leads to neuron senescence and death tion of p25 and ultimate neuronal death and senescence. The discovery of this pathway has implications for the pathogenesis 123

Chow et al. (2019) used cortical and hippocampal neurons from the IR mouse model to observe cellular changes associated with senescence. A key player known to drive senescence is the proto-oncogene β-catenin (Adams & Enders, 2008), which was found in increased levels following chronic insulin exposure and accumulation of p25 (Fig. 2a). Furthermore, IR mice with increased levels of senescent neurons were found to have poorer spatial memory and cognitive function, as measured by their performance in the Morris water maze and Y-maze. Interestingly, p25 is also known to cause neuronal death by forming CDK5p25, a neurotoxic dyad (Fischer et al., 2005). Chow et al. (2019) investigated this idea further by modulating levels of CDK5 in a mouse neuronal cell line treated with insulin. Overexpression of CDK5 increased the number of dying neurons, whereas the opposite effect was observed upon CDK5 reduction or blocking its activity with roscovitine. Chow et al. also observed the effects of CDK5 modulation on cell senescence. CDK5 overexpression reduced the fraction of senescent neurons in culture, and while CDK5 knockdown increased this fraction, blocking CDK5 activity with roscovitine had no effect (Fig. 2b). This finding suggests that the physical presence of CDK5 is required to inhibit senescence rather than its enzymatic activity.

major significance as there is controversy regarding the presence of p25 in AD brains. A study conducted by Tseng et al. (2002) show that the frontal cortex, inferior parietal cortex, and hippocampus of AD individuals have higher levels of p25 than control groups, and that accumulation of p25 may contribute to AD pathogenesis. However, another study by Tandon et al. (2003) observed no change in p25 in AD brain homogenates. In light of this controversy, Chow et al.’s work is interesting because it reveals a previously unknown p25-modulating pathway, as well as downstream effects that support Tseng et al.’s proposal. Additionally, Chow et al. (2019) demonstrate that insulin-induced p25 accumulation functions in two different pathways to trigger neuronal senescence and cause death. βcatenin and associated cellular senescence has been observed in previous studies (Hiyama et al., 2010). Chow et al. (2019) further underscore this connection by showing that IR mice with increased levels of β-catenin-induced senescent neurons have poorer cognitive function and spatial memory than their non-IR counterparts. However, the link between p25 and βcatenin dysregulation is a relatively novel discovery that has only been studied once before (Chow et al., 2014). Furthermore, the finding that CDK5-p25 leads to cell death (Chow et al., 2019) agree with many other studies that have also made the same connection (Cruz et al., 2003; Saito et al., 2007). Interestingly, Chow et al. (2019) demonstrate that CDK5 knockdown increases senescence, but blocking CDK5 activity has no effect. Therefore, the authors suggest that the physical presence of CDK5 blocks senescence, rather than its enzymatic activity. The mechanism underlying this novel finding has not been elucidated. CRITICAL ANALYSIS

Figure adapted from Chow et al. (2019). Nature neuroscience, 22(11), 1806-1819. Figure 2. a, Western blots of β-catenin, p35, and p25 following insulin treatment for the indicated amount of time. b, Percentage of SA-β-ga in neurons treated with roscovitine, CDK5 siRNA, or overexpression of GFP-CDK5. SA-β-ga is a known biomarker of senescence and is used to quantify cellular senescence. All in vitro treatments were performed on insulin-treated mouse neuronal cells. CONCLUSIONS/DISCUSSION The research done by Chow et al. (2019) elucidates mechanisms underlying the effects of hyperinsulinemia on the brain. The authors uncover a key link between hyperinsulinemia and cellular ageing by demonstrating that impaired glycolysis and reduced HK2 leads to p25 accumulation. This result is of

Chow et al. (2019) are the first to uncover a mechanism underpinning the connection between hyperinsulinemia, cognitive decline, and neurodegeneration. The authors used a mouse hippocampal neuronal cell line to observe the accumulation of p25. While the use of mouse models is standard for AD research, it would be interesting to know if p25 accumulation would have occurred under the same conditions in a human neuronal cell line, or in cell lines of different brain regions. In the study conducted by Tandon et al. (2003), neither human nor mouse whole brain homogenates show any change in p25 levels. However, this allows for the possibility that p25 changes are region-specific, and that certain brain regions could exhibit higher p25 levels, while other brain regions exhibit unchanged or potentially lower p25 levels. Indeed, Tseng et al. (2002) show that p25 is increased specifically in the hippocampus, frontal cortex, and inferior parietal cortex. More research should be conducted to better understand changes in cellular p25 in re-

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sponse to hyperinsulinemia, and how this response may differ pathology of neurodegeneration that may be slightly different across brain regions. than the classic age-related neurodegeneration. Additionally, examining p25 accumulation in the insulin treatment group Chow et al. (2019) show that insulin induces senescence should indicate which brain region is most affected by hyperinin neurons via a CDK5-independent pathway. Conversely, Alexsulinemia. This information may give us a better understanding ander et al. (2003) demonstrated that CDK5 is actually required of the neural changes and cognitive symptoms first experienced for cellular senescence, as driven by tumour-suppressive pathby a prediabetic. ways. However, it is very possible that hyperinsulinemia drives a pathology of senescence that is independent and unconnectMore research should be conducted on CDK5 interaced to the pathologies moderated by tumour suppressors. That tions to better understand the role of this enzyme in insulinbeing said, Chow et al. (2019) found that CDK5 knockdown in- induced senescent cells. Since Chow et al. (2019) demonstrated creases the proportion of senescence in insulin-treated cells, that enzymatic activity is not required for this protein to inhibit which is the expected effect since CDK5 knockdown also reduc- senescence, it is possible that CDK5 is inhibiting other proteins es cell death. However, the authors did not provide a mecha- or participating in more transient interactions. Therefore, it is nism that can explain why blocking CDK5 activity does not reca- crucial to use a method that can detect weaker protein-protein pitulate the same effect. While this result implicates that only interactions. BioID is a relatively new screening method that is the physical presence of CDK5 is required for inhibiting senes- used to detect proteins based on proximity to the protein of cence, understanding the mechanism responsible for this effect interest (Roux et al., 2013). The first step of this method is to could point towards other proteins or pathways that modulate create a fusion protein with BirA (biotin protein ligase) and the senescence. Research on CDK5 interactions should be conduct- protein of interest. Upon providing the cell with biotin, BirA will ed to better understand this enzyme’s role in the pathology of biotinylate proteins in close proximity to the protein of interest. insulin-induced senescence. Biotinylated proteins can then be purified using streptavidin beads and identified with mass spectrometry. Performing this assay in insulin-treated cells with CDK5 as the protein of interFUTURE DIRECTIONS est would yield a list of all proteins that interact with CDK5. The The chronic insulin exposure treatment that Chow et list can be trimmed down by omitting proteins that are known al. (2019) performed could be repeated across neuronal cell to be involved with CDK5 kinase activity. The resulting list lines of different brain regions to see if p25 accumulation would include all CDK5-interacting proteins that are candidates differs across regions. The authors should focus on six brain for senescence modulation. A literature search for these proregions that are known to have metabolic or histopathologic teins’ function, cellular location, and response to insulin signalsignificance to AD: the entorhinal cortex, superior frontal gyrus, ling could reveal which proteins are most likely to be responsihippocampus, primary visual cortex, middle temporal gyrus, ble for the effect described by Chow et al. (2019). It is possible and posterior cingulate cortex (Liang et al., 2008). In a previous that some of these proteins are already known to be associated study, Liang et al. (2008) identified that CDK5 and p25 are with cellular senescence, giving a strong indication as to which differentially expressed across these regions in AD brains. Conproteins are most relevant. Finally, standard overexpression/ sequently, we can expect chronic insulin exposure to have knockdown experiments can be performed on each protein to differential effects on p25 accumulation, potentially acting in a observe its effects on cellular senescence. This experiment will region-specific manner. A control group should be WT, neugive us a better understanding of the role of CDK5 in insulinronal cell lines from each of the six brain regions, with no insuinduced cellular senescence, and it may even reveal novel prolin treatment. The treatment group, also including each of the teins that function under this pathway. six brain regions, should undergo 48 hours of chronic insulin exposure as per Chow et al.’s protocol (2019). Finally, a third group of AD neuronal cell lines should be used, without treatment. Since Chow et al. (2019) have already shown that p25 accumulation leads to neuronal death and senescence, examining the differences in p25 accumulation between the insulin treatment group and the AD group could shed more light on the connection between hyperinsulinemia and neurodegeneration. Similar changes in p25 for both groups would further support the mechanism proposed by Chow et al. While different changes between the groups would not invalidate Chow et al.’s work, this finding would indicate that hyperinsulinemia drives a 125

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Assessment of the Link between Plasma Cytokines and Rapid Eye Movement Sleep Behavior Disorder. Seyedeh Ghazal Fooladi

Rapid Eye Movement (REM) Sleep Behavior Disorder (RBD) is a neurological disorder marked by the loss of naturally occurring muscle paralysis during REM sleep. RBD is closely associated with synucleinopathies and has been hypothesized to have a synucleinopathic nature. While neurodegeneration and neuroinflammation have been reported as correlates of RBD, association of this neurological disorder with peripheral inflammation has yet to be investigated. Kim et al, were first to assess the peripheral inflammatory cytokines of patients diagnosed with idiopathic RBD (iRBD) and to analyze them in relation to patients’ clinical symptoms. They collected plasma samples from 54 iRBD patients without comorbid Parkinson’s disease or dementia, and from 56 healthy individuals. Samples were used to assess levels of interleukin (IL)-1β, IL-2, IL-6, IL-10 and tumor necrosing factor- α (TNF- α), using Meso Scale Discovery V-Plex proinflammatory panel 1 kit. RBD patients were divided into different risk factor groups based on their symptoms for motor, olfactory, autonomic and cognitive deficits as well as RBD severity. Results of the Kim et al. study demonstrated no statistically significant difference between IL-1β, IL-2, IL-6 and TNF- α levels of the patient group and healthy controls. IL-10 levels were significantly elevated in iRBD group, the difference however, did not withstand Bonferroni correction. No association was found between patients’ number of risk factors and their plasma cytokines. Overall, results of Kim et al.’s study did not support a link between iRBD and peripheral cytokines. However, their study was limited in terms of design, control for potential confounding factors, as well as the sample size. Therefore, further studies with larger sample sizes, assessing a broader spectrum of plasma cytokines, are required to determine the existence of a link between RBD and peripheral inflammation. Key words: idiopathic rapid eye movement sleep behavior disorder, synucleinopathy, peripheral inflammation, plasma cytokines

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INTRODUCTION Rapid eye movement (REM) sleep is a sleep stage characterized by rapid eye movements, vivid dreaming and temporary paralysis of skeletal muscles also known as REM atonia (Peever and Fuller, 2017). REM sleep behavior disorder (RBD) is a sleep disorder and a neurological condition marked by pathological loss of REM atonia (McKenna and Peever, 2017). Idiopathic RBD (iRBD) is a form of RBD that happens in the absence of relevant neurological disorders. Loss of atonia in RBD causes excessive motor behaviors that are often aggressive in nature and can lead to severe injuries to the patients or their bed partners. However, the most clinically concerning aspect of this disorder is that 80-90% of the patients develop either Parkinson’s disease (PD), dementia with Lewy bodies (DLB) or multiple systems atrophy, which are collectively known as synucleinopathies1. Synucleinopathies are a group of disorders characterized by accumulation of alpha-synuclein (α-syn) aggregates inside the cytoplasm of neurons and glia (Marti et al, 2003) and are associated with neurodegeneration (Visanji et al, 2016).

There are multiple lines of evidence suggesting an association between RBD and synucleinopathies, thereby providing support for the hypothesis that RBD stems from synucleinopathy in the REM atonia generating circuit of the brain. The majority of RBD patients are diagnosed with a form of synucleinopathy within 615 years of their initial diagnosis (Iranzo, et al, 2014). Postmortem analysis of the brains of patients with RBD and comorbid PD, has shown neuronal loss and α-syn pathology in the sublaterodorsal tegmental nucleus (SLD) and ventromedial medulla (vM), which are the main brain regions responsible for generation of REM atonia (Iranzo et al, 2013). Neuro-imaging studies have also shown signs of neurodegeneration in the vM and SLD of RBD patients (Garcia-Lorenzo et al, 2013; Boucetta et al, 2016). The idea of α-syn pathology causing RBD is consistent with Braak’s staging model of PD. This model posits that synucleinopathy starts in the gut, ascends through the vagus nerve and passes the brain stem where the REM atonia generating circuit is located before reaching substantia nigra and causing the classical PD symptoms (Peever and Fuller, 2017).

Neuroinflammation has been shown to be an important factor in initiation and progression of various neurodegenerative diseases including synucleinopathies (Kempuraji et al, 2016). Neuroinflammation and glial activation are known to both contribute to and be caused by neuronal death which is a robust correlate of synucleinopathies (Kempuraji et al, 2016). Pathogenic αsyn aggregates have proinflammatory activity and can activate glia (Wang et al, 2015; Grimming et al, 2016). Glial activation has been detected in the brains of PD patients as well as the animal models of PD (Wang et al, 2015). Studies have shown that there is a bidirectional communication between the peripheral immune system and the brain (Kempuraji et al, 2016), and that the systemic and neuro-inflammation are related (Perry et al, 2007). Moreover, presence of pathogenic α-syn has been detected in the periphery of patients with synucleinopathy ( Mollenhauer et al, 2011; Malek et al, 2014; El-Agnaf,

2006), and administration of α-syn fibrils has been shown to stimulate cytokine release from peripheral monocytes (White et al, 2018). Studies have also demonstrated peripheral inflammation in both PD and DLB patients (Williams’Garry et al, 2016; Surendranathan et al, 2018).

The above-mentioned studies provide evidence for associations between RBD and synucleinopathic neurodegeneration, neurodegeneration and neuro- and systemic inflammation, as well as synucleinopathy and peripheral inflammation. Therefore, it is reasonable to hypothesize that iRBD patients exhibit signs of systemic inflammation, which is related to prodromal symptoms of α-syn pathology. Kim et al (2019) were first to examine plasma inflammatory cytokines of iRBD patients and to assess them in relation to patients’ clinical symptoms. Results of their study showed no relationship between iRBD and peripheral cytokines which could potentially be due to their limited sample size or exclusion criteria. their study opens a new line of investigations that could have a significant impact on the understanding and management of iRBD and synucleinopathies. Detection of systemic inflammation in iRBD patients can provide evidence for a possible role of peripheral inflammation in RBD progression and may have diagnostic potential with regards to the stage of synucleinopathy. MAJOR RESULTS Kim et al. compared venous plasma cytokines of 54 iRBD patients without comorbid PD or dementia, with that of 56 age and sex matched healthy controls. The assessed cytokines included interleukin (IL)-1β, IL-2, IL-6 and tumor necrosing factorα (TNF- α), which are proinflammatory, as well as IL-10 which is an anti-inflammatory cytokine. While results of their study showed no significant difference between proinflammatory cytokine levels of iRBD patients and that of healthy controls, IL10 levels were significantly elevated in the patient group (Fig 1). The IL-10 difference, however, did not withstand Bonferroni correction.

Figure Adopted from Kim R. et al. (2019), Movement Disorders. DOI: 10.1002/ mds.2784. Figure 1. Comparison between plasma cytokines of iRBD patients with that of healthy controls. Out of all assessed cytokines, only IL-10 levels were significantly elevated (P = 0.022). Cytokine levels are expressed on a logarithmic scale.

Kim et al. also divided iRBD patients into different groups, based on their risk for neurodegeneration. Clinical assessments focused on levels of motor, olfactory, autonomic and cognitive

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deficits as well as RBD severity, were performed for stratifica- showed no increased inflammation associated with higher tion of patients into different risk factor groups. Contrary to number of risk factors. their initial hypothesis, plasma cytokines were not correlated with clinical variables. CRITICAL ANALYSIS Results of the Kim et al study were not consistent with their initial hypothesis and with the available literature. No significant elevation was observed in inflammatory cytokines of iRBD patients and no link was shown between peripheral inflammation and the stage of iRBD. The most important limitation of this study was its exclusion criteria. No assessment was made regarding the disease states of participants or the use of medications with potential to alter plasma cytokines, such as antiinflammatory or immunosuppressant drugs. All conclusions made in this study rely heavily on the assumption that changes in plasma cytokines are solely due to RBD and its implications. Therefore, infections or intake of drugs with anti or proinflamFigure Adopted from Kim R. et al. (2019), Movement Disorders. DOI: 10.1002/ matory effects can act as confounding factors and affect the mds.2784. results. Figure 2. Comparison between plasma cytokines of iRBD patients with different number of risk factors. No association was found between plasma cytokines and clinical symptoms.

DISCUSSION Results obtained by Kim et al rejected all of their initial hypotheses. There was no significant difference between proinflammatory cytokines of iRBD patients and that of healthy individuals. However, a trend towards elevated IL-10 levels was observed in the patient group. While there are no other studies assessing serum inflammatory cytokines of this specific patient population, a study conducted by Mondello et al (2018) illustrated increased levels of C-reactive protein (CRP) in the serum samples of iRBD patients. CRP is a plasma protein whose concentration increases in response to inflammation and is used as a marker for inflammatory states in the clinic (Black et al, 2004). Previous studies have demonstrated that CPR’s expression is controlled by IL-6 and IL-1β (Kushner et al, 1995). Since neither of those cytokines were increased in the iRBD group of Kim et al.’s study, it can be concluded that results obtained by Kim et al are not consistent with those obtained by Mondello. While the authors were unsure of the reason behind lack of an increase in the assessed proinflammatory cytokines of iRBD patients, they proposed that the elevated IL-10 levels could be due to compensatory mechanisms against inflammation induced by α-syn accumulation.

As mentioned above, onset of synucleinopathy can range from 6 to 15 years after the RBD diagnosis, which highlights the heterogenous nature of synucleinopathies’ progression. This heterogeneity might account for lack of an association between peripheral inflammation and iRBD, observed in the study. Since samples were only collected once in the study, there remains the possibility of peripheral inflammation having a role in RBD neurodegeneration and progression at a different time and stage of the disease. However, assessment of the plasma cytokines of iRBD patients with different number of risk factors

Clinical examination of the study participants revealed that overall, 73% of the iRBD patients used either clonazepam, melatonin, or both. While effects of clonazepam on plasma cytokines is not yet clear, melatonin has been shown to have antioxidant and anti-inflammatory properties (Chahbouni et al, 2010; Favero et al, 2017). However, additional analysis done by Kim et al, showed that there was no significant difference between plasma cytokines of patients receiving melatonin, those who did not and the healthy controls. Both clonazepam (Fantini, 2012) and melatonin (McGrane, 2015) alleviate symptoms of RBD (St Louis and Boeve, 2017). Therefore, although use of melatonin by the patient group was not likely to significantly affect the results in terms of cytokine levels, use of either of the drugs could have potentially affected the risk factor score. Both type and dosage of the RBD drugs should have been controlled for.

Cross sectional design of this study can also be considered a limitation. Since progression of synculeinopathies can vary widely between individuals, it is highly important to assess disease symptoms and inflammatory cytokines on an ongoing basis and to use both within and between subject designs. Meso Scale Discovery V-Plex proinflammatory panel 1 kit was used for plasma cytokine analysis. This kit had been successfully used by other studies before (Kerrin et al, 2017). However, Kim et al were unable to detect IL-1β and IL-2 in 6 and 4% of the participants respectively, which biased the final results of their study. Use of an alternative tool with higher sensitivity such as Impracer (Adler et al, 2009) could have resolved this issue. While the assessed cytokines were selected based on the results of studies assessing plasma cytokines of PD patient, they might not have captured the RBD’s immune response. Extending the analysis to include other inflammatory markers such as interferon gamma, or IL-17A, which are also elevated in PD patients (Mount et al, 2007; Green et al, 2019) might have

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been helpful. The low statistical power which was due to the small sample size can be considered another factor limiting Kim et al.’s study.

system stimulation would experience a faster progress of disease.

FUTURE DIRECTIONS While Kim et al.’s study failed to provide evidence for a link between peripheral inflammation and iRBD, significance and potential applications of such a link justify more studies in this field. Further studies on human subjects, that assess a broader spectrum of plasma cytokines and incorporate regular follow ups can help determine whether there is a link between peripheral inflammation and RBD. Patients using medications with potential for altering plasma cytokines should be excluded and neuroimaging should be done to assess the extent of cell loss in the vM and SLD. The neuroimaging results should be taken into consideration when assigning patients to different risk factor groups. While a major limitation of Kim et al.’s study was lack of control for confounding factors such as medications used by the participants, both Kim et al. and Mondello et al (2018) studies suffered from a small sample size. Stricter exclusion criteria further limit the sample size and thereby lower the statistical power. Use of animal models of RBD may be a potential solution to that problem. The brain circuit responsible for generation of REM sleep atonia has been extensively investigated and SLD and vM have been shown to be the main regions involved (Peever and Fuller, 2017). Synucleinopathy can be induced in specific regions of the brain using viral vectors carrying either wild type or mutated forms of α-syn gene (Visanji et al, 2016; Conway et al, 2000). Therefore, animal models of RBD can be generated by inducing synucleinopathy in their SLD and vM. These models allow for assessment of the peripheral inflammation as a consequence of RBD, and of its effect on RBD severity and synucleinopathy progression. Plasma cytokines should be compared between animals with α-syn pathology in their REM circuit and controls. Disease severity can be compared between animal models who receive anti-inflammatory drugs, those whose peripheral immune system has been activated to release cytokines, and control models of RBD. Kim et al did not find a link between RBD and plasma cytokines and no study has yet discovered whether peripheral inflammation plays a role in pathogenesis of iRBD. However, the illustrated inflammation in the periphery of patients with PD and DLB, makes it reasonable to hypothesize that mice with α-syn pathology in their REM atonia circuit will show elevated levels of plasma cytokines. The positive association between concentration of certain plasma cytokines and severity of PD (Green et al, 2019) and the potential use of inflammatory pathways as therapeutic targets for PD (Want et al, 2015), make it reasonable to hypothesize that animals receiving anti-inflammatory drugs would experience a slower disease progression compared to controls, and that those who are subject to peripheral immune

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The Neurobiological Role of Mifepristone as a Potential Treatment for Anorexia Nervosa Mâ&#x20AC;&#x2122;Kayha Fortella

This review focuses on the drug mifepristone and its efficiency as a potential treatment for weight restoration in anorexia nervosa (AN). The study consisted of rats divided into three groups: rats on dietary restriction (control), rats injected with methylphenidate (MET group) and rats injected with methylphenidate and mifepristone (MET + Mif group). Rats were injected with 5 mg/kg/day of methylphenidate to induce a 15-25% weight loss through hyperactivation of the HPA axis. The MET + Mif group showed an increase in mean body weight, closer to the control rats body weight; however, food intake was not affected. Additionally, the MET + Mif group expressed lower levels of the POMC and adiponectin receptors than the MET group. The mifepristone allowed rats to lower their plasma adiponectin concentrations and increase their adipose mass, thus, increasing their body weight through changes in receptor expression in the cerebral and insular cortex. Future studies are required to determine if mifepristone has the same effect on weight restoration in individuals that suffer from both AN and depression. Key words: anorexia nervosa (AN), methylphenidate (MET), mifepristone (Mif), POMC receptor, adiponectin receptor, glucocorticoid receptor, Hypothalamus-Pituitary-Adrenal (HPA) axis, insular lobe, weight restoration

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BACKGROUND/ INTRODUCTION Anorexia Nervosa (AN) is a potentially fatal eatingdisorder that involves extreme dieting and results in the inability to maintain a normal body weight (Yager and Anderson, 2005). AN is a multifactorial disease because it has both a genetic component and environmental component. Recent studies have shown that 50-80% of AN cases are due to genetic heritability, although no genes have yet to be linked to the cause of this disorder (Oberndorfer et al., 2013). The main goal of most AN patients is to sustain a low body weight. A study by Kaye et al., (2013) indicate that areas of the forebrain vital for reward, mood and motivational drives may be dysregulated during AN, resulting in altered eating patterns. It is suggested that eating patterns are associated with the mood of the individual; hence, individuals with AN tend be depressed which correlates with a decreased appetite and weight loss (Eckert et al., 1982). Ultimately, eating food or thinking about food raises anxiety levels in individuals with AN. Changes in the brain have been implicated in many eating disorders. It has been hypothesized that the anterior insular lobe assists in the maintenance of AN (Kim et al., 2012). Functions of the insular lobe include hand control, swallowing, gastric motility, food intake, food imagination, satiety, odor and taste aversion learning and self-image perception (Mutschler et al, 2009). Similarly, the hypothalamic-pituitary-adrenal (HPA) axis, a key regulator during stress, has been shown to play a role in AN development and maintenance. The clinical symptoms of AN, including abnormal temperature, abnormal growth hormone release and abnormal eating, are due to a dysregulated pituitary and hypothalamus (Licinio et al., 1996).

Rats exposed to mifepristone had an increase in mean body weight (figure 1). Initially, the MET + Mif group displayed a weight loss similar to the MET group of 15-25% weight loss (Khalil et al., 2018); however, once the mifepristone was introduced â&#x20AC;&#x201C; around 40 days into the intervention â&#x20AC;&#x201C; there is an increase in their body weight that resembles the control group rather than the MET group whom had the smallest average weight after the intervention phase.

Figure 1: A plot graph depicting the change in body weight over time in control rats, MET rats and MET + Mif rats. Food intake increased in the treatment groups vs control group Dietary food intake remained highest in the MET mice and MET + Mif mice compared to the control mice at the end of the experiment (Figure 2). These results were statistically significant.

Mifepristone (RU486) is a synthetic steroid that functions as a glucocorticoid and progesterone receptor antagonist (Khalil et al., 2018). A study by Kling et al. (1993) used mifepristone in a randomized clinical trial (RCT) to test its effects on AN. The participants were divided into two groups: healthy weight individuals (control) and underweight individuals (AN group). Prior to intervention, the AN group displayed higher cortisol levels compared to the control group. After mifepristone administration, the cortisol levels in AN individuals was much lower compared to controls. This suggests that mifepristone can be Figure 2: Food consumption in control mice, MET mice a possible treatment in AN via reducing HPA axis hyperactivaand MET + Mif mice. tion through reduction of cortisol levels; however, research is Figure Adapted from Khalil et al. Neuroscience Restill required to determine if it influences weight restoration in search, 135, 46-53. AN. The paper discussed in this review, by Khalil et al. (2018), focuses on determining whether weight loss induced by HPAaxis hyperactivation and the insular lobe during AN can be reversed using mifepristone. The experiment consisted of three Mifepristone lowered Adiponectin and Pro-opiomelanocortin groups: control, MET group (5 mg/kg/day of methylphenidate) (POMC) receptors expression and increased expression of Proand the MET + Mif group (5 mg/kg/day of methylphenidate and gesterone and Glucocorticoid receptors 10 mg/kg/day dosage of mifepristone). The expression of receptors in the cerebral cortex and MAJOR RESULTS insular lobe varied between the controls, MET group, and MET + Mif group (Figure 3). The MET group displayed the highest Mifepristone increased mean body weight level of POMC receptor expression, while the control group and MET + Mif group expressed lower levels. The MET + Mif group 135

displayed the highest glucocorticoid and progesterone receptor expression, followed by the MET group expressing the next highest number of these receptors. The MET group expressed the greatest adiponectin gene receptors, while both the control and MET + Mif group expressed lower levels. Both the MET group and MET + Mif group expressed high levels of TNF-alpha receptors.

Figure 4: A graphic map displaying how the hypothalamus and insula contribute to the effects of AN (blue) and mifepristone reverses these effects (red). Figure Adapted from Khalil et al. Physiology & Behaviour, Figure 3: Gene expression of POMC, glucocorticoid, progesterone, adiponectin and TNF-alpha receptor in the control, MET, MET + Mif groups. Figure Adapted from Khalil et al. Neuroscience Research, 135, 46-53.

surgery, researchers saw an inverse relation between plasma adiponectin concentrations and changes in anthropometric values, such a BMI and hip circumference. Specifically, the researchers recorded that a 21% decrease in BMI was associated with a 46% increase in adiponectin levels (Yang et al., 2018). Therefore, a decrease in weight results in increased adiponectin plasma concentrations. Thus, adiponectin acts on the hypoCONCLUSION/ DISCUSSION thalamus to initiate the increased release of CRH (Qi et al., 2004); however, mifepristone, inhibits increased adiponectin Based on the results from the study, Khalil et al., levels in the brain; thereby, reducing hypothalamic release of (2018) conclude that mifepristone can be a possible treatment CRH and promoting weight gain. for reversal of AN. Mifepristone induces neurobiological changes in the rat brain via reduction of the HPA axis, resulting in a POMC is vital for hunger and satiety regulation and decrease in gene expression of two receptors, POMC and adi- reflects ACTH gene expression in the brain. ACTH is one of the ponectin, normally elevated in expression in anorectic mice. eight peptides that form from POMC processing (Ehrlich et al., Thus, the reduction of these receptors is enough to induce 2010). Since HPA axis activity involves increased ACTH and mifweight recovery in these mice through upregulation of adipose epristone reduces HPA axis activity, this statement validates mass. In a prior study by Khalil et al. (2017), the authors re- the MET + Mif group displaying reduced POMC receptor exvealed that the active HPA axis interacts with the insular lobe to pression. create the emotional and behavioural aspects of anorexia nervosa (figure 4). Normally, in AN, the hypothalamus acts on the The authors recorded high levels of progesterone and insular lobe to induce a decreased appetite due to disgusts to- glucocorticoid receptor expression in the MET + Mif group. wards self-image. Additionally, the hypothalamus will release Stress activates the HPA axis resulting in elevated plasma glucocorticotropin-releasing hormone (CRH) which stimulates the corticoids, such as cortisol, which bind to the glucocorticoid pituitary gland to release adrenocorticotropic hormone (ACTH). receptors (Oomen et al., 2007). Methylphenidate administraThe increased levels of ACTH will act on the adrenal gland to tion induces stress, consequently, increasing glucocorticoid increase glucocorticoids. All these neural changes contribute to receptor expression. Although the MET + Mif group display a decreased appetite and decreased adipose mass resulting in a increased glucocorticoid receptor expression, the mifepristone lower body weight in anorectic individuals. binds to these receptors, preventing cortisol from binding to the receptor; thus, preventing weight loss in these mice (Khalil et al., 2018; Oomen et al., 2007). Similarly, the increase in proOne of the major findings in this study was mifepristone gesterone receptor expression seen in the Met + Mif group is lowered adiponectin receptor expression at the insular and blocked by mifepristone. Mifepristone blocks the binding of cerebral level (Khalil et al., 2018). The mifepristone adminis- certain progesteroneâ&#x20AC;&#x2122;s to these receptors, thereby, inducing tered to mice enabled them to decrease their adiponectin plas- weight gain (Khalil et al., 2018). ma concentrations, which is a hormone secreted by adipose tissue. Similarly, a study by Yang et al. (2001) focused on deterAlthough mice in the MET group exhibited decreased mining the biological link between plasma adiponectin levels body weight gain, their increased food intake results following and obesity after gastric bypass surgery. After gastric bypass methylphenidate administration were shocking and did not 136

coincide with the results from prior studies. For example, the study by Elfers and Roth (2011) examined the impact of methylphenidate on hypothalamic obesity. Their results indicated methylphenidate injection results in a significantly decreased food intake in obese rats (figure 5). A possible explanation for the opposing results obtained by each study is the dose of methylphenidate used in the two experiments, as 20 mg/kg/ day was used in the study by Elfers and Roth, while only 5 mg/ kg/day was used in the present study.

play in these mice that’s counteracting the normal activity of mifepristone. Thus, before mifepristone administration for AN, it is important to determine the status and medical history of the person receiving it to ensure that the drug functions the way researchers proposed for it to function. FUTURE DIRECTIONS

It is known that anorectic individuals are severely underweight, and this is primarily due to a decreased appetite; however, in the following study the mice administered methylphenidate did not decrease their food intake. Future studies should be conducted on comparing different levels of methylphenidate and average food intake to see if there is a specific methylphenidate dosage that contributes to an animal induced AN model. For example, the experiment should consist of giving some animals in the study 10 mg/kg/day and other animals 15 mg/kg/day and comparing over a 30-day period. If the rats receiving the 15 mg/kg/day dosage decrease their food intake, it validates this dosage as appropriate for creation of an AN animal model via decreased food intake. Also, future studFigure 5: Average food intake (FI) in obese rats exposed ies should focus on discerning the effects of mifepristone in to methylphenidate versus control rats. depressed, anorectic individuals. If the drug functions how it WHITE bar – obese rats without MET; BLACK bar – obese would in normal AN, results will show a weight increase; howrats with MET. ever, if it functions like how it would in depression, results will Figure Adapted from Elfers and Roth. Front. Endocrinol., 2, show a weight decrease. Nonetheless, these results will show 2-5. whether mifepristone is a more suitable treatment for depression or anorexia. Additionally, future experiments on lesions in the insular cortex of mice or human patients can be done to CRITICAL ANALYSIS determine if this is enough to induce AN behaviour, potentially offering another method of an induced-AN model. If ablation of The authors concluded mifepristone could be a possi- the insula results in AN behaviour, it confirms the insula’s role ble treatment for AN through stabilizing the HPA axis and in- in causing this disorder, thus, providing a potential target area ducing weight gain through increased adipose mass. Focusing for treatment. on the cause of a disorder or disease is important because it provides a reason behind the diseases occurrence and makes it easier to prevent its appearance as researchers now have a known target of the drug. Thus, a study by Lawson et al. (2013) exert that mifepristone disrupts the maintenance of AN rather than targeting the cause. Another vital factor the authors should have examined is the effect of mifepristone in individuals that suffer from depression and AN coincidingly. Individuals who suffer from eating disorders also tend to suffer from depression. In comparison to eating disorders, brain images of a depressed individual display increased glucocorticoid and progesterone receptor expression, indicating that the person is experiencing stress. Hence, mifepristone may be associated with the treatment of symptoms of depression relating to AN symptoms. However, the effects of the drug in anorectic rats versus depressed rats may differ from how it works in rats suffering from both depression and AN. In the study by Beebe et al. (2006), the authors examine the effects of mifepristone on weight gain in rats that are currently on the antipsychotic, olanzapine. The results of this study show that mifepristone counteracts the activity of olanzapine in these rats, resulting in a decreased weight gain. This is important because it reveals that something else is at 137

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Beebe, K.L., Block, T., DeBattista, C., Blasey. C., and Belanoff, J.K. (2006). The efficacy of mifepristone in the reduction and prevention of olanzapine-induced weight gain in rats. Behav. Brain Res., 171(2), 225-229. doi: https://doi.org/10.1016/ j.bbr.2006.03.039 Eckert, E.D., Goldberg, S.C., Halmi, K.A., Casper, R.C., and Davis, J.M. (1982). Depression in anorexia nervosa. Cambridge University Press, 12(1), 115-122. doi: https://doi.org/10.1017/S003329170004335X Elfers, C.E. and Roth C.L. (2011). Effects of methylphenidate on weight gain and food intake in hypothalamic obesity. Front. Endocrinol., 2, 2-5. doi:10.3389/fendo.2011.00078 Ehrlich, S., Weiss, D., Burghardt, R., Infante-Duarte, C., Brockhaus, S., Muschler, M.A., Bleich, S., Lehmkuhl, U., and Frieling, H. (2010). Promoter specific DNA methylation and gene expression of POMC in acutely underweight and recovered patients with anorexia nervosa. J. Psychiat. Res., 44(13), 827-833, doi:https://doi.org/10.1016/j.jpsychires.2010.01.011 Kaye, W.H., Wierenga, C.E., Bailer, U.F., Simmons, A.N., and Bischoff-Grethe, A. (2013). Nothing tastes as good as skinny feels: the neurobiology of anorexia nervosa. Trends Neurosci., 36(2), 110-120. doi: https://doi.org/10.1016/ j.tins.2013.01.003 Khalil, R.B., Smayra, V., Saliba, Y., Hajal, J., Bakhos, J.J., Souaiby, L., Richa, S., Tamraz, J., and Farès, N. (2018). Mifepristone reduces hypothalamo-pituitary-adrenal axis activation and restores weight loss in rats subjected to dietary restriction and methylphenidate administration. Neurosci. Res., 135, 46-53. doi: 10.1016/j.neures.2017.12.008 Khalil, R.B., Souaiby, L., and Farès N. (2017). The importance of the hypothalamo-pituitary-adrenal axis as a therapeutic target in anorexia nervosa. Physiol. Behav., 171, 13-20. doi: ttp://dx.doi.org/10.1016/j.physbeh.2016.12.035 Kim, K.R., Ku J., Lee J.H., and Jung Y.C. (2012). Functional and effective connectivity of anterior insula in anorexia nervosa and bulimia nervosa. Neurosci. Lett., 521(2), 152-157. doi: 10.1016/j.neulet.2012.05.075 Kling, M.A., Demitrack, M.A., Whitfield Jr., H.J., Kalogeras, K.T., Listwak, S.J., DeBellis, M.D., Chrousos, G.P., Gold, P.W., and Brandt, H.A. (1993). Effects of the glucocorticoid antagonist RU 486 on pituitary-adrenal function in patients with anorexia nervosa and healthy volunteers: enhancement of plasma ACTH and cortisol secretion in underweight patients. Neuroendocrinol., 57, 1082–1091. doi: 10.1159/000126474

Lawson, E.A., Holsen, L.M., DeSanti, R., Santin, M., Meenaghan, E., Herzog, D.B., Goldstein, J.M., and Klibanski, A. (2013). Increased hypothalamic-pituitary-adrenal drive is associated with decreased appetite and hypoactivation of food motivation in anorexia nervosa. Eur. J. Endocrinol., 169(5), 639-647. doi:10.1530/EJE-13-0433

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Usage of Botulinum Toxin in Understanding Facial Feedback

Chanel Fu

Emotions are a hallmark of human experience. The process of an emotional experience and emotional understanding is thus an area of current research in psychology and neuroscience. Since its proposal, a popular theory of emotional processing, the Facial Feedback Hypothesis, has continued to develop. The hypothesis separates different processes in the experience of emotion such as distinct neural networks for each emotion and the effect of depth of processing. As such, the mechanism of proprioceptive facial feedback and its influence on perception of emotions is an area of contention. Inhibiting the contraction of certain muscles in facial expressions via botulinum toxin injection is one method used to isolate the effects of facial feedback. The present study furthered botulinum toxin studies by injecting botulinum toxin to inhibit the contraction of muscles used in furrowing the brow in human subjects. The study used a pre- post-treatment design with a dynamic change detection paradigm of gradually changing faces. Participants indicated when they detected a change in the facial expression. Both the control and treatment group detected the change in happy faces faster in the second session and the control group detected the change in angry faces faster in the second session. The treatment group did not show a decrease in reaction time to angry faces in the second session. This suggests that the inability to contract the muscle affects the ability of the treatment subjects to improve at the task for the anger condition only, and that facial feedback is specific to the muscle and emotion pairing. These results further the understanding of the physiological process of emotional perception and processing. Key words: botulinum toxin, emotion, anger, Facial Feedback Hypothesis, change detection, emotion perception

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The psychological and physiological origins of emotion have long been theorized, and more currently, studied empirically. In the 1800s, Charles Darwin proposed that expression of an emotion will intensify its experience, and suppression would dull it. Later, Fritz Strack and his co-researchers profoundly changed the field of emotion research with the Facial Feedback Hypothesis (Strack, Martin, & Stepper, 1988). They proposed the Facial Feedback Hypothesis after their study found that inhibiting a smile resulted in a less intense response to humour, whereas encouraging a smile by holding a pen in the mouth more intense response. Though other studies have failed to replicate their findings, the idea that physiological affect modulates emotional processing persists through other anecdotal and experimental evidence (Wagenmakers et al., 2016). The field has become more subdivided since the Facial Feedback Hypothesis was proposed. Emotional contagion, the concept that one person’s emotions can be transmitted to another subconsciously, and facial mimicry, describing matching facial expressions in another person are separate terms that are considered two facets of understanding emotional processes

(Alam, Barrett, Hodapp, & Arndt, 2008). In more current laboratory research, there are a number of methods being used to understand how emotion is experienced. Neuroimaging studies, for example, have suggested that perceiving and experiencing emotion have overlapping neural bases. Experience, perception, and retrieving emotions and memories of emotions involve similar and overlapping neurons (Niedenthal, 2007). Furthermore, the amygdala was activated when participants were presented angry faces versus happy faces (Kim et al., 2014). Experiments have also been conducted using botulinum toxin, commonly used for cosmetic treatment and known as Botox. Botox temporarily inhibits acetylcholine signalling thus facial muscles are unable to flex (Hambleton, 1992). By selectively inhibiting facial muscles, researchers aim to isolate the effect of facial feedback on various emotional processes. It has been shown that activation of the left amygdala was reduced in botulinum toxin treated individuals upon imitating an angry face compared to control subjects (Hennenlotter et al., 2009). Sentence reading, a more behavioural approach to understanding emotion, showed that Botox patients rated slightly emotional sentences and facial expressions less emotional than before their Botox treatment (Baumeister, Papa, & Foroni, 2016). Reading time was also slowed when botulinum toxin was inject-

ed in a region that is required to convey the emotion evoked by a sentence (Havas, Glenberg, Gutowski, Lucarelli, & Davidson, 2010). Davis and colleagues (2010) injected Botox in the glabellar region, involved in furrowing the brow, as well as the orbicularis oculi muscles involved in ‘laugh lines’ in some participants, and hyaluronic acid, a filler with no effect on muscle contraction, in the control participants. They found using a pre- posttreatment design that participants with the Botox treatment rated the intensity of their emotional experience less strongly from Session 1 to Session 2 after watching video clips (Davis, Senghas, Brandt, & Ochsner, 2010). Another botulinum toxin study revealed that compared to matched controls who had dermal fillers that do not impair muscle activation, Botox recipients were less accurate in identifying emotions using a ‘Reading the Mind in the Eyes Test’ design (Neal & Chartrand, 2011). Apart from understanding facial feedback, botulinum toxin studies can be adapted clinically. As suggested by Kruger and Wollmer (2015) and Coles, Larsen, Kuribayashi, and Kuelz (2019), the use of Botox may also be used in treatments of depression. The main paper in focus by Bulnes and colleagues (2019), similarly, used botulinum toxin to inhibit facial muscles to investigate how it affects reaction time on a task pre- and posttreatment. However, the injection of Botox was limited to the corrugator supercilii muscle of the glabellar region, which is necessary to express anger via furrowing the brow, but not happiness (Jäncke, 1996). This addressed the potential confound of inhibiting other facial muscles, an issue in previous studies noted by the authors. Furthermore, unlike other Botox studies on emotion, the task did not rely on higher mental processes of emotion such as comprehension nor introspection. By using gradually changing displays of faces from neutral to either angry or happy, the investigators targeted short term cognition rather than semantic information retrieval, while also introducing the concept of temporal change in emotional detection (Bulnes, Mariën, Vandekerckhove, & Cleeremans, 2019). This study hence furthers research in emotional experience by isolating muscular mechanisms and introducing other forms of testing that may be more indicative of how emotional processes occur. MAJOR RESULTS Participants were seated in front of a computer display and shown a face which gradually changed from neutral to either anger or happiness. They were instructed to press a button to indicate when they detected a change in the emotion of the face. This task was completed during two sessions. The treatment was given several weeks before the second session. The study found a significant decrease in the reaction times from Session 1 to Session 2 in both groups for the detection of happiness, and in the control group for the detection of anger. The treatment group did not exhibit a significant decrease in reac-

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tion times from Session 1 to Session 2 in both groups for the detection of happiness, and in the control group for the detection of anger. The treatment group did not exhibit a significant decrease in reaction time for anger. Furthermore, both groups showed improvement in identification accuracy from Session 1 to Session 2 and were more accurate identifying happiness overall. Taken in consideration with pre-existing literature, this suggests there are two routes to emotional perception – one that is changed with Botox treatment which is primarily relying on facial mimicry, and other that is semantically driven that was not investigated in this paper by Bulnes and colleagues (2019).

Figure 1. Reaction times for indicated detection of change in emotion in both treatment and control groups across both sessions, taken from original article. Figure adapted from Bulnes, L. C., Mariën, P., Vandekerckhove, M., & Cleeremans, A. (2019). Scientific Reports, 9(1), 1–13.

DISCUSSION The authors found that Botox treated subjects did not improve on the change detection task when the display showed anger, unlike their performance on happiness. The control subjects showed improvement in reaction time across both emotions. This finding is significant in understanding how facial feedback may be involved in perceiving emotion in another individual and the experience of emotion more broadly. The results are congruent with other botulinum toxin studies that inhibited muscles involved with a negative emotion versus a positive emotion in that the response to the negative emotion was affected by Botox (Baumeister et al., 2016; Davis et al., 2010). More precisely, the study was specific to the corrugator supercilii muscle, unlike previous studies which were less precise in Botox application, and thus demonstrated a more specific relationship between the corrugator muscle and anger. Bulnes and co-authors (2019) proposed a dual-route mechanism to processing other people’s facial expressions. They extrapolated that one route in facial feedback is experience independent, which is impacted by deafferentation of motor movements and therefore was impaired with botulinum toxin. The other route is built upon semantic knowledge of emotions which was not impelled in the task. As a novel proposal, the authors suggest that facial feedback may be compared to visual information and are integrated for the perception of emotion. This is influential to the field of emotion psychology and neuroscience because it provides insight to the mechanism of facial feedback and what aspects of emotional perception are specifically affected by facial feedback. These findings may be the basis of further investigation to explore the neural bases of facial feedback.

CRITICAL ANALYSIS The study by Bulnes and colleagues (2019) contributes to exploring further dimensions of emotional perception that have not been previously studied. Specifically, the authors used a change detection paradigm which prohibited participants from using semantic information to guide their reactions, unlike the sentence reading design used by Baumeister et al. (2016) and Havas et al. (2010). Using dynamic stimuli is important to this field because emotional perception occurs across time, and it is an important factor that is involved in how one perceives emotion. Furthermore, because the design requires participants to react to a change but not the nature of the change, it does not rely on explicit mental representations of emotion but rather implicit sensitivity to changes. Bulnes and colleagues (2019) introduced temporal effects on emotional perception of another person – a new factor to the field of emotional processing. However, in their design there are a few considerations which may be of concern in drawing conclusions about the mechanism in which emotional perception occurs. As noted in their research, deafferentation effects may be due to the general inability to furrow the brow or the impossibility to mimic the expression on the display, so it is unclear whether the lack of facial movement was the cause of the observed result or the blocked afferent signal. Also, the injection of botulinum toxin was not controlled with subjects who also underwent a cosmetic procedure. Bulnes et al. (2019) argued that the pre- post-treatment design allowed for each group to be compared to itself. However, there may be attributes of the individuals who chose to have the cosmetic procedure which may affect their reaction to emotion detection that are not considered, therefore participant recruitment is a limitation in this study. Although the existing research shows similar effects to Bulnes et al. (2019) using both used non-Botox cosmetic treatments such as fillers and no treatments, placebo effects remain a confound to the results (Baumeister et al., 2016; Davis et al., 2010; Havas et al., 2010; Hennenlotter et al., 2009). The researchers defend this choice as the design measured implicit mentalization of the emotion. In terms of further areas of study, the authors should consider testing using the same change detection paradigm with other emotions, especially sadness and fear as it also involves the corrugator supercilii muscle and therefore the extent to which the corrugator supercilii muscle is involved in the perception of these emotions may be explored (Davis et al., 2010). Isolating the eyes and eyebrows for the design may also be worth consideration to separate the facial feedback mechanism in eye expressive muscles. Eyes are especially salient features for the detection of fear, but the saliency in anger is yet to be determined (Elsherif, Saban, & Rotshtein, 2017).

FUTURE DIRECTIONS The use of botulinum toxin in emotion research studies is to selectively inhibit certain muscles that are known to contribute to facial expressions. However, a general limitation with botulinum toxin studies is the concern about proper control subjects and conditions. Bulnes et al. (2019) identified the injection of fillers in Neal and Chartrand (2011) as a potential confound, but also did not have a control that underwent a cosmetic procedure which may induce placebo effects in the Botox treated individuals. The authors should

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therefore consider a design in which all participants seek Botox treatment. Participants would then receive either a Botox injection or a saline injection between sessions and be informed that they may not be receiving Botox upon registering for the study. After the experiment is complete, all participants who received saline will receive Botox to ensure they receive the treatment. This design addresses the concern regarding the effect of an injection and the possibility that those who seek a Botox treatment may be predisposed to altered emotion detect is by only recruiting participants who seek the treatment. Based on the current research, it is expected that a similar result would occur in that the participants who received Botox would not show the improvement effect in anger that is shown in happiness and in the controls for both emotions. If the results of Bulnes et al. (2019) are not replicated using a saline control, it may highlight an issue in the assumption that the botulinum toxin is effective in deafferentation. Alternatively, the saline controls may also be indicative of placebo effects in facial feedback, in which further studies could be done. Theoretically, if the participants with saline demonstrate a placebo effect, then the two-route mechanism suggested by Bulnes et al. (2019) would be under contention as the belief of hindered facial feedback should not affect the implicit recognition of the task the authors designed.

choline but TMS would prevent the excitation of the afferent neuron, this could help clarify which part of the facial feedback pathway is relevant for observing the results in Bulnes et al. (2019). Further studies to then analyze the important of timing in this pathway could then be conducted. Emotional perception research is promising as future tools are developed.

It is also possible that the injection of any solution that creates pressure on the facial muscles may have an unexpected effect on facial feedback. Therefore, another proposal to sever the pathway of facial feedback is to use transcranial magnetic stimulation (TMS) to selectively inhibit the contraction of certain muscles. TMS is a noninvasive procedure that is relatively new and has not yet been used in facial feedback research. It can be used to selectively inhibit facial muscles motor production to map language production areas (Weiss Lucas et al., 2019). Unfortunately, representational topography of facial musculature has yet to locate the area of the motor cortex that may relate to the corrugator supercilii (SĂ¤isĂ¤nen et al., 2015). When further knowledge of cortical mapping of the facial muscles is developed, TMS could be a powerful tool for the researchers using the change detection paradigm. TMS may be used in lieu of botulinum toxin to inhibit afferent signaling, thus permitting the researchers to recruit participants from a greater portion of the population. Furthermore, using TMS would eliminate the confound due to the effect of the cosmetic procedure, and could be used as verification of the results found by Bulnes et al. (2019). A study using TMS could entail a similar design to Bulnes et al. (2019) with pre- post-treatment but participants could be split into three separate groups. The control group could have a limb muscle inhibited that would not interfere with the task, and one treatment condition could have the corrugator supercilii inhibited (Group A) and the other treatment group could have the zygomaticus major inhibited (Group B). As the zygomaticus major is important in forming a Duchenne smile, it would be expected that Group B would not show improvement on the happiness detection in Session 2, and Group A would not show improvement in anger detection in Session 2 (Ekman, Davidson, & Friesen, 1990). These results would align with pre-existing research and thus would provide further evidence for the results and assumptions of botulinum toxin studies. If the results are not replicated, this would provide more information about the mechanism of facial feedback when compared with botulinum toxin research. As botulinum toxin inhibits the acetylcholine signaling by preventing the exocytosis of acetyl-

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What Happens to the Brain Following Sleep Loss?

Shahd Fulath Khan

While researchers agree that sleep constriction has adverse effects on the brain, it is unknown whether these effects are due to sleep loss or prolonged wakefulness. It is also unknown how these effects vary during the different phases of the circadian cycle. An emerging study demonstrates that sleep constriction, even without extended wakefulness, can have detrimental effects on cognitive performance. These harmful effects are still observed during the next day, despite the arousalpromoting function of the circadian cycle. These results inform the current debate on whether or not sleep constriction results in a disturbance of the homeostatic balance of the sleep-wake cycle. The findings show that sleep loss alone, even without extended wakefulness, can affect the homeostasis of the sleep-wake cycle and cause cognitive deficits. A critical analysis of the study shows that the design is limited as it does not replicate the experiences of sleep loss in everyday life and it uses a small sample size. However, the design uses reliable measures for observing the physiological and behavioral effects that occur due to sleep constriction. Future directions include studying the different effects of sleep constriction and sleep deprivation by using brain imaging techniques like fMRI and measuring other behaviors, such as mood. Key words: chronic sleep restriction, sleep, extended wakefulness, forced desynchrony, homeostasis, circadian rhythm, psychomotor vigilance task, cognitive performance

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BACKGROUND or INTRODUCTION. Sleep loss has detrimental effects on brain functioning and behavior. Studies have shown that sleep loss affects cognitive functioning, attention, mood, and physical health (Killgore, 2010). However, it is not clear whether those effects are attributed to sleep constriction or extended wakefulness. Sleep constriction is defined as sleep that is less than the minimum amount of time (< 6 hours) required for healthy functioning (Chaput, Després, Bouchard, & Tremblay, 2007), while extended wakefulness refers to the state of being awake for greater than 16 consecutive hours (McHill, Hull, Wang, Czeisler, & Klerman, 2018). Researchers have suggested that the observations in individuals who experienced sleep loss may be due to staying awake for too long during the day, which causes a buildup of sleep pressure on the brain (Van Dongen, Maislin, Mullington, & Dinges, 2003). Moreover, previous studies are susceptible to the confounding effects of circadian rhythm (Belenky et al., 2003; Van Dongen et al., 2003). Since circadian phases promote arousal and sleep at different times during the 24-hour cycle (Wright, Lowry, & LeBourgeois, 2012), cognitive performance could be influenced by the circadian phase of the measurement. Therefore, there is a need to design an experiment that a) separates the effect of sleep loss from extended wakefulness, and b) measures the difference in the effect of sleep loss throughout the circadian cycle.

The study by McHill et al. (2018) aims to address these questions by investigating the effects of sleep constriction and circadian phases independent from extended wakefulness. It is important to note that there is a difference between sleep constriction and sleep deprivation. Sleep constriction consists of a shortened amount of sleep, whereas sleep deprivation consists of no sleep at all (McHill et al., 2018). In McHill et al.’s (2018) study, the researchers focused only on sleep constriction. To determine whether there is a difference between the effects of sleep constriction and extended wakefulness, the researchers used a forced-desynchrony protocol to separate the effects (McHill et al., 2018). Forced desynchrony refers to the separation of circadian rhythm from the time spent awake and asleep. The researchers used a 20-hour day inpatient protocol to restrict the amount of extended wakefulness. Extended wakefulness was defined as being awake for more than 16 hours. Both the chronic sleep constriction (CSR) group and the control group avoided extended wakefulness by staying awake less than 16 hours (McHill et al., 2018).

McHill et al. (2018) demonstrated that sleep loss causes increased lapses in attention, increased reaction time, and no change in subjective alertness. These observations worsened throughout the circadian night and did not recover during the circadian day. The PVT allowed for the measurement of the average reaction time of individuals, which is the operationalization of vigilance and attention among participants.

Vigilance is Decreased in CSR Group

In Figure 1, the data indicates that individuals who experienced chronic sleep constriction (CSR) had a longer reaction time in comparison to individuals without sleep constriction (McHill et al., 2018). These effects worsened during the circadian night, as demonstrated by the two peaks in the adjacent graph. During the circadian day, the results did not return to baseline levels, even though the body system promotes arousal during the day phases.

Figure 1: The median reaction time (RT) was obtained from the psychomotor vigilance task. The results show a significant increase in RT among individuals who experienced chronic sleep restriction. Adapted from “Chronic sleep curtailment, even without extended (>16-h) wakefulness, degrades human vigilance performance.” By A. W. McHill, J. T. Hull, W. Wang, C. A. Czeisler, 2018. Proceedings of the National Academy of Sciences of the United States of America, 115(23), 6071.

Attention is Decreased in CSR Group

In Figure 2, each individual’s lapses of attention were recorded using the PVT. Lapses of attention were defined as an RT > 500ms (McHill et al., 2018). Participants who experienced chronic sleep restriction (CSR) had significantly longer lapses in attention compared to participants who had sufficient sleep (McHill et al., 2018). Similar to their reaction times, their lapses of attention were worsened during the night and were not regained during the following McHill et al. (2018) measured vigilance, attention, and alertness day. using different parameters to assess overall cognitive performance. The parameters used to study cognitive performance are the Psychomotor Vigilance Task (PVT) to measure vigilance and attention, the Visual Analog Scales (VAS) to measure subjective alertness, and the Karolinska Sleepiness Scale (KSS) to measure subjective sleepiness (Akerstedt & Gillberg, 1990; Wyatt, Ritz-De Cecco, Czeisler, & Dijk, 1999). The PVT is a computer-based task that measures vigilance and attention by recording the length of time it takes for a participant to respond to a visual stimulus (Killgore, 2010). These measures were collected throughout the circadian day and night Figure 2: The PVT was used to record lapses of attention among participants. Individuals with chronic sleep constriction had increased lapses until the end of the protocol in order to examine how the effects of of attention compared to individuals without sleep constriction. Adapted sleep constriction differ throughout the circadian cycle. from “Chronic sleep curtailment, even without extended (>16-h) wake-

MAJOR RESULTS

fulness, degrades human vigilance performance.” By A. W. McHill, J. T. Hull, W. Wang, C. A. Czeisler, 2018. Proceedings of the National Academy of Sciences of the United States of America, 115(23), 6071.

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Subjective Alertness is the Same Between CSR and Control Groups

In Figure 3, the participants in both the CSR group and the control group showed no statistically significant difference between their subjective alertness (McHill et al., 2018). All individuals reported the same level of subjective alertness, despite having different performances in the neurobehavioral tasks.

Figure 3: The Visual Analog Scale measured the subjective alertness of each individual. The data show no significant difference between the self-reported alertness of individuals with sleep constriction and individuals with sufficient sleep. Adapted from â&#x20AC;&#x153;Chronic sleep curtailment, even without extended (>16-h) wakefulness, degrades human vigilance performance.â&#x20AC;? By A. W. McHill, J. T. Hull, W. Wang, C. A. Czeisler, 2018. Proceedings of the National Academy of Sciences of the United States of America, 115(23), 6071.

DISCUSSION This study expands on previous findings by demonstrating the difference between the role of circadian rhythms and the time spent asleep. Circadian rhythms regulate sleep-wake cycles by activating arousal-promoting and sleep-promoting mechanisms at different times in the 24-hour cycle (Pavlova, 2017). Past studies did not separate the effect of circadian rhythm from sleep loss (Belenky et al., 2003; Van Dongen, Maislin, Mullington, & Dinges, 2003). This significantly undermines the generalizability of the findings from previous studies. To address this problem, McHill et. al (2018) used a forced desynchrony protocol that allowed for the separation of the effects observed from chronic sleep restriction and those observed from sleep-pressure build-up throughout the circadian cycle. Forced de-synchrony was achieved by using 20-hour days instead of 24-hour days. This allowed the researchers to avoid extended wakefulness (being awake for > 16 hours) among all participants. Individuals in the CSR group slept for 4.67 hours and were awake for 15.33 hours, whereas individuals in the control group slept for 6.67 hours and were awake for 13.33 hours. The study duration lasted for 20 calendar days, or 24 sleep-wake cycles.

The findings by McHill et al. (2018) inform the debate on the effects of sleep loss by illustrating that sleep constriction alone causes deficits in cognition, attention, and alertness. The measurements were conducted at different times in the circadian cycle to determine the effects of arousal-promoting and sleep-promoting mechanisms on performance. Even though the circadian phase promotes arousal in the brain during the day, the deficits observed in cognitive performance are still not recovered in the day following sleep constriction (McHill et al., 2018). Therefore, these findings indicate that even without extended wakefulness, sleep deficiency can cause detrimental changes in cognitive function. This also informs the debate

on whether sleep constriction alone causes a disturbance in the homeostasis of the circadian rhythm.

The difference in the results from the subjective alertness and objective alertness tasks are consistent with findings from the literature. Previous studies have identified that although individuals who experienced CSR preform worse in objective tasks, they report the same level of alertness as the control group (Akerstedt & Gillberg, 1990; Kosmadopoulos et al., 2017). This has important implications on everyday activities that require more accurate subjective judgements in alertness, such as driving. Individuals who have impaired attention due to sleep loss may to decide to drive despite being in a poor cognitive condition.

The behavioral findings are consistent with the current research on the biological mechanisms that occur in the brain during sleep. In mice studies, researchers showed that constricting sleep results in a build-up of microglia that are not inhibited during the night (Diering et al., 2017), causing fatigue and poor performance in memory tasks. Other researchers showed that there are increased levels of extracellular adenosine and adenosine receptors in sleep deprived brains, which has been linked to deficits in cognitive performance (Basheer, Bauer, Elmenhorst, Ramesh, & McCarley, 2007; Krueger, 2008; Porkka-Heiskanen, Zitting, & Wigren, 2013). Therefore, the behavioral findings by McHill et al. (2018) are consistent with the underlying biological processes that have been documented to occur in the brain.

CRITICAL ANALYSIS One major limitation of this study is that it does not accurately model sleep loss that occurs in the real world. Individuals experience a range of emotional and physical stressors in addition to sleep constriction. However, the design recruited participants in a 20-day inpatient protocol where the participants rested in private suites and engaged in sedentary activities like reading and watching movies (McHill et al., 2018). So, the researchers did not replicate the normal conditions of individuals who experience sleep loss in everyday life. This can be improved by collecting information about the nature of participantâ&#x20AC;&#x2122;s usual environment, such as regular work hours, mental illness diagnosis, number of dependent children, or their daily tasks. These variables could be used in statistical analyses to adjust for confounding effects.

Another limitation is the small sample size that has been used in the study. The sample consisted of 10 females and 7 males, aged 18 to 35 years old. The CSR group was comprised of 9 individuals, while the control group was comprised of 8 individuals. This negatively affects the external validity (i.e. generalizability) of the results, rendering them less statistically powerful than studies with larger sample sizes.

While the study is limited in its imitation of real-world sleep loss and small sample size, it uses a design that has increased accuracy of measuring the physiological and behavioral effects observed in sleep loss. By accounting for prior sleep patterns, measuring several beat cycles (i.e. time it takes to complete one circadian cycle and

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sleep-wake schedule), and including female participants, McHill et the brains of individuals who experienced acute sleep deprivation al. (2018) provide a more comprehensive design compared to previ- and those who experienced chronic sleep restriction (Maric et al., ous studies. 2017). Another possible approach is to use functional Magnetic Resonance Imaging (fMRI) to examine the differences in the brains of individuals who experienced CSR (sleeping less than 5 hours), Unlike other sleep dose-response studies (Belenky et al., 2003; Van individuals who experienced sleep deprivation (no sleep at all), and Dongen et al., 2003), McHill et al. (2018) accounted for self-induced individuals who experienced sufficient sleep (sleeping more than 6 CSR behaviors among their participants by adjusting the sleep hours). The research question would test the hypothesis of whether patterns of participants at least three weeks prior to the start of the or not brain areas involved in cognitive function have decreased protocol. All the participants were required to sleep for 10 hours blood flow following different kinds of sleep loss. Therefore, using each night, and their compliance was verified by wrist actigraphy different methods like fMRI can expand our understanding of the and other physiological measures (McHill et al., 2018). While difference between sleep deprivation and sleep constriction. Belenky et al. (2003) allowed participants to get sufficient sleep for three days before the protocol, that design does not balance the effects of individuals who experienced CSR for more than three The difference between sleep deprivation and sleep constriction consecutive days as their circadian rhythm would not be able to can also be examined through behavioral studies that measure recover within three days (Pavlova, 2017). emotions, eating behaviors, and physical performance. The link between cognitive performance and sleep loss has been extensively studied in the literature (Alizadeh Asfestani et al., 2018; Hagewoud, The researchers also recorded the effects of CSR on cognitive per- Bultsma, Barf, Koolhaas, & Meerlo, 2011; Kaplan et al., 2019; Lo et formance throughout four beat cycles instead of one cycle al., 2012; McHill et al., 2018). However, other effects such as chang(Kosmadopoulos et al., 2017; McHill et al., 2018). This uncovered es in eating behaviors, mood, or physical function have not been the effects of a) the different circadian phases (circadian day and studied as greatly as cognition. This could be due to the developnight) and b) the accumulation of sleep debt on vigilance and atten- ment of valid and reliable tools like the PVT and VAS. Therefore, tion. Previous studies did not show a clear distinction between the researchers should invest time in developing tools that allow us to effects of time spent asleep, extended wakefulness, or circadian critically examine the changes in other behaviors due to sleep loss. rhythms. Therefore, McHill et al. (2018) introduce an important finding to the current debate on sleep debt and sleep loss.

McHill et al. (2018) used reliable tools and expanded on previous studies by adjusting the participant pool. The PVT has been validated as a reliable and valid measure of vigilance (Lim & Dinges, 2008). Therefore, McHill et al. (2018) used the task as it accounts for sleep loss independent of intelligence levels among participants. Additionally, the participant pool included female participants, unlike previous studies that tested sleep loss on males only (Kosmadopoulos et al., 2017). This accounts for the physiological differences across the sexes that could occur following sleep loss.

FUTURE DIRECTIONS â&#x20AC;&#x201C; The findings of the study provide important insights for the field of sleep and cognition. If this study did not work, the results would indicate that extended wakefulness could be a plausible contributor to the negative effects observed in neurobehavioral tasks. This has implications on the effect of biological mechanisms of arousal and wakefulness. Insignificant results would indicate that constricting sleep may not be as detrimental as prolonged wakefulness. By contrast, McHill et al. (2018) showed that sleep constriction is the determining factor for the cognitive deficits that are observed.

One area of improvement is to use different methods to examine the effects of sleep loss and extended wakefulness on cognitive performance. A possible approach is using brain imaging to examine the differences in processes and structures that are involved in cognitive performance. Brain scans can provide a more detailed explanation to the behavioral observations that are common in individuals who experienced sleep loss. Maric et al. (2017) used electroencephalogram (EEG) studies to demonstrate the differences between

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Examination of Flammulina velutipes polysaccharides: A Gut Microbiota Modulation Resulting in Learning and Memory Improvements Julia Gallucci

Cognitive impairments such as Alzheimerâ&#x20AC;&#x2122;s disease (AD) have both a high incidence and morbidity rate, making them a great challenge in the neuroscience field. There are various factors contributing to the prevalence of AD, many of which were thought to have a heredity component such as genes and family history. However, the rapid increase in AD incidence cases over the years has raised the question as to whether environmental factors, such as diet, have been underestimated. Flammulina velutipes is an edible mushroom that contains many bioactive compounds, such as polysaccharides. This study showed Flammulina velutipe polysaccharides (FVP) was effective in reversing scopolamine- induced learning and memory impairments in mice. Fecal microbiota transplantation derived from FVP fed mice appeared to have improved learning and memory function compared to common microbiota derived from control mice. The improvements were assessed using behavioral measures such as Morris Water Maze. FVP appeared to have an underlying mechanism, where the polysaccharides had an impact on the gut microbiota composition, significantly decreasing the abundances of Clostridia and Bacilli while increasing Bacteroidia, Erysipelotrichia and Actinobacteria. Furthermore, FVP reduced neuroinflammation through the suppression of pro-inflammatory cytokines (IL -1beta, IL-6 and TNFalpha) while promoting anti-inflammatory cytokine IL-10. These findings suggest regulation of the gut microbiota and neuroinflammation through diet, may have a causal role in improving learning and memory related impairments. Key words: Learning, Memory, Diet, Flammulina velutipes, Polysaccharides, Inflammatory Cytokines, Gut Microbiota, Morris Water Maze

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BACKGROUND or INTRODUCTION.

MAJOR RESULTS

Alzheimer’s disease (AD) is a progressive neurodegenerative disease, and is thought to be one of the most common cognitive impairments in the central nervous system (Hu et al. 2018; Kumar A & Tsao JW, 2019). In fact, statistics show that the incidence rate of AD has been increasing each year, where in 2016, 700,000 Americans aged 65 and up had died of an AD - related disease (Gaugler, James, Johnson, Scholz & Weuve, 2016). Typically, there has been a genetic focus on these cognitive deficits related to Alzheimer’s disease, where specific genes such as the APOE and genegene interactions have been emphasized as the underlying cause (Liu, C.-C., Kanekiyo, T., Xu, H., & Bu, G., 2013.). Therefore, there was a large proportion of research regarding environmental factors that had been neglected. Although aging, family history and susceptibility genes have been traditionally considered as the most important factors, in recent studies, it has been hypothesized that environmental factors are just as, if not more, important than genetic factors in AD (Hu, Wang & Fin ,2016).

In Hu et al.’s research (2018), mice underwent scopolamineinduced learning and memory impairments. Mice were then split into 5 groups. Group 1 and 2 being control and model group, respectively, group 3 was fed FVP, and group 4 and 5 received a fecal microbiota transplant, either from control mice (group 1) or FVP fed mice (group 3) respectively. Hu et al. (2018) found that there was a change in gut microbiota composition, where FVP treated groups (either fed or fecal transplant) had Clostridia and Bacilli significantly decreased and Bacteroidia, Erysipelotrichia and Actinobacteria increased compared to controls. Figure 1 from Hu et al., (2018) clearly illustrates this modulation, where the effects of FVP can be visualized through the bacterial taxonomic profiling data. Another major finding involved inflammatory cytokines, where FVP treated groups had a suppression in pro-inflammatory cytokines (IL -1beta, IL-6 and TNF-alpha) and a subsequent promotion of the anti-inflammatory cytokine IL-10 compared to controls. Figure 2 illustrates the effect of FVP treatment on the concentrations of cytokines, where the emphasis is on comparing FVP to control groups. Lastly, when analyzing the behavioral effects that FVP treatment has on learning and memory, Hu et al., (2018) found FVP treated mice had improved behavioral performance given the Morris Water Maze test (Figure 3). These collective findings suggest that FVP is a valid candidate for improving memory and learning related impairments. To summarize, FVP, given through diet as well as microbiota transplant, was able to effectively reduce the neuroinflammation associated with a mouse model of AD, by changing the gut microbiota composition and reducing proinflamatory cytokines. Hu et al. (2018) were also able to prove this modulation, as changes in diet had corresponding behavioral effects, improving learning and memory in a behavioral assay.

Literature reviews have extensively looked at in vitro as well as animal studies, and have identified the toxic effects that environmental factors have at a biological level, specifically revealing alterations of pathways associated with AD, emphasizing the need for further investigation (Beach, Manivann, Halden, Yegambaram ,2015). Scientists have now shifted their focus to environmental influences that are involved in shaping one’s health. There has been an increasing amount of studies suggesting that changes to the gut microbiota composition resulting from modulations such as an alteration in diet or exercise may reflect corresponding changes in brain function (Hooper, Dan & Macpherson 2012; Korem et al., 2015). Researchers have additionally found that the gut microbiota has been implicated in regulating neuroinflammation, where neuroinflammatory responses seem to involve signals from the gut microbiota (Challis et al., 2016). In Alzheimer’s disease, neuroinflammation has been thought to contribute as much as, or even more to pathogenesis as the genetic factors that result in FVP altered microbiota composition plaques and tangles (Bodea et al., 2014). FVP had a profound effect on the gut microbiota composiFlammulina velutipes is an edible mushroom that contains many bioactive compounds, one being polysaccharides (Yeh MY, Ko WC, Lin LY, 2014). A previous study has shown that Flammulina velutipe polysaccharides (FVP) is an effective agent in improving laboratory induced impairment of learning and memory in mice given the Morris Water Maze paradigm (Yang et al., 2015). Yang et al.’s (2015) results suggested FVP significantly decreased the total swimming distance of mice in the hidden platform test, and additionally increased the number of platform crossings during the probe test. However, the mechanism behind the F. velutipes polysaccharides’ mode of action remained unknown.

tion, where through 16 S rRNA sequencing, there was a notable distinction between FVP-treated and control groups. Specifically, Figure 1 shows the distinction, where given FVP treated groups, the relative abundance of Clostridia and Bacilli significantly decreased, while Bacteroidia, Erysipelotrichia and Actinobacteria levels were increased compared to controls.

In Hu et al.’s research (2018), scientists aimed to identify the functional mechanism behind FVP, where the emphasis was on how dietary changes impact gut microbiota composition, inflammatory cytokines and determining if these modulations correspond to changes in learning and memory impairments. Specifically, mice induced with learning and memory impairments underwent either FVP derived or control microbiota fecal transplantation. Scientists then used DNA extraction and high throughput sequencing to identify the microbiota composition changes associated with the transplantation, along with modulations to the levels of inflammatory cytokines. Lastly, to look at the consequent learning and memory changes, the Morris Water Maze paradigm was used as a behavioral assay.

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Figure 2. Illustration of FVP effects on the concentration of cyto- Figure Adapted from Hu et al. (2018). The Royal Society of Chemiskines. (A): indicates the concentration of TNF-alpha (B): indicates try, 9, 1424–1432. the concentration of IL-6 (C): indicates the concentration of IL1beta (D): indicates the concentration of IL-10. The letter a and b over the bar signifies the difference is significant compared to con- CONCLUSIONS/DISCUSSION trol or model group respectively. The outstanding conclusion of the scientific article by Hu et al. Figure Adapted from Hu et al. (2018). The Royal Society of Chemistry, 9, 1424–1432. (2018) had corroborated the fact that environmental factors such FVP treatment improved behavioral performance in Morris Wa- as diet can have a profound impact on improving learning and memory impairments. Here, direct diet of F. velupies or transplanter Maze Task tation of gut microbiota derived from mice fed F. velupies polysacMice were assessed for behavioral improvements by using the charides resulted in the enhancement of cognitive functions. These classic Morris Water Maze paradigm. Each group was trained before induced impairments for 3-days to find the platform that was results, as concluded by the authors, propose that perhaps FVPsubmerged in the water. The probe test was used to determine if caused modulation of gut microbiota and the inhibition of neurointhe mice had learned the platform location, based off how much flammation is related to the improvements in induced learning and time is spent in the area which the platform previously resided in. memory impairments in mice, where these alterations may be the After learning and memory impairments were induced, the swimmajor mechanisms (Hu et al.,2018). Additionally, the authors ming paths of the control mice and their search strategies were characterized as random and irregular, displaying the impairments. acknowledge that their results corroborate previous studies findHowever, the FVP treated groups performed much better than the ings, claiming that diet has an impact on cognitive impairments. controls, where their spatial search strategies were consistent with For instance, in a study conducted by Zhang, Yang & Jin (2016), the platforms original location (Figure 3). authors showed that polysaccharides from the edible mushroom Pleurotus ostreatus also displayed improvements in cognitive impairments of AD rat models. Also, consistent with the findings, studies have showed inflammation to be the cause of many cognitive diseases (Hu et al.,2018), and FVP to have anti-inflammatory properties (Zhao, S., Li, B., Chen, G., Hu, Q., & Zhao, L., 2016). Unlike the literature previously reviewed in this paper, Hu et al.’s (2018) research provides a plausible alternative to genetics as a leading factor to cognitive disease, where diet is of interest. Further, Hu et al.’s (2018) experimental approach, unlike previously reviewed papers revealed diet may have a direct impact on the gut microbiota, which can in turn influence the prevalence and slow down the progression of learning and memory impairments. This research goes a step further by testing pro-inflammatory cytokine levels that have been linked to AD like cognitive disease, ultimately opening the door for further research that links diet’s effects on gut microbiota and inflammation to neurodegenerative disorders. The previously reviewed research has had a greater focus on genetics and uncovering the genes in which underlie cognitive impairments, along with a profound focus on the brain, rather than applying novel techniques and looking at alternative paths such as the gut microbiota as a target site effected by the condition. According to Hu et al. (2018), the authors acknowledge that it is still uncertain whether their effects in learning and memory are a direct result to changes in the gut microbiota and further exploFigure 3. Illustration of FVP effects on Morris Water Maze task performance (A): illustrates the swimming paths of each treatment ration is needed. However, the results do demonstrate a strong group (B): represents how much time each group spent around the correlation, advocating the idea that behavior is related to gut platform (C): represents the number of times mice in each group crossed the platforms’ old location during probe test. The letters a microbiota alteration. Additionally, this study was the first to and b over the bar indicates a significant difference between the test whether microbiota alteration may be necessary for learncontrol group or model group respectively.

ing and memory improvements (Hu et al.,2018)

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CRITICAL ANALYSIS

FUTURE DIRECTIONS

The authors of the paper concerning FVP diet and its significance on the gut microbiota composition and inflammatory cytokines offers alternative routes for research regarding cognitive disorders such as Alzheimer’s Disease (Hu et al., 2018). This research on FVP reveals the need for subsequent studies on environmental factors such as one’s diet’s involvement in neurodegenerative diseases as well as the correlation between gut microbiota and learning and memory. Future research could compare the effectiveness of FVP in human subjects, and observe any improvements in AD- affected variables, such as learning and memory. The experimenters only used animal models, neglecting to show how these diet induced changes could translate to AD patients. Nevertheless, Hu et al.’s (2018) research provided a framework for subsequent studies, as the results proved to be promising with a lack of diet based side effects. These findings allow experimenters to continue this research on human samples without any ethical concern. Although we as humans share many similarities to animals, model organisms are not completely parallel to humans in terms of their functions, especially cognition. Due to humans’ unique features, it has been proven difficult to model and generalize human neurological disorders in animal models (Russell JJ, Theriot JA, Sood P, et al., 2017). Thus, further research is needed to confirm the idea that human behavior is related to diet and gut microbiota alteration.

Scientists interested in FVP acting as a dietary supplement in human participants have consistently found that there is a lack of toxic effects as well as clear benefits to human health (Yeh MY, Ko WC, Lin LY, 2014). Researchers should now resort to testing a FVP based diet on human participants with regards to aiding in the elevation of AD symptoms, as there appears to be clear benefits to the diet and no toxic concerns in the mice models as well as prior human participants. In turn, these experiments will be able to confidently confirm the findings Hu et al., (2018) made, as well as demonstrate the importance of environmental factors in human cognitive deficits. A strength in Hu et al.’s (2018) experimental methods was their use of microbiota transplantation. By transferring gut microbiota of FVP fed mice to mice with induced impairments, experiments were able to show a reversal of the impairment. If they had just used the FVP fed mice, the findings may have been viewed as more correlational rather than causal. Another potential experiment could use a genetic model to test for diet-induced reversal of cognitive impairments. For instance, a model organism with the APO gene that has been emphasized as an underlying cause of Alzheimer’s disease. Results from this experiment can either show that environment has a greater effect on Alzheimer’s disease than genetics, or the reverse. If scientists would be able to display reversal of the APO model’s cognitive impairments, this may be convincing enough to state that environmental effects are perhaps more important in the prevalence of the disease than genetic factors. However, if FVP supplementation had no effect on APO model’s cognitive impairments, this may result in the presumption that genetic influence is more impactful than environment.

The authors used scopolamine induced mice to model cognitive learning and memory impairments. Scopolamine has been readily used in literature as a reliable psychopharmacological model of Alzheimer’s disease, where scopolamine alters brain connectivity similarly to that reported in AD (Bajo R, et al., 2015). This suggests that it was ideal for Hu et al. (2018) to choose scopolamine-induced models in determining the effecLastly, to confirm there are no confounding variables tiveness of modulation of diet for treating AD, as this model has affecting the result of this study, a hypothetical experiment been previously used in literature which looked at potential could compare an anti-inflammatory drug to FVP’s behavioral novel therapies for treating such cognitive diseases (Brazell C, effects on those with Alzheimer’s disease. If scientists could Broks P, Preston G, Stahl S, Traub M, 1988). display a significant difference in participants given FVP comThe authors propose that FVP treatment reduced neu- pared to those treated with anti-inflammatory drugs, one could roinflammation as well. Inflammation is the cause of many neu- reliably rule out the cognitive benefit of FVP is solely its antirological diseases (Amor S, Puentes F, Baker D, van der Valk P, inflammatory properties. However, if those treated with FVP 2010). Thus, this fact alone opens up the doors for further re- supplementation had no improvements in cognitive perforsearch. Through the modulation of diet and consequential re- mance relative to those given anti-inflammatory drugs, one duction of pro-inflammatory cytokines, cognitive disorders may may conclude FVP’s effects are likely due to its antiinflammatory properties, rather than its effect on the gut mibe effectively treated or perhaps even prevented. The interaction between environment and genetics crobiome composition. with regards to AD is poorly understood in literature (Wlassoff, V., 2017). Scientists are aware of environment playing a role in cognitive diseases, and that certain genes can predispose one to such disease, however the interplay between the two factors is often neglected. Hu et al. (2018) focused on diet, but overlooked how this environmental factor may interact with genetic factors. For instance, the author left the readers with remaining questions, such as if a patient has a genetic susceptibility, will changing their diet to be rich in FVP still show equivalent improvements as the induced models. 152

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Investigating the Mechanisms of REM Sleep Control: Connecting the Hypothalamus and Brainstem REM Switch.

Gianluca Guglietti

REM sleep makes up a quarter of our nightly sleep yet itâ&#x20AC;&#x2122;s function and mechanisms by which it is controlled are still debated in the scientific community. Recent years have elucidated some of the mechanisms of the REM sleep switch identifying nuclei in the brainstem responsible for the NREMREM sleep switch but higher inputs into this system have yet to be fully investigated. In the article we will be discussing today Chen et al. utilize retrograde tracing and calcium imaging to identify two distinct populations of neurons in the Dorsal Medial Hypothalamus (DMH) that were both differentially active in NREM versus REM and projected to different brain regions. One population was made up of neurons that projected to the preoptic area (POA) in the hypothalamus and were more active in NREM sleep; the other projected to the raphe pallidus (RPA) in the brainstem and were more often active during REM sleep. They then used optogenetics to selectively activate, with channelrhodopsine, and suppress, with iC++, these subpopulations showing that each population was both sufficient and necessary for sleep stage transition. This study was able to identify the neurons potentially responsible for connecting the circadian and homeostatic regulators of sleep in the hypothalamus to the brainstem NREM-REM sleep switch previously identified in the literature. This research may prove to have significant translational value in understanding a variety of parasomnias associated with abnormal sleep stage transitions such as rem sleep behavioral disorder (RBD), narcolepsy, and Hypnogogic and hypnopompic hallucinations. The optogenetic control of sleep stage transition also acts as a powerful tool for investigating the function of sleep. Key words: Non Rapid Eye Movement (NREM) Sleep, Rapid Eye Movement (REM) Sleep, Galanin, GABA, Sleep Stage Switch, Dorsal Medial Hypothalamus, Preoptic Area, Raphe Pallidus, Retrograde Tracing, Calcium Imaging, Optogenetic

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Introduction REM sleep was first defined by Eugene Aserinsky and Nathaniel Kleitman in 19531 and has since been a topic of extreme interest in the field of sleep biology and neuroscience. REM is characterized by, as the name suggests, rapid eye movement but also low amplitude high frequency EEG waves and complete muscle atonia. These periods of REM occur regularly in the sleep cycle and account for approximately 20-25% of oneâ&#x20AC;&#x2122;s nightly sleep, increasing in frequency and duration as the night goes on2. REM sleep has been theorized to be associated with a variety of phenomena and functions including dreaming 3, memory consolidation4, and neural ontogeny5. Much research has been done on the regulation and mechanism of the wake to sleep switch, giving us a strong understanding of how we change behavioral states. Despite this our understanding of the transition between the sleep stages of NREM and REM is less well studied. It is currently believed that two nuclei in the upper pons are responsible for the switch from NREM to REM and back, the ventrolateral periaqueductal grey (vlPAG) and the subcoeruleus (SubC), also known as the sublaterodorsal nucleus6. These two nuclei from a mutually inhibitive circuit via GABAergic inputs meaning when one is active the other will be silenced (Figure 1). The SubC is referred to as the REM-on nucleus as when it is active it in turn activates REM sleep via glutamatergic Precoeruleus neurons which gives REM its distinctive EEG pattern7. In addition, the SubC excites the GABAergic neurons of the ventromedial medulla which results in inhibition of motor neurons and muscle atonia. Despite this switch being identified decades ago the neural mechanisms controlling the switch have yet to be fully explained. Many of the nuclei that regulate sleep lie in the hypothalamus, not brainstem, such as the suprachiasmatic nucleus (SCN) which controls circadian rhythms and the ventrolateral preoptic nucleus (VLPO) which is the target of a variety of homeostatic mechanisms 8. In addition a variety of changes in the hypothalamus tend to encourage REM sleep such as the production of melanin concentrating hormone in the lateral hypothalamus9. The question remains, how do these sleep regulatory neurons communicate with the sleep switch in the brainstem?

of a bistable switch resulting in changes of sleep state with no overlap time.

The authors of this paper have Identified the dorsal medial hypothalamus (DMH) as a target for investigation as it has been shown as a relay point for both the SCN and VLPO to other sleep promoting regions10. Chen et al. utilize calcium imaging to identify neurons in the DMH that are selectively active during REM sleep. They then use retrograde tracing to verify their targets, in this case the raphe pallidus in the brainstem. They then use optogenetics to show that these neurons when activate are able to flip the switch between NREM and REM and induce REM sleep. Major Results11

Calcium imaging and Retrograde Tracing reveals two distinct Galaninergic neuron populations in the DMH. The authors first used microendoscopic calcium imaging to determine the differential activity of neurons in the dorsal medial hypothalamus in wake, NREM, and REM sleep. They first investigated galenergic neurons, a subpopulation of GABA neurons, using a GAL-cre expression of GCaMP6f, a calcium indicator. They found a strong bimodal relationship in the galanin neurons of the DMH along the NREM-REM axis, with one population firing predominantly during NREM and the other during REM (Fig. 2). This is in contrast with the GABAergic neurons in general which, using GAD2-cre mice, showed a unimodal distribution with more heterogeneity. In both cases they observed a unimodal distribution in the wake-NREM modulation index, showing no preference in activity between the two.

Figure 1. NREM-REM Flip-Flop: This Diagram from Saper et al.6 illusFigure 2. REM-NREM Activity Differences: this graph shows the distritrates the basic anatomy of the REM-NREM switch with the vlPAG and the SubC forming a mutually inhibitive circuit allowing for the formation bution of activity of various neurons in the DMH. The grey circles repre-

155

represent galanin negative GABA neurons and the black dots represent galanin positive GABA neurons. The galanin positive neurons segregate into two groups, one predominantly active during REM and the other primarily active during NREM forming a bimodal distribution.

After discovering these two populations the authors attempted to see if they could differentiate between them by their synaptic target. To do this they applied eYFP anterograde staining showing that the galaninergic neurons synapsed on wide ranging targets including the preoptic area (POA), raphe pallidus (RPA), lateral hypothalamus (LHA), dorsomedial region of the thalamus, periaqueductal gray (PAG), and dorsolateral pons. They then performed retrograde staining to identify the neuronal populations whose axons projected to these regions, starting with the POA due to its importance in sleep regulation. It was found that these POA projecting neurons also had axonal projections to the LHA but not the RPA. They then did simultaneous retrograde staining of both the POA and RPA and found they both had projections from the DMH, as expected, but had very little overlap in the neurons projecting to each distinguishing two distinct subpopulations of DMH neurons (Fig. 3).

projecting neurons from the DMH were sufficient for suppressing REM and facilitating NREM. The inverse was noted when these neurons were inhibited using iC++, showing that of POA projecting neurons from the DMH are required for NREM.

The same set of experiments using optogenetic modulation were used on the RPA neurons, and revealed the opposite results (Figure 4b). Activation of RPA projecting neurons suppressed NREM and activated REM sleep, showing that these neurons were both necessary and sufficient for REM transition and maintenance in sleep.

Figure 4 Optogenetic Activation of Sleep State Change: Each graph shows relative time spent in REM, NREM, and wake. (A) change with optogenetic activation of POA projecting DMH neurons, the grey bar is the period where the laser light is activated and thus the neurons are more active resulting in increase NREM at the expense of REM. (B) change with optogenetic activation of RPA projecting DMH neurons, revealing an increase in REM during activation and decrease in NREM. Figure 3. Retrograde Tracing Reveals Two Distinct Neuronal Populations: Retrograde tracing from the POA with eGFP and RPA with mCherry reveals neurons in the DMH that project to each region. Interestingly when overlaid there seems to be very little overlap suggesting these two neuronal populations are distinct.

Conclusion

The authors of this paper were able to identify two distinct REM-on and REM-off neuronal subpopulations in the Finally, they married these two techniques and perDMH. They were also able to show that the REM-on neurons formed calcium imaging on these two distinct populations using had GABAergic projections to the RPA in the brainstem a nucle12 retrograde staining and GAL-cre dependent GCaMP6f expresus known to be REM-off . They conclude that these sion in the DMH. This revealed that the POA projecting neurons DMH neurons act through the RPA to promote REM were largely suppressed in REM sleep showing decreased activ- sleep. The Authors have less definite conclusions regarding the ity; in contrast the RPA projecting neurons showed increased REM-off DMH neurons as they could be acting through a variety activity in REM sleep. Thus the two subpopulations as defined of mechanisms such as inhibition of MCH producing neurons in by their axonal targets seemed to overlap with REM-on versus the LHA or REM promoting neurons in the POA. REM-off functional groups.

Optogenetic Activation of DMH Subpopulations Shows a Causal Relationship Between Activity and Sleep Stage.

Critical Analysis

Despite the relative strength and impact of this paper there are a few confounds and limitations the authors have not The authors then employed optogenetic modulations of fully accounted for. The first and most glaring is the possibility these subpopulations to investigate the causal nature of this of off target activation due to their imprecise method of chanrelationship. They first used channelrhodopsine to optogenelrhodopsine expression. They used a retro virus to express netically activate the POA projecting subpopulation. They obchannelrhodopsine in all galanin expressing neurons projecting served increased time in NREM during activation with deto the POA or RPA. Despite targeting the DMH specifically with creased time in REM and no change to wakefulness (Figure 4a). laser light this does not preclude the possibility of activating This was shown to both be due to decreased REM initiation and passing axons of neurons from other regions of the hypothalamaintenance. This result demonstrates that activity of POA mus. Due to the high concentration of sleep modulating neu156

neurons in the hypothalamus, as discussed above, this represents a significant confound that was not accounted for by the authors. Additionally, Despite the authors concluding that these neurons are able to promote REM sleep through inhibition of the RPA they failed to back up this claim with experimental evidence. At no point did they show that optogenetic activation of DMH REM-on neurons results in decreased RPA activity, this is a limitation of their research that requires further investigation. It is theoretically possible that these DMH neurons are synapsing on inhibitory interneurons in the RPA which would result in a double negative effect and increased activity in the RPA.

ing of spatial memory, a type of memory closely associated with REM sleep4, and then allowed to sleep. We would the split the rats into groups which are allowed to sleep normally, as well as rats with optogenetically inhibited and increased REM sleep. I theorize that the rats given increased REM sleep would later be better able to remember the maze and complete more quickly in comparison to controls and REM inhibited rats. Similar experiments could be formulated to test a variety of hypothesized REM sleep functions and provide stronger experimental evidence for its purpose than we currently have in the literature.

Future Directions The discoveries of this article open the doors to a variety of exciting new experiments. Firstly, the authors may consider further elucidating the mechanisms of NREM-REM sleep state control by testing the neurons responsiveness to hormones and neuropeptides associated with sleep state switching 13 such histamine , orexin, and melanin concentrating

hormone14. These experiments could help place these neuronal subpopulations in the complex network of sleep regulating nuclei in the hypothalamus. Although a more interesting opportunity made available by this research would be the direct investigation of REM sleep using the optogenetic tools with the identified neurons. Research into the function of sleep is often done through a sleep deprivation paradigm where animal models are subjected to total sleep deprivation extrapolating the function of sleep from the deficits present in its absence. This provides two main problems, the first being the high levels of stress induced by sleep deprivation which is difficult if not impossible to control 15 for , and the second being that any observations may be due to the consequences of increased wakefulness and not the consequence of decreased sleep. In recent years researchers have attempted to move toward induced sleep paradigms, using selective activation of sleep-inducing neurons to investi16 gate itâ&#x20AC;&#x2122;s benefits . These challenges in sleep research study design are shared by REM sleep research where the most common model is the flowerpot method in which a rat is placed on a platform above a pool of water. As the rat slowly transitions into REM sleep it will experience muscle atonia falling into the water be17 low and waking back up . This has the obvious problems previously outlined above regarding high stress and may be less about the disruption of REM sleep as much as it is a function of regularly interrupted sleep in general. The mechanisms outlined by Chen et al. provides investigators a way to regulate REM sleep directly and reliably without waking the animal or causing undue stress. As REM sleep is theorized to be associated with memory consolidation4 it would be interesting to test this theory with these newfound tools. I propose and experiment in which rats are taught a complex maze task requiring the encod157

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Binge eating disorder (BED) in both humans and rodents includes a change in emotional profile following binge eating, which can be assessed via behavioral and neurophysiological investigation. Kiana Habibagahi

Binge eating disorder (BED) is one of the most common kinds of eating disorders characterized as having regular binge eating episodes and is often comorbid with other psychiatric disorders such as depression, anxiety and compulsivity. This review focused primarily on Satta et al.â&#x20AC;&#x2122;s paper, which assessed BEDâ&#x20AC;&#x2122;s emotional characteristics. Authors used a female rodent BED model, where they induced binge eating via intermittent access to margarine, a high-fat, palatable food. Post-binge, BED rats had lower depressive symptoms in forced swim tests, less anxious behavior in the elevated plusmaze. No difference was found for marbles buried in the marble-burying test post-binge, though they buried more marbles pre-binge compared to controls, suggesting higher compulsive-impulsive behavior. Results suggested improvement in emotional profile following binge. Despite their key findings, several elements of the paper demanded further investigation. Some studies, for instance, have reported that BED does not always improve anxious or depressive symptoms. This review also investigated why BED may be more prevalent in women, including whether their menstrual cycle plays a role. It also encouraged investigating the disorder in men, and what changes BED would cause in their body. Finally, neurological findings such as neuroimaging studies and staining assays were discussed. These studies investigated changes in anxious, depressive and impulsive behavior, findings that could provide a biological basis of BED to increase understanding of its nature and whether it influences or is influenced by mood states. Key words: Binge eating disorder, depression, anxiety and compulsivity, high-fat.

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BACKGROUND Binge eating disorder (BED) is categorized as having regular binge eating episodes, where the subject consumes large amounts of food that another individual would not normally eat within a similar time-frame or setting1,2. It has an approximate worldwide lifetime prevalence of 1.9 %, and is found to be more common than other eating disorders 2, 3. The disorder has been officially recognized since 20131, with key characteristics such as experiencing a loss of control (LOC) that consequently leads to binge eating episodes2. Other attributes of the disorder include eating more quickly than usual, eating until uncomfortably full and eating even in the absence of hunger; such symptoms must occur at least once a week over a duration of three months4. Further, BED is not associated with unhealthy compensatory behaviors, such as excessive exercise or vomiting, which could counter excessive caloric intake5. Consequently, BED individuals face a higher risk of weight gain and 70% of BED cases lead to obesity5. Obesity may introduce other health complications including type II diabetes, cardiovascular disease and hypertension6.

HR displayed significantly different eating behavior: they had higher daily consumption compared to both groups by the 2nd week, they had greater consumption compared to LR during the three days of limited access to margarine and they weighed more compared to controls by the 5th week. Similar results were also observed in mice models15. These results are relevant as they demonstrate that binge eating, referring to a loss of control over eating behaviour, can contribute to or lead to weight gain. The subject may crave and binge on the temporarily available, highly palatable food selectively, despite having constant access to chow. This behaviour was evident in HR, who consumed margarine quicker and in greater amounts compared to LR. This has been reported in previous publications as well16. Intermittent access to highly palatable food may therefore drive binge eating, rather than being driven by metabolic needs such as hunger. This finding also aligns with several human studies, where subjects indulged in bingeeating in the absence of hunger 1,17.

Despite the overlap between obesity and BED, studies have suggested that they may be neurologically distinct disorders7,8, making BED an independent health condition that can be investigated and treated differently than obesity in the absence of BED. For instance, compared to normal weight and obese individuals without BED, BED individuals report higher reward-sensitivity, increased activity of the medial orbitofrontal cortex, anterior cingulate cortex, and insular cortex upon viewing high-caloric food7. Another contrast between BED and obesity is that stress and emotion dysregulation play significant roles9,10,11. A distinct emotional profile may therefore be associated with BED, one that is characterised by deficits in emotion regulation2,12,13.

HR’s emotional profile also changed post-binge, and was assessed using various rodent behavioral tests. Tests were performed both 1.5h before and after binge eating. The tests included the forced swim test (FST), marble burying (MBT), elevated plus maze (EPM) and testing Spontaneous locomotor activity (LA). FST assesses depressed behavior, where higher immobility corresponds with more depressive-like symptoms. MBT investigates repetitive, obsessivecompulsive and anxious behaviour in rodents18, where the total number of marbles buried may positively correlate with these behaviours. EPM measures anxious-like behavior, with lower entry and time spent in the open-arms of the plus-maze signifying higher anxious behavior. Finally, LA may have been assessed with a similar apparatus used in open field tests, though this test was not explicitly stated. This test assesses general locomotor activity, which may correspond to exploratory and anxious behavior.

Authors such as Satta et al.5 have therefore sought to investigate BED’s emotional profile, the impact it can have on weight and the symptoms it may include. Emotion regulation is defined as the attempt to control and influence which emotions are felt and expressed, when they are felt and how they are expressed; however, since BED is associated with a lack of emotion regulation, the disorder can also undermine a patient’s ability to regulate eating4. This eating behaviour may arise due to intense emotions, ranging from negative ones such as anger and sadness to unspecific emotional states such as emotional stress4. Not surprisingly, BED is comorbid with psychological disorders and psychological distress2, becoming a possible motivation for the authors to further investigate BED’s emotional profile. Such distressed states include depression, anxiety and compulsiveness. BED has also been reported as being more prevalent in females5, another finding that calls for further investigation.

MAJOR RESULTS Satta et al. observed several major findings, including changes in eating behavior and the emotional profile of binge-eating rats. Female Sprague Dawley rats were categorized into three different diet groups: Low restriction (LR), High restriction (HR) and Control. LR had chow and water available ad libitum, and 2-hour (h) access to margarine, a palatable food, every day of the week. HR differed, in that they only accessed margarine on Mondays, Wednesdays and Fridays; HR was therefore used as a validated BED model14. Finally, controls had chow and water but no margarine.

In post- binge, for instance, the FST revealed that HR spent decreased time immobile, and the elevated plus maze (EPM) displayed that they spent more time in the open arms. Both results suggest elevated mood in test subjects, with the former highlighting lower depressive symptoms and the latter highlighting lower anxious symptoms. Differences between HR and the other two groups was mainly observed during pre-binge: compared to both groups, HR buried more marbles pre-binge in the marble-burying test (MBT) and spent less time in the open-arms compared to LR for EPM. The former may highlight more compulsive behavior, and the latter more anxious behavior. However, no difference was detected between HR and control during pre-binge EPM, during post-binge MBT or during either phases of locomotor testing. Thus, exploration and locomotion did not differ between groups.

CONCLUSIONS The behavioral tests suggested that there was a change in emotional profile of female rats both upon approaching food and following binge eating. HR showed lower anxiety and depressive symptoms compared to groups in post-binge and weighed more

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than both groups. HR also had higher obsessive-compulsive behavior than groups during pre-binge. However, no difference in locomotion existed between groups and therefore could not have driven differences in eating. The following results have several implications. For instance, anxious symptoms may have been reduced due to ingestion of the high-fat, palatable food (margarine); high intake of high-fat food may have anxiolytic effects by altering cholesterol metabolism and the hypothalamic-pituitary-adrenocortical (HPA) axis that regulates stress responses. The authors’ insight that palatable food can lower overall stress responses has been previously observed: mice that underwent social defeat stress (SDS) did not display social avoidance following high-fat caloric intake; however, inadequate intake of palatable foods failed to provide anxiolytic benefits19. Though the authors did not investigate this, quantity of caloric intake may also be an important factor that regulates anxious behavior.

pressive behaviors after being given palatable food. However, a mother rat’s high-fat diet may impose health risks upon future offspring; their high-fat diet may intensify stress responses by altering offspring neuroendocrine systems, including increasing glucose transporter 2 mRNA and protein expression29.

CRITICAL ANALYSIS

Although several behavioural tests were performed to assess BED’s emotional profile, some of the paper’s findings require further study. Primarily, the authors can elaborate or explore why BED may be more prevalent in women, rather than merely stating it. Various metabolic or hormonal differences between males and females may cause this discrepancy. Notably, women may be more likely to comfort eat compared to men, a habit that can vary throughout their menstrual cycle30; their sex and the estrous cycle may therefore be important factors affecting binge eating. Animal studies can be performed to investigate how different stages of the menstrual cycle could affect binge eating, along with how overFurther, high-fat ingestion is a common compensatory behavior all stress responses of regulatory systems such as the HPA axis may used in society to reduce or counter social stress20. Though posialso be impacted. The authors can also perform neuroimaging and tive changes including reduced anxiety were observed following staining assays as a next step, helping them find neurological corhigh-fat ingestion, encouraging this eating habit may introduce relates that could support their claims. For instance, they can inhealth risks. High-fat administration to mice increases caloricvestigate how BED may increase activity in certain brain regions or intake and corticosterone levels, which could negatively impact the what neurotransmitters might be released; these findings may metabolic profile. High corticosterone associated with high-fat help the authors formulate a physiological or neurological profile intake may therefore induce metabolic disease development20. of BED, in addition to examining its emotional profile. Moreover, the authors should research BED in male populations, since BED continues to affect men’s health. In addition to performDepression and BED are also commonly comorbid in humans 21, ing animal studies, they may also investigate different trends that making the study’s findings clinically relevant. Depression has been occur in both genders, through cognitive-emotional surveys such found to directly affect binge eating, particularly among women, as impulsivity tests and neuroimaging studies. Finally, the authors which also makes the authors’ investigation of female rats relecan investigate which negative emotional state is a stronger driving vant22. Negative affect is a key cognitive-behavioural component of force for binge eating, helping them better understand its elusive binge-eating, and higher levels of negative urgency and affect can underlying neuropathophysiology. BED has often been theorized also predict paediatric binge-eating21. Some theorize that bingeas an impulsive compulsive disorder, since a loss of control is one eating serves as an “escape from awareness,” where negative of its key characteristics5,31. Distinguishing between the roles of emotions become less salient due to the cognitive narrowing that hedonics and motivation in BED may also help further characterize happens while eating23. Contrastingly, some obesity models that the disorder, such that it can be treated more effectively. encourage high-fat consumption correlated with depressive symptoms. This contrast may have occurred due to differences in speFUTURE DIRECTIONS cies used (mice VS rats) or differences in protocol24. This finding may also suggest that food has a threshold or limit as to how well To further understand BED in women, studies can investigate feit can alleviate negative mood. male rats undergoing various stages of the menstrual cycle. Researchers can apply the limited sucrose intake (LSI) paradigm, where rats access sucrose twice daily. In females, LSI may the disObsessive behavior pre-binge is also comparable to addictive betinction is behaviour, which can be measured using EPM; LSI also havior, where addicts undergo similar negative emotional states reduced HPA axis stress responses during the menstrual cycle’s when undergoing drug withdrawal. However, in pre-binge phase proestrus/estrus (P/E) phase30. To assess HPA activity, researchers before EPM, HR did not display higher anxiety compared to concan collect blood samples and measure plasma adrenocorticotrols. This contrasted with other studies where rats showed anxiotropic hormone (ACTH) via radioimmunoassay (RIA). cFos immuno25,26 genic behavior when withdrawn from palatable food . This dislabeling, which marks neuronal activation, may also show that LSI crepancy between studies may have occurred due to different increases activity in the central amygdala medial subdivision (CeM) protocols to induce binge eating, the EPM design or rat strain and bed nucleus stria terminalis posterior subnuclei (BSTp) during used. P/E. Researchers can therefore highlight regulatory roles of estrous cycles, CeM and BSTp in BED. Similarly, males with LSI may Withdrawal from palatable food has also been shown, in the literadisplay lower anxious behaviour and HPA axis activity following ture and in this study, to induce depressive symptoms in the literaLSI30 Future studies could investigate BED neurophysiology in hu27,28 ture , particularly in the pre-binge state of FST. Highly palatable mans; researchers can use similar assays, neuroimaging techniques food can also alleviate some of the negative impacts that follow and anxiety questionnaires such as the Beck anxiety inventory psychologically stressful events. For instance, mother rats that had (BAI)32 to assess the extent to which animal results mirror findings been separated from their pups displayed lower anxious and de-

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in human studies. Additionally, although BED’s neurophysiology is largely unknown, human studies often highlight impulsivity’s strong role in the disorder. To replicates previous studies and further investigate impulsivity in BED, patients can perform standardized impulsivity questionnaires. Results may show that BED patients score higher for impulsivity than both normal and non-BED obese individuals on both Barratt (BIS-11) and UPPS impulsiveness scales31,33,34,35,36; higher BIS-11 scores are also likely to positively correlate with correlate with higher food intake9. Researchers can use functional magnetic resonance imaging (fMRI) or record magnetoencephalography (MEG) signals to asses neural correlates of increased impulsivity in BED, specifically during inhibitory control 31. Inhibitory control, or response inhibition, is an executive function that helps subjects inhibit natural impulses37. Results may show hypo-activation of the prefrontal network during response inhibition and a food-centred decline in inhibition. This may occur due to an increased reward response for food and inadequate resources required to active the prefrontal network involved in response inhibition31. Such findings may therefore help further characterise BED as a loss-of-control eating disorder. Finally, behavioural tests and neurological assays can investigate food’s hedonic effects in binge eating38. Behavioural tests include runway and maze tests, which can assess how rewarding subjects find the food. In these two tests, animals must navigate the food reinforcement (goal) compartment by either passing another narrow compartment or a maze, respectively. Task-completion speed indirectly measures food’s reinforcing–rewarding value. These tests can highlight how hedonics could drive binge eating; they can also help disassociate pleasure from motivation in BED and how addictive food can be. Opioid signalling, for instance, may refer to pleasure and could be measured through a rat’s orofacial expression. Antagonist binding may also reveal hedonic hotspots in the medial nucleus accumbens (NAcc)38. However, dopamine signalling may tie with the desire or motivation to binge-eat. Likewise, behavioural tests can help deduce whether an animal’s depressed-like mood could lead to binge eating. Chronic Social Instability Stress protocols may be used to induce depression in female rats39 and their binge eating can be measured and compared with healthy control. Immunohistochemistry including measuring changes c-Fos expression can also be performed on brain slices of both healthy and stress-induced rats. Compared to controls, there may be higher c-Fos mRNA levels in hippocampal regions following chronic stress and a lower immunoreactivity at pyramidal cell layer40. Dissecting the neural basis of BED may therefore be another way to investigate other factors involved in BED, creating opportunities for future treatment interventions.

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Psychoplastogens: a growing class of compounds that promote long lasting structural, functional, and behavioral changes after only a single dose

Adel Halawa

Mood disorders are on the rise in the developed world, and current treatment remains un-efficacious. Recent clinical studies have found a single dose of psilocybin or ketamine under a therapeutic setting to have long lasting antidepressant and anxiolytic effects. The neurological mechanism underlying this medicinal use was hypothesized to be an increase in prefrontal cortex plasticity, a region known to hypofunction in mood disorders. Pyramidal neurons cultured from rat prefrontal cortices were treated with three serotonergic psychedelic compounds; 2,5-Dimethoxy-4-iodoamphetamine (DOI), N,Ndimethyltryptamine (DMT), and Lysergic Acid Diethylamide (LSD). Their effects on Rats and Drosophila in vivo were tested, and so was electrophysiology ex vivo. Results showed significant increases in dendritic complexity, and a function increase in EPSCs. Several pharmacological antagonists were used to conclude that this effect was dependant on the 5-HT2A receptor, Tropomyosin receptor kinase B, and mTOR. The authors termed this new class of compounds psychoplastogens. They are different from other plasticity inducing compounds in that their effects are achieved in a single dose. These compounds have great neurotherapeutic potential, both in mood disorders and in other degenerative or trauma related injuries, where the brain would greatly benefit from increased plasticity. Studies are now investigating if non-hallucinogenic compounds with similar effects can be produced while maintaining similar efficacy. This could present an exciting new avenue for the aid of patients suffering from treatment resistant pathologies. Key words: mood disorders, psychedelics, plasticity, prefrontal cortex, ketamine, serotonin, Brain derived neurotrophic factor, mTOR.

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INTRODUCTION. Mood disorders, such as major depressive disorder (MDD), generalized anxiety, substance abuse, and PTSD are currently on the rise, along with suicide rates1,2. There continues to be active debate in the field regarding the etiology of this trend, but all agree on its severity3. These disorders debilitate a patient’s quality of life, and can present broader burdens on society, health care costs, work productivity and the economy4,5. After the serendipitous clinical discovery of antidepressants like Selective Serotonin Reuptake Inhibitors (SSRIs) and monoamine oxidase inhibitors, Serotonin imbalances were implicated as a hypothesized cause of MDD6. We now know that such a hypothesis is overly simplistic; mood disorders are much more complicated and treatment is highly individualistic. A recent resting state fMRI study concluded that there exist at least four different subtypes of depression7. Unfortunately, only one third of patients respond to these antidepressants, and even those that do suffer many unwanted side-effects8. With repeated dosing, they slowly stimulate plasticity in these patients. However on an ever longer timescale, when a patient develops tolerance, their neurophysiology will shift its homeostasis such that trying to get off of these drugs can result in more severe MDD8. This evidence, combined with an epidemiological rise in prevalence, all urgently point to the need for a better understanding of mood disorders and new avenues for treatment.

professional14-16. This bypasses many of the aforementioned consequences of taking pharmaceuticals for long periods of time. Patients in these trials commonly report this being one of the most “spiritual” experiences in their life, and a change in perspective that remains salient long after15,16. These reports mirror those of ketamine, therefore it is likely that both act in a similar fashion17.

Multiple brain regions have been associated with these pathologies, including the amygdala, hippocampus, Nucleus Accumbens, and Basal Ganglia9. Of particular relevance to this review is the abnormal functioning of the Prefrontal cortex (PFC); more specifically the medial, ventral, and subgenual PFC9-11. Human postmortem studies have found increased neuronal atrophy in these regions among MDD patients, while animal data points to a loss of dendritic spines, implying long term depression9. Ketamine, an NMDA antagonist weaker effects on other neurotransmitter systems, is currently in clinical trials for its antidepressant qualities 12. Astonishingly, it has been efficacious in reducing symptoms of treatment resistant populations, as well as alcohol withdrawal, heroin addiction, and PTSD12. Neurological mechanisms were later elucidated; after only a single dose, the drug increases structural and functional plasticity in the pyramidal frontal cortical-hippocampal circuits13. While it sounds counterintuitive at first that an NMDA antagonist induces greater plasticity, it has been shown to work by elevating levels of brain derived neurotrophic factor (BDNF) and mTOR in these regions of interest13. Relevantly, post-mortem studies have found both of these compounds to be downregulated in patients suffering from depression13.

In order to gain an idea about the effects of psychedelics, primary cortical neurons from rat brains were plated and treated with three psychedelic compounds; 2,5Dimethoxy-4-iodoamphetamine (DOI), N,Ndimethyltryptamine (DMT), and Lysergic Acid Diethylamide (LSD). Dose was 10µM because this was the dose used in the ketamine studies, except DMT, which was used at a concentration of 90µM. This was done to more accurately mimic concentrations in the cerebrospinal fluid after an intraperitoneal antidepressant dose (10mg/kg in rats)18,19. These three were chosen because each is derived from a different chemical family, which are amphetamines, tryptamines, and ergolines respectively. Sholl analysis, a commonly used method to quantify morphological features, was then used to analyze the data. All three compounds resulted in an increased area under the curve, an operational measure of dendritic arbour complexity (Figure 1B). This was largely a result of an increase in the total number of dendritic branches, number of primary dendrites, and total dendritic length (Figure 1C-F). There was no difference in the length of the longest dendrite. These effects can also qualitatively be seen in figure 1A.

To investigate this hypothesis, researchers from UC Davis tested the effects of serotonergic psychedelics on neurons derived from adult Sprague-Dawley rats, Drosophila larvae, and zebrafish embryos17. The authors explicitly replicated much of the design used to study Ketamine in order to highlight their similarities17. Treatment with these drugs increased structural plasticity, and was found to be dependant on Tropomyosin receptor kinase B (TrkB), mTOR, and the 5HT2A receptor17. The group found the results to hold true both in vivo and in vitro, and confirmed that these structural changes also resulted in measurable functional changes ex vivo17. This emerging class of compounds, termed psychoplastogens, act on a conserved signaling mechanism to rapidly induce plasticity in regions of interest. This has huge potential for clinical use, not solely limited to mood disorders.

MAJOR RESULTS Primary cortical neuron cultures derived from rat brains:

Further analysis of treated cortical neurons under superresolution structured illumination microscopy allowed the authors to observe an increase in the number of dendritic spines Recently, similar clinical benefits have been observed for serotonergic psychedelics. Studies from John Hop- per unit length (Figure 1G, 1I). Notably, LSD almost doubled the amount of spines (Figure 1G). A shift in spine morphology kins, MAPS, and other institutes have found psychedelics to ratios was also seen, with an increase in the number of fillipoaid with MDD, cigarette addiction, and the existential anxiety diums and a decrease in mushroom subtypes (Figure 1H. of terminally ill patients14-16. The efficaciousness of these Other observations include an increase in synaptic density drugs is greater than any other antidepressant previously but not size, and an increase in VGLUT1 but not PSD-95 studied, and has warranted much interest in their mechanism (Figures K-M). This implies increased synaptogenesis and number of glutamate vesicles, but not glutamate receptors. of action14. Uniquely, these drugs are not prescribed, but instead given only once in a therapeutic setting guided by a

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The antagonist completely blocked the psychedelic’s effects, confirming their hypothesis. This was repeated with DMT and LSD, and the same results were replicated. Lastly, treatment with rapamycin and the consequential blockade of mTOR, a downstream signalling molecule activated by TrkB signalling, also inhibited neuritogenesis and spinogenesis. Previous work has shown this to be the same pathway through which ketamine acts13.

In-vivo analysis on Drosophila, Sprague-Dawley rats, and Zebrafish

Investigating the pathways involved: The efficaciousness of different psychedelics was tested and quantified as a percentage relative to the arbour complexity produced by 10µM of ketamine. This test was done in a dose dependant manner (Figure 2C). While all of these compounds are known to bind to 5-HT2A receptors, the authors found that none had hill slopes of 1.0, suggesting polypharmacology (Figure 2C). To confirm that this receptor was responsible for the structural changes, they co-treated this neurons with ketanserin, a selective 5-HT2A antagonist. This abrogated the ability of any of these psychedelics to induce an increase in the area under the sholl curves (Figure 2A). The authors further observed that a compound’s potency at inducing structural plasticity correlated with its affinity for the 5-HT2A receptor, with LSD being the most potent.

Encouraged by their in-vitro results, authors treated Drosophila larvae during both the first and third instar with vehicle, LSD, or DOI. Results indicated that class I sensory neurons increased their number of branches with treatment, with no change in dendritic length. This was independent of how mature the neurons were (first or third instar). Zebrafish embryos were also treated with the compounds of 6 days, but no statistically significant difference in morphology was observed.

Rats were given an intraperitoneal injection of 10mg/ kg DMT, after which PFC neurons were imaged using a Golgi -cox stain. This dose was chosen because it had been shown to improve symptoms in animal models of depression, and is the same dose used during studies of ketamine19,21. Yet Since ketamine is known to increase levels of BDNF, again, a significant increase in dendritic spine density was authors also measured this neurotrophin’s mRNA and protein observed. Animals were then sacrificed, and the electrophysilevels after treatment using droplet digital PCR (ddPCR) and ological properties of their neurons were investigated ex vivo ELISA. Tthere was no change in mRNA levels, and a statistiusing voltage clamped whole cell recordings. Results showed cally non-significant doubling of protein levels. This does not that both amplitude and frequency of spontaneous excitatory align with other studies that found an increase in BDNF postsynaptic currents increased. This implies that the struc20 mRNA expression after agonism of the 5-HT2A receptor . tural changes are accompanied by functional ones, and that Authors then treated cortical neurons with BDNF, DOI, or these persist for hours after the DMT had already been broboth in order to investigate if the two had synergistic or addiken down in the body. tive effects. Sholl analysis indicated that all three treatment groups had comparable effects (Figure 2B), suggesting that DISCUSSION: the two compounds work through similar pathways. In order The study’s results show that three different psycheto confirm this hypothesis, cortical neurons were treated with delics were able to increase structural and functional plasticity DOI or a combination of DOI and ANA-12, a TrkB antagonist.

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points examined, since Vaidya et al. fixed the brains 3 hours after treatment, whereas this study treated cultures for 24h before extracting RNA for ddPCR17,20,. Emotional regulation largely involves the prefrontal cortex, which is hypofunctioning in people with mood and stress disorders9-11. As people age, they become better at controlling emotions, and this tracks well with the development of the PFC22. This region is also important in decision making, working memory, goal-directed learning, and regulation of attention22. It is thus reasonable to presume that any molecule increasing structural and functional plasticity in this region should also result in cognitive benefits. The literature supports this, as animals models have shown improved learning when animals are treated with DMT, LSD, or ketamine19,23-25. While clinical trials to date have only measured the effects of these compounds on mood, this suggests that they can also alleviate cognitive deficits associated with depression. Other compounds have with similar psychoplastogenic effects have been found, including mGluR2/3 antagonists, TrkB agonists, and the muscarinic antagonist scopolain vitro and in vivo. They observed that this effect was demine17. This suggests a common mechanism, where drugs pendant on the 5-HT2A receptor and subsequent TrkB and mTOR signalling. These findings are very significant, as they acting on different receptors all convergently increase TrkB are the first to report a detailed account of common molecular signalling. Compellingly, exercise also has potent antidepressant and anxiolytic properties, and has also been shown to mechanisms through which different psychedelics act. The increase BDNF levels26,27. These compounds warrants further authors coined the term “psychoplastogens” to describe investigation, as BDNF supplements do not cross the blood these compounds and their ability to induce neuritogenesis and spinogenesis. While these studies were done on rats, the brain barrier in vivo and are thus not feasible for treatment28. same effect was seen in Drosophila suggesting that this sig- The ability to induce rapid plasticity has greater implications beyond mood disorders. Therapeutic potential includes neunalling is evolutionarily conserved. It is thus likely that the rodegeneration, stroke, trauma, etc. same signalling mechanisms are at work in humans. While these psychedelics were shown to be polypharmaceutical, both the use of receptor antagonists and the correlation reported between receptor affinity and psychoplastogenic potency provides compelling evidence that this is the pathway through which signalling occurs. Furthermore, changes in EPSC are seen long after treatment, supporting the single-dose model currently being practiced in clinical trials. Notably, human clinical trials formerly mentioned have used psilocybin as the psychedelic of choice, which was not tested in this study14-16. However, the fact that psilocybin also binds the 5-HT2A receptor, and the fact that it also works using a single-dose model, makes it likely that it also acts through this common mechanism. The authors followed the same protocol used to study Ketamine in order to highlight their similarities, and actually found LSD to be more potent17,21. Treatment of neurons with just serotonin does not have the same effects on structure and function, which is interesting since this would presumably also to bind the 5HT2A receptor. This suggests that the effects of psychedelic compounds are more nuanced and cannot be explained by a simple increase in monoamine transmission. This paper found that treatment with psychedelic compounds had no effect on BDNF mRNA levels, but this was discordant with the literature. Vaidya et al. found both increased expression in the neocortex and decreased expression in the hippocampus after rats were intraperitoneally injected with DOI20. This could be due to the differential time

CRITICAL ANALYSIS This study very thoroughly addressed and confirmed it’s hypothesis. Many experiments were conducted to elucidate the molecular, morphological, and functional changes seen at the cellular level, which might explain the mental and behavioural changes reported in the clinic. All methods used, such as sholl analysis, golgi-cox stains, and patch clamps, are well established key techniques in neuroscience. That being said, some are quite dated. More advanced techniques, such as two photon microscopy, could have been used for in vivo dendritic analysis29. However, by following the same protocol used to study ketamine, the authors have created a standard through which all future pscyhoplastogens can be tested17,21. While doses were used to maintain internal validity with ketamine experiments, they were still inordinately high relative to concentrations reached in humans. Maximum human blood concentrations of LSD after a 200µg dose is 13.9nM, almost 1000x less than the 10µM concentration used in this study30! The authors included an analysis of dose dependant responses in the supplementary data, where it is evident that a 13.9nM dose would roughly be 60% as efficacious in increasing arbour complexity (Figure 2C). Regardless, cell cultures were treated with the compounds for 72 hours, which is extremely physiologically inaccurate. Many of these drugs have short half lives, i.e 3 hours for LSD and 1219 minutes for DMT30,31. The in vivo data accounts for this

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confounding variable, but the dose given to the rats was also very high. Using conversion equations described under FDA guidelines, a 10mg/kg dose in rats roughly equates to 1.6mg/ kg in humans32. Contransingly, studies on Ketamine and DMT in humans use a maximum dose of only 0.5 and 0.4mg/kg respectively33,34. The external validity of these experiments is thus questionable, and more data must be collected using physiologically accurate paradigms to determine if the results are still applicable. Indeed, one experiment using only 1ÂľM of DOI found only a transient increase in spine size that reversed soon after, and no effect on spine density in cortical neurons35. It can be argued that Ly et al., the authors of the study under review, are also too quick to conclude that this increased plasticity is necessarily a good thing. There are many examples were increased complexity of spines and dendrites are associated with negative outcomes. For example, even though the nucleus accumbens is associated with reward, chronic stress results in increased spinatogensis in that region36. Ly et al. thus rely too heavily on linear top-down processing when assuming that increased plasticity in a region associated with positive outcomes will necessarily accentuate these positive outcomes. To further this point, in vivo rat experiments have shown that cocaine, nicotine, and Phencyclidine all increase the number of dendrites and spines in the medial PFC37. Notably, this was after multiple exposures and not a single dose, but the point holds true; the increased plasticity induced by these other drugs is unlikely to result in behavioural benefits.

FUTURE DIRECTIONS

There is currently debate in this field regarding whether the hallucinogenic effects of these compounds are necessary for their antidepressant and anxiolytic properties40. As aforementioned, other psychoplastogens exist, and structurally similar compounds that affect plasticity without altering the nature of perception are being investigated and have been tested on animals17. In order for this debate to be settled, the effectiveness of these drugs must be tested in clinical trials on humans. If it mirrors the antidepressant effects of ketamine and Psilocybin, then it will mean that the hallucinoThere are many reports of people having extremely genic effects are not necessary. This would open the doors traumatizing experiences while under the influence of psychefor a completely novel approach for the treatment of mood delic compounds, and long-lasting adverse effects on mental disorders. health38. Researchers in the clinical side of this field stress the importance of cultivating a safe and welcoming mind set One interesting finding reported by Ly et al. was that as well as a therapeutic environment that minimizes the serotonin did not produce the same psychoplastogenic efchance of an aversive experience. The authors of this paper fects as the other 5-HT2A agonists, presenting an interesting do not take this into account, and found plasticity to increase avenue for future research17. According to the theoretical in all rats and neuron cultures treated with these compounds, pharmacologist Terry Kenakin, G-protein coupled receptors suggesting this effect to be independent from subjective excan have multiple different activated conformational states, periences17. One common feature of human psychedelic reeach activating different pathways (Figure 4) 41. Moreover, ports is that they are extremely salient and affect behaviour binding of ligands can favor different conformation states, for a long period of time. For example, one study found that potentially explaining the variation in agonist activity42. The use of psilocybin resulted in a significant increase in opencurrent hypothesis in the literature is that LSD shows preferness, one of the five factor personality traits, that persisted for ence for the PLA2-AA pathway, whereas serotonin activation an entire year39. The psychoplastogenic effects of these com- results in more signalling via the PLC-IP pathway42. There are pounds might better explain the salience and persistence of multiple ways to test this; the structure of serotonin bound 5these experiences, rather than their antidepressant qualities. HT2A receptors can be investigated using Lipidic cubic phase Effects on mood are likely more contingent on the valence of crystallization and compared to already existing crystallized the subjective experience one would have under the influence images of this receptor bound to LSD43. Also, the response of these compounds. Much more testing is needed to parse of Gi/o and Gq/11 knockout models can be compared in reout these two different possibilities. With the recent desponse to LSD and serotonin treatment. Gi/o is involved in the stigmatization of psychedelics and the renaissance of rePLA2-AA pathway, therefore if the hypothesis is correct, Gi/o search papers investigating them, it will be exciting to see the KO would render subjects insensitive to the hallucinogenic progression of this field in the coming decades. effects of this compound, and Gq/11 KO should have a minimal effect42. The use of a Cre-lox system to only induce knockouts in the serotonergic system would result in less confounds. Lastly, RNA-seq or RT-qPCR can be used to analyze the differential transcriptomes of cortical cells in response to either serotonin or LSD, which can implicate the activation of

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different signalling pathways. For example, increased expression of MAPK1 and MAPK3 would implicate Gi/o signalling, and if this is seen in cells treated with LSD then it would help confirm this hypothesis42. RNA-seq can also be used to study the different transcriptomes of cells treated with ketamine, scopolamine, and LSD. These three bind different receptors, yet cause similar psychoplastogenic effects. By comparing the similarities and differences in signalling, it could give a better idea as to how they convergently result in increased TrkB stimulation. After investigating that associative data, knock out models or GFP tracking studies can be used to confirm proposed signalling mechanisms44.

While this paper focused on the molecular effects, future studies can implement a systems approach to investigate antidepressant mechanisms of these compounds. Data garnered from fMRI studies in humans show that psychedelics reduce activity in the default mode network, which consists of the posterior cingulate cortex and medial prefrontal cortex45. This is interesting, because the same pattern of decreased BOLD signalling is seen in expert meditators, an activity that has also been shown to have potent antidepressant and anxiolytic activity46. To test causality, animals studies can be conducted were optogenetics are used to inhibit this network. Viral injections should be placed at one site i.e PCC, but optrodes should be implanted at the distal site i.e mPFC in order to selectively inhibit communication between these two regions, and not just bluntly inactivate the entire regions. Archaerhodopsin should be used instead of halorhodopsin in order to induce more long lasting inhibitions and thus increase the external validity of this experiment47. Afterwards, the forced swim test and the sucrose anhedonia test can be used to investigate the hypothesis; which is that inhibition of this network will reduce behavioural symptoms of depression. Lastly, a trend currently gaining traction is microdosing, where subjects are taking extremely low doses of these compounds many times a week48. Practitioners of this trend report similar mood benefits along with an increase in creativity. Using the same standard now established by Ly et al., an experiment can be conducted to investigate whether repeated low dose exposure results in similar effects on plasticity. Molecular signalling is nuanced such that varying concentrations of the same signalling molecule can have very different downstream effects. That being said, an increase in plasticity would still be the expected result after multiple low dose exposures.

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The Effect of Zeta Inhibitory Peptide on Behavioral Patterns and Synaptic Plasticity Caused by Cocaine Use

Dorottya Harangi

One of the reasons that cocaine users have cravings during abstinence is cocaine-induced plasticity, which means that cue-evoked cravings still persist, even in the absence of drug use. An important factor underlying addiction is the inability to eliminate memories associated with taking the drug. Zetainhibitory peptide (ZIP) has been used in memory studies and has been shown to disrupt cocaineinduced synaptic potentiation and cocaine sensitization. However, the specifics of the ability of zetainhibitory peptide to reverse altered behavioral patterns and synaptic plasticity have not yet been explored in extensive detail. To elucidate the properties of ZIP, the researchers examined the ability of ZIP to alter cocaine-induced behavioral activity and synaptic plasticity and the effect of ZIP on long term depression (LTD) in the nucleus accumbens (NAc) after cocaine self-administration. The researchers found that a single intra-accumbal injection of ZIP can block cocaine reinstatement (RI) up to a week later and these effects of ZIP do not generalize to natural rewards as RI of sucrose seeking was not affected. Using intra-accumbal slices from control and cocaine-experienced mouse brains, they also found that ZIP rescues cocaine-induced deficits in NMDA and mGlur5 dependent LTD without affecting saline controls as well as lowering the increased AMPA/NMDA ratio. Therefore, it is possible that the mechanism for zeta-inhibitory peptide is related to its ability to weaken synaptic connections and restore LTD, which would lead to the extinction of cocaine-primed reinstatement. Key words: zeta inhibitory peptide, LTD, nucleus accumbens, synaptic plasticity, reinstatement, behavioral activity

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INTRODUCTION

MAJOR RESULTS

Cocaine dependence is a very serious addiction that is categorized as a chronically relapsing disorder, as many individuals with the addiction relapse even after decades of abstinence (Deutschmann, Lenz, McGrath, & Briand, 2019). There are a variety of different variables that contribute including molecular changes in the brain as well as demographics such as age, race and socioeconomic status (Bickel, Snider, Quisenberry, Stein, & Hanlon, 2016). The nucleus accumbens (NAc) is a part of the brain important in addiction studies as it is part of the reward pathway and plays a role in the development and expression of drug related behaviors (Dong & Nestler, 2014). The use of cocaine induces long term changes in the structure of the NAc, which causes it to preferentially respond to drug related cues and relay them into behaviors (Dong & Nestler, 2014). Neurons in the NAc undergo changes, such as an increase in Îą-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, that make this brain region more susceptible to cocaine cues, even after months of abstinence (Wolf, 2016). Extracellular dopamine concentrations have been found to increase in the NAc in cocaine addiction and this modification of dopamine concentrations contributes to the abusive properties of cocaine (Di Chiara & Imperato, 1988).

ZIP reduces cocaine-cued but not sucrose-cued reinstatement To test the effects of ZIP on cocaine seeking activity, rats first underwent 18 days of cocaine self-administration followed by 7-10 days of extinction. 24 hours after reaching extinction they were given a cocaine-primed RI test where the rats exhibited significant RI of cocaine seeking. 2 hours after they reached extinction criteria, they were given either ZIP or SCR-ZIP, which is a scrambled inactive form of peptide, and then were tested again either 24 hours or 1 week later on cocaine-primed RI. The rats that had received the ZIP microinjection did not exhibit RI in the 24 hour or 1-week group, while the SCR-ZIP group did. To test if the effects of ZIP applied to natural rewards, such as sucrose, as well, a separate group of rats underwent 18 d of sucrose self-administration and extinction and were then exposed to a sucrose primed RI. 2 hours after rats reached extinction, they were given ZIP or SCR-ZIP. 24 hours later, the rats were tested again on sucrose primed RI, and both groups exhibited significant RI on the post test. Another study done in 2012 also found that ZIP was able to get rid of conditioned place preference (CPP) caused by cocaine by inhibiting PKMz expression in the NAc shell specifically, which is where extracellular dopamine levels are increased during addiction (Shabashov, Shohami, & Yaka, 2012). The NAc shell is where drug cravings and relapse are controlled and similarly to the current study they were able to reduce drug cued behavior (Shabashov et al., 2012).

Zeta inhibitory peptide (ZIP) is a protein kinase C (PKC) specific inhibitor that is capable of abolishing certain types of memories as well as long term potentiation (Deng, Lubinski, & Page, 2015). Studies done with hippocampal ZIP infusions in mice have shown impairment in the ability to retrieve recent spatial memories, leaving remote spatial memories unaffected (Hales, Ocampo, Broadbent, & Clark, 2016). ZIP has been used a various studies to show that PKC isoform protein kinase M-z (PKMz) is necessary for long term potentiation (LTP) maintenance and long term memory, however other studies have shown that ZIP is able to reverse LTP in both PKMz and PKMz-KO mice, proving that the effect of ZIP is not dependent on PKMz (Volk, Bachman, Johnson, Yu, Figure 1 (Deutschmann et al., 2019): The effect of ZIP and & Huganir, 2013). SCR-ZIP on reinstatement in cocaine and sucrose primed mice It is known that cocaine alters the structure of the C) ZIP prevents cocaine-cued reinstatement (RI) up to a week NAc which causes increased response to cocaine cues and later in mice, while SCR-ZIP has no effect and RI persists D) reduces long term depression (LTD) in D2-dopamine receptor ZIP and SCR-ZIP administered 24 hours prior to post-test have expressing neurons in the NAc (Dong & Nestler, 2014) no effect on sucrose RI (Heinsbroek et al., 2017). The authors were looking at the ability of ZIP to alter cocaine-induced behavioral activity and ZIP able to block RI in PKMz-KO mice synaptic plasticity and the effect of ZIP on LTD in the NAc after cocaine self-administration. The authors examined the effect that ZIP had on the reinstatement (RI) of cocaine addicTo examine if the effects of ZIP were dependent on tion in rats and mice that were allowed to self-administer the PKMz, researchers used wild-type and PKMz-KO mice that drug. They also looked at the effect a bath application of ZIP had 10 days of food training followed by 10 days of cocaine had on LTD in NAc slices. They found that ZIP administration self-administration, then extinction. These mice where then was able to reduce cocaine-cued behavior as well as rescue given 30 mmol injections of ZIP or SCR-ZIP 2 hours after the LTD in the NAc. last extinction, and then underwent cue induced RI. Both wildtype and knockout mice that had received the ZIP mi176

-croinjection did not exhibit RI, while the SCR-ZIP groups did. LTD levels in control, saline-yoked mice B) The application of ZIP helps to restore the levels of NMDAR-LTD in NAc brain slices of cocaine-experienced mice that were reduced due to cocaine self-administration CONCLUSIONS/DISCUSSION

The authors of the current 2019 paper found that a single intra-accumbal injection of ZIP can block cocaine reinstatement (RI) up to a week later and these effects of ZIP do not generalize to natural rewards as RI of sucrose seeking was not affected. They also found that ZIP rescues cocaineinduced deficits in NMDAR and mGlur5 dependent LTD without affecting saline controls. The researchers concluded that PKMz was not necessary for the effects of ZIP and that it was Figure 2 (Deutschmann et al., 2019): The effect of ZIP and able to disrupt both the behavioral and synaptic plasticity SCR-ZIP on wildtype mice and mutant (PKMz-KO) mice A) that is associated with cocaine use. They suggested that the both wildtype and mutant mice show no response to coability for ZIP to disrupt cocaine-associated behavior was caine, demonstrating extinction B) after cocaine-priming both wildtype and mutant mice given ZIP show no reinstate- due to its capability to weaken long term depression ment (RI) while both groups of mice given SCR-ZIP show sig- (Deutschmann et al., 2019). nificant RI There have been a few other papers showing that the administration of ZIP can erase reward memory in the Bath application of ZIP rescues NMDAR and mGluR5 mediat- form of conditioned place preference in mice that underwent cocaine self-administration (Howell et al., 2014), and ed LTD one paper done in 2013 specifically examined PKMz-KO mice and found that the effect of ZIP was still present in these The researchers looked at two different types of mice (Lee et al., 2013). The study done in 2019 is important LTD, N-methyl-D-aspartate receptor (NMDAR) dependent and mGluR5-dependent. For both types they took brain slic- because it validates these earlier studies and provides further evidence in support of their findings. In addition, the es of NAc from control, saline yoked C57BL/6J mice which current paper also examined two specific types of LTD in the exhibited robust LTD and added ZIP via a bath application. They found that the addition of ZIP did not alter LTD expres- NAc and found that both NMDAR and mGluR5-dependent sion. The authors then took brain slices of NAc from cocaine- LTD were rescued via a bath application of ZIP which was a novel finding (Deutschmann et al., 2019). addicted mice that underwent 10-14 days of selfadministration and 10-14 days of withdrawal. In these mice both NMDAR and mGluR5-mediated LTD was very low or gone. When a bath application of ZIP was administered and a low train frequency stimulus (1 Hz, 10 min) was applied, both types of LTD were rescued in the NAc. Similar to an earlier study done in 2014, they found that AMPA receptor density was reduced when ZIP was applied (Howell et al., 2014), reversing the increase in AMPA/NMDA ratio that was found in the NAc of cocaine-experienced mice.

The importance of these findings is that there is currently no drug that has been shown to be effective at treating cocaine use disorder (Shorter, Domingo, & Kosten, 2015). Although there have been various drug treatments proposed, they have all had various problems such as addictive liability, medical safety concerns or a variety of side effects (Shorter et al., 2015). Given that cocaine abuse can be considered a global burden and this burden has increased quite significantly over the past 2 decades, it is important that mechanisms be developed to treat this disorder (Degenhardt et al., 2014). CRITICAL ANALYSIS

The authors examined some of the limitations such as the lack of explanation in their article about the mechanisms underlying how ZIP works to restore both NMDAR and mGluR5-mediated LTD. The authors proposed that it would Figure 3 (Deutschmann et al., 2019): The effect on long be related to the removal of AMPA receptors from the synterm depression (LTD) of a bath application of ZIP on nucleus apses, but this is a question that would require further study accumbens (NAc) slices from control and cocaine experito understand the specific mechanisms underlying the two enced mice A) the application of ZIP does not affect regular types of LTD and the common mechanism that allows ZIP act 177

on both of them (Deutschmann et al., 2019). The authors also used forced abstinence as opposed to active extinction in their electrophysiological experiments. There have been some proposed differences between the two methods that suggest differences in mGluR5 trafficking which leads to blunted LTD only in extinguished rats, not abstinence rats (Knackstedt et al., 2010). This study showed LTD reinstatement under the conditions of forced abstinence, so follow up studies would need to be performed to see if ZIP would have the same effect on extinction studies as it does on abstinence as the mGlu5R trafficking and the protein expression in the NAc is somewhat different (Knackstedt et al., 2010). In addition, although the authors found similar results with PKMz-KO mice as they did with mice that had PKMz intact, it is still possible that these mice have developed compensatory mechanisms such as PKCÎš/Îť which would suggest that some molecule for long term memory storage is present that is responsive to ZIP (Tsokas et al., 2016). Therefore, the authors cannot conclude with certainty that ZIP is completely independent of the PKMz pathway.

hypothesized led to an inhibition in cocaine seeking behavior in these mice as compared to abstinence mice (Knackstedt et al., 2010). If ZIP is able to reverse plasticity to the same extent in extinction and abstinence mice, then they can conclude that although there are differences in presentation the two conditions are similar enough to be used interchangeably. However, if there are major differences in the extinguished versus abstinence mice, such as the inability of ZIP to rescue extinction based LTD, which can be mimicked via DHPG-LTD, then this would show that there are major differences in the way that the two conditions manifest and futures studies would need to ensure they justify their use of a specific condition.

An important thing to keep in mind is that individual variations in animals such as impulsivity, saccharin intake, and age and gender, are important and can greatly influence how they react to drugs and drug treatments (Regier, Claxton, Zlebnik, & Carroll, 2014), therefore it is possible that some of the variation in results can be attributed to such characteristics. For example, only male rats were used in the experiments and it has been found that a greater percentage of male rats reach acquisition criteria for cocaine selfadministration and they meet this acquisition criteria faster, however once the criteria are met, female rats selfadminister a greater amount of cocaine than males during the maintenance phase (Swalve, Smethells, & Carroll, 2016). Another group also found that male rats have higher cocaine methyl esterase and ethyl transferase activity than female rats do, indicating a sex-based difference not only in behavior but in cocaine metabolism as well (Zhang, Dean, Brzezinski, & Bosron, 1996). FUTURE DIRECTIONS

A possible future experiment would be to look at NAc brain sections of extinction versus abstinence mice when doing the electrophysiology experiments with LTD to see if (S)-3,5-dihydroxyphenylglycine-induced (DHPG) LTD is also disrupted. The experiment would have 3 conditions, salineyoked controls, mice that self-administered cocaine then underwent active extinction and mice that self-administered cocaine then underwent forced abstinence. The electrophysiology experiments performed in the article would be repeated, looking at mGluR5-mediated LTD specifically, as that is the type of LTD that DHPG induces, to see if there are any differences between the two cocaine administered conditions. Previous studies have shown that in extinguished mice there is reduced mGluR5-mediated LTD which the authors 178

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Examination of the Effects of Acetaminophen on Empathic Behaviour and the Neuropeptides involved in Empathy

Eisha Haroon

Empathy is a psychological construct that involves identifying another personâ&#x20AC;&#x2122;s affective states and responding appropriately. Current research has shown that painkillers, such as acetaminophen, have detrimental effects on complex social behaviours and processes like empathy. In particular, studies have displayed that those given acetaminophen show blunted responses to emotional stimuli, and decreased empathy towards others experiencing pain. The specific mechanism of action has yet to be elucidated. Oxytocin and vasopressin are thought to play crucial roles in the process of empathy, specifically in the prefrontal cortex and the amygdala, two brain regions shown to be active during empathy evaluations and behaviours. Kandis et al. studied the effects of different dosages (100, 200, 400mg/kg) of acetaminophen on empathic behaviours in rodents and examined changes oxytocin and vasopressin levels in the prefrontal cortex and amygdala in 2018. The authors found a linear dose-response relationship between acetaminophen and empathy-like behaviours. A single high dose of acetaminophen (400mg/kg) caused a significant delay in empathy-like behaviour. Furthermore, subsequent administrations of acetaminophen (200 and 400mg/kg) administered daily also corresponded with a decrease in empathy behaviours. The authors found that all doses of acetaminophen were correlated with decreases in oxytocin in the prefrontal cortex and amygdala compared to control groups of rodents given equal amounts of saline. In addition, acetaminophen was also correlated with decreased levels of vasopressin in the amygdala for all doses, and in the prefrontal cortex for rodents given 200 and 400 mg/kg of acetaminophen. These results are significant as they shed light on the neurological underpinnings of empathy processing and provide further insight into how acetaminophen negatively impacts empathy in rodents, which is applicable to the understanding of human empathy. This is crucial information as acetaminophen is one of the biggest over-the-counter analgesics available, and clearly has unwanted effects on social behaviours. Key words: empathy, prosocial behaviours, acetaminophen, oxytocin, vasopressin, prefrontal cortex, amygdala

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BACKGROUND and INTRODUCTION Empathy is multi-faceted psychological construct that impacts social interactions and prosocial behaviour in everyday life (Bernhardt & Singer, 2012; Lamm, Rütgen, & Wagner, 2019). Empathy can be defined as the ability to accurately perceive and identify another person’s (or target’s) affective state, and appropriately respond to that state (Barraza & Zak, 2009; Kandis et al., 2018). For instance, upon recognising another person is sad, this can lead to sympathising for the other person, which can result in prosocial behaviour towards the person experiencing sadness (Bernhardt & Singer, 2012). Prosocial behaviour can be approached as a behaviour that is done to intentionally benefit others (Eisenberg, Fabes, & Spinrad, 2007). There are three distinct components that have been identified to be involved in human empathy: mentalizing, experience sharing, and prosocial concern (Zaki & Ochsner, 2012). Mentalizing is the cognitive aspect of empathy and considers the ability to make inferences about mental states, whereas experience sharing is the capacity to affectively share the emotional experience of others (Zaki & Ochsner, 2012). The final component is the ability to use the information garnered from the other two processes and act in manner that helps the other person (Zaki & Ochsner, 2012). The brain regions implicated in empathy include the medial prefrontal cortex, which is specifically involved in the process of mentalizing, and the amygdala, which is part of the emotional and affective component of empathy (Kandis et al., 2018; Zaki & Ochsner, 2012). Evidence suggests that two of the main neuropeptides involved in the process of empathy are oxytocin and arginine vasopressin, which is similar in structure to oxytocin (Hurlemann et al., 2010; Kandis et al., 2018; Meyer-Lindenberg, Domes, Kirsch, & Heinrichs, 2011). In particular, oxytocin has been found to be involved in bonding, trust, and emotion recognition, and vasopressin has been implicated in the processes of emotional empathy and prosocial behaviours (Hurlemann et al., 2010; Meyer-Lindenberg et al., 2011; Wu, Shang, & Su, 2015).

The capacity for empathy and prosocial behaviour is not limited to humans. Previous research has shown that animals can experience emotional empathy, whereas cognitive empathy has only been observed in humans, primates, and rodents (Sivaselvachandran, Acland, Abdallah, & Martin, 2018). In light of this, rodent models have been used in empathy and pain research due to the shared capacity between rodents and humans to engage in cognitive empathy (Sivaselvachandran et al., 2018). Furthermore, the same brain regions and neuropeptides involved in human empathy have been shown to be involved in empathic processes in rodents. In previous psychology studies, it has been shown that painkillers, such as acetaminophen (paracetamol) have the ability to blunt emotional responses and reduce empathic behaviours in humans (Durso, Luttrell, & Way, 2015; Mischkowski, Crocker, & Way, 2016, 2019). This is concerning as acetaminophen is one of the largest over-the-counter analgesics available, and its mechanism of action and impact on empathy is still relatively unknown (Kandis et al., 2018).

ther understand the impact acetaminophen has on the neuropeptides implicated in empathy (oxytocin and vasopressin) by studying changes in prosocial behaviours in Sprague Dawley rats given acetaminophen at different doses. In this study, two rats (that were harbored together in the same environment) were placed in a cage (the Helping Behaviour Test adapted from (Sato, Tan, Tate, & Okada, 2015) where one rat was placed in some water, and the other rat was behind a door that connected to the water-filled area (Figure 1). Rats learned to open the door to save the rat in distress in water over 12 days (Kandis et al., 2018). The time taken to open the door to help the other rat acted as a behavioural measure of empathy. This “empathy” test has been used in a previous study which displayed that rats do show prosocial behaviours to help other rats in distressed, i.e. soaked in water (Sato et al., 2015). The “helper” rats were given different doses of acetaminophen (100, 200, and 400 mg/kg) with equal doses of saline given to the control group; 30 minutes after the dose was administered, empathic behaviour was tested, and this process was repeated for 11 days (Kandis et al., 2018).

Figure 1. Empathy behaviour test equipment used in Kandis et al., 2018 Kandis et al., adapted the protocol from the Helping Behaviour Test from Sato et al., 2015. The time taken for the rat on the right side of the cage (not in water) to open the door to save the other rat was used as a behavioural measure of empathy. The longer the time taken to open the door, the less empathy the helper rat displayed.

Blood and brain tissue samples were obtained to evaluate changes in oxytocin and vasopressin after exposure to acetaminophen, and whether any changes correlated with differences in empathy behaviour (Kandis et al., 2018). Through this method, the effects of acetaminophen on the neuropeptides directly involved in empathy were assessed. The results showed that high doses of acetaminophen decreased empathy-like behaviour, and continual acetaminophen administration, at relatively low doses, also decreased empathy behaviours (Kandis et al., 2018). Furthermore, acetaminophen was found to decrease levels of oxytocin and vasopressin in the prefrontal cortex and amygdala (Kandis et al., 2018). These results can have implications on our understanding of acetaminophen as an analgesic, and how it could negatively influence human social interactions.

MAJOR RESULTS Behavioural Results

In a study conducted in 2018, Kandis et al. attempt to fur181

Kandis et al., judged changes empathic behaviour based

on the mean latency for opening the door in the Helping Behaviour Test (Figure 1). The authors found that after being given a single high dose of acetaminophen (400mg), the mean latency for opening the door was significantly increased compared to other dosage groups (Figure 2) (Kandis et al., 2018). Continual daily administrations of acetaminophen in all test groups were correlated with significant increases in time taken for the helper rats to open the door to save the other rat (Kandis et al., 2018). The time it took for the helper rat to open the door was found to be significantly increased for all 11 days of testing in the 400mg/kg group, and significantly increased since Day 5 in the 200mg/kg group (Figure 3) (Kandis et al., 2018). The authors also tested for changes in anxiety in rats treated with acetaminophen using the Open Field Test and the Elevated Plus Maze Test, and assessed any possible changes in motor coordination using the Rotarod Performance Test (Kandis et al., 2018). No significant differences in anxiety were found between test groups and control in both tests (Kandis et al., 2018). In addition, locomotor activity was not affected by the acetaminophen dosage (Kandis et al., 2018). As such, any changes in mean latency can be linked directly to the acetaminophen dosage and not an intermediate effect of change in locomotor ability or anxiety. Therefore, based on the results, it is evident that acetaminophen decreases empathy-like behaviours in rodents (Kandis et al., 2018). Furthermore, it was found that there was a linear dose-response relationship, where increases in acetaminophen dose were correlated with increases in mean latency for opening the door in the Helping Behaviour Test (Kandis et al., 2018). This follows the results from a previous human psychology study that displayed that acetaminophen reduced empathy for pain (Mischkowski et al., 2016).

were significantly lower in the amygdala tissue in all experimental groups compared to the control (Figure 5). In the prefrontal cortex, vasopressin levels were only significantly lower in rats given 200 and 400 mg/kg of acetaminophen (Figure 5) (Kandis et al., 2018).

Figure 3. Mean latency for opening the door in the Helping Behaviour Test for rats given different doses of acetaminophen across 11 testing days (Kandis et al., 2018) Continual high doses (200 and 400 mg/kg) of acetaminophen were correlated with increased time taken to open the door.

Figure 4. Changes in oxytocin levels in the prefrontal cortex and amygdala of rats given different doses of acetaminophen (Kandis et al., 2018) Rats in all experimental groups exhibited significant decreases in oxytocin in the amygdala and prefrontal cortex (PFC)

Figure 2. Mean latency to open the door of the Helping Behaviour Test in rats given different single-dose administrations of acetaminophen (Kandis et al., 2018) Only in the 400mg/kg group was mean latency for opening the door significantly increased after a single dose of acetamino-

Biochemical Results Kandis et al., also measured levels of oxytocin and vasopressin—neuropeptides that have been found to be involved in empathy and prosocial behaviour—in the prefrontal cortex and the amygdala, both of which have been shown to be active during empathy judgements (Hurlemann et al., 2010; Kandis et al., 2018; Meyer-Lindenberg et al., 2011). The authors found that oxytocin levels were significantly lower in both the prefrontal cortex and the amygdala in all test groups compared to control (Figure 4) (Kandis et al., 2018). Vasopressin levels

A significant negative correlation was found between the level of oxytocin in the prefrontal cortex and empathy behaviour: the mean latency for opening the door in the Helping Behaviour Test (Kandis et al., 2018). Kandis et al., were one of the first studies to establish the direct negative impact acetaminophen has on the neuropeptides associated with empathy in the crucial brain regions (2018). The authors results follow previous evidence that oxytocin and vasopressin are integral in emotion cognition and complex social processes behaviours like empathy (Hurlemann et al., 2010; Kandis et al., 2018; Meyer-Lindenberg et al., 2011). They also established a correlational relationship between oxytocin levels in the prefrontal cortex and empathy-like behaviour (Kandis et al., 2018). The prefrontal cortex has not only been shown to be involved in the mentalizing aspect of empathy, but the level of activity in the prefrontal cortex has been found to be predictive of prosocial behaviour (Majdandžić, Amashaufer, Hummer, Windischberger, & Lamm, 2016; Zaki & Ochsner, 2012). Therefore, the results from Kandis et al. (2018) illustrate the importance of oxytocin in the prefrontal cortex in empathy processes, and display that acetaminophen seems to have a direct negative effect on oxy-

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oxytocin levels, which in turn negatively impacts empathy be- social behaviour; this supports the findings found by Kandis et haviours in rodents. al., that oxytocin plays an integral role in empathy and prosocial behaviours (Meyer-Lindenberg et al., 2011). The results from Kandis et al. in tandem with other research could have implications for research into the treatment of populations with social impairments, such as people with schizophrenia or autism spectrum disorder, and whether increasing oxytocin and vasopressin in the brain could help these populations (Kandis et al., 2018; Meyer-Lindenberg et al., 2011). The authors hypothesize that acetaminophen interferes with the endocannabinoid system, and could potentially affect the serotoninergic system if this were to be the case (Kandis et al., 2018). The novel results from this study shed light into the mechanism Figure 5. Changes in vasopressin levels in the prefrontal cortex of action of acetaminophen, and how it could impact the mechand amygdala of rats given different doses of acetaminophen (Kandis et al., 2018) anisms of empathy. Rats in all experimental groups exhibited significant decreases in vasopressin in the amygdala. Rats given 200 and 400 mg/kg of acetaminophen had decreased levels of vasopressin in the prefrontal cortex (PFC)

CONCLUSIONS/DISCUSSION Kandis et al., found that administrations of a single high dose of acetaminophen, and continual doses 200 and 400mg/ kg of acetaminophen led to less empathy-like behaviour in Sprague-Dawley rats (Figure 2, Figure 3). There appeared to be a linear dose-response relationship (Kandis et al., 2018). Furthermore, any administration of acetaminophen appeared to directly cause a decrease in oxytocin in the prefrontal cortex and the amygdala, and a decrease in vasopressin in the amygdala (Figure 4, Figure 5). Only acetaminophen dosages of 200 and 400mg/kg seemed to cause a decrease of vasopressin in the prefrontal cortex (Figure 5). Crucially, a negative correlation was found between the level of oxytocin found in the prefrontal cortex of the helper rat, and the time it took for the helper rat to save the distressed rat (Kandis et al., 2018). These findings are significant to both empathy research and analgesic research as they emphasize the potential negative side effects of acetaminophen by highlighting that acetaminophen is hindering the neurological mechanisms involved in empathy. This has further implications in human research as this study was one of the first studies to provide direct evidence of the role oxytocin plays in empathy and prosocial behaviours.

CRITICAL ANALYSIS The study conducted in 2018 by Kandis et al., tested the effects of acetaminophen on empathy-like behaviours in rodents, and assessed changes in neuropeptides associated with empathy (oxytocin and vasopressin) in the two significant brain regions active during empathy processes that include both cognitive empathy and affective empathy (the prefrontal cortex and the amygdala). Kandis et al. had two distinct components to their study that other experiments have not previously examined: changes in neuropeptides in rodents given acetaminophen, and changes in empathy-like behaviour across time. One of the advantages of a rodent model in neuroscience research is the ability to take brain tissue samples immediately after the experiment is over. This allowed tests on the concentrations of neuropeptides specifically in brain regions found to be involved in empathyâ&#x20AC;&#x201D;a task which is difficult to accomplish in human studies. The authors were able to directly highlight the importance of oxytocin in the prefrontal cortex during empathy processes and behaviours (Kandis et al., 2018). Moreover, the authors tested the effects of repeated acetaminophen administration on empathy behaviours, which is a novel approach to understanding the potential long-term side effects of acetaminophen (Kandis et al., 2018). As mentioned before, acetaminophen is the largest over-the-counter analgesic available, and taken by close to a quarter of adults in the United States (Kandis et al., 2018; Mischkowski et al., 2016). The unwanted social side-effects of acetaminophen were not established until recently in neuroscience and psychology research, and Kandis et al. were the first to investigate if there could be long lasting impacts if acetaminophen is taken daily; the authors found that relatively small doses of acetaminophen (200mg/kg) when taken daily are correlated with a decrease in empathy-like behaviour across time (Figure 3) (Kandis et al., 2018). The next steps would be to study if repeated acetaminophen exposure would cause irreversible impairment of social behaviours like empathy. The authors noted that the rodents tested had never been exposed to acetaminophen before, which is obviously not possible to maintain when studying human subjects, and is one of the advantages of the rodent model (Kandis et al., 2018).

The results from this study follow that of previous behavioural studies from the field of psychology that showed that acetaminophen blunts emotional responses to both positive and negative stimuli and has shown to decrease the human empathic response to pain (Durso et al., 2015; Mischkowski et al., 2016). Acetaminophen is taken by close to 25% of the adult population in the United States, and these results are serious as a physical painkiller clearly has unwanted side-effects on social behaviours (Mischkowski et al., 2016). Moreover, these results illustrate the importance of the neuropeptide oxytocin in empathy processes like perspective-taking, and its role extends to more species than just rodents and humans (Decety, Bartal, Uzefovsky, & Knafo-Noam, 2016; Kandis et al., 2018). In a study conducted in 2011, oxytocin was administered intranasally, and was found to improve emotion recognition and pro183

There were a few issues to potentially consider in the study conducted by Kandis et al. in 2018. The rats were harbored in the same environment before the experiment. Therefore, it is unclear as to whether there would still be an empathic response in rats had they had no exposure to the rat in distress prior to the experiment. The authors tried to control the concern of emotional contagion by keeping rats separate from each other to limit contact during the experiment, but it is possible that this could have still had an impact on the results, where mean latency to open the door could have been even more increased in helper rats that were unfamiliar with the distressed rat (Kandis et al., 2018). There is the potential criticism that rats have to learn how to operate the door in the Helping Behaviour Test (Figure 1) before the experimental manipulations can be administered, and whether this is truly a measure of empathy-like behaviour. However, an earlier study conducted in 2015 which used the same experimental design (the Helping Behaviour Test), found that rats would still come to the aid of a distressed rat before opening a similar door in the same chamber which led to food (Sato et al., 2015). This thus displays that rats do retain the capacity for empathic behaviour. Sato et al. also found that rats that have previous experience of being soaked were quicker to help distressed rats (2015). Kandis et al. placed both sets of rats in the pool area of the helping behaviour test (Figure 1) during the learning phase of the experiment (2018). It would be interesting to explore if the results found by Kandis et al. in 2018 replicate when the helper rats have had no experience of being soaked before, and whether they would show any empathic behaviour at all if given acetaminophen. In addition, acetaminophen has previously been shown to impact anxiety, however, in this study acetaminophen appeared to have no effect on anxiety levels (Kandis et al., 2018). Considering the authors highlighted the possible link to the endocannabinoid system, we would expect to see a change, specifically a decrease, in anxiety levels in rats given acetaminophen, especially since acetaminophen has anxiolytic effects (Kandis et al., 2018). This remains to be further investigated.

FUTURE DIRECTIONS First and foremost, based on the results from Kandis et al. in 2018, it would be pertinent to further investigate the mechanism of action of acetaminophen in regard to its effects on the neuropeptides oxytocin and vasopressin. As mentioned before, the authors suggest that the endocannabinoid system is involved in the neurological mechanisms of acetaminophenâ&#x20AC;&#x2122;s effect on empathy, as both oxytocinergic and vasopressinergic neurons have cannabinoid receptors at their axon terminals, and are inhibited by activation of those receptors (Kandis et al., 2018). It would be interesting to explore potential knockouts of the gene that encodes the CB1 receptor (cannabinoid receptor) at axon terminals: the CNR1 gene (CNR1 Geneâ&#x20AC;&#x201D;GeneCards, 2019; Kandis et al., 2018). As previous research has indicated that acetaminophen activates the CB1 receptor (Kandis et al., 2018), which would inhibit oxytocinergic and vasopressinergic neurons, we would expect acetaminophen to then have no effect on empathic behaviours in rodents where the CB1 recep-

tor is not expressed, as the CNR1 gene has been knocked out, compared to wildtype rodents. If there is no difference between wildtype and knockout rodents, then there is clearly another neurological system involved that has previously not been explored. Furthermore, if the CB1 receptor activation is the reason that acetaminophen dampens prosocial behaviour in rodents, there should also be a decrease in serotonin levels (which the authors did not directly test in 2018) observed becaused serotonergic neurons also have the CB1 receptor at the axon terminals (Kandis et al., 2018). The aim of these experiments is to further understand the underlying mechanisms of complex social behaviours that acetaminophen seems to negatively impact. Kandis et al. did not study real time changes in brain activity in rodents while they engaged in the empathy-like behaviour (2018). It would be prudent to investigate whether there are changes to activity in the prefrontal cortex in rats given acetaminophen compared to control when placed in the Helping Behaviour Test (Figure 1). Given the changes to empathy-like behaviour, we would expect that the prefrontal cortex shows less activity in acetaminophen-treated rats compared to control when undergoing empathy-inducing tasks, due to the fact that Kandis et al. displayed a correlation between oxytocin levels in the prefrontal cortex and empathy behaviours (2018). One of the novel findings from Kandis et al.â&#x20AC;&#x2122;s study in 2018 is that repeated acetaminophen administration negatively affected empathy behaviours. Therefore, the next question is to test whether this change is irreversible. It would be interesting to replicate the exact experiment and continue to test empathy behaviours in rodents given acetaminophen for 11 days, testing whether the behaviours continue after administration of acetaminophen to the rats has stopped. If reversal of the effect of decreased empathy does not occur, there should be much more research into the unwanted side effects of acetaminophen in humans and whether it should continue to be sold as an over-the-counter analgesic. It is important to note that rats can metabolise acetaminophen much faster than humans (Kandis et al., 2018), therefore all results from research using rodent models are not necessarily generalizable to the human population. Overall, the insights Kandis et al. provide further understanding of the neuropeptides involved in empathic processes and prosocial behaviours, and the brain regions in which they are involved (2018). In addition, the authors also suggest potential neurological mechanisms impacted by acetaminophen which cause detrimental effects to complex social behaviours (Kandis et al., 2018).

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Durso, G. R. O., Luttrell, A., & Way, B. M. (2015). Over-the-Counter Relief From Pains and Pleasures Alike: Acetaminophen Blunts Evaluation Sensitivity to Both Negative and Positive Stimuli. Psychological Science, 26(6), 750–758. https:// doi.org/10.1177/0956797615570366

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Kandis, S., Ates, M., Kizildag, S., Camsari, G. B., Yuce, Z., Guvendi, G., … Uysal, N. (2018). Acetaminophen (paracetamol) affects empathy-like behavior in rats: Dose-response relationship. Pharmacology Biochemistry and Behavior, 175, 146–151. https:// doi.org/10.1016/j.pbb.2018.10.004

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Alzheimer’s Disease Mouse Models Show Scanning Ultrasound Treatment Removes Amyloid-β Peptides and Restores Performance on Memory Tasks Ryan Huang

Alzheimer’s disease (AD) is a progressive neurodegenerative disease that causes problems with memory, thinking and behavior. It is known that the amyloid-β (Aβ) peptide plays a role in the development of AD. Although neurological research has been conducted to look for possible causes for AD and potential treatments, little progress has been made in finding a non-invasive treatment specific to the Aβ peptide. Leinenga and Götz conducted a study to investigate the effect of repeated scanning ultrasound (SUS) treatments on a mouse model of AD through its ability to remove Aβ peptides. Ten male APP23 mice, which contain the Aβ plaques and show deficits in memory function, were treated with SUS treatments over a period of six weeks. Through the use of confocal microcopy and threedimensional reconstruction, it was shown that activated microglia internalized Aβ peptides into their lysosomes after being subjected to SUS. It was found that SUS treatment reduced the cortical area occupied by Aβ plaques by 56%. The study also showed that SUS treated mice performed better on memory tasks through improved results on the Y maze, the novel object recognition (NOR) test and the active place avoidance (APA) task. The results are relevant as they suggest that SUS treatment is a useful and harmless way of restoring memory in AD patients through the removal of Aβ plaques and should be further explored for its noninvasive potential in AD therapy. Key words: Alzheimer’s Disease, Amyloid-β, Scanning Ultrasound, Microglia, Memory

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INTRODUCTION AD is the most common cause of dementia with memory loss as one of its greatest symptoms. There have been decades of research in the field of neuroscience directed towards understanding the physiological mechanisms of the disease as well as possible treatments (Pistollato et al. 2016). Recently, great progress has been made in comprehending the biological mechanisms of AD due to advances in molecular and cell biology techniques (Selkoe 2001). It is generally accepted that the Aβ peptide plays an essential role in the development of AD (LaFerla et al. 2007). These peptides clump into extracellular deposits called amyloid plaques. The plaques are found at a high level in individuals with AD because of increased peptide production and decreased peptide removal (Necula et al. 2007). Although therapeutic strategies centered around Aβ peptides have been explored, they have been invasive and led to negative side-effects (De Strooper et al. 2010). As well, none of these proposed treatments have been able to be translated into successful therapeutic procedures for AD patients. The study by Leinenga and Götz aimed to establish a noninvasive therapy for AD using SUS treatment through multiple linked experiments (Leinenga & Götz 2015). First, C57BL/6 non-Tg wild-type mice were anesthetized, injected with microbubbles and Evans blue indicator dye, and then subject to SUS treatment. It was found that SUS treatment can open up the blood brain barrier (BBB) without causing any harm. Next, Aβ plaque forming AP23 mice were treated with SUS over a six-week period, and they were evaluated for their memory skills using the Y maze. It was revealed that the transgenic mice show increased defections between maze arms in the Y maze. Another batch of transgenic mice were tested for memory functions using the NOR test and the APA test where it was shown that SUS-treated mice performed better on both tests. Following the memory tests, confocal microscopy was used to examine microglia during SUS treatment, showing that the microglia play a role in Aβ clearance. Currently, devices that release ultrasounds capable of penetrating the brain are being tested on humans. Lipsman and his colleagues used an ultrasound guided by magnetic resonance to successfully treat tremor and chronic pain (Lipsman et al. 2013). The investigation of the use of SUS to remove Aβ plaques as a noninvasive treatment for AD comes at the perfect time where ultrasounds are undergoing clinical trials on humans.

and an indicator dye called Evans Blue. Evans Blue dye binds to a protein known as albumin, which is normally excluded from the brain. Therefore, if the dye showed up in the brain, it would show that the BBB is open, allowing albumin to enter the brain. After SUS treatment, the dye was able to enter the brain showing BBB opening (Figure 1). The results were compared to a control group who received the sham treatment, involving placing the mice under the ultrasound transducer but emitting no ultrasound.

Figure Adapted from (Leinenga % Götz 2015). Science Translational Medicine, 7(28): 278-291 Figure 1. Opening of the BBB. A clear entry point was marked with Evans Blue dye after SUS treatment shown by the blue mark. The dye is able to enter the brain bound to albumin after SUS treatment. No entry point was found in the sham treatment.

Leinenga and Götz then moved the beam across the entire forebrain of the mouse and were able to open the BBB throughout the brain (Leinenga & Götz 2015). Using fluorescence imaging, the brain slices showed great amounts of Evans Blue dye after SUS treatment (Figure 2). The opening of the BBB was proved to be non-invasive. The mouse brain tissue was analyzed four hours and one day after treatment using an acid stain and found no evidence of ischemic damage.

MAJOR RESULTS In Leinenga and Götz’s study, there were five major experiments that yielded significant results (Leinenga & Götz 2015). First, they demonstrated focused opening of the BBB through SUS treatment. Next, they showed a reduction in the number of Aβ plaques following SUS treatment. Then, they demonstrated through two separate experiments that SUS treatment improved memory using the Y maze, NOR test and APA task. Lastly, they highlighted a key role of microglia in the removal of Aβ plaques and made a connection between SUS treatment and the activation of the microglia. SUS Treatment Can Safely Open the BBB

Figure Adapted from (Leinenga % Götz 2015). Science Translational Medicine, 7(28): 278-291

In the first experiment, Leinenga and Götz showed in Figure 2. Widespread Opening of the BBB Throughout the Brain. A LI-COR C57BL/6 non-Tg mice that SUS treatment can open up the BBB scanner was used to obtain brain slices of the brain after SUS treatment throughout the brain. The slices show great concentrations of Evans Blue. (Leinenga & Götz 2015). Before treating with SUS, they anesthetized the mice and injected them intravenously with microbubbles

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Reduction in Aβ plaques following SUS treatment At the age of 12 months, the APP23 mice used in the study were shown to have a great number of Aβ plaques (Leinenga & Götz 2015). Leinenga and Götz treated a cohort of 10 male APP23 mice with SUS five times over six weeks. A control group was also tested, who received the sham treatment. After SUS treatment, the authors found that the cortical area taken up by the Aβ plaques were reduced by 56% (Figure 3). Additionally, they found that the average number of plaques in each cortical section was reduced by 52% (Figure 3).

Figure Adapted from (Leinenga % Götz 2015). Science Translational Medicine, 7(28): 278-291 Figure 4. Performance of APP23 mice on the Y-maze. Non-Tg sham mice are the wild type showing a high level of alternation between the arms of the maze. In APP23 Sham mice with plaque buildup, arm alternation is reduced. Wild type levels are restored in APP23 mice treated with SUS.

Figure Adapted from (Leinenga % Götz 2015). Science Translational Medicine, 7(28): 278-291 Figure 3. Comparison of Aβ plaques in Control Sham and SUS Treated Mice. A, The plaque burden was significantly reduced by 56% in SUS treated mice. B, The number of plaques per cortical section were reduced in SUS treated mice by 52%.

SUS treatment can restore memory function

The authors treated a second cohort of APP23 mice and 10 non-Tg mice to perform more robust memory examinations (Leinenga & Götz 2015). The APP23 mice were divided into two groups and received weekly SUS or sham treatment for seven weeks. Then the mice were first subjected to the APA task, which is a test focused on the spatial learning of the hippocampus. Mice learned to avoid a shock zone in the test. The authors found that SUS-treated mice showed better learning and avoidance of the shock zone with less total shocks (Figure 5). The authors also conducted the NOR test, which measures the time spent with a new object compared with a familiar object. After analyzing the results, the authors reported that both the non-Tg mice and the SUS treated mice spent more time with the novel object, meanwhile the APP23 sham mice spent more time with the familiar object (Figure 6).

The APP23 mice were subjected to a spatial memory task known as the Y-maze (Leinenga & Götz 2015). If the spatial memory of the mice were up to wild-type standards, they should show a preference to alternate between the arms of the maze and not remain in one arm. The authors showed that SUS treatment was able to return the number of arm alternations to wild type levels, while the APP23 mice that were not treated with SUS mice showed significantly less alternations (Figure 4). Figure Adapted from (Leinenga % Götz 2015). Science Translational Medicine, 7(28): 278-291 Figure 5. The Results of the APA test. The SUS-treated mice show better spatial learning in the hippocampus as they were able to better avoid the shocks than sham-treated mice. However, they still had more shocks than the wild

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Figure Adapted from (Leinenga % Götz 2015). Science Translational Medicine, 7(28): 278-291

type non-transgenic mice.

Figure 7. Aβ Internalization in sham treated and SUS-treated mice. Microglia in SUS-treated mice contain twice as much Aβ than sham treated control mice.

CONCLUSIONS/DISCUSSION

Figure Adapted from (Leinenga % Götz 2015). Science Translational Medicine, 7(28): 278-291 Figure 6. The Results of the NOR test. Non-Tg mice show a clear preference for the novel object. This preference disappeared in APP23 mice with the sham control treatment but were restored in APP23 mice treated with SUS.

SUS treatment induces microglia activation to aid in plaque clearance The authors used spinning disk confocal microscopy on the initial cohort of 10 male APP23 mice to study the action of the microglia (Leinenga & Götz 2015). Through this technique, they were able to visualize the microglia in SUS-treated brains actively fragment and engulf the Aβ plaques. Specifically, they found that microglia in SUS-treated mice contained twice the amount of Aβ plaques (Figure 7). The authors also found that the microglia in the SUS-treated group were more activated as analysis showed that they contained less branches. In comparison, microglia in sham treated groups were more highly branched. These morphological changes upon microglia activation are understood well in the literature. It is known that the activation status of microglia is characterized by a transformation from a highly branched shape to an amoeboid cell body shape (Heindl et al. 2018).

Leinenga and Götz showed that SUS treatment leads to the activation of microglia and promotes microglial Aβ plaque uptake (Leinenga & Götz 2015). This significantly reduces the number of Aβ plaques found in the brain of the mouse model of AD and restores spatial memory functions as shown in the NOR test, APA test and Y-maze. The authors also showed that the SUS treatment safely opens up the BBB, providing another treatment option where SUS can be paired with other treatments for AD that require access to the brain such as anti-Aβ antibodies. The authors’ experiments in finding a non-invasive treatment for AD are in agreement with the literature in terms of the great role that Aβ plaques play in AD. Most neurologists agree with the “amyloid cascade” hypothesis, which states that Aβ aggregation initiates a cascade of events leading to the formation of neurofibrillary tangles, severe neurodegeneration and eventually dementia (Allsop & Mayes 2014). It is with this hypothesis in mind that the authors proposed SUS to remove Aβ plaques as a treatment for AD and they successfully showed an improvement in AD disease symptoms (Leinenga & Götz 2015). Although methods targeting Aβ plaque removal have been previously explored, Leinenga and Götz’s research was different as their method of SUS treatment didn’t require additional therapeutic agents such as antibodies. Previous research has been focused on using therapeutic agents such as antibodies that were often invasive and could lead to immune attack from the host. Previously, Rasool and his colleagues successfully reduced amyloid load and demonstrated improved cognition by using monoclonal antioligomeric antibodies that target Aβ (Rasool et al. 2013). In this study, Leinenga and Götz showed that they were able to achieve the same level of Aβ reduction through SUS treatment as the levels achieved by Rasool and his colleagues using antibodies in passive immunization (Leinenga & Götz 2015). Therefore, the authors were able to improve on an area of research that has been previously explored by presenting a new non-invasive method capable of achieving the same results.

CRITICAL ANALYSIS The authors of the paper examined SUS treatment as a therapy for AD and were able to provide evidence supporting SUS as an effective non-invasive therapy in mice models. (Leinenga & Götz 2015). However, there are additional questions to be asked and further research required to fully understand the physiological processes of the treatment and to be able to advance to human models. The authors showed that SUS treatment is able to transiently open the BBB to allow microglia from the spinal cord in addition to just the microglia in the brain to remove Aβ plaque deposits in the brain. Nevertheless, it is possible that the opening of the BBB can lead to more deposits of newly generated Aβ in the brain, which is not considered by the authors. More research needs to be conducted on the ramifications of opening the BBB and other harmful substances that may enter the brain along with Aβ while the BBB is open. If this is a long-term treatment plan, it is also nec-

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essary to determine if repeated opening of the BBB will produce negative effects on the brain. Similar experiments have been conducted to evaluate safety, but only on specific areas of the brain and not the opening of the entire BBB. For instance, Downs and his colleagues established that repeated opening of the BBB at the basal ganglia was safe for up to twenty months without any longterm negative consequences (Downs et al. 2015). Next, since the authors are targeting a human audience for this treatment, they need to consider the differences between the human brain and mouse models. Murphy and Levine showed that Aβ is more easily cleared from the brain in animal models than the human brain and they showed that there are physical and biochemical differences between Aβ deposits in mice and the Aβ plaques found in AD (Murphy & Levine 2010). Since Aβ-depositing animal models do not have the full AD pathology, it is also difficult to measure actual disease outcomes of AD in mice models. For instance, it is hard to determine in mice models the SUS effect on massive neuronal loss, which would normally be observed in AD patients. Instead, the mice model only allows the author to conclude that there are improvements in memory function with Aβ plaque removal. In addition to the differences in the animal model, the human brain is also larger in general with a thicker skull and therefore requires additional testing. As well, the SUS treatment was also only conducted on mice models that showed a great amount of plaque growth. In order to evaluated SUS as a therapy, additional experiments should be conducted to determine its efficacy under additional conditions such as those of just a little plaque growth. Moreover, Aβ is not the only hallmark feature of AD. The microtubule binding protein tau is also an important player in the development of AD neuron degeneration (Holtzman 2008). In Holtzman’s research, he found that neuron degeneration was still observed even if Aβ was removed as long as tau was present (Holtzman 2008). Therefore, just Aβ plaque clearance alone as proposed by Leinenga and Götz, might not be enough and further research needs to be completed to understand the interactions between Aβ and the tau protein in AD pathogenesis (Leinenga & Götz 2015).

greater impact on AD pathogenesis as Aβ. Since the authors are proposing SUS treatment as a therapy for AD, it is essential to understand how to optimize its results. Previously, researchers showed that despite lower amounts of Aβ, a high amount of tau could still lead to end stage dementia (Holmes 2008). In this case, the authors should perform an extension to their experiment and also consider tau levels. They should perform SUS treatment on the APP23 mice, who also express high or low levels of tau and determine how their spatial memory is affected in the NOR task, APA task and Y maze. If there is a significant difference in spatial memory capability between the groups, the authors know that SUS treatment to remove Aβ plaques alone may not be enough to treat AD, and they will have to focus on combining their treatment with antibodies that target the tau protein. Ultrasounds have been previously coupled to deliver proteins to the brain as demonstrated by Kinoshita and colleagues, who delivered Herceptin to the brain across the BBB using an ultrasound (Kinoshita et al. 2006). The authors could expand this combination of antibody delivery and ultrasound treatment with a focus on Aβ plaques. Next, the authors only performed experiments on mice that had a heavy Aβ plaque burden (Leinenga & Götz 2015). The authors should perform SUS treatment tests on mice of varying amounts of Aβ peptide levels and determine if there are improvements of spatial memory in each case. This would provide better guidelines for the use of SUS treatment and determine if there are thresholds of Aβ levels in the brain that are required before SUS treatment is effective and actually increases brain function. These future experiments will allow the results of Leinenga and Götz’s study to be better generalized to human AD pathways as opposed to just mice models and explores methods to better understand and maximize the effect of the treatment.

FUTURE DIRECTIONS The authors conducted their research on APP23 mice models, which contain mutations to overexpress Aβ peptide in the brain (Leinenga & Götz 2015). However, as explained in the critical analysis, Aβ deposits in these mice show different physical and biochemical properties than the Aβ plaques found in AD patients, making it difficult to generalize these results. Therefore, a future experiment that can be conducted would be to test SUS treatment on models that better resemble AD. For instance, nonhuman primates (NHPs) have identical Aβ sequences to humans and contain numerous of the same human biochemical pathways (Martin et al. 1991). If the SUS treatment used by Leinenga & Götz produce similar improvements in spatial memory upon Aβ plaque clearance in these primate models, researchers can have a greater confidence that they will successfully work as a therapy for human AD, since the primates have much more similar biochemical pathways to humans. If similar improvements in spatial memory are not observed, or if there are problems with Aβ clearance using SUS treatment in these models, it shows that the therapy may work on mice models but will likely not work on humans. Next, the authors could perform an extension on their experiment that also takes into account interactions with the tau microtubule binding protein, which many researchers believe have just as great if not a

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The transcription factor Zfp189 orchestrates a gene network in the prefrontal cortex that mediates stress resilience and harbors antidepressant effects Lazar Joksimovic

Stress engages major homeostatic changes to the brain and body that when appropriately regulated are beneficial to human functioning. However, chronic stress accompanied by inappropriate psychological, cognitive and behavioural responses constitute a major trigger of major depressive disorder. Increasing attention has been paid to the importance of genome-wide transcriptional changes in resilience to stress. Understanding the specific transcriptional networks that regulate resilient vs. susceptibility responses to stress is critical for identifying urgently needed effective antidepressant targets. In their 2019 Nature Neuroscience study, Lorsch et al. conducted gene co-expression analyses on previously acquired RNA-seq data from brains of mice resilient to the chronic social defeat stress paradigm to uncover a novel transcriptional network engaged in resilience. The group identified a novel gene network, defined as the pink module, that is preferentially activated in the prefrontal cortex in resilient mice by the highest-ranked driver gene, Zfp189, an uncharacterized zinc finger transcription factor. Overexpression of Zfp189 in PFC neurons of mice reversed depression-associated behaviours in susceptible mice, suggesting antidepressant effects. Through upstream pathway analysis, the authors identified CREB as the transcription factor that regulates expression of Zfp189. While CREB knockout and locus-specific targeting of dCas9-G9a suppressed Zfp189 expression, dCas9-CREB binding to CRE motifs upstream of Zfp189 preferentially induced expression of pink module genes. Altogether, the authors identified a pro-resilient transcriptional hierarchy that can be therapeutically harnessed in major depressive disorder through manipulation of Zfp189, either directly, or through enhancing CREB expression. Key words: major depressive disorder, chronic social defeat stress, stress resilience, transcriptional gene networks, zinc finger protein 189 (Zfp189), cAMP responsive element binding protein (CREB)

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seen in susceptible mice24,26. The relationship between the differentially regulated genes in the resilient brain, however, remains unknown. Understanding the transcriptional programs that are activated in resilient mice exposed to stress is critical for uncovering novel desperately needed antidepressant targets.

Introduction Background Major depressive disorder (MDD) is a complex debilitating disease that is characterized by distressing behavioural, cognitive and emotional symptoms, such as avolition, memory impairments and anhedonia, respectively1. With a lifetime prevalence of 6.7%, MDD is currently the leading cause of disability worldwide2, and increases the overall risk of mortality by 6080% 3. Despite the numerous available psychotherapies and pharmacological modalities, roughly a third of patients fail to experience remission of their symptoms, even after undergoing multiple treatments4. Numerous causes of MDD have been proposed, including a 35% genetic component 5; alterations in regional brain volumes, most notably decreases in hippocampus size6; abnormal connectivity patterns of multiple brain networks7,8; hyperactivation of the hypothalamic–pituitary–adrenal axis9; increased neuroinflammation10; dysfunctional synaptogenesis and plasticity11, as well as decreased neurogenesis9; and epigenetic changes in animal models and post-mortem MDD brain tissues10. Despite the major advances in our understanding of the multifactorial biology of MDD, a unifying mechanistic understanding of the disease remains unknown.

Zfp189 as a novel protective factor in major depressive disorder

This review will discuss the findings from the 2019 Nature Neuroscience study published by Lorsch et al.27, in which the authors decipher a novel pro-resilient transcriptionally active network in the prefrontal cortex (PFC) of driven by Zfp189, a gene encoding a putative zinc finger transcription factor whose function has not been previously implicated in stress resilience. By clustering co-transcriptionally regulated genes into modules based on differential expression data from their recently established RNA-sequencing (RNA-seq) dataset of mice exposed to CDSD, Lorsch and his team identified a unique resilient module, which they termed the pink module30. By reconstructing the gene regulatory network based on differential expression of genes (DEG), gene-gene co-expression patterns and predicted upstream transcriptional regulators, Zfp189 was identified as the only driver gene that recapitulated the pink module differential gene expression profile in the PFC30. Interestingly, mRNA levImportantly, stressful life events have been strongly implicated els of the human ortholog of Zfp189 (ZNF189) were also found in the onset of MDD11–14. The body’s homeostatic response to to be decreased in the PFC of post-mortem MDD brains comstress is known as allostasis15, and is adaptive to human func- pared to controls, suggesting clinical relevance30. tioning. However, chronic stress or inadequate physiological and psychological responses to stress, defined as allostatic load16, can wreak havoc on the body’s homeostatic systems, The causative role of Zfp189 in stress resilience was confirmed Zfp189 via herpes simplex virus (HSV) vecimpairing normal brain functioning. While some individuals are by overexpressing 30 tor in PFC neurons . Notably, overexpression of Zfp189 in PFC susceptible to the negative consequences of stress, others exhibit in stress resilience, defined as a dynamic process that leads to positive neurons reversed depression-associated behaviours 30 17 susceptible mice, revealing antidepressant effects . The authors adjustment in biological, behavioural and cognitive responses . By studying how animals and humans respond in highly adverse then employed clustered regularly interspersed short palindromenvironments, scientists have uncovered some of the neuro- ic repeats and a mutant form of Cas9 lacking enzymatic activity chemical18, hormonal18,19, genetic20, and epigenetic21 changes (dCas9) to uncover cAMP responsive element (CRE) binding protein (CREB) as the putative upstream regulator of Zfp189 30. that differentiate susceptibility and resilience to stress. CREB knockout increased stress susceptibility, while dCas9CREB targeting to the Zfp189 locus induced its transcription Transcriptional networks in stress resilience and in turn, downstream activation of pro-resilient pink module genes30. In conclusion, the authors reveal how transcriptional changes governed by a hierarchical regulatory structure govern In order to probe the distinct biological mechanisms that distinstress-resilient behaviour30. guish the resilience and susceptibility responses, scientists commonly use mice subjected to chronic social defeat stress (CSDS), a validated model of depression22,23. In this model, a Major Results proportion of mice exhibit phenotypes resembling those of humans with MDD and are defined as “susceptible”, while the remaining portion are classified as “resilient”. In recent years Zfp189 regulates a resilient-specific gene network in the PFC scientists have discovered several maladaptive gene expression programs within limbic circuits in mice susceptible to CSDS24,25. Conversely, resilience has been associated with brain Lorsch et al. identified transcriptional changes engaged in stress -specific active transcriptional responses different from those resilience by conducting weighted gene co-expression network

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analysis (WGCNA) on their previously acquired RNA-seq dataset from mice exposed to CSDS (Figure 1). WGCNA is a systems biology tool used for mining functional clusters based on pairwise correlations between co-expressed genes. Complemented with analysis of known protein-protein interactions, this analysis revealed 12 modules that were present across four brain regions previously shown to be associated with MDD pathophysiology26,28: the PFC, the nucleus accumbens (NAc), the basolateral amygdale (BLA), and the ventral hippocampus (VHIP) (Figure 1). This analysis revealed 12 biologically relevant modules expressed across the four brain regions30. In order to identify modules which were transcriptionally active and resilient-specific, module differential connectivity (MDC) was employed to compare module structure between RNA-seq data from resilient, susceptible and control mouse brains (Figure 1). MDC was supplemented with downstream enrichment analysis to determine the degree to which resilient module-specific genes were differentially regulated across the 4 brain regions 48 hours post-CSDS30. These bioinformatic approaches ultimately revealed the pink module as transcriptionally active across the analyzed brain regions uniquely in resilient mice (Figure 1).

Zfp189 was the only one that recapitulated the differentially expressed gene enrichment profile of the pink module ((also uniquely upregulated in the PFC of resilient mice (Figure 1)). Although little is known about the mechanism of action of Zfp189 and no previous studies have linked the gene with psychiatric disease pathophysiology, Odeberg et al. cloned the Zfp189 gene in 1988 and found that it encodes for KrĂźppelassociated box (KRAB) protein29. Later, in 2015, Najafabadi, H.S. et al. discovered that the human ortholog binds to DNA regulatory sites30, suggesting it functions as a transcription factor. Importantly, the identification of a gene network transcriptionally engaged in the PFC of resilient mice corroborates earlier findings by Covington et al. demonstrating reversal of depression-associated behaviours in CSDS-exposed mice via optogenetic stimulation of the medial PFC (mPFC)31. Moreover, studies by Radley et al.32 and Covington et al.31 have shown that chronic restraint stress and chronic social defeat stress induce pathological alterations and decrease neuronal activity, respectively in the mPFC. On the human side, by conducting a genome-wide meta-analysis, Howard et al. discovered 102 variants associated with depression. Many of these genes were enriched for expression in the prefrontal cortex. Taken together, these findings demonstrate a critical role of PFC in the pathophysiology of depression in both mice and humans. Zfp189 exerts pro-resilient effects in the PFC and reverses depressive behavioural features

Figure 1. Adapted from Lorsch et al. (2019) a) Outline of the CSDS protocol, characteristic behaviour in the social interaction zone test for resilient vs. susceptible mice, and the four brain regions used for RNA-seq analysis. b) Coloured bars represent resilient modules identified through WGCNA. Specificity of resilience is shown through MDC of resilient modules in susceptible and control mice, with colours demonstrating statistical significance (FDR q < 0.05). Internally, only significant (P < 0.05) differential expression of genes (DEG) is shown in colour. Only pink module demonstrates resilience MDC specificity as well as DEG enrichment across the four brain regions. c) Gene interaction network of pink module depicts Zfp189 as top driver gene (contains the most connections). d) Relationship between pink module DEG, scaled by -log10(P-value), and Zfp189 expression, illustrates that both pink module and Zfp189 are differentially expressed in resilient vs. susceptible mice in the PFC.

Exploring the significance of ZNF189 in human resilience, Lorsch et al. found decreased ZNF189 mRNA levels in the PFC of post-mortem MDD brain tissues compared to control samples. Once human relevance of Zfp189 was confirmed, Lorsch et al. sought to identify the specific cell populations in which the hub gene is actively expressed. By conducting RNAScope, an in-situ hybridization assay for detecting specific RNAs in intact tissue, on mouse PFC sections, the authors observed neuronally enriched expression of Zfp189 27, corroborating findings from Zeisel et al.â&#x20AC;&#x2122;s study on single cell-RNA sequencing of mouse somatosensory cortical and hippocampal cells33. Through several in vivo experiments, the scientists confirmed a causal function of Zfp189 in stress resilience 27. By injecting mice subjected to an accelerated social defeat paradigm with HSV-Zfp189 vectors, which selectively target neurons, the scientists were able to show that overexpression of Zfp189 in the PFC increases resilience to CSDS (Figure 2). Precisely, mice that were treated with HSV-Zfp189 spent significantly more time in the social interaction zone, a measure of fear; showed greater sucrose preference, a measure of anhedonia; and did not show anxiety-like behaviour in the open-field test ( a measure of anxiety), compared to HSV-GFP mice (Figure 2).

Through reconstruction of the pink module according to genegene correlations and validated upstream transcription factors, the authors identified multiple driver gene candidates, of which 194

antidepressant effects30. CREB is an upstream regulator of Zfp189-driven proresilient behaviour Through a combination of bioinformatic algorithms complemented with robustly controlled in vivo experiments, Lorsch et al. found that CREB is an upstream regulator of the pink module. By combining the HOMER algorithm, used for discovery of regulatory motifs in largescale omics data (in this case, RNAseq data set) with ingenuity pathway analysis, which hunts for upstream regulators in -omics data, the scientists revealed CREB as the only hit shared by both analytical tools30. Interestingly, other studies have found that certain genetic variations and reduced functioning of the CREB gene is associated with human MDD and depressive behaviour in mice34,35,36 and that enhanced CREB expression harbors antidepressant effects37. Wilkinson, M.B. et al. previously discovered through chromatin immunoprecipitation that binding of active Ser133phosophorylated CREB to Zfp189 CRE sites was reduced following CSDS in the NAc, a region of the brain responsible for processing reward, and that treatment with the tricyclic antidepressant, imipramine, increased levels of phosphorylated CREB38.

Figure 2. Adapted from Lorsch et al. (2019) f) Experimental timeline showing Zfp189 overexpression before CSDS and subsequent experiments that measure depression-associated behaviour in mice. g) Mice injected with HSV-Zfp189 in PFC spend significantly more time in interaction zone with the presence of a target mouse compared to socially defeated HSV-GFP. h) Overexpression of Zfp189 in PFC significantly increases sucrose preference, a measure of anhedonia. i) Experimental timeline depicting Zfp189 injections into PFC of susceptible mice and subsequent behavioural experiments. j) Zfp189 overexpression in PFC of susceptible mice significantly reverses depression-associated social withdrawal compared to HSVGFP controls. k) Susceptible mice injected with HSV-Zfp189 demonstrate significantly increased sucrose preference compared to HSV-GFP mice. Interestingly, it was found that HSV-mediated delivery of Zfp189 into the PFC reversed chronic social stress-induced behavioural deficits in susceptible mice (Figure 2). RNA-seq of virally infected tissue revealed that overexpression of Zfp189 in the PFC activated transcription of 33.1% of pink module genes, many of which were closely integrated in the network30. Pink module network activation was observed in both previously susceptible and control mice (not exposed to social defeat stress)30. Altogether, these findings confirmed that Zfp189driven transcriptional changes in the PFC exert pro-resilient and

Figure 3. Adapted from Lorsch et al. (2019) a) Experimental timeline used to investigate effects of CREB KO in the PFC of mice exposed to a subthreshold social defeat. b) Cre-mediated KO of CREB in PFC causes statistically significant decreased time in interaction zone with presence of target mouse, demonstrating a pro-resilient effect of CREB. c) CREB mRNA levels and d) Zfp189 mRNA levels are decreased in CREB KO, suggesting a functional interaction between the two genes. e) Experimental timeline depicting interventions used to probe relationship between CREB and Zfp189 and their effects on resilience to subthreshold social defeat stress. f) Zfp189 overexpression in PFC rescues behavioural deficits in CREB KO mice, as demonstrated by significantly increased time in interaction zone. g) Zfp189 overexpression also significantly increases sucrose preference in CREB KO mice. The authors validated CREB as an upstream regulator of Zfp189 expression through several experiments. In the first experiment, they injected adeno-associated viral (AAV) vectors expressing Cre recombinase plus GFP or GFP alone into the PFC of CREBfl/fl mice subjected to a subthreshold social defeat test

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(Figure 3). AAV-Cre mice developed social avoidance in the social interaction test, while AAV-GFP mice did not, which confirmed that CREB has a positive effect on resilience (Figure 3). In the AAV-Cre mice, Zfp189 mRNA levels were significantly reduced compared to the control group, which provided evidence for CREB-mediated regulation of Zfp189 (Figure 3).

which is in turn transcriptionally regulated by CREB. The authors provide conclusive evidence for the causative relationship between expression of specific neuronal genes and largescale pro-resilient transcriptional changes in PFC, a region of the brain whose function is impaired in MDD7,34,39.

The identification of a pro-resilient transcriptional network and its driver gene bears fruit to the pressing need for more effective therapeutic targets in MDD30. Understanding why most individuals overcome psychological stressors while others succumb to debilitating psychiatric conditions like MDD remains a critical question. Over the past several decades, scientists have uncovered numerous social, psychological and biological factors linked to MDD. Given the complex interplay of numerous genes, epigenetic modifications, neurotransmitter systems, immune system perturbations and hormonal changes observed in animal models of depression, it is likely that more effective therapies will need to target transcriptional networks that mediate the abnormal molecular, cellular and circuit-wide changes. Future investigations are required to bridge the mechanisms through which environmental sensitivities, genetic variations, Figure 4. Adapted from Lorsch et al. (2019) e) Combined deliv- epigenetic modifications, and downstream transcriptional netery of constitutively active CREB (CREBS133D) to the CRE motif works interact to influence the pathophysiology and course of of Zf9189 via HSV-Zfp189-sgRNA significantly increased MDD. Zfp189 mRNA levels compared to untargeted CREB S133D-dCas9, and Zfp189-targeted dCas9 lacking a functional domain. This Several other studies have also examined how stress-induced suggests that CREB binding to Zfp189 CRE regulatory motifs transcriptional changes mediate complex anxiety and depression upstream of the transcription start site activates expression of -associated behaviours in mice exposed to CSDS. A 2007 study Zfp189. f) Experimental timeline used to probe the effects of by Krishnan et al. was the first to investigate how genome-wide dCas9-targeted constitutively active CREB to the Zfp189 pro- expression profiles in the mesolimbic reward circuit are signifimoter in PFC neurons. g) CRISPR-mediated CREBS133D target- cantly different between mice resilient vs. susceptible to ing to the Zfp189 promoter significantly increased the time so- CSDS23. Specifically, PFC and amygdala projections to the cially defeated mice spent with the target mouse, conclusively NAc orchestrated resilient behaviour, while VHIP projections to revealing the pro-resilient effects of CREB-Zfp189 interactions. the NAc had the opposite effect26. The authors also found that The scientists further validated their predictions through CRISPR-Cas9 driven locus-specific editing of the CRE motif in the Zfp189 promoter30. Constructs containing single guided RNAs (sgRNAs) targeting the Zfp189 CRE motif together with a nuclease-dead, RNA-guided, DNA-binding Cas9 protein fused to CREBS133D, a constitutively active phosphomimetic version of CREB (dCas9-CREBS133D) were injected in the PFC of one group of mice, while the control groups independently received the above construct with a phospho-null version CREBS133A, non-targeting sgRNA, or dCas9 lacking a functional moiety. Only the HSV-dCas9-CREBS133D + HSV-Zfp189-sgRNA construct caused a significant increase in expression of Zfp189 as well increased resistance to CSDS-induced social defeat (Figure 4).

more genes were differentially regulated than shared in susceptible vs. resilient mice in the major components of the mesolimbic circuit, the NAc and VTA, illustrating distinct transcriptional responses in resilience26.

A later study by Bagot et al. in 2016 profiled patterns of genome-wide differential gene co-expression in four interconnected regions involved in depression, the PFC, the VHIP, the NAc, and the VTA in resilient vs. susceptible mice exposed to CSDS, revealing distinct transcriptional networks between the two groups of animals26. Their finding of increased differential connectivity in susceptible gene modules in contrast to an increased number of DEGs in resilient brains, illustrated the important distinction between the two metrics - differential expression of genes and module differential connectivity - used to characterDiscussion and Conclusions ize complex gene expression data sets. An increased differential connectivity in susceptibility gene networks suggests a greater alteration of biologically relevant pathways, while a greater In their 2019 study, Lorsch et al. uncovered a novel pro-resilient number of resilient DEGs exemplifies a larger number of differtranscriptional hierarchy in the PFC that is preferentially actientially upregulated and downregulated genes without fundavated by the neuronally-enriched transcription factor Zfp189, 196

mental alterations in molecular pathway assemblies. These critical findings beg the question whether overexpression of Zfp189 in the PFC is mechanistically sufficient to reverse susceptible differential connectivity networks in socially defeated animals. Interestingly, Bagot et al. observed a greater number of DEGs at 48 hours post-CSDS in resilient vs. control mice compared to susceptible vs. normal mice in all analyzed brain regions. However, after re-exposing mice 28 days post-CSDS to an acute social defeat stress procedure for 1 hour, the scientists detected a greater number of DEGs in susceptible vs. control mice than in resilient vs. control mice in all analyzed brain regions, except for the PFC. This suggests that in the acute phase of the stress response, resilient brains exhibit stronger transcriptional activity, reflective of an active adaptive response. Then eventually the neural circuits may decrease their responsiveness to stress, apart from the PFC, which has been shown to protectively minimize stress-induced overactivation of other brain regions40. In addition to revealing unique transcriptional modules differentially expressed in susceptible mouse brains, the team’s investigation into the inter-regional variation in gene co-expression revealed transcriptome-mediated circuit-level changes in resilient vs. susceptible mice29. By manipulating differentially regulated susceptibility hub genes, the scientists uncovered direct effects of differential gene expression patterns on synaptic and circuit functioning29. However, the authors did not probe the function of any resilient hub genes, which bears fruit to the importance and novelty of Lorsch et al.’s findings. While multiple studies have analyzed transcriptional networks in stress-resilience26,26,41 and susceptibility29, this study was the first to uncover a pro-resilient transcriptional hierarchy regulated by a hub gene, Zfp18927. Manipulating Zfp189 alone or its interaction with its upstream regulator, CREB, influenced expression of 281 genes in the pink module, highlighting a hierarchy in which a drive gene regulates expression of numerous resilient-specific genes in response to chronic stress27. Overall, the authors’ elucidation of a PFC-specific hierarchical transcriptional organization and its bridge with complex depression-associated behaviour in mice reveals a novel molecular mechanism of stress resilience 27. This finding has major implications for the development of future therapies for MDD.

by injecting HSV vectors expressing Zfp189 or GFP (as a control) in the PFC of mice exposed to an accelerated social defeat test27. To measure anxiety- or depression-like behaviour, the scientists exposed the mice to numerous behavioural tests, including the social interaction test, open-field test, forced-swim test, and sucrose preference test30. The use of multiple behaviour tests that evaluate various phenotypes related to depression, such as anhedonia, fear, anxiety, and learned helplessness, the authors conclusively showed that Zfp189 expression in the PFC has a proresilient effect30. Further, the scientists tested whether Zfp189 overexpression in the PFC could reverse CSDS-associated depressive behaviour in susceptible mice via the same HSVmediated delivery of Zfp18930. By observing a reversal of chronic stress-associated social defeat in susceptible mice injected with HSV-Zfp189 compared to controls, it was concluded that Zfp189 exerts both pro-resilient and antidepressant effects30. While the anti-depressant effects of CREB have been investigated in previous studies37,38,39, Lorsch et al. determined a previously uncharacterized depression-relevant interaction between CREB and Zfp18927. The scientists manipulated this interaction through carefully designed experiments, including knocking out CREB and over-expressing Zfp189, as well as CRISPR-Cas9 mediated locus editing of the CREB-binding motifs in the Zfp189 promoter27. By robustly dissecting the different components of the proresilient regulatory interaction between CREB and Zfp189, the authors identified a potential mechanism to upregulate a network of pro-resilient genes: through direct pharmacological upregulation of Zfp189 or via CREB-mediated manipulation27. The elucidation of a Zfp189-regulated transcriptional network (pink module) in the PFC with complex behavioural outcomes corroborates Bagot et al.’s findings of the ability of a hub gene to regulate region-specific gene expression patterns and stress responses26. While Lorsch et al. did not investigate functional connectivity between well-characterized depression-associated brain regions, the VHIP, the PFC, the NAc and the VTA, Bagot et al. investigated circuit-level transcriptional changes induced by susceptibility genes in these brain regions in response to stress 29. It remains to be elucidated how overexpression of pro-resilient genes like Zfp189 influences functional connectivity with these brain regions as well as other depression-relevant areas of the brain like the amygdala13.

Critical Analysis The study by Lorsch et al. describes a novel transcriptional proresilient network with antidepressant effects activated by the hub gene Zfp189 and regulated upstream by CREB27. By integrating differential gene expression and differential module connectivity networks analyses, the scientists obtained a comprehensive understanding of transcriptional responses activated in mice resilient to chronical social stress activates. In addition, Lorsch et al. probed the causative pro-resilient effects of Zfp189 and Zfp189CREB interactions through numerous robustly controlled experiments. First the scientists manipulated the expression of Zfp189

Importantly, given that thousands of genes have been found to be differentially regulated in resilient vs. susceptible mice in response to stress13,26, it is likely that the 281 genes in the pink module interact with many other disease-relevant molecular pathways. Therefore, it remains to be investigated how the various genome-wide transcriptional changes interact and influence synaptic function and circuit-level changes in depression. Future Directions

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While Lorsch et al. demonstrate that Zfp189, a putative transcription factor, activates a pro-resilient transcriptional network in the PFC, they did not investigate the mechanism by which Zfp189 regulates expression of pink-module genes. Further, given that thousands of genes have been shown to be differentially regulated in response to resilience29, deciphering the genome-wide DNA binding sites of Zfp189 could reveal novel gene-gene coexpression patterns potentially amenable to therapeutic interventions. Chromatin immunoprecipitation followed by wholegenome sequencing (ChIP-seq) would be conducted in parallel with RNA-seq to probe the genome-wide regulatory binding sites of Zfp189 and the resultant transcriptional changes. Briefly, mice would be divided into four groups (n=10/group): resilient, susceptible, control (unexposed to CSDS) and Zfp189 KO mice. ChIP-seq and RNA-seq would be conducted on individual samples of neurons dissociated from the PFC. Based on the prediction that Zfp189 functions as a transcription factor, it would be expected that due to its increased expression in resilient mice, ChIP-seq would detect a broader engagement of DNA sites as well as more defined signal peaks for Zfp189 target DNA sites in resilient mice compared to susceptible, control and KO groups. In addition, a sound hypothesis would be that Zfp189-activated genes (as demonstrated by ChIP-seq) are more highly expressed in resilient vs. control groups (due to lack of active stress-induce engagement of Zfp189 expression) and in resilient vs. KO and susceptible mice. One would also imagine that in addition to activating expression of pro-resilient genes, Zfp189 might simultaneously repress transcription of susceptibility network genes uncovered by Bagot et al.29 Integration of ChIP-seq with RNA-seq would enable detection of the specific susceptibility gene networks that might be repressed by Zfp189. If Zfp189 is shown to repress susceptibility gene networks, this would add another mechanistical layer to the ability of a resilient hub gene to reverse depression-associated behaviours. Finally, genes found to be directly regulated by Zfp189 would then be annotated to specific biological pathways through ingenuity pathway analysis, and the molecular pathways referenced to the already discovered molecular, epigenomic and genomic perturbations in resilient and susceptible mice.

slices of PFC injected with HSV-Zfp189 (to overexpress Zfp189) or HSV-GFP (control). Based on the well-characterized stressprotective function of the PFC, overexpression of Zfp189 would be predicted to increase spontaneous excitatory post-synaptic currents in PFC neurons. These cellular and circuit-level investigations would be integrated with the above described RNA-seq and ChIP-seq experiments to unravel a comprehensive biological understanding of Zfp189-induced pro-resilient changes in the PFC and functionally associated regions.

In addition to validating transcription factor activity for Zfp189, it is important to characterize its effects at the cellular and neural circuit level. Given that depression involves substantial synaptic and circuit-level alterations8,26,28,39, elucidating specific brain region architectural changes induced by Zfp189 would unravel the macroscopic mechanism through which a pro-resilient hub gene mediates stress resilience. Uncovering how Zfp189 expression levels influence synaptic architecture as well as functional connectivity between depression-relevant brain regions, would enrich our mechanistic understanding of how transcriptional networks influence complex depression-associated behaviours. To investigate this link, functional magnetic resonance imaging (fMRI), which is used to quantify functional connectivity based on neural time series deducted from blood oxygenation-levels, would be performed on resilient, susceptible, control and Zfp189 KO mice. Synaptic function would be investigated in these same mice through voltage and current clamp recordings on coronal 198

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A New Technique to Examine the Importance of Infralimbic Cortex in Retrieval of Extinction Memory: Optogenetics. Tuana Kant

Infralimbic (IL) subregion of the medial prefrontal cortex plays an important role in the retrieval of extinction of auditory fear conditioning. However, the temporal precision and neuronal specificity of the previous studies are still remaining as a question. Moreover, the necessity of the IL activity during the retrieval of extinction has also not been tested directly. In the research study conducted by Do-Monte et al. (2015), the authors activated or silenced the IL activity in rats during the tones of extinction training or extinction retrieval. The authors used optogenetic approach with channelrhodopsin and halorhodopsin channels expressed in different mice. They found that activation of IL neurons during extinction tones reduced freezing and strengthened extinction of fear memory. On the other hand, silencing of IL neurons during extinction tones increased freezing and reduced extinction of fear memory. Interestingly, the silencing of IL neurons during retrieval tones did not have any effect on extinction of memory or on the fear response. These results show that IL activity is necessary for the learning of extinction however it is not necessary for the retrieval of extinction memory. This study is significant as it is the first study that modifies the neurons during the retrieval of extinction memory using optogenetics techniques which improve the temporal precision and neuronal specificity. The results from this study can be used as a framework to treat anxiety disorders in humans which have the base of retrieval of extinction fear memory. Key words: anxiety disorders; amygdala; extinction; fear conditioning; infralimbic subregion; memory; optogenetics; retriev

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BACKGROUND and INTRODUCTION Infralimbic subregion of the medial prefrontal cortex (IL) plays an important role in the consolidation of extinction memory in retrieval of extinction (Burgos-Robles et al. 2007; Mueller, Bravo-Rivera, & Quirk, 2010; Marek, Strobel, Bredy, & Sah, 2013). The importance of IL in extinction of conditioned fear is due to its communication with basolateral amygdala complex (BLA) (Lingawi, Laurent, Westbrook, & Holmes, 2019). During the extinction retrieval, IL activates GABAergic cells (ITCs) in the amygdala which has a feed-forward inhibition to the output from the amygdala (Royer and Pare, 2002). Therefore, during the extinction training, IL neurons activate the ITC neurons which then inhibit the output of fear response (Royer et al. 1999). By inactivating or lesioning IL during the extinction training, different studies found that IL is not necessary for the conditioned freezing response during the extinction training but is necessary for the retrieval of extinction memory 24 hours later (Quirk et al., 2000; Rhodes and Killcross, 2004; Laurent and Westbrook, 2009; Mercado, Coreano, & Quirk, 2011). Studies using single-cell recording similarly also found that cells in the IL only signaled during the retrieval phase and did not show an activation during the conditioning or extinction phases (Milad and Quirk, 2002). The results showed that the activation of IL responses can indicate the success of good extinction in the retrieval tests (Milad and Quirk, 2002; Burgos-Robles et al., 2007). Different electrical stimulation studies showed that stimulation of IL during the extinction tones increased the success of retrieval of extinction on next day (Milad et al., 2004) and reduced the fear in the retrieval test (Vidal-Gonzalez et al., 2006). However, as electrical stimulation can back propagate and activate different fibers of passage there can be confounding factors in these results (Hamani et al., 2010). Other studies also showed that facilitation of IL enhanced extinction memory. Different studies used different facilitators of IL such as brain-derived neurotrophic factor (Peters et al., 2010), GABA antagonist picrotoxin (Thompson et al., 2010; Change & Maren, 2011) and microsimulation (Kim et al., 2010) and found enhancement in extinction memory in a cued fear conditioning task. Moreover, other studies used inhibitors of IL such as D2 antagonist raclopride (Mueller et al., 2010) and inactivation of IL with muscomil (Laurent & Westbrook, 2009; Sierra-Mercado et al., 2011) and found a decrease in extinction memory in cued fear conditioning tasks. However, all of these studies are correlation as they do not manipulate IL during the retrieval of extinct fear memory. The research by De-Monte et al. (2015) has important implications for humans. Anxiety disorders such as PTSD and phobias are usually the consequences of triggering traumatic events (Bukalo, Pinard, & Holmes, 2015). IL plays an important role in the inhibition of behaviors that can result in anxiety inducing outcomes (Jinks and Gregor, 1997). Animals with their IL lesioned remember the conditioned fear less and have a higher frequency of putting themselves in a previously anxiety created situation (Jinks and Gregor, 1997). Moreover, rats tend to perform high anxiety behaviors when their IL cortex and BLA connections are hypoactive and tend to perform less anxiety behaviors when their IL cortex and BLA connections are hyperactive (Knapska et al., 2012).

found that under the fMRI, there is an increased activity in vmPFC during the retrieval of extinction and the activity is correlated with the success of extinction retention (Phelps et al., 2004; Kalisch et al. 2006). Therefore, as the activation of vmPFC increased, the inhibition control thus the extinction recall increased as well (Hartley et al. 2011). Even though there are many previous studies that shows the importance of IL for the retrieval of extinction, they are lacking the temporal precision and neuronal specificity due to their methods of stimulation or inactivation. Furthermore, none of the previous studies directly manipulated the IL during the retrieval of extinction therefore could only make correlational links between the importance of IL and retrieval. In the research by De-Monte et al. (2015), the researchers used a new technique called optogenetics to stimulate or inactivate the IL with laser and light channels in order to answer the question of the necessity of IL region during the extinction and retrieval phases. They found that stimulation of IL during the extinction of tones has important implications on the retrieval of memory as it increases the retrieval of extinct memory. Furthermore, they also found that silencing of IL during extinction of memory reversely decreased the retrieval of extinct memory. This study was the first study to find that IL does not have an important role during the retrieval of memory as silencing of IL during this phase did not effect the retrieval of extinct memory. As De-Monte et al. (2015) found that IL plays an important role in extinction but not during the retrieval of extinction, the current treatments for anxiety disorders in humans can be readjusted and can target the vmPFC during the extinction phases of treatments.

MAJOR RESULTS In Do-Monte et al. (2015)â&#x20AC;&#x2122;s research, the authors found that activation of IL neurons during extinction tones decreased freezing and increased extinction of memory. On the other hand, another major finding is that inactivation of IL neurons during extinction tones decreased extinction of memory (Do-Monte et al., 2015). Interestingly, the authors also found that silencing of IL neurons during retrieval did not have an effect on the retrieval of extinction (Do-Monte et al., 2015).

Activation of IL Neurons During Extinction Tones (ChR2) Reduces Freezing Response and Strengthens the Retrieval of Extinction: In this part of the study, the authors first injected these rats with the Adeno-Associated Viruses (AAV) which was to express Channelrhodopsin (AAV5:CaMKII :: hChR2(H134R)-EYFP). Channelrhodopsin is a channel which increases the depolarization as it gets activated with blue laser through optic fibres. De-Monte et al. (2015) followed a 3 day procedure to test the extinction retrieval. During this procedure the rats first received 1 day of foot shock (NS) and tone (CS) conditioning. After the conditioning, in day 2 the rats received CS without the US for the extinction training. Furthermore, for day 3 the CS was given again without the NS to test the extinction retrieval. During this study, the authors found that activation of IL neurons during extinction tones deceased freezing and increased extinction of memory. The authors first tested if the activation of ChR2 channels was sufficient enough to increase the firing rate of The region in humans that is subsequent of IL in rats is known to be neurons (Figure 1) and found that 72% of the neurons expressing ventromedial prefrontal cortex (vmPFC). Previous studies have ChR2 showed increase in their firing rates when they were manipulated with blue laser light.

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Figure Adapted from DoMonte et al. (2015). J Neurosci, 35(8), 3607– 3615. Figure 1. The graph showing that 72% of neurons expressing the ChR2-eYFP gene have increased firing rate when they are activated by the blue laser.

The authors then activated the ChR2 neurons with blue laser on day 2 and tested their freezing responses to represent their extinction of memory. They found that ChR2 neurons had a reduced freezing in extinction training than the control group when the blue laser was on during this training (Figure 3). This shows that the optogenetic activation of IL increased the extinction of memories. The authors then tested if the freezing response was different in the retrieval phase (day 3) without the blue laser and found that without any laser on day 3, the rats who had optogenetic manipulation in day 2 still showed a reduced freezing response thus increased retrieval of extinction compared to the control group (AAV5:CaMKII::eYFP) (Figure 2). This fits in line with previous studies that showed increased retrieval of extinction when IL was electrically stimulated during extinction tones (Milad et al., 2004, VidalGonzalez et al., 2006). Lastly, the authors tested a new group of rats with optogenetic IL activation during day 3. When the IL of rats were optogenetically stimulated during the retrieval of extinction tones, the experimental rats showed reduced freezing than the control rats (Figure 3). This shows that the activation of IL during retrieval of extinction tones is sufficient enough to increase the retrieval of extinction and reduce the freezing response.

Figure Adapted from Do-Monte et al. (2015). J Neurosci, 35(8), 3607–3615. Figure 3. The graph showing that the rats expressing ChR2 whose IL were activated by blue laser during the retrieval training showed a decrease in their freezing response during the retrieval of extinction which represents a better retrieval

Silencing IL Neurons During Extinction Tones (eNpHR) Increases Freezing Response and Impairs the Retrieval of Extinctio: De-Monte et al. (2015) then injected the rats with the Adeno-Associated Viruses (AAV) which was to express Halorhodopsin (AAV5:CaMKII ::eNpHR3.0-eYFP). Halorhodopsin is a channel which increases the hyperpolarization of neurons thus decreases the likelihood of firing when it gets activated with yellow laser through optic fibers. To test this, the authors first activated IL neurons with yellow laser and found that 47% of the neurons showed reduced firing rate (Figure 4). The authors then conditioned a group of rats and then activated their IL with yellow laser during the extinction training in day 2. They found that silencing IL neurons during extinction training did not have any effect on conditioned freezing and they did not show any different freezing response than the control group during the extinction training (Figure 5). On the other hand, silencing IL neurons during extinction increased the freezing response during the extinction (Figure 5) which indicated that the retrieval of extinction memory was impaired, and the rats had a harder time remembering when their IL was silenced during the extinction training. These results are in line with previous studies which showed inactivation of IL during extinction training did not have any effect on extinction during the session but led to worse retrieval of extinction in day 3 (Quirk et al., 2000; Rhodes and Killcross, 2004; Laurent and Westbrook, 2009; Mercado, Coreano, & Quirk, 2011).

Figure Adapted from Do-Monte et al. (2015). J Neurosci, 35(8), 3607–3615. Figure 2. The graph showing that the rats expressing ChR2 whose IL were activated with blue laser during the extinction training showed a decrease in their freezing response during the retrieval of extinction which repre-

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Figure Adapted from Do-Monte et al. (2015). J Neurosci, 35(8), 3607–3615. Figure 4. The graph showing that 47% of neurons expressing the eNpHR-eYFP gene have decreased firing rate when they are activated by the yellow laser.

Figure Adapted from Do-Monte et al. (2015). J Neurosci, 35 (8), 3607–3615. Figure Adapted from Do-Monte et al. (2015). J Neurosci, 35(8), 3607–3615. Figure 5. The graph showing that the rats expressing eNpHR whose IL were silenced by yellow laser during the extinction training showed an increase in their freezing response during the retrieval of extinction which represents a worse

Figure 7. The graph showing that the rats whose IL were inactivated by muscomil during the retrieval training showed no change in their freezing response during the retrieval of extinction which represents that IL is not necessary during the re-

CONCLUSIONS/DISCUSSION

From their studies, De-Monte et al. (2015) found that activation of Silencing IL Neurons During Retrieval Tones (eNpHR) Does Not IL neurons during extinction tones reduced freezing and increased Change the Retrieval of Extinction: the extinction of fear memory. Meanwhile, silencing of IL neurons during extinction tones increased freezing and reduced extinction Last but not least, the authors wanted to test if IL neurons are necessary during the retrieval of memory. To test this, the au- of fear memory. The authors also found that silencing of IL neurons thors first optogenetically silenced the IL neurons with yellow laser during retrieval tones did not have any effect on extinction of during day 3 which is the retrieval test. They found that silencing IL memory or on the fear response; which was a novel finding. These neurons during the retrieval did not have any effect on the retrieval results are important to highlight the importance of IL signaling of extinction (Figure 6). To rule out the possibility that the light during the learning of extinction of fear memories. The results also might silence the Glutamatergic neurons without affecting the in- provide a new finding to the literature which shows that IL is not hibitory neurons the authors also used a nonspecific pharmacologi- necessary during the retrieval of extinction memory phase. Even cal inhibition with fluorescently labeled muscimol. Muscimol was though the finding from this study that IL is important for extinction used to enhance GABA receptor activity thus to inactivate the IL of fear is supported by previous evidences in the literature; previ(Sierra-Mercado et al., 2011). By silencing IL neurons by GABA agonist during extinction retrieval, they found that there was no effect ous studies only showed correlations between IL activity and reon retrieval on day 3 and also on day 10 (Figure 7). These results trieval of fear extinction (Quirk et al., 2000; Rhodes and Killcross, show that IL activity is not necessary at the retrieval phase and they 2004; Laurent and Westbrook, 2009; Mercado, Coreano, & Quirk, 2011). Therefore, this is the first study to show a causal importance only play an important role during the extinction phase. of IL activity. One of the possibilities of why IT has important implications on initial learning of extinction but not during the retrieval might be because of the induced plasticity in downstream targets. The ITC cells in amygdala gets activated by an incredibly powerful depolarizing current from IL. This powerful depolarization of ITC cells can induce plasticity and can affect the behavior in the next day during the retrieval phase. This shows that the activation of IL during the retrieval phase is not necessary as the changes induced by IL during the initial learning of extinction can create the effects (Amir et al., 2011). Figure Adapted from Do-Monte et al. (2015). J Neurosci, 35 (8), 3607–3615. Figure 6. The graph showing that the rats expressing eNpHR whose IL were silenced by yellow laser during the retrieval training showed no change in their freezing response during the retrieval of extinction which represents that IL is not neces-

Besides the similarities between previous papers, there are also discrepancies between this paper and previous literature. This study shows that IL activity is not necessary during the time of retrieval however previous studies show that IL is actually necessary (Laurent and Westbrook, 2009; Sangha et al., 2014). In these studies, the authors used a contextual fear paradigm and found that pharmacological inactivation of IL during retrieval tests impaired retrieval of contextual fear extinction. The possible reason for this

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discrepancy might be because of the different designs of these experiments. The fear memory has two different types thus two different networks and measurement techniques. One of them is tone fear conditioning and the other is the contextual fear conditioning (Jacobs, Cushman, & Fanselow, 2010). In contextual fear conditioning, IL is known to communicate contextual information for retrieval of extinction (Zelikowsky et al., 2013) and therefore might be playing an important role in retrieval of extinction. However, in the current study, IL might be less important during the retrieval of extinction memory as the design is based on an auditory fear conditioning task and tone fear conditioning does not require IL activity (Jacobs et al., 2010).

There are few elements of the paper that require further study. First element is the small sample size in the experiments. The three different studies all include 6 to 10 rats as the group sample sizes which results in smaller samples.

The other element that needs to be improved is the resultsâ&#x20AC;&#x2122; representation of the real life. In this study, there is a constant time between each action potential when the neurons with ChR2 are being stimulated with blue laser. However, in real life, there is a different burst patterns in IL where retrieval of extinction increases the degree of bursting in IL (Burgos- Robles et al., 2007). Therefore, the authors should take that into consideration when stimulating the neurons with blue laser and should modify their laser patterns The results from De-Monte et al. (2015) are very relevant to hu- according to the natural patterns. mans. As the region in humans that is subsequent of IL in rats is ventromedial prefrontal cortex (vmPFC) (Phelps et al., 2004; Ka- The final limitation that needs further study is the method of fear lisch et al. 2006), these results can help to target the vmPFC in conditioning. In the study by De-Monte et al. (2015), the authors anxiety disorder treatments. The circuit between vmPFC and amyg- only used one type of fear conditioning which is auditory fear condala is essential in fear response and it can be altered by different ditioning. As there are two types of fear conditioning methods methods to modify the fear extinction (Bukalo, Pinard, & Holmes, (Jacobs et al., 2010); this limits the generalizability and applicability 2015). As the results from De-Monte et al. (2015) show that IL cor- of the results. Therefore, the same study should be replicated by tex is important in rats to modulate the extinction of fear memory using a contextual fear conditioning paradigm instead of an auditoduring the extinction learning phase, new research can target ry fear conditioning. vmPFC during the extinction learning phase and increase its activity to increase the quality of the extinction trial. With this way, anxiety FUTURE DIRECTIONS â&#x20AC;&#x201C; disorders such as PTSD and phobia can be treated as they are usually caused by triggering traumatic events similar to conditioned Previous studies showed that tone fear conditioning and contextufear responses (Bukalo, Pinard, & Holmes, 2015). al fear conditioning recruit different networks in the brain (Jacobs et al., 2010) and suggest using different methods to test these two types of fear. As a future study, researchers should test the contexCRITICAL ANALYSIS tual fear conditioning by manipulating IL activity with optogenetics. The paper by De-Monte et al. (2015) demonstrates an out- As this study only tested tone fear conditioning and previous studstanding experiment to show the necessity of IL during the retriev- ies with contradicting results did not use optogenetics as their al of extinction memory by directly stimulating IL during retrieval. methods (Laurent and Westbrook, 2009; Sangha et al., 2014), the Optogenetics is a new technique to control the brain which has a researchers should replicate these studies with optogenetics to see high spatial and temporal sensitivity. The researchers are able to if the results were because of lack of temporal precision or was express the light-sensitive proteins selectively in specific cell types due to the contextual fact. The methods to test contextual fear or locations. The researchers are also able to control the time of conditioning includes returning the animal to the original training the stimulation with optic fibers as the neurons of interest are acti- context (Jacobs et al., 2010), which is different than the tone fear vated or silenced only when light is received (LaLumiere, 2011). In testing. The researchers should replicate the methods of the study electrical stimulation, the activation can back propagate and acti- by Laurent and Westbrook (2009), where the animals were put in vate different fibres of passages which can create confounding four different chambers for the contextual fear extinction test and factors (Hamani et al., 2010). Moreover, in knockout models, the their IL was inactivated by muscomil during the retrieval of extincresearchers do not have high temporal control and they cannot tion. In this study instead of muscomil, the IL should be inactivated easily activate a specific structure in the brain. Knockout models by optogenetic techniques when the animals are returned back to can also have other physiological abnormalities due to the missing the original training context. genes (Nelson, 1997). Therefore, optogenetics has several advantages over electrical stimulations and knockout models as it can The outcomes expected from this study would be that the retrieval provide an accurate causal link between the behavior and brain of contextual fear memory will be impaired when IL is silenced regions. In this study, the optogenetic approach gives the authors using optogenetics. As IL is known to communicate contextual inmore control over the area and temporal precision as they can formation for retrieval of extinction (Zelikowsky et al., 2013), the control when they want IL to be activated and when they want IL inactivation of IL will disrupt the extinction of memory in a contexto be silenced. As previous studies did not stimulate the IL during tual setting. An impaired retrieval of extinction will show that IL the retrieval of tones and only found correlations between IL activ- plays an important role in contextual memory and therefore ity and retrieval of extinction (Quirk et al., 2000; Rhodes and Kill- affects the retrieval of extinction. cross, 2004; Laurent and Westbrook, 2009; Mercado, Coreano, & If results were different and showed no impairment in the retrieval Quirk, 2011), this study gives an opportunity to find causal results of extinction fear memory, it would indicate that IL plays an imas well as the necessity of IL activity for the retrieval of auditory portant role in retrieval of extinction in both tone and contextual fear extinction. fear conditioning. It will also show that the reason for different results that previous researchers got (Laurent and Westbrook,

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2009; Sangha et al., 2014) were not because of ILâ&#x20AC;&#x2122;s contribution to the contextual information. It will indicate that the reason for discrepancies between the results were due to the lack of temporal precision and neural specificity. As optogenetics provide better temporal precision and specificity than pharmacological studies, the results of previous studies might change if there were any confounding effects. This study and its results would improve the knowledge about IL cortex and its contribution to the retrieval of extinction in different types of fear conditionings. It will provide a better idea about ILâ&#x20AC;&#x2122;s role in contextual fear and if IL is necessary for retrieval of extinction in general. This will fit into the entire framework of the literature about extinction memory and the circuits for retrieval of memory. There are other few studies that the authors should perform in the future to increase the reliability of the results. As the study by DeMonte et al. (2015) had small sample sizes (n=6-10); in order to increase the effect size, reduce the effect of any outliers and get more reliable results, the authors should replicate these studies with bigger sample sizes. If the results change, it will indicate that the significant results by De-Monte et al. (2015) might be due to possible outliers. On the other hand, if the results stay the same as the previous studies, it will show that the conclusions are reliable and generalizable. The authors should also replicate the study by De-Monte et al. (2015) using an alternative neurostimulation technique of deep brain stimulation (DBS). In this study, the authors used optogenetics to inhibit the IL during the retrieval of extinction (De-Monte et al., 2015). As previous studies did not inhibit IL during the retrieval of extinction, this study should be replicated with an alternative method in order to increase the reliability of the results. DBS is used to dissociate input and output signals thus inhibit the region of interest (Chiken and Nambu, 2014). The future study should inhibit the IL by using DBS. It would be expected to see no change in the extinction of memory, replicating the study by DeMonte et al. (2015). If the retrieval of extinction changes due to the inhibition, it might indicate two possible reasons: First is that DBS and optogenetics have different mechanisms of inhibition and the study with DBS had confounding factors that affected the extinction of memory. The second is that IL might actually be important for fear extinction and the study with optogenetics should be replicated to see if the results are replicable. This study will give a better understanding about the necessity of IL and different methods to inhibit a region of interest. These future studies should be considered to increase the reliability and the applicability of the results.

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Zelikowsky M, Bissiere S, Hast TA, Bennett RZ, Abdipranoto A, Vissel B, FanselowMS (2013) Prefrontalmicrocircuitunderliescontextuallearn- ing after hippocampal loss. Proc Natl Acad Sci U S A 110:9938 –9943. CrossRef Medline

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Exercise and Neuroplasticity in Brain Injury Bryan Handoyo Kartono

Neuroplasticity typically refers to the nervous systemâ&#x20AC;&#x2122;s ability to alter, reshape, and reorganize its structure, function, and connections in response to an intrinsic or extrinsic stimulus in order to adapt better to novel situations. The fact is that neural networks, depending on experiences, are not fixed, but dynamically occurring and disappearing throughout the lifetime. Intensive training in motor and cognitive skills and imagination of brain injury movements is thought to contribute to the reorganization of brain activity patterns, which in turn normalizes brain function. Numerous scientific articles have been discussing the significance of neuroplasticity on recovery following brain injury. The study conducted by Griesbach et al. observed that exercise induces neuroplasticity through the BDNF-related pathway. Studies conducted by Szulc-Lerch et al., Juenger et al., and Chin et al., shows that, in human subjects, exercise induces neuroplasticity in brain damage caused by radiation in brain tumor patients, congenital hemiparesis patients, and traumatic brain injury patients, which shows that exercise induces neuroplasticity in various kind of brain injury. With substantial data that shows cellular and cognitive effects caused by exercise in animal models and results to support the impact of exercise in patients with different types of brain injury, good evidence is available to support the role of exercise in inducing neuroplasticity in patients with brain injury. Key words: neuroplasticity, exercise, brain damage, brain tumor, congenital hemiparesis, traumatic brain injury, brain-derived neurotrophic factor

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INTRODUCTION Neuroplasticity is the incredible ability of the brain to change and adapt. It generally refers to the nervous systemâ&#x20AC;&#x2122;s ability to change, remodel, and reorganize its structure, function, and connections in response to an intrinsic or extrinsic stimulus for the purpose of better ability to adapt to new situations1. Even though the idea of neuroplasticity just came out recently, it is one of the most remarkable discoveries in the field of neuroscience. The fact is that neural networks, depending on experiences, are not fixed, but dynamically occurring and disappearing throughout the lifetime2. While we repeatedly practice one activity, neuronal circuits are being developed, leading to a better ability to perform the practiced task with less energy2. In contrast, once we ceased to practice one activity, the brain will reroute these neuronal circuits by a â&#x20AC;&#x2DC;use it or lose itâ&#x20AC;&#x2122; principle2. Research showed that various aspects of the brain could be reconstructed even through adulthood 3. However, the developing brain exhibits a higher degree of plasticity than the grown-up brain3. Neuroplasticity ranges from microscopic changes in individual neurons to larger-scale changes such as cortical reorganization in response to brain injury4. The concept of neuroplasticity allows one to explain various phenomena, such as habituation, sensitization to a specific position, medication tolerance, and even recovery following brain injury 2. As a matter of fact, it is known to be the basis for much of many of the cognitive and physical rehabilitation treatment following brain injury1. It is believed that intense training of motor and cognitive tasks and imagination of movements following brain injury leads to the reorganization of brain activity patterns, which in turn normalizes brain function5. The purpose of this review paper is to discuss the efficacy of exercise in inducing neuroplasticity in subjects with brain injury. Numerous scientific articles have been discussing about the significance of neuroplasticity on recovery following brain injury in animal models. For instance, by evaluating the effects of exercise following experimental TBI, Griesbach et al. concluded that, in the rats' traumatic brain injury (TBI) model, voluntary exercise could endogenously upregulate brain-derived neurotrophic factor (BDNF) and improve recovery when applied at the appropriate post-injury time window, as they found out that exercise dramatically increases the levels of hippocampal phosphorylated synapsin I and phosphorylated cyclic AMP response element-bindingprotein (CREB), as well as phosphorylated synapsin I and BDNF 6. A study conducted by Yamamoto et al. focused on the redevelopment of neural pathway following brain injury and found out that following a motor exercise for three months, adult primate brains with motor lesions can rearrange an extensive network to allow motor recovery by enhancing the coupling of sensorimotor and motor commands through rewired frontocerebellar connections8 Numerous studies also support the role of exercise on neuroplasticity in human subjects. In the research study by Szulc-Lerch et al., after examining the anatomical T1 magnetic resonance imaging (MRI) data and multiple behavioral outcomes, they concluded that exercise in radiation-treated pediatric brain tumor patients has a benefit on the brain as it was associated with increase pre and postcentral gyri, left temporal pole, left superior temporal gyrus, and left parahippocampal gyrus cortical thickness9. Exercise also improves the cognitive function of the brain following brain injury. In a research study by Chin et al., the experimenters examined the effect of aerobic exercise on TBI patients. They found out that, as

seen from several cognitive and psychological tests, aerobic exercise improves various aspects of cognitive function10. In a research study conducted by Juenger et al., the experimenters examined ten patients with congenital hemiparesis which was caused by unilateral cortico-subcortical infarctions in the middle cerebral artery territory that received a 12-day intervention of constraintinduced movement therapy (CIMT)11. Following an observation by functional MRI (fMRI), the experimenters concluded that exercise could promote changes of cortical activation in congenital hemiparesis, as they found out that increases in cortical activation during paretic hand movements in the primary sensorimotor cortex of the affected hemisphere as well as improved task performance11.

MAJOR RESULTS Exercise Increases BDNF in Animal Models The

Figure 1 - BDNF immunostaining on day 21 of the postinjury. BDNF was primarily distributed in the hippocampus region of the dentate gyrus and CA3. Exercise resulted in an increase in the number of BDNF6.

study conducted by Griesbach et al. observed that exercise leads to the increment in BDNF (Figure 1), as well as its downstream effectors, CREB, and synapsin I6. However, the experimenters also noted that when the exercise is administered to soon after TBI, it will not yield the same result as the molecular response to exercise is disrupted, which will lead to a delayed recovery time6. It will instead lead to a lower level of CREB and phosphorylated synapsin I, as well as the failure to upregulate BDNF6. The experimenters also noted that the changes in the amount of BDNF and enhanced synaptic plasticity caused by exercise might improve cognitive performance6. This finding came from the fact that mice from delayed -exercise group perform better in the Morris water maze test than the early-exercise group6. Exercise in Long-Term Pediatric Brain Tumor Survivors Szulc-Lerch et al. main research focus is to examine the effectiveness of exercise practice in 28 radiation-treated long-term pediatric brain tumor survivors for neural and behavioral rehabilitation9. By using unbiased automated vertex-wise analyses, as seen in Figure 2A, the experimenters found out that exercise leads to an increase in cortical thickness for the right precentral and postcentral gyri8. They also recorded an increase in cortical thickness in the left temporal pole, left superior temporal gyrus, and left parahippocampal gyrus8. As seen in Figure 2B, Szulc-Lerch et al. also

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found out that, based on the Deformation Based Morphometry in activation in the interhemispheric fissure, in the primary sen(DBM) results, exercise resulted in increased WM volume underly- sorimotor cortex of the contralesional hemisphere and in the cereing the right motor and somatosensory cortices, as well as in the bellum. parietal lobe8. Szulc-Lerch et al. noted that the areas where they found an increase in WM volume in motor and premotor cortices, which also showed an increase in cortical thickness in response to exercise8.

Figure 2 – (A) Comparison of the thickness in the right precentral and postcentral gyrus cortex before (Pre) and after (Post) exercise visualized by boxplots. (B) Increased white matter (WM) volume underlying the motor cortex, somatosensory cortex, and parietal lobe visualized by boxplots8.

Exercise in Congenital Hemiparesis Patients

Figure 3 – fMRI activation before and after CIMT during active and passive movements of the paretic hand. ∆ indicates significant increases in activation. The brain surfaces were flipped, such that the left side of the picture is the left side of the patients11.

Figure 4 – Changes in the brain after 12 days of CIMT visualized by (a) fMRI, (b) transcranial magnetic stimulation (TMS), (c) magnetoencephalography (MEG). These data shows that the type of corticospinal reorganization in congenital hemiparesis affects the CIMT-induced diverging cortical changes16.

A later study by Juenger et al. found out that the type of corticospinal reorganization in congenital hemiparesis affects the CIMTinduced diverging cortical changes16, such that, as seen in Figure 4, patients with ipsilateral corticospinal projections showed a reduced transsynaptic primary motor cortex (M1) excitability and reduced synaptic activity during paretic hand active movements after 12 days of CIMT, whereas patients with crossed contralateral corticospinal projections showed an increment in those parameters16. Exercise in Traumatic Brain Injury Patients Chin et al. main research focus is to examine the effect of exercise, specifically aerobic exercise, on cognitive function of TBI patients10. Following supervised intensive aerobic exercise program, TBI patients showed significant increment in cognitive function 10. These cognitive functions include the domains of processing speed, executive functioning, and overall cognitive function. The experimenters also noted that the magnitude of the improvements observed may be influenced by the adaptability of patient’s cardiorespiratory system to the aerobic exercise10. The positive effect of aerobic exercise on cognitive function was also reported on various study. Voss et al. found a strong association between the improvements in cardiorespiratory fitness and cognitive function in older adults13, while Kluding et al. concluded that exercise is associated with an improvement in cognitive function in stroke patients17.

Assessing the impact of CMIT on ten patients with congenital hemiparesis which was caused by unilateral cortico-subcortical infarctions in the middle cerebral artery is the collective focus of DISCUSSION Juenger et al. study11. As seen in Figure 3, FMRI during paretic Based on the available data showing effects that were induced hand movement revealed an increment in activation of primary by exercise in animal models, together with findings to support the sensorimotor cortex of the affected hemisphere, in this case, the effect of exercise in patients with various kind of brain injury, left hemisphere. In addition, the researcher also found an increase there is significant evidence to support the role of exercise in in-

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ducing neuroplasticity in patients with brain injury. The study conducted by Griesbach et al. concluded that, in animal model, voluntary exercise could endogenously upregulate brain-derived neurotrophic factor (BDNF) and improve recovery when applied at the appropriate post-injury time window6. This study specifically highlights the importance of exercise on BDNF, as exercise leads to the increase of BDNF, as well as its downstream effectors, CREB, and synapsin I, and it will, in turn, promotes brain recovery 6. However, it is known that the effect is time-dependent, as they observed that there is a delayed recovery time when exercise is administered too soon after injury time. They suggested that it might be caused by metabolic alterations that occur during this post-injury period6. A study conducted by Lee et al. suggested that there is a lower concentration of the ATP, which is the primary source of cellular energy, following TBI, and Lifshitz et al. indicated that there is a structural alteration of the mitochondria following TBI18,19. Therefore, it is suspected that if exercise is administered too soon, it may enhance ATP loss or redeploy it from functions that are vital for recovery, such as producing synaptic plasticity molecules, by introducing an unnecessary additional amount of energy needed at a time when the brain is exhausted. The experimenters also noted that the changes in the amount of BDNF and enhanced synaptic plasticity caused by exercise might improve cognitive performance6. It is presumed that the lack of BDNF, synapsin I, and CREB increases reflect changes in selected molecular systems that influence cognitive performance6. It is unclear, however, if the same mechanism also applies to the human being. Szulc-Lerch et al. concluded that exercise in pediatric brain tumor patients has a benefit on the brain as it was associated with increase the pre and postcentral gyri, left temporal pole, left superior temporal gyrus, and left parahippocampal gyrus cortical thickness, as well as increased WM volume underlying the right motor and somatosensory cortices, as well as in the parietal lobe9. This finding is supported by a study conducted by Chaddock-Heyman et al., in which they found out that fitness level is directly proportional to cortical thickness in children and adults12. The exercise-induced cortical thickness increment may represent brain recovery processes in the context of injury, although it does not follow the development pattern that is normally observed in healthy children 12. Szulc-Lerch et al. observation on the WM volume is also supported by a study by a study by Voss et al., in which, in their research, they observed training-related changes in white-matter structure in human adult13, as well as a study by Scholz et al., in which they found out that the increase in WM volume is typical following motor-skill learning. Szulc-Lerch et al also thinks that the increase in WM volume in motor and premotor cortices may be caused by the increased neural activity during exercise, which in turn may induce activitydependent myelination or axonal sprouting and branching, which may lead to increased WM volume that was seen in this study2. While Juenger et al. did not look at brain tumor patients, they also concluded that exercise could induce changes in the brain cortex; in this case, it induces changes in cortical activation in patients with congenital hemiparesis11. They suggest that an increment in activation of primary sensorimotor cortex of the affected hemisphere indicates that even severe lesions acquired early during brain development do not seem to hinder the development of neural circuits that is critical to neuroplasticity in this part of the brain 11. This study is the first study to observe neuroplasticity activity predominantly in the affected hemisphere, as the contralesional hemisphere is known to be the major player during the re-organization of hand functions15. Therefore, this study brought up the possibility that neuroplasticity activity of motor recovery would also occur in the contralesional hemisphere11. In contrast, Chin et al. concluded that exercise improves the cognitive function of TBI patients10. It is suspected that cardiorespiratory fitness gains may be a determinant of the observed improvement in cognitive function10.

All in all, the current existing human studies, including studies from Szulc-Lerch et al., Juenger et al., and Chin et al., showing enhanced neurocognitive outcomes for patients with various kind of brain injury who participated in exercise supports the need for a supervised prospective study looking on the effect of exercise on cognitive recovery. While the exact mechanism underlying the neuroplasticity in the human brain following brain injury is unclear, it is possible that, as proposed by Griesbach et al. and Chytrova et al., the BDNF, synapsin I and CREB play a significant role in promoting brain recovery6,7. These studies have some limitations. Most of these studies have small sample sizes. A small sample size leads to several problems, such as a higher probability that the results obtained from the study are due to chance, as well as the study does not represent the general population, which can render the study useless. Another limitation is that most of these studies also did not look at carryover effects, which means that we did not know whether the changes observed in this study ceased or maintained after completion of the training.

CRITICAL ANALYSIS Several types of research indicated that neurogenesis following brain injury in rodents was correlated with the upregulation of the BDNF, an omnipresent growth factor of the central nervous system (CNS), in the rodent hippocampus and that this process was associated with enhanced performance in temporospatial memory tasks 6,7. The link found between cellular regeneration and enhanced neuroplasticity makes the signaling pathways by which exercise promotes hippocampal cell growth and in the potential for newly-born neurons to perform in the context of traumatic cell loss interesting. This also raised the possibility of the role of exercise in enhancing neuroplasticity to promote recovery following brain injury as a therapeutic intervention. Many kinds of research have helped to explain the molecular and cellular changes that occur following exercise in the hippocampus. Current work indicates that exercise promotes neuroplasticity by affecting the cellular regeneration mechanism that occurs through BDNF upregulation through a pathway that includes a CREB and synapsin I6. More research also indicates that the environment of exercise upregulates several proteins that plays a role in energy metabolism and synaptic plasticity if administered at the right time6. An increasing number of retrospective population-based research also supports the role of exercise on neuroplasticity in human studies. Nevertheless, as convincing as these studies are, they only demonstrate correlation, not causation. Research on exercise and neuroplasticity following brain injury in human provide important insights into the potential positive effects of exercise on neuroplasticity for several important reasons. Despite the variation of exercise, time after injury, and study parameters, the current literature implies that patients with a brain injury can participate safely in exercise. Several studies on human subjects also investigated the effect of exercise on various kinds of brain injury. It has been proven that exercise work on brain damage caused by radiation in brain tumor patients, congenital hemiparesis patients, and traumatic brain injury patients9, 10, 11. However, given the lack of research examining the role of exercise in promoting brain recovery, there is no way, based on current literature, to state whether and how exercise will improve recovery mechanisms such as plasticity, regeneration, etc.

FUTURE DIRECTIONS Current available studies on exercise and neuroplasticity accentuate our narrow knowledge of the mechanism through which neuroplasticity takes place and suggest that further investigation in this area is critical. Researchers should resort more to testing the effect of exercise on a specific brain mechanism that is known to promote brain recovery. For instance, the researcher can opt to investigate BDNF in humans with brain injury, as according to animal models, the upregulation of BDNF and its related downstream

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effectors plays a role in promoting neuroplasticity, which in turn helps brain recovery. Results from this experiment might provide us an insight into how exactly exercise might induce neuroplasticity in patients with brain injury. While we expect to see an increase in the amount of BDNF in the brain, a negative result does not necessarily mean the study is a failure; it just simply eliminates the possibility that exercise induces neuroplasticity through BDNFrelated pathway. It is essential to specify which kind of brain injury were the researcher investigate as, from the previous sections, we know that exercise induces a different type of effect depending on the type of brain injury, thus different kind of brain injury might have its own specific recovery mechanism that is unique from one another. We can also do an experiment in BDNF-knockout mice. As mentioned earlier, the upregulation of BDNF and its related downstream effectors is essential in promoting brain recovery through neuroplasticity. While we expect to see little-to-none improvement on BDNF-knockout mice following brain injury, a different result does not mean that the study is controversial; it means that there might be another pathway that promotes brain recovery, and this leads to more thorough research on identifying that particular pathway. We can also test this at a different time period of when the exercise was administered as based on Griesbach et al., exercise will upregulate the expression of BDNF and its related downstream effectors only if administered at the right time6. It is suspected that we will not see an increase in BDNF and its related downstream effectors if exercise is administered too soon after the brain injury occur. However, a negative result might mean either time is not important in human subjects or exercise does not affect neuroplasticity through the BDNF-related pathway. It is therefore essential to remove the possibility of exercise acting on the BDNF-related pathway by doing this study after a considerable amount of time, considering the result of the study by Griesbach et al6. It is also important to observe carryover effects that might be present following the exercise training. It is also interesting to test the effect of a different kind of exercise on brain plasticity following brain injury. The studies mentioned in this paper have different types of exercise, such as aerobics, cardio, and even a therapy that has a specific set of a different kind of exercise. Different kinds of exercise might exert a different kind of effect, and even if they did exert the same kind of effect, it might affect the brain through a different kind of pathway.

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The Highs and Lows of Pregnancy: Effects of Prenatal Cannabis Exposure on Dopaminergic Neurons in the Ventral Tegmental Area Haleema Khan

With the increasing legalization of cannabis, there are many misconceptions around the safety of use for pregnant women. Cannabis is often used as a therapy for pregnancy-related conditions, such as morning sickness, raising questions about the potential impact of use on offspring. Rising evidence suggests that prenatal cannabis exposure (PCE) might predispose offspring to various neuropsychiatric disorders, linked to atypical dopaminergic activity. The current state of knowledge does not elucidate the underlying physiological effect of PCE on dopaminergic function in the ventral tegmental area (VTA), a brain region which plays a prominent role in reward pathways, cognition, and attention, all of which are involved in many neuropsychiatric disorders. In order to investigate this, the present study exposed rat dams to Î&#x201D;9-tetrahydrocannabinol (THC) and observed increased behavioural sensitivity in male but not female PCE offspring acutely exposed to THC during pre-adolescence. Marked outcomes of increased behavioural sensitivity included impaired sensorimotor gating and increased risk-taking in various motor tasks in PCE rats. This shift in behaviour was found to occur due to extensive molecular and synaptic changes in the dopaminergic neurons of the VTA; PCE induced a hyperdopaminergic state in VTA neurons. A US Food and Drug Administration (FDA) approved drug called pregnenolone, restored normal synaptic properties and re-established baseline dopaminergic activity, suggesting a promising therapy for offspring exposed to THC prenatally. These findings highlight the importance of cautioning pregnant women about the potential harms of prenatal cannabis use to the developing fetus. Key words: cannabis, dopamine, prenatal, offspring, behaviour, ventral tegmental area, fetus

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INTRODUCTION:

Overtime, public attitudes regarding cannabis have varied, with adults increasingly viewing cannabis as harmless (Hasin, 2018). In North America, the use of cannabis continues to grow in pregnant women (Alpar et al., 2015). Particularly, past-month cannabis use increased by 62% from 2002-2014 among pregnant women (Brown et al., 2017) and the overall use of cannabis during pregnancy ranges between 25% (Obstetrics & Gynecology, 2015). Notably, cannabis use is most prevalent among 18-25-year-old pregnant women, indicating that young women are at the greatest risk (Brown et al., 2017). With this growing acceptance and use of cannabis, has come a plethora of concerns regarding the safety of use for pregnant women. The American College of Obstetricians and Gynecologists recommends that doctors discourage the use of cannabis for medicinal purposes during preconception, pregnancy, and lactation, to avoid adverse side effects to the mother and the fetus (Obstetrics & Gynecology, 2015). Despite this, crosssectional analyses indicate that cannabis continues to be recommended to pregnant women as a therapy for morning-sickness (Dickson et al., 2018). Moreover, while cannabis use is legalized in several regions, there is no legal warning against the harmful sideeffects of use during pregnancy (Volkow et al., 2017). Further, healthcare professionals often fail to inform patients about the risk of cannabis use during pregnancy (Volkow et al., 2017). Î&#x201D;9-tetrahydrocannabinol (THC), a type of cannabinoid, is the main psychoactive ingredient in cannabis, which has profound effects on the endocannabinoid system in the brain (Galve-Roperh et al., 2013). The endocannabinoid system is involved in cell proliferation, differentiation, growth, and survival with many possible outcomes depending on the signalling pathway involved (Galve-Roperh et al., 2013). Cannabinoids such as THC bind to two members of the G-protein coupled receptor family, cannabinoid receptors 1 (CB1R) and 2 (CB2R), primarily found on the cell membrane, which mediate downstream signals (Zou & Kumar, 2018). CB1R is the main subtype within the central nervous system, while CB2R is mainly found in the peripheral nervous system (Zou & Kumar, 2018). THC can readily

bind to both cannabinoid receptors producing a wide range of physiological effects on mood, appetite, chronic pain, learning and memory, and motor control among many other systems (Zou & Kumar, 2018). The prenatal brain is highly sensitive to maternal drug use because of its critical development period, making the fetus susceptible to neuropsychiatric disorders which may appear later in life (Morris et al., 2011). Literature in psychology has widely posited that both genetic and environmental factors play important roles in the development of neuropsychiatric disorders. Genetic background and/or environmental conditions can impact the brain in a way that increases susceptibility to the onset of psychiatric symptoms at a later time (Tsuang et al., 2004). As such, understanding the changes to the brain circuit from PCE might help prevent the emergence of potential disorders. Studies show that prenatal cannabis exposure (PCE) predisposes individuals to a wide range of behavioural and cognitive deficits such as hyperactivity, compulsive activity, loss of sustained attention, and enhanced sensitivity to substance abuse (Morris et al., 2011, Huizink et al., 2014). Importantly, all of these outcomes are closely tied to the dopaminergic system (Volkow et al., 2011). When dopamine function becomes significantly disrupted with drug use, an individual may have impaired salience attribution and motivation, leading to compulsive behaviours (Volkow et al., 2011). This marked disruption is seen in the orbitofrontal cortex and cingulate gyrus, which are brain areas critically involved in inhibitory control and attention (Volkow et al., 2011). The ventral tegmental area (VTA) is another brain region which plays a key role in the reward circuit, motivation, and cognition and may be affected by PCE (Ansari et al., 2010). While the impact of cannabis use has been investigated in other brain regions, the physiological impact of PCE on the VTA of offspring and subsequent behavioural outcomes have yet to be elucidated. The present study tested the effects of PCE on behaviour as well as molecular and synaptic changes in the VTA of rat offspring. The authors found that PCE leads to behavioural deficits in offspring which are explained by physiological changes in the VTA,

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marked by a hyperdopaminergic state. Postnatal administration of pregnenolone rescued the hyperdopaminergic state of PCE rats and restored baseline synaptic and behavioural properties. These results illuminate the problematic effects of prenatal cannabis use on fetal development and subsequent susceptibility to neuropsychiatric disorders in offspring. As such, it is important to emphasize the risks of prenatal cannabis use in clinical settings to avoid possible harms to the fetus.

were performed using male rat offspring only (Figure a-b). *all figures retrieved from primary article (Frau et al., 2019)

MAJOR RESULTS:

PCE Behavioural Phenotype PCE rats were modeled by introducing 2 mg per kg of THC daily, to rat dams from day 5 of gestation to day 20. This low dose was given to ensure that dams would not elicit cannabinoid tolerance after repeated exposure while also avoiding a substantial impact on maternal behaviour. Offspring were tested in various behavioural tasks basally and following acute THC exposure during the third and fourth post-natal weeks which align with human pre-adolescence, a time when clinical markers of neuropsychiatric disorders begin to show. Using pre-pulse inhibition (PPI) of the acoustic startle reflex, the experimenters tested whether PCE alters sensorimotor gating, which is a marker of schizophrenia and other psychiatric disorders. Acute THC administration was found to disrupt PPI parameters in the PCE group in the male offspring only (p = 0.037) (Figures a-b). In a cerebral micro-dialysis test, it was found that the nucleus accumbens (NAc), a target of the VTA, had larger dopamine levels in the PCE offspring following acute THC exposure and that PPI and dopamine levels in the NAc were inversely proportional (p < 0.05) (Figures c-d). Further, behavioural disinhibition was assessed in a dopamine-dependent wire-beam bridge task, which revealed that PCE rats acutely exposed to THC were more prone to cross the bridge and display impaired risk evaluation compared to control rats (p = 0.0006) (Figure e). This impulsive behaviour following THC exposure is in line with a study by Morris and colleagues (2011), which demonstrates that PCE offspring show signs of compulsive behaviour compared to controls. These results were only consistent in male rat offspring, so subsequent studies

Figure a-b: PCE affects PPI startle response; response only seen in male rats.

Figures c-d: PCE increases dopamine levels in nucleus accumbens. PPI values inversely proportional to NAc dopamine levels in PCE mice.

Figure e: PCE rats demonstrate an increased tendency to cross wire-beam. Increased Dopamine Neuron Excitability in VTA of PCE Offspring The experimenters performed a patch-clamp recording of the portion of the VTA which projects to

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the NAc and found that dopamine neurons from PCE offspring spontaneously fired at a higher frequency (p = 0.001) and showed a depolarized resting membrane potential (RMP) (p = 0.01) (Figure fh). Additionally, dopamine neurons from PCE showed increased excitability due to somatically injected currents (p = 0.0001) (Figure i) and a shorter delay in action potential firing (p < 0.001) (Figure j). Notably, in comparison to the control offspring, a larger proportion of dopamine neurons fired action potentials in PCE offspring (80% versus 24%) (Figure j).

Figure f-h: PCE dopamine neurons show firing at higher frequency and depolarized RMP.

Synaptic Changes in PCE in Favour of Excitation Next, the experimenters assessed how PCE effects the synaptic properties of VTA dopamine neurons. They saw a marked increase of excitatory dopaminergic neurons compared to inhibitory neurons (p = 0.002) (Figure k). This was explained by a reduced neurotransmitter release probability at inhibitory synapses, as calculated by the decreased coefficient of variation2 value of inhibitory postsynaptic currents (IPSCs). This was further clarified as the researchers found an increased level of bassoon density in the GABAergic synapses (Figure l). Bassoon is a cytomatrix protein which inhibits the recruitment of voltage-gated calcium channels required for vesicle release at the presynaptic terminal (Glebov et al., 2017). They used confocal and stochastic optical reconstruction microscopy (STORM) to quantify bassoon density and measured a significant increase of bassoon in the GABAergic neurons of the PCE rats (p = 0.03). These data illustrate that PCE rats have a change in the presynaptic proteins which decrease vesicle release in inhibitory neurons, consequently increasing the excitation of dopamine neurons.

Figure i: Increased excitability in PCE dopamine neurons due to somatically injected currents. Figure j: Reduced action potential firing latency in dopamine neurons in PCE rats, as well as a larger proportion of dopamine neurons firing in PCE rats. Figure k: increased excitatory to inhibitory ratio of dopaminergic neurons in VTA. 218

has been known to reverse the effects of anxiety, depression, and impaired cognition (Vallee et al., 2016). It has also demonstrated capabilities in reducing the effects of cannabinoids by limiting downstream intracellular pathways of the CB1R (Vallee et al., 2016). As such, pregnenolone offers itself as a promising therapy to reverse the effects of PCE-induced changes in the function of dopamine neurons in the VTA and the subsequent behaviour of rats. To assess pregnenolone as a therapy, the researchers administered 6 mg per kg of pregnenolone Additionally, PCE rats showed a larger AM- once per day for 9 days. Following administration, PA/NMDA receptor ratio compared to controls in the pregnenolone showed to reverse dopamine neuron VTA dopamine neurons (p < 0.0002) (Figure m). excitability in PCE brain slices as assessed by resting Moreover, the researchers observed a longer decay in membrane potentials (p = 0.0006) and neuron firing AMPA currents in excitatory post-synaptic neurons activity (p < 0.01) (Figure o). Pregnenolone also redopamine neurons in PCE rats (p = 0.0004) (Figure stored normal synaptic properties of dopamine neun). These results share a resemblance with a study rons as seen through the EPSC/IPSC (p = 0.0002) and which showed the increase of NMDA and AMPA AMPA/NMDA (p = 0.0001) ratios (Figure p). Critireceptors in VTA dopamine neurons of offspring ex- cally, pregnenolone rescued the disruption of somaposed to other drugs such as alcohol (Hausknecht et tosensory gating functions seen in the PCE offspring al., 2015). Altogether, this explains how prenatal drug in the behavioural tasks (p = 0.032) (Figure q). exposure may increase glutamatergic receptors in dopamine neurons in the VTA.

Figure m: Increased ratio of AMPA/NMDA receptor Figure o: Pregnenolone restores normal RMP and ratio in PCE dopamine neurons compared to controls. neuronal firing activity in PCE rats.

Figure n: Longer decay of AMPA currents in PCE excitatory post-synaptic dopamine neurons compared Figure p: Pregnenolone restores normal synaptic to controls. properties in dopaminergic neurons as seen through EPSC/IPSC ratio and AMPA/NMDA receptor ratio. Pregnenolone Restores Normal Dopamine Function in PCE Rats Pregnenolone is an FDA-approved steroid that 219

attention (Dickson et al.; 2018; Volkow et al., 2017). Moreover, during pregnancy, the fetal brain is in a critical development period making it highly vulnerable to external agents such as THC. Consequently, it is important to take precautions against use to not harm the fetus or predispose it to psychiatric disorders later in life. CRITICAL ANALYSIS: Figure q: Pregnenolone rescues somatosensory gating In this study, following the behavioural task, impairments seen in PCE rats in behavioural task. all subsequent studies were performed only on male rats because this population showed a distinct endoDISCUSSION: phenotype. If these effects translate onto human models, then this study only explains half of the story. FeThe present study provides evidence for the impact of prenatal THC exposure on offspring. PCE male gonadotropic hormones such as estrogen and induces synaptic changes in the VTA of male rat off- progesterone have been shown to modulate basal dospring, leading to a hyperdopaminergic state. Conse- pamine levels in the striatum and NAc (Becker, quently, sensorimotor gating and risk assessment are 1999). Therefore, there may be differences in the VTA dopaminergic response to PCE in male and feimpaired due to the excessive amounts of dopamine in the VTA. Pregnenolone, an FDA approved steroid male offspring. It is worthwhile to investigate the differences between male and female rats in terms of drug, rescues this hyperdopaminergic state and redopaminergic excitability, synaptic changes, and stores baseline dopamine levels and normalizes bepregnenolone rescue to elucidate why these phenotyphaviour. Altogether, these findings reveal the longterm impact of cannabis use during pregnancy and the ic results are different between the two populations. Moreover, the study is not entirely clear on potential threats to the developing fetal brain from whether the timing of THC exposure in the gestationsuch exposures. Mesostriatal dopamine dysfunction is particu- al period makes an impact on the outcome of the fetal larly prominent in the pathobiology of schizophrenia brain. In the study, the rat dams were exposed from (McCutcheon et al., 2019). Previous studies have also day 5 to day 20 of gestation, but the authors did not clarify what this period corresponds to in humans or made a correlative link between a predisposition to whether this time frame was critically chosen as a THC-induced psychosis and dopamine release (Dâ&#x20AC;&#x2122;Souza et al., 2005). This sheds light on the possi- sensitive period in fetal brain development. Clinicalbility of PCE as a risk factor for episodes of psychosis ly, women might still be able to employ cannabis as a during adolescence. In PCE rats, the hyperdopaminer- therapy for pregnancy-related sickness, but by limiting its use to a given period of gestation to not harm gic state led to marked phenotypic differences compared to controls as evidenced by various behavioural the fetus. This would be a useful element to investitasks. PCE rats, when exposed to THC acutely during gate in order to establish limits of cannabis use. pre-adolescence, showed dysfunctional sensorimotor gating and impaired risk evaluation. Maladaptive behaviour is a hallmark of psychosis, eluding to the possible link between PCE and psychosis onset. This study also elucidates the importance of cautioning pregnant women regarding the use of cannabis. As mentioned previously, cannabis is often used as a remedy against morning-sickness by pregnant women and does not receive much cautionary

FUTURE DIRECTIONS: To close the remaining gaps of the study, the experimenters should investigate the behavioural and molecular changes in the VTA of female offspring as a result of PCE. Only the behaviour tasks were studied in both male and female offspring. Thus, the experimenters should investigate whether PCE dopamine neurons demonstrate increased excitability in

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the VTA using patch-clamp recordings. By replicating the studies investigating the male rats in females, the experimenters should observe the synaptic changes in the VTA in PCE rats. Specifically, they should investigate the ratios of excitatoryinhibitory neurons, bassoon density using STORM, and glutamatergic receptor ratios, as performed in the male rats. Finally, the researchers should also investigate the effect of pregnenolone on restoring baseline dopaminergic activity and consequent behaviour. Only then, can the study elicit the effects of PCE on female offspring. If the outcome of this experiment does not replicate the studies performed on male rats, then there may be other variables such as female gonadotropic hormones (Becker, 1999) that modulate dopamine levels in the VTA. Performing this study would further increase the translational value of the present study in human models, as it would explain the underlying physiology in the female PCE brain. In order to establish whether the timing of the gestational period is important in cannabis exposure, this experiment could further investigate PCE on different periods of rat dam gestation corresponding to a human gestational timeline. PCE could be introduced following each trimester in gestation as the fetal brain achieves different developmental milestones such as neural induction, proliferation, and migration in each trimester (Herschkowitz, 1988). This would help to further clarify whether a specific period in gestational brain development is most sensitive to cannabis exposure. Moreover, this could help establish the hard limits of cannabis use in pregnancy. If this study shows no variation between time of PCE and impact on the offspring, then we may conclude that PCE at any time during pregnancy causes harm to the fetus.

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6. D’Souza, D. C., Abi-Saab, W. M., Madonick, S., Forselius-Bielen, K., Doersch, A., Braley, G., . . . Krystal, J. H. (2005). Delta-9tetrahydrocannabinol effects in schizophrenia: Implications for cognition, psychosis, and addiction. Biological Psychiatry, 57(6), 594-608. doi:10.1016/j.biopsych.2004.12.006 7. Dickson, B., Mansfield, C., Guiahi, M., Allshouse, A., Borgelt, L., Sheeder, J., . . . Metz, T. (2018). Recommendations from cannabis dispensaries about first-trimester cannabis use. Obstetrics & Gynecology, 131(6), 1031-1038. doi:10.1097/AOG.0000000000002619 8. Frau, R., Miczán, V., Traccis, F., Aroni, S., Pongor, C. I., Saba, P., . . . Melis, M. (2019). Prenatal THC exposure produces a hyperdopaminergic phenotype rescued by pregnenolone. Nature Neuroscience, 22(12), 1975-1985. doi:10.1038/s41593-019-0512-2 9. Galve-Roperh, I., Chiurchiù, V., Díaz-Alonso, J., Bari, M., Guzmán, M., & Maccarrone, M. (2013). Cannabinoid receptor signaling in progenitor/stem cell proliferation and differentiation. Progress in Lipid Research, 52(4), 633-650. doi:10.1016/j.plipres.2013.05.004 10. Glebov, O. O., Jackson, R. E., Winterflood, C. M., Owen, D. M., Barker, E. A., Doherty, P., . . . Burrone, J. (2017). Nanoscale structural plasticity of the active zone matrix modulates presynaptic function. Cell Reports, 18(11), 2715-2728. doi:10.1016/j.celrep.2017.02.064 11. Hasin, D. S. (2018). US epidemiology of cannabis use and associated problems. Neuropsychopharmacology : Official Publication of the American College of Neuropsychopharmacology, 43(1), 195-212. doi:10.1038/npp.2017.198 12. Hausknecht, K., Haj-Dahmane, S., Shen, Y., Vezina, P., Dlugos, C., & Shen, R. (2015). Excitatory synaptic function and plasticity is persistently altered in ventral tegmental area dopamine neurons after prenatal ethanol exposure. Neuropsychopharmacology : Official Publication of the American College of Neuropsychopharmacology, 40(4), 893-905. doi:10.1038/npp.2014.265 13.

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Electrochemical and Structural Assessment of Neuron-Glioma Interactions Farbod Khorrami

Gliomas are the most frequently observed tumors of central nervous system and can arise from any class of glial cells found in the brain. When glial cells become cancerous, they are classified based on the cell line they originated from such as astrocytic tumors, oligodendroglial tumors and ependymal tumors. Several genetic and environmental factors have been suggested for the etiology of glioma such as genetic backgrounds, ionizing radiation and allergies. In addition, studies have determined several causes for glioma progression and migration including hypoxia-induced PLOD2, tumor associated microglia and angiogenesis. However, it is unknown how glioma are involved in neural networks and what types of pathways in a neural microenvironment contribute to glioma proliferation. In this paper, a set of experiments designed by Venkatesh and colleagues were performed to understand the interconnectivity between neurons and glioma cells. GFP+ glioma cells were observed on postsynaptic side of synapses in human glioma xenografts in mice. In addition, co-localization of synaptic proteins was apparent between neurons and glioma, confirming synaptic structures between glioma cells and neurons. Neuronal activity also lead to glioma proliferation, as well as inward currents and prolonged currents that were blocked by NBQX and TTX respectively. The results indicated the dependency of glioma cells on their neural microenvironment and signify receptors and interactions that are potential targets for glioma therapy. Key words: glioma proliferation, oligodendroglial precursor cells (OPCs), AMPA receptors (AMPAR), GluA2 subunit, postsynaptic density protein-95 (PSD95), Neuroligin 3 (Nlgn3), gap junctions, tumor microtubules, neuronal hyperexcitability

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INTRODUCTION

MAJOR RESULTS

Glioma is the most common form of the central nervous system (CNS) cancers dedicating nearly one third of brain and spinal tumors to itself 1. Glial cells are responsible for supporting the neurons by myelination, reuptaking of neurotransmitters and modifying blood supply depending on neural activity 2. So far, glioma formation and progression have not been thoroughly understood; but there are several explanation on how cancerous cells are formed, maintained and progress through nervous tissue. There are studied genetic causes that are influential in glioma such as hypoxia induced procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) and B7-H6 that promote tumor migration and proliferation. PLOD2 is responsible for collagen synthesis in the extracellular matrix by hydroxylating lysine residues. In hypoxia induced cases and in patients with glioma PLOD2 had an increased expression suggesting its close relationship with hypoxia-inducible factor-1Îą (HIF-1Îą). Moreover, PLOD2 is involved in activating PI3K pathway which is crucial in cancer proliferation and spread 3. Another examined genetic factor is B7-H6, a transmembrane protein closely associated with natural killer (NK) cells and important in immune response. This protein is overexpressed in glioma and results in tumor invasion to other tissues. Its upregulation also causes dysfunction in cell cycle and apoptosis leading in increased cell division 4.

Synaptic structures between neurons and glioma cells depend on NLGN3

Venkatesh et. al used several different cell lines of glioma cell types obtained from adult and paediatric patients such as H3K27M+ diffuse midline glioma (DMG), also known as DIPG. Upon inspecting these cells, a cell heterogeneity was seen that consisted of glioma sub populations resembling oligodendroglial precursor cells (OPCs) and others resembling astrocytes. The OPC-like glioma had high expression of genes for glutamate receptors and other postsynaptic constructs while astrocyte-like cells did not8 (Figure 1a).Later, the authors attempted to physically observe the synapses between glioma cells and neurons. To achieve this, they xenografted green fluorescent protein (GFP) -labelled DIPG cells in mouse hippocampus and observed them using immune electron microscope; after which they identified GFP+ cells in 10% of the synapses 8. Venkatesh et al. had already established in previous studies that NLGN3 production due to activity of presynaptic neurons fosters expression of synaptic genes 8. To confirm the role of NLGN3 in synaptogenesis, glioma cells with fluorescent PSD95 and neurons WT or Nlgn3KO were cultured together. It was observed that co-localization of PSD95 and KO 8 Non-genetic factors also have been identified as facilitators of glio- presynaptic synapsin was decreased with Nlgn3 neurons (Figure ma growth. Glioma cells secrete glial cell-derived neuropathic factor 1b). This is in concordance with Lie et. al where higher NLGN3 levels in deeper regions of the brain is associated with glioma relapse after (GDNF) that recruits microglia to their surrounding environment. surgery 9. Then, microglia synthesize numerous substances that intensify the 5 growth and movement of glioma cells . Stress-inducible protein 1 b a (SIT 1) is an example of these microglial products that enhances cell 6 migration . Additionally, these microglia release cytokines such as IL -6 which facilitates vasculogenesis and angiogenesis, aiding in glioma growth and motility 7. The current body of literature considers many genetic and environmental aspects for etiology of glioma. However, there is not much data available on how glioma cells are integrated in neural networks. It is unclear how healthy neurons interact with tumors and affect them functionally and structurally. In this research paper, authors designed and performed a series of experiments to determine whether functional synaptic connections exist between neurons and glioma and if they affect each other through depolarization. First, they discovered synaptic genes in a subpopulation of glioma and using immune microscopy observed GFP+ postsynaptic glioma cells 8. They also noticed diminished colocalization of presynaptic and postsynaptic proteins in neuronglioma synapses which had Nlgn3KO, highlighting the significance of NLGN3 in synapse viability. Then, they observed xenografted glioma display excitatory post synaptic currents (EPSCs) in response to depolarization and show prolonged EPSCs after rise in extracellular potassium concentration, showing that synapses are electrophysiologically functional. Even though they established the importance of AMPARs for glioma proliferation, they realized AMPAR blockers only reduce glioma proliferation while they are in their neural microenvironment. This further emphasizes on necessity of neuron-glioma synapses in tumor progression. These finding provide another mechanism for understanding how glioma cells grow and describe possible pathways and receptors/junctions that can be focused on for therapeutic purposes.

Figure 1. a. Among the glioma subpopulations, those that are similar to OPCs had the highest expression of synaptic genes. b. Lower presynaptic and postsynaptic protein colocalization in neuron-glioma synapses with Nlgn3KO neurons.

AMPARs allow synaptic activity between neurons and glioma cells After identifying synaptic structure the authors took on characterizing the currents that would be an indication of an electrochemically active synapse. In order to accomplish this, fluorescent tagged glioma cells were placed in CA1 region of mouse hippocampus and were allowed to grow and proliferate. Then, these cells were patchclamped so their response to activation of axons in CA3 region of hippocampus could be recorded (Figure 2a). Measurements depicted inward depolarizing EPSCs in glioma cells that had a lower amplitude after being treated by TTX 8 (Figure 2b). Furthermore, the reversal potential for the currents was measured close to 0 mV while glioma EPSCs were blocked in response to drugs NBQX and noticeably decreased in response to NASPM, both of which are AMPARs antagonists (Figure 2c,d,e). lastly, in order to further investigate the existence of synapse functionality, extracellular Ca2+ was substitut-

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with strontium which allowed allochronic presynaptic vesicle secretion. As expected, no action potential was observed in post synaptic glioma cells; however, small inward currents were detected that resembled minuscule EPSCs (Figure 2f) 8.

a b

d d

Figure 3. a. In addition to normal EPSCs, glioma cells had prolonged EPSCs that resemble the activity-dependent currents seen in astrocytes. b. The prolonged EPSCs are artificially produced as extracellular potassium concentration increased and neuron activity was blocked. c. recording of glioma cells matches with activity of neurons d. Unlike AMPARs and classic EPSCs, prolonged currents are not blocked by NASPM e. TTX blocked prolonged currents of glioma cells

Figure 2. a. Observed activity of xenografted cells in CA3 region of mouse hippocampus by stimulation of Schaffer collaterals and commissural neurons. b. TTX blocks the inward currents of glioma cells c. reversal potential of the currents is close to 0 mV implying it is an AMPAR d,e. NASPM and NBQX result in reduced amplitude of EPSCs f. Replacing extracellular calcium with strontium result in random release of synaptic vesicles and small EPSCs

Gap junctions connect glioma cells through tumor microtubes

Glioma have AMPAR-independent activity In addition to normal EPSCs, the authors observed prolonged EPSCs. Instead of being related to synaptic activity, these currents were indicative of neuronal activity with similar characteristics of astrocytic currents observed in neuronal activity (Figure 3a) 8. Similar to behavior of astrocytic currents, an increase in extracellular potassium concentration while blocking neurons pharmacologically resulted in eliciting these prolonged currents in glioma cells (Figure 3b). In fact, these current followed a similar pattern as axon stimulated surrounding neurons (Figure 3c). Interestingly, unlike normal aforementioned EPSCs these prolonged currents were not blocked by NASPM but were blocked by TTX (Figure d,e).

As mentioned before, the authors noted cellular heterogeneity in the tumor tissue, with 10% of glioma cells displaying classic EPSCs and 40% produce prolonged currents as a result of microenvironment neuronal activity. Of course, there are differences between these cells. For instance, cells with prolonged EPSCs have remarkable lower resistance than other subtypes, similar to how normal astrocytes have lower resistance than other healthy neurons as reported in the literature (Figure 4a) 11. It was hypothesized that this occurs due to gap junctions that connect glioma cells together. To investigate this, biocytin dye was injected into cells and later observations showed this dye moving between cells in a network created by gap junctions (Figure 4b) 8. In addition, applying carbenoxolone and meclofenamate resulted in the decrease of prolonged EPSCs (Figure 4c) 8. Moreover, using GCaMP6 which is a calcium tracer fluorescent marker, Venkatesh et. al identified simultaneous currents in connected glioma cells that were in phase with each other (Figure 4d) 8.

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Figure 5. a. glioma cell expression ChR2-YFP were placed and grown in mouse cortex b,c. stimulation of glioma cells with ChR2-YFP increased their proliferation, while the control group did not have any changes in growth.

Furthermore, the authors aspired to know how GluA2 subunit of AMPARs affect glioma proliferation. Three groups of mice were xenografted with overexpressed GFP-labelled GluA2 subunit, GFP and GFP-labelled dominant negative GluA2 (GluA2-DN). After a period of time, mice with over overexpressed GluA2 glioma cells had higher mortality than other two groups (Figure 6a) 8. This is because glioma cells with GluA2-DN simply are not viable. This was confirmed after a tissue culture consisting of GluA2-DN glioma cells and non-GluA2-DN glioma cells only the latter remained viable after a period of growth (Figure 6b) 8. In addition cultures of glioma cells and neurons contributed to increased proliferation of glioma cells and NBQX resulted in their blockage as expected. Surprisingly, this was not observed with isolated glioma cells. Without neurons, their proliferation was low and remained at the same level even after NBQX treatment (Figure 6c) 8. Finally, to asses therapeutic options, Venkatesh et. al treated glioma mice with AMPAR blocker perampanel and gap junction blocker meclofenamate and observed that compared to control group, glioma cell viability was halved (Figure 6d) 8.

Neurons and glioma cells have bidirectional relationship It was demonstrated that neuronal activity facilitates glioma proliferation. Glioma cells also influence neurons and cause hyperexcitability in them. Glioma cells produce abnormally high levels of glutaFigure 4. a. glioma cells with prolonged EPSCs have profoundly lower resistance mate which leads to overstimulation of cortical neurons and prob. movement of biocytin in the tumour indicates a network connecting glioma duces seizures 13. To further confirm this, the authors performed cells. c. Gap junction blockers CBX and Mec lower the amplitude of prolonged EEG on adults with high grade glioma and detected high gamma glioma EPSCs d. Glioma cells exhibit allochronic in-phase currents made possible by gap junctions. activity outside and close to the tumor in the surrounding area (figure 7a) 8. This occurs due to release of glutamate and synaptogenic factors and downregulating inhibitory neurons. Same neural Glioma progression is dependent on depolarization by neurons hyperactivation was seen in mice with xenografts of paediatric glioma (Figure 7b) 8. It has been shown that depolarization in many regions of the brain, particularly hippocampus, leads to neurogenesis 12. Therefore, it was hypothesized that glioma cells may behave similarly. To asses this, control and channelrhodopsin-2 (ChR2) yellow-fluorescent protein (ChR2-YFP) expressing glioma cells were xenografted into mouse cortex (Figure 5a) 8. Shining blue light on cells led to depolarization of ChR2-YFP expressing glioma cells and their proliferation while control group remain unchanged (Figure 5b,c) 8.

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Venkatesh et. al identified that there is structural and electrochemical connection between glioma cells and the neurons in their microenvironment. They observed NLGN3-dependent synaptic structures that displayed classical EPSCs and were electrochemically active due to AMPARs, especially their GluA2 subunit. On the other hand, other glioma cells exhibited prolonged currents that were potassium dependent and were blocked by TTX. These cells were also connected to each other through gap junctions, giving them low resistance, and creating a network that allows synchronicity between glioma cells. Finally, a bidirectional relationship was identified between glioma and healthy neurons, in which glioma depolarization increased their proliferation and glioma activity hyperexcited the neurons of microenvironment. However, glioma proliferation only occurred while neurons were present.

These findings are significant because they elucidate how glioma cells operate in a microenvironment containing neurons. most importantly they suggest a novel mechanism by which glioma cells progress in the brain. Based on these results, neuron depolarization and presynaptic release of vesicles contribute to proliferation of glioma cells through activation of AMPARs as they release glutamate, while the glioma cells conduct currents within a network. Similar results is observed in a recent study where glutamate secretion increased glioma growth and AMPARs facilitated calcium currents 14. This provides incredible prospective for localized targeting of these AMPARs and thus cessation of disease progression. In addition, gap junctions are another possible method of preventing glioma activity, since they amplify the prolonged EPSCs 8. As reported in the literature, gap junctions of glioma are essential to their invasive capabilities by transferring microRNAs 15. Therefore, identifying these network-forming gap junctions and blocking d them is very important in restricting glioma migration and restricting the disease. Moreover, it is critical to acknowledge that glioma growth does not occur without a neural microenvironment. Thus, pharmacologically isolating glioma cells can be another therapeutic approach to eliminating them. Furthermore, since GluA2 subunit is essential for survival of glioma cells, destroying this particular subunit could serve as cancer treatment. This is tested for in a study where propofol is used to selectively block the GluA2 subuFigure 6. a. mice with GluA2-DN glioma cells survived longer than mice with 16 overexpressed GluA2 glioma cells b. After a period of time a tissue with a mix of nit of glioma AMPARs and decreased its viability . Therefore, this GluA2-DN and non-GluA2-DN cells only had viable non-GluA2-DN cells. c. Glio- study introduces several treatment options that can repress glioma ma cells only displayed proliferation and NBQX-induced decreased proliferation invasion.

in the presence of neurons. d. Treating glioma tissues with Perampanel and meclofenamate resulted in reduced disease progression

CRITICAL ANALYSIS b

This paper presented novel information on integration of glioma cells inside neural microenvironments. However, this study was focused only on the relationship between neurons and glioma cells and was in a sense one dimensional. In other words, the authors ignored other types of interaction that may exist and affect neurons and glioma cells. For instance, microglia also enhance glioma growth and migration by releasing numerous factors 5. Further research can investigate the simultaneous effect of microglia and neurons in a tissue sample on glioma cells. it has been demonstrated that glioma cells could lead to neurodegeneration by facilitating 17 Figure 7. a. Areas around the tumor have higher gamma power. b. Xenografted membrane-type 1 metalloproteinase on microglia . Considering a glioma cells cause hyperexcitation in its microenvironment. noticeable portion of glioma tumors consist of microglia, it could be possible that neurons may not be able to exert the same effect on glioma cells in vivo. Another important point regarding environCONCLUSION ment is the anatomical location of where glioma cells were xenografted. In this paper, glioma cells were placed in hippocampus

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and cerebral cortex. However, close to 40% of glioma cases occur at frontal lobe1. It is unknown if disease progression in frontal lobe follows the same mechanisms as the temporal lobe, or if neurons of that region of the brain influence the glioma cells differently, if at all. Moreover, even though the paper points out the significance of GluA2 subunit of AMPARs in tumor viability, the molecular pathway for this observation is not explained. Other studies have reported that GluA2 subunit in lung cancer is involved in apoptotic pathways through caspase-3 and p53 pathways 18. It is unclear if this is the case for glioma but further investigation in this matter is necessary because revealing this pathway may result in easier targets for treatment. Additionally, the authors only focused on glioma subpopulations that resemble OPCs and therefore make functional synaptic structures with healthy neurons 8. However, other glioma types are not discussed here because they do not form clear synaptic connections. Yet, the authors delineated prolonged EPSCs that were non-synaptic and had considerable importance on tumor progression. Similarly, other types of glioma may be proliferating by the same principle or other methods. As a result, integration of other subtypes of glioma in neural circuits must be studied as well. It is also unclear if other glioma types form connecting networks using gap junctions. Lastly, the authors did not investigate how inhibiting glioma cells will change hyperexcitability of surrounding neurons. It may be possible that suppressing glioma activity profoundly affects seizure frequency and intensity. This should be studied in epilepsy-focused studies in order to seek for seizure treatments. Therefore, the results of the study by Venkatesh et. al not only provides an opportunity for alleviating glioma, but also fosters a chance to treat seizures that maybe caused by this disease.

be hyperpolarized while voltage gated sodium channels are blocked to prevent depolarization and generation of action potentials. Vanadate can be used as a potential agent to hyperpolarize glioma cells 19. After a period of engraftment, the viability of control and experimental groups will be compared. If prolonged hyperpolarization glioma cells decreases their proliferation, there will be reduced fluorescent light observed in the experimental group. Otherwise, if hyperpolarization, unlike depolarization, does not affect glioma growth, there should be little to no difference in tumor burden between the control and experimental groups. Furthermore, the researchers should use their results in a more treatment-based manner and translate their results to human trials. Inhibiting the GluA2 subunit of AMPARs is an excellent way to determine drugs for glioma treatment. By having control and experimental groups of mice and using GluA2 antagonist drug CRNN, it can be determined whether specifically inhibiting this subunit has any therapeutic consequences 20. If AMPAR blocking is an effective treatment, the experimental group will have a higher survival rate compared to control group.

FUTURE DIRECTIONS There are several important experiments that can be performed in the future. First, it should be determined whether neurons still exert the same effect on glioma cells in other parts of the brain and in vivo, as opposed to solely examining the interaction of neuron and glioma in cell cultures. To understand how other cells present in the microenvironment, such as microglia, modify glioma proliferation two categories of mice, each with subgroups, need to be chosen and compared to a culture of glioma and neurons used as control. Each subgroup in category 1 will have glioma cells xenografted in a unique anatomical region of mouse brain so glioma cells can be in the presence of microglia. Category 2 will be treated the same but the microglia in the microenvironment surrounding the glioma will be overactivated by injecting activating cytokines to that region. Glioma proliferation in these two categories will be compared with each other and the cell culture to understand the importance of location of incidence and other cells types present in microenvironment. By taking the neurodegenerative capabilities of microglia into account, it can be hypothesized that neurons will not be able to contribute to glioma growth in vivo. Thus, glioma present in cell culture will most likely show the highest growth levels. Additionally, an experiment should be designed as a method to test if glioma depolarization leads to their growth, their subsequent hyperpolarization or inhibition will lead to reduced tumor burden as well. To achieve this, in a prospective study two groups (control and experimental) of GFP-labelled glioma cells will be xenografted into mouse cortex. Then, the experimental group will

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1. Ostrom Q, Gittleman H, Farah P, Ondracek A, Chen Y, Wolinsky Y, Stroup N, Kruchko C, Barnholtz-Sloan J. CBTRUS Statistical Report: Primary Brain and Central Nervous System Tumors Diagnosed in the United States in 2006-2010. Neuro-Oncology. 2013;15(suppl 2):ii1ii56. 2. Halassa M, Fellin T, Haydon P. The tripartite synapse: roles for gliotransmission in health and disease. Trends in Molecular Medicine. 2007;13(2):54-63. 3. Song Y, Zheng S, Wang J, Long H, Fang L, Wang G, Li Z, Que T, Liu Y, Li Y et al. Hypoxia-induced PLOD2 promotes proliferation, migration and invasion via PI3K/Akt signaling in glioma. Oncotarget. 2017;8(26). 4. Jiang T, Wu W, Zhang H, Zhang X, Zhang D, Wang Q, Huang L, Wang Y, Hang C. High expression of B7-H6 in human glioma tissues promotes tumor progression. Oncotarget. 2017;8(23). 5. Hambardzumyan D, Gutmann D, Kettenmann H. The role of microglia and macrophages in glioma maintenance and progression. Nature Neuroscience. 2015;19(1):20-27. 6. Carvalho da Fonseca A, Wang H, Fan H, Chen X, Zhang I, Zhang L, Lima F, Badie B. Increased expression of stress inducible protein 1 in glioma-associated microglia/macrophages. Journal of Neuroimmunology. 2014;274(1-2):71-77. 7. Zhu C, Kros J, Cheng C, Mustafa D. The contribution of tumor-associated macrophages in glioma neo-angiogenesis and implications for anti-angiogenic strategies. Neuro-Oncology. 2017;19(11):1435-1446. 8. Venkatesh H, Morishita W, Geraghty A, Silverbush D, Gillespie S, Arzt M, Tam L, Espenel C, Ponnuswami A, Ni L et al. Electrical and synaptic integration of glioma into neural circuits. Nature. 2019;573(7775):539-545. 9. Venkatesh H, Johung T, Caretti V, Noll A, Tang Y, Nagaraja S, Gibson E, Mount C, Polepalli J, Mitra S et al. Neuronal Activity Promotes Glioma Growth through Neuroligin-3 Secretion. Cell. 2015;161(4):803-816. 10. Liu R, Qin X, Zhuang Y, Zhang Y, Liao H, Tang J, Pan M, Zeng F, Lei Y, Lei R et al. Glioblastoma recurrence correlates with NLGN3 levels. Cancer Medicine. 2018;7(7):2848-2859. 11. McKhann G, D’Ambrosio R, Janigro D. Heterogeneity of Astrocyte Resting Membrane Potentials and Intercellular Coupling Revealed by Whole-Cell and Gramicidin-Perforated Patch Recordings from Cultured Neocortical and Hippocampal Slice Astrocytes. The Journal of Neuroscience. 1997;17(18):6850-6863. 12. Urbach A, Baum E, Braun F, Witte O. Cortical spreading depolarization increases adult neurogenesis, and alters behavior and hippocampus-dependent memory in mice. Journal of Cerebral Blood Flow & Metabolism. 2016;37(5):1776-1790. 13. Buckingham S, Robel S. Glutamate and tumor-associated epilepsy: Glial cell dysfunction in the peritumoral environment. Neurochemistry International. 2013;63(7):696-701. 14. Venkataramani V, Tanev D, Strahle C, Studier-Fischer A, Fankhauser L, Kessler T, Körber C, Kardorff M, Ratliff M, Xie R et al. Glutamatergic synaptic input to glioma cells drives brain tumour progression. Nature. 2019;573(7775):532-538. 15. Hong X, Sin W, Harris A, Naus C. Gap junctions modulate glioma invasion by direct transfer of microRNA. Oncotarget. 2015;6(17). 16. Wang X, Li Y, Wang H, Zhu M, Guo D, Wang G, Gao Y, Yang Z, Li T, Yang C et al. Propofol inhibits invasion and proliferation of C6 glioma cells by regulating the Ca 2+ permeable AMPA receptor-system x c − pathway. Toxicology in Vitro. 2017 [accessed 2019 Dec 8];44:57-65. 17. Langenfurth A, Rinnenthal J, Vinnakota K, Prinz V, Carlo A, Stadelmann C, Siffrin V, Peaschke S, Endres M, Heppner F et al. Membranetype 1 metalloproteinase is upregulated in microglia/brain macrophages in neurodegenerative and neuroinflammatory diseases. Journal of Neuroscience Research. 2013;92(3):275-286. 18. Zhang H, Yang W, Lu J. Knockdown of GluA2 induces apoptosis in non-small-cell lung cancer A549 cells through the p53 signaling pathway. Oncology Letters. 2017;14(1):1005-1010. 19. Lichtstein D, Mullikin-Kilpatrick D, Blume A. Hyperpolarization of neuroblastoma-glioma hybrid NG108-15 by vanadium ions. Proceedings of the National Academy of Sciences. 1982;79(13):4202-4206. 20. Qneibi M, Hamed O, Natsheh A, Fares O, Jaradat N, Emwas N, AbuHasan Q, Al-Kerm R, Al-Kerm R. Inhibition and assessment of the biophysical gating properties of GluA2 and GluA2/A3 AMPA receptors using curcumin derivatives. PLOS ONE. 2019;14(8):e022113

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Rifampicin: A Light in the Search for Therapeutics Against Alzheimer’s Disease Hyeok Jun Lee

Alzheimer’s Disease (AD) is a common form of dementia whose prevalence is projected to increase. Despite its characterization in the 1900s, the mechanisms underlying this disease are partially understood, but is thought to involve the aggregation of amyloid-beta (Aβ) protein. Aβ aggregates are also known to accelerate other common AD pathologies such as neurofibrillary tangles of tau, neuroinflammation, and neurotoxicity. With numerous failed clinical trials, five drugs are currently available to offset symptoms, but they only modulate downstream pathways without altering the progression of disease itself. Therefore, a need for a new therapeutic has never been more urgent. Umeda et al. (2016) contribute to the search by focusing on drugs that were previously described to have anti-aggregating properties on amyloidogenic proteins. Through cell cultures, cell-free conditions, and mice models, they found that the therapeutic rifampicin can effectively decrease AD pathology such as Aβ oligomerization, tau hyperphosphorylation, microglial activation, and synapse and memory loss. These results confirm previous claims on the effectiveness of rifampicin in AD and its ability to target the underlying pathology. Key words: Alzheimer’s disease, rifampicin, therapeutic, amyloid-beta, tau, neuroinflammation, neurodegeneration, anti-aggregating, Aβ oligomers, amyloid cascade hypothesis

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Figure 1. Overview of the amyloid cascade hypothesis. The generation of Aβ accelerates other pathologies and symptoms of AD. (Adapted from Briggs et al., 2016)

BACKGROUND

Alzheimer’s Disease (AD) accounts for 75-80% of dementia cases and is characterized by the progressive decline of cognitive abilities including memory, language and executive function MAJOR RESULTS (Weller & Budson, 2018; Briggs, Kennelly, & O’Neill, 2016). As Umeda et al. (2018) focused on both in vitro and in vivo AD the prevalence of AD is projected to increase three-fold by 2050 models when experimenting the anti-aggregating properties of (Naj, Schellenberg, & ADGC, 2017), a need for an effective treatrifampicin. More specifically, the Osaka mutation of APP (APPOSK) ment is urgent. was used in cell cultures and mice models as it caused the accumulation of Aβ oligomers intracellularly without plaques. They addiDespite continual efforts, candidate therapeutics have had a 99.6% failure rate and only five drugs have been approved for use tionally considered Tg2576 mice which produce amyloid deposi(Briggs et al., 2016): cholinesterase inhibitors (donepezil, rivastig- tions. In both transfected cell cultures and mice models, rifampicin mine, galantamine, and tacrine) and a NMDA antagonist treatment decreased Aβ oligomerization intracellularly and extra(memantine). However, Briggs (2016) noted that about 33% of cellularly, but did not disrupt Aβ production itself. The presence of patients would show side effects to cholinesterase inhibitors, and extracellular Aβ suggested the restoration of clearance mechathese drugs show very limited clinical effects without altering the nisms such as autophagy-lysosomal function which they later concourse of neurodegeneration in AD. This is likely because cholines- firmed with p62/sequestosome-1 analysis. Even though of its terase inhibitors and memantine target downstream effects of AD effect on oligomers, rifampicin slightly increased amyloid deposi(Briggs et al., 2016), not the main underlying pathology. tions/plaques in Tg2576 mice, meaning they exhibited no disaggregating effects on larger fibrils. Furthermore, NMR studies suggestLike ed that rifampicin did not hinder Aβ monomers as well. Despite other their results, Meng et al. (2008) conflictingly showed that rifampicneuroin did not prevent human islet amyloid polypeptides (IAPP) aggregation previously.

degenerative diseases, AD is characterized by the aggregation of misfolded protein, specifically amyloid-beta (Aβ), in the CNS and its accumulation is known to occur years before clinical symptoms (Briggs et al., 2016). The amyloid cascade hypothesis states that the abnormal processing of amyloid precursor protein (APP) leads to the production of free Aβ and the formation of Aβ plaques underly neurotoxicity. However, recent evidence show that amyloid plaques do not correlate well with AD pathogenesis (Socias et al., 2018). Instead, it is now proposed that Aβ oligomers propagate cell-to-cell (Umeda et al., 2016) and disrupt membranes to induce neurotoxic effects (Socias et al., 2018). Other pathologies seen in AD include hyperphosphorylation of tau which interact synergistically with Aβ to produce neurofibrillary tangles (NFT) (Weller & Budson, 2018), neuroinflammation, and neurotoxicity due to oxidative stress (Briggs et al., 2016). Since Aβ supposedly underly the main pathology and accelerate other characteristics of AD, therapies that inhibit Aβ seem most efficient and effective.

Other than Aβ, Umeda et al. (2016) were also interested in other pathologies. They found less hyperphosphorylated tau in both free -cell conditions and mice, restoration of synapse loss, and the prevention of microglial activation in APPOSK mice. The ability of rifampicin to prevent tau aggregation suggest that they exhibit antioligomerizing properties on a broad family of amyloidogenic proteins. Rifampicin also protected cells from mitochondrial damage and oxidative stress as it lowered cytosolic cytochrome c levels and caspase 3 activation. Oxidative stress due to inflammatory responses and free radical production has been observed in AD, so rifampicin’s protective effects may be attributed to its antioxidant and free radical scavenging effects (Yulug et al., 2018).

Umeda et al. (2016) attempted to justify that rifampicin, a widely used antibiotic, is an effective drug against AD that targets the amyloid cascade. They used immunohistochemical assays on cell cultures and mice models expressing human Aβ to show that rifampicin inhibits Aβ oligomerization, tau hyperphosphorylation, synapse loss, and microglial activation.

Figure 2. Level of Aβ monomers, dimers and trimers in cells with or without rifampicin treatment. The largest decrease is seen in dimers (small number oligomers). (Adapted from Umeda et al., 2016)

When evaluating memory using Morris Water Maze, the APPOSK mice treated with monthly oral rifampicin improved their

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memory in a dose-dependent manner. In addition, older mice showed improved memory, but to lesser degrees than younger mice with progressively lower doses. This established a relationship between the progression of the disease and the time treatment was employed.

infections in clinical trials (Gosztyla & Robinson, 2018). As Aβ production was persistent, this potential innate immune function is likely preserved. Furthermore, the produced monomers could associate with pre-existing fibrils directly without having to form toxic oligomeric species (Umeda et al., 2016), explaining the increase of amyloid depositions which could be regarded as beneficial. Another important result was that rifampicin seemed to restore clearance mechanisms. The accumulation of Aβ is not only because of its overproduction, but also the failure to eliminate them. With rifampicin treatment, Umeda et al. (2016) showed that the aberrant intracellular proteins were successfully cleared to the extracellular environment. Furthermore, since this antibiotic was previously shown to upregulate the expression of efflux proteins such as P-glycoprotein in the BBB (Yulug et al., 2018), the cleared amyloid proteins could then be transported to the periphery for protein degradation. Despite this recent study, the antiaggregating effect of rifampicin was noted before and there exist few studies that contradict these findings. Meng et al. (2008) showed that rifampicin did not prevent IAPP fibril formation which is another type of amyloidogenic protein possibly linked to AD. However, difference in methodologies and aims may account for this discrepancy and many recent studies, including Umeda et al. (2016), agreed that rifampicin does have beneficial effects in AD (Yulug et al., 2018).

CRITICAL ANALYSIS

Figure 3. Escape performance of APPOSK mice in Morris Water Mize. In 12-month old mice (A), rifampicin dosage performed as well as nontransgenic mice in memory task. In 18-month old mice (C), performance improved in dose-dependent manner. (Adapted from Umeda et al., 2016)

CONCLUSIONS/DISCUSSION To summarize, Umeda et al. (2016) confirmed that rifampicin could potentially treat AD by inhibiting Aβ oligomerization. Furthermore, rifampicin inhibited other AD pathologies such as hyperphosphorylation of tau, neurotoxicity, and microglia activation and improved clinical symptoms such as memory in mouse models. This is helpful to the search of therapeutics because rifampicin is a relatively safe and already widely used antibiotic that is known to cross the blood-brain barrier (BBB), allowing for oral administration. Umeda et al. also found that Aβ levels did not change which may be beneficial. Recently, researchers have proposed that Aβ may function as an antimicrobial peptide (AMP) due to the similar effects they have on membranes and the noticeable correlation between Aβ production inhibition and susceptibility to

As stated previously, Meng et al. (2008) argued that rifampicin had negligible anti-aggregating properties on IAPP. However, Umeda et al. (2016) similarly found that rifampicin had negligible effects on amyloid fibrils in plaques. The anti-aggregating effects were most pronounced on low number oligomers which exhibit stronger toxicity than fibrils through membrane disruption (Socias et al., 2018). Furthermore, Umeda et al. used different antibodies and methods when staining for protein aggregates instead of Thioflavin T assays which Meng et al. found led to false positive results after rifampicin treatment. Nevertheless, the study did not attempt to explain how rifampicin interacts with these oligomers. The authors used NMR to show the lack of interactions with monomers, but this method cannot be employed on oligomers. They instead showed that Aβ oligomers have more extended beta sheets compared to oligomeric GST and suggested this as the main interaction site. If this is confirmed, this may partially explain the negligible effects rifampicin has on Aβ fibrils since their beta-sheet structures are mostly buried while they are exposed in oligomers (Verma, Vats, & Taneja, 2015). Furthermore, studying the interactions would reveal whether the interaction domains are also found in physiological amyloidogenic proteins needed in normal function (Umeda et al., 2016). If so, then rifampicin may have a risk of disrupting necessary processes regulated by these proteins. Tg2576 and APPOSK mice used by the authors both express human APP with known mutations found in familial AD (Sasaguri et al., 2017; Tomiyama et al., 2010), so the results are more generalizable to humans. However, mice models generate only three of the six human isoforms of tau which are not commonly found in NFT of AD (Sasaguri et al., 2017). The authors attempted to account for rifampicin’s effect on human tauopathies

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using tau609 mice. Although tau aggregation decreased that the synergistic mechanisms between Aβ and tau pathology with treatment, these mice models would not account for the syn- could not be offset by rifampicin. ergetic effect Aβ has on tau accumulation (Weller & Budson, 2018). Furthermore, this model required a FTD-related mutation of tau (Umeda et al., 2016) whereas NFT of AD contain non-mutated tau. Transgenic mice expressing both human APP and tau can be considered, but it would still require an unrelated tau mutation and exhibit tau aggregation independent of Aβ accumulation (Sasaguri et al., 2017). The authors did not examine whether rifampicin relieves clinical symptoms in humans diagnosed with AD, but this has already been studied. Leob et al. (2004) previously showed that daily doses of oral rifampicin with doxycycline slowed cognitive deterioration in mild and moderate AD patients. However, a similar study done by Molloy et al. (2013) conflictingly concluded that rifampicin, doxycycline, nor their combined use had any effect on clinical symptoms. A reason for these conflicting findings may be due to experimental design. A more recent study by Iizuka et al. (2017) concluded that a minimum dose of 450 mg daily for 1 year was needed for significant improvement in AD which was not met by previous studies. Furthermore, another reason why previous clinical studies may have failed is because of late timing of treatment. Umeda et al. (2016) found that older AD-model mice showed less improvement in memory tests after rifampicin treatment than younger mice. As clinical symptoms occur years later after disease pathology, some patient in failed studies may have required a higher dosage than they were given.

FUTURE DIRECTIONS As the specific interaction of rifampicin with Aβ are unknown, other amyloidogenic proteins such as ACys could be tested for anti-oligomerizing effects under cell-free conditions by rifampicin and those whose aggregation is inhibited could be used to elucidate binding domains through homology analysis. If none of the other amyloidogenic proteins are inhibited, then there likely exists a unique domain in Aβ that rifampicin targets. If a certain domain is suspected, then mutagenesis studies should be employed for confirmation. Furthermore, surface plasmon resonance could be used to directly confirm whether rifampicin interacts with fibrils, oligomers or monomers (Frenzel et al., 2014) as the authors’ claims on binding partners were mainly elucidated. If rifampicin interacts with fibrils or monomers, then this would contradict the authors’ suspicions on rifampicin’s binding partners and further studies would need to be employed to justify rifampicin’s ineffectiveness against plaques. In addition, similar in vivo experiments using TgF344-AD rats should be employed to confirm rifampicin’s effect of human tau aggregation influenced by Aβ accumulation. Similar to human AD, rats have six isoforms of tau and increased NFT with the appropriate isoforms when Aβ accumulates without a mutation in tau (Cohen et al., 2013). Hence, the model is useful when experimenting Aβ -mediated tauopathy. Rifampicin should decrease both Aβ and tau pathology according to current findings. As authors systematically argued for the anti-aggregating properties of rifampicin on Aβ, a negligible decrease in Aβ oligomerization would likely reflect a problem with experimental design. However, if rifampicin shows no effect on tau aggregation, then this might suggest

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Myopathic changes and motor neuron degeneration observed in a Matr3 overexpression transgenic mouse model of ALS Jooyun Lee

Mutations in over thirty genes have been associated with causing amyotrophic lateral sclerosis (ALS), including the gene encoding the nuclear matrix protein, Matrin3 (Matr3). However, the mechanisms by which mutated forms of MATR3 elicit multisystem proteinopathy like ALS remains unclear. Therefore, C57BL/6 mice were introduced to human WT or mutant MATR3 (S85C) through adenoassociated viruses (AAV) and transgenic mice with the overexpression of mutant MATR3 were generated to observe muscle and nervous system pathology. Upon histological analysis, mice injected with human WT or mutant MATR3 in the tibialis anterior (TA) muscles showed sarcoplasmic aggregation of MATR3, and smaller myofibers with internalized nuclei. Additionally, autophagy markers such as p62 were increased in the mice injected with AAV-mutant MATR3 compared to the control. As for the transgenic mutant MATR3 mice (TG), at 12 weeks, hindlimb clasping, rotarod, and gait abnormalities were present. Similar to the AAV-mice, the MATR3 TG mice also showed fibre-size variation and larger, internalized myonuclei. Furthermore, MATR3 was localized in the sarcoplasm of TG mice while MATR3 was localized in the nucleus for the non-transgenic mice (NTG). Lastly, mutant S85C TG mice showed motor neuron degeneration and increased glial activation in the spinal cord. These mice models resemble the clinical features seen in ALS patients and may be used as tools to study therapeutic approaches. Key words: Matrin3, amyotrophic lateral sclerosis, transgenic mouse model, motor neurons, muscle

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BACKGROUND or INTRODUCTION. Amyotrophic Lateral Sclerosis (ALS) is a fatal, progressive disease characterized by motor neuron degeneration in the brain or spinal cord.14,18 ALS affects the upper and lower motor neurons that innervate muscles leading to deteriorating motor function, paralysis and death, typically within 3-5 years of diagnosis.9,14,18The majority of ALS cases appear to be sporadic with no underlying genetic or environmental cause.9However, 10% of ALS cases are familial, and mutations in approximately 30 genes have been directly linked to disease onset.9,18

In familial ALS, motor neuron degeneration has been associated with abnormalities in genes involved in RNA binding, oxidative stress, and protein homeostasis.15Therefore, ALS has been identified as a proteinopathy as protein abnormalities appeared to precede neurodegeneration.15,16 More specifically, protein misfolding, aggregation and mislocalization due to mutations in the Tar DNA binding protein (TARDBP), fused in sarcoma (FUS), and superoxide dismutase-1 (SOD1) genes have been linked to ALS pathology.11,14 Similarly, several mutations in Matrin3, which encodes a nuclear matrix protein that regulates alternative splicing, were found to be linked to ALS.5 In ALS patients carrying the mutant MATR3 S85C, strong nuclear staining and cytoplasmic aggregates of the mutant MATR3 were present in motor neurons, suggesting MATR3-dependent neurotoxicity and protein abnormalities.10 Furthermore, these patients had rounded myofibers, internalized myonuclei, and rimmed subsarcolemmal vacuoles in their muscles which resembled hallmark features of ALS myotoxicity.10 Subsequently, primary rat cortical neurons with the overexpression of human MATR3 S85C exhibited cell death induced by MATR3 sequestration into aggregates, strongly supporting the pathogenic effects of mutant MATR3.7

In a study by Rayaprolu et al., differential expression levels of MATR3 were observed in various murine tissues and the central nervous system.13 The highest expression levels were found in reproductive organs, while the lowest levels were found in the spinal cord and muscles.13 Interestingly, the low levels of MATR3 expression were found in areas affected by ALS and were implicated in increasing susceptibility to the pathological effects of MATR3 mutations.13 However, the mechanism by which the MATR3 mutations may elicit ALS has not been thoroughly investigated. Therefore, to clarify the mechanism by which MATR3 S85C causes multisystem proteinopathies like ALS, two mouse models overexpressing mutant MATR3 S85C were generated and examined for their

clinicopathological features.17 H&E staining on muscle tissues injected intramuscularly with AAV-MATR3 WT or AAV-MATR3 S85C revealed that overexpression of the wildtype or mutant MATR3 gene elicited similar levels of myotoxicity.17 Likewise, immunofluorescence staining of transgenic MATR3 S85C mice tissues showed myopathic histological changes, motor neuronal loss and glial activation, supporting the importance of this novel model for analyzing MATR3-related ALS pathogenesis.17 MAJOR RESULTS Intramuscular Injections of AVV vectors expressing WT or mutant MATR3 caused TA muscle pathology

Zhang et al. intramuscularly injected AAV-vectors expressing human WT and mutant MATR3 (S85C) into the tibialis anterior (TA) muscles of C57BL/6 mice to determine if differences in muscle toxicity were elicited by the different gene variants. Muscle samples injected with the WT and mutant MATR3 genes underwent H&E staining to exhibit myofiber size variation and increased levels of internalized nuclei compared to the control expressing AAV-GFP. Furthermore, muscles injected with AAVmutant MATR3 demonstrated sarcoplasmic MATR3 aggregates, enlarged nuclear staining, and increase levels of autophagy-related proteins such as p62.

Figure 1. H&E staining of 10 weeks old TA muscles intramuscularly injected with AAV vectors containing either GFP, WT MATR3 or MATR3 S85C. In the WT and mutant MATR3 muscles, myofibers (red) appeared smaller in diameter than the GFP controls and their enlarged myonuclei (blue) were internalized in the sarcoplasm. (Top row scale bar: 500 Îźm; Bottom row scale bar: 50Îźm)

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Figure 2. Quantification of immunofluorescence staining against MATR3 and p62 show increased levels of (B) MATR3 and (C) p62 sarcoplasmic aggregates in the WT and mutant MATR3 TA muscles. No aggregates were found in the PBS control. (p <0.01; ****p <0.0001).

Abnormal changes in body weight and motor function seen in mutant MATR3 TG mice Additionally, the authors generated transgenic mice that stably overexpressed MATR3 S85C through a CMV early enhancer/chicken b-actin (CAG) promoter. The clinical phenotypes of these mice were observed with rotarod, hindlimb reflex and footprint tests. In comparison to nontransgenic mice (NTG), the transgenic MATR3 mice (MATR3 TG) displayed slower motor activity, hindlimb clasping, and gait abnormalities. Moreover, MATR3 TG mice had lower body weights and reduced life spans compared to the NTG mice.

Figure 3. (A-B) H&E staining of 20 weeks old mutant MATR3 TG mice and (E-F) non-transgenic mice GC muscles (Scale bar: 100 Îźm). (B-F) Zoomed in images of A and E. (A-B) Smaller myofiber diameter (red), internalized nuclei (blue) and rimmed vacuoles (clear) were exhibited by mutant MATR3 TG mice compared to NTG mice (E-F). (J,K,L,N,O,P) Immunofluorescence staining for TDP43, p62 and LC3 in mutant MATR3 TG and NTG GC muscles (Scale bar:100 Îźm). (J,K,L) Sarcoplasmic aggregates of TDP-43, p62, and LC3 were visible in GC muscles of mutant TG mice but (N,O,P) no sarcoplasmic aggregates were seen in NTG mice.

Histological analysis of mutant MATR3 TG mice muscles Degeneration of motor neurons and the activation of present pathology and increased levels of proteins deg- glial cells seen in mutant MATR3 TG mice spinal cords radation

Upon histological analysis of 20 weeks old mice, gastrocnemius (GC) muscles of the MATR3 TG mice displayed myopathic changes such as reduced myofiber sizes, internalized nuclei and rimmed vacuoles as seen in a similar MATR3 F115C study by Moloney et al. Furthermore, immunofluorescence staining of the muscles revealed sarcoplasmic aggregates of MATR3, p62, LC3 and TDP-43, resembling mutant MATR3 neuroglioma cell models. However, no sarcoplasmic inclusions were found in the NTG mice, and MATR3 was solely detected in their myonuclei. Subsequently, proteomic analysis of the GC muscle showed that proteins related to nuclear function, protein degradation, stress responses, and chaperones were upregulated in the mutant MATR3 TG mice. 237

Lastly, H&E and immunohistochemical staining of the anterior horn in the spinal cords of MATR3 TG mice showed decreased number of motor neurons, and increased degeneration of axons. Similar to SOD1 transgenic mice generated by Hall et al., mutant MATR3 TG mice showed increased levels of ionized calcium-binding adaptor protein1(Ibal-1)-positive microglia cells and glial fibrillary acidic protein (GFAP)-positive astrocytes. In the motor neurons, MATR3 levels were concentrated in the cytoplasm for the mutant MATR3 TG mice while nuclear localization of MATR3 was seen in the NTG mice

ate ALS pathology may be conducted. Furthermore, discovering new drug therapies would make a significant impact on ALS patients by potentially alleviating symptoms such as the inability to walk, talk, eat, swallow and breathe to increase the quality of life.

Figure 4. Quantification of immunohistochemistry for (C) motor neurons (SMI-32: nonphosphorylated neurofilament H), (D) astrocytes (GFAP), and (E) microglial cells (Iba-1) in the anterior horn of 20 weeks old spinal cords show decreased numbers of motor neurons and increased numbers of activated glial cells in mutant MATR3 TG mice. (*p <0.05).

CONCLUSIONS/DISCUSSION In this study, two different mouse models were generated to understand the function of MATR3 and its implications in causing muscle and motor neuron pathology that resembles ALS. The overexpression of human WT or mutant MATR3 was observed to cause myopathic changes such as smaller myofibers, internalized nuclei, and the upregulation of protein degradation molecules.17Likewise, the overexpression of the pathogenic gene caused the degeneration of the motor neurons in the spinal cord, and activated glial cells, clarifying the mechanism by which MATR3 causes the onset of ALS.17 Therefore, due to the significant resemblance between mutant MATR3 S85C and ALS patient tissue pathology, the authors have described these models to be helpful for analyzing ALS pathogenesis.

Additionally, several studies have shown the pathological effects of other MATR3 mutations such as the F115C mutation.9However, Zhang et al. developed the first transgenic mouse model expressing the human form of the most common mutation found on the MATR3 gene.17 As a result, this study supported the findings of previous studies observing ALS patients with the MATR3 S85C mutation, by demonstrating that the disruption of one gene caused rather severe muscle and spinal cord phenotypes.1,10 Furthermore, this study showed that the effects of mutant MATR3 were tissues specific which may imply that mechanisms involved in regulating MATR3 may differ between tissues and influence the susceptibility to mutant MATR3.17 Lastly, many papers have discussed the importance of RNA binding proteins such as FUS, and TDP-43 which account for approximately 30% of familial ALS cases. 11,12 However, MATR3 is a relatively new gene to be linked to ALS, resulting in the lack of research in this field.5 Therefore, this paper provided the essential tools for further studies to explore MATR3 localization, regulation, and other molecular mechanisms to potentially understand ALS pathogenesis in MATR3 S85C patients and provide insight into therapeutic approaches.

CRITICAL ANALYSIS The results from this study bear significant relevance to understanding ALS. Although ALS is the most common motor neuron disease, currently only two treatments: riluzole and edaravone, have been approved by the Food and Drug Administration.4 Riluzole is known to extend the lifetime of ALS patients by solely 2-3 months while edaravone works by slowing down the disease for only selective individuals, presenting the need for new treatments in the field of ALS.4 Unlike most of the previous literature focusing on oxidative stress-related ALS models such as the SOD1 transgenic mice to discover treatments, Zhang et al. developed a ALS model representing RNAbinding protein abnormalities.8,17 By generating a mouse model that resembles the clinicopathological features of ALS patients with RNA-binding abnormalities, pharmacodynamics studies to find potential drug targets to allevi-

In this study, Zhang et al. provided more concrete data to support the pathogenic nature of the MATR3 S85C mutation by generating two different ALS-associated mouse models and by conducting experiments in a coherent manner.17 After the authors observed phenotypic changes in AAV-MATR3 injected muscles, they validated and confirmed the effects of mutant MATR3 with a transgenic MATR3 model.17 This allowed the authors to avoid having low expression levels of the MATR3 gene in the tissues due to host-immunity against adenoviruses and allowed for a more stable MATR3 mouse model.6 Although these in vivo models provide a starting point to investigate MATR3-induced ALS pathologies, the data may not be completely translatable to humans. The au-

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thors failed to recognize that there are currently no known human cases of ALS caused by the overexpression of MATR3.9 Therefore, by overexpressing the gene in mice, more exaggerated phenotypes in the muscle and spinal cord could have been developed, failing to accurately resemble the disease. Additionally, the mere action of overexpressing a gene may induce downstream effects leading to increased or decreased production of certain proteins.9 Thus, the disease phenotype seen in these mice may potentially be caused by different mechanisms compared to ALS patients with the S85C mutation. Moreover, the authors did not incorporate wildtype MATR3 TG mice as a control. Hence, when comparing the phenotypes of only the mutant MATR3 TG mice with the NTG mice, the significant differences in tissue histology may partially be attributable to transgenesis.6Furthermore, the brain, which is an organ greatly affected in ALS, was not analyzed for pathology, lowering the reliability of these mice to model ALS. Another major flaw in this study was the lack of data interpretation. In the tissues of both the mutant MATR3 mouse models, an upregulation of proteins related to protein degradation and glial cells were found, leading to the possibility that MATR3 may be affecting these pathways.17 However, the authors fail to mention the relevance of this data, and how activating protein degradation or neuroinflammatory pathways could potentially be the molecular mechanism MATR3 is involved in to induce pathology. FUTURE DIRECTIONS Currently, only two drugs, riluzole and edaravone, have been approved as treatments to slow down ALS pathogenesis.4 Therefore, with the transgenic model already developed, these mice could be used to test out new therapeutic drugs or treatments. For instance, Rapmycin has been investigated as a potential treatment for ALS in a transgenic mutant TDP-43 model.11,17 Similar to the MATR3 S85C, mice with severe phenotypes exhibited sarcoplasmic aggregates of the mutated protein. 11However, with the administration of Rapamycin, which is known to activate autophagy and downregulate neuroinflammation, TDP-43 mislocalization was rescued.11Therefore, by administering this drug on the MATR3 S85C transgenic mice, a rescue in their phenotype may imply that MATR3 is a part of the autophagy and inflammatory pathway. In contrast, if no rescue is observed, it may implicate that MATR3 S85C is affecting different pathways to elicit myotoxicity and neurotoxicity. However, if the MATR3 S85C mice show an improved phenotype, optimization of the drug dosage and duration may be investigated, resulting

in a possible therapeutic drug for MATR3 S85C ALS patients. In recent literature, an emphasis on ALS progressing from the muscles and “dying back” towards the brain has been found.12 Furthermore, in studies with transgenic ALS SOD1, and FUS mice, Martineau et al. and Picchiarelli et al., observed neuromuscular junction abnormalities such as smaller NMJs preceding lower motor neuron degeneration.11,12 Therefore, the authors of this present study could observe if abnormal neuromuscular junction morphology or size preceded motor neuron degeneration in the transgenic MATR3 S85C mice. Muscle, spinal cord, and brain tissues from various time points in disease progression should be collected, stained with antibodies targeting the presynaptic and postsynaptic regions of the NMJ, and quantified. Smaller NMJs, followed by denervation of the lower motor neuron axons would demonstrate the trajectory pathogenic MATR3 affects, supporting the “dying back” hypothesis. However, if abnormalities in either the spinal cord or brain precede muscle pathology, MATR3 may be an exceptional protein that doesn’t affect the conventional pathway or pathogenesis of ALS may appear through different developmental routes. Nevertheless, by investigating the start and endpoint of MATR3 S85C-induced pathology, a better understanding of the mechanism by which the mutation causes ALS could be determined. Furthermore, to study the pathological effects of the MATR3 S85C mutation in a more physiologically relevant system, a MATR3 S85C knock-in mouse model should be generated using CRISPR-CAS9. In contrast to the transgenic model, the mutant MATR3 gene in the knock-in mice would be inserted into a specific locus in the animal’s genome.6 This would allow the MATR3 gene to be expressed under natural expression patterns and at an endogenous level.6 The knock-in mice would show similar muscle and nervous system phenotypes to the MATR3 S85C transgenic model if the S85C mutation is specifically responsible for causing ALS-linked pathologies. Furthermore, data interference due to the nature of transgenesis exaggerating pathological effects would also be minimized with this model.6 Thus, this knock-in MATR3 S85C model would more accurately exhibit the mechanisms by which MATR3 causes ALS and may provide data more translatable to human ALS patients.

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1. Barp A, Malfatti E, Metay C, Jobic V, Carlier RY, Laforet P. The first French case of MATR3-related distal myopathy: Clinical, radiological and histopathological characterization. Revue Neurologique. 2018;174(10):752–755. doi:10.1016/j.neurol.2017.08.004 2.Butti Z, Patten SA. RNA Dysregulation in Amyotrophic Lateral Sclerosis. Frontiers in Genetics. 2019;9. doi:10.3389/fgene.2018.00712 3.Gallego-Iradi MC, Clare AM, Brown HH, Janus C, Lewis J, Borchelt DR. Subcellular Localization of Matrin 3 Containing Mutations Associated with ALS and Distal Myopathy. Plos One. 2015;10(11). doi:10.1371/journal.pone.0142144 4.Jaiswal MK. Riluzole and edaravone: A tale of two amyotrophic lateral sclerosis drugs. Medicinal Research Reviews. 2018;39(2):733–748. doi:10.1002/med.21528 5.Johnson JO, et al. Mutations in the Matrin 3 gene cause familial amyotrophic lateral sclerosis. Nat. Neurosci. 17, 664-670 (2014). 6.Justice MJ, Dhillon P. Using the mouse to model human disease: increasing validity and reproducibility. Disease Models & Mechanisms. 2016;9(2):101–103. doi:10.1242/dmm.024547 7.Malik AM, Miguez RA, Li X, Ho Y-S, Feldman EL, Barmada SJ. Matrin 3-dependent neurotoxicity is modified by nucleic acid binding and nucleocytoplasmic localization. eLife. 2018;7. doi:10.7554/elife.35977 8.Martineau É, Polo AD, Velde CV, Robitaille R. Dynamic neuromuscular remodeling precedes motor-unit loss in a mouse model of ALS. eLife. 2018;7. doi:10.7554/elife.41973 9.Moloney C, Rayaprolu S, Howard J, Fromholt S, Brown H, Collins M, Cabrera M, Duffy C, Siemienski Z, Miller D, et al. Analysis of spinal and muscle pathology in transgenic mice overexpressing wild-type and ALS-linked mutant MATR3. Acta Neuropathologica Communications. 2018;6(1). doi:10.1186/s40478-018-0631-0 10.Müller TJ, Kraya T, Stoltenburg-Didinger G, Hanisch F, Kornhuber M, Stoevesandt D, Senderek J, Weis J, Baum P, Deschauer M, et al. Phenotype of matrin-3-related distal myopathy in 16 German patients. Annals of Neurology. 2014;76(5):669–680. doi:10.1002/ana.24255 11.Paraskevas GP, Bourbouli M, Zaganas I, Kapaki E. The emerging -43 proteinopathy. Neuroimmunology and Neuroinflammation. 2018;5 (5):17. doi:10.20517/2347-8659.2018.18 12.Picchiarelli G, Demestre M, Zuko A, Been M, Higelin J, Dieterlé S, Goy M-A, Mallik M, Sellier C, Scekic-Zahirovic J, et al. FUS-mediated regulation of acetylcholine receptor transcription at neuromuscular junctions is compromised in amyotrophic lateral sclerosis. Nature Neuroscience. 2019;22(11):1793–1805. doi:10.1038/s41593-019-0498-9 13.Rayaprolu S, Dalton S, Crosby K, Moloney C, Howard J, Duffy C, Cabrera M, Siemienski Z, Hernandez AR, Gallego-Iradi C, et al. Heterogeneity of Matrin 3 in the developing and aging murine central nervous system. Journal of Comparative Neurology. 2016;524(14):2740– 2752. doi:10.1002/cne.23986 14.Rowland, L. P. & Shneider N. A. Amyotrophic lateral sclerosis. The New England Journal of Medicine. 2001;344,1688-1700 15.Strong M, Rosenfeld J. Amyotrophic lateral sclerosis: A review of current concepts. Amyotrophic Lateral Sclerosis and Other Motor Neuron Disorders. 2003;4(3):136–143. doi:10.1080/14660820310011250 16.Strong MJ, Kesavapany S, Pant HC. The Pathobiology of Amyotrophic Lateral Sclerosis: A Proteinopathy? Journal of Neuropathology and Experimental Neurology. 2005;64(8):649–664. doi:10.1097/01.jnen.0000173889.71434.ea 17.Zhang X, Yamashita S, Hara K, Doki T, Tawara N, Ikeda T, Misumi Y, Zhang Z, Matsuo Y, Nagai M, et al. A mutant MATR3 mouse model to explain multisystem proteinopathy. The Journal of Pathology. 2019;249(2):182–192. doi:10.1002/path.5289 18.Zhao M, Kim JH, van Bruggen R, & Park J. RNA- binding proteins in amyotrophic lateral sclerosis. Molecules and Cells. 2019;41, 818-829

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Characterization of Newborn Dentate Granule Cell Morphology and Loss of Innervation in Frontotemporal Dementia and Activity-Dependent Rescue Lauren Levy1

The hippocampus is one of the few regions of the brain that continues neurogenesis well into adulthood. This is known as adult hippocampal neurogenesis, or AHN. Mutations in GSK-3β, the main tau kinase, and tau, have been implicated in many neurodegenerative diseases and have been shown to arrest adult hippocampal neurogenesis (Eriksson et al., 1998). The exact impact of AHN on behaviour is not thoroughly understood but is believed to be related to high levels of potential interference and subsequent pattern separation, as an element of learning, memory, and plasticity (Eriksson et al., 1998). While new dentate granule cells appear to enable different responses to similar inputs, mature DGCs are associated with more stable memory storage and pattern completion (Eriksson et al., 1998). Loss of AHN, which occurs in neurodegenerative diseases like Alzheimer’s, may correspond to a loss of plasticity (Eriksson et al., 1998). Recently, María Llorens-Martín and colleagues have shown that FTD and TauVLW newborn DGC are disconnected from other regions of the brain, as well as morphological changes such as increased inhibitory synapses and reduced connectivity of adult-born DGCs using retroviral and monosynaptic retrograde rabies virus tracing (Kempermann et al., 2018). They used both post-mortem tissue from the posterior end of the anterior hippocampus from patients with frontotemporal dementia, or FTD, and healthy controls, and TauVLW mice, popular models of FTD, to explore this phenomenon. By injecting one of three RGB viruses that selectively integrate into presently dividing cells at different times, they were able to isolate three populations of adult-born DGC. In addition, they demonstrate rescue of neurodegeneration in mice with enriched environments and chemoactivation, highlighting potential therapeutic considerations.

Key words: adult hippocampal neurogenesis, dentate granule cells, frontotemporal dementia, TauVLW mice, enriched environment, tauopathy, DREADD, clozapine-N-oxide

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BACKGROUND In 1998, Eriksson et al. first showed evidence of adult hippocampal neurogenesis, or AHN, in the human dentate gyrus, using post-mortem hippocampal tissues (Eriksson et al., 1998; Kempermann et al., 2018). Since then, much has been learned about AHN and now it is understood that new-born dentate granule cells, or DGC, derive from radial glia-like cells in the sub-granular layer of the dentate gyrus. These cells exhibit three distinct modes of self-renewal: asymmetric division yielding a new intermediate progenitor cell, asymmetric division yielding a new RGL, or symmetric division yielding two RGL (Bonaguidi et al., 2011). The exact function of this neurogenesis is not entirely understood, but it is believed to be related to high levels of potential interference and subsequent pattern separation. In simple terms, when two inputs are very similar but require different outputs, this separation of patterns may be enabled by adult neurogenesis. Furthermore, a sharp drop in adult hippocampal neurogenesis is associated with Alzheimer’s disease, as well as frontotemporal dementia, while remaining present in neurologically healthy adults (Moreno-Jiménez et al., 2019). Much of this research has come from the Llorens-Martín laboratory. For the first time, Julia Terroros-Roncal et al., part of the Llorens-Martín lab, have shown that in both frontotemporal dementia human samples and TauVLW, or FTD-model, mice, new-born DGC exhibit altered morphology, particularly reduced connection to distal regions and a higher ratio of inhibitory innervation (Terreros-Roncal et al., 2019). Additionally, they have shown that chemoactivation with designer receptors and drugs can completely reverse these changes, while activity promoted by an enriched environment can partially rescue these effects, in TauVLW mice. Frontotemporal dementia is the third mostcommon group of dementias, and a leading cause of dementia in patients under the age of 65. FTD is an umbrella term that describes multiple dementia variants and appears to be strongly linked to amyotrophic lateral sclerosis, such that FTD and ALS may represent extremes of a spectrum of disorders. This theory is based on shared pathological, genetic, and eventually behavioural features (Lattante, Ciura, Rouleau, & Kabashi, 2015). FTD is defined by selective degeneration of the frontal and temporal cortices (Bang, Spina, & Miller, 2015). While the exact causes of ALS and FTD are not understood and treatment does not yet exist, FTD has been modelled in mice with the TauVLW transgenic mouse, which expresses three single nucleotide polymorphisms that promote hyperphosphorylation and subsequent prefibrillar tau aggregates, reflecting chromosome-17 linked FTD with parkinsonism. These mice recapitulate key pathological features of FTD, particularly tau aggregation and hyperphosphorylation. Activity and enriched environments have been studied as therapies to reduce neurodegeneration and are further applied here. Not long before this study was published, Balthazar et al. demonstrated that an enriched environment reduced density of amyloid-β plaques, markers of neurodegeneration, in the dorsal portion of the hippocampus in transgenic-APP, or amyloid precursor protein, mice (Balthazar, Schöwe, Cipolli, Buck, & Viel, 2018). Additionally, Lahiani-Cohen et al. showed

in 2011 that moderate environmental enrichment reduced neurofibrillary tangle aggregation, a pathological hallmark of tauopathies like Alzheimer’s, in transgenic mice models of tauopathy (Lahiani-Cohen et al., 2011). There is thorough evidence to suggest that environmental enrichment and activity, especially unrestricted access to running, delays onset of pathological traits associated with major dementias, especially in rodent models. Chemoactivation of DGC was also shown to reverse the effects of the mutant tau on newborn DGC morphology and connectivity. Chemoactivation was induced using Designer Receptors Exclusively Activated by Designer Drugs, or DREADDs. This involved stereotaxically-injected hM3D:GFP, which combines a modified excitatory human muscarinic M3 receptor and green fluorescent protein. This designer receptor selectively responds to clozapine-N-oxide, a synthetic ligand that is introduced orally. Terroros-Roncal et al. not only characterize morphological changes in DGC induced by TauVLW and FTD, particularly reduced connectivity, but demonstrate two protocols that rescue those change. This describes and quantifies altered adult hippocampal neurogenesis, particularly newborn DGC disconnection from distal regions, in FTD for the first time.

MAJOR RESULTS Terroros-Roncal et al. have shown previously that in TauVLW mice, there is less proliferation of neural precursor cells (Llorens-Martin, Hernandez, & Avila, 2011). However, there is still proliferation and they have gone on to highlight morphological differences in the new-born DGC. With the hippocampus of five control subjects and three FTD subjects, Golgi staining revealed increased proximal branching and reduced distal branching. Based on the expression of GAD65+ and Gephyrin, markers of presynaptic and postsynaptic inhibitory innervation, the authors found increased inhibitory innervation. Additionally, both the length of the primary apical dendrite (U(1,7) = 1329.000; p ≤ 0.001) and total dendritic length (U(1,7) = 483.000; p ≤ 0.001) were reduced. The authors found similar results in their TauVLW mice, though total dendritic length was not reduced. Using retroviruses that encode either PSD95:GFP, or post-synaptic density protein 95, which reflects afferent connectivity of cells, the authors found that both the number and density of PSD was reduced, suggesting reduced afferent connectivity. To study efferent connectivity, the authors focused on Syn:GFP, which reflects the efferent connectivity of mossy fibre terminals of DGC, and found that like afferent connectivity, efferent connectivity was also reduced, suggesting that functional maturation of TauVLW DGC is impaired. Additionally, the mice also showed increased inhibitory innervation in the dentate gyrus. The authors go on to test two possible therapies in mice and found that both an enriched environment and chemoactivation of new-born DGC rescues both the morphological alteration and, partially or completely, respectively, restores connectivity of DGC. By providing the mice with rotating collections of toys and regular new bedding, the mice were subjected to an enriched environment protocol for 8 weeks. Using DREADDs, or Designer Re ceptors Exclusively Activated by Designer Drugs, the authors selectively activated the new-born DGC with a combination of a

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modified human muscarinic receptor and clozapine-Noxide. Both reversed morphological alterations in the DGC of the mice, while an enriched environment partially rescues otherwise-lost connectivity and chemoactivation completely restores connectivity.

Figure 1, cont. I, Schollâ&#x20AC;&#x2122;s analysis of Golgi-stained murine DCGs. G-H: Golgi staining in the hippocampus of WT (G) and TauVLW mice (H) via confocal tilescan. J, Comparison of healthy and TauVLW mouse for total dendritic length. Unlike human cells, results were not statistically significant. K, Comparison of murine primary apical dendrite. L, Murine cells with more than one primary apical dendrite.

Figures Adapted from Terreros-Roncal, et al. (2019). The Journal of Neuroscience, 39(29), 5794. Figure 1. Morphological alterations of new-born DGC in patients with FTD and mice with TauVLW are similar based on Golgi -staining analysis. A,B: Golgi-staining of hippocampal DGC in the hippocampus of (A) a healthy control subject and (B) a patient with FTD via confocal tilescan. C, Shollâ&#x20AC;&#x2122;s analysis of Golgistained DGCs, comparing number of dendritic intersections to distance from the cell body. D, Comparison of total dendritic length. E, Length of the primary apical dendrite of DGCs. F, Cells with more than one primary apical dendrite, as a percentage.

Figure Adapted from Terreros-Roncal, et al. (2019). The Journal of Neuroscience, 39(29), 5794 Figure 2 (G). Altered afferent connectivity of newborn Tau VLW DGC. Afferent connectivity ratio of local (DG), proximal (HC) and distal brain regions based on RV-Syn-GTRgp retroviruses stereotaxic injection into the hippocampus, followed by EnvA-G -MCh rabies virus stereotaxic injection four weeks later, to trace the cells innervating starter cells.

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Figure Adapted from Terreros-Roncal, et al. (2019). The Journal of Neuroscience, 39(29), 5794.

Previous literature has shown that neurogenesis declines with age, mainly as consequence of decreased available progenitor cells. However, increased rates of adult neurogenesis, including dendritic growth, spine formation, and neuronal integration is associated with chronic voluntary running and some enriched environment protocols (Trinchero, Herrero, & Schinder, 2019). Running acts at two levels to promote neurogenesis: raising the rate of progenitor cell proliferation (Henriette van Praag, Kempermann, & Gage, 1999; H. van Praag, 2005) and accelerating the maturation and subsequent functional integration of these cells (Trinchero et al., 2017). Indeed, adult hippocampal neurogenesis has long been established, albeit at reduced levels relative to young brains. Additionally, activity and various types of exercise are known to promote neuroprotective factors. However, the mechanisms by which this occurs are less understood, as is the precise role of neurogenesis and maturation and integration of new-born cells, but they may enable subsequent pattern separation or influence synaptic plasticity. Interestingly, recent discoveries in computational theoretical neuroscience highlight a possible impact of dendritic length on plasticity. In 2017, Bono and Clopath constructed biophysically realistic neuron models to better understand rules that govern spike-time dependent potentiation, or STDP, a biological mechanism for adjusting synapse strength that enables long-term potentiation and subsequently, learning. Bono and Clopath found that increased distance from the soma and dendritic length relates to resistance to reduced STDP (Bono & Clopath, 2017). This is supported by experimental results from Letzkus, Kampa, and Stuart that demonstrate in vitro evidence suggesting that that the potentiation window decreases with distance from the soma. This is attributed to the attenuation of the back-propagating action potential over distance. Furthermore, distal and proximal synapses appear to have different action potential timing requirements (Letzkus, Kampa, & Stuart, 2006). Broadly, there is evidence suggesting that distal neurons are more resistant to plasticity than proximal neurons. Altered cell morphology, particularly reduced dendritic length under disease state conditions, appears to have a direct impact on synaptic plasticity. Terroros-Roncalâ&#x20AC;&#x2122;s study applied methods used for quantifying cell morphology and nerve connectivity to disease-state, specifically tau aggregate-prone mice and hippocampal tissue from FTD patients, then both a direct and indirect protocol for promoting hippocampal dentate granule cell activity. This combination of methods shows for the first time the changes in new-born TauVLW and FTD DGC, and that established methods for promoting neurogenesis remain applicable to this condition. The study is limited by a low sample size, as only three subjects with FTD are tested. Considering that FTD is an umbrella term that describes multiple, distinct variants, the study would have benefitted from a greater sample size, with better attention to different FTD variants. However, considering that storage of samples may affect quality of samples and results, obtaining a larger sample would have been especially difficult. Finally, while the presence of human AHN is well-established, many factors related to are not well understood because of inconsistency in protocols that may affect results, such as storage method and markers used.

Figure Adapted from Terreros-Roncal, et al. (2019). The Journal of Neuroscience, 39(29), 5794 Figure 3(B-G). Representative images of adult-born DGCs transduced with Venus-(B,E) (2 week old),mCherry-(C,F) (4 week old), or Cerulean(D,G) (8 week old). TauVLW show altered morphology, such as reduced total dendritic length and distal innervation. CONCLUSIONS/DISCUSSION For the first time, the authors thoroughly charsacterize morphological alteration in new-born DGC, a hallmark of AHN, in humans with FTD compared to non-FTD controls and mice with TauVLW, a human tau protein that is prone to hyperphosporylation and enables partial modelling of FTD and related tauopathies, compared to control mice. The authors found reduced afferent and efferent connectivity, especially to distal regions, and a higher proportion of inhibitory innervation. The authors go on to show that acti- CRITICAL ANALYSIS vation of DGC through chemoactivation and an enriched The authors of the paper concerning altered morpholoenvironment can reverse these morphological changes, ingy and connectivity of newborn dentate gyrus cells in FTD and forming treatment plans to reduce FTD. 244

TauVLW propose models of rescue, using both chemoactivation and enriched-environment protocols (Terreros-Roncal et al., 2019). However, their method of chemoactivation, which relies on designer receptors and ligands, may have had unintended side effects. While the ligand, clozapine-N-oxide, is believed by some to be biologically inert, other studies have shown that it may be reverse-metabolized to clozapine under physiological conditions (Manvich et al., 2018). Clozapine is an atypical antipsychotic, and is a known serotonin antagonist, with high affinity for several dopaminergic receptors. As such, clozapine-Noxide may not be biologically inert within the nervous system but acting as clozapine. This issue may be resolved by repeating the experiment with alternative DREADD systems, or by including a control series that receives clozapine and clozapine with the designer receptor, to be compared to the DREADD and clozapine-N-oxide experiments. While optogenetics has become a popular method for selective activation of neurons, it is possible that the hippocampus posed issues because it is too deep within the brain, though optogenetic stimulation of the hippocampus is well-documented. Additionally, while enriched environment protocols have some standardization among studies, exercise amount was not controlled or quantified. As such, the strength of the effects of an enriched environment may not have been fully explored, and better highlighted by different protocols. Improving on this will be explored in the Future Directions. The study is limited by a low sample size, as only three subjects with FTD are tested. Considering that FTD is an umbrella term that describes multiple, distinct variants, the study would have benefitted from a greater sample size, with better attention to different FTD variants. However, considering that storage of samples may affect quality of samples and results, obtaining a larger sample would have been especially difficult. Despite some of the weaker parts of this study, it is nonetheless a remarkable example of elegant experimental design. Identification of new-born DGC via RGB retrovirus, afferent and efferent connectivity via PSD95:GFP or Syn:GFP retroviruses, and innervation via monosynaptic retrograde rabies collectively represent innovative, thorough methods to elucidate the changes in cell morphology and connectivity. The authors provide very strong evidence of visible change in Tau VLW and FTD hippocampal dentate granule cells. Their methods ought to be applied to future studies to further understand this phenomenon. Though their methods of rescue may not have been as sound, their approach to characterizing altered morphology and connectivity was brilliant. Though the conclusion that there is activity-dependent reconnection could have been made stronger using greater attention to the enriched environment protocols and better characterization of the activity, the authors thoroughly demonstrate altered cell morphology, reduced distal connectivity, and alterations in the type of connectivity. FUTURE DIRECTIONS â&#x20AC;&#x201C; One of the major findings of Terroros-Roncal et al.â&#x20AC;&#x2122;s study was the establishment that neurogenesis alone is not a marker of brain health, since connectivity and morphology may still be stunted. Recent years have seen immunotherapy for hyperphosphorylated tau move beyond proof-of-concept studies to clinical trials in the arms race to claim pharmaco-

logical therapy for major dementias like Alzheimerâ&#x20AC;&#x2122;s disease. Developing a streamlined protocol that addresses core components of this study for application to tauopathy therapies would prevent the further study of therapies that support neurogenesis but fail to support maturation or connectivity of new-born neurons. Such a study would focus on total dendritic length, length of the primary apical dendrite, whether innervation is distal or proximal, and ratio of inhibitory to excitatory innervation and might be distributed like a kit with a checklist of elements to consider. Not only would this reveal therapies that fail to promote normal neurogenesis, it would better reveal the behavioural effects of this altered morphology and its variation across different variants of both FTD and other dementias. This might also focus on the effect of this impaired morphology on long-term potentiation and depotentiation. After all, the study that this review focuses on does not identify a deleterious effect on cognition caused by or related to this morphology but rather shows that it follows tau hyperphosphorylation and FTD diagnosis. Repeating these studies with more attention to FTD variant, other dementias, lifestyle and genome analysis, and across other mouse models might allow scientists to predict who will develop this altered morphology and study cognitive decline associated with it. This would continue in both rodent and human tissues. Additionally, since the enriched environment did not entirely reverse the effects, testing variations of the protocol to better optimize it for future studies would be ideal, as it ought to reveal limits to the protocol. Running and aerobic exercise has long been isolated as a component of an enriched environment that promotes neurogenesis, including increased levels of BDNF, or brain-derived neurotrophic factor (Trinchero et al., 2019). Additionally, since newborn DGC are strongly associated with spatial distinction, better understanding the molecular mechanisms and behavioural components that rescue dysconnectivity would better inform both pharmacological therapy and preventative behaviour. This would mean comparing different enriched environment elements, such as toys used and games or tests implemented, as well as variations on durations and frequency of changes in elements. The goal of this would be to identify features that have a greater impact on both cognition and prevention of altered morphology of newborn DGC, ultimately informing programming in nursing homes and among elderly populations.

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REFRENCES

1. Balthazar, J., Schöwe, N. M., Cipolli, G. C., Buck, H. S., & Viel, T. A. (2018). Enriched Environment Significantly Reduced Senile Plaques in a Transgenic Mice Model of Alzheimer’s Disease, Improving Memory. Frontiers in Aging Neuroscience, 10, 288. https:// doi.org/10.3389/fnagi.2018.00288 2. Bang, J., Spina, S., & Miller, B. L. (2015). Frontotemporal dementia. The Lancet, 386(10004), 1672–1682. https://doi.org/10.1016/ S0140-6736(15)00461-4 3. Bonaguidi, M. A., Wheeler, M. A., Shapiro, J. S., Stadel, R. P., Sun, G. J., Ming, G., & Song, H. (2011). In vivo clonal analysis reveals self-renewing and multipotent adult neural stem cell characteristics. Cell, 145(7), 1142–1155. https://doi.org/10.1016/j.cell.2011.05.024 4. Bono, J., & Clopath, C. (2017). Modeling somatic and dendritic spike mediated plasticity at the single neuron and network level. Nature Communications, 8(1), 706. https://doi.org/10.1038/s41467-017-00740-z 5. Eriksson, P. S., Perfilieva, E., Björk-Eriksson, T., Alborn, A.-M., Nordborg, C., Peterson, D. A., & Gage, F. H. (1998). Neurogenesis in the adult human hippocampus. Nature Medicine, 4(11), 1313–1317. https://doi.org/10.1038/3305 6. Kempermann, G., Gage, F. H., Aigner, L., Song, H., Curtis, M. A., Thuret, S., … Frisén, J. (2018). Human Adult Neurogenesis: Evidence and Remaining Questions. Cell Stem Cell, 23(1), 25–30. https://doi.org/10.1016/j.stem.2018.04.004 7. Lahiani-Cohen, I., Lourbopoulos, A., Haber, E., Rozenstein-Tsalkovich, L., Abramsky, O., Grigoriadis, N., & Rosenmann, H. (2011). Moderate Environmental Enrichment Mitigates Tauopathy in a Neurofibrillary Tangle Mouse Model. Journal of Neuropathology & Experimental Neurology, 70(7), 610–621. https://doi.org/10.1097/NEN.0b013e318221bfab 8. Lattante, S., Ciura, S., Rouleau, G. A., & Kabashi, E. (2015). Defining the genetic connection linking amyotrophic lateral sclerosis (ALS) with frontotemporal dementia (FTD). Trends in Genetics, 31(5), 263–273. https://doi.org/10.1016/J.TIG.2015.03.005 9. Letzkus, J. J., Kampa, B. M., & Stuart, G. J. (2006). Learning rules for spike timing-dependent plasticity depend on dendritic synapse location. The Journal of Neuroscience : The Official Journal of the Society for Neuroscience, 26(41), 10420–10429. https:// doi.org/10.1523/JNEUROSCI.2650-06.2006 10. Llorens-Martin, M., Hernandez, F., & Avila, J. (2011). Expression of frontotemporal dementia with parkinsonism associated to chromosome 17 tau induces specific degeneration of the ventral dentate gyrus and depressive-like behavior in mice. Neuroscience, 196, 215– 227. https://doi.org/10.1016/j.neuroscience.2011.08.057 11. Manvich, D. F., Webster, K. A., Foster, S. L., Farrell, M. S., Ritchie, J. C., Porter, J. H., & Weinshenker, D. (2018). The DREADD agonist clozapine N-oxide (CNO) is reverse-metabolized to clozapine and produces clozapine-like interoceptive stimulus effects in rats and mice. Scientific Reports, 8(1), 3840. https://doi.org/10.1038/s41598-018-22116-z 12. Moreno-Jiménez, E. P., Flor-García, M., Terreros-Roncal, J., Rábano, A., Cafini, F., Pallas-Bazarra, N., … Llorens-Martín, M. (2019). Adult hippocampal neurogenesis is abundant in neurologically healthy subjects and drops sharply in patients with Alzheimer’s disease. Nature Medicine, 25(4), 554–560. https://doi.org/10.1038/s41591-019-0375-9 13. Terreros-Roncal, J., Flor-García, M., Moreno-Jiménez, E. P., Pallas-Bazarra, N., Rábano, A., Sah, N., … Llorens-Martín, M. (2019). Activity-Dependent Reconnection of Adult-Born Dentate Granule Cells in a Mouse Model of Frontotemporal Dementia. The Journal of Neuroscience, 39(29), 5794. https://doi.org/10.1523/JNEUROSCI.2724-18.2019 14. Trinchero, M. F., Buttner, K. A., Sulkes Cuevas, J. N., Temprana, S. G., Fontanet, P. A., Monzón-Salinas, M. C., … Schinder, A. F. (2017). High Plasticity of New Granule Cells in the Aging Hippocampus. Cell Reports, 21(5), 1129–1139. https://doi.org/10.1016/ j.celrep.2017.09.064 15. Trinchero, M. F., Herrero, M., & Schinder, A. F. (2019). Rejuvenating the Brain With Chronic Exercise Through Adult Neurogenesis. Frontiers in Neuroscience, 13, 1000. Retrieved from https://www.frontiersin.org/article/10.3389/fnins.2019.01000 16. van Praag, H. (2005). Exercise Enhances Learning and Hippocampal Neurogenesis in Aged Mice. Journal of Neuroscience, 25(38), 8680–8685. https://doi.org/10.1523/JNEUROSCI.1731-05.2005 17. van Praag, Henriette, Kempermann, G., & Gage, F. H. (1999). Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus. Nature Neuroscience, 2(3), 266–270. https://doi.org/10.1038/6368

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Alzheimer’s disease: Consequences of neurofilament light protein gene deletion in APP/PS1 mice model Fei Li

Alzheimer’s disease (AD) is a progressive neurodegenerative disease that causes the impairment of many cognitive abilities. AD pathology is characterized by amyloid-beta (AB) plaques and neurofibrillary tangles (NFTs). AB plaques are known to cause inflammatory responses, synaptic dysfunction, and neuronal death. NFTs are composed of accumulations of hyperphosphorylated Tau which leads to neuronal degeneration. Less research has been based on cytoskeletal proteins such as neurofilaments (NFs) and their roles in neuronal pathology. The study by Fernandez-Martos et. al. (2015), investigated the contribution of neurofilament light (NFL) protein, a subunit to neurofilament triplet proteins in AD pathology. The study used aging transgenic APP/PS1 mice with AD on a background of a neurofilament light knockout (NFL KO) to analyse NFL involvement with amyloid-beta plaque, dystrophic neurites, synapse vulnerability, and glial responses. Using tissue extraction and immunohistochemical procedures, experimental results showed that APP/PS1 NFL -/- mice had increased levels of AB plaque deposition, dystrophic neurites, microgliosis, and lost or decreased sizes of synapses. The findings of this study suggest that the role of NFL involve neurite protection and regulation of AB plaque formation. This review provides an overview of the neurofilament subunit, neurofilament light protein as a biomarker and its involvement in the pathology of Alzheimer’s disease. Key words: Alzheimer’s disease (AD), neurofilament light (NFL) protein, amyloid-beta (AB), dystrophic neurites (DNs), microgliosis, synapse density

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showed increased AB plaque, DN, and microgliosis, as well as lowered synapse density for NFL knockout (NFL KO) mice compared to NFL present mice. It is then proposed that NFL proteins protect Alzheimer’s disease (AD) is the most common type of progressive the neuron and regulates AB plaque formation. neurodegenerative disease. AD is a global public health priority and a major cause of dementia, one of the world’s leading cause of death (Lane et al. 2018). Since the year 1906 when Alois Alzheimer MAJOR RESULTS presented AD, various studies have been done concerning the pathology and the possible treatments of AD (Liu, Xie, Meng, & Kang, 2019). No treatment has yet to be found to stop the progres- Amyloid accumulation sion of AD. But, two major clinical drug treatments, acetylcholinesterase inhibitors (AChEIs) and the antagonist of N-methyl-D- APP/PS1 NFL -/- mice displayed significant increase in neocortical aspartic acid (NMDA) receptor have been found to relieve some AB depositions compared with APP/PS1 NFL +/+ mice (Fernandezsymptoms of AD (Liu et al. 2019). Martos et al. 2015). The analysis was done using immunohistochemical procedures performed on dissected sections of the neoAD is a cognitive disease caused by neuronal death (Gaugler, cortex and hippocampus in APP/PS1 NFL -/- and APP/PS1 NFL +/+ James, Johnson, Scholz, & Weuve, 2016). The loss or damage of mice. Tissue was stained with Thioflavin-S which detects AB nerve cells cause problems majorly in brain regions, hence AD is described as a cognitive disease. Major symptoms to AD consist of plaques. MOAb-2 antibody was also used to detect for AB and exthe inability to remember new information, the loss of memory, cluded amyloid precursor protein (APP) detection. The plaque size difficulties completing daily tasks, and problems with communica- did not appear to show significant causation concerning plaque tion (Gaugler et al. 2016). Multiple risk factors have been found to load. Plaque number showed causation for plaque load. The sigcontribute to AD, such as age, heredity, and inheritance of the AP- nificant increase in AB plaque load was only detected in neocortex OE gene (Gaugler et al. 2016). AD increases with age and people analysis and not in hippocampus analysis. The results showed that with first-degree relatives with AD have been found to be at higher NFL KO mice increased AB depositions which agreed with more risk (Gaugler et al. 2016). The APOE allele has been found to be a determinant of neuritic plaques (NPs) and neurofibrillary tangle recent studies which found that NFL as a good biomarker to detect neurodegeneration in AB positive subjects (Bos et al. 2019). (NFT) (Tiraboschi et al. 2004).

BACKGROUND and INTRODUCTION

Previous studies have generally focused research on theories such as the amyloid cascade hypothesis, the neurotransmitters hypothesis, or the tau propagation hypothesis, as the causing factors to neuronal death. Studies have identified various indicators for AD, such as increased amyloid-beta plaque, tau hyperphosphorylation, and increasing neurofibrillary tangles. Neurofibrillary tangles are a known indicator of AD as seen through past findings. Findings have shown that the neurons that harbour NFT are more likely to degenerate (Bussière et al. 2003). Nonphosphorylated neurofilament protein-containing neurons were previously tested to show peak NFT formation as dementia progressed through its disease stages, and thus concluded that NFT formation was associated with the degeneration of neurons (Bussière et al. 2003). Neurons with NFT are considerably more vulnerable. Major research has been done on the tau propagation hypothesis which propose that the formation of neurofibrillary tangles is caused by AD hyperphosphorylated tau (AD P-tau) protein (Alonso, Grundke-Iqbal, & Iqbal 1996). AD P-tau act as nucleation centers for normal tau aggregation and cause the formation of long bundles of neurofilaments. AD P-tau disassembles microtubules that are normally assembled by normal tau through tubulin competition (Alonso et al. 1996). The competition causes increased tau binding and forms the neurofibrillary tangles that lead to neuronal death (Alonso et al. 1996). Figure Adapted from Fernandez-Martos et al. (2015). Neurobiology of Aging, 36(10), 2757–2767. Figure 1. Amyloid-beta deposition analysis. A-C. Thioflavin-S staining at the neocortex. Significant increase in load and number, but not size. D-F. Thioflavin-S staining at the hippocampus. No difference reported in the hippocampus. G-I. MOAB-2 staining at the neocortex. Significant increase in load and number, but not size. J-L. MOAB-2 staining at the hippocampus. No difference reported for hippocampus.

As seen in past studies for neurofibrillary tangles, research has been focused on the tau propagation hypothesis and less research has been done on other cytoskeletal components of the neuron in regard to AD involvement. Neurofilament proteins are one the major types of intermediate filaments found in myelinated axons of neurons (Fernandez-Martos, King, Atkinson, Woodhouse, & Vickers, 2015). The neurofilament light (NFL) protein is a subunit of the neurofilament (NF) triplet proteins (Fernandez-Martos et al. 2015) and has been proposed by the current study by Fernandez-Martos et Dystrophic neurites al. (2015) to be greatly impactful in AD pathology. This study used techniques of immunohistochemistry to test amyloid-beta (AB) deposition levels, dystrophic neurite (DN) pathology, synapse den- APP/PS1 NFL -/- mice were found to show increases in dystrophic sity, and glial response on transgenic AD (APP/PS1) mice with a neurites in the neocortex. APP/PS1 NFL -/- and APP/PS1 NFL +/+ neurofilament light gene homozygous knockout (NFL -/-). Results

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mice tissue was obtained from the neocortex and stained with Thioflavin-S (Fernandez-Martos et al. 2015). Using immunohistochemistry, the DN populations were immunolabeled with synaptophysin and a-internexin. APP/PS1 NFL -/- mice immunolabeled with synaptophysin resulted in increased average DN size and increased percentage of DN load compared to APP/PS1 NFL +/+. APP/PS1 NFL -/- mice immunolabeled with a-internexin resulted in increased average DN size and decreased DN number compared to APP/PS1 NFL +/+. Limited research has been done on DN association with NFL to compare with current study results.

Figure Adapted from Fernandez-Martos et al. (2015). Neurobiology of Aging, 36(10), 2757–2767. Figure 2B. Dystrophic neurite analysis. A-F. Showed DN populations (aINT labelled) around AB plaques (Thio-S labelled). G-I. Significant DN size increase, number decrease, but no load difference for APP/PS1 NFL -/- mice compared to APP/PS1 +/+.

Synapse Vulnerability APP/PS1 NFL -/- mice showed greater significant reduction in bouton density and size compared to the APP/PS1 NFL +/+ mice. Both transgenic mice types were shown to have reduction compared to the wild-type. The analysis was conducted by staining with Thioflavin-S on neocortical tissue and colabeling for synaptophysin. Images were collected using a spinning disk UltraView confocal microscope and bouton number and size found using ImageJ watershed algorithm (Fernandez-Martos et al. 2015).

Figure Adapted from Fernandez-Martos et al. (2015). Neurobiology of Aging, 36(10), 2757–2767. Figure 2A. Dystrophic neurite analysis. A-C. Demonstration that immunolabels labelled separate DN populations. D-I. Showed DN populations (SYN labelled) around AB plaques (Thio-S labelled). J-L. Significant load and size increase of DNs, but not number of APP/PS1 NFL -/- mice compared to APP/PS1 +/+ .

Figure Adapted from Fernandez-Martos et al. (2015). Neurobiology of Aging, 36(10), 2757–2767. Figure 3. Synapse vulnerability analysis. A. Region immunolabelled with SYN and used for analysis for APP/PS1 NFL -/-. B. Region of APP/ PS1 NFL +/+ immunolabelled with SYN. C-F. Significant decreased in density and bouton size for R1 regions, but no difference for R2 comparing APP/PS1 NFL -/- to APP/PS1 NFL +/+.

Glial Response APP/PS1 NFL -/- mice showed increase in microgliosis and no change in astrogliosis compared to APP/PS1 NFL +/+ mice. The

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tissue was stained with Thioflavin-S and co-labeled with GFAP for astrocytes and Iba1 for microglia. Images of the tissue were collected with a spinning disk UltraView confocal microscope and densitometry analysis was done to find area that contained astrocytes and microglial cells (Fernandez-Martos et al. 2015).

(Fernandez-Martos et al. 2015). Authors are unclear of the cause of the decrease and have predicted that it may be due to the particular population to be more vulnerable to dystrophy. Studies have separated DNs into many forms such as neurofilament, tau, and chromogranin A immunolabeled forms, or used in the study, synaptophysin and a-internexin immunolabeled forms (Dickson et al. 2015). Different DNs populations may be at different locations proximate to the plaque and have different responses to NFL KO (Sharoar, Hu, Ma, Zhu, & Yan, 2019). APP/PS1 NFL -/- mice compared to APP/PS1 NFL +/+ mice show decreased synapse density and decreased size of boutons for the R1 area. For both R1 and R2 areas, both transgenic types observed deduction compared to the WT. Authors conclude that more loss of synapse is observed in the immediate surroundings of AB plaques (Fernandez-Martos et al. 2015). This study had made great contribution to the investigation of NFL as a promising biomarker for detection of AD.

CRITICAL ANALYSIS Figure Adapted from Fernandez-Martos et al. (2015). Neurobiology of Aging, 36(10), 2757â&#x20AC;&#x201C;2767. Figure 4. Glial response analysis. A-B. GFAP labelling for astrocyte. Similar astrocyte distribution between APP/PS1 NFL -/- and APP/PS1 NFL +/+ mice. D-E. Iba1 labelling for microglia. Significant increase in microglia around plaques in APP/PS1 NFL -/- compared to APP/PS1 NFL +/+. C-F. Significant area covered by microglia for APP/PS1 NFL -/-.

CONCLUSIONS/DISCUSSION This study investigates the neurofilament light protein as a biomarker for AD. Several experiments have been conducted to make conclusions that NFL is a promising biomarker. NFL KO mice has been found to show increase in neuronal degeneration factors and indicators such as AB deposition and DNs. The NFL protein can thus be be considered a biomarker and protein levels can be associated with measurement of the severity of AD.

In the study, Fernandez-Martos et al. (2015) have conducted most experiments in the neocortex and have found no changes within the hippocampus. Upon closer examination, of Figure 1., results seem to show lower load and number of AB plaques for APP/PS1 NFL -/compared to the higher values for APP/PS1 NFL +/+. This data could suggest that NFL KO leads to a lowering of harmful plaque in the hippocampus. The hippocampus has been found to harbour the earliest presence of neurofibrillary tangles observed in AD patients using magnetic resonance imaging (MRI) (Mungas, Tractenberg, Schneider, Crane, & Bennett, 2014). The authors should have included the hippocampus in more of their experiments to see if NFL KO really had no effect on the brain component. The sample size for this study only included five mice, which is a very small sample. Small samples are allowed if the variable measured shows large and reliable changes to make the results significant (Button et al. 2013). But there is still lower statistical power for small samples and lower reliability. The authors should have used a larger sized sample.

Previous biomarkers suggest AB plaque levels, tau protein levels, and inherited mutations for proteins such as Presenilin-1 and Presenilin-2 as indications of future AD development (Mantzavinos & Alexiou, 2017). What makes the NFL protein an important biomarker, is that the degenerating neurons that decreasing NFLs appear in are associated with DNs and AB plaques that occur very early in the stages of AD (Dickson, King, McCormack, & Vickers, 1999; Fernandez-Martos, 2015). This makes NFL an important biomarker for detection of AD in patients early on so that there can be early preparation for treatments.

In the study, two populations of DNs were used for experimentation concerning DN pathology. Studies have shown that different DN populations occur in different locations and can differ in the association with NFLs (Su, Cummings & Cotman, 1994). It is thus suggested that the authors are to be more specific in their experiments about which types of DN are tested. The authors have suggested that the SMI 312 neurites may be a subtype that has more vulnerability to dystrophy than other populations. This vulnerability should be addressed and tested before experimentation. There should be identification and analysis on all DN populations being APP/PS1 NFL -/- mice compared to APP/PS1 NFL +/+ mice has measured. been observed to increase AB deposition, indicated through increased load and number (Fernandez-Martos et al. 2015). The authors conclude that the high number of AB contributed to the depo- A final suggestion for improvement may be to take focus from mice sition load and that the size of AB did not affect the load. to human research. Mice and humans share similar genes but still have vast differences in genome, protein coding and expression (Lin et al. 2014). Results from mice may not be conserved for huAPP/PS1 NFL -/- mice were found to increase DN population surman AD pathology. Research should be made using human subjects rounding AB plaques compared to APP/PS1 NFL +/+, indicated in the future. through increased size affecting load for synaptophysin labeled populations (Fernandez-Martos et al. 2015). For a-internexin labeled populations, a slight increase was observed for APP/PS1 NFL Research addressing these areas could help clear up unknowns and -/- compared to APP/PS1 NFL +/+, as indicated by an increase in get a more in depth picture of Alzheimerâ&#x20AC;&#x2122;s disease. average size. An unexpected result for APP/PS1 NFL -/- also showed decrease of DN number compared to APP/PS1 NFL +/+

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FUTURE DIRECTIONS The study by Fernandez-Martos et al. (2015) showed impactful results, but it had only a focus on mice. Future studies could have some focus on humans as well as deeper investigations into the hippocampus, DN population, and the localization of the synapse. For research into humans, two sets of subjects would be recruited for the study, one set with individuals diagnosed with AD and a second set with individuals who are not carriers for mutations causing high risk of AD (Weston et al. 2017). Measurements of NFL concentration could be taken from each individual using ultrasensitive immunoassay on Simoa platform (Weston et al. 2017). Individuals with low risk of AD can act as a control for future experimentation. NFL levels can be sorted for low NFL and high NFL levels. To research NFL impact on hippocampus, subjects can be subjected to MRI scans on the hippocampus. Based on the current study, authors reported no change for amyloid load between NFL KO mice and NFL present mice (Fernandez-Martos et al. 2015). Therefore, the expected result should show higher neuronal degeneration in NFL low subjects compared to NFL high subjects (Scheltens, Fox, Barkhod, & De Carli, 2002). Subjects with no AD should not show degeneration in MRI scans. In the study, two populations of DNs were used for experimentation concerning DN pathology. Studies have investigated DN population growth using two-dimensional and three-dimensional (3D) electron microscopy (EM) (Sharoar et al. 2019). Using this technology, the two population could be investigated as to where and when these DNs appear and how they affect AD pathology. This study also tested areas of R1 and R2 which were characterized by differences in distance proximate to the AB plaque. As observed in the results, it was concluded that at the immediate proximity there appeared to be the greatest synapse loss, which decreased in severity moving away from the plaque. Future experiments could attempt using similar techniques to measure the average distance of synapse which could help predict damage done on synapses. Synapse density decline has been associated as a consequence of AB plaques with AB oligomers present in presynaptic boutons and terminal axons (Terry, 2006). Synapse density is measured in vitro and not in vivo, therefore future investigation can be on the average proximity from plaques that show synapse loss, therefore predicting possible damage using AB plaque markers instead of measuring synapses (Terry, 2006). As amyloid formation can be detected in vivo (Villar-Pique, Espagaro, Ventura, & Sabate, 2014). Lastly, the experiments in this study should be reproduced with larger sample sizes or meta-analyses done on similar studies to confirm reliability of results (Button et al. 2013). With metaanalyses less bias is made and less results affected by confounding factors.

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The gut microbiome and the brain: how Akkermansia muciniphila can alleviate hippocampal-associated deficits that result from high fat diets Melanie Lopez

In recent years, the gut microbiome has been of particular interest in relation to brain health. This is a result of associations between neurological diseases/disorders (like depression, Alzheimerâ&#x20AC;&#x2122;s and Parkinsonâ&#x20AC;&#x2122;s disease) and changes in the gut microbiome being made. These changes can result from many things, including a high fat diet (HFD), and can cause inflammation in the brain (which involves changes in microglial cells as well as the transmembrane protein toll-like receptor 4, also known as TLR4). A recent study1 investigated the relationship between hippocampal functions like learning and memory and HFDs. This was done by testing the cognitive abilities of two groups of mice on different diets (a normal diet and an HFD respectively). After investigating changes in populations of the gut microbiome, it was found that Akkermansia muciniphila was one of the bacterial populations that decreased in HFD mice. This group of mice was then given A. muciniphila and saw significant improvements and memory tests, as well as decreased hippocampal inflammation. Key words: high fat diet, gut microbiome, Akkermansia muciniphilaÂ¸ hippocampus, neuronal inflammation, obesity, TLR4, learning, memory, fear conditioning, mice model

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BACKGROUND and INTRODUCTION Obesity has long been associated with negative effects on human health, with recent studies estimating that it affects over 36% of people worldwide2. In both mouse3 and human models4, clear distinctions between bacterial populations of the gut have been demonstrated when comparing those of healthy versus overweight individuals. One main item of interest has been the ratio of gut microbiome Bacteroidetes and Firmicutes, with both mouse3 and human models4 seeing an increase in Firmicutes (although Bacteroidetes still dominate in human models). Furthermore, connections have been made between the gut microbiome and brain health, with studies finding that depression correlates positively with microbiome uniformity5. As obesity has been found to alter the gut microbiome, it consequently has been associated with negative effects on the brain, including increased inflammation as well as increased risk for Alzheimer’s6. One hypothesis is that irregularities in the gut microbiome, such as the aggregation of alphasynuclein that is seen in Parkinson’s disease, travel to the brain via the vagus nerve, inducing neurological disorders.7 Overall, obesity appears to negatively affect brain health by causing changes in the gut microbial flora and fauna. When looking at specific populations in the gut microbiome in which changes result in neuronal abnormalities, Akkermansia muciniphila has been identified as a potential bacterium. Although functions of A. muciniphila are still being explored, it appears to have a role in reducing inflammation, as research has shown that individuals with inflammatory bowel diseases like Crohn’s disease and ulcerative colitis have significantly lower levels of it compared to healthy individuals8. Furthermore, it has been shown that A. muciniphila can help decrease lipopolysaccharide levels in the bloodstream (which are especially saturated in overweight individuals)9, which has been identified as a cause of hippocampal inflamation10. Inflammatory responses, which are associated with microglial activation, can impede functions related to the hippocampus as well as prevent the development of new hippocampal neurons11. Toll-like receptor 4 (TLR4), which is found in microglial cells, binds lipopolysaccharides and activates immunogenic-response related pathways12. In the primary study1, researchers hypothesized that the excess fat intake of the HFD mice was impacting the TLR4-related pathways via the gut microbiome and the vagus nerve. A. muciniphila treatment appears to be a viable option to alleviate the aforementioned impairments. In the main paper to be discussed1, scientists looked at the effect of obesity on the brain by comparing test performance of mice, which were divided into two groups based on diet: a normal, chow diet versus a high fat diet (HFD). The two groups underwent testing – either fear conditioning (for which freezing was used as the measure of fear response) and the Barnes’ circular maze test (which used time spent near escape route among other factors to measure memory). Stool samples were also taken to be used for 16S rRNA sequencing to assess changes and diversity in the gut microbiome. Administration of A. muciniphila was used in an attempt to rescue the HFD mice, as its levels decreased in the aforementioned group. Measurements of action potentials in neurons of the hippocampus, as well as visualizations of hippocampal neurons, were taken to determine differences between normal diet mice, HFD mice, and HFD mice post-A. muciniphila administration. After receiving A. muciniphila treatment, HFD mice saw significant improvements in hippocampal-related functions.

MAJOR RESULTS In this experiment1, the mice that were used were 3 weeks of age (all male) and were kept and cared for under the same conditions. The diet that was assigned (either normal or HFD) was fed to the mice for 6 weeks before testing was done. Additionally, there was a separate group of mice which were given antibiotic treatments (Abx mice) to get rid of the gut microbiome; this group was then further subdivided into two and were given transplants of the gut microbiome (FTM mice) from either the normalfed mice group or the HFD-fed mice group. In all experiments, the FTM mice showed similar results to their non-FTM counterparts. Two tests were done on the mice to analyze their memory and learning capabilities: fear conditioning and the Barnes circular maze test. For the first test, scientists conditioned the mice to associate a tone with a light shock while in a specific environment (i.e. a box). Freezing was measured both in response to being placed in the same box again and in response to hearing the same tone in a novel box. HFD mice showed significantly less freezing in the original box, but no significant difference in response when the tone was played in the new box (Figure 1). For the second test, mice were placed in a circular maze and escape times as well as time spent near escape route were measured (note that the maze had visual cues which the mice could use to determine their orientation in relation to the escape route). Once more, the HFD mice were impaired in this task, making more mistakes and spending significantly less time near the escape route in comparison to normal-diet mice (Figure 2). Overall, the normal mice showed better results (i.e. results that demonstrated better hippocampal-related capabilities) in comparison to HFD mice.1

Figure Adapted from: Yang Y, Zhong Z, Wang B, Xia X, Yao W, Huang L, Wang Y, Ding W. Early-life high-fat diet-induced obesity programs hippocampal development and cognitive functions via regulation of gut commensal Akkermansia muciniphila. Neuropsychopharmacology. 2019;(June). http:// dx.doi.org/10.1038/s41386-019-0437-1. doi:10.1038/s41386-0190437-1) Figure 1. Results from the fear conditioning test, demonstrating that HFD mice showed less freezing when placed in the original box (A) but no notable difference between groups when the conditioned tone was played in a novel box (B). (Note that insignificant differences are annotated using n.s., while asterisks (*) indicate significance level, with more asterisks meaning more significance).1

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pal-related defects, specifically in learning and memory. Treatment with A. muciniphila in HFD mice was able to alleviate hippocampal-related afflictions. Connections were made to the activation of microglial cells, specifically related to TLR4 receptors, which are known to bind lipopolysaccharides, resulting in immunogenicpathway activation and subsequently inflammation. This finding is supported by previous literature, which have observed similar impairments in HFD mice models.6,10 Earlier work found increased inflammation in HFD mice, but did not make any specific/direct connections to a certain cell type10. Figure Adapted from: Yang Y, Zhong Z, Wang B, Xia X, Yao W, Huang L, Wang Y, Ding W. Early-life high-fat diet-induced obesity programs hippocampal development and cognitive functions via regulation of gut commensal Akkermansia muciniphila. Neuropsychopharmacology. 2019;(June). http:// dx.doi.org/10.1038/s41386-019-0437-1. doi:10.1038/s41386-0190437-1)

This study1 also built further upon the connection between TLR4 and HFD, along with the connection to A. muciniphila. By using antagonists against TLR4, the authors1 were able to show the link between immunogenic responses and hippocampal impairments. The relationship between TLR4 and inflammation has been demonstrated previously as well, with immunogenic responses Figure 2. The layout of the Barnes maze test (A), along with the qualitative being 12causally linked to neurogenerative diseases like Alzheimeasurement of how long the mice spent in the various areas of the maze (B), mer’s . with significant differences being observed in the number of errors made (C), the amount of time it took the mice to find the escape hole (D), and the The results of this study1 are important as the authors not amount of time they spent near the escape (E). No significant difference was only identified A. muciniphila as a possible method of treatment in observed in speed between groups (F). (Note that insignificant differences are diseases where neuroinflammation is prevalent (such as Alzheiannotated using n.s., while asterisks (*) indicate significance level, with more mer’s disease6), but also the possibility of targeting TLR4 as well. asterisks meaning more significance).1

16S rRNA ribosomal sequencing was used to analyze the gut microbiomes of the two groups, with the following significant changes in HFD mice: a decrease in Bacteroidetes, an increase in Firmicutes, a decrease in Verrucomicrobia and an increase in Proteobacteria. When investigating further into specific populations, significant decreases in Akkermansia muciniphila and Lactobacillus reuteri were observed.1 HFD mice were administered A. muciniphila in order to see the associated effects. When these mice performed the fear conditioning and Barnes circular maze test, results were more similar to normal-diet counterparts. Additionally, others were given Lactobacillus reuteri or heat-killed A. muciniphila in order to confirm that the improvement in symptoms was attributed only to A. muciniphila. To test for placebo effect, some HFD mice were administered only PBS instead of the mixture of A. muciniphila and PBS. The improvements seen with A. muciniphila were significantly better than those seen in the placebo group. Furthermore, both quantitative and qualitative attributes of hippocampal neurons (e.g. action potential amplitudes, growth, size, etc.) in HFD mice that received A. muciniphila treatment were more similar to normal diet mice than to HFD mice who did not receive treatment).1 Toll-like-receptor 4 (TLR4), which is part of the pathway responsible for immunogenic responses (and subsequently inflammation), is known to be affected by levels of lipopolysaccharides as they are TLR4 ligands12. In this study1, treatment with TLR4 antagonists were given in HFD mice and resulted in significant improvements in fear conditioning and the Barnes circular maze test; the normal diet mice demonstrated no changes in response to TLR4 antagonists. Wiping out the HFD mice’s gut microbiome using antibiotics also resulted in improvements.1

CONCLUSIONS/DISCUSSION

The researchers showed the association of TLR4 and HFD as normal diet mice did not show any effects in response to being administered TLR4 antagonists, while the HFD mice did1. This supported their hypothesis of a connection between the gut and the brain, which has also been explored in previous work7.

CRITICAL ANALYSIS One surprising result that was observed in this study 1 was that despite the fact that A. muciniphila administration did help the HFD mice regain hippocampal-related capabilities and approach levels comparable to the normal-chow diet mice, no significant change to the gut microbiome was observed. This idea was not discussed in more detail. If there was no significant change to the gut microbiome, it’s hard to determine whether or not the administration of A. muciniphila will help in the long term. Although tests were conducted for 7 weeks1, it’s difficult to say whether or not these effects will continue on afterwards. In terms of the length of the A. muciniphila administration, the mice received it twice during 24hour cycles over 4 weeks. The mice should be observed for an extended period of time after the treatment has been stopped. If the effects of A. muciniphila are no longer exhibited once treatment has stopped, then this would suggest that permanent changes in the gut microbiome are needed for long-term alleviation of HFD-related symptoms. If the HFD manifestations are no longer present even after treatment has stopped, despite the fact that no changes in their gut microbiome were observed1, then this would suggest that A. muciniphila made permanent alterations elsewhere in the mice that don’t need to be maintained by A. muciniphila bacterial populations. Overall, the paper1 was supported by previous research6,10 and provided interesting insights into the role of A. muciniphila and the effects of HFD.

The major finding of this study1 was that high fat diets FUTURE DIRECTIONS change the gut microbiome, with the most significant change being a decrease in A. muciniphila. This change results in hippocam-

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Since the researchers in the study did not observe any changes in the gut microbiome populations, despite seeing improvements when testing HFD mice who received A. muciniphila1, it would be interesting to explore this idea further, as mentioned previously. Another study13, which looked at ALS mice models, found that an increase in A. muciniphila was also associated with an increase in nicotinamide. Nicotinamide is often used in acne medication as it has been found to reduce redness, which is an inflammatory response14. More research should be done to see if changes in nicotinamide levels is the sole cause of improvement, especially considering that no significant changes of the gut microbiome were observed despite HFD miceâ&#x20AC;&#x2122;s recovery.1 An experiment could be conducted that combines both the methods of the main article that was discussed 1 as well as the ALS mice model study13. HFD mice models should be separated into two groups, in which they either receive A. muciniphila treatment or only nicotinamide. Then, inflammatory responses measurements as well as hippocampus function-related tests should be performed. Comparisons should be made between the two groups to see if there are significant differences in their performance as well as any changes observed in hippocampal neurons. It is possible that A. muciniphila plays other roles that are important to maintaining/improving brain health, so it may prove to be the better treatment option overall. Differences in improvements observed between HFD model mice given nicotinamide versus A. muciniphila should be investigated and more research should be done in both of the aforementioned treatmentsâ&#x20AC;&#x2122; roles in brain health.

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Ng M, Fleming T, Robinson M, Thomson B, Graetz N, Margono C, Mullany EC, Biryukov S, Abbafati C, Abera SF, et al. Global, regional, and national prevalence of overweight and obesity in children and adults during 1980-2013: A systematic analysis for the Global Burden of Disease Study 2013. The Lancet. 2014;384(9945):766–781. doi:10.1016/S0140-6736(14)60460-82.

Ley RE, Bäckhed F, Turnbaugh P, Lozupone CA, Knight RD, Gordon JI. Obesity alters gut microbial ecology. Proceedings of the National Academy of Sciences of the United States of America. 2005;102(31):11070 LP – 11075. http://www.pnas.org/ content/102/31/11070.abstract. doi:10.1073/pnas.05049781023.

Ley RE, Turnbaugh PJ, Klein S, Gordon JI. Human gut microbes associated with obesity. Nature. 2006;444(7122):1022–1023. https://doi.org/10.1038/4441022a. doi:10.1038/4441022a

Winter G, Hart RA, Charlesworth RPG, Sharpley CF. Gut microbiome and depression: What we know and what we need to know. Reviews in the Neurosciences. 2018;29(6):629–643. doi:10.1515/revneuro-2017-0072

Kothari V, Luo Y, Tornabene T, O’Neill AM, Greene MW, Geetha T, Babu JR. High fat diet induces brain insulin resistance and cognitive impairment in mice. Biochimica et Biophysica Acta - Molecular Basis of Disease. 2017;1863(2):499–508. http:// dx.doi.org/10.1016/j.bbadis.2016.10.006. doi:10.1016/j.bbadis.2016.10.006

Rietdijk CD, Perez-Pardo P, Garssen J, van Wezel RJA, Kraneveld AD. Exploring Braak’s Hypothesis of Parkinson’s Disease. Frontiers in neurology. 2017;8:37. https://www.ncbi.nlm.nih.gov/pubmed/28243222. doi:10.3389/fneur.2017.00037

Png CW, Lindén SK, Gilshenan KS, Zoetendal EG, McSweeney CS, Sly LI, McGuckin MA, Florin THJ. Mucolytic bacteria with increased prevalence in IBD mucosa augment in vitro utilization of mucin by other bacteria. American Journal of Gastroenterology. 2010;105 (11):2420–2428. doi:10.1038/ajg.2010.281

Everard A, Belzer C, Geurts L, Ouwerkerk JP, Druart C, Bindels LB, Guiot Y, Derrien M, Muccioli GG, Delzenne NM, et al. Cross-talk between Akkermansia muciniphila and intestinal epithelium controls diet-induced obesity. Proceedings of the National Academy of Sciences. 2013;110(22):9066 LP – 9071. http://www.pnas.org/content/110/22/9066.abstract. doi:10.1073/pnas.1219451110

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Chesnokova V, Pechnick RN, Wawrowsky K. Chronic peripheral inflammation, hippocampal neurogenesis, and behavior. Brain, Behavior, and Immunity. 2016;58:1–8. http://dx.doi.org/10.1016/j.bbi.2016.01.017. doi:10.1016/j.bbi.2016.01.017

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Blacher E, Bashiardes S, Shapiro H, Rothschild D, Mor U, Dori-Bachash M, Kleimeyer C, Moresi C, Harnik Y, Zur M, et al. Potential roles of gut microbiome and metabolites in modulating ALS in mice. Nature. 2019;572(7770):474–480. https://doi.org/10.1038/ s41586-019-1443-5. doi:10.1038/s41586-019-1443-5

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Exploring metformin potential as treatment for stroke recovery in different ages and sexes by examining neuronal progenitor cell pool expansion and cognitive recovery Anais Lupu

Interruption of blood flow when strokes occur can result in neuronal loss without immediate intervention. These neuronal losses may affect motor and cognitive function of the individuals affected. One possible target aimed to aid stroke recovery involves the replacement of damaged neurons by the recruitment and differentiation of stem cells. Metformin (met), a common diabetic treatment with relatively small side effects, has been shown to reduce the incidence of ischemic stroke in diabetic patients taking the medication. Although the mechanism behind met is not well understood, one of the pathways suggested involves the activation of the neural precursor cells (NPCs) and expansion of neural stem cell (NSC) pool to replace the damaged brain cells. The ability of met to aid motor recovery after stroke in mice models has been shown in literature, however, cognitive recovery has not been looked at. The research study conducted by Ruddy et. al (2019) showed cognitive recovery in neonatal stroke mice on met by using puzzle box task. Furthermore, they looked at the age- and sexdifferences as a factor which affects the performance of met in stroke recovery. The main results of their research suggested that the role of met in NPC expansion is inhibited by the presence of testosterone, and the lack of estradiol. These results demonstrated the importance of taking both sexes, and age, into consideration when conducting research trials. Key words: metformin (met), mice model, neonatal stroke, stroke recovery, NPC expansion, age effect, sex hormones effect, testosterone, estradiol

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INTRODUCTION Metformin (met) is a commonly used drug to improve insulin sensitivity and cardiovascular health of those living with type 2 diabetes, with minimal side effects. Diabetic patients are at a higher risk for stroke incidence (Jia et al., 2015), and several epidemiological studies observed lower severity and better stroke outcome in diabetic patients when they are on met (Mima et al., 2016; Turner et al., 1998; Cheng et al., 2014). This prompted a closer look at the mechanism by which met may have contributed to better stroke recovery seen in diabetic patients (Abdelsaid et al., 2014), and whether this also applied to the nondiabetic population (Venna et al., 2014; Abbasi et al., 2018). Met can cross the blood-brain barrier (Lv et al., 2012), and its pleiotropic effect works on multiple molecular pathways to give its neuroprotective property post-stroke (Mima et al., 2016; Jia et al., 2015; Li et al., 2010; Dadwal et al., 2015; Arbeláez-Quintero et al., 2017). In both diabetic (Abdelsaid et al., 2014) and nondiabetic (Venna et al., 2014) mice models, the restoration of blood flow via enhanced angiogenesis was seen with post-stroke administration of met. This angiogenesis was the consequence of increased vascular endothelial growth factor (VEGF) expression due to chronic adenosine monophosphate-activated protein kinase (AMPK) activation (Venna et al., 2014; Jia et al., 2015; Jin et al., 2014). In addition, this enhanced AMPK activation also skewed the microglial ratio towards the M2 phenotype which reduces inflammation and enhances postinjury tissue repair, therefore possibly reduces brain atrophy after experimental stroke (Jin et al., 2014; Li & Mccullough, 2009; Fang et al., 2017). Evidence of reduced infarct damage can be seen in mice models given chronic met administration (Jia et al., 2015). Another possible pathway that met acts upon is neurogenesis (Liu et al., 2014; Dadwal et al., 2014; Fatt et al., 2015). Typically, after injury, there is an increase of NSC differentiation into new neurons and oligodendrocytes, however, this process is very limited (Ikeda et al., 2015). Dadwal et al. (2015) demonstrated the enhancement in endogenous neural precursor cells (NPCs) activation, expansion, and cells migration to injury site, following met application in a neonatal mice stroke model or mice with perinatal hypoxia ischemia (H-I) insult, a model of childhood brain injury. In addition, met has also been shown to drive neural stem cells (NSCs) towards differentiation (Dadwal et al., 2014; Ould-Brahim et al., 2018; Fatt et al., 2015). Furthermore, Dadwal et al. (2014) showed an improvement in sensory-motor function following met application in neonatal mice. The potential neurogenerative ability marked met as a promising therapeutic approach to aid stroke recovery. Nonetheless, the cognitive recovery post-stroke is yet to be demonstrated. Moreover, met has been shown in the instance of spinal cord injury (Gilbert & Morshead, 2018), and colorectal cancer (Park et al., 2017) that there are differences in outcome between sexes, where females typically show better recovery. Ruddy et al. (2019) set out to explore the sex- and agedependence effect of met after stroke, specifically on the cognitive outcome. They used the C57BL/6 neonatal stroke mice model and looked at the number of neurospheres, where the increase in neurospheres indicates NSC expansion, and at puzzle box task scores, and found that NPC expansion along with met-induced cognitive recovery depend upon the presence of sex hormones. MAJOR RESULTS

The paper by Ruddy et al. (2019) looked at the number of neurospheres taken from mice of different ages and sexes, before and after receiving the met treatment, in order to determine NPC expansion. They found that met injection does enhance NPC activation and NSC expansion, but only in neonatal mice (P8 to P12), and in adult (over 6 weeks of age) female mice (Figure 1).

Figure 1. Bar chart illustrating the fold change in NSC pool before and after treatment between vehicle and met group. (A) shows female mice. (B) shows male mice. Neonates were given 20 mg/kg of met (or vehicle in the control) via subcutaneous injection daily from P9 to P12. Juvenile and adult mice were given 200 mg/kg of met (or vehicle for control) via intraperitoneal injection daily (from P21 to P27 for juvenile group; P50 to P56 for adult mice group). Figure Adapted from Ruddy et al. (2019). Science Advances. 5(9), 1–10.

NPC expansion seen in neonates in Ruddy et al. (2019) was also seen in Dadwal et al. (2014) which used a similar experimental design. In both Dadwal et al. (2014) and Ruddy et al. (2019), H-I C57BL/6 neonates were created by ligating the left common carotid artery at P8, followed by 1 hour of hypoxia. In both experiment, neonates were given subcutaneous injection of 20 mg/kg of met. Dadwal et al. (2014) took it a step further to demonstrate that the increased NPC activation does in fact reflects in the number of cells differentiated into new oligodendrocytes and neurons (Figure 2).

Figure 2. Bar chart showing the linage the stem cell differentiated into. (A) O4 marker was used to identify newly formed oligodendrocytes from the expanded neurosphere population. (B). beta III marker was used to mark new neurons formed from the expanded neurosphere population. Compared with the control group, the met group showed several fold increase in number of oligodendrocytes and neurons formed. Figure Adapted from Dadwal et al. (2014). Stem Cell Reports. 5(2), 166–173.

NPC expansion enhancement is associated with better cognitive recovery In adult mice, only H-I female with long-term met treatment solved the eighth puzzle box task significantly faster, which reflects their ability to acquire skills used to solve novel tasks faster than the H-I controls which were given vehicle and solved the task slower (Ruddy et al., 2019) (Figure 3). In fact, the performance of female H-I mice given met was almost at the same level as for unlesioned controls. However, it should be noted that in both vehicle

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control groups, female H-I mice scored slightly better diol. In addition, injection of testosterone in neonates of both sexes resulted in the loss of NSC expansion (Ruddy et al., 2019). than male H-I mice. Ruddy et al. (2019) tagged neurospheres from each sex using yellow-fluorescence-protein tags, and conducted a set of in vitro experiments to establish that neurospheres of male and female mice, once removed from their niche, will both respond similarly to only testosterone or estradiol. The presence of the sex hormone alone (testosterone or estradiol) does not result in NPC expansion. Furthermore, the adult male neurospheres, when placed into a female environment, can expand equally as the female neurospheres. On the other hand, the female neurospheres, when placed into a male environment, will not show the same NPC expansion as they do in the female environment. The sex differences in met performance fits in with Gilbert & Morshead (2019) who found that only in neurosphere assay taken from the spinal cord of adult females, and not males, was NSC expansion observed when met was given.

CONCLUSIONS/DISCUSSION In addition to reproducing the results that were in line with the literature body such as met’s treatment ability to expand the NPC in H-I neonate models (Dadwal et al., 2014). And that the sex differences in met mediated NPC expansion that has been reported in cases of spinal cord injury (Gilbert & Morshead, 2019), was also seen in NSC from the brain (Ruddy et al. 2019). Ruddy et al. (2019) added to the current literature by highlighting the role of sex hormones in the ability of met to mediate NPC expansion.

Figure 3. Plots represent the time it took for mice to complete the puzzle box task which involves the removal of cardboard plug to access the goal boxes, 48 days post-H-I injury, and 2 weeks post met injection. The number on the x-axis denotes the number of boxes the mice were able to solve the novel puzzle task inside. H-I lesion increased latency in the time it took to complete the eighth task when compared to naïve uninjured mice. Five weeks of met injection led to higher pass rate in Specifically, Ruddy et al. (2019) found that the NPC exfemales on the eighth box, but not in males, when compared to vehicle control. pansion seen in met treatment is dependent upon the presence of

female hormone estradiol, and the absence of male hormone tesFigure Adapted from Ruddy et al. (2019). Science Advances. 5(9), 1– tosterone. They also showed that this NPC expansion observed in 10. female mice corresponds to better cognitive recovery after H-I

Met’s NPC expansion is dependent upon the presence of estradiol, and absence of testosterone Figure 4 shows that in a loss-of-function test, adult female mice which underwent ovariectomy to remove circulating estradiol resulted in losing the NPC expansion observed in wild type adult female mice (Ruddy et al., 2019). On the other hand, castration in adult male mice which removed circulating testosterone resulted in the gain of NPC expansion which was only observed in intact adult female mice.

stroke when compared to male mice (Ruddy et al., 2019). The presence of sex hormones differ at each life stage in mice (Ruddy et al., 2019). In neonates, there is a period of time where estradiol peaks. In juveniles, the circulating sex hormones are quite low; this changes once rats undergo puberty when sex hormones peaks once again. Adult male and female rats have the same level of estradiol, however, they mostly diverge in terms of testosterone level: in males, there is a high level of testosterone circulating in the system, as opposed to low testosterone levels in females. Therefore, the varying NPC observed in different ages and sexed corresponded to the varying levels of sex hormones. The identification of testosterone and estradiol as factors that affects met NPC expansion ability is a novel finding (Ruddy et al., 2019). This is a signal to revisit past experiments, and for future experiments to consider the age and sex of the mice used.

CRITICAL ANALYSIS Unlike Dadwal et al. (2014) (Figure 2), Ruddy et al. (2019) Figure 4. Illustration showing the loss of function test in adult male did not confirm whether the newly expanded NSC pool seen in and female mice. The mice were given daily injection of 200 mg/kh of different ages and sexes translated into the differentiation of new met for 1 week from P50 to P57. The presence of testosterone in male mice, and the loss of estradiol in female mice, resulted in no NPC ex- oligodendrocytes and new neurons. pansion, whereas the presence of estradiol in female mice, and the loss Furthermore, they did not offer a pathway explanation of testosterone in male mice resulted in NPC expansion.

which ties in sex hormones with varying met-induced NPC expansion shown in the result. The authors hypothesized that sex hormones may affect the expression of factors and other proteins When exploring juvenile mice (P17 to P28), by giving implicated in the met response such as transporter in which met female juvenile mice estradiol, NSC expansion can be seen (Ruddy et al., 2019). However, no such NSC expansion could be uses to enter the cell, or the downstream pathways activated by seen in the group of male juvenile mice which were given estra- met. Figure Adapted from Lupu (2019).

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Although, the body of literatures has shown that met was able to work in environments where Ruddy et al., (2019) showed that enhanced NPC expansion did not occur (Venna et al., 2014; Jin et al., 2014). In focal cerebral ischemia model induced by transient middle cerebral artery occlusion (MCAO), male mice in the age which Ruddy et al. (2019) observed no NPC expansion when treated with met, showed motor function recovery (Venna et al., 2014; Jin et al., 2014). Venna et al. (2014) used C57BL/6N male mice each weighed about 20-25 g, which puts the mice at the age approximately around late-juvenile to adult age by Ruddy et al. (2019) definition (“C57BL/6 Mouse Model Information Sheet”, 2011). Venna et al. (2014) was able to show that met contributed to stroke recovery measured by improved motor and coordination scores in tasks such as Adhesive-tape removal, and Apomorphineinduced rotational activity when compared to vehicle controls. Another similar experiment was done by Jin et al. (2014), but they did use CD-1 male mice instead of C57BL/6N. The mice used were around 25-30 g, which falls within the juvenile age (“CD-1 IGS Mouse Model Information Sheet”, 2011). These male mice given met injections were able to show improvement on motor and coordination test such as in the Corner and Rotarod test, when compared to vehicle controls. Both Venna et al. (2014) and Jin et al. (2014) did not look at NPC expansion, nor did they measure sex hormone levels; they looked at the increase in VEGF being transcribed, and the new blood vessels being formed with the application of met. In cases with transient occlusion such as in these two papers, the restoration of blood flow via angiogenesis was enough to recover motor function. However, the main take away from Venna et al. (2014) and Jin et al. (2014) was that they showed that met was able to activate AMPK in order to increase the level of VEGF, despite having the presence of testosterone, and insufficient estradiol level. Therefore, the focus should be on the met-activated downstream pathway. Fatt et al. (2015) suggested a linkage between met-activated AMPK pathway, and NPC expansion. Fatt et al. (2015) and Wang et al. (2012) showed that met enhanced NPC proliferation, self-renewal, and neural differentiation processes by increasing the transcription factor TAp73, and atypical protein kinase C-mediated Ser436 phosphorylation of CBP (aPKC-CBP). Specifically, met acts on the AMPK-aPKC-CBP pathway to promote neuronal differentiation (Wang et al., 2012; Fatt et al., 2012), while TAp73 promotes self-renewal or proliferation. Moreover, Ruddy et al. (2019) concluded that sexdependent effects of met on NPC are correlated with functional recovery after stroke based on their study of H-I lesion and the degree of cognitive performance in adult mice treated with and without met. However, this experiment was quite limited as they only included H-I adult mice model with intact gonads, therefore, only correlational relationship can be assumed about the sex hormones presence, and cognitive recovery after met treatment.

FUTURE DIRECTIONS

pathways in which met is involved is responsible for this expansion. Moreover, building on Fatt et al. (2015) who suggested that met increases TAp73 and activates aPKC-CBP pathways to promote neural precursor proliferation and differentiation, the amount of TAp73 should be compared using immunohistochemistry in the presence and absence of testosterone and estradiol. If the presence of testosterone drives either the decrease of TAp73 level, and/or the decrease of phosphorylated CBP, this would link testosterone with at least one of the molecules. Similar experiments should be done using estradiol; if estradiol is involved in either TAp73 and/or aPKC-CBP pathways, there should be an increase of these molecules. In addition, another thing to consider is the amount of sex hormones needed to affect these pathways, so varying concentrations of testosterone and estradiol should be tested. Lastly, to ensure that the cognitive functional recovery seen in the H-I model is from the met-induced NPC expansion, which is effected by estradiol and testosterone presence, future experiments should look at cognitive recovery in adult female mice that underwent ovariectomy, and adult male mice that underwent castration. In addition, immunohistochemistry on the newborn cells should be visualized to confirm that the stem cells in the expanded NPC have differentiated into new neurons and oligodendrocytes over time. If the NPC are not differentiating, there might be other factors which are contributing to the cognitive recovery seen, for example, increased angiogenesis. Labeling of the new blood vessels formation could be done using the co-labeling of cluster of differentiation 31 (CD31) with fluorescein isothiocynateconjugated lectin. The difference between the puzzle box task performance in adult female mice with sham surgery, and in adult female mice with ovariectomy, is the circulating estradiol. If mice without circulating estradiol showed worse cognitive performace than the sham control with circulating estradiol, this will support the conclusion of Ruddy et al. (2019) about the permissive effect of estradiol on the ability of met to aid cognitive recovery. However, if female mice without circulating estradiol showed better, or similar recovery as the sham, this would not support the role of estradiol in cognitive recovery, and other pathways must be considered. A similar experiment design must be used with male mice and circulating testosterone. To support the conclusion of Ruddy et al. (2019) about the inhibitory effect of testosterone, testosterone absent in castrated males must be shown to boost their cognitive performance when treated with met. However, if castrated males did not perform better, or they performed as poorly as the sham, this would mean that testosterone did not contribute to cognitive recovery seen in the initial experiment. If the end result deviated from the expected hypothesis, this would suggest that there are other differences between adult male and female mice that would result in different cognitive recovery.

Future experiments may look at AMPK α-2 knock-out mice to determine whether NPC expansion will still be seen in adult female knock-out mice treated with met or not. AMPK α-2 knock-out will prevent AMPK activity (Venna et al., 2014). If knockout mice did not show NPC expansion, this will suggest that metinduced NPC expansion are mediated by AMPK signaling. However, if NPC expansion still occurs, this suggests the presence of other

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Metformin Acts on Two Different Molecular Pathways to Enhance Adult Neural Precursor Proliferation/Self-Renewal and Differentiation. Stem Cell Reports, 5(6), 988–995. doi: 10.1016/j.stemcr.2015.10.014 Gilbert, E. A. B., & Morshead, C. M. (2019). Metformin Activates Neural Stem and Progenitor Cells in the Spinal Cord and Improves Functional Outcomes Following Injury. FASEB, 33(1). Ikeda, T., Iwai, M., Hayashi, T., Nagano, I., Shogi, M., Ikenoue, T., & Abe, K. (2005). Limited differentiation to neurons and astroglia from neural stem cells in the cortex and striatum after ischemia/hypoxia in the neonatal rat brain. American Journal of Obstetrics and Gynecology, 193(3), 849–856. doi: 10.1016/j.ajog.2005.01.029 Jia, J., Cheng, J., Ni, J., & Zhen, X. (2015). Neuropharmacological Actions of Metformin in Stroke. Current Neuropharmacology, 13(3), 389–394. doi: 10.2174/1570159x13666150205143555 Jin, Q., Cheng, J., Liu, Y., Wu, J., Wang, X., Wei, S., … Zhen, X. (2014). Improvement of functional recovery by chronic metformin treatment is associated with enhanced alternative activation of microglia/macrophages and increased angiogenesis and neurogenesis following experimental stroke. Brain, Behavior, and Immunity, 40, 131–142. doi: 10.1016/j.bbi.2014.03.003 Li, J., & Mccullough, L. D. (2009). Effects of AMP-Activated Protein Kinase in Cerebral Ischemia. Journal of Cerebral Blood Flow & Metabolism, 30(3), 480–492. doi: 10.1038/jcbfm.2009.255 Li, J., Benashski, S. E., Venna, V. R., & Mccullough, L. D. (2010). Effects of Metformin in Experimental Stroke. Stroke, 41(11), 2645–2652. doi: 10.1161/strokeaha.110.589697 Lv, W.-S., Wen, J.-P., Li, L., Sun, R.-X., Wang, J., Xian, Y.-X., … Gao, Y.-Y. (2012). The effect of metformin on food intake and its potential role in hypothalamic regulation in obese diabetic rats. Brain Research, 1444, 11–19. doi: 10.1016/ j.brainres.2012.01.028 Mima, Y., Kuwashiro, T., Yasaka, M., Tsurusaki, Y., Nakamura, A., Wakugawa, Y., & Okada, Y. (2016). Impact of Metformin on the Severity and Outcomes of Acute Ischemic Stroke in Patients with Type 2 Diabetes Mellitus. Journal of Stroke and Cerebrovascular Diseases, 25(2), 436–446. doi: 10.1016/j.jstrokecerebrovasdis.2015.10.016 Ould-Brahim, F., Sarma, S. N., Syal, C., Lu, K. J., Seegobin, M., Carter, A., … Wang, J. (2018). Metformin Preconditioning of Human Induced Pluripotent Stem Cell-Derived Neural Stem Cells Promotes Their Engraftment and Improves Post-Stroke Regeneration and Recovery. Stem Cells and Development, 27(16), 1085–1096. doi: 10.1089/scd.2018.0055 Park, J. W., Lee, J. H., Park, Y. H., Park, S. J., Cheon, J. H., Kim, W. H., & Kim, T. I. (2017). Sex-dependent difference in the effect of metformin on colorectal cancer-specific mortality of diabetic colorectal cancer patients. World Journal of Gastroenterology, 23(28), 5196. doi: 10.3748/wjg.v23.i28.519 Venna, V. R., Li, J., Hammond, M. D., Mancini, N. S., & Mccullough, L. D. (2014). Chronic metformin treatment improves poststroke angiogenesis and recovery after experimental stroke. European Journal of Neuroscience, 39(12), 2129–2138. doi: 10.1111/ejn.12556 Wang, J., Gallagher, D., Devito, L. M., Cancino, G. I., Tsui, D., He, L., … Miller, F. D. (2012). Metformin Activates an Atypical PKC-CBP Pathway to Promote Neurogenesis and Enhance Spatial Memory Formation. Cell Stem Cell, 11(1), 23–35. doi: 10.1016/j.stem.2012.03.016

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Review article: Potential contribution of gut dysbiosis to Alzheimerâ&#x20AC;&#x2122;s Disease through the gut-brain axis Wing Yau Kimberly Mak

Alzheimerâ&#x20AC;&#x2122;s disease (AD) is a neurodegenerative disease in which AD symptoms will progressively develop into dementia-related conditions. A major hallmark of the AD brain is the deposition of betaamyloid (A ) which forms amyloid plaque, resulting in neuronal death and cortical atrophy. Despite advances in scientific research, unclear AD etiology continues to be one of the major healthcare concerns in an aging population. However, it is known that the complex interaction between genetics and the environment contributes to AD pathogenesis. Gut microbes can be modified by environmental factors such as diet and lifestyle. The latest research suggested that alterations in the gut microbiome are associated with AD progression. Hence, the presence of a gut-brain axis might allow bidirectional communication between the enteric nervous system and the brain. In their study, Vogt et al. (2017) found that the composition of gut microbiota was significantly different between AD patients and normal healthy individuals through 16s ribosomal RNA (rRNA) sequencing. The primary findings of this study revealed that AD participants have reduced microbial diversity and altered bacterial abundance compared to healthy gut microbiota. Moreover, the gut microbiota of AD individuals displayed a decreased abundance of Firmicutes and Actinobacteria while Bacteroidetes increased in abundance. Vogt et al. (2017) and other studies have supported the idea that gut dysbiosis is associated with AD pathogenesis. This evidence provides a new direction for future research to investigate neuropathological conditions through the lens of the gut-brain axis. Key words: Alzheimerâ&#x20AC;&#x2122;s Disease, gut microbiome, gut-brain axis, neurodegeneration, inflammation, microbial diversity, bacterial abundance

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BACKGROUND or INTRODUCTION.

tion of microglia, impairing its ability to phagocytose fibrillar A (Lin, Zheng, & Zhang, 2017; Lue, Kuo, Beach, & Walker, Alzheimer’s disease (AD) is a condition where the 2010). Hence, unresolved neuroinflammation will sustain the brain undergoes progressive degeneration, and the risk of developing AD increases with age (Hill et al., 2014). AD brain release of proinflammatory cytokines such as interleukin-1 (IL is characterized by the deposition of beta-amyloid (A ) which -1) and tumor necrosis factor-alpha (TNF-a) which can injure blood-brain barrier (BBB) (Lin, Zheng, & Zhang, 2017; Hardy forms amyloid plaque, resulting in neuronal apoptosis and & Selcoe, 2002; Lue, Kuo, Beach, & Walker, 2010). Similar procortical atrophy (Bronzuoli, Iacomino, Steardo, & Scuderi, inflammatory response is observed in people who experience 2016). One of the biggest mysteries and a controversial topic dysbiosis due to antibiotic treatment, enteric infection and in the field of neuroscience is to discover the underlying mechanism of AD, thereby enabling a better understanding of exposure to a novel environment (David et al. 2014). neurodegenerative disease to improve diagnostic tools and Firmicutes and Bacteroidetes are the dominant phyla in establish prevention protocols (Folch et al., 2015). Currently, gut microbiota (Sochocka et al., 2018; Hill et al., 2014). Gut the scientific community does not have a consensus view on a microbiome in healthy individuals is relatively stable particular agent or pathway causing AD, and whether it origi- throughout a lifetime, and these microorganisms mediate the nates from an infectious source or it is merely a form of eninteraction with their human host and other environmental dogenous dysregulation of brain homeostasis (Hill et al., 2014; factors (Sochocka et al., 2018). Dysbiosis, characterized by disSochocka, Zwolińska, & Leszek, 2017). However, it is known turbance to normal microbial distribution, could be a contribthat only 5% of AD diagnosis is solely due to genetics, while uting element to AD pathogenesis by inducing neuroinflammore studies are showing that AD risk is influenced by many mation and microglia activation (Sochocka et al., 2018; Heneenvironmental aspects and epigenetics (Hill et al., 2014; Xu & ka et al., 2015). David et al. (2014) identified acute and extenWang, 2016). sive microbiota changes after individuals were exposed to It was predicted that the prevalence of AD will continue to rise as the aging population grows exponentially (Lin, Zheng, & Zhang, 2017). If AD persists as an epidemic, more weight is added to the public health system (Lin, Zheng, & Zhang, 2017). The presence of effective intervention and diagnostic markers will alleviate the burden on public health, and halt AD progression by implementing prevention early on (Lin, Zheng, & Zhang, 2017; Sochocka et al., 2018). At present, the treatments that are available to AD patients target the amyloid cascade pathway which encourages the clearance of A plaques in the brain. However, currently available medications failed to exert promising results as it is effective against resolving symptoms without understanding the fundamental issue that underlies AD symptoms. Reduced neuroinflammation and A dissolution are only short-lived effects when the initial goal of the drug is not designed to target the root of AD (Bronzuoli, Iacomino, Steardo, & Scuderi, 2016).

unconventional events such as intestinal infection or spontaneous lifestyle changes including diet. Chronic gum infection homes oral pathogens to the mouth, and continuous ingestion of these oral pathogens alters the composition of gut microbiota (Sochocka et al., 2018). Persistent gum infection can cause microbiota imbalance and increased release of proinflammatory cytokines in the gut (Sochocka et al., 2018). Altogether, recurring infection and unresolved inflammation will heighten the host's immune response on a systematic level, which could contribute to neuroinflammation and neurodegeneration indicated in AD (Sochocka et al., 2018). Vogt et al. (2017) have notably demonstrated that AD individuals possess an altered gut microbiome. Bacterial DNA was isolated from fecal samples of healthy and AD participants, and 16s ribosomal RNA (rRNA) gene sequencing was used to analyze the compositional difference of gut microbiota between individuals with and without AD (Vogt et al. 2017). Microbial richness and abundance were quantified by operational taxonomic units (OTU). The major findings revealed that AD participants had a decreased abundance of Firmicutes and Actinobacteria but increased in Bacteroidetes abundance compared to healthy participants (Vogt et al. 2017). More research, including Vogt et al.’s (2017), has started to validate the idea that microbiome alterations are associated with AD pathology. Incorporating the inflammatory hypotheses into this equation can further explain how microbiome regulates the inflammatory response and design new therapeutic approaches that target the inflammatory pathway and reconstruct a healthy microbiome in AD patients (Sochocka et al., 2018).

In the 1990s, the dominant theory adopted by researchers to explain the potential causes of AD was the amyloid cascade hypothesis (Liu, Xie, Meng, & Kang, 2019). Hardy and Higgins (1992) proposed that accumulation of A fibrils is the culprit of pathological symptoms observed in AD such as neurofibrillary tangles, extensive neuron death, and dementia. Due to the mutated amyloid precursor protein (APP) gene, APP is continuously cleaved into A which aggregates into A fibrils (Hardy & Selcoe, 2002). This pathway is central to the amyloid cascade hypothesis (Hardy & Higgins, 1992). However, more studies have rejected the amyloid cascade hypothesis as some individuals do not exhibit cognitive impairment or other pathological markers involved in MAJOR RESULTS AD despite the presence of A plaque. Modern AD research Vogt et al. (2016) examined the microbiota composihas shifted to understand the inflammatory hypothesis which tion using 16s rRNA gene sequencing technique after isolating states that chronic microbial infection leads to constant activa-

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bacterial DNA from the fecal samples of AD and healthy participants. In figure 1 and 2, a reduction of bacterial richness and diversity was presented by the AD gut microbiome in comparison to their sex- and age-matched healthy individuals (Vogt et al., 2017). In Vogt et al.â&#x20AC;&#x2122;s (2016) study, they also reported a compositional difference of the microbial taxa between healthy and AD cohorts, and specifically, Firmicutes and Actinobacteria decreased and Bacteroidetes increased in abundance (Figure 3). Figure 2 indicated an increase of Bacteroides in the Bacteroidetes phyla and a decrease of Actinobacteria in the Bifidobacterium phyla. The differential expression of taxa was coupled with cerebrospinal (CSF) markers to assess the association between microbiota dynamics and AD pathogenesis (Vogt et al., 2017). Altogether, experimental results have supported the presence of the gut-brain axis which is relevant to the inflammatory hypothesis (Vogt et al., 2017). Future therapeutic interventions should aim to modulate the gut microbiome based on the gut-brain hypothesis (Vogt et al., 2017).

differential expression of gut microbes. All changes in AD gut are relative to control gut. OTU above 0 means that family or genus increased abundance in AD gut compare to control, while below 0 means reduced abundance.

Figure retrieved from Vogt et al. (2017). Scientific Reports, 7(13537), 111. Figure 3. AD individuals exhibited different abundance in Firmicutes, Bacteroidetes and Actinobacteria relative to control individuals. Reduction of Firmicutes and Actinobacteria is accompanied with increased Bacteroidetes in AD gut microbiome.

Gut Dysbiosis correlates with inflammatory symptoms in AD Contrasting bacterial composition between healthy and AD cohorts was reported in Vogt et al.â&#x20AC;&#x2122;s (2017) study. Instead of human studies, Harach et al. (2015) approached the problem from another perspective and decided to examine the difference in gut microbiome between healthy control mice and transgenic mice with the APP gene. In accordance with the results from Vogt et al. (2017), the mouse study stated alFigure retrieved from Vogt et al. (2017). Scientific Reports, 7(13537), 1- terations in the gut microbiome of APP-transgenic mice and indicated that 8-months-old transgenic mice exhibited a sig11. nificant decrease in Firmicutes and Actinobacteria content Figure 1. Bacterial diversity is reduced in AD gut microbiota compare to control participants. Faithâ&#x20AC;&#x2122;s phylogenetic diversity measures biodiversi- while Bacteroidetes abundance was elevated (Harach et al. 2015). Moreover, some studies observed declining Firmicutes ty of the gut microbiome in AD and control individuals. abundance in individuals diagnosed with type 2 diabetes (T2D) and obesity (Alam et al. 2014). Both obesity and T2D induces chronic inflammation as a result of altered microbiota, and the inflammatory response observed in T2D could be linked to insulin resistance (Alam et al., 2014; Vogt et al., 2017; Seo & Holtzman 2019). In regards to AD, there is no agreement with what underlies neuroinflammation but David et al. (2014) outlined the impact of host lifestyle influences gut microbiome. Once the gut microbiota is disturbed by abrupt changes to its daily experience, microbiome instability will weaken normal immune response and heighten host sensitivity to the environment, resulting in an inability to clear A plaque and neuroinflammation as observed in AD (David et al. 2014; Lin, Zheng & Zhang 2017). Despite inconsistent directional changes in bacterial composition, obesity, T2D and AD Figure retrieved from Vogt et al. (2017). Scientific Reports, 7(13537), 1- studies all indicated that disease-state microbiota was modified. Gut dysbiosis alone can trigger an inflammatory re11. sponse, and the changes to specific taxa might not be as imFigure 2. Overall lower abundance of gut microbes in AD participants. portant as a global alteration of microbiome dynamics. Hence, Each dot represents an OTU, in which all closely related gut microbes different experiments collectively suggested that gut dysbiosis were clustered into 1 family or 1 genus. Log2 Fold Change depicts the

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coincides with inflammatory diseases such as AD (Vogt et al., 2017; Alam et al. 2014; Seo & Holtzman 2019).

fy the abundance of specific taxa within the gut microbiota (Cattaneo et al., 2017). 16s rRNA gene sequencing, the experimental approach used by Vogt et al. (2017), enables an overall CONCLUSIONS/DISCUSSION comparison of bacterial composition to determine the relationIn this study conducted by Vogt et al. (2017), they concluded that AD individuals experience alterations to their ship between gut microbes and how abundance distortion gut microbiome. In terms of microbiota diversity and richness, plays a role in AD pathology. CRITICAL ANALYSIS both were significantly reduced in the AD gut compared to Although Vogt et al. (2017) reported a significant relahealthy individuals (Vogt et al., 2017). Moreover, AD microbitionship between altered microbial abundance and CSF biota displayed an increase of Bacteroidetes and a decrease of omarkers in AD participants, a similar trend was observed in Firmicutes and Actinobacteria relative to healthy individuals the non-demented control cohort. CSF biomarkers used in this (Vogt et al., 2017). These findings presented by Vogt et al. study measured A deposition, neurofibrillary tangles and (2017) support the presence of the gut-brain axis, in which microglia activation, which are common clinical endpoints in malfunction during the bidirectional communication between AD pathology (Vogt et al., 2017). Yet, the control cohort disthe gut and brain might underlie AD mechanism. played a correlation between CSF biomarkers and bacterial Interestingly, Vogt et al. (2017) concluded that abundance and these healthy individuals did not have any AD increased Bacteroidetes and decreased Bifidobacterium abunprofile (Vogt et al., 2017). The control group should display a dance might trigger a proinflammatory response. Bacteroidedifferent trend from AD participants if appropriate bites is classified as gram-negative bacteria, and the primary omarkers was used for this circumstance. Vogt et al. (2017) constituents of its outer membrane comprise lipopolysacchashould have addressed why bacterial abundance in healthy ride (LPS) (Vogt et al., 2017). LPS evokes an innate immune participants is associated with CSF biomarkers, despite the response to produce proinflammatory cytokines, which is then absence of an AD background. Moreover, AD participants did released into the blood circulation, allowing the body to gennot stop taking AD medication during their participation in erate a systemic response to fight off infection (Vogt et al., Vogt et al’s (2017) study. This becomes a caveat of the study 2017; Mancusoa & Santangelo, 2017; Heneka et al., 2015). Since because the effect of AD medication on the gut microbiome is aging increases permeability of gut epithelia and BBB, LPS can unclear (Vogt et al., 2017). escape from the gut and translocate to the brain. LPS triggers Since Vogt et al. (2017) collected fecal samples from neuroinflammation while activated microglia tries to clear LPS humans, experimental findings are applicable to humans, in the vicinity of the brain (Vogt et al, 2017). Similarly, the rewhich is a major advantage of this study. Previous studies duction of Actinobacteria from the Bifidobacterium phyla used mouse models to study the gut microbiome-brain hycould promote microbial translocation, because Bifidobactepothesis (Harach et al., 2015; Bäuerl, Collado, Cuevas, Viña, & rium possesses anti-inflammatory properties, in turn, lowers Martínez, 2018). But the results cannot be generalized to huLPS count and ensures gut epithelia is impermeable to mimans, simply because humans and mice are utterly different crobes (Arboleya, Watkins, Stanton, & Ross, 2016). A mouse species and observation in mouse model might not be replicatstudy conducted by Romond et al. (2008) suggested that the ed in humans. Hence, findings from Vogt et al.’s (2017) study relationship between Bacteroidetes and Bifidobacterium are were more relevant to humans, regardless of different alteranegatively correlated. While more Bacteroidetes promotes tions to gut microbiome reported by other mouse model studmicrobial translocation, the latter suppresses microbial transies (Harach et al., 2015; Bäuerl, Collado, Cuevas, Viña & Marlocation (Romond et al, 2008). Romond et al. (2008) and Cattatínez, 2018). neo et al. (2017) suggested that tight regulation of the ratio of Bacteroidetes to Bifidobacterium in the gut is critical for the Another human study conducted by Cattaneo et al. suppression of systemic proinflammatory response and reduc- (2017) was able to show results that were in contradiction to tion of leaky intestinal membrane. Changes to microbial ratio Vogt et al.’s (2017) findings. Cattaneo et al. (2017) presented an will release proinflammatory cytokines from the gut into ciropposite trend of bacteria abundance, in which Bacteroidetes culation, and proinflammatory cytokines that reach the brain decreased and Firmicutes increased in AD humans. However, could cause neuroinflammation. Hence, neuroinflammation in Cattaneo et al. (2017) only investigated six bacterial taxa that AD pathology is either induced by the release of proinflamma- were relevant to AD pathology according to other scientific tory cytokines by gut microbes or LPS translocation (Cattaneo literature, and qPCR technique was utilized instead of 16s et al., 2017). rRNA sequencing. Catteneo et al. (2017) did not study the Relating back to Vogt et al’s (2017) paper, the differential abundance of all existing bacterial taxa in the gut interpretation of their findings is supported with the papers because qPCR is designed to study specific taxa. published by Cattaneo et al. (2017) and Romondet al. (2008). Essentially, Vogt et al. (2017) and Catteneo et al. Nevertheless, Vogt et al. (2017) is the first to study broad (2017) concluded gut dysbiosis is associated with increased changes across bacterial taxa between AD and healthy gut AD risk. An interesting step in future experiments is to exusing 16s rRNA gene sequencing. Whereas, previous research plain the oppositional trends of gut microbes distribution in used quantitative polymerase chain reaction (qPCR) to quanti-

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the study by Cattaneo et al. (2017) and Vogt et al. (2017). Vogt et al. (2017) should further investigate the mechanistic aspect of the gut-brain axis because it is unclear whether AD causes gut dysbiosis or gut dysbiosis causes AD, or other modifiable environmental factors cause dysbiosis which results in AD.

mental results reject the proposed pathway of the gut-brain axis, it might indicate that gut microbiome alterations caused by infection do not induce an inflammatory response in the brain, and microglia are activated by a separate, unknown mechanism in AD individuals.

FUTURE DIRECTIONS â&#x20AC;&#x201C; In a prospective experiment, the usage of AD medication should be substituted entirely with selective serotonin reuptake inhibitors (SSRI). As SSRI does not alter gut microbiota, AD participants should take SSRI in place of other AD medications with unknown effects (Vogt et al., 2017). Since AD is a progressive disorder, it takes years or even decades to develop AD symptoms to its full effect. If possible, extending the length of a study will facilitate the analysis of longitudinal data by taking fecal samples every year. Investigators should expect changes in gut microbiota over time, which will correspond to different stages of AD progression. This will allow scientists to understand the implication of microbiota dynamics in the progression of AD. The initial attempt of Vogt et al. (2017) study was to identify the variation of gut profile between AD and healthy non-demented humans. Luckily, results enabled Vogt et al. (2017) to establish a correlational relationship between the gut microbiome and brain in AD pathogenesis, but it did not address the underlying mechanism of the gut-brain axis. In other words, this study successfully suggested the presence of a gut-brain axis but the pathway that enabled bidirectional communication remains uncertain. The inflammatory hypothesis, which describes the relationship between bacterial infection and neuroinflammation, can serve as a starting point of understanding the communication pathway in the gut-brain axis. By introducing bacterial infection to the mouse model, investigators should note how the microbial composition changes upon infection because an altered ratio might imply the roles played by each gut microbes during inflammation. For instance, feeding the mouse with a diet containing pathogens should trigger an immune response in the gut, in turn changing the distribution of microbial abundance and gut profile. Proinflammatoryassociated gut microbes should increase in abundance to release proinflammatory cytokines into blood circulation, thereby informing other body parts to prepare their defense against the pathogen. Eventually, proinflammatory cytokines travel to the brain and activate proinflammatory microglia (M1) in the brain. CD11b and CD68 are common biomarkers used to test M1 activation (Gu et al., 2018). If the proposed mechanism was valid, results should show an increased abundance of proinflammatory-associated gut microbes, accompanied by increased proinflammatory cytokines in blood and the expression of CD11b and CD68 on activated M1 microglia (Gu et al., 2018). Altogether, the aforementioned results contribute to chronic neuroinflammation and neuronal cell loss as seen in the AD brain. If the experi266

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Elucidating the Role of mTORC2 Signalling in Syndromic Autism Spectrum Disorder. Meghan Maulucci

:Biologically-targeted treatments for autism spectrum disorder (ASD) remain one of the biggest unmet needs in pharmacology today. It is known that ASD has a strong genetic component, yet specific molecular mechanisms are poorly-understood. It has been suggested that dysregulation of mammalian target of rapamycin (mTOR) signalling is involved in abnormal neurodevelopment of individuals with ASD. However, the roles of mTORâ&#x20AC;&#x2122;s two main pathways, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), in producing ASD symptoms have not been clarified. In this study, Chen et al. (2019) use a mouse model with genetic deletion of phosphatase and tensin homolog (PTEN), a negative regulator of mTOR signalling, as a model of syndromic ASD with hyperactive mTOR signalling. Then, by genetically knocking out the activity of either mTORC1 or mTORC2, the authors found that hyperactive mTORC1 signalling is responsible for macrocephaly, whereas hyperactive mTORC2 is responsible for many of the other core symptoms of ASD including social deficits. The authors then attempted to develop a therapeutic approach to target the core symptoms of ASD in the Ptendeficient mouse model by inhibiting mTORC2 activity. Using a single-stranded antisense oligonucleotide (ASO) to promote mRNA degradation of one of the key proteins in mTORC2, it was found that a single intracerebrovasular injection was able to rescue core symptoms in the mouse model of ASD. Key words: Syndromic, idiopathic, autism spectrum disorder (ASD), mammalian target of rapamycin (mTOR), phosphatase and tensin homolog (PTEN), signalling, mouse model

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BACKGROUND Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impaired social skills, as well as restrictive and repetitive interests (American Psychiatric Association, 2013). Current treatments for ASD include behavioural therapy and medication; however, there are no targeted drugs that are effective in treating all of the core symptoms in ASD (Perisco et al., 2015). Anti-epileptic and anti-psychotic drugs are used off-label for treating ASD, although their mechanism of action is poorly understood. Therefore, there is a large unmet need to develop biologically-targeted therapies for ASD. ASD can be broadly grouped into syndromic ASD or idiopathic ASD. Idiopathic ASD accounts for over 80% of autism cases, for which the specific etiology of the disorder is unknown (Benvenuto et al., 2009). Syndromic ASD is observed in specific genetic mutations and metabolic disorders, with distinct phenotypes that strongly resemble ASD. Thus, understanding the mechanisms underlying syndromic ASD, for which genetic cause of the neurological disorder is known, may provide a path to understanding idiopathic ASD and potential targets for therapy.

macrocephaly, epilepsy, and autism associated with Pten deficiency, and whether inhibition of mTORC2 may be useful as a target for autism therapies. MAJOR RESULTS Elucidating the impact of hyperactive mTORC2 signalling The authors of the study began by creating a mouse model with knockout of the Pten gene (Pten-KO). As expected, in Pten-KO mice, phosphorylation of the downstream target of mTORC1 and mTORC2 were both increased, indicating that Pten deficiency leads to hyperactive signalling of mTORC1 and mTORC2. These mice exhibited the typical ASD syndrome phenotypes associated with Pten deficiency including decreased survival, macrocephaly, seizures, behavioural

One example of syndromic ASD is seen in individuals with loss-of-function mutations in the phosphatase and tensin homolog (PTEN) gene. Approximately 10% of ASD patients present with PTEN mutations (Varga et al., 2009). There are several other neurological disorders which have been known to produce syndromic ASD, including Fragile X syndrome and tuberous sclerosis complex (TSC), which all present with similar ASD phenotypes such as imFigure adapted from Chen et al. (2019). Nature Medicine, Epub paired social skills (Winden et al., 2018). Interestingly, dysregulaahead of print. tion of mTOR signalling is common to a large portion of these genetic conditions (Muhlebner et al., 2019). Therefore, there is a Figure 1: a) This figure outlines the main sites of downstream good possibility that mTOR signalling is at least partly responsible phosphorylation for mTORC1 and mTORC2. In hyperactive for the syndromic ASD phenotype. mTORC1 signalling, there is expected to be increased phosphorylaThe mTOR signalling pathway is involved in important cellu- tion of S6. In hyperactive mTORC2, there is expected to be inlar functions including growth, proliferation, and protein synthesis creased phosphorylation of AKT. b) Through western blot analysis, (Laplante & Sabatini, 2012). In developing neurons, this has impli- it is demonstrated that in Pten-KO mice, there is increased cations for differentiation of precursor cells into neurons, as well amounts of phosphorylated S6 and AKT, indicating that mTORC1 as synaptic plasticity. The mTOR signalling pathway has two main and mTORC2 are both hyperactive. In Rptor-KO mice, there is a components, mTORC1 and mTORC2. PTEN is a negative regulator reduction in the amount of phosphorylated S6, consistent with a of mTOR signalling, and in mice with knockouts in the PTEN gene reduction of mTORC1 signalling. In Rictor-KO mice, there is a re(Pten-KO), the mTOR signalling pathways become overactive and duction in the amount of phosphorylated Akt, indicating reduced mTORC2 signalling. result in ASD phenotypes (Zhou & Parada, 2012). The hyperactivation of mTORC1 is inhibited by acute treatment with rapamycin, whereas it has been suggested that mTORC2 is resistant to rapamycin treatment (Sarbassov et al., 2004). Previous studies by Zhou et al. (2009) demonstrated that in Pten-KO mouse models, mTORC1 activity is increased, but chronic administration of rapamycin is able to partially rescue the macrocephaly, social deficits, and seizures associated with Pten deficiency. Thus, the current view in literature is that overactive mTORC1 signalling is the underlying mechanism for ASD syndrome in Pten deficiency (Sato, 2016). However, more recent studies have suggested that both mTORC1 and mTORC2 signalling are increased in Pten-KO mice, and that mTORC2 is also inhibited by chronic, but not acute, treatment of rapamycin (Nguyen et al., 2015; Schreiber et al., 2014). These new findings warrant further consideration as to whether mTORC2 signalling may also play a role in ASD syndrome observed in Pten deficiency. A study by Chen et al. (2019) seeks to identify whether hyperactive mTORC2 signalling also plays a role in

The authors then selectively reduced activity of either mTORC1 or mTORC2 by knocking out Rptor or Rictor, respectively, which serve as key proteins in either complex. Knocking out mTORC1, but not mTORC2, was able to prevent the Pten-KO mouse from developing macrocephaly. This data suggests that mTORC1 may be responsible for the underlying mechanisms leading to macrocephaly in syndromic ASD patients. One of the most significant findings of this paper is that deletion of Rictor, and thus reduction of mTORC2 signalling, was able to rescue all other phenotypes associated with Ptendeficiency, while deletion of Rptor had a less significant effect. Rictor-KO mice showed improved social behaviour (see figure 3), reduced epilepsy, improved memory, and less rigid or repetitive behaviour. This contrasts the findings of a large portion of previous literature which focused only on mTORC1 as the main mechanism behind syndromic ASD. This new evidence presented by Chen and

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colleagues warrants further investigation into the role of mTORC2 would be useful to elucidate whether the role of mTORC2 has been regulation, which was largely overlooked in many past studies. overlooked in these syndromes as well (Winden et al., 2018). Developing a therapy Based on the evidence that hyperactivity of mTORC2 is involved in Pten-deficient syndromic ASD, the authors developed a potential therapy to inhibit overactivity of mTORC2 signalling. A single-stranded antisense oligonucleotide (ASO) was designed to target Rictor; the ASO binds Rictor mRNA, leading to its degradation by RNase H in the cell. This was expected to have similar effects to genetic knockout of Rictor. The ASO was delivered by a single intracerebrovasular (ICV) injection. As expected, the singe ASO injection rescued social deficits, long term memory, epilepsy, and repetitive behaviours in Pten-KO mice, similar to the results observed in Pten-Rictor-KO mice. These findings represent a step in the right direction, towards developing a biologically-target therapy to treat all of the core symptoms of ASD. CONCLUSIONS AND CRITICAL ANALYSIS The findings of this study represent a significant advancement in our understanding of the molecular mechanisms underlying ASD. The authors have highlighted that Pten deficiency leads to over activation of mTORC1 and

Syndromic ASD provides a great opportunity to study the mechanisms behind autism; however, syndromic ASD accounts for less than 20% of autism, leaving the remaining 80% of idiopathic autism cases poorly understood (Benvenuto et al., 2009). It is important to consider whether findings from studies on syndromic ASD can be extrapolated to cases of idiopathic ASD. Autism has a strong genetic component, and it is currently believed that over 50% of susceptibility to developing idiopathic ASD is due to genetic factors (De Rubeis & Basbaum, 2015). In genomic studies of individuals with ASD, no single genetic cause was identified; however, over 100 possible genetic links have been identified. It is possible that many of these genes may be involved in a common pathway such as mTOR signalling. This is consistent with findings that syndromic ASD can be caused by a number of genetic mutations, many of which involve mTOR signalling (Winden et al., 2018). There are limited models of idiopathic ASD, but some studies have confirmed that mTOR signalling is also dysregulated in non -syndromic cases (Ganeshan et al., 2019). The mechanisms of mTOR dysregulation are less understood in these cases; some studies find increased phosphorylation of downstream targets (Onore et al., 2017), and others suggest that downregulation of mTOR signalling leads to development of idiopathic ASD (Ganeshan et al., 2019; Nicotine et al., 2015).

mTORC2, and that mTORC2 is actually responsible for many of the phenotypes that had originally been attributed to overactivation of mTORC1. This opens an opportunity to develop more effective FUTURE DIRECTIONS drugs now that appropriate targets, such as those involved in the mTORC2 signalling pathway, can be identified and explored. Future studies should first investigate whether mTORC2 signalling assumes a similar role in producing ASD phenotypes in It is important to consider the reproducibility and interpre- other types of syndromic and idiopathic ASD. Other forms of syntation of the findings. In the social novelty test, preferential inter- dromic ASD include Fragile X syndrome and Tuberous Sclerosis action with a novel mouse over a familiar mouse was compared. Complex (Fernandez & Scherer, 2017). Fewer models exist for idioControl mice preferred interacting with the novel mouse, while pathic ASD; however, it has been well-documented that maternal Pten-KO mice showed little differentiation, serving as a model of exposure to valproic acid (VPA) during pregnancy increases the risk social impairment observed in ASD. In the long-term memory test, of autism in children. Exposing rodents to VPA in utero provides mice were presented with a familiar object and a novel object. one of the best current models of idiopathic autism (Nicolini & Control mice preferentially interacted the novel object, yet Pten- Fahenstock, 2018). If regulating mTORC2 is a good option for fuKO mice showed no differentiation. It was assumed that long term ture therapies, hyperactive mTORC2 should play a universal role in memory impairments prevented the Pten-KO mice from recogniz- producing ASD phenotypes, and inhibition of mTORC2 should reing the familiar object. This poses the question as to whether mice verse these phenotypes. in the social novelty test simply did not remember the familiar mouse, since both the familiar mouse and the familiar object were Upon confirming the broad applicability of mTORC2 therapresented 24 hours before their respective tests. It is, therefore, pies in multiple animal models of syndromic and idiopathic ASD, also difficult to conclude whether inhibition of mTORC2 signalling studies may begin investigating the safety and efficacy of potential rescued social deficits, long term memory, or both. For future stud- therapies. Rictor ASO injections have been suggested as a prelimiies, it would be useful to conduct additional tests on sociability, nary treatment for regulating mTORC2 signalling, and should be such as olfactory habituation and dishabituation, tube dominance tested for safety and efficacy in multiple animal models of syntests, or ultrasonic vocalization test in order to separate these dromic and idiopathic ASD, as well as in humans with ASD. effects from the effects on long term memory (Stanford Medicine). Other forms of syndromic ASD need to be investigated as well. As demonstrated by Busch et al. (2019), individuals with Pten mutations tend to exhibit distinct forms of autism, and this may warrant special consideration. Although mTOR signalling has been identified as a key component in many ASD syndromes, it cannot be assumed that the same mechanisms underly all forms of syndromic ASD. Additionally, some literature has suggested that mTORC1 plays a key role in many of these syndromes, thus it

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Fecal Microbiota Transplantation as a Therapeutic Strategy for Alzheimer’s Disease Dhruva Nilakantan

Alzheimer’s disease (AD) is neurodegenerative disease and the leading cause of dementia worldwide. It is marked by cognitive deficits, amyloid beta plaques, tau hyperphosphorylation and neuroinflammation. The gut microbiota has been explored as a target for therapies for neural diseases. Fecal microbiota transplantation (FMT) involves the removal of an affected individual’s gut microbiota and replacing it with a healthy individual’s microbiota. FMT has been used in individuals with autism, Parkinson’s disease and multiple sclerosis and has shown encouraging results. AD is associated with an irregular gut microbiota, therefore Sun et al. (2019) explored how FMT would affect symptoms in transgenic AD mice. FMT reversed cognitive deficits, decreased amyloid beta plaques & tau hyperphosphorylation, increased synaptic plasticity, decreased neuroinflammation, and readjusted gut microbiota composition as well short chain fatty acid (SCFA) levels. The results are relatively significant as they show that FMT could be used a therapeutic strategy with Alzheimer’s disease, however further experiments are needed to elucidate specific metabolite signaling mechanisms and to determine the effect of FMT with humans. Key words: Alzheimer’s disease (AD), fecal microbiota transplantation (FMT), gut microbiota, cognitive deficits, amyloid beta (Aβ), tau hyperphosphorylation, neuroinflammation, short chain fatty acids (SCFAs)

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BACKGROUND AND INTRODUCTION

microbiota composition, and SCFA signaling were investigated (Zhou et al., 2019). The results were compared across three Dementia – a progressive cognitive impairment – is one of groups: Tg mice, Tg mice with a FMT (Tg + FMT), and healthy wild the leading causes of impairment and mortality worldwide, cur- type (WT) mice. It was hypothesized that Tg + FMT mice would rently affecting 44 million people (Wortmann, 2015). This number have reversed the AD symptoms seen in the Tg mice. is set to double by 2030 (Wortmann, 2015). Alzheimer’s disease (AD) is the leading cause of dementia and is developed in one per- MAJOR RESULTS son every 66 seconds in the United States (Alzheimer’s AssociaFMT treatment of Tg + FMT mice involved administration of tion, 2016). While a small number of cases of AD are genetic, the antibiotics: vancomycin, ampicillin and metronidazole. The mice vast majority of cases are spontaneous (Bateman et al., 2010). In were then administered a fecal solution from the WT mice over 4 both genetic and spontaneous cases, pathology is marked by amyweeks (Sun et al., 2019). loid beta (Aβ) plaques, tau hyperphosphorylation, and neural inflammation. Aβ plaques are developed due to the improper processing of amyloid precursor protein (APP), leading to 40- and 42- Effects of FMT on Cognitive Impairments amino acid long variants of by-products known as Aβ40 & Aβ42. The cognitive abilities of mice were tested using the Morris Regularly, APP cleavage and processing results in numerous fragWater Maze (MWM) test and the Object Recognition Test (ORT). ments, which have been implicated in synaptogenesis, signal transThe MWM test assesses spatial learning and memory, while the duction, and cell adhesion, however mutations in the APP gene ORT assesses object discrimination. The MWM test involves the lead to Aβ development (Zhang, Thompson, Zhang & Xu, 2011). use of a cylindrical water tank with four quadrants. One of the The cause of tau hyperphosphorylation is unclear, however, it leads to aggregation and neurofibrillary tangles (NFTs), which in- quadrants contains a hidden platform that the mice are trained to find over a period of time. After training is complete, the hidden duces neurotoxic effects (Reilly et al, 2017). Finally, microglial actiplatform is removed, and the amount of time spent in the quadvation occurs due to Aβ deposition and NFTs, leading to neuroinrant which used to contain the hidden platform – the target quadflammation (Serrano-Pozo, Frosch, Masliah & Hyman, 2011). These rant – is recorded (Sun et al., 2019). The ORT involves training so markers of AD together cause losses in synaptic plasticity as well as that the mice are habituated to an object with a certain material cognitive impairment (Serrano-Pozo et al., 2011). for a period of time. Following training, the mice are placed in a The gut microbiota has received much attention in the neu- room with the familiar object and a novel object, and time spent roscience community due to increasing examination of the gut- with each object is recorded (Sun et al., 2019). brain axis. The phylogenetic composition and enterotype of the gut, as well as the metabolites used in communication with the brain have been implicated in some disease states. After fecal transplantation from mice with AD, Germ free (GF) mice have significantly increased Aβ, suggesting gut colonization can affect AD pathology (Jiang, Li, Huang, Liu & Zhao, 2017). Certain enterotypes of the gut microbiota are associated with improved cognitive function, specifically, Lactobacillus helveticus NS8 has been shown to induce resiliency to stress-related depression (Liang et al., 2015). In transgenic AD mice, it has been shown that Acidobateria and Bifidobacterium together can slow AD progression (Bonfili et al., 2018). The most studied metabolites involved in gut-brain axis communication are short chain fatty acids (SCFAs), including acetate, butyrate and propionate. Butyrate, specifically, has been shown to reverse hippocampal and cognitive deficits due to chronic stress in mice (Han, Sung, Chung & Kwon, 2014). Fecal microbiota transplantation (FMT) involves the removal of the gut microbiome from a diseased individual and replacing it with bacteria from a healthy donor. It has been used in scientific studies with small sample sizes to treat autism, Parkinson’s disease and multiple sclerosis, with promising results (Evrensel & Ceylan, 2016). The effects of FMT had yet to be tested on individuals with AD. Recent research has shown that AD is associated with an abnormal gut microbiota (Jiang et al., 2017). Sun et al. (2019) used a FMT to see its effects on APPswe/PS1dE9 transgenic (Tg) mice. These mice have a mutant APP gene and a mutant presilin 1 (PSEN1) gene, which codes for a subunit of the γ-secretase enzyme. γ-secretase is involved in the cleavage of APP, and its dysfunction is associated with AD pathology (Zhou et al., 2019). The effects of FMT on cognitive deficits, amyloid beta plaques, tau hyperphosphorylation, synaptic plasticity, neuroinflammation, gut

The Tg mice had the longest escape latencies of all groups, and spent the least time crossing the target quadrant and the least time within the target quadrant (Sun et al., 2019)(Figure 1A). The Tg + FMT mice showed significantly shorter escape latencies by the end of the trial (Sun et al., 2019)(Figure 1A). Tg + FMT mice also spent significantly more time crossing the target quadrant and within the target quadrant (Sun et al., 2019)(Figure 1B/C). In other MWM tests, it has been shown that valproic acid (VA) treated mice have shorter escape latencies and spend more time in the target quadrant than mice with AD (Bromley-Brits, Deng & Song, 2011). VA has been shown to have pharmaceutical potential for AD therapy (Bromley-Brits, Deng & Song, 2011). Therefore, the MWM results correspond with past literature. FMT reversed spatial memory and learning deficits, as Tg + FMT mice had significantly better results than Tg mice (Sun et al., 2019). The Tg + FMT mice also performed better on the ORT, as they had a significantly better discrimination index than the Tg group (Figure 1D). Transgenic AD mice have been shown to have deficits in the ORT due to their cognitive impairments (Ramirez, 2015). These results therefore also correspond with past literature, as Tg mice had the worst results of all three groups, while the FMT reversed the cognitive impairments on the ORT.

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Figure 1: (A) Escape latency times across training and testing. (B) The amount of time spent crossing the target quadrant across all three groups. (C) The amount of time spend in the target quadrant across all three groups. (D) Discrimination indices across all three groups.

Figure 2: (A) Aβ40 across the three groups. (B) Aβ42 levels across the three groups (C) Phosphorylated tau at threonine-231 ratios with β-actin across the three groups. Figure adapted from Sun et al., 2019. Translational PsychiaFigure adapted from Sun et al., 2019. Translational Psychi- try, 9(1). atry, 9(1). Effects of FMT on Synaptic Plasticity Effects of FMT on Aβ Plaques and Tau Hyperphosphorylation

Synaptic plasticity was assessed using immunochemistry and Western blotting. The levels of PSD-95 and Synapsin-I were assessed across the three groups, once again compared as a ratio with β-actin in the WT group. Results showed a significant increase in both PSD-95 and The number of Congo red patches were counted in Synapsin I in the Tg + FMT group, as compared to the Tg the cortex and in numerous areas of the hippocampus, group (Sun et al., 2019)(Figure 3A/B). Both PSD-95 and showing where compact Aβ deposition was occurring. The Synapsin I are downregulated in APP/PS1 mice (Zhou et Tg group had a higher number of Congo red patches than al., 2016), therefore FMT treatment reversed Alzheimer’sthe WT group, while the Tg + FMT group had less patches like pathologies in synaptic plasticity. than the Tg group (Sun et al., 2019). The Tg group also B A had the highest amount of Aβ40 and Aβ42 in the brain, while the Tg + FMT group had significantly less (Sun et al., 2019)(Figure 2A/B). Similar differences in Congo red staining (Lok et al., 2013) and in Aβ40 and Aβ42 levels (Han et al., 2017) between WT and APPswe/PS1dE9 mice have been reported. Therefore, FMT significantly reversed Aβ plaque deposition (Sun et al., 2019). Amyloid beta plaques and tau hyperphosphorylation were assessed across the three groups using Congo red staining and Western blotting, respectively.

Western blotting was used to compare the levels of tau phosphorylation on threonine-231 across the three groups. Phosphorylation levels were compared as a ratio to the amount of β-actin in the WT group. Phosphorylation at the threonine-231 position was significantly decreased in the Tg + FMT group (Sun et al., 2019)(Figure 2C). Other pharmacological therapies on similar APP/PS1 mice also report a significant decrease in the treatment group by Western blotting, where the non-treated group had the highest amount of phosphorylated tau (Zhou et al., 2016). FMT treatment significantly decreased tau phosphorylation. A

Figure 3: (A) PSD-95 levels across the three groups. (B) Synapsin I levels across the three groups. Figure adapted from Sun et al., 2019. Translational Psychiatry, 9(1). Effects of FMT on Neuroinflammation

Neuroinflammation was assessed by Western blotting and immunochemistry. COX-2, a protein important in the process of inflammation was Western blotted by comparing to a WT ratio once again, this time using a reference of GAPDH (Sun et al., 2019). The levels of a microglial marker – CD11b – were assessed by immunochemistry. COX-2 (Figure 4) and CD11b levels were significantly lower in the Tg + FMT group as compared to the Tg group, which had the highest amounts of all three groups (Sun et al., 2019). These results corroborate with literature, where APP/PS1 mice with an Alzheimer’s-like phenotype have elevated levels of COX-2 and CD11b (Zhu et al., 2017), however inhibition of inflammation significantly decreases COX-2 (Cui et al., 2012). 275

in an AD brain in contrast with a healthy brain (Mosconi, Pupi & Leon, 2008). FMT treatment reverses SCFA signaling changes in an AD brain. A

Figure 4: Changes in COX-2 levels across all three groups compared as a ratio to the WT GAPDH. Figure adapted from Sun et al., 2019. Translational Psychiatry, 9(1). Effects of FMT on Gut Microbiota Composition

Phylotypes of the groups were compared using 16S rRNA sequencing. Tg mice had higher Proteobacteria and Verrucomicrobia; while having lower Bacteroidetes at the phylum level. Tg + FMT mice had completely opposite changes in their gut microbiome at the phylum level (Sun et al., 2019)(Figure 5). Similar changes in the Tg mice were also seen at other levels of taxonomy, however, the Tg + FMT mice generally reversed all these changes (Sun et al., 2019). Similar changes in microbiota composition in transgenic APP/PS1 mice have been reported (Sun et al., 2019). A treatment using ingestion of fructooligosaccharides also reversed these changes in the same study (Sun et al., 2019). FMT treatment reversed changes in gut microbiota composition that occurs in a n Alzheimer’s-like phenotype.

Figure 5: Phylum composition across the three groups. Figure adapted from Sun et al., 2019. Translational Psychiatry, 9(1). Effects of FMT on SCFA Signaling

Acetate, propionate and butyrate were the three SCFAs analyzed using NMR spectroscopy. The levels of acetate and propionate were not changed across the three groups (Figure 6A/C). However, butyrate levels were significantly decreased in the Tg group, and this change was significantly reversed in the Tg + FMT group (Sun et al., 2019)(Figure 6C). Glucose metabolism is significantly decreased in AD, therefore these results corroborate with past literature, as less butyrate would be seen

Figure 6: (A) Acetate levels across the three groups. (B) Propionate levels across the three groups. (C) Butyrate levels across the three groups. Figure adapted from Sun et al., 2019. Translational Psychiatry, 9(1). CONCLUSIONS/DISCUSSION

The major conclusions drawn by Sun et al. (2019) were that FMT can reverse changes caused by AD onset, including cognitive deficits, amyloid beta plaques, tau hyperphosphorylation, synaptic plasticity, neuroinflammation, gut microbiota composition, and SCFA signaling. The authors concluded that the brain changes may have occurred due to microbiota alterations following FMT, leading to modified SCFA signaling (Sun et al., 2019). The abnormal gut microbiome in AD (Jiang et al., 2017) may be the cause of the brain symptoms such as Aβ plaques, rather than the other way around. These results also show that both a molecular change in the brain – APP/ PS1 mutation – and an abnormal gut microbiome are needed for AD pathology. These findings also corroborate with earlier findings, when GF mice were transplanted with fecal microbiota from AD mice, leading to AD pathology (Jiang et al., 2017), as this study further strengthens the link between gut colonization and AD. Previous literature has associated Acidobateria and Bifidobacterium with a slowing of AD progression (Bonfili et al., 2018), however data on these were not collected in this study. The protective effects of butyrate on hippocampal and cognitive deficits may also be implicated, (Han et al., 2014). Butyrate is decreased in the Tg mice, but is significantly increased back to WT levels after FMT, so butyrate itself may be a key metabolite involved in anti-AD processes. The findings from this paper are all novel, as a FMT has never been explored for AD. Therefore, these findings are relatively significant as they may be used a medical therapy. However, as the study is very novel, it needs to be replicated in order to ensure FMT’s reliability. The external validity of the results could also be increased in future human studies.

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CRITICAL ANALYSIS

There are still many details to be elucidated following the study from Sun et al. (2019). The authors concluded that butyrate may be a key metabolite involved in SCFA signaling to protect against AD. However, future experiments need to be performed to elucidate the mechanism of this neuroprotection. The authors also made no connection between what bacterial phyla may be specifically involved in butyrate signaling. Clostridium butyricum, a species of bacteria that produces butyrate, has been shown to have protective effects in those with vascular dementia, the second most common type of dementia (Liu et al., 2015). The Clostridium genus was not explored during 16S rRNA sequencing, therefore no conclusions can be made on whether this bacteria has neuroprotective effects on AD through butyrate signaling. The mice used in this experiment also do not account for any senescence-related changes in the brain that often precede AD in humans. Senescence accelerated mouse prone 8 (SAMP8) and APP/PS1 models have been generated in a previous study (Lok et al., 2013). These senescence-adjusted models show significantly more AÎ˛ deposition than regular APP/PS1 lines (Lok et al., 2013), however this line of mice was not explored in this study. SCFAs are also not the only method of gut-brain signaling. The vagus nerve is very important in regulating the gut microbiota, and vagotomies have been shown to block changes in depressive behaviors (Sandhu, Sheerwin, Schellekens, Stanton, Dinan & Cryan, 2017). Lipopolysaccharides (LPS) are also released by the gut, for peripheral immune activation and have been shown to lead to depressive behaviors (Sandhu et al., 2017). The gut microbiota also synthesizes many neurotransmitters (Sandhu et al., 2017). However, any changes in these gut-brain signaling mechanisms and their links to AD were not explored in this study. On the other hand, this paper is the first to show the effects of a FMT on AD. It also demonstrates the FMT positively alters the major hallmarks of AD, including cognitive deficits, AÎ˛ deposition, tau hyperphosphorylation and neuroinflammation. Other neurodegenerative diseases such as Parkinsonâ&#x20AC;&#x2122;s disease and multiple sclerosis have had promising results following FMT (Evrensel & Ceylan, 2016), so this study may also prove to be an effective therapeutic strategy.

sis should be monitored, especially in the hippocampus. If there is an increase in synaptogenesis, butyrate may indeed be the key metabolite involved, and could be used as in future AD therapy. Further experiments may include a vagotomy study to see if the vagus nerve is involved in gut-brain communication and AD pathogenesis. If a vagotomy does pathogenesis in SAMP8-APP/PS1 mice, then it could be involved in AD signaling, and could be used in a future therapeutic strategy. In a similar FMT study, LPS and neurotransmitter levels could also be monitored to see if there are any changes in SAMP8-APP/PS1 mice preand post- FMT. If there are changes, then they may be involved in the AD signaling mechanism. Further experiments could then involve administration of LPS or neurotransmitters and monitoring of synaptogenesis to understand the brain mechanisms of the metabolites. If the metabolites improve synaptogenesis, they could be used as future therapeutic strategies. To understand which precise gut bacteria species are involved in signaling, specific antibiotic experiments could be done with SAMP8-APP/PS1 mice. The initial experiments could start out on a larger scale, by using antibiotics to remove a phylum. If the phylum removal of Bacteroidetes, for example, caused decreased AD symptoms in SAMP8-APP/PS1 mice, then that phylum contains the bacteria involved in AD signaling. From there, further experiments could continue down the taxonomical scale, in order to find the specific genus or species responsible for signaling. That species could then be targeted in therapies. Eventually, clinical trials are needed involving humans to see if an FMT is an appropriate AD therapy. As other brain disorders have produced promising results following FMT (Evrensel & Ceylan, 2016), this procedure has potential as a future therapy for one of the leading causes of mortality worldwide.

FUTURE DIRECTIONS

In forthcoming experiments, researchers should look to better elucidate metabolites and signaling pathways involved in gut-brain communication. Experiments involving butyrate administration to SAMP8-APP/PS1 mice should be conducted, and changes in synaptogene277

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Axon Demyelination and Degeneration in White Matter As Biomarker for Traumatic Brain Injury. Nicholas Pagano

Traumatic brain injury (TBI) is suffered when the head experiences a sudden blow or jolt causing brain damage. TBI includes many different types of injuries to the head and brain, including CoupContrecoup injury, brain contusions, and most commonly, concussions. The severity of many types of TBI has historically been underestimated, with many still believing that injuries such as concussions are no more than a nuisance. Among the many symptoms are reduced attention/concentration and information processing challenges. In the brain, white matter in the corpus callosum (CC) is critical to the communication and coordination between left and right hemispheres, required for physical movement and complex cognitive processing. In the research study conducted by Marion et al. (2018), it is proposed that information processing declines as a result of reduced action potential (AP) conduction in white matter axons. The main results suggest that damage to both white matter axons and surrounding myelin are incurred from TBI, reducing the effectiveness of insulative myelin in neuronal communication, further resulting in a loss of AP conduction speeds and communication through the CC. These findings can prove critical to the developing field of TBI treatment and diagnosis, acting as a biomarker for various TBIs. Many such injuries, like concussions, lack a biological diagnostic indicator for medical professionals to rely on, and development of this technology can aid in not only recognition, but timely treatment of demyelinated axons to reduce the processing deficits seen in patients. Key words: Traumatic brain injury (TBI), concussion, corpus callosum (CC), myelin, action potential (AP), diagnosis, axon, demyelination, degeneration, white matter

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BACKGROUND or INTRODUCTION. Traumatic brain injury (TBI) is a global epidemic, with an estimated sixty-nine million individuals affected each year (Dewan et al., 2018). TBI can be incurred from vehicular collisions, sports injuries, falls, and more. However, many hold the outdated belief that many forms of TBI are not severe or dangerous. This is largely due to the symptoms associated, such as confusion, memory loss, headaches, fatigue, dizziness, and more; few of these indicate long-term effects or debilitation. However, recent studies have found that patients exhibit many symptoms persisting beyond the initial injury (McMahon et al., 2015; Theadom et al., 2016; McInnes et al., 2017). Equally significant are the challenges medical professionals face when diagnosing TBIs, as these symptoms are common to many conditions and cannot conclusively indicate a TBI with certainty. Doctors have had no distinct and accessible biomarker for concussions and other TBI, making it difficult to diagnose confidently (Powell et al., 2008; Graham et al., 2014; Kulbe et al., 2016). Two seemingly unrelated symptom that are seen to persist in adults after suffering from a TBI are cognitive deficiencies/ slowed information processing, and physical coordination (McInnes et al., 2017). Patients find difficulty concentrating and focusing, and it takes longer than normal to process and understand complex information. It also becomes a challenge to coordinate different parts of the body in complicated ways. One region of the brain involved in both complex processing and physical coordination is the corpus callosum (CC), the largest structure of white matter (Fitsiori et al., 2011). It has been seen in previous studies that white matter does lose structural integrity after TBI (Nakayama et al., 2006; Rutgers et al., 2008). This region is crucial in communication between the two hemispheres of the brain, and any damage to its neurons can result in coordination and processing deficits. In the research study by Marion et al., published in 2018, the experimenters induced concussive TBI in mice, and compared it to control or “sham” mice who underwent the same procedure lacking the concussion. Mice had their scalp cut along the midline, and the skull was impacted at 4m/s. After three days, the brain was excised and visualized using the full-brain clearing CLARITY process. White matter axonal damage was illustrated using Thy1YFP-16 mouse models, which showed significant axonal swelling in the CC and cingulum. Conduction in white matter axons was tested using ex vivo slices of the brain, stimulating axons crossing the CC using electrodes and measuring the speed at which the signal was conducted. There was a general reduction in the velocity of conduction three days after TBI was induced, but after two weeks and six weeks it was seen that conduction speeds significantly increased. The status of axon myelination and damage in the CC was evaluated through electron microscopy, which showed that after TBI, demyelination and paranodal damage occurred and persisted for up to six weeks. The findings of this study demonstrate the lingering effects of TBI up to 6 weeks beyond the initial injury. Demyelination and damage of white matter axons in the CC directly impacts conductivity, which was seen to decrease immediately through the electrode stimulation. This leaves opportunity to develop treatments for TBI to protect, maintain, or reconstruct myelin on CC white matter neurons, reducing the symptoms of processing deficiency and the risk of long-term consequences.

MAJOR RESULTS In Marion et al.’s research (2018), mice were raised socially, and pairs of littermate mice were separated as “sham” or “TBI”. Both had their scalp excised, with the TBI mice experiencing an induced concussion. The scalp was closed, and after three days, mice were euthanized. Their brains were removed and analyzed. Researchers were blinded to groupings until after analysis was complete. Axon Swelling in CC After TBI Mice expressing the Thy1-YFP gene were analyzed for swelling in axons of the CC using the CLARITY process. The sham mice showed little deviation in the size of their CC axons. TBI mice, however, demonstrated significant swelling in the CC, as seen in Figure 1. In both hemispheres of the CC, inferior to the cortex and superior to the lateral ventricle, there is a high density of expressed YFP, indicating axonal swelling. This finding agrees with Yu et al. (2017), both reflecting the ability to use YFP in detecting CC axon damage after TBI. These injured axons span the CC, but there is much lower density in the center. With the frontal cortex as a region of complex cognitive function, damage to the CC near this area would reduce or inhibit communication between the left and right hemispheres of the frontal cortex, leading to a deficit in complex cognitive function and focus. This is one of the most recognizable symptoms of concussions and other TBI, allowing for the possibility of YFP being used to detect TBI via CC axonal damage.

Figure Adapted from Marion et al. (2016). Journal of Neuroscience , 30 (41), 8723-8736. Figure 1. A. The CC of a TBI mouse 3 days after injury. The yellow fluorescent protein (YFP) shows high density of axonal swelling between stimulating electrodes. B. High density was seen superior to the lateral ventricle. C. The cingulum also expresses damaged and swollen axons

AP Conduction in White Matter after TBI The conductivity of axons were measured by stimulating ex vivo slices of the excised mouse brains with electrodes. In white matter, we see two types of axons: N1 axons are myelinated and have high conduction speeds; N2 axons are non-myelinated and are slower in conducting. Myelin is known to be an insulator and protector of axons, and increases the speed and efficiency of APs in neurons (Susuki, 2010). After TBI, there was a change in the conductivity of CC axons, shown in Figure 2. In sham mice, N1 conduction remained significantly faster than N2 axon speeds, but in TBI mice after three days, conduction speed in N1 axons was greatly reduced. Not only this, but the amplitude of N1 signaling reduc-

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es, while N2 signaling amplitude increases. This indicates that some N1 axons have lost their myelin sheath immediately after TBI, thus becoming N2. This demyelination is likely the cause of slower signaling during coordination and information processing. After two weeks, there is a significant increase in N1 velocity, possibly indicating some remyelination occurring. However, N1 amplitude remains lower in TBI mice than sham even after six weeks. This again indicates long-term effects of TBI, and a target for treatment of symptoms. As another control, researchers conducted the electrode test in another, more ventral white matter region in the same slices, the anterior commissure. It was found that after TBI, the anterior commissure did not demonstrate any change in AP velocity. This shows that TBI has significant impact on the CC and its function, while other areas of the brain remain relatively unscathed.

Axon demyelination, damage, and paranode abnormalities Electron microscopy was used to evaluate damage to CC axons and paranodes. The nodes of Ranvier between myelin sheathes is critical for saltatory conduction, necessary for increasing the velocity of conduction in neurons. Any disruption in the structure of the nodes of Ranvier can have consequences on the efficiency of AP conduction. The area where myelin attaches to axons is called the paranode domains. For those N2 axons which are not protected by myelin, they are more likely to suffer damage and axonal degeneration. N1 axons could experience degeneration as well as demyelination (Mierzwa et al., 2015).

Figure Adapted from Marion et al. (2018). Journal of Neuroscience, 30(41), 8723-8736. Figure 3. A. Sham mice have unaffected axons, with only some non-myelinated. B. 3 days after TBI, degeneration of axons (shown by green arrows) and demyelination (shown by blue arrows and blue highlight) is seen in the CC. C. 6 weeks after TBI, both degenerated and demyelinated axons are still present. D. Paranodes (white arrows and yellow highlight) formed by myelin loops in sham mice. Taken from coronal section of the CC. E. TBI mice had areas of unaffected axons and myelin in the CC. F,G. These intact axons were directly neighbouring damaged axons (blue highlight), paranodal abnormalities (red highlight), and demyelination. H. 1 week after TBI there is still evidence of damaged axons and Figure Adapted from Marion et al. (2018). Journal of Neuroscience, 30 afflicted paranodes. I,J. 6 weeks after TBI, axonal degeneration and paranode (41), 8723-8736. damage persists. Figure 2. A. N1 and N2 conductivity speed between sham and 3-day TBI mice. N1 in sham is shown to be much faster than N2 in conduction, but after TBI there is a drastic reduction in conduction. B. N1 conduction velocity after 3 days (red), 2 weeks (blue), and 6 weeks (green). After 3 days there is a dramatic reduction in N1 signaling between sham and TBI mice, but at 2 weeks the TBI conductivity recovers significantly, not quite returning to baseline until after 6 weeks. C. N2 conduction velocity in sham and TBI mice. There is no signs of slowed velocity in N2 conduction after TBI over any time period. D. N1 amplitude of signaling is reduced greatly after 3 days, but recovers after 2 weeks, indicating a demyelination followed by partial remyelination. E. N2 amplitude of signaling increases 3 days after TBI, indicating newly demyelinated N1 axons fall to N2 wave. F. Combined amplitude of N1 and N2 axons shows that after 6 weeks there is significant loss of conduction. G,H. N1 and N2 conduction velocities in the anterior commissure shows no change between sham and TBI.

There is seen to be a decrease in the combined amplitude of N1 and N2 axons in mice experiencing TBI over six weeks. Once again, this indicates persistent effects after TBI is suffered. The demyelination of axons in the CC is particularly influential in the symptoms experienced after incurring TBI, and there presents a clear critical period within the first two weeks of injury for remyelination to occur, possibly reducing the lasting effects of TBI.

Axonal damage and degeneration has been connected with cognitive and coordinative function (Johnson et al., 2013; Yin et al., 2016). This experiment demonstrated that damage and demyelination to axons is a persistent condition after six weeks, as well as paranode abnormalities. This can lead to detached or misshapen myelin loops, affecting the conductivity of the neurons involved.

CONCLUSIONS/DISCUSSION The most significant conclusions of the article by Marion et al. (2018) involve the demyelination of axons in the CC white matter as a driving force for the symptoms experienced by TBI patients. The understanding of TBI pathophysiology is limited, making it very difficult for medical professionals to both diagnose and treat patients. This article provides significant information towards biomarkers, and allows for the development accessible and non-

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invasive methods of identifying them. The authors developed an ethical and efficient experiment to evaluate multiple components involved in the pathophysiology of TBI, using multiple controls to compare against. The article identifies short-term recovery after TBI that turns to later degeneration, applying it to persisting conditions seen in TBI patients. Prior studies have evaluated individual components of this one, such as Yu et al.â&#x20AC;&#x2122;s (2017) use of YFP for identifying axonal swelling, but the overall encompassing of both axonal degeneration, damage, and demyelination provides a full understanding of the causes for axonal conduction deficits, provides a relation to its specificity to the CC, and its impact on overall cognitive and coordinative function. These findings can pave the way for therapies to reduce demyelination and axonal damage, or provide repair in the critical two-week period identified.

CRITICAL ANALYSIS The authors of this paper identified some limitations in the study. The evaluation of conductivity and electron microscopy were done ex vivo, and do not account for the many varying conditions in an active, living, functioning brain. It is currently unknown if a functioning brain has alternate methods of communication between hemispheres beyond the CC, or if the very principle of demyelination is counteracted by another condition or factor. If this is the case, then the symptom of complex processing deficiency is rooted elsewhere. If an in vivo experiment can support the findings of this article, it can be confidently said that the demyelination of axons in the N1 wave reduce the speed, amplitude, and efficiency of communication in the CC. Another concern of the study is the molecular basis of each of these issues. There are questions that are not addressed, such as how, on a molecular scale, axons become demyelinated through TBI, or what causes the swelling. The molecular scale may provide insight to treatments or therapies to discourage demyelination and degeneration, or at the very least a deeper understanding of the injury of study. The authors do address the concern of technological limitations, and suggest a further evaluation to the possible redistribution of ion channels through demyelination and degeneration. This would be best studied in vivo, to understand how the body reacts to such conditions, and how to best support or encourage repair. Being an ex vivo experiment, it is difficult to study persisting conditions on a more applicable scale. The maximum time frame of evaluation was six weeks, which is negligible in an average human lifespan. Many patients do not experience long-term symptoms of TBI until years later, such as chronic traumatic encephalopathy (CTE) patients being diagnosed over 8 years after initial injury (Tharmaratnam et al., 2018). With more time of evaluation in vivo, researchers can better understand the true lingering effects of TBI, rather than extrapolating from a comparatively fractional amount of time.

sudden shake of the sample, to re-evaluate the structure as the axon is demyelinated. This understanding can provide key information to be applied to treatments or therapies in preventing or repairing demyelination and axon degeneration during the critical period of approximately two weeks. A therapy that can target the CC during demyelination has the potential to prevent cognitive deficiencies in patients both immediately and persistently. An in vivo version of the conduction experiment should be conducted, in order to evaluate the true impact of TBI in CC axonal conduction velocity. Using a sham and TBI mouse, stimulating the CC in vivo with electrodes will better demonstrate the speed of communication, accounting for neuroplasticity and an adapting and active brain. A challenge that will be experienced is the possible conflict of signals in a live animal, so sedatives will likely be required to minimize cognitive function. If this demonstrates a reduced speed, it can be more confidently said that the cognitive processing deficiencies experienced are likely a result of demyelination in the CC, and communication between the hemispheres of the cortex is being reduced. If this does not demonstrate such results, this may indicate an alternative pathway the brain is using, or a molecular factor that mitigates the negative effects of demyelination; this means that the cognitive impairment is a consequence of a different issue in the brain which may not have yet been considered. Producing these evaluations over a more significant amount of time is crucial for the application. A lengthier evaluation would require a different test species, but would provide a better understanding of how persistent demyelination and degeneration of axons is, and how that can pertain to long-term TBI such as CTE and Second-Impact syndrome. It is expected that there would be further degeneration with more time, as it was seen that after six weeks degeneration and demyelination persisted, with a decreased conduction amplitude of N1 and N2 axons combined. If this is the case, it may become evident as to what is the cause of lingering TBI symptoms, and in conjunction with studies of molecular structure can lead to therapies and treatments. If over time there is recovery of myelination and axonal structure, this indicates the persistent symptoms are a result of another cause, which would warrant its own investigation.

FUTURE DIRECTIONS Future studies should focus to answer what molecular functions are involved in the demyelination and degeneration of axons after TBI. The sudden force experienced by the brain can have many effects, but it is critical to understand the molecular reactions along the neuronal membrane and paranodes to understand how the myelin sheath is removed. This can be done with an in vitro experiment, studying the molecular structure of the membrane-myelin interaction, then inducing a TBI-like event such as a

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Graham, R., Rivara, F. P., Ford, M. A., Spicer, C. M., Youth, C. on S.-R. C. in, Board on Children, Y., … Council, N. R. (2014). Concussion recognition, diagnosis, and acute management. Retrieved from https://www.ncbi.nlm.nih.gov/books/ NBK185340/

Johnson, V. E., Stewart, W., & Smith, D. H. (2013). Axonal pathology in traumatic brain injury. Experimental Neurology, 246, 35 –43. https://doi.org/10.1016/j.expneurol.2012.01.013

Kulbe, J. R., & Geddes, J. W. (2016). Current status of fluid biomarkers in mild traumatic brain injury. Experimental Neurology, 275, 334–352. https://doi.org/10.1016/j.expneurol.2015.05.004

Marion, C. M., Radomski, K. L., Cramer, N. P., Galdzicki, Z., & Armstrong, R. C. (2018). Experimental traumatic brain injury identifies distinct early and late phase axonal conduction deficits of white matter pathophysiology, and reveals intervening recovery. Journal of Neuroscience, 38(41), 8723–8736. https://doi.org/10.1523/JNEUROSCI.0819-18.2018

McInnes, K., Friesen, C. L., MacKenzie, D. E., Westwood, D. A., & Boe, S. G. (2017). Mild Traumatic Brain Injury (Mtbi) and chronic cognitive impairment: A scoping review. PLOS ONE, 12(4), e0174847. https://doi.org/10.1371/journal.pone.0174847

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Mierzwa, A. J., Marion, C. M., Sullivan, G. M., McDaniel, D. P., & Armstrong, R. C. (2015). Components of myelin damage and repair in the progression of white matter pathology after mild traumatic brain injury. Journal of Neuropathology and Experimental Neurology, 74(3), 218–232. https://doi.org/10.1097/NEN.0000000000000165

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Gut Microbiota Dysbiosis Impact On Memory in Mice

Samantha Parmasar

It has been established that gut microbiota dysbiosis results in gut inflammation known as colitis. Gut microbiota dysbiosis has been linked to neurological disorders such as Alzheimerâ&#x20AC;&#x2122;s disease, Parkinsonâ&#x20AC;&#x2122;s disease and autism. This study focuses on whether colitis derived gut microbiota dysbiosis influences memory functioning in mice. The hypothesis was tested by applying 2,4,6trinitrobenzenesulfonic acid (TNBS) treatment, lipopolysaccharide (LPS) treatment and Escherichia coli (EC) treatment to healthy mice and evaluating their memory and learning via Y-maze and passive avoidance tasks. The three treatments resulted in alteration of gut microbiota levels, particularly an increase in Lactobacillus johnsonii (LJ). Gut microbiota dysbiosis subsequently resulted in learning and memory impairment. Further tests with LJ treatment to EC and TNBS affected mice resulted in restoration of the normal gut microbiota composition and corrected memory functioning. These results indicate that disruption of the normal microbiota composition or dysbiosis caused by gut inflammation or colitis inducers such as TNBS and LPS impairs memory in mice and can subsequently be alleviated by LJ treatments. This provides evidence for how the gut affects the brain in the gut-brain axis. Key words: Colitis inducers, Lactobacillus johnsonii, memory impairment, inflammation, colitis, 2,4,6trinitrobenzenesulfonic acid, lipopolysaccharide, gut microbiota dysbiosis, Y-maze, passive avoidance tasks.

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BACKGROUND or INTRODUCTION. Stress affects gut microbiota by stimulating the brain to produce hormones which cause gut microbiota dysbiosis and the subsequent production of toxic LPS through colitis. LPS secretion promotes inflammation via activation of toll-like receptor 4 (TLR4)/NF -ᴋB signaling and production of pro-inflammatory cytokines such as TNF-α, interlukin-1(IL-1) and interlukin-2 (IL-2).1,8,10 LPS induced pro-inflammatory cytokine production can affect memory tasks involving the hippocampus via interruption of neural circuit functioning.2 Previous research has shown increased amounts of activated astrocytes and microglia in the cortex and hippocampus of mice as a result of increased LPS levels.8 Additionally, research done by Valero et al in their 2014 article, observed decreased doublecortin-positive neurons, decreased synaptic contacts in new neurons as well as decreased dendritic volume in mice treated with LPS thereby affecting production of hippocampal neurons and subsequently affecting memory.9 LPS and other endotoxins produced from gut microbiota disturbance, interrupt neurotransmitter and cytokine secretion responsible for CNS communication with the gut.3 Colitis inducers such as TNBS also cause gut microbiota dysbiosis and increased LPS blood levels. This study aimed to evaluate if changes to gut microbiota composition, caused by colitis inducers such as TNBS, could cause similar colitis and memory impairment in mice. This experiment is relevant as research is limited pertaining to the impact of colitis inducers on memory and how gastrointestinal inflammation can affect neural circuits pertaining to memory tasks.

MAJOR RESULTS TNBS Treatment: Mice received intrarectal injection of TNBS solution into the colon and subsequently held in a vertical position for 30s in order to distribute TNBS in the colon.3 The control group received saline treatment. TNBS treatment to mice resulted in colon shortening, increased NF-ᴋB activation, increased inflammatory markers, increased EC and decreased LJ, increased blood and gut microbiota LPS levels and TNF-α levels in the hippocampus. Y-maze and passive avoidance tests carried out on TNBS affected mice showed reduced learning and memory. Additional outcomes of TNBS treatment included hippocampal NF-ᴋB activation, inhibited brainderived neurotrophic factor (BDNF) expression and phosphorylation of CREB.3 Passive avoidance task was carried out using a twocompartment acrylic box connecting a lit section to a dark section by a hole. The mouse was originally placed into the lit section and upon entering the dark area, received a 0.3mA electrical shock for 2s. Measurements for latency time were taken with respect to reentering the dark compartment. The Y-maze consisted of a threearm horizontal maze where the mouse was assigned one arm and recordings were taken for the number of entries into the arm within 8 minutes by each mouse.3

Previous research provides supplementary evidence for the impact on colitis inducers on memory in mice. Researchers Lim et al in their 2017 article, explore how LJ ameliorates memory impairment caused by TNBS, a colitis inducer as well as how LJ inhibits the LPS production and its downstream inflammatory effects.4 This research is relevant as neurodegenerative diseases such as Alzheimer’s disease involve gut microbiota disturbance. New therapies involving restoration of normal gut microbiota composition in such diseases are worth exploring and developing. This study also reiterates the fact that research is limited on the relationship between gut microbiota and memory impairment.4 In this study, the researchers Jang et al, applied a TNBS treatment, EC treatment and LPS treatment on normal mice and evaluated their learning and memory using Y-maze and passive avoidance tasks.3 The control mouse was treated with glucose. EC and TNBS affected mice then underwent LJ treatment in attempt to restore the normal gut microbiota composition and learning and memory was once again evaluated using Y-maze and passive avoidance tasks. EC, TNBS and LPS treatments resulted in colitis, increased LPS blood levels, decreased LJ, increased EC and decreased Firmicutes. Affected mice were evaluated as memory impaired using the Y-maze and passive avoidance tasks. The glucose control mouse showed no evident colitis, gut microbiota dysbiosis nor memory impairment. LJ treatment applied to EC and TNBS affected mice resulted in restoration of normal gut microbiota composition and corrected memory functioning.3 This research is relevant as research is limited with respect to the relationship between colitis and memory function and this study shows how using bacteria to restore normal gut microbiota balance could repair cognitive function and perhaps be incorporated into therapies for other neurodegenerative diseases such as Alzheimer’s disease.

Figure 1 adapted from Jang et al (2017) Nature: Muscoal Immunology. 11, 369-379 Figure 1: (a) Decreased colon length in TNBS affected mice in comparison to the control mice, (b) MPO activity higher than control mice levels, (c) measurements taken in colon of NF-ᴋB activation, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and tight junction protein expression, (d) significant increase in EC when measuring Enterobacteriaceae levels, (e) decreased levels of Bifidobacteria and Lactobacilli, (f) increase fecal LPS levels, and (g) increased blood LPS levels both measured using Limulus assay, (h) increased blood TNF-α levels and (i) increased hippocampal TNF-α levels both measured using enzyme-linked immunosorbent assay,

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(j) Y-maze showed decreased spontaneous attenuation levels, (k) passive avoidance task showed decreased latency time for TNBS affected mice, (l) BDNF expression and NF-ᴋB expression in hippocampus measured by immunoblotting. LPS Treatment: LPS treated mice showed increased blood and gut microbiota LPS levels, increased hippocampal TNF-α and memory impairment as assessed by Y-maze and passive avoidance tasks. LPS similarly inhibited hippocampal BDNF expression and phosphorylation of CREB.3 Authors Zhao et al similarly showed in their 2019 article how LPS-induced neuroinflammation causes cognitive impairment via increased microglia activation, neuronal cell death in hippocampus, increased TNF-α, COX-2, iNOS and increased NF-ᴋB activation.12 Additionally, researchers Zarifkar et al in their 2010 article, similarly explored the impact of agmatine on LPS-induced memory impairment with the basis that LPS results in neuroinflammation subsequently causing cognitive impairment.13

showed how the Lactobacillus pentosus var. plantarum 29 of the same Lactobacillus genus attenuated memory impairment in mice via restoration of doublecortin, BDNF expression, NF-ᴋB activation in the brain and promoted anti-inflammatory effects on LPS affected mice.11 Additionally, Lim et al in their 2017 article, similarly demonstrated how LJ attenuated LPS-induced memory impairment in mice via inhibition of colon shortening, MPO activity, NFᴋB, decreased LPS levels and increased BDNF expression.4 These findings support the theory that gut microbiota possess the ability to attenuate colitis and memory impairment.

CONCLUSIONS/DISCUSSION

The researchers Jang et al concluded that gut microbiota dysbiosis affected neuroactive molecule secretion in the brain and subsequently gastrointestinal inflammation which resulted in memory impairment. The researchers also highlighted that maintaining the normal gut microbiota balance plays a role in maintaining correct cognitive function such as memory.3 This is shown LJ Attenuation: through the LJ treatment to TNBS and EC affected mice resulting in Mice received oral administration of LJ once a day for 5 days amelioration of memory impairment and colitis. This research is after 72 hours of having their final treatment with TNBS or EC.3 Y- important as it provides evidence for a relationship between gastrointestinal inflammation and memory impairment. The results maze and passive avoidance task were carried out 2 hours after obtained by Jang et al agree with the results of other research final LJ treatment. LJ treatment to TNBS and EC affected mice resulted in increased BDNF expression and phosphorylation of CREB, done by Lim et al that explores how LJ can attenuate colitis and memory impairment in mice via LPS inhibition. Similar results from supressed hippocampal NF-ᴋB, decreased blood LPS levels, deLim et al study show that LJ increased tight junction expression creased blood and hippocampal TNF-α levels and ameliorated and supressed NF-ᴋB activation in Caco-2 cells both of which were memory impairment evaluated using the Y-maze and passive 3 affected by LPS.4 The article by Jang et al similarly showed LJ to avoidance tasks. increase tight junction expression and decreased NF-ᴋB activation.3 Additionally, research done by Lee et al on LJ used to ameliorate scopolamine-induced memory impairment in mice showed similar results.5 Such results showed that LJ suppressed NF-ᴋB activation and increased BDNF expression in neuronal cells both of which were affected by LPS and thus alleviating memory impairment.5 Researchers Jeon et al in their 2010 paper similarly showed how L. helveticus, cultured in fermented milk, was able to attenuate memory impairment and APP metabolism induced by scopolamine treatment.14 Additionally, research done by Mohammadi et al in their 2019 article, found a combination of L. helveticus R0052 and B. longum R0175 being able to ameliorate LPS-induced hippocampal apoptosis.15 Their study provides further supporting evidence for the ability of gut microbiota to alleviate cognitive impairments. The culmination of results from these different articles show that overall gut microbiota plays an important role in the gut -brain axis and how probiotics show promising potential as a therapy for memory impairment associated with gut dysbiosis.5

CRITICAL ANALYSIS The authors discuss how increased LPS blood levels are Figure 2 adapted from Jang et al (2017) Nature: Muscoal Immunolobserved in diseases involving gut permeability issues such as inogy. 11, 369-379 flammatory bowel disease.3 Further experiments should be perFigure 2: (a) Y-maze used to assess learning and memory, (b) imformed that evaluate the potential of gut microbiota restoration in munoblot measuring BDNF and NF-ᴋB hippocampal levels, (c) ob- alleviating abnormal gut permeability in the context of inflammaserved decrease in blood LPS levels, (d) decreased blood TNF-α tory bowel disease. The authors mention Alzheimer’s disease (AD) levels observed for LJ treated mice and (e) decreased hippocampal throughout their paper however the experiments conducted are TNF-α levels, (f) colon length restored to value seen in normal not done using AD models but rather in the context of general mice, (g) decreased MPO activity, (h) measured levels of NF-ᴋB, colitis inducers. The authors should therefore perform the same iNOS and COX-2 (i) measures of tight junction expression. experiments using AD models and inflammatory bowel disease models. Additionally, the authors should address if this is a permaResearch done by authors Jeong et al in 2015, similarly nent solution to memory impairment involved with colitis or if it is

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only short term and there exists the possibility of a relapse. Furthermore, the authors should address if any side effects were observed to be involved in their original study. In comparison to the other referenced articles, there appear to be no discrepancies in results. The authors also mention how diet could also affect cytokine levels in circulation which can also impact brain functioning.3 Since Jang et al has already established that gut microbiota plays a role in alleviating colitis and by extension memory impairment, further experiments should be carried out to investigate the impact of diet on cognitive function. Authors Noble et al investigated the impact of a Western Diet on cognitive functioning in a review article and reveal that Enterobacteriaceae is associated with colitis and impaired cognitive function.6 Therefore, the authors Jang et al could build further on their results by using mice models for different diets to observe the impact, if any, on gastrointestinal inflammation and then Y-maze and passive avoidance tasks for memory function.

vation and the impact on cognitive function. Researchers Chunchai et al investigated restoration of cognitive function by prebiotics, probiotics and synbiotics via decreased microglial activation.7 Their findings showed that prebiotics, probiotics and synbiotics decreased microglial activation, subsequently alleviating cognitive impairment.7 Further research needs to build upon this to evaluate specifically the mechanism behind LPS microglial activation.

FUTURE DIRECTIONS â&#x20AC;&#x201C; The role of gastrointestinal inflammation in the context of a neurodegenerative disease such as Parkinsonâ&#x20AC;&#x2122;s disease (PD), Alzheimerâ&#x20AC;&#x2122;s disease (AD) or dementia is still limited in research. Future research needs to incorporate similar tests with LJ or other bacteria affected by increased LPS as a treatment to affected animals to investigate its ability to restore cognitive function in the context of AD or PD. It would also be interesting to investigate the effect of irritable bowel syndrome (IBS) on memory or any side effect on cognitive function and to evaluate potential alleviation with bacteria that can restore the gut microbiota balance. A possible experiment would involve using a mouse model for IBS and investigating memory and learning through Y-maze and passive avoidance tasks. Results indicating cognitive impairment caused by IBS would further result in analysis of fecal pellets for gut microbiota levels. The bacteria found at abnormal levels would be orally administered to IBS affected mice and further tests on memory and learning would be carried out using the Y-maze and passive avoidance tasks. If there is indeed an impact of gut microbiota on memory in IBS affected individuals, memory and learning tests would show alleviation of cognitive impairment upon treatment with deficient bacteria. Furthermore, future research needs to be done on whether gut microbiota can ameliorate memory impairment associated with AD and whether it can be used as a long-term treatment. As AD is a neurodegenerative disease that progressively worsens, it would be interesting to explore whether probiotic treatments can serve as a permanent solution or if it must continually be used as a short-term solution and if there is the possibility of a relapse in memory impairment. Also, if any side effects would be involved with the usage of probiotics. An experiment to test this would involve using AD mouse models and performing the same methods previously mentioned above however over a longer trial period such as a year. The researchers Jang et al mention in the discussion that it is known that increased LPS induced proinflammatory cytokine production via activation of microglia. However, they state that it is unknown whether this microglia activation by LPS is direct or indirect.3 Future research needs to address this gap by investigating the relationship between gut microbiota and microglia acti-

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Jang, S. E., Lim, S. M., Jeong, J. J., Jang, H. M., Lee, H. J., Han, M. J., & Kim, D. H. (2018). Gastrointestinal inflammation by gut microbiota disturbance induces memory impairment in mice. Mucosal immunology, 11(2), 369.

Lim, S. M., Jang, H. M., Jeong, J. J., Han, M. J., & Kim, D. H. (2017). Lactobacillus johnsonii CJLJ103 attenuates colitis and memory impairment in mice by inhibiting gut microbiota lipopolysaccharide production and NF-κB activation. Journal of functional foods, 34, 359-368.

Lee, H. J., Lim, S. M., & Kim, D. H. (2018). Lactobacillus johnsonii CJLJ103 Attenuates Scopolamine-Induced Memory Impairment in Mice by Increasing BDNF Expression and Inhibiting NF-κB Activation. Journal of microbiology and biotechnology, 28(9), 1443-1446.

Noble, E. E., Hsu, T. M., & Kanoski, S. E. (2017). Gut to brain dysbiosis: mechanisms linking western diet consumption, the microbiome, and cognitive impairment. Frontiers in behavioral neuroscience, 11, 9.

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Badshah, H., Ali, T., & Kim, M. O. (2016). Osmotin attenuates LPS-induced neuroinflammation and memory impairments via the TLR4/NFκB signaling pathway. Scientific reports, 6, 24493.

Valero, J., Mastrella, G., Neiva, I., Sánchez, S., & Malva, J. O. (2014). Long-term effects of an acute and systemic administration of LPS on adult neurogenesis and spatial memory. Frontiers in neuroscience, 8, 83.

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Jeong, J. J., Woo, J. Y., Kim, K. A., Han, M. J., & Kim, D. H. (2015). Lactobacillus pentosus var. plantarum C29 ameliorates age‐ dependent memory impairment in F ischer 344 rats. Letters in applied microbiology, 60(4), 307-314.

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Zhao, J., Bi, W., Xiao, S., Lan, X., Cheng, X., Zhang, J., ... & Fu, Y. (2019). Neuroinflammation induced by lipopolysaccharide causes cognitive impairment in mice. Scientific reports, 9(1), 5790.

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Zarifkar, A., Choopani, S., Ghasemi, R., Naghdi, N., Maghsoudi, A. H., Maghsoudi, N., ... & Moosavi, M. (2010). Agmatine prevents LPS-induced spatial memory impairment and hippocampal apoptosis. European journal of pharmacology, 634(1-3), 8488.

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Mohammadi, G., Dargahi, L., Naserpour, T., Mirzanejad, Y., Alizadeh, S. A., Peymani, A., & Nassiri-Asl, M. (2019). Probiotic mixture of Lactobacillus helveticus R0052 and Bifidobacterium longum R0175 attenuates hippocampal apoptosis induced by lipopolysaccharide in rats. International Microbiology, 22(3), 317-323.

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Understanding the Mechanism through which Type 2 Diabetes Mellitus Causes Alzheimer’s Pathophysiology Rudra Patel

Alzheimer’s disease is a neurodegenerative disorder that causes memory loss. Its pathology includes increased beta-amyloid plaques and tau tangles in various regions of the brain, specifically the hippocampus. Type 2 diabetes mellitus results from insulin insensitivity that restricts the cellular uptake of glucose. Individuals with Type 2 diabetes are more likely to acquire early onset Alzheimer’s, but the relation between the two diseases is unknown. Caveolin-1 (Cav-1) is a receptor in the neuroendothelium that regulates the neuronal uptake of insulin. It has previously been discovered that there is a downregulation of Cav-1 receptors in diabetes; hence it is possible that a decrease in this receptor may be contributing to the Alzheimer’s phenotype. Bonds et al. (2019) restored Cav-1 levels in diabetic mice using viral injections and observed a reversal of the Alzheimer’s pathology in the hippocampus, as well as a restoration of memory functions. This study provides novel evidence to suggest that the downregulation of Cav-1 in diabetes contributes to Alzheimer’s symptoms, and that a potential therapy to reverse Alzheimer’s related cognitive impairments could involve restoration of receptors via viral injections. Key words: Caveolin-1, Alzheimer’s Disease, Type 2 Diabetes Mellitus, Amyloid Plaque Precursors, Beta-Amyloid, Tau, BACE-1, eNOS, Viral injections

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c INTRODUCTION Type 2 diabetes mellitus (T2DM) is characterized by insulin resistance, which inhibits the cellular uptake of glucose, resulting in hyperglycemia (Wang et al., 2016). Interestingly, individuals with T2DM have a 57% increased chance of acquiring Alzheimer’s disease (AD) compared to healthy counterparts (Gudala et al., 2013), but the direct mechanism by which T2DM increases susceptibility to AD remains unknown. Features of T2DM induced AD includes behavioral impairments such as memory deficits, as well as pathological hallmarks such as increased amyloid plaque precursors (APP), BACE-1, β-amyloid (Aβ), and hyperphosphorylated tau (Jack et al., 2018). In the brain, glucose crosses the blood brain barrier through GLUT1 receptors, and then enters neurons through GLUT3 receptors, both of which operate independent of insulin signaling (Gray et al., 2014). Hence, insulin insensitivity in T2DM does not inhibit the entry of glucose into neuronal cells like it does elsewhere in the body. Considering the most evident outcome of insulin insensitivity is inapplicable in the brain, it can be inferred that T2DM increases susceptibility of AD through means other than the inhibition of glucose uptake. Unlike glucose, insulin requires interaction with neuroendothelial receptors to enter the brain. In order to cross the blood brain barrier, insulin must phosphorylate the Caveolin-1 (Cav-1) receptor which in turn creates a caveolae that helps endocytose the hormone (Mastick et al, 1995). These receptors, along with many others that are involved in the processing of amyloid-plaque precursors (APP), are located within lipid rafts (Vetrivel et al., 2004). The close proximity of enzymes in the lipid rafts suggests that potential interaction between Cav-1 receptors and enzymes such as BACE-1 that are involved in the processing of APP may contribute to the AD phenotype. Previous studies have shown that T2DM results in a downregulation of Cav-1 receptors which ultimately inhibits neural insulin uptake (Wang et al., 2011). Since Cav -1 may interact with enzymes that process AD hallmark proteins, its downregulation in T2DM could contribute to AD pathophysiology.

lobes of human patients with T2DM were compared with samples from age-matched, non-diabetic counterparts. Western blots using primary antibodies against Cav-1 indicated a significant decrease in the levels of the receptor in patients with T2DM (Figure 1A), which coincided with an increase in β-amyloid (Aβ) (Figure 1B), a hallmark of AD. The researchers quantified the amount of protein by densitometric measurements. Western blots also revealed decreased epithelial nitric oxide synthase (eNOS) (Figure 1C), suggesting cerebral vasoconstriction (Bonds et al., 2019). Previous studies imply that eNOS is responsible for creating nitric oxide which is a vasodilator (Wang et al., 2009); hence its downregulation increases the susceptibility of cerebral infarctions in the hippocampus, causing cognitive impairments in AD (Tan et al., 2015). Next, Bonds et. al (2019) performed Western blots on hippocampal protein lysates of db/db mice. Assays revealed that decreased Cav-1 receptors (Figure 2A) coincided with an increase in β-secretase 1 (BACE-1) (Figure 2B) which is a primary protease involved in the processing of APP (Bonds et al., 2019). The upregulation of BACE-1, consequently, suggests that a loss of Cav-1 increases APP cleaving to create more Aβ. Last, Western blots indicated an increase in total tau (DA9) (Figure 2C), as well as phosphorylated taus (CP13 and AT8) (Figure 2D, 2E) in db/db mice. Importantly, there was a significant increase in the CP13/DA9 ratio (Figure 2F), but no observable difference in the AT8/DA9 ratio (Figure 2G). This implies that Cav-1 depletion is correlated with a disproportionate hyperphosphorylation of Ser202 to create greater levels of CP13 (Bonds et al., 2019). Other studies have shown that an increase in CP13 causes tau deposition commonly observed in human AD neuropathology (Fiock et al., 2019).

Bonds et al. (2019) attempted to test this hypothesis in three specific ways. First, they determined the level of Cav-1 as well as AD biomarkers in the brains of T2DM human and mice models. Next, they determined if AD pathophysiology can be rescued by restoring Cav-1 levels using viral injections in diabetic (db/ db) mice models. Last, they determined whether Cav-1 affected the processing of APP by comparing the ratios of different Aβ, with higher Aβ42 compared to Aβ40 being indicative of neurotoxicity (Klein et al., 1999). Ultimately, this study revealed that T2DM does indeed lead to depletion of Cav-1 levels which subsequently contributes to AD pathophysiology, a phenomenon that includes increased Aβ, APP, BACE-1 and phosphorylated tau (Bonds et al., 2019). Notably, Bonds et al. found that restoration of Cav-1 receptors through viral injections reverses many of the AD symptoms to a degree that is comparable to control subjects. A strong understanding of the mechanism in which T2DM induces AD is essential for the development of potential AD therapies.

MAJOR RESULTS Part 1: Characterizing a Correlation Between T2DM and AD Physiology in Humans and Mice In Bond et al. (2019), protein lysate from the temporal

Figure 1 (Figures adapted from Bonds et al. (2019). The Journal of Neuroscience 39(43), 0730–19): Western blot analysis of the quantity of A: Cav-1, B: Aβ, and C: eNOS in the temporal lobe tissue of human patients with T2DM compared to age-matched, nondiabetic individuals

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Next, in Bond et al. (2019), hippocampal protein lysates were analyzed using Western blots for specific AD markers. First, the db/db mice with restored Cav-1 had decreased expression of APP (Figure 3B) and BACE-1 (Figure 3C). Normally, BACE-1 cleaves APP into Aβ, causing amyloid plaque aggregates (Tamagno et al., 2006); hence the observed decrease suggests a downregulation of the AD pathology. Second, mice with restored Cav-1 receptors had a decreased ratio of AT8/tau5 (Figure 3D), which is indicative of lower tau phosphorylation in the presence of Cav-1. Combined, these results suggest that restoration of Cav-1 receptors through viral injections can reverse AD pathophysiology by rescuing memory loss and reducing AD biomarkers such as Aβ and phosphorylated tau.

Figure 2 (Figures adapted from Bonds et al. (2019). The Journal of Neuroscience 39(43), 0730–19): Western blot analysis of the quantity of A: Cav-1, B: BACE-1, C: DA9, D: CP13, E: AT8, F: CP13/ DA9, and G: AT8/DA9 in the hippocampal tissue of db/db mice compared to wild-type (WT) mice

Figure 3 (Figures adapted from Bonds et al. (2019). The Journal of Neuroscience 39(43), 0730–19): A: performance in novel object recognition, and levels of B: APP, C: BACE-1, and D: ratio of AT8/ Tau5 in mice with restored Cav-1 levels

Part 3: Determining How Depletion of Cav-1 Causes Changes in APP Processing

Once it was confirmed that depletion of Cav-1 increases Aβ, Bonds et al. (2019) attempted to determine whether APP processing pathways were affected by Cav-1 levels. Their research drew on several precedents. Galvao et al. (2019) suggests that APP Upon establishing the correlation of depleting Cav-1 reprocessing through the amyloidogenic pathway produces different ceptors with increased AD protein hallmarks in db/db models, Bonds et al. (2019) attempted to restore the levels of Cav-1 recep- variation of Aβ which creates toxic aggregates (Galvao et al., tors through viral injections. A cohort of db/db mice were injected 2019). Specifically, Klien et al. (1999) indicate that Aβ42 has significantly higher neurotoxic properties which increase the risk of oxiwith Ad-CMV-Cav-1 to restore Cav-1 receptors, and a control dative damage compared to Aβ40. Bonds et al. (2019) first manipugroup was established by injecting Ad-CMV-GFP which did not alter Cav-1 levels (Bonds et al., 2019). The hippocampal integrity of lated Cav-1 levels in human embryonic kidney (HEK) cells that exboth groups was tested in a novel object recognition task. All mice press APP mutant form using viral injections. Cav-1 levels were were first accustomed to a specific object. After the initial training, downregulated using LV-shRNA-Cav-1, and upregulated using AdCMV-GFP. Control samples were established by injecting LV-shRNA the mice were placed in a cage with both a novel object and the object they were previously familiar with. Cohen et al. (2015) have -scrambled and Ad-CMV-GFP. ELISA analysis indicated that HEK inferred that since rodents have an intrinsic drive for exploration, cells with downregulated Cav-1 secreted significantly more Aβ (Figure 4A). Upregulation of Cav-1 in mutant HEK cells did not the mice that spent more time with the novel object have hippochange the amount of Aβ secreted (Figure 4A), but it decreased campal integrity that permits them to remember and avoid the previously familiar object. In Bond et al., it was observed that db/ the amount of Aβ42 to a degree that was indistinguishable from the control samples (Figure 4B). Since the Aβ42/Aβ ratio decreases db mice with restored Cav-1 levels spent significantly more time when Cav-1 is restored, it can be inferred that Cav-1 receptors with the novel object (Figure 3A), thus suggesting hippocampal inhibit the amyloidogenic pathway to decrease AD pathology. integrity.

Part 2: Establishing A Causal Relationship Between Depleting Cav-1 Levels and AD Pathophysiology

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Next, the location of the increased Aβ was determined using density analysis with subsequent Western blotting. Previous studies indicate that the processing of APP occurs in the cell membranes where integral proteins convert APP into Aβ that form toxic aggregates (Butterfield and Lashuel, 2010). Bonds et al. (2019) discovered an abundance of APP in membrane lipids compared to other cellular compartments in Cav-1 knockout mice, suggesting that depleting Cav-1 mislocalizes APP to the membrane and increases the likelihood of it being cleaved to Aβ.

provide an avenue for preventing the onset of AD in patients with T2DM by using viral injections to restore Cav-1 receptor levels invivo.

CRITICAL ANALYSIS This paper has two major limitations. First, the effect of increased Aβ was not properly applied to AD models. In mice, Aβ does aggregate like it does in humans, so it cannot be assumed that Aβ forms clusters that contribute to AD phenotype (KastyakIbrahim et al., 2013). Similarly, neurofibrillary tangles formed by hyperphosphorylated tau are not consistently observed in mice models (Kastyak-Ibrahim et al., 2013). While the hallmarks AD proteins are visible in mice models, they do not consistently create the same pathophysiology that is responsible for the neurological deterioration in AD. This means that mice models are not adequate in replicating the deficits observed in human AD patients. Consequently, it is necessary to apply these observations to alternative models that better mimic human models. This can potentially be done by using alternative models such as pluripotent stem cells from human AD patients. Lee et al. (2016) created neurospheroid models of AD using induced pluripotent stem cells from Alzheimer’s patients. They were able to use this model to detect changes in AD biomarkers such as BACE-1 and Aβ upon testing of AD treatments. This model provides exclusive advantage because the pathophysiology mimics the characteristics found in human AD models. Second, the results in the mice model were assumed to be applicable to human models because both diabetic models had Figure 4: (Figures adapted from Bonds et al. (2019). The Journal of decreased Cav-1 levels and increased AD pathophysiology. This Neuroscience 39(43), 0730–19) ELISA analysis of the amount of generalization may be ineffective because depleted Cav-1 levels A: Aβ and B: Aβ42 in mice with upregulated or downregulated Cav- were observed in the temporal lobe of human model, whereas the 1 levels. C: Western Blots that indicate the relative amount of APP analysis was done on the hippocampus of the mice. Primarily, in lipid membrane (BF = buoyant fraction) and other cellular com- there are intrinsically lower amounts of endothelial cells in the temporal lobe compared to the hippocampus, hence decreased partments (HF = heavy fractions). Cav-1 levels in human samples may be a result of decreased neuCONCLUSIONS/DISCUSSION roendothelial cells (Wilhelm et al., 2016). Additionally, age and AD Overall, the experiment conducted by Bonds et al. (2019) causes progressive deterioration of the blood brain barrier in the has three major implications. First, it confirms that diabetic huhippocampus, but the temporal lobe remains fairly intact throughmans and mice have a decreased amount of Cav-1 receptors which out lifetime (Wilhelm et al., 2016). Therefore, the authors should coincide with increased AD hallmarks such as BACE-1, APP, and Aβ. standardize the examined region of the brain in order to effectiveSecond, they show that restoring Cav-1 levels in db/db mice modly make comparisons between mice and human models. Since the els rescues memory deficits, thus confirming that hippocampal authors ultimately used the novel object recognition task to anaimpairments are caused by Cav-1 depletion. In human HEK-FAD lyze the behavioral effects of AD, they should confirm that Cav-1 is cells, restoring Cav-1 levels decreases Aβ and in turn rescues AD downregulated in the hippocampus of humans with T2DM. pathophysiology. Third, depletion of Cav-1 receptors promotes the FUTURE DIRECTIONS amyloidogenic pathway by increasing the amount of cytotoxic Bonds et al. (2019) suggested that downregulation of Cav-1 Aβ42. This finding suggests that restoring Cav-1 inhibits the pathway that creates cytotoxic amyloid that is responsible for causing alters the amyloidogenic pathway and is responsible for alternate cognitive impairments in AD. processing of APP into cytotoxic Aβ42. A similar explanation that While the correlation between depleting Cav-1 and increas- highlighted mechanistic changes underlying increased tau phosing AD pathology has been well established in previous literature, phorylation was lacking. In order to address this, the relation beBonds et al. (2019) provide unique evidence suggesting that Cav-1 tween Cav-1 and various enzymes that phosphorylate tau must be may be actively protecting the brain from AD characteristics. This explored. Two primary kinases involved in this process are PKA and is specifically determined when HEK cell samples with depleted MAPK (Razini and Lisanti., 2001). PKA is located within the caveolae, hence its proximity to Cav-1 may allow its activity to be inhibitCav-1 levels had higher levels of cytotoxic Aβ42 levels, thus suged (Razani and Lisanti, 2001). Similarly, Cav-1 controls the Ras-Rafgesting that the amyloidogenic pathway was altered in T2DM models that had depleted Cav-1. The authors restore Cav-1 levels MEK-ERK pathway which involves MAPK, hence the downregulausing viral injections and observe a direct reversal of both hallmark tion of Cav-1 can affect the level of phosphorylation performed by MAPK (Ferrer et al., 2006). Considering that both of these pathproteins, and behavioral impairments commonly found in AD brains. This finding suggests that depleting Cav-1 in diabetes is not ways that are involved in tau phosphorylation are controlled to correlated with AD pathophysiology, but in fact causes these char- some degree by Cav-1, perhaps its downregulation in T2DM is reacteristics. The results from this paper are important because they sponsible for hyperactivity of these enzymes. The activity of these 292

two enzymes can be examined in isolation by introducing protein knockout (KO). This can be accomplished by having the three following KO models: Cav-1 KO and MAPK KO, Cav-1 KO and PKA KO, and Cav-1 KO, PKA KO and MAPK KO. If the knockout model still produces hyperphosphorylated tau, it can be inferred that the pathway that was knocked down was not implicated in phosphorylating tau. If a knockdown model decreases the amount of hyperphosphorylated tau, then the mechanism by which Cav-1 inhibits hyperphosphorylation can become apparent. Furthermore, qPCR of the transcriptional levels of the genes that create these two enzymes in each KO model should be conducted in order to determine changes in gene expression of different enzymes involved in tau phosphorylation pathways in Cav-1 knockout models. This would provide more insight into the cellular changes that occur due to Cav-1 depletion. Finally, the direct interaction between Cav-1 and the enzymes involved in APP processing and tau phosphorylation was never explicitly determined. Insight into these interactions are critical to confirm that depletion of Cav-1 in T2DM causes the AD phenotype through the interactions of enzymes that create the AD hallmark proteins. Kumagai et al. (2015) genetically engineered GPCRs by fusing a fluorescent HaloTag to the N-terminal of the receptor. Next, they introduced a HaloTag ligand which binds to the genetically engineered receptor, allowing illumination. A similar pulse-chase experiment can be established by genetically engineering the Cav-1 receptor with bioluminescent tags. If the receptor interacts with enzymes involved in processing AD protein hallmarks, then it can be concluded that Cav-1 plays an inhibitory role that prevents processing of APP and tau. This is important in confirming the means through which Cav-1 protects the brain from an AD phenotype.

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Potential Biomarkers for Alzheimer’s Disease in Human Retina Zeel Patel

Alzheimer’s disease (AD) is the most common type of dementia, a neurodegenerative disease which impairs cognitive function, and affects about 45 million people globally. Presently, no cure exists and with the ageing population, AD is expected to impose a severe burden on the healthcare system. For treatment options to be viable, non-invasive, in vivo diagnosis of this disease in the preclinical stages, before the appearance of symptoms, is needed. Several hallmark characteristics in the brain are common in AD patients. The aggregation of proteins such as amyloid beta (Aβ) plaques and phosphorylated tau (pTau) tangles in addition to neurodegeneration is observed in human AD patients as well as in mouse models. Recently, activation of pro-inflammatory, neurotoxic astrocytes and microglia have also been implied in AD pathology. In an attempt to devise a diagnostic method to detect AD, Grimaldi et. al (2019) sampled slices of human retina tissues from AD patients and their agematched controls to observe features of AD pathology using histological analysis, immunofluorescence, and RNA fluorescence in situ hybridization (FISH). This study found that hallmark features of AD pathology such as Aβ and pTau aggregation, activation of pro-inflammatory astrocytes and microglia, and activation of apoptotic pathways involved in neurodegeneration are elevated in human retinal slices of patients with AD compared to their age-matched controls. Indeed, sampling of human retinal slices could provide non-invasive detection of AD in vivo which would provide early diagnosis necessary for potential treatments. Key words: Alzheimer’s disease, neurodegeneration, microglia, astrocyte, retina, diagnosis, amyloid beta, phosphorylated tau, caspase-3, pro-inflammation

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INTRODUCTION

Aβ and pTau aggregation in AD retina

AD is an irreversible neurodegenerative disease and the most common cause of dementia (Sosa-Ortiz, Acosta-Castillo, & Prince, 2012). Symptoms of AD often include cognitive impairment in areas such as memory, language, motor function, personality, and behavior(Weller & Budson, 2018). Hallmark characteristics of AD include aggregation of Aβ plaques and fibrils of pTau (Weller & Budson, 2018). It has also been demonstrated that Aβ plaques induce the activation of microglia (Tu et al., 2015) and astrocytes (Hu, Akama, Krafft, Chromy, & Van Eldik, 1998). Furthermore, the activation of both of these cells have been associated with elevated pro-inflammatory cytokines such as IL-1β (Currais et al., 2016; Hu et al., 1998).

Sections of retina were stained for Aβ and pTau by Grimaldi et al. (2016) to detect protein aggregation characteristic of AD pathology. A similar study previously conducted by den Haan et al. (2018) reported increased aggregation of pTau in AD retina. However, in this study, AD retinal slices contained significantly greater aggregation of both Aβ (Figure 1A) and pTau (Figure 1B) when compared to age-matched controls (Grimaldi et al., 2016). Since Aβ and pTau aggregation is a hallmark feature of AD, observing increased numbers of these proteins in AD retina is a promising result for early diagnosis.

Currently, no cure exist for AD. Several drugs have been made available to try and combat the symptoms associated with AD. For example, the loss of acetylcholine neurons has been mitigated by drugs such as donepezil, rivastigmine, and galantamine (Yiannopoulou & Papageorgiou, 2013). However, although these drugs have been effective in reducing neuronal death associated with AD, they still only treat the symptoms, and not the actual progression of the disease itself. This may be due to the amalgamation of the various previously mentioned pathologies that contribute to the progression of the disease. Since symptoms of Alzheimer’s disease are only noticed until protein aggregation has already significantly accumulated, ear- Figure 1. Quantification of protein aggregates in human retinal ly diagnosis is imperative for effective treatment (Mueller et al., samples. FOV: field of view (A) Aβ levels; **: p < 0.01; t-test, n 2005). = 5 patients, 45 total fields (B) pTau levels; ***: p < 0.001; tTo detect AD early in its progression, several biomarkers test, n.= 6 patients, 38 total fields. Figure adapted from Grimalhave been investigated as potential diagnostic tools. In an di et al. (2019) Frontiers in Neuroscience, 13(1), 925. effort to discover an early diagnosis using AD biomarkers, Grimaldi et al. (2019) considered the human retina. Since retinal Neurodegeneration ganglion cells originate from a similar embryological region as In 1981, Terry et al. recorded neuronal loss in AD pathe brain, it has been implied that there could be shared physitients in the frontal and temporal cortices. Since then, many ological processes that can then be traced back to the early studies have noted the loss of cholinergic neurons specifically development of AD (Heavner & Pevny, 2012; Javaid, Brenton, and its role in cognitive impairment (Mufson, Counts, Perez, & Guo, & Cordeiro, 2016). To test this hypothesis, Grimaldi et al. Ginsberg, 2008). Therefore, Grimaldi et al. (2019) investigated (2019) obtained post-mortem human retinal samples from 10 whether there was neuronal loss in the retinal ganglion layer, AD patients and 10 age-matched controls. They then persimilar to that of AD pathology. Neuronal loss can be measured formed histological analysis, immunofluorescence, or RNA FISH through the upregulation of primary molecules involved in to detect the presence of the AD pathology. Indeed, AD retinal apoptotic pathways. One such molecule is the caspase-3 prosamples contained elevated levels of extracellular Aβ plaques, tein, a member of the caspase protein family which is involved intracellular pTau neurofibrillary tangles, activation of proin protease-mediated apoptosis (Louneva et al., 2008). As such, inflammatory microglia and astrocytes, and increased molefinding elevated levels of caspase-3 in retinal ganglion cells cules associated with apoptotic pathways. These results are would indicate neuronal cell loss and therefore, serve as an significant because, if retina AD pathology can be used to deindicator for AD pathology. Indeed, the inner layer of human tect the AD in early stages, then non-invasive techniques such retinas had increased levels of caspase-3 compared to ageas visual testing or retinal imaging can be utilized to diagnose matched controls when detected by immunofluorescence with AD in an early timepoint, where treatment may prove to be an anti-caspase-3 antibody (Figure 2B; Grimaldi et. al, 2019). most effective. Since visual impairments have been reported in AD patients (Sadun, Borchert, DeVita, Hinton, & Bassi, 1987), neuronal loss MAJOR RESULTS mediated by caspase-3 pathways, specifically in the retinal ganglion layer, could explain these symptoms. As mentioned previously, 10 human retinal slices from post-mortem AD patients as well as 10 age-matched control samples with no history of eye disease were collected for the Microglia Activation and Neurotoxicity study completed by Grimaldi et al. (2016). Staining for AD paMicroglia have been reported to have dual effects in thology was then performed on all samples. neurodegenerative brains. This is due to the activation of mi297

croglia into either the M1 or M2 state (Tang & Le, 2016). M1 microglia are characterized by the release of pro-inflammatory cytokines, particularly IL-1β (Tang & Le, 2016). Conversely, M2 microglia activation is reported to have opposite, antiinflammatory effects in their milieu (Tang & Le, 2016). In AD, the presence of pro-inflammatory cytokines such as IL-1β has been shown to shift microglia activation from the M2 to the M1 state (Koenigsknecht-Talboo & Landreth, 2005). Therefore, Grimaldi et. al (2019) measured the microglia density using Iba1 staining and the presence of the pro-inflammatory cytokine IL1β and upregulation of triggering receptor expressed on myeloid cells-2 (TREM-2) in retina samples. The study found that microglia density was increased in AD retina samples compared to their age-matched controls (Figure 2A; Grimaldi et al., 2019). Furthermore, IL-1β levels were also elevated in AD human retinal samples compared to their age-matched controls (Figure 2B; Grimaldi et al., 2019). Since IL-1β is a pro-inflammatory cytokine which has been reported to induce the shift in microglia activation from an M2 to an M1 state (Tang & Le, 2016), this finding supports the activation of neurotoxic microglia. Furthermore, TREM-2 has been implicated in regulating inflammatory responses, phagocytosis of Aβ, and proliferation of microglia (Gratuze, Leyns, & Holtzman, 2018). As such, upregulation of the TREM-2 could translate to AD progression. RNA FISH was used to measure TREM-2 mRNA expression by Grimaldi et al. (2019) because a suitable antibody for TREM-2 is not yet available. Indeed, TREM-2 was upregulated in AD retinal samples compared to their age-matched controls (Figure 2D; Grimaldi et al., 2019). Therefore, Grimaldi et al. (2019) concluded that human AD retinal samples exhibit microglia activation and neurotoxicity similar to the pathology observed in the brain.

Figure 2. Quantification of pro-inflammatory and apoptotic markers in human retinal samples. (A) Number of microglia stained per FOV; ***: p < 0.001; t-test, n = 6 patients, 44 total fields. (B) Number of microglia expressing IL-1β; *: p < 0.05; ttest n = 5 patients, 31 total fields (C) Number of astrocytes stained per FOV; ****: p < 0.001; t-test, n = 6 patients, 44 total fields. (D) Number of microglia expressing TREM-2 per FOV; *: p < 0.05; t-test, n = 4 patients, 31 total fields. (E) Number of retinal ganglion cells expressing caspase-3 per FOV; *: p < 0.05; ttest, n = 4 patients, 17 total fields. Figure adapted from Grimaldi et al. (2019) Frontiers in Neuroscience, 13(1), 925. Astrocyte Activation and Neurotoxicity It has been reported that astrocytosis is prominent in areas surrounding Aβ plaques and pTau neurofibrillary tangles in the temporal neocortex of AD patients (Serrano-Pozo et al., 2011). Additionally, astrocyte phenotypes also change during the progression of AD (Henstridge, Hyman, & Spires-Jones, 2019). Therefore, astrocyte proliferation and phenotype could serve as a biomarker for AD progression. Based on this rationale, Grimaldi et al. (2019) investigated astrocyte proliferation by quantifying astrocyte density in glial fibrillary acidic protein (GFAP) staining in AD human retinal samples. They discovered that there was an increased density of astrocytes in AD retina compared to age-matched controls (Figure 2C; Grimaldi et al., 2019). Additionally, to determine if neurotoxic astrocyte activation was present in AD retina, Grimaldi et al. (2019) also measured the levels of complement component (C3). NF-κB signalling pathways in astrocytes release C3 in AD patients (Lian et al., 2015). It has been demonstrated that increased levels of C3 induces dendritic disruption and synaptic loss (Lian et al., 2015). Thus, observing increased levels of C3 in AD retina could indicate an activation of neurotoxic astrocytes. Immunofluorescence staining and subsequent quantification of C3 in retina samples revealed increased levels of C3 in AD samples compared to their age-matched controls (Figure 2E; Grimaldi et al., 2019). Therefore, astrocyte proliferation and neurotoxicity were consistent with AD pathology in the retina and the brain. CONCLUSIONS/DISCUSSION Ultimately, Grimaldi et al. (2019) concluded that the hallmark features of AD pathology observed in the brain can also be found in AD human retina. Aβ and pTau aggregation, neurodegeneration, neurotoxic microglia activation, and neurotoxic astrocyte activation were all increased in AD human retinal samples compared to their age-matched controls (Grimaldi et al., 2019). By the time clinical symptoms are visible in AD patients, pathology such as protein aggregation has accumulated to the point where treatments cannot control disease progression (Javaid et al., 2016). Unlike previous literature reviewed in this paper, Grimaldi et al. (2019) investigated biomarkers beyond Aβ and pTau aggregation. Since the aggregation of these proteins may also be present in other neurodegenerative diseases, such as in macular degeneration (Matsubara et al., 2010), Aβ and pTau accumulation alone may not serve as a specific marker for AD. Therefore, in addition to protein aggregation, Grimaldi et al. (2019) also revealed, for the

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first time, other AD-specific markers such as IL-1β elevation and activation of neurotoxic microglia and astrocytes. These findings recapitulate the pathology observed in human AD patients as well as in AD mouse models, such as 5xFAD (Henstridge et al., 2019). The detection of these features in the human retina could also provide an explanation for the visual deficits observed in some AD patients clinically (Javaid et al., 2019). As such, these results provide a more comprehensive screening for AD pathology in human retina and could provide an effective diagnostic tool for AD progression in humans. These findings are particularly interesting because the accessibility of the human retina enables diagnosis to be completed in a noninvasive procedure. Matching visual screening or ocular testing to these molecular markers could therefore provide an interesting avenue for early AD diagnosis. CRITICAL ANALYSIS The primary findings obtained from the study completed by Grimaldi et al. (2019) indicated hallmark features of AD pathology in the human retina. The detection of Aβ and pTau aggregation was consistent with previous literature that assessed for these proteins in AD human retina (den Haan et al., 2018). However, Grimaldi et al. (2019) acknowledged in their paper that they could not distinguish which area of the retina they were examining from the retinal slices they obtained from the Human Eye Biobank for Research. This is particularly important for pTau measurements because the authors also admit that only a selection of the retinal slices showed positive staining for anti-pTau antibodies. As such, pTau could be differentially expressed across the retina and therefore, it would be beneficial to further investigate the specific retina locations associated with pTau aggregation to ensure accurate diagnosis. Furthermore, RNA FISH was used to observe an increase in TREM-2 expression because the authors of this study claimed that there was no available antibody for TREM-2. Although RNA FISH is a useful technique to measure mRNA levels, the fact that Grimaldi et al. (2019) observed TREM-2 mRNA expression in areas without Iba1 microglial staining indicated that TREM-2 does not strictly co-localize with microglia expression. Denaturing effects due to inherent limitations of the protocol is stated as a possible explanation for mis-localization of TREM-2 in this paper; however, the visible expression of TREM-2 in both normal and AD retina samples could suggest an alternate explanation that TREM-2 is normally expressed in the human retina. Additional research that either develops a suitable antibody for TREM-2 or elucidates the mis-localization properties of TREM-2 is needed to ensure the validity of TREM-2 as a biomarker for AD diagnosis.

(Osteopontin) were used by Grimaldi et al. (2019) to determine microglia and astrocyte neurotoxicity. Although all of these markers were present in the AD retina samples examined, these markers alone may not serve as accurate determinants of neurotoxic activation. For example, A1 astrocyte activation is characterized by the secretion of IL-1α, TNFα and C1q, (Liddelow et al., 2017) whereas M1 microglia have been associated with IL-1β, TNFα, and IL-6 secretion. As such, the aforementioned molecules measured by Grimaldi et al. (2019) may not be the most precise indicators of neurotoxic microglia and astrocyte activation. Lastly, the use of human retina as a valid location for early AD diagnosis is only plausible if the proposed biomarkers can be detected in the early stages of AD and that these biomarkers can be detected in a non-invasive manner. The retinal samples analyzed by Grimaldi et al. (2019) were all from obtained post-mortem. As such, it is difficult to conclude from this study alone that AD pathology appears in the early stages of AD. It could also be possible that AD pathology spreads from the brain into the human retina during later stages. As such, further experiments that trace the spread of AD pathology, perhaps using animal models may better verify the use of human retina for early diagnosis. Furthermore, the molecular biomarkers identified by Grimaldi et al. (2019) may not be suitable for non-invasive diagnosis as retinal samples are required for staining protocols. Therefore, assessing neurodegeneration using retinal imaging techniques may be a more practical application of these findings in a clinical setting. Future experiments that can measure microglia and astrocyte activation or cytokine levels in the human retina in vivo and non-invasively would be beneficial for clinical diagnosis. FUTURE DIRECTIONS

Research that develops early diagnostic tools for AD is imperative for novel treatments that can act before AD pathology is irrepressible. The findings presented by Grimaldi et al. (2019) suggests that future research can focus on completing a more thorough screening of markers for AD pathology in the human retina. As previously mentioned, the activation states of microglia and astrocytes can be better defined by assessing for increases in specific cytokines. This can be accomplished utilizing the same protocol used by Grimaldi et al. (2019) but using additional assays such as enzyme-linked immunosorbent assay (ELISA) to detect for cytokines characteristic of A1 astrocytes and M1 macrophages. It would be expected that IL-1α, TNFα, and C1q levels would be elevated in astrocytes, marking a A1 phenotype (Liddelow et al., 2017) and IL-1β, TNFα, and IL-6 levels would be elevated in microglia, marking a M1 phenotype Another aspect of AD pathology that Grimaldi et al. (Tang et al., 2016). (2019) investigated was microglia and astrocyte activation, and Furthermore, if TREM-2 is to be used as a marker for M1 their associated neurotoxicity. Iba1 and GFAP staining provided microglia activation, then a more suitable method for measurevidence for increased proliferation of microglia and astrocytes ing its expression beyond RNA FISH is necessary for more accurespectively (Grimaldi et al., 2019). However, since both microrate results. To eliminate the possibility of any inherent TREM-2 glia and astrocyte can have different activation states (Liddelow expression in the human retina, an effective antibody for TREM et al., 2017; Tang & Le, 2016), it important to characterize their -2 is needed. After repeating the experiments done by Grimaldi phenotype beyond proliferation. IL-1β, TREM-2, C3 and OPN 299

et al. (2019) with a suitable antibody, it would be expected that TREM-2 is indeed exclusively co-localized with activated microglial; however, if this is not the case, then TREM-2 may not be an appropriate indicator for AD pathology in the retina, perhaps due to endogenous expression of TREM-2 in the retina. Alternatively, a protein detection method that does not utilize antibodies such as tandem mass spectroscopy could also be applied to detect biomarkers, including TREM-2. It would be expected that in retinal samples, such as the ones obtained by Grimaldi et al. (2019), TREM-2 levels would be elevated in AD patients compared to their age-matched controls.

ty of retinal sample analysis for clinical use. However, better mouse models for AD are still needed since current models such as 5xFAD mice do not present with all AD clinical symptoms. For example, IL-1Î˛ expression is increased in 5xFAD astrocytes, but not in human AD patients (Grimaldi et al., 2019; Rosenzweig et al., 2019). Additionally, innocuous dyes that can be applied directly in eye testing and measure molecular markers, such as pro-inflammatory states of microglia and astrocytes, could also be a potential focus of future research. Ultimately, a variety of markers need to be assessed to discriminate AD pathology from other diseases.

In the study done by Grimaldi et al. (2019), the AD retinal samples were compared to control samples that did not have any history of eye disease. However, future experiments that compare AD retinal samples to retinal samples with ageassociated eye diseases would provide a more distinct characterization of biomarkers associated with AD pathology against other eye diseases. This could be completed using the same protocol as the one used by Grimaldi et al. (2019), but simply adding another sample of patients that had history of eye disease, but not AD. Ideally, only AD retinal samples would present with the aforementioned cytokines and AD pathology. However, if the eye disease samples also shared AD pathology, such as increased TREM2 expression, for example, then that would indicate that a more thorough screening with additional, more specific biomarkers would be needed for clinical applications. Moreover, the directionality of AD pathology transmission between the brain and the retina should be better elucidated to validate using pathology observed in the human retina for early AD diagnosis. This could be supported by future studies that correlate characteristics such as early retinal ganglion thinning to later AD diagnosis in humans. This could be tested on AD animal models, such as the 5xFAD mouse model (LaFerla & Green, 2012). For example, pathological markers similar to the ones investigated by Grimaldi et al. (2019) could be measured in specific areas of the brain and retina in mice models. Sacrificing 5xFAD mice at various time points during disease progression and analyzing samples of the brain and retina by staining or biochemical analysis could aid in determining the path of AD pathology progression. If indeed, pathology in the retina is observed prior to pathology in the brain or at a time where treatments may still be successful, then these findings would support the use of retina samples for AD diagnosis. Lastly, for these results to be applicable clinically, analysis of these biomarkers needs to be conducted in vivo and noninvasively. Non-invasive retinal imaging methods, such as optical coherence tomography, can be used to detect measurable changes such as retinal ganglion layer thinning. Experiments that provide evidence for co-pathology of increased ADassociated cytokine levels and neurodegeneration, such as the one presented by Grimaldi et al. (2019), are needed to validate clinical screening approaches for AD diagnosis. Recapitulating these findings in 5xFAD mice or other AD mouse models during various stages of disease progression would support the validi300

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Evaluating Probiotic Supplementation as a Potential Treatment for Age-Related Long-Term Potentiation Loss and Increased Neuroinflammation Matthew Petrei

There is increasing evidence that suggests an intimate relationship between the gut and the neuronal function of the brain. In recent years scientists have begun to understand how the composition of an individualâ&#x20AC;&#x2122;s gut microbiome can influence important factors like neuroinflammation and resulting diseases such as Alzheimerâ&#x20AC;&#x2122;s. Deficits in memory brought on by neuroinflammation can be assessed in relation to various physical properties. A decline in long-term potentiation (LTP), indicative of memory consolidation, is often used as an early indicator of neurodegeneration. With more attention being paid to the gut and its influence on neuronal function, researchers like Distrutti et al. (2014) investigated whether intentional changes to the gut microbiota composition in aging rats had any effect on their ability to maintain LTP. Distrutti et al. (2014) demonstrated that changes in the gut microbiome composition following treatment with the probiotic VSL#3 resulted in an increase in LTP and a decrease in pro-inflammatory processes, including the alteration of gene activity associated with inflammation and cell death. These findings suggest the gut as a potential new target for therapies looking to ameliorate the deficits in cognition related to aging. The results put forth by Distrutti et al. (2014) open possible avenues for further study, especially into the metabolic products of the microbiome. Experiments that examine the effects these metabolites have on the neuronal function could reveal potential therapies and elucidate the underlying mechanisms that drive age related cognitive decline. Key words: gut microbiota, long-term potentiation, neuroinflammation, brain derived neurotropic factor, probiotic, bifidobacterium

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Introduction

Distrutti et al.’s (2014) study establishes whether or not the composition of an individual’s microbiome can be deliberately altered through probiotic (VSL#3) administration and how these changes affect cytokine production in the brain and influence LTP. Through RNA microarray analysis the authors analyzed the relative changes in bacterial phyla composition and link these changes to the up or downregulation of specific proteins like BDNF, which is involved in LTP, and genes like PLA2G3, which is associated with oxidative distress (MartínezGarcía et al., 2010).

It is a well known but unfortunate fact that aging is coupled with a decline in cognitive function. This decline has been attributed to the onset of neuroinflammation, which plays a role in the deterioration of neuronal properties such as longterm potentiation (LTP) and consequently, spatial memory (Distrutti et al., 2014). With the average human lifespan having increased significantly over the last two centuries (Salazar, Valdés-Varela, González, Gueimonde, & de los Reyes-Gavilán, 2017), finding novel methods of combatting this decline has become an incredibly urgent issue. Major Results Neuroinflammatory processes have been linked to an array of neurodegenerative disorders including Alzheimer’s disease and Schizophrenia. These inflammatory reactions are widely mediated by, the release of cytokines from neural immune cells known as microglia, which have been shown to attenuate LTP (Rezaei Asl et al., 2019). These pro-inflammatory cytokines, specifically interleukin 1β (IL-1β) and tissue necrosis factor α (TNF-α), are produced globally by immune cells and are found to be upregulated in patients experiencing cognitive impairments (Maqsood & Stone, 2016). Significantly, the upregulation of these proinflammatory cytokines corresponds with an increase in oxidative stress within the brain and lower levels of brain derived neurotropic factor (BDNF), which have both been linked to a loss of LTP (Heyck & Ibarra, 2019). BDNF has been shown to play an active role in synaptic development and is closely associated with increased synaptic activity (Rex et al., 2006). Importantly, it has been shown in multiple in vivo studies, that decreases in BDNF reduce neurogenesis in certain areas of the hippocampus (Taliaz, Stall, Dar, & Zangen, 2010). These proinflammatory cytokines have been shown to increase as a result of aging, often alongside microglial activation (Lynch, 2010). Recent discoveries have drawn more attention to the role of the gut in human health providing researchers with targets for novel therapies. There has been ample evidence which suggests that the gut microbiota plays a role in mediating neuroinflammatory processes through the gut-brain axis (Maqsood & Stone, 2016). Studies similar to the investigation carried out by Distrutti et al. (2014), aim to observe the effect of the supplementation of probiotics to the gut on neuronal function. Two common bacterial genera used in probiotic cocktails are Lactobacillus and Bifidobacterium (Feng et al., 2019). Various strains of these genera are used in the probiotic VSL#3 which has been shown to alleviate inflammatory activity, such as colitis, in in vivo rat models (Wang et al., 2019). Importantly, in vivo tests have shown that the supplementation of Lactobacillus and Bifidobacterium containing probiotics leads to changes in neurometabolites (O’Hagan et al., 2017) as well as increased LTP, as shown in Distrutti et al.’s (2014) study. These effects are attributed to the production of bacterial metabolites like butyrate, a short chain fatty acid, which is known to have a role in limiting inflammatory activity (Heyck & Ibarra, 2019).

Changes in Gut Microbiota Composition In their study using rat models, Distrutti et al. (2014) attempted to see whether or not the gut microbiome of an individual could be intentionally altered to improve their cognitive function. After a forty-two-day period during which they administered VSL#3 to aged and young rats they found stark differences in enterotype composition between the control and treatment aged models (Distrutti et al., 2014). Using 16s rRNA sequencing of fecal pellets Distrutti et al. (2014) were able to determine that VSL#3 did affect the microbiota composition in aged rat models. There was a large change in Actinobacteria, the phyla encompassing Bifidobacteria, which decreased in the aged control group and increased in the aged VSL#3 treatment group (Distrutti et al., 2014). The changes in the aged VSL#3 treatment group mirrored the microbial changes in both young rat models (Distrutti et al., 2014). Firmicutes, the phyla containing Lactobacillus, are shown to decrease in all groups except for the aged control rats, where they increase in population percentage (Distrutti et al., 2014). There was a reduction in Proteobacteria levels across all groups and a general increase in relative Bacterodetes as well. This data suggests that there are spontaneous age-related changes to the gut microbiota composition. It also shows that probiotic supplementation does in fact alter gut microbiome composition and shifts the relative proportions to those found in in younger individuals.

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Figure 1: Relative Ratios of Bacterial Phyla in Rat Models Before and After Treatment. The graphs represent the relative abundance of the major phyla in the rat gut microbiota before (A) and after (B) the 42 day VSL#3 treatment. Phyla in order from top to bottom of the bar: Actinobacteria, Firmicutes, Proteobacteria, Bacteriodetes.

transcription has been shown to decline with age (Distrutti et al., 2014). The differences in gene expression that occur with advanced age indicate that the spontaneous changes in gut microbiota composition could be an initiator of the inflammatory processes that lead to the attenuation of LTP. Distrutti et Figure adapted from Distrutti, E., O’Reilly, J. A., McDonald, C., al. (2014) noted that the extended treatment with VSL#3 reCipriani, S., Renga, B., Lynch, M. A., & Fiorucci, S. (2014). Modulation of intestinal microbiota by the probiotic VSL#3 resets brain gene versed these age-related changes in transcription, thereby reducing the inflammatory effects within the brain which would expression and ameliorates the age-related deficit in LTP. PLoS pose a risk to LTP. A similar approach to addressing transcripThis method of determining the relative composition tional changes in the aging brain was done by Simpson et al. of microbiota phyla is used in similar studies, such as Yang, Qi- (2009) who used microarray analysis to look at white matter an, Xu, Song, & Xiao, (2018). lesions. Analysis of LTP and Inflammation LTP decline has been closely associated with increased neuroinflammation, which both increase with age (Distrutti et al., 2014). Distrutti et al. (2014) selected CD68 and CD11b mRNA levels as markers of inflammation as they are indicators of microglial activation and have been associated with reduced LTP (Distrutti et al., 2014). It was found that both CD68 and CD11b levels increased in both aged (control and VSL#3 treated) models when compared to the young models (Distrutti et al., 2014). However, CD68 and CD11b mRNA levels in VSL#3 treated aged rats were lower in comparison to the control (Distrutti et al., 2014). The difference in CD11b levels was not deemed significant by Distrutti et al. (2014) and therefore is not indicative of any significant difference in inflammatory activity. These changes were coupled with a drastic increase in BDNF mRNA, which has been associated with increased neurogenesis LTP formation (Distrutti et al., 2014). CD68 levels are a common marker for microglial activation and are commonly used in studies like one done by Yang, Wang, Wu, & Jiao, (2018) to assess neuroinflammation. Distrutti et al. (2014) inserted an electrode into the perforant pathway of the dentate gyrus. After high frequency stimulation it was seen that both of the young groups and the VSL#3 treated aged group had higher levels of excitatory postsynaptic potential (EPSP), indicating greater LTP (Distrutti et al., 2014). This reinforces the hypothesis that decreased inflammation enhances the brain’s ability to perform LTP and that VSL#3 has the potential to rescue LTP in the aging brain. A similar study examining the effects of probiotic supplementation by Romo-Araiza et al. (2018) used electrodes to measure changes in LTP.

Figure 2: A) Graph showing the EPSP slopes of the perforant pathway for each test groups. Point of high frequency stimulation indicated by arrow. Legend: Black circle represents Aged control, white circle represents young control, black triangle represents Aged VSL#3 treatment, white triangle represents young VSL#3 treatment. B) Relative CD68 mRNA levels in the various test groups. Figure adapted from Distrutti, E., O’Reilly, J. A., McDonald, C., Cipriani, S., Renga, B., Lynch, M. A., & Fiorucci, S. (2014). Modulation of intestinal microbiota by the probiotic VSL#3 resets brain gene

Alterations in Gene Transcription One of the major components in this study was to look at the ways in which shifts in microbiota composition altered gene transcription in brain tissue. Distrutti et al. (2014) performed a microarray on cortical tissue from the various treatment groups and looked at trends in gene regulation. Researchers noted that genes involved in inflammatory processes were unregulated in the aged control rat model, specifically PLA2G3 and Alox15, in comparison to the young control rat model (Distrutti et al., 2014). This was coupled with the downregulation of Nid2, a membrane protein that has been associated with the prevention of β amyloid plaque formation and whose

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c and Alox15, in comparison to the young control rat model (Distrutti et al., 2014). This was coupled with the downregulation of Nid2, a membrane protein that has been associated with the prevention of β amyloid plaque formation and whose transcription has been shown to decline with age (Distrutti et al., 2014). The differences in gene expression that occur with advanced age indicate that the spontaneous changes in gut microbiota composition could be an initiator of the inflammatory processes that lead to the attenuation of LTP. Distrutti et al. (2014) noted that the extended treatment with VSL#3 reversed these age-related changes in transcription, thereby reducing the inflammatory effects within the brain which would pose a risk to LTP. A similar approach to addressing transcriptional changes in the aging brain was done by Simpson et al. (2009) who used microarray analysis to look at white matter lesions.

cline, Distrutti et al. (2014) examined the larger trends in gene transcription and translation in response to this shift in microbial composition. Specifically looking at the upregulation of genes like PLA2G3, which has been shown to be involved in cell apoptosis and Alzheimer’s disease (Martínez-García et al., 2010), spontaneously occur with age and alterations to the gut microbiota. Other studies mainly focused on BDNF production and the roles of bifidobacterium in ameliorating inflammatory processes. Critical Analysis

In their paper Distrutti et al. (2014), provided information on the genetic and biochemical consequences of an age induced change in microbiota composition. While providing integral information as to how the gut microbiota may affect the brain, the study fails to address some gaps in its methodology that prevent its findings from being widely comDiscussion prehensive and reliable. Notably Distrutti et al. (2014) used in Through their study Distrutti et al. (2014) were able to vivo rat models, yet they perform no behavioral assays in their demonstrate that the composition of the gut microbiome experiment. changes with age and that this change induces alterations in The use of behavioral assays is necessary in experiments inflammatory gene regulation. The study concluded that these regarding changes to brain chemistry and gene transcription as changes, which coalesce and result in the decline of LTP, can they provide context to these shifts. The role of BDNF in LTP be ameliorated and even partially reversed by the consistent formation and memory consolidation has been well estabadministration of the probiotic VSL#3 (Distrutti et al., 2014). lished (Taliaz et al., 2010). However, it is unclear from the The application of the VSL#3 probiotic can be used to intenstudy conducted by Distrutti et al. (2014) what forms of tionally shift the composition of the gut microbiota to attenumemory were altered by the VSL#3 induced increase in BDNF. ate the destructive aspects of age-related neuroinflammation. For example, in Romo-Araiza et al.’s (2018) study of the effects The authors note that the results of this study fall in line with of combination probiotic and prebiotic supplementation, they what has been previously written about the altered gut microfound that while the spatial memory improved in treated rats, biome and it’s wide-spanning effects on the metabolic and there was no significant change in associative memory. This genomic activity in the brain (Distrutti et al., 2014). suggests that the increase in BDNF does not have universal Distrutti et al.’s (2014) VSL#3 study is an example of effects in the brain and that Distrutti et al.’s (2014) analysis of the early exploration into the role of microbiota in neurogene- the relationship between memory and the gut microbisis and its relationship to aging. It has been cited as one of the ome may be incomplete. Behavioral assays allow researchers original research papers which examined the therapeutic to better understand the practical consequences of these bioeffects that changing the gut microbiome has on markers of logical changes and provide much needed information on the age-related cognitive decline, like neuroplasticity (Ticinesi et efficacy of specific interventions. al., 2018). Several other studies and reviews have since corAlthough Distrutti et al. (2014) tracked the changes in roborated the results found by Distrutti et al. (2014). A review common markers of neuroinflammation (e.g. CD68) and neuropaper by Heyck and Ibarra (2019), outlined the ways that neugenesis (e.g. BDNF), they neglected to look at compounds roinflammation is linked to age and how alterations to the gut widely associated with inflammation and regulation of LTP, ILmicrobiota, specifically an increase in certain strains of 1β, and TNF-α (Heyck & Ibarra, 2019). These cytokines have a bifidobacteria, has been shown to attenuate these inflammatowell-characterized and universal role in inflammatory processry processes and increase production of BDNF. A study by Roes throughout the body and as a result become an important mo-Araiza et al. (2018), also linked the administration of probifactor when evaluating this treatment. This exclusion ignores a otics to a shift in gut bacteria composition (increased proporkey area of study with regards to the microbiome and its tions of bifidobacteria), which was shown to ameliorate some effects on common inflammatory pathways in the body. of the learning deficits present in middle-aged rats. However, this study, unlike Distrutti et al.’s (2014), also looked at the When looking at the composition of the gut microbieffects of probiotics in combination with prebiotics. Some ome, Distrutti et al. (2014), should have gone into more specistudies like Rezaei Asl et al.’s (2019) even cite Distrutti et al. ficity than simply looking at the relative proportions of bacteri(2014), and expand on the conclusions made in the paper, ap- al phyla. As Tian et al. (2019), demonstrate in their paper, plying them to models of Alzheimer’s disease. different strains of bifidobacterium. longum subspecies can have varying effects on BDNF production (Tian et al., 2019). Unlike many of the other papers concerning the Therefore, by only addressing the changes in phyla Distrutti et effects the gut microbiota has on neuronal function and deal. (2014), ignore the complex interactions between subspecies 306

that play an important role in inflammatory activity. There have also been studies that look at the specific relationship to genera of Actinobacteria and Firmicutes. For example, a paper by Feng et al. (2019), suggests that bifidobacterium levels in the gut increase in abundance in response to the presence of lactobacillus. Further it has been shown that butyrate, a metabolite implicated in increased BDNF production and limiting the production of cytokines like TNF-α and IL-1β, is produced by Clostridium butyricum, part of the Firmicutes phyla (Heyck & Ibarra, 2019). These butyrate-producing bacteria feed off the acetate made by bifidobacteria (Alessandri, Ossiprandi, MacSharry, van Sinderen, & Ventura, 2019). Therefore, it is important to understand the relationship between bacterial groups as they perform complementary activities that facilitate many important biological processes. Simply showing relative changes in phyla gives too broad a picture and obfuscates complex microbial interactions. Future Directions Distrutti et al. (2014) established that changes in the composition of the gut microbiome of an individual can have a profound effect on neuronal function, ameliorating the effects of age-related neurodegeneration. Subsequent studies established that this is likely the result of bacterial interactions within the gut, which leads to the production of certain metabolites, specifically short chain fatty acids (SCFAs) such as butyrate. Therefore, it would be beneficial to design experiments that determine the bacterial compositions within the gut responsible for producing specific metabolites to better understand how these specific metabolites affect gene transcription. Specifically, these tests should look at the main genes discussed in Distrutti et al.’s (2014) paper, BDNF, Nid2, PLA2G3 and Alox15, in addition to common neuronal markers of inflammation like TNF-α and IL-1β. Understanding which metabolites alter the transcriptional activities within the brain will provide the foundational knowledge necessary for further investigation into the direct cellular mechanisms governing neural decline and open up pathways for clinical use of probiotics in the future. Following the general protocol for rat models laid out by Distrutti et al. (2014), researchers could select group of young and aged wild-type rats and assess their spatial memory using the Morris Water Maze test, as outlined in Romo-Araiza et al. (2018). Once the rat model’s spatial memory has been evaluated, then their stool samples can be collected. As shown in Heaney (2019), mass spectrometry can be used to analyze stool samples and determine microbial metabolic activity, especially the production of SCFAs. This data can then be used to determine which metabolites, especially SCFAs, are most common to young and aged conditions and in what combinations. Assigning metabolite profiles to various behaviours and enterotypes will help researchers better understand what molecular and cellular interactions underlying changes in neuronal function, especially in spatial memory.

searchers to get and understanding of how the metabolites administered affect neuronal function. Protocols outlined in Hoban et al. (2016) provide suitable methods for the administration of antibiotics in the development of models. Metabolites found in young rat models displaying intact spatial memory will be administered to aged rat models. Meanwhile young rat models will be given the metabolites produced by the microbiota of the older rat models. Brain tissue from the dentate gyrus, the area of the brain associated with spatial memory, can then be taken and put through quantitative real-time PCR (RT-qPCR) with primers for the specific genes of interest. This will provide specific data on the genes of interest and allow researchers to easily determine whether the application of the specific metabolites influenced transcription. The results of this sequencing are also an indication of whether the mechanisms involved in these transcriptional changes are purely the product of bacterial metabolic output. If the theory that the bacterial metabolites are driving these changes in the transcriptome is correct then researchers should see similar effects in the regulation of BDNF, Nid2, PLA2G3 and Alox15 that were seen in Distrutti et al. (2014). They should also see a downregulation of TNF-α and IL-1β in aged brains and an upregulation in younger brains. However, if these transcriptional changes are the result of other mechanisms that require the presence of living, dynamic bacteria, then the changes might be less pronounced or may not occur at all. This would require further study into the specialized functions and interactions living bacteria have in the gut beyond their established metabolic roles. It would also be beneficial to test the ability of the aged rats treated with metabolites to perform LTP. This can be done using the same protocol outlined in Distrutti et al. (2014) as it is measuring the same property under the same rational. Aged models treated with the metabolites of younger rat models should display a higher propensity for LTP formation while the young models should show a decreased ability to perform LTP. If this is not the case it is possible that the metabolite alterations in gene transcription were not significant enough to induce a phenotypic change or that they had no effect at all. Further tests would then need to be done into the role living gut microbiota play in LTP formation.

Distrutti et al. (2014) put forth possible connections between the gut and the brain that could explain their results: the vagus nerve and the immune system. Arboleya et al. (2016) note that bifidobacterium metabolites are known to have anxiety reducing abilities mediated through the vagal pathways. Therefore, researchers should repeat the protocol outlined in Distrutti et al. (2014), where one group is an aged rat model with an intact vagus nerve treated with VSL#3 and the other is an aged rat model with a severed vagus nerve treated with VSL#3. This would allow researchers to determine if the vagus To determine if the changes found in Distrutti et al. nerve plays a role in producing the results witnessed in the (2014) were driven solely by the metabolic output of the rat original paper. model’s microbiota researchers will need wild-type models who have been allowed to age naturally. They then must deplete their microbiomes using antibiotics. This will allow re307

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Overexpression of RGS8 Protein Correlation to Elongation of Primary Cilia Induces Antidepressant-like Phenotype Mila Phan

Regulator of G protein signaling (RGS) proteins are known to negatively regulate G protein coupled receptors (GPCR). The main GPCR in mice are melanin-concentrating hormone receptor 1 (MCHR1). Knock out MCHR1 experiments on mice models are known to induce antidepressant-like phenotypes. RGS8 protein act to negatively regulate MCHR1, so there is speculation of it also inducing an antidepressant-like phenotype. Kobayashi et al (2018) created a mice model of overexpressing RGS8 gene called RGS8tg and compared how it performs in several behavioural tests measuring for depression and anxiety-like behaviours to the wild type (WT). They found reduced immobility duration during forced swimming test (FST) and reduced stress-induced hyperthermia. When RGS8tg was treated with SNAP94847, a MCHR1 antagonist, during FST there was no change in immobility duration. However, treatment of desipramine, a norepinephrine reuptake inhibitor, induced further reduced immobility duration. This indicated that RGS8tg already acts to block MCHR1, through similar mechanisms as SNAP94847. This also indicates RGS8 protein isnâ&#x20AC;&#x2122;t involved in the monoaminergic system when dealing with depression since there were effects when treated with desipramine. RGS8tg mice also had increased RGS8 mRNA expression at the cerebellum and hippocampus and increased RGS8 protein levels specifically at the hippocampal CA1 where there was also elongated primary cilia. Overall, the results showed RGS8tg displayed antidepressant-like phenotype and the authours credit this possibly due to the relationship between RGS8 and elongated primary cilia. Therefore, the authours concluded that with these results, there is room for future RGS8 regulation for treating mood disorders.

Key words: regulator of G protein signaling protein 8 (RGS8), melanin concentrating hormone (MCH), melanin concentrating hormone receptor 1 (MCHR1), hippocampus, cerebellum, anxiety, depression, treatment, desipramine, SNAP94847, forced swimming test (FST), stress-induced hyperthermia test, primary cilia, elongation

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BACKGROUND or INTRODUCTION Heterotrimeric G protein coupled receptors (GPCRs) contain an alpha subunit which is known for its slow intrinsic GTPase activity [1]. Due to the alpha subunit’s slow activity, cells require mechanisms in which there is rapid activation and inactivation. This is where regulator of G protein signaling (RGS) proteins play a role by negatively regulating GPCRs by binding to their alpha subunits and stimulating quicker GTPase activity [2]. In rodents, the main GPCR is melanin-concentrating hormone receptor 1 (MCHR1) which is activated by a neuropeptide called melanin-concentrating hormone (MCH) [2]. The MCH-MCHR1 system has previously been implicated in depression and anxiety in studies using MCHR1knock out (KO) rodent models where they’re found to have an antidepressant/anxiolytic-like phenotype [3, 4]. Since MCHR1 may be involved in depression, this has led to research in what type of protein-protein interactions occur in this system. MCHR1 has been found to interact with a variety of RGS protein such as RGS2 [5] and RGS8 [6]. Particularly, RGS8 protein inhibit specific G-alpha subunit GTPase activity thus acting as an antagonist for MCHR1 [6]. In Kobayashi et al (2018) the authours used the established relationship between MCHR1 and RGS8 to examine how overexpression of RGS8 may lead to regulation of anxiety or depression-like behaviours. The authours created a transgenic overexpressing RGS8 mouse model (RGS8tg) and compared its behaviour in a variety of tests against the wild type (WT). Behavioural tests measured for anxiety/depression-like behaviours and included the social interaction test, stress-induced hyperthermia, forced swimming test (FST), sucrose-consumption test, and the open-field test. In addition to the FST, the mice were administrated either SNAP94847, a MCHR1 antagonist, or desipramine, a selective norepinephrine reuptake inhibitor, to compare its effects. The authours found no major differences between the mice for the open-field test, social interaction test and sucrose-consumption test. However, comparing the mice during stress-induced hyperthermia, RGS8tg seemed to have reduced hyperthermia after stress. As well as during FST, the RGS8tg mice had reduced immobility duration, further reduced immobility duration when treated with desipramine, and no change from treatment of SNAP94847. The authours found increased RGS8 mRNA expression in both the Purkinje layer of the cerebellum and pyramidal layer of the hippocampus, in which RGS8 mRNA expression here is found basally [7]. Furthermore, they also found increased RGS8 protein expression in RGS8positive neurons of the hippocampus in RGS8tg mice which also correlated with elongated primary cilia. Overall, based on their findings, the authours were able to conclude that RGS8tg had induced an antidepressant-like phenotype where future modulation of RGS8 can be involved in the treatment of mood disorders.

other, however, no differences in behaviour were found between the mice. The stress-induced hyperthermia test was conducted by measuring rectal-temperatures before and after the FST, which was the stress event. This test involved measuring anxiety levels by looking at the physiological state of the mouse using temperature, and found that RGS8tg had reduced hyperthermia after stress compared to WT. These results indicate that RGS8tg undergoes a type of reduced physiological signaling under stress, however, this anxiolytic behaviour doesn’t present itself through locomotive or social defective behaviours.

Figure Adapted from Kobayashi et al. (2018)Neuroscience, 383, 160169. Figure 1. Stress-induced hyperthermia test after FST. White bar (left): indicates WT. Black bar (right): indicates RGS8tg. Stress-induced hyperthermia is a measure of anxiety-like behaviour. RGS8tg had reduced stress-induced hyperthermia.

Antidepressant-like Phenotype in RGS8tg After FST

The authours measured depression-like behaviours using 2 different tests, this includes the sucrose-consumption test and FST. Sucrose-consumption test was conducted by measuring sucrose water intake before and after FST, which was the stress event. The authours found no difference in sucrose water intake between the mice. FST was conducted by placing the mice in a deep tank for 6 minutes where they’re forced to swim to stay afloat. Depression-like behaviours were measured based on how much movement the mouse displayed under these conditions. This means because RGS8tg was found to have reduced immobility, it implies a less depressed phenotype. This finding of reduced depression-like behaviour has been similarly replicated in studies using KO MCH and injection of MCHR1 receptor antagonist [3]. Sucrose consumption test indicates that there is no difference in depression-like behaviours, however, FST indicates there is. This discrepancy can’t be due to locomotion defects in the WT, as we know based on the findings in the open-field test. However, previous studies have found that administration of MCH, thus activation of the MCH-MCHR1 system, induces a nonspecific eating behaviour [8], which may explain why there is no overeating in the RGS8tg mice since MCH-MCHR1 system isn’t activated. Therefore, MAJOR RESULTS since FST involves locomotion and we know locomotion isn’t Anxiolytic Phenotype in RGS8tg After Stress-induced Hyperthermia affected by an external system, it proves to be a better measureTest ment for depression-like behaviour. The authours measured anxiety-like behaviours using 3 different behavioural tests, this includes the open-field test, social interaction test, and stress-induced hyperthermia. The open-field test was based off the number of times the mouse would spend in the middle of a cuboidal apparatus, this was to measure exploratory drive and anxiety levels using locomotion as the parameters. The authours found no differences in behaviour and locomotion between the mice during this test. The social interaction test was conducted by placing two mice of similar weight in an apparatus where they were scored for their social behaviours with one an-

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cFigure Adapted from Kobayashi et al. (2018)Neuroscience, 383, 160ed to be longer, this was not seen in CA3 region. This suggests 169. there is a correlation between RGS8 protein levels and the length Figure 2. Immobility durations during FST. White bar (left): indicates of primary cilia. In a previous study, it’s been shown that treatment WT. Black bar (right): indicates RGS8tg. FST is a measure of depression- of MCH thus activation of MCHR1 for a few hours induced primary like behaviour. RGS8tg had reduced immobility duration therefore cilia shortening [6]. This gives rise to the idea that RGS8tg may reduced depression-like behaviour. have reduced cilia length modulation, thus producing elongated cilia. Alternatively, there is another study linking the relationship Administration of SNAP94847 Doesn’t Affect RGS8tg During FST, mice could either be administrated SNAP94847 between cilia length perturbation in neocortical pyramidal neurons or desipramine. The authours found that when RGS8tg mice were and impaired dendrite growth [9]. This could mean that RGS8tg given SNAP94847 there was no change in their already reduced elongated cilia may relate to stronger synaptic signaling due extenimmobility, but given desipramine, the mice had further reduced sive dendrite growth. Unfortunately, the authours haven’t disimmobility. Meanwhile, the WT reduced immobility similarly to cussed why protein levels and cilia length are unaffected at the RGS8tg without any treatment when given either SNAP94847 or CA3 region even though it also contained AC3/MCHR1 double posidesipramine. The main result from this experiment is that tive cilia. SNAP94847 didn’t affect RGS8tg depression-like behaviour, this is likely due to the overexpression of RGS8 which is known to block MCHR1 already. In this manner, RGS8tg phenotype is equivalent to being repeatedly treated with SNAP94847. Desipramine influencing RGS8tg immobility duration indicates that RGS8 works independently of the monoaminergic system and isn’t associated with the MCH-MCHR1 system. Change in immobility duration for desipramine treatment in RGS8tg also shows that the lack of change during SNAP94847 treatment can’t be due to locomotive differences between the mice.

Figure Adapted from Kobayashi et al. (2018)Neuroscience, 383, 160169.Figure 4. (A) Shows RGS8 protein level differences through fluorescent activity in the hippocampal CA1. White bar (left): indicates WT. Black bar (right): indicates RGS8tg. RGS8tg has higher levels of RGS8 protein in CA1. (B) Shows primary cilia length differences in the CA1 region. White bar (left): indicates WT. Grey bar (right): indicates RGS8tg. RGS8tg has elongated primary cilia in CA1. (C) Shows RGS8 protein levels in the cerebellum and CA1 region with either a WT or RGS8tg background using antisense probes. Top: indicates wild type. Bottom: indicates RGS8tg. RGS8tg has higher RGS8 protein levels in the cerebellum and CA1 region. Figure Adapted from Kobayashi et al. (2018)Neuroscience, 383, 160169. CONCLUSIONS/DISCUSSION Figure 3. Mice administrated with either SNAP94847 or desipramine and their immobility durations during FST. White bar: indicates no treatment. Grey bar: indicates treatment of SNAP94847. Black bar: indicates treatment of desipramine. SNAP94847 is a MCHR1 antagonist, desipramine is a norepinephrine reuptake inhibitor. RGS8tg displays no change and further reduced immobility durations for SNAP94847 and desipramine, respectively.

Based on having reduced stress-induced hyperthermia and decreased immobility time in FST, RGS8tg seems to confer an antidepressant-like and anxiolytic-like phenotype. This phenotype may be due to the increase of RGS8 protein and elongated primary cilia at the hippocampal CA1 region. Based on these results, the RGS8tg Have Increased RGS8 Protein Levels and Elongated Primary authours have concluded that RGS8 may be a potential candidate for the development of antidepressants. Cilia at Hippocampal CA1 The authours found relative increases in RGS8 mRNA expression in areas such as the cerebellum and hippocampus. However, using immunohistochemistry (IHC) staining in the cerebellum there was non-significant change in levels of RGS8 protein, but levels were increased in the hippocampal CA1. Although the authours didn’t address the discrepancy between increased mRNA expression and no change in protein levels at the cerebellum, it can be assumed there was some type of post-translational dysregulation in RGS8tg occurring only at the cerebellum. To see how increased protein expression at CA1 in RGS8tg may play a role in the antidepressantlike phenotype and interactions with MCHR1, the authours first had to find where MCHR1 was localized. MCHR1 was co-stained with a marker for primary cilia called AC3 and from there, the authours found that AC3/MCHR1 double-positive cilia in CA1 tend-

Many studies have indicated the importance of RGS protein in multiple central nervous system disorders such as RGS2 being involved in anxiety [5] or RGS4 being involved in schizophrenia [10]. However, aside from Kobayashi et al (2018), there aren’t many studies indicating relationships between disorders and RGS8 protein. Furthermore, much of the current research on RGS8 doesn’t explore its potential impact on behavioural performance or how it may relate to the MCH-MCHR1 system, even though many studies have highlighted its significance in depression-like behaviours [3, 4, 5, 11]. Kobayashi et al (2018) helps expand our knowledge of the MCH-MCHR1 system and specifically highlights the importance of MCHR1 activation in depression-like behaviours, as well as it is the first study to link an antidepressant-like pheno-

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FUTURE DIRECTIONS to elongated cilia. Although, this is not the first study to show that there may be a correlation between elongated primary cilia and other disorders. A previous study has shown treatment of lithium, which is used as an anti-manic for bipolar disorder, correlated with elongated primary cilia in the dorsal striatum and nucleus accumbens [12]. In a separate study, we see elongation of primary cilia in the dorsal striatum of Parkinson’s models after degeneration of dopaminergic neurons, which the authours attributed this cilia elongation to lack of dopaminergic inputs [13]. This study similarly gives rise to the idea that RGS8tg may have elongated primary cilia due to lack MCHR1 inputs which reduces primary cilia length modulation [6, 11].

CRITICAL ANALYSIS When the authours measured for anxiety-like behaviours, they used three parameters: locomotion, social behaviour, and physiological temperature. Looking at locomotion-related and social behaviour-related parameters, we don’t see discrepancy between anxiety levels, so this may indicate that RGS8 doesn’t affect motor input or exploratory drive but may require some type of stressing signal for RGS8 activation as seen in the stress-induced hyperthermia test. Based on previous studies, activation of the MCH-MCHR1 system by injection of MCH elevates plasma ACTH and corticosterone levels [11]. This may imply that because RGS8 blocks activation of the MCH-MCHR1 system, there will be lower ACTH and corticosterone levels. Lower stress hormone levels may explain why RGS8tg only displays reduced anticipatory anxiety physiologically but is unaffected through other measures such as locomotion. In order to test for this, the authours can reperform the stressinduced hyperthermia test in RGS8tg and WT but also measure for stress hormone levels in order to see if there is less hypothalamicpituitary-adrenal axis activation that may occur solely in RGS8tg. Alternatively, even though there is an increase in RGS8 protein in RGS8tg, it does not indicate that the protein is actively working, as it could have post-translational modifications. This may mean that RGS8tg may only display anxiolytic-like behaviours under a specific stress that signals and removes any post-translational modifications that will in turn affect physiological conditions such as temperature. In order to test for this, the authours have to measure if there are increased levels of post-translational modifications in RGS8tg under normal conditions, and decreased levels during/after stress conditions. In RGS8tg mice the authours found increased levels of mRNA expression in both the cerebellum and hippocampus but no increased levels of protein in the cerebellum. This may be explained due to post-translational modifications occurring specifically in the cerebellum. RGS protein are often modified by phosphorylation or being marked for proteasomal/lysosomal degradation [14]. In order to test this, the authours can measure for levels of phosphorylation and degradation in the cerebellum of RGS8tg and then compare it to the levels in the hippocampal CA1. To assess the importance of modulating RGS8 protein levels, the authours can attempt to microinject high levels of RGS8 into the cerebellum of WT and observe if there are any differences in behaviour and primary cilia length. However, a limitation would be that modulation of RGS8 protein in the cerebellum may be there for a reason, whereby microinjection of high levels of RGS8 may be lethal.

In Kobayashi et al (2018) the authours conclude that in the future, they would like to observe if RGS8 protein plays a role in non -neuronal cells and whether it also affects their primary cilia lengths. They also noticed other RGS proteins such as RGS2 or RGS6 are involved in anxiety-like and depression-like behaviours by interacting with serotonin. Thus, there is potential for there to be a link between RGS8 and serotonin that may also help build the case that RGS8 is a potential candidate for development of antidepressants. In order to confirm that post-translational modifications are the cause of normal RGS8 protein levels in the cerebellum, the authours can perform a pulse chase assay to measure degradation. Pulse chase assay can be performed by using radiolabeled RGS8 protein and microinjecting it into the cerebellum and CA1 of WT to observe and compare rates of degradation. If in the cerebellum there is a higher rate of degradation, then we can assume there are mechanisms in which high levels of RGS8 protein is unwanted and must be rapidly degraded. If the cerebellum and CA1 have the same rate of degradation, then this means turnover rate is not the main mechanism of which RGS8 protein levels are the same in RGS8tg and WT cerebellums. Alternatively from turnover rate, the authours can measure phosphorylation modification levels which act to inactivate RGS protein [14]. Phosphorylation modifications can be identified by performing a western blot, first by separating the protein sample from the cerebellum from both RGS8tg and WT with SDSpage and then loading the sample on a gel where it’s later transferred to a membrane. On these two membranes we will introduce the phosphorylation antibody in order to compare the membranes for levels of phosphorylation. If RGS8tg has higher phosphorylation levels, then this means the cerebellum doesn’t have use for increased levels of RGS8 protein so it will inactivate it through phosphorylation modifications. If RGS8tg has the same levels of phosphorylation, then we can assume there is another mechanism in which post-translational modifications are occurring in RGS protein that hasn’t been highlighted yet. The authours make their conclusions assuming RGS8 protein is linked to elongated primary cilia, but there haven’t been experimentations done to confirm this relationship. What’s known is in the Purkinje cerebellar layer there is normal expression of RGS8 as well as primary cilia containing MCHR1 [15]. In order to confirm the relationship between RGS8 protein and primary cilia length, the authours can attempt to perform a gene knockdown experiment on RGS8 using RNA interference specifically in the Purkinje layer of the cerebellum of WT. By comparing the control WT and gene knockdown primary cilia lengths, the authours can measure if there is shortening of the gene knockdown’s primary cilia length. If cerebellar primary cilia are shortened, we can better confirm the authour’s conclusion that there is a relationship between elongated primary cilia and increased RGS8 expression, thus an antidepressant-like phenotype. If the cerebellar primary cilia are the same, then either the authour’s conclusion is disproven or there is another mechanism specifically in the cerebellum interfering with what usually occurs in the hippocampus. This experiment can only be done assuming reduced levels of RGS8 protein in the cerebellum isn’t lethal, but if it is lethal, we can perform the same experiment but at a different brain region that also contains AC3/MCHR1 double positive neurons and where RGS8 mRNA expression is normal, for example, ideal brain regions could be the dorsal raphe neurons or the hippocampal CA3 [7, 16].

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REFRENCES 1. Koelle MR. A new family of G-protein regulators â&#x20AC;&#x201C; the RGS protein. Current Opinion in Cell Biology. 1997; 9:143-147. 2. Kobayashi Y, Takemoto R, Yamato S, Okada T, Iijima M, Uematsu Y, Chaki S, Saito Y. Depression-resistant phenotype in mice overexpressing regulator of G protein signaling 8 (RGS8). Neuroscience. 2018; 383:160-169. 3. Georgescu D, Sears DM, Hommel JD, Barrot M, Bolanos CA, Marsh DJ, Bednarek MA, Bibb JA, Maratos-Flier E, Nestler EJ, DiLeone RJ. The hypothalamic neuropeptide melanin-concentrating hormone acts in the nucleus accumbens to modulate feeding behaviour and forced-swim performance. The Journal of Neuroscience. 2005; 25:2933-2940. 4. Roy M, David NK, Danao JV, Baribault H, Tian H, Giorgetti M. Genetic inactivation of melanin-concentrating hormone receptor subtype 1 (MCHR1) in mice exerts anxiolytic-like behavioral effects. Neuropsychopharmacology. 2005; 31:112-120. 5. Matsubara-Miyamoto M, Chung S, Saito Y. Functional interaction of regulatory of G protein signaling-2 with melanin-concentrating hormone receptor 1. Annals of the New York Academy of Sciences. 2010; 1200:112-119. 6. Hamamoto A, Yamato S, Katoh Y, Nakayama K, Yoshimura K, Takeda S, Kobayashi Y, Saito Y. Modulation of primary cilia length by melanin-concentrating hormone receptor 1. Cellular Signaling. 2016; 28:572-584. 7. Gold SJ, Ni YG, Dohlman HG, Nestler EJ. Regulators of G-protein signaling (RGS) proteins: Region-specific expression of nine subtypes in rat brain. The Journal of Neuroscience. 1997; 17:8024-8037. 8. Duncan EA, Proulx K, Woods SC. Central administration of melanin-concentrating hormone increases alcohol and sucrose/quinine intake in rats. Alcoholism: Clinical and Experimental Research. 2005; 29:958-964. 9. Guadiana SM, Semple-Rowland S, Daroszewski D, Madorsky I, Breunig JJ, Mykytyn K, Sarkisian MR. Arborization of dendrites by developing neocortical neurons is dependent on primary cilia and type 3 adenylyl cyclase. The Journal of Neuroscience. 2013; 33:2626-2638. 10. Mirnics K, Middleton FA, Stanwood GD, Lewis DA, Levitt P. Disease-specific changes in regulator of G-protein signaling 4 (RGS4) expression in schizophrenia. Molecular Psychiatry. 2001; 6:293-301. 11. Smith DG, Davis RJ, Rorick-Kehn L, Morin M, Witkin JM, McKinzie DL, Nomikos GG, Gehlert DR. 11. Melanin-Concentrating Hormone-1 Receptor Modulates Neuroendocrine, Behavioral, and Corticolimbic Neurochemical Stress Responses in Mice. Neuropsychopharmacology. 2005; 31:1135-1145. 12. Miyoshi K, Kasahara K, Miyazaki I, Asanuma M. Lithium treatment elongates primary cilia in the mouse brain and in cultured cells. Biochemical and Biophysical Research Communications. 2009; 30:757-762. 13. Miyoshi K, Kasahara K, Murakami S, Takeshima M, Kumamoto N, Sato A, Miyazaki I, Matsuzaki S, Sasaoka T, Katayama T, Asanuma M. Lack of dopaminergic inputs elongates the primary cilia of striatal neurons. Public Library of Science. 2014; 9:e97918. 14. Alqinyah M, Hooks SB. Regulating the regulators: epigenetic, transcriptional, and post-translational regulation of RGS proteins. Cellular Signaling. 2018; 42:77-87. 15. Chizhikov VV, Davenport J, Zhang Q, Shih EK, Cabello OA, Fuchs JL, Yoder BK, Millen KJ. Cilia proteins control cerebellar morphogenesis by promoting expansion of the granule progenitor pool. The Journal of Neuroscience. 2007; 27:9780-9789. 16. Nino-Rivero S, Torterolo P, Lagos P. Melanin-concentrating hormone receptor-1 is located in primary cilia of the dorsal raphe neurons. Journal of Chemical Neuroanatomy. 2019; 98:55-62.

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Apelin-36: An Overlooked Peptide with Promising Effects on Parkinson’s Disease Andrea Pinto

Parkinson’s Disease (PD) is the second most prevalent progressive neurodegenerative disease. It is attributed to a gradual loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) due to the accumulation of misfolded α-synuclein proteins. As a result, common symptoms of PD include rigidity of the muscles, a resting tremor, a shuffling gait and bradykinesia, all of which vary in severity among diagnosed individuals. This variance produces a lack of understanding on how to reverse the disease pathology. Zhu et al. (2019), attempts to study the mechanism by which Apelin-36 prevents endoplasmic reticulum stress (ERS) to ameliorate the common symptoms associated with PD. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridin (MPTP) was injected into C57BL/6 mice to induce neurotoxicity mirroring the hallmark effects of PD. Injection of Apelin-36 into the SNpc of MPTPtreated mouse models improved motor deficits. The treatment also decreased levels of α-synuclein expression but rescued the progressive loss of dopaminergic neurons, SH-SY5Y cells, and tyrosine hydroxylase (TH) expression. Furthermore, a suppression of GRP78, CHOP and the cleaved caspase12 is detected, indicating an aversion of ERS. Considering these results, Apelin-36 establishes as a promising treatment for PD by improving the major clinical symptom, motor disturbances. This approach can also be considered as a treatment option for other neurodegenerative disorders with similar symptoms such as Alzheimer's Disease and amyotrophic lateral sclerosis. Moreover, the results reveal a greater understanding of the mechanism by which Apelin-36 can induce neuroprotective effects. Key words: Parkinson’s disease, neurodegeneration, MPTP-induce mouse models, SH-SY5Y cells, substantia nigra, endoplasmic reticulum stress (ERS), Apelin-36, treatment

315

INTRODUCTION

Parkinson’s Disease (PD) is becoming an increasingly concerning neurodegenerative disease due its forecasted prevalence. The disease is further compounded by its complexity, involving a multitude of risk factors as well as affecting both motor and psychiatric function at varying severities (Bietz, 2014). Due to this complexity and variability in symptoms, there has been a low yield in promising treatments. Motor dysfunctions such as bradykinesia, have been associated with the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) as result of α-synuclein aggregation. Zhu et al. (2019) attempts to inhibit endoplasmic reticulum stress (ERS) to alleviate both the loss of dopaminergic neurons and α-synuclein aggregation, thus improving motor deficits. The mechanism by which this is achieved, involves examining the neuroprotective effects of Apelin-36 on MPTP-treated mice, which reveals its potentiality in treating PD (Zhu et al., 2019). An arduous, yet crucial step to developing a treatment, is to establish a model for the disease. A common chemically induced model for PD is an intranigral injection of MPTP into C57BL/6 mice . MPTP is capable of crossing the blood brain barrier due to its lipophilic properties, where it is then modified into its toxic form, 1-methyl-4-phenylpyridinium, also known as MPP+. The toxic metabolite has a strong selectivity for dopamine receptors, causing an efficient uptake into dopaminergic neurons. Upon its uptake, MPP+ concentrates within the mitochondria to produce reactive oxygen species (ROS), becoming toxic to the neurons (Langston, 2017). Loss of dopaminergic neurons, consequently decreases levels of tyrosine hydroxylase (TH), a ratelimiting enzyme involved in the synthesis of dopamine (Wang et al., 2017). These effects are also demonstrated in SH-SY5Y cells, a neuroblastoma cell line commonly used to study neurodegenerative diseases (Ko et al., 2018; Xicoy et al., 2017). Similarly to the development of PD, MPTP conveniently targets dopaminergic neurons of the SNpc exclusively (Langston, 2017). Although this selectivity remains unclear, it is apparent that MPTP-induced mice models are a valuable model for PD by mirroring its pathophysiology.

Blotting was conducted to detect expression of TH and αsynuclein in SNpc. Lastly, cell viability was assessed using CCK8 assay whereas cell apoptosis was measured by flow cytometry and TUNEL staining. The results of these experiments indicated, intranigral injection of Apelin-36 was capable of dissipating neurotoxic effects by promoting survival of MPP+- treated SHSY5Y cells, preventing further loss of dopaminergic neurons, restoring TH expression, and improving motor functions of MPTP-induced mice models. Apelin-36 also had the added benefit of inhibiting α-synuclein expression thus preventing further neurodegeneration. Furthermore, a mitigation of ERS was observed in Apelin-36 treated MPTP-induced mouse models. Effects on ERS were concluded by noting a decrease in GRP78, CHOP and cleaved caspase-12 in SH-SY5Y cells, which overall inhibited apoptosis (Zhu et al., 2019).

MAJOR RESULTS

Dissipation of Neurotoxicity Apelin-treated MPTP-induced mouse models displayed a dissipation in neurotoxicity, as measured by the survival of SH-SY5Y cells, dopaminergic neurons, TH expression, and motor function. SH-SY5Y cells were initially treated with Apelin-36 followed by MPP+. CCK-8 assay shows higher dosages of Apelin-36 (1µM) is correlated with a greater cell viability. On the other hand, quantitative analysis of flow cytometry (Fig. 1) reveals decreased levels of cell apoptosis, corroborating the increased cell viability revealed by the CCK-8 assay. Although the effects of Apelin-36 on SH-SY5Y cells have yet to be studied by other groups, the study of Jiang et al . (2018) also noted a gradual increase in the survival of MPP+-treated SH-SY5Y cells after treatment with its isoform Apelin-13.

Another significant contributor to the loss of dopaminergic neurons, are the consequences of endoplasmic reticulum stress (ERS) (Martinez et al., 2017). ERS is triggered by an accumulation of α-synuclein within the ER which activate ER transmembrane protein kinases, PERK and IRE1α (Ryu et al., 2002). A signalling cascade is then initiated, producing transcription factors to promote apoptosis. ERS serves an important interference point to which loss of dopaminergic neurons can be averted, Fig. 1: Quantitative analysis of flow cytometry indicates a decrease in cell apopthus decelerating neurodegeneration within individuals. tosis in MPP+ treated SH-SY5Y cells, occurring in a dose-dependent manner. To reverse the effects of neurodegeneration, Apelin-36 Figure Adapted from Zhu et al. (2019) Brain Research, 1721. emerges as a potential treatment option. Apelin-36 is a neuropeptide derived from Apelin, an endogenous ligand of a G-protein An attenuation of neurotoxicity is also indicated by an increase in coupled receptor (GCPR) (Sakamoto et al., 2016). Most studies dopaminergic neurons in the Apelin-36+MPTP mice group comare currently examining the ability of Apelin to protect brain inju- pared to the MPTP group, as presented by immunohistochemistry ry caused by ischemia. Apelin is naturally produced within the and immunofluorescence of TH. Increased levels of TH expresnervous system and has associated neuroprotective properties sion reflects increased prevalence of dopaminergic neurons. Likewise to the previous experiment, Apelin-13 treatment on 6(Ishimaru et al., 2017). hydroxydopamine treated mice also revealed an attenuation of Examination of these neuroprotective aspects highlights dopaminergic neuron death (Haghparast et al, 2019). Apelinits importance in reversing Parkinsonian symptoms. The study of 36+MPTP mice further demonstrated increased TH expression Zhu et. al consisted of four groups, which received injections of compared to the MPTP mice group. Due to the lack of experisaline, MPTP and/or Apelin-36 for 5 days. The control group was ments conducted using Apelin-36, these results are unable to be injected with saline. The Apelin-36 group was injected with Ape- confirmed by current literature. However, the results align with lin-36 and saline. The MPTP group was injected with MPTP and the improved motor function of Apelin-36+MPTP group, as a saline. Finally, the Apelin-36+MPTP group was injected with significantly longer time was spent on the rotarod compared to Apelin-36 and MPTP . Motor disturbances were measured using MPTP group (Fig. 2). Considering these results, Apelin-36 disa rotarod test. Immunohistochemistry and immunofluorescence plays an ability to dissipate neurotoxicity and improve Parkinwere used to evaluate loss of dopaminergic neurons. Western sonian symptoms . 316

Fig 2: MPTP group shows significant loss in motor function, which was reversed after Apelin-36 treatment, as seen by an increased time spent on the rotarod. Figure Adapted from Zhu et al. (2019) Brain Research, 1721.

Regulation of α-synuclein A decelerating loss in dopaminergic neurons suggests an involvement between Apelin-36 and α-synuclein. Western blotting reveals increased α-synuclein in the MPTP group which was significantly repressed after Apelin-36 treatment in the mice models and SH-SY5Y cells. This repression in α-synuclein is consistent with a previous study conducted by Zhu et. al. (2019) using identical experimental procedures . The ability for Apelin-36 to prevent α-synuclein aggregation in SNpc, suggests ERS may also be mitigated. Mitigation of ERS Effects of Apelin-36 on ERS was the focus in the study of Zhu et al. (2019), as it suggests to be the main target in preventing progressive neurodegeneration. Expression of GRP78, CHOP and cleaved caspase-12 were used as to reflect the severity of ERS. Both Western Blotting and immunofluorescent staining revealed increased expression of GRP78 (Fig. 3-A), CHOP (Fig.3-B), and cleaved capse-12 (Fig. 3-C) in the MPTP group, but expression of all three indicators were inhibited by Apelin36 treatment. Jian et. al. (2017) also found decreased levels of GRP78, CHOP and cleaved caspase-12 in MPTP-treated mice which was associated with an attenuation of ERS. However, this study involved Apelin-13 treatment as opposed to the Apelin36. Nevertheless, a reduction in GRP78, CHOP and cleaved caspase-12 mitigated ERS, emphasizing the relevance of the ER in cell viability.

Fig 3: Quantitative analysis of the Western Blotting reveals decreased expression of GRP78 (A), CHOP (B), and cleaved capase-12 (C) in SH-SY5Y cells treated with MPP+, indicating an overall mitigation of ERS. Figure Adapted from Zhu et al. (2019) Brain Research, 1721.

DISCUSSION From the experiments conducted by Zhu et. al. (2019), intranigral injection of Apelin-36 into MPTP-treated mouse models was capable of dissipating neurotoxicity by promoting survival of SH-SY5Y cells, presence of dopaminergic neurons, expression of TH, and motor function. Furthermore, the treatment prevented aggregation of α-synuclein in not only the mouse models but as well as the MPP+ treated SH-SY5Y cells. Most importantly, Apelin-36 mitigated ERS by decreasing expression of its indicators, GRP78, CHOP, and cleaved caspase12. Taking these results into consideration, the authors highlight the neuroprotective abilities of Apelin-36 as well as the inhibition of ERS, causing an overall reversal of Parkinsonian symptoms. Previous studies have only investigated the role of Apelin (Ishimaru et. al, 2017) or Apelin-13 (Jiang et. al, 2018) in neurodegenerative disorders. Ishimaru et al. (2017) reveal an acceleration in retinal neuronal degeneration in Apelin deficient mice, as a result of downregulation in its GCPR signaling. On the other hand, Jiang et al. (2018), demonstrates the protective effects of Apelin-13 on MPP+-treated SH-SY5Y cells by promoting its survival. Both studies strengthen the correlation between presence of Apelin and the prevention of neuronal death. However, there exists a lack of knowledge whether the success of Apelin and Apelin-13 in decelerating neurodegeneration also extends to Apelin-36. Zhu et al. (2019) attempt to fill this gap by introducing Apelin-36 as a possible treatment option for PD. The treatment has produced promising results both in vitro using MPP+treated SH-SY5Y cells and in vivo using MPTP-induced mouse models. This neuropeptide sets itself apart from other treatments such as Amantidine, by its ability to combat several mechanisms

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involved PD pathobiology, as opposed to a single target (Paquette et al., 2013). This is significant considering individuals diagnosed with PD experience several symptoms at varying severities. Although significant research has been published on the molecular pathway of ERS (Ryu et al., 2002) and its consequences (Martinez et al., 2017), Apelin-36 appears to be one of the few drugs able to attenuate this response. As a result, this study creates an opportunity to identify certain targets to suppress within the ERS. Additionally, it shifts the focus of researchers to an entirely unexplored area of neuroscience requiring further attention to the specific molecular mechanisms by which Apelin-36 induces these neuroprotective effects. By considering this avenue for treatment, it generates a new perspective through which researchers can approach other neurodegenerative diseases as well.

CRITICAL ANALYSIS As alluded to previously, the results of the experiment examining the effects of Apelin-36 on dopaminergic neurons, α-synuclein, TH levels, motor function, SH-SY5Y cells and ERS, are all heavily supported by existing literature. This becomes extremely crucial as Zhu et al. (2019) are one of the few groups investigating Apelin-36 treatment through the lens of PD. By performing experiments on already heavily researched topics, the authors rationale for the peptide’s neuroprotective effects becomes increasingly plausible. Another favourable aspect of the study was that its experimental procedures were conducted both in vitro and in vivo. An in vitro experiment performed on MPP+-treated SH-SY5Y cells, allows to examine the effects of the treatment on a molecular basis as well as its relevance to human cells (Xicoy et al., 2017). An in vivo experiment was also performed using the MPTP-induced mouse models, allowing to observe the effects of the treatment in the context of the whole organism. Furthermore, the experiment itself involved a localized injection of Apelin-36 into the SNpc, ensuring only the cells of the SNpc are affected (Machado et al., 2011). Contrastingly, MPTP was systemically introduced via an intraperitoneal injection. As a result, the neurotoxin is able to naturally reproduce the pathophysiology similarly displayed in individuals diagnosed with PD. This includes the aggregation of α-synuclein, the pattern of dopaminergic neuron loss, and some behavioural deficits (Meredith et al., 2011). Despite the benefits of the MPTP-induced mouse model, much controversy persists surrounding its use to accurately recapitulate PD pathology as observed in humans. Zeng et. al (2018) contest the benefits of this model due to its inability to generate Lewy-body inclusions, a major hallmark of PD. This issue becomes most prominent if MPTP is introduced intraperitoneally, as performed by the Zhu et al (Gibrat et al, 2009). Beyond this, PD is often diagnosed in humans approximately at the age of 60 years or older. Unfortunately, the life span of mice make it challenging to replicate this onset naturally and is not feasible for researchers to experiment using aged mice (Potashkin et al, 2011) To overcome this issue, a future study should examine alternative models that are more representative of human PD pathophysiology. A major limitation in the study of Zhu et. al, was the lack of reasoning for the use Apelin-36 over other isoforms of Apelin such as Apelin-12, Apelin-13, or Apelin-17 (Luo et al, 2019). Ishimaru et al. (2017), specifically emphasizes the biological potency of Apelin-13 in comparison to Apelin-36. This claim suggests if Apelin-13 demonstrates identical neuropro-

tective effects, then a smaller dose of Apelin-13 could be equivalently effective as a larger dose of Apelin-36. If true, Apelin-13 can be a more beneficial treatment in terms of costeffectiveness and the safety of the patient. Additionally, to improve the plausibility of the results from Zhu et al. (2019), the timeline of the study must be extended. Effects of the Apelin-36 treatment models were only studied 5 days after the first injection on mouse models and a 24h incubation period on SH-SY5Y cells, making it unclear if there was a retention in neuroprotective effects. Future investigations should consider monitoring the consequences of Apelin-36 on mice and SH-SY5Y cells periodically over months as opposed to days. Another potential dilemma is the method of administration. Although no significant concerns arise from intranigral injections into mouse models, this method lacks specific targeting of dopaminergic neurons. Accordingly, Apelin-36 is also taken up by surrounding cell types such as astrocytes and microglia. Considering the potency Apelin isoforms, this form of administration may lead to potential off-target effects (Ishimaru et al., 2017). Therefore, alternative techniques of administration must be explored to ensure only dopaminergic neurons are targeted. FUTURE DIRECTIONS Using the appropriate mouse model is an ongoing and heavily debated issue. Commonly used mouse models are treated with environmental toxins such as 6-hydroxydopamine (6-OHDA), MPTP, paraquat or rotenone (Potashkin et al., 2011). As mentioned earlier, MPTP is most capable, amongst these options, of reflecting PD pathophysiology as seen in humans. However, they lack the ability of reproducing an important hallmark of PD, Lewy Body inclusions. A study conducted by Gibrat et al (2009), revealed a 14-day chronic intraperitoneal infusion of MPTP into mice is able to generate these inclusions. Therefore, identical experimental procedures can be conducted on chronically administrated MPTP-induced mouse models to determine if Apelin-36 is also capable of attenuating Lewy Body inclusions. As for the other limitations such as age of onset, nonhuman primates appear to be a more representative model. These primates not only have similar brain anatomies to humans but also display the most accurate pathophysiology for PD, especially when administered MPTP. Nonhuman primates clearly exhibit muscular rigidity and bradykinesia, behavioural deficits not demonstrated in mouse models or other simple organisms (Zeng et al., 2018). Due to the astounding success of Apelin-36 in MPTP-induced mouse models, this treatment should also be investigated in nonhuman primates to study its extent in alleviating behavioural deficits. To compare the efficacy of neuroprotective effects between Apelin-36 and Apelin-13 a study must be conducted such that it is identical to the standards of Zhu et al. (2019). All experimental procedures will be identical, except MPTPinduced mouse models and SH-SY5Y cells will be treated with Apelin-13 as opposed to Apelin-36. From the results presented by Jiang et al., Apelin-13 is expected to reverse Parkinsonian symptoms in a similar manner to Apelin-36. Accordingly, it is reasonable to expect the dissipation of neurotoxicity as demonstrated by an increased survival of SH-SY5Y cells, appearance of dopaminergic neurons, expression of TH, and improved motor function. An enhanced regulation of αsynuclein should also be expected to be displayed by both the mouse model and SH-SY5Y cells. Additionally, Apelin-13 318

will likely downregulate the expression of GRP78, CHOP, and cleaved caspase-12, thus mitigating ERS. As mentioned earlier, Ishimaru et al. (2017) showed Apelin-13 is biologically more potent compared to Apelin-36, suggesting these results would be amplified under identical dosage levels. Although these results are foreseeable, a possibility exists where contradictory results are received. If this is the case, the results then suggest Apelin13 activates an alternative pathway that does not contribute to the neuroprotective effects of neurons in SNpc.

are also contributing to the neuroprotective effects through its interaction with Apelin-36. As a result, subsequent studies can then examine the involvement of other cells to provide a more reliable and effective treatment for the future.

In order to accurately examine the efficacy of the Apelin36 treatment, a similar study to Zhu et al. (2019) must be conducted but with an extended timeline. This prospective study would involve injecting Apelin-36 or saline once during the beginning of the experiment and monitoring the appropriate markers for approximately 2 months. Due to the potency of Apelin36, it suggests a single injection produces long-lasting effects (Ishimaru et al, 2017). Therefore, it would be expected that there will be a significant increase in neuroprotective effects before reaching a plateau. On the other hand, a decline in survival of dopaminergic neurons, TH expression, and motor function, or an increase in Îą-synuclein aggregation, GRP78, CHOP, and cleaved caspase-12, may also occur. These results imply a continuous re-administration of the treatment is required in order to maintain the neuroprotective effects. The method of administration is also another point of contention, as intranigral injections lacks specificity to dopaminergic neurons. Introduction of cre-dependent adenosineassociated virus (AAV) vectors in MPTP-induced mouse models may be able to overcome this hurdle. The gene encoding for Apelin-36 will be initially inverted and be flanked by 2 loxP sites orientated towards each other. Only cells exposed to a Crerecombinase will recognize the orientation of the loxP sites, reverting the position of the gene such that it is no longer inverted, thus resulting in its transcription driven under the elongation 1Îą promoter. This construct will be introduced by AAV serotype 9 by intranigral injection. The purpose of this injection is to avoid possible transduction of the AAV9 into cells of other brain regions, minimizing off-target effects. A major advantage of this administration, is the high tropism AAV9 vectors innately possess for the SNpc. To ensure the vector is expressed in dopaminergic neurons of the SNpc, only these neurons will exclusively contain a Cre-recombinase capable of inverting the gene to allow for its transcription, which can be achieved by developing Cre-transgenic animals (Grames et al., 2018). Before Apelin-36 can be introduced, transfection into dopaminergic neurons can be confirmed by replacing the Apelin-36 gene with a green fluorescent protein (GFP) gene, in a separate mice group. The results can then by analyzed by immunohistochemistry. Other factors studied by Zhu et al. can also be examined to determine if this method alters the effectiveness in the reversal of PD symptoms. Due to the similarity of the experiment performed by Grames et al. (2018), it is expected that the immunohistochemistry reveals localized expression of GFP and Apelin-36 in the mice receiving AAV9 vectors to exclusively dopaminergic neurons of SNpc. The MPTP group will not show any expression as no treatment was introduced. Based on the results presented by Zhu et al. (2019), it would be hypothesized that the MPTPinduced mice models treated with the AAV9, will have increased TH expression but decreased GRP78, CHOP and cleaved caspase-12 expression. Also, these groups will show improved motor function as indicated by longer times spent on the rotarod. On the other hand, the MPTP group will expect to show contrasting results in all experiments performed. If these expectations are not observed, it suggests other cells of the SNpc 319

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Grames, M. S., Dayton, R. D., Jackson K. L., Richard, A. D., Lu, X., Klein, R. L. (2018) Cre-dependent AAV vectors for highly targeted expression of disease-related proteins and neurodegeneration in the substantia nigra. FASEB J, 32(8), 4420-4427 doi:10.1096/ fj.201701529RR

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Bilateral Lesioned Hippocampus Inhibits Retrieval of Previously Learned Memories but Not New Postoperative Memories Janai Puckett

Primates and rodents are exemplars when studying hippocampal lesions and the role the hippocampus plays in memory. Froudist-Walsh et al. (2018) studied rhesus monkeys with bilateral hippocampal lesions and tested them on scenes learned preoperatively and postoperatively to look at the difference in memory retrieval. Lesioned monkeys were impaired at retrieving recent or remote preoperative memories but had no difficulties learning new scenes postoperatively; there was no difference in rate of acquisition or percent error between control and experimental monkeys. They concluded that the experimental and control monkeys performed the same postoperatively due to other surviving hippocampal tissue compensating for the function of the lesioned hippocampus. These results allot more evidence the role of the hippocampus in memory formation and retrieval. Key words: hippocampus, memory, lesion, rhesus monkey, visual memory, medial temporal lobe (MTL), remote memory, recent memory, amnesia, learning

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INTRODUCTION

surrounding hippocampal area is critical for memory (Kim, et al., 2015). Bird (2017) demonstrated that surrounding hippocampal Memory is ubiquitous to everyone, as well as its recon- areas are insufficient in recognition memory if the information was structive characteristic. Memory is how one maintains information previously seen, however new information can be learned indeover time, and it can be encoded in numerous ways (visually, pho- pendent of the hippocampus if it is unfamiliar. nologically, spatially, etc.). There are also multiple different types There are two types of amnesia individuals can suffer of memory: short-term, long-term, explicit, implicit, sensory, and from which involve the hippocampus: anterograde and retrograde. more. Each type of memory plays a different role in memory funcAnterograde amnesia is the inability to form new explicit memories tion. after damage to the MTL. This is the type of memory impairment that patient H.M. suffered from. However, retrograde amnesia is The hippocampus is known to play a significant role in the inability to remember recent events once suffering from some memory (Tulving & Markowitsch, 1998); it not only consolidates sort of head trauma. Retrograde amnesia seems to be temporally memory, but also aids in things such as spatial processing and navi- graded in humans, as shown by Kritchevsky et al. (1989) case studgation. There are two prominent theories in the psychology field ies. Anagnostaras et al. (1999) also found evidence of retrograde based around memory consolidation. The first one is the Standard amnesia being temporally graded in rats with hippocampal lesions. Consolidation Model (SCM) which proposes that memories are dependent on the hippocampus for encoding, but independent for The hippocampus is involved in different types of learnretrieval (Squire et al., 1995). Squire et al. (1995) created this mod- ing to some extent. Covington et al. (2018) looked into the role of el with the idea that at first, the hippocampus plays a major role in the hippocampus in statistical learning and concluded that it was scaffolding the memory to support memory formation. As time necessary. However, if the hippocampus was affected in any way goes on, reactivation of memories allows cortical-hippocampal (disease, atrophy etc.), learning was not eliminated completely. interactions to strengthen the memory, and eventually the cortex would be able to maintain the connections in the absence of the Froudist-Walsh et al. (2018) researched the role of the hippocampus. Ritchey et al. (2013) found that an individual needs hippocampus in learning and memory from before and after receivboth hippocampal and cortical connections for memory formation ing a hippocampal lesion. A group of rhesus macaque monkeys and retrieval, they are not sufficient individually. Kritchevsky et received bilateral hippocampal lesions and were tested on scene al. (1989) studied many patients with hippocampal damage that memories created before the lesions. These monkeys were also exhibited temporally graded retrograde amnesia; meaning that more exposed to new scenes following the lesion to distinguish the role recent memories were forgotten, but remote memories were retriev- of the hippocampus in encoding and retrieval of memories. They able. More remote memories are able to be retrieved, according to found that scenes encoded prior to the lesion were impaired postopthe SCM, because the hippocampus is no longer involved. The eratively, however, monkeys were still able to learn new scenes second prominent theory is the Multiple Trace Theory (MTT) following the lesion. These findings are relevant to memory as it which was created a few short years after the SCM. This theory exemplifies that the hippocampus may not always be critical in suggests that only semantic memories are independent from the memory formation and retrieval. hippocampus during retrieval, and that episodic memories are dependent on the hippocampus during both encoding and retrieval (Nadel et al., 1997). Nadel et al. (1997) proposed this model be- MAJOR RESULTS cause they found evidence that patients with large hippocampal Froudist-Walsh et al. (2018) used an MRI-guided techlesions displayed no apparent gradient for the previous three decades and their autobiographical memory was little to none. Accord- nique to bilaterally lesion the hippocampus, described by Hampton ing to SCM, the memories within those previous three decades et al. (2004). Once the experimenters collected their MRI scans, should have been fully integrated into the cortex, but that was not they placed the monkeys under anesthesia and used a midline incithe case. With each reactivation of a memory, there is reconsolida- sion to inject 0.09 M of ibotenic acid, an excitotoxin, into the hiption and a new memory trace is created. With multiple memory pocampus. They discovered that when lesioning the hippocampus, traces, it would be difficult to locate a memory without some sort monkeys were unable to retain memories acquired preoperatively of direction. The hippocampus acts like an index during retrieval of regardless of whether they were remote or recent. However, conepisodic memory as it searches the traces and picks out which trace trols had no trouble remembering previously learned scenes. Surprisingly, lesioned monkeys were able to form new memories of is relevant for the memory the individual is trying to retrieve. scenes learned postoperatively; there was no significant difference One case study that has been extremely influential in stud- in the error or the rate of acquisition between control and lesioned ying the role of the hippocampus in memory is Patient H.M. (Henry monkeys. Molaison) who received a bilateral hippocampal removal. They discovered postoperatively that his epilepsy improved, but he Hippocampal Lesion showed extreme memory impairment (Larry, 2009). Henry was Authors observed that the hippocampal volume of the lesioned unable to remember anything for more than a few seconds. He was monkeys was smaller compared to controls (Fig. 1), which is a an exemplar case demonstrating that the hippocampus is critical for possible reason for why monkeys had difficulty remembering previously learnt scenes. They also found that monkeys with hipponot only memory formation, but recent memory retrieval. campal lesions had significantly more errors than controls when The medial temporal lobe (MTL) has also been shown to remembering scenes learned preoperatively compared to postoperabe important in memory. This area consists of the hippocampus, tively (Fig. 2). Controls had no effect between postoperatively and entorhinal cortex, perirhinal cortex, and parahippocampal cortex preoperatively learned scenes (Fig. 2). These findings are important (Simon & Spiers, 2003). These areas are known to be critical for because they demonstrate that when the hippocampus is lesioned, long-term processing of memory, especially in declarative memory both remote and recent memories are impaired due to the memory (facts and events). These areas were lesioned during Henryâ&#x20AC;&#x2122;s hippo- being irretrievable. As Ritchey et al. (2013) showed, in order to campal removal, which allowed researchers to functionally link the retrieve a memory, there must be hippocampal and cortical interacMTL to long-term memory. Rats which have had their hippocampal tions; without one, the individual is unable to retrieve a previously formation (areas surrounding the hippocampus) lesioned have learned memory. shown impaired memory for both contextual and spatial features of their experiences (Mumby et al., 2002). Other studies have looked Postoperative New Learning into the importance of the MTL in memory tasks and concluded the

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Monkeys with bilateral hippocampal lesions showed no difference from controls when learning new scenes (Fig. 3). Not only did they make the same amount of errors as controls in preoperative learning, but they also had similar acquisition rates of learning. Also, lesioned monkeys learning rate preoperatively was similar to their learning rate postoperatively. Authors observed no difference between the controls and the lesioned monkeys in both postoperative learning and acquisition of scenes (Fig. 3). These findings are important as they show that there are other areas that can compensate for the loss of the function of one’s hippocampus and memories may still be able to form. Covington et al. (2018) results align with this finding, as they showed that a damaged hippocampus alone does not inhibit an individual from creating new memories. As Bird (2017) demonstrates, when the hippocampus is damaged, there is still an ability to acquire new memories as long as there are no previous associations that are needed to be retrieved.

retrieve recent and remote memories postoperatively, but are able to acquire new memories at the same rate and with the same percent error as controls. These findings demonstrate that the hippocampus is critical for retrieving information already learned, but is not the determining factor for whether individuals can make new memories. Remaining hippocampal formation tissue and other areas of the MTL are able to take over the function of the hippocampus and allow individuals to continually learn new information. These results are relevant to the ambiguity of the role of hippocampus in memory. Previous studies show that the hippocampus is critical in both encoding and retrieval of memories (Squire et al., 1995), however other studies show that the hippocampus is not always necessary in new learning (Covington et al., 2018). Previous studies have shown the hippocampus is involved in statistical learning (Covington et al., 2018), but these results demonstrate that the hippocampus is not needed in visual scene learning which was unique. These results are important as it shows that there may be a chance for not only new learning post-hippocampal damage, but also learning at the same rate and to the same extent as individuals without hippocampal damage. The conclusions Froudist-Walsh et al. (2018) drew were not completely novel but made a large impact on the existing evidence in regard to memory and the hippocampus.

CRITICAL ANALYSIS

Figure 1. Illustration of hippocampal volume between control and experimental monkeys. Red color: the unlesioned hippocampal volume. Black color: the remining volume of the hippocampus of the monkeys with lesions. This picture depicts a shrinkage in monkeys receiving hippocampal lesions. Figure adapted from Froudist-Walsh et al. (2018). The Journal of Neuroscience, 38(36), 7800 – 7808.

Figure 2. A. Represents the retrograde memory impairment in monkeys with bilateral hippocampal lesions. These monkeys made more recognition errors on previously learned scenes postoperatively. B. Represents the control group that had no significant difference between percent recognition error preoperatively or postoperatively. Figure adapted from Froudist-Walsh et al. (2018). The Journal of Neuroscience, 38(36), 7800 –7808.

Following this experiment, there still remains uncertainty as to what the exact role of the MTL is when the hippocampus is damaged. The finding of the rhesus monkeys are able to form new explicit memories once their hippocampus was lesioned was believed to be due to surviving tissue (Froudist-Walsh et al., 2018). These results contradict what (Larry, 2009) found in regard to Patient H.M.; he was unable to form new explicit memories when his hippocampus was removed. The novelty of this finding may oppose Patient H.M., but it aligns with Bird (2017) and how his participants learning was not completely abolished with hippocampal damage. These results also challenge the Multiple Trace Theory of memory consolidation as it is believed that episodic memory is fully dependent on the hippocampus for retrieval (Nadel et al., 1997). Since these monkeys were able to form new episodic memories without their hippocampus, these results may better support the Standard Consolidation Model of memory consolidation that states that any type of memory is independent of the hippocampus for retrieval. However, these results challenge both theories in their statement that the hippocampus is necessary for encoding memories; the monkeys were able to learn new scenes postoperatively without their hippocampus. Previous studies both support and refute the findings of Froudist-Walsh et al. (2018) conclusions. Something that requires further study in this paper is the time period allotted for monkeys to learn each scene. The researchers gave the monkeys as much time as they needed to learn the scenes to 90% proficiency which varied between one and four months, depending on the monkey. If this experiment were to be replicated, the time period for learning should be controlled because that could also impact the results of the role the hippocampus plays.

FUTURE DIRECTIONS

Figure 3. C. Demonstrates that there was no anterograde memory impairment between control and lesioned monkeys. D. Shows the standard error of means between both groups preoperatively and postoperatively. This graph depicts that there was no significant difference between the groups and their acquisition rates both preoperatively and postoperatively. Figure adapted from FroudistWalsh et al. (2018). The Journal of Neuroscience, 38(36), 7800 –7808.

CONCLUSIONS/DISCUSSION The conclusions drawn by Froudist-Walsh et al. (2018) were that monkeys with bilateral hippocampal lesions are unable to

The role of the hippocampus needs to be looked at in other forms of memory. This paper was strictly on visual scene memory, and there was evidence that if the hippocampus is damaged, new explicit learning can still persist. However, other types of memory, such as phonological and implicit, were failed to be addressed. Further experiments on different kinds of memory can investigate if new learning may occur when patients receive bilateral hippocampal regions. If a similar experiment were done on primates but with phonological memory, the expected outcome would be the same as what was found in Froudist-Walsh et al. (2018) study; even though the hippocampus is lesioned, the surviving MTL tissue should be able to compensate for the function. However, if these results were not found, this would indicate that the hippocampus plays a different role in alternate forms of encod-

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ding memories. When encoding phonologically, the same corticalhippocampal interactions may not occur, or at least not to the same extent, which requires the hippocampus to be fully functional at all times. Another experiment that researchers could conduct is to see if this experiment yields similar results when using semantic memory instead of solely using episodic memory. As the MTT suggests, semantic memories will be independent of the hippocampus at retrieval (Nadel et al., 1997). So instead of comparing the preoperative learning to postoperative learning, one could compare strictly preoperative learning of semantic and episodic memories. Researchers would present different scenarios (episodic memories) to subjects as well as present various semantic facts that can be memorized. Once they are presented with both episodic and semantic information to 90% proficiency, they will receive bilateral hippocampal lesions using the same technique as Hampton et al. (2004). Following the lesion, they will participate in retrieval tasks by prompting the subject and looking at what they report. The expected outcome of that experiment would be that participants are unable to retrieve episodic memories but will have no trouble with semantic retrieval. Those findings would support MTT fully, and SCM to an extent as the SCM does not differ between episodic and semantic memories. However, if these results show that participants could not retrieve either episodic or semantic memories, this disproves both the MTT and SCM theories. That would indicate that the role of the hippocampus in memory formation and retrieval is more critical than previously thought, regardless of the circumstance. Perhaps the processing of semantic and episodic memory is not as different as they are portrayed by the MTT.

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Bird, C. M. (2017). The role of the hippocampus in recognition memory. Cortex, 93, 155-165. doi: 10.1016/j.cortex.2017.05.016

Covington, N. V., Brown-Schmidt, S., & Duff, M. C. (2018). The Necessity of the Hippocampus for Statistical Learning. Journal of Cognitive Neuroscience, 30(5), 680-697. doi: 10.1162/jocn_a_01228

Froudist-Walsh, S., Browning, P., Croxson, P., Murphy, K., Shamy, J., Veuthey, T., Wilson, C., & Baxter, M. (2018). The Rhesus Monkey Hippocampus Critically Contributes to Scene Memory Retrieval, But Not New Learning. The Journal of Neuroscience, 38(36), 7800 â&#x20AC;&#x201C;7808.

Hampton, R. R., Hampstead, B. M., & Murray, E. A., (2004). Selective hippocampal damage in rhesus monkeys impairs spatial memory in an open-field test. Hippocampus 14,808 â&#x20AC;&#x201C; 818.

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Determining optimal rTMS protocols for treatment of PTSD Amelie Raedler

Post-traumatic stress disorder is very prominent in todayâ&#x20AC;&#x2122;s society, especially in the veteran community. The disorder is characterized by the four symptom clusters of re-experiencing, avoidance, arousal, and negative cognitions and mood. Current treatments include medications such as antidepressants and psychotherapy such as cognitive processing therapy, but no improvement in symptoms is seen in about 1/3 of the affected population. Repetitive transcranial magnetic stimulation is a non-invasive form of neurostimulation mainly used in the treatment of depression. It is administered via a wire coil placed and held on the exterior of the skull for up to half an hour per day. Depending on stimulation frequency, it can either increase or decrease excitability in the underlying cortex. Lately, this form of treatment has also been studied as an alternative treatment for PTSD, but there are still large discrepancies between the optimal site and frequency of stimulation. A recent experiment by Ahmadizadeh et al (2018) compared stimulation of the right dorsolateral prefrontal cortex to bilateral stimulation of both sides. The study also included a sham condition as a control. Active stimulation was concluded to be more effective in PTSD treatment than sham; however, no significant difference was seen between uni- and bilateral stimulation. A future, large-scale experimental study in this field is recommended to reconcile the discrepancies between these results and existing research. Key words: repetitive transcranial magnetic stimulation; rTMS; post-traumatic stress disorder; PTSD; veterans; bilateral; unilateral; DLPFC

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BACKGROUND and INTRODUCTION. Post-traumatic stress disorder (PTSD) is triggered by experiencing or witnessing a traumatic event that involves a real or perceived threat to the self or others. Its symptoms are categorized into four main groups: re-experiencing, avoidance, negative feelings, and hyperarousal. These symptoms create a debilitating condition that affects up to 8% of the overall population (21). It is especially prevalent in the veteran community, where between 1120% suffer from the symptoms. Current treatments consist of medications (primarily antidepressants) and psychotherapy. While these treatments are able to reduce symptoms in about two thirds of the affected population, this leaves millions of individuals to suffer. Researchers are seeking out new modalities to help these treatment-resistant cases, with a major field of interest being repetitive transcranial magnetic stimulation (rTMS). This non -invasive technique has been FDA approved in the treatment for depression since 2008. In a recent network meta-analysis on the use of this modality to treat depression, no large difference was found in efficacy of different stimulation sites and frequencies. However, the paper suggested that low frequency, bilateral stimulation of the dorsolateral prefrontal cortex (DLPFC) was slightly favoured over the other conditions (23). Due to the disparate underlying mechanisms, the optimal protocol will likely be different for PTSD. rTMS entails a pulsating electromagnetic field creating waves of electrical current. These currents pass through the scalp and skull, stimulating the underlying cortical neurons. rTMS interventions can be highly varied, and its effects on cortical neurons depends both on the site of stimulation and the frequency. High frequency stimulation tends to have an excitatory effect, while low frequency stimulation has been seen to cause cortical inhibition (22). PTSD is known to heavily affect the right DLPFC, so this has been the main stimulation site in rTMS protocols. A substantial amount of past experiments compared low frequency stimulation of the right DLPFC to a sham condition. Other trials have adapted this protocol to include different frequencies and/or stimulation sites, such as left DLPFC. Further studies have also used the same protocol with simultaneous psychotherapy (10,24). A recent paper by Ahmadizadeh et al (2018) aimed to address the question of optimal stimulation site. This study used high frequency stimulation at 20Hz and consisted of three treatment groups; unilateral right, bilateral, and sham (control). The DLPFC was targeted in all participants, and the full intervention consisted of ten sessions, once a day per week (weekdays only) for two weeks. The study found that rTMS is an effective treatment for PTSD but did not conclude a disparity between the two active groups.

MAJOR RESULTS Ahmadizadeh et al (2018) performed double-blinded rTMS on a total of 58 male veterans diagnosed with PTSD in clinical interview using the DSM-4 criteria. These participants were randomized by a statistician into three treatment groups: bilateral, unilateral right,

and sham. As seen in Table 1, no significant differences were seen in demographic and clinical data between these three groups. Table 1. Participant demographic and clinical characteristics. Figure Adapted from Ahmadizadeh et al. (2018). Brain Research Bulletin, 140, 338-340. Patient PTSD symptoms were self-evaluated using the PTSD checklist- military version (PCL-M) at three timepoints; baseline, after five sessions, and after ten sessions. This scale has been found to be very consistent with other measures for PTSD, and a score of 50 has previously been determined as a reasonable lower cut-off for PTSD (3). rTMS treatments The DLPFC was targeted in every treatment group. Ahmadizadeh et al (2018) used only high frequency stimulation (20Hz), which has been found to be most effective in previous studies. Stimulation of the right DLPFC has shown to be more effective than the left side â&#x20AC;&#x201C; however, bilateral stimulation has not been studied. For this reason, Ahmadizadeh et al compared bilateral to unilateral right stimulation. The third group was control sham treatment, so these patients were not subjected to any active currents. Prior to beginning of treatment, the motor threshold of participants in all treatment groups was determined. This is done using TMS stimulation of the motor cortex and is defined as the stimulus strength creating a muscle response in the finger in at least 5/10 stimuli. Following this, all participants were stimulated at 100% of their individual motor threshold. Sham treatment was administered using a sham coil that looks and sounds the same as the active coil; however, no magnetic energy passed from the coil into the cortex. Due to the double blinded nature of the study, neither participants nor the diagnosing clinicians were cognisant of the treatment condition. The rTMS administrator was not blinded, since they had to switch between the active and sham coil. Treatment Response Ahmadizadeh et al (2018) defined a response to treatment as an improvement of 2 or more standard deviations from the baseline PCL-M measure, consistent with previous literature definitions of response (7). Overall, Ahmadizadeh et al (2018) found the proportion of responders to be higher in the active bilateral as well as the active unilateral condition, compared to sham treatment. Sham had a 0% response rate, while bilateral and unilateral had a rate of 62.5% and 41.2%, respectively. Bilateral stimulation appeared significantly more effective after the first week of treatment, but by the end of the second week, no significant difference was noted between the two active conditions. The PCL-M ratings at each of the three timepoints for each group are represented in Table 2. Consistent with previous research, the results showed that rTMS is an acceptable therapy in the treatment of PTSD. Safety and tolerability Three participants in the bilateral group complained of adverse side effects in the form of a slight headache or discomfort following the treatment. One participant in the unilateral group reported warmth sensation, and no adverse events were reported in the sham group. However, the treatment was generally well tolerated, and the three treatment groups did not differ significantly in these adverse effects.

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Table 2.

PCL-M: Means and standard deviations at three timepoints. Figure Adapted from Ahmadizadeh et al. (2018). Brain search Bulletin, 140, 338-340.

Re-

CONCLUSIONS/ DISCUSSION Ahmadizadeh et al (2018) concluded that active, high frequency rTMS is more effective in the treatment of PTSD than sham rTMS, without a significant difference in tolerability. In contrast to the author’s hypothesis that bilateral stimulation would produce superior results, the two active groups did not differ significantly from one another. These discoveries are important because current treatments are unable to fully alleviate symptoms in approximately two thirds of PTSD patients (8). The conclusion that rTMS is an effective treatment for PTSD is consistent with the majority of previous research that has been conducted in this field. Much of the preceding research compared low frequency stimulation of the right DLPFC to different conditions, including sham. Two of these recent studies examined the use of rTMS with simultaneous psychotherapy. Osuch et al (2009) tested low frequency stimulation to the right DLPFC with simultaneous exposure therapy but did not find an effect. Kozel et al (2018) used the same experimental design with cognitive processing therapy, which was significantly superior to sham. The Ahmadizadeh paper is the first incorporating bilateral stimulation, but this technique did not show a significant difference to unilateral. In two imaging studies (11,12) and an animal study (13), PTSD was associated with the right DLPFC. None of these studies showed a connection between PTSD and the left DLPFC, which could explain why bilateral stimulation was not seen to be more effective than unilateral right. CRITICAL ANALYSIS rTMS has been shown to be effective in the treatment of PTSD. However, neither the optimal stimulation site nor frequency have been determined. Previous research has shown that high frequency stimulation (>1Hz) is superior to low frequency (; however, the Ahmadizadeh et al paper (2018) did not address the question of optimal high frequency, only considering 20Hz. Another study should be conducted comparing the effect of various high frequencies. The reviewed study was double blinded to both the clinician and the participant. However, the rTMS administrator was previously notified of the treatment assignment, and their interaction could have led the participant to assume their treatment group. Also, the sham coil used in this experiment did not create any physical stimulation. rTMS is often reported to be quite uncomfortable, so participants in the sham condition may have been able to assume their group assignment through the absence of sensation. To produce the most accurate results, the study should be repeated with a sham coil that also reproduces scalp sensation, such as the one used in the pilot trial by Fryml et al (2019). In this case, there is no need for the administrator to change the coil: it changes between active and sham depending on the individualized participant chip placed within. This triple blinding could further increase the external validity of the research.

While Ahmadizadeh et al’s (2018) results were consistent with the bulk of previous research, some studies have shown rTMS to not be an effective treatment for PTSD (5,10,14,15,16). However, two of these studies used only low frequency stimulation (5,10). Rosenberg et al (2002) likely did not find a result because they applied high frequency stimulation to the left DLPFC only. A very recent pilot study by Fryml et al (2019) tested high frequency stimulation of the right side against sham and did not find a significant result. However, this lack of difference can likely be attributed to the small sample size of only eight participants. Another very current paper by Kozel et al (2019), which had a sample size of 35, found rTMS to be effective in the treatment of PTSD, but did not see a significant difference between high and low frequency stimulation of the right DLPFC. These discrepancies show that further research is required to interpret the results, or the lack thereof. At least one randomized controlled trial of large sample size should be conducted incorporating all the different treatment arms to allow for direct comparison of efficacy. FUTURE DIRECTIONS – Due to persisting discrepancies, some research has been conducted in this field using animal models. Two recent studies (17,18) recreated the afore-mentioned methods in rat and mouse models, respectively, and have shown similar improvements of PTSD symptoms. Both of these novel studies used high frequency stimulation; however, they targeted sites in the prefrontal cortex other than the dorsolateral aspect. This decision can most likely be attributed to the underlying differences between the human and rodent brains but make it more challenging to compare these results to human trials. It has been widely accepted that PTSD has a large heritable component. For this reason, a critical next step is to create a transgenic animal model for the disorder, but this is impossible while the underlying genetic mechanisms are still largely unknown (19). Additionally, the large diversity of symptoms defined as PTSD makes it difficult to create a non-transgenic rodent model of high construct validity. For these reasons, finding the underlying mechanisms of PTSD should be a priority. In the meantime, researchers should focus on conducting a large-scale clinical trial using treatment arms of sham, low and high frequency to both the right and left DLPFC. The hypothesized outcome of this study would be significantly higher efficacy of right high-frequency stimulation, which could lead to this protocol becoming an approved form of treatment. From previous research, the sham condition would be anticipated to create the lowest results. However, if one of the other treatment arms was seen to be the most effective, it would demonstrate that we do not understand the mechanisms underlying PTSD. One theory behind this would be the heavy impact of the disorder on the amygdala. This brain area is deeper than the cortex and is not stimulated during rTMS. One of the most prominent treatments for the amygdala is psychotherapy, or “recoding the memory” (20). The next step after these results might be to try rTMS and psychotherapy simultaneously, targeting both the cortex and amygdala at once.

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Ahmadizadeh, M. J., & Rezaei, M. (2018). Unilateral right and bilateral dorsolateral prefrontal cortex transcranial magnetic stimulation in treatment post-traumatic stress disorder: A randomized controlled study. Brain Research Bulletin 140, 334–340.

Ballenger, J.C., Davidson, J., Lecrubier, Y., Nutt, D.J., Foa, E.B., et al. (2000). Consensus statement on posttraumatic stress disorder from the International Consensus Group on Depression and Anxiety. J. Clin. Psychiatry 61, 60–66.

Weathers, F.W., Litz, B.T., Herman, D.S., Huska, J.A., Keane, T.M., 1993. The PTSD checklist (PCL): reliability, validity, and diagnostic utility San Antonio, TX. Paper Presented at the Annual Convention of the International Society for Traumatic Stress Studies.

Boggio, P. S., Rocha, M., Oliveira, M. O., Fecteau, S., Cohen, R. B., et al. (2010). Noninvasive brain stimulation with high-frequency and low-intensity repetitive transcranial magnetic stimulation treatment for posttraumatic stress disorder. J. Clin. Psychiatry, 71(8), 992–999.

Cohen, H., Kaplan, Z., Kotler, M., Kouperman, I., Moisa, R., et al. (2004). Repetitive transcranial magnetic stimulation of the right dorsolateral prefrontal cortex in posttraumatic stress disorder: A double-blind, placebo-controlled study. Am. J. Psychiatry, 161(3), 515–524.

Watts, B. V., Landon, B., Groft, A., & Young-Xu, Y. (2012). A sham controlled study of repetitive transcranial magnetic stimulation for posttraumatic stress disorder. Brain Stimulation, 5(1), 38–43.

Jacobson, N. S., Truax, P (1991). Clinical significance: A statistical approach to defining meaningful change in psychotherapy research. J Consult Clin Psychol 59, 12-19.

Steenkamp, M.M., Litz, B.T., Hoge, C.W., Marmar, C.R., (2015). Psychotherapy for military- related PTSD: a review of randomized clinical trials. JAMA 314(5), 489–500.

Nam, D. H., Pae, C. U., & Chae, J. H. (2013). Low-frequency, repetitive transcranial magnetic stimulation for the treatment of patients with posttraumatic stress disorder: A double-blind, sham-controlled study. Clin. Psychopharm. Neu, 11(2), 96–102.

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Osuch, E. A., Benson, B. E., Luckenbaugh, D. A., Geraci, M., Post, R. M., & McCann, U. (2009). Repetitive TMS combined with exposure therapy for PTSD: a preliminary study. Journal of Anxiety Disorders, 23(1), 54–59.

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Fryml, L. D., Pelic, C. G., Acierno, R., Tuerk, P., Yoder, M., et al. (2019). Exposure Therapy and Simultaneous Repetitive Transcranial Magnetic Stimulation: A Controlled Pilot Trial for the Treatment of Posttraumatic Stress Disorder. Journal of ECT 35(1), 53–60.

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Rosenberg, P.B., Mehndiratta, R.B., Mehndiratta, Y.P., Wamer, A., Rosse, R.B., et al. (2002). Repetitive transcranial magnetic stimulation treatment of comorbid posttraumatic stress disorder and major depression. J. Neuropsychiatry. Clin. Neurosci. 14(3), 270– 276.

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Kozel, F. A., Van Trees, K., Larson, V., Phillips, S., Hashimie, J., et al. (2019). One hertz versus ten hertz repetitive TMS treatment of PTSD: A randomized clinical trial. Psychiatry Research 273(1), 153–162.

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Wang, H.-N., Bai, Y.-H., Chen, Y.-C., Zhang, R.-G., Wang, H.-H., et al (2015). Repetitive transcranial magnetic stimulation ameliorates anxiety-like behaviour and impaired sensorimotor gating in a rat model of post-traumatic stress disorder. PLOS ONE 10(2).

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Griffin, J. (2005). PTSD: why some techniques for treating it work so fast. Human Givens Journal 12(3).

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Zwanzger, P., Steinberg, C., Rehbein, M.A., Bröckelmann, A.K., Dobel, C., et al. (2014). Inhibitory repetitive transcranial magnetic stimulation (rTMS) of the dorsolateral prefrontal cortex modulates early affective processing. Neuroimage 101, 193–203.

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Elevated levels of plasma neurofilament light chain concentration in patients with anorexia nervosa. Retaj Ramadan

Anorexia Nervosa (AN) is a severe psychiatric disorder in which restrictive caloric intake leads to extreme weight loss and associated secondary issues (Treasure et al., 2015). It is known that a multitude of biological, psychological, cultural, and social factors play a critical role in this disorder (Treasure et al., 2015). The neurobiological underpinnings of AN are equally critical to understanding the disorder and ultimately developing effective treatment plans for patients. One such neurobiological factor is the possibility that neurodegeneration, or loss of neuronal cells in the brain, contributes to AN. In their 2019 study, Nilsson et al. use single molecule array (a highly effective method of quantitative molecular analysis of plasma) to find elevated levels of plasma neurofilament light chain (NfL) concentrations in patients with AN in comparison to recovered patients and non-Anorexic controls (Nilsson et al., 2019). NfL is a cytoskeletal protein marker for neurodegeneration, released by neuronal cells following acute neuroaxonal injury into the cerebrospinal fluid (CSF) and plasma (Khalil et al., 2018). Its presence in the plasma of AN patients reveals important advances in understanding the neurobiological dynamics of AN and monitoring treatment courses. The relevance of these findings also touches on broader implications, ultimately putting forth the role of neurodegeneration as a possible avenue for further research in understanding the complexities of Anorexia Nervosa. Key words: Anorexia Nervosa, Neurofilament light chain, Neurodegeneration, Neuroaxonal injury.

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INTRODUCTION Anorexia Nervosa (AN) is a severe psychiatric disorder in which restrictive food intake leads to extreme weight loss and associated secondary issues that accompany extreme weight loss such as endocrinological disturbances and severe disruption in normal metabolic processes (Schorr & Miller, 2017). AN is the most lethal mental disorder, with the highest mortality rate among all known psychiatric disorders (Arcelus et al., 2011). AN is generally diagnosed in women of prepubertal age (Treasure et al., 2015), although a small fraction of men are also diagnosed with AN (Strother et al., 2012). Individuals with AN display varied behavioural conduct; caloric restrictions, extreme and unhealthy exercise, obsession with numerical values of weight, denial of sick state, and purging are among common indicators of the disorder (Schorr & Miller, 2017). Ultimately, this behaviour leads to severe weight loss which effectually shuts down critical metabolic processes of the body and results in death if left untreated (Treasure et al., 2015). The exact pathophysiology of AN is unclear (Treasure et al., 2015); biological, psychological, social, and cultural factors are all thought to play a crucial role in the disorder (Schorr & Miller, 2017). A focus on the psychological and social underpinnings of AN is especially salient in the literature (see Serpell et al., 1999; Kezelman et al., 2015; Rodgers et al., 2014). However, given the multi-faceted nature of the disorder, research into the neurobiological foundations of AN is equally important in understanding the disorder and ultimately developing effective treatment plans for patients. One such avenue is research into the neurobiology of Anorexia Nervosa, with a specific emphasis on the neurodegenerative characteristics of the disease (Nilsson et al., 2019). Neurodegeneration is broadly classified as neuronal cell death and loss (Wyss-Coray, 2016), exhibited in conditions such as Multiple Sclerosis (MS), Alzheimer’s Disease (AD), and Parkinson’s Disease (PD) (Khalil et al., 2018). In the study of neurodegeneration, a relatively new and invaluable tool has been the use of neurofilament protein levels in the cerebrospinal fluid (CSF) and the plasma. Neurofilaments are cytoskeletal intermediate protein filaments present in neuronal cells (Khalil et al., 2018). Their concentrations are generally stable within the axon and exhibit low turnover rates (Khalil et al., 2018). However, axonal injury results in a release of neurofilaments in the CSF and (to a lesser extent) the plasma (Reiber, 1994). Thus, the levels of neurofilaments in the CSF and plasma indicate neuroaxonal damage (Khalil et al., 2018), acting as a biomarker for neurodegeneration. Indeed, neurofilament levels have been found to be increased in conditions of neurodegeneration such as Multiple Sclerosis (Disanto et al., 2007), Alzheimer’s Disease (Petzold et al., 2007), Parkinson’s Disease (Constantinescu et al., 2009), Stroke (Zanier et al., 2011), Bipolar Disorder (Jakobsson et al., 2014), and following Traumatic Brain Injury (TBI) (Shahim et al., 2017). In AN, neurodegeneration can equally be assessed via neurofilament levels providing fascinating insight into the possible neurobiological underpinnings of the disorder. Nilsson et al.’s 2019 study assesses the neurofilament

concentration in female patients with AN, specifically looking at neurofilament light chain concentration. The study finds that plasma neurofilament light chain (NfL) concentration is significantly elevated in AN patients in comparison to controls and in comparison to AN patients who are >1 year recovered (Nilsson et al., 2019). NfL concentrations are measured via single molecule array (Simoa), a reliable neurofilament assay method that shows the highest quantitative sensitivity among currently used analytic methods such as enzyme-linked immunosorbent assays (ELISA) and electrochemiluminescence-based assays (ECLA) (Kuhle et al., 2015). The relevance of these findings is difficult to overstate; a possible biological marker for Anorexia Nervosa is invaluable not only in diagnosis of the condition, but also in monitoring the success of treatment options. Additionally, the study highlights the neurophysiological facet of AN, putting forth neurodegeneration as a possible avenue for further research in understanding the complexities of this disorder. MAJOR RESULTS The study finds that Anorexic patients (AN) show significantly increased NfL plasma concentrations in comparison to recovered Anorexics (AN-REC) and controls (CTRLS). Results are based on a group of 12 anorexic patients, 11 recovered anorexics, and 12 age-matched controls. Venous blood samples were acquired from all participants and these samples were then analyzed via the Single Molecule Array assay method (Simoa). Specifically, this method uses paramagnetic beads tagged with antibody capture agents sensitive only to neurofilament light chain proteins (SimoaTM, 2013). Simoa is especially powerful because beads tagged with the capture agents contain upwards of 250 000 attachments sites for the NfL proteins, allowing researchers to identify incredibly small amount of the protein in the plasma samples (SimoaTM, 2013). In fact, Simoa is able to detect single molecule concentrations in the sample (SimoaTM, 2013). The concentration of NfL analyzed via Simoa reveal that AN patients average an NfL concentration of 24.8 pg/ml in comparison to AN-REC patients, who average 9.2 pg/ml and CTRLS, who average 7.8 pg/ml. In a second replication study comprised of different patients in the same AN, AN-REC, and CTRLS groups, similar findings come to light: AN patients consistently display significantly higher NfL plasma concentrations than recovered anorexics and controls. Note that while recovered Anorexics display higher NfL plasma concentrations than the control group, this finding was not significant. Therefore, according to these findings, recovered Anorexics are showing some normalization of NfL plasma levels comparable to controls. These results ultimately suggest that Anorexia Nervosa’s disease profile includes acute neuronal damage which can be assessed using quantitative analysis of plasma neurofilament light chain concentrations. These results also align appropriately with what is acknowledged in the literature on neurodegenerative disease; increased neurofilament concentrations have been implicated as a marker for neuronal damage in a number of neurodegenerative diseases such as Alzheimer’s Disease (Petzold et al., 2007), Parkinson’s Disease (Constantinescu et al., 2009), and

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Bipolar Disorder (Jakobsson et al., 2014). CONCLUSIONS/DISCUSSION The study finds that NfL plasma concentrations are significantly elevated in the Anorexic patient group, in comparison to recovered Anorexics and controls. Recovered Anorexics display higher NfL levels than controls, but lower levels than the Anorexic patient group, further suggesting that NfL levels normalize with recovery. These findings are significant because they point to NfL plasma levels as a potential biomarker for AN. This meaningful discovery allows for a more comprehensive understanding of Anorexia Nervosa as one that includes neurodegeneration. Additionally, it provides a way in which clinicians can diagnose or monitor progression of the disorder as well as possible treatment courses. The authors conclude that the presence of elevated NfL plasma levels in Anorexics point to some acute neuronal damage associated with Anorexia Nervosa, although the exact origin of neuronal damage remains unclear. In recovered Anorexics, NfL levels are significantly lower than the Anorexic patient group, further leading the authors to conclude that recovery plays a role in reversing the elevated protein levels (Nilsson et al., 2019). The current study addressing increased NfL concentrations in Anorexia Nervosa is not only recent, but also the first of its kind. As such, there remains a gap to be filled in the literature on addressing and validating its specific results. However, neurofilament concentration in neurodegenerative disease more broadly is relatively well documented (Khalil et al., 2018). Specifically, neurofilament concentration has been implicated as a marker of acute neuronal injury in neurodegenerative diseases like AD, PD, TBI, and Bipolar Disorder (Khalil et al., 2018). These have been touched on briefly in the introduction, but in light of the current paper’s results, one particular study on Bipolar Disorder warrants further discussion. Jakobsson et al. found that neurofilament light chain concentrations were increased in patients with Bipolar Disorder in comparison to controls (Jakobsson et al., 2014). Comparable to Anorexia Nervosa, Bipolar Disorder is also a psychiatric condition with unclear pathogenesis and a multi-faceted disorder profile (Eddy et al., 2008). Consequently, the finding that neurofilament light chain concentrations are equally elevated in the patient population of Bipolar Disorder (as per the Jakobsson et al. study) provides significant support for the findings of the current study on Anorexia Nervosa.

CRITICAL ANALYSIS While the findings of this paper are fascinating and introduce a novel way in which to conceptualize the underpinnings and consequences of Anorexia Nervosa, the results are not without possible limitations that must be further addressed. Consider first that the NfL levels listed for the AN group are only in comparison to AN-REC group and the CTRLS in the same study. While it can be concluded that NfL values are significantly elevated in the AN patient group in comparison to CTRLS or AN-REC, there is no universally established physiologically “normal” level of NfL in the plasma recognized in the literature or in current clinical practice (Khalil et al., 2018). The subsequent problems arising from this fact are reflected entirely in the study’s replication cohort (a second trial measuring the same outcome with a new patient group). In this case, the results show higher levels of CTRLS NfL (9.3 pg/ml) than the first discovery cohort values for CTRLS (7.8 mg/ml). These discrepancies are expected (the individuals in each group are different) but troublesome because understanding the broader implications of these results are limited by the fact that no collective values of normal, age and gender matched NfL plasma levels have been established. Next, consider that NfL concentrations are not the sole biomarker of neurodegeneration. Other neuronal damage markers include glial fibrillary acidic protein (GFAP) and neuronal specific enolase (NSE). Elevated levels of these proteins also indicate acute neuronal damage, similar to neurofilament light chain concentration (Ehrilich et al., 2008). According to a study by Ehrilich et al. in 2008, however, both GFAP and NSE were not found to be elevated in AN patients in comparison to controls (Ehrilich et al., 2008). It would be expected that if neurodegeneration is found in Anorexia Nervosa, research would yield similar results across multiple acute neuronal damage markers, and not only NfL plasma levels. This raises further questions about what is causing the elevation in the NfL marker, if it is not neurodegeneration as suggested by the study under review. Furthermore, consider that NfL levels have been shown to increase with age (Khalil et al., 2018). Studies have implicated NfL levels (in both the plasma and the CSF fluid) in the normal process of ageing, stipulating a 2.5-fold increase in NfL levels between 20 and 50 years of age (Yilmaz et al., 2017). Therefore, the use of NfL levels as solely a marker of neurodegeneration in disease is limited; clearly, it is equally implicated in more routine processes like ageing. Note that the study under review does correct for age in statistical analysis and that the control groups are age matched. However, even statistical corrections are limited if the effect of ageing on NfL levels is not fully understood in the first place (Khalil et al., 2018). For example, the study in question set inclusion criteria that broadly recruited AN patients over the age of 18 years old, with a 5-year onset for the condition. Granted, even these broad parameters are relatively difficult to meet, and participant recruitment may be a serious challenge in such studies given the exclusivity of the patient groups under study. However, the age discrepancies in patient groups may present a possible drawback, especially given the revealing (albeit limited)

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research into the association between NfL levels and age (Khalil et al., 2018). As an aside, this limitation also has substantial, broader implications for the marker’s usefulness in neurodegenerative diseases that are often associated with an increase in age. One possible concern to further consider is that NfL levels may not accurately measure what they are currently postulated to measure in diseases such as Alzheimer’s, which has a clear age association (Masters et al., 2015). Finally, consider again that Anorexia Nervosa is a multifaceted mental disorder thought to be influenced by psychological, genetic, and social factors, among many others (Treasure et al., 2015). The disorder profile may vary between individuals. For example, the specific caloric restriction behaviour of Anorexics differs between individuals with AN, with some engaging in purging and others in obsessive exercise. Equally, then, biological markers for neurodegeneration (such as NfL) can play different roles across the patient population, possibly important in some and less so in others. FUTURE DIRECTIONS Further research addressing the presented limitations is needed in order to understand the undoubtedly complex association between NfL levels and Anorexia Nervosa. Possible direction of this research will now be presented. First, in addressing the reliability of NfL concentrations as a marker of neurodegeneration, it is critical that the research develop sound and acknowledged NfL reference concentrations. Specifically, this can be addressed via analyses comprised of large samples stratified by age, gender, and health status. This review suggests NfL concentrations be measured in these groups via Simoa in order to develop universal, published reference values of the biomarker. Additionally, because the marker is detectable in the CSF and the plasma (Khalil et al., 2018), future studies must take into consideration that levels of NfL may differ in both, and thus require separate examination (in the CSF and plasma). One way to address this is a replication study measuring NfL concentrations in the CSF in patient groups of anorexics, recovered anorexics, and controls to determine whether the same relative levels are observed in both the CSF and the plasma. One possible method to measure the NfL concentration in the CSF is enzyme linked immune-absorbent assay (ELISA), a secondgeneration immunoassay (Khalil et al., 2018). Next, in addressing the lack of other biomarkers such as NSE and GFAP as mentioned earlier, this paper suggests a replication of the current study under review, in which NSE and GFAP are measured, as well. Simoa may be an appropriate assay method in this case to detect even small amounts of these biomarkers. The patient groups in this experimental outline are similar to those in the text under review: anorexics, recovered anorexics, and controls. Keeping in mind that Ehrlich et al. did not test for NfL concentration, it could be found that the NSE and GFAP neurodegenerative markers are elevated only in the presence of NfL in Anorexic patients. Furthermore, the molecular mechanism for GFAP, NSE and (once again) NfL must be further explored; neurodegenerative biomarkers may not all be associated in the same process-

es. For example, GFAP is a damaged glial cell marker and may not be expected to increase if Anorexia Nervosa acts specifically on neuronal cells (Ehrilich et al., 2008). Characterizing the structure and function of these biomarkers with respect to Anorexia Nervosa (using protein chromatography) is one way to address this problem. With a greater understanding of the molecular structures and roles of GFAP, NSE, and NfL, researchers can better appreciate what their concentrations reveal about neurodegeneration. This review next proposes a more specific research design in order to address the inherent, intricate, and often complex limitations that come with the multi-faceted nature of Anorexia Nervosa. Future research must comprise a long-term approach to patient groups with Anorexia Nervosa. Instead of comparing the AN patient group to different, age-matched ANREC patient group, researchers can track the same AN patients as they enter 1 year recovery to become AN-REC patients. This study would begin with measurements of the AN patient’s plasma NfL concentrations before admittance into a recovery program (this program may be the same local Centre for Eating Disorders from which the original AN-REC patient group was recruited). Measurements are then taken periodically throughout treatment, perhaps every month or so. With successful recovery and based on the findings of the paper under review, this current review predicts that levels of NfL in the plasma should decrease with ongoing recovery, as neuronal recovery was observed in the original study. However, this review proposes that such long-term follow-ups comparing NfL plasma levels in the same individual would yield more comprehensive and specific results on the role of NfL in AN. Finally, and in the case that financial limitations are not obstructive, this review proposes corresponding neural imaging studies of the followed patients, which may shed light on the structural changes in the brain following neurodegeneration as proposed by elevated NfL plasma concentrations. Based on studies that show AN results in reduced brain volume (Phillipou et al., 2014), such accompanying neuroimaging methods in the proposed future study could allude to the root cause of elevated NfL levels (which may be a reduction in brain volume or some other, currently unidentified structural change). Such a study’s methodology can include the use of Magnetic Resonance Imaging (MRI) on anorexic, recovered anorexic, and control patient groups in order to compare these structural changes. To conclude, Anorexia Nervosa is a severe psychiatric condition and the overlap of numerous factors in the disorder make its study both challenging and fascinating. In developing appropriate treatment plans and expanding our understanding of the disease, it is critical that research considers the neurobiological underpinnings of the disorder. Such understanding comes with an examination of the role of neurodegeneration in AN, and how biomarkers like NfL and their elevated concentrations can characterize this fact. The paper under review is certainly an exciting primer in this regard and helps in driving the appeal for more research and discovery in the area of neurodegeneration and psychiatric conditions such as Anorexia Nervosa.

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MDD: Investigating Microglial Polarization and Endoplasmic Reticulum Stress Using a Chronic Social Defeat Stress Mouse Model Kayla A. Romao-Penacho

The role of neuroinflammation in the pathogenesis of major depressive disorder (MDD) is becoming an increasingly important topic. Recent evidence illustrates the connection between inflammation of the central nervous system (CNS) and MDD patients. However, analysis into microglia polarization and endoplasmic reticulum (ER) stress signaling has not yet been explored. Tang et al. (2018) began their investigation into this topic by creating a mouse model of MDD through chronic social defeat stress (CSDS). The MDD model was verified through behavioral examinations during the social interaction test (SIT), forced swimming test (FST), and sucrose preference test (SPT). The depression model was also confirmed by examining decreased hippocampal spine density and postsynaptic density protein 95 (PSD95) expression in CSDS mice. Microglial polarization and ER stress was then analyzed using Western blot and real-time polymerase chain reaction (PCR), and a comparison was made between CSDS and control mice. Analysis at the transcriptional level revealed expression increases in M1 microglial markers (inducible nitric oxide synthase (iNOS), CD16, CD86, CXCL10) in CSDS mice compared to control. There was no difference in M2 microglial markers (Arginase and CD208) between CSDS and control mice. At the protein level, hippocampal staining revealed increased iNOS+ cells in CSDS mice, with its level of Arginase+ cells insignificantly different from control. A significant increase in ER stress signaling factors (protein kinase RNA-like ER kinase (PERK), phosphorylated a-subunit of eukaryotic translation initiation factor 2 (p-eIF2), C/EBP homologous protein (CHOP), X-box binding protein (XBP1)) was also observed in MDD mice compared to control. Thus, the results found by Tang et al. (2018) suggests the influence of M1-mediated neuroinflammation and increased ER stress activation on MDD development. Key words: neuroinflammation, major depressive disorder (MDD), microglia, endoplasmic reticulum (ER) stress, chronic social defeat stress (CSDS

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c INTRODUCTION MDD is one of the most prevalent mental disorders around the world, affecting approximately 17.3 million adults in the United States alone (Center for Behavioral Health Statistics and Quality, 2018). Selective serotonin reuptake inhibitors (SSRIs) are some of the most commonly prescribed medications for MDD, however they can cause major side effects and loose effectiveness over time. As MDD rates continue to increase within the population (Klerman & Weissman, 1989), the need for new therapeutic treatments for MDD is rising. Many studies are now revealing links between neuroinflammation and depression. MDD patients have been found to have higher levels of inflammatory markers like IL-6 (Maes et al., 1995), as well as a greater number of circulating leukocytes (Maes et al., 1992). Depressed patients also have a higher risk of inflammatory diseases such as diabetes and heart disease, which may reflect a proinflammatory nature of MDD (Fenton & Stover, 2006). Some SSRIs have been shown to have anti-inflammatory effects, which constitute a successful treatment in many MDD patients (De Kock, Loix, & Lavand’homme, 2013). Activation of microglia/macrophages has also been reported in both human and animal MDD models (Bayer, Buslei, Havas, & Falkai, 1999; Pan, Chen, Zhang, & Kong, 2014). Understanding the role of neuroinflammation in MDD pathogenesis will reveal possible therapeutic targets for depressive patients. Activated microglia are polarized towards one of 2 different states: M1 and M2 (Orihuela, McPherson, & Jean Harry, 2016). These states have antagonistic functions and responses to stimuli. M1 microglia are pro-inflammatory, releasing pro-inflammatory cytokines such as TNF-α and IL-1β in response to endogenous stimuli. They also express high levels of iNOS for NO production (Gordon & Taylor, 2005; Villalta, Nguyen, Deng, & Tidball, 2009). Opposite to this, M2 microglia are anti-inflammatory and release anti-inflammatory cytokines such as IL-10 and arginase-1 (Arg1). Associations between microglial polarization state and neuroinflammation have been studied in neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, Huntington disease, and multiple sclerosis (Orihuela, McPherson, & Jean Harry, 2016). However, microglial polarization state within CSDS-induced MDD mice has not yet been investigated. Within cells, the ER is involved in protein synthesis, protein folding, storage of intracellular Ca2+, and the lipid synthesis. Perturbations in any of these homeostatic processes due to inflammatory responses can lead to ER stress. ER stress involves the accumulation of misfolded proteins, which is toxic to cells. It can also trigger the unfolded protein response (UPR), which aims to reestablish homeostasis while inducing more inflammatory signaling (Schröder, 2008). Thus, ER stress may also be implicated in MDD pathogenesis. Investigation into the role of ER stress in Alzheimer’s disease, Parkinson’s disease, and bipolar disorder have been recorded, but its connection to MDD has not yet been explored. The purpose of the study by Tang et al. (2018) was to examine the role of inflammation in the pathogenesis of MDD. This was done through analysis of microglial polarization and ER stress. The study began by creating a CSDS mouse model of MDD and verifying it through behavioral tests, spine density levels and synaptic protein expression in the hippocampus. Microglial polarization and ER stress was the compared between depressed and control mice via analysis at the transcriptional and protein level. The results of this

study found that CSDS mice had increased expression M1 microglial markers and increased activation of ER signaling pathways compared to control. This suggests the involvement of M1 microglia and ER stress in the development and progression of MDD, as they both promote inflammation.

MAJOR RESULTS CSDS mics elicit MDD behavioral phenotype In this study, the CSDS treated mice underwent multiple behavioral tests to confirm their development of MDD. These tests included the SIT, FST, and SPT, all following the 10 consecutive days of social defeat. During the SIT, the social interaction ratio was significantly lower in CSDS compared to control mice (Fig. 1a). During the FST, CSDS mice spend significantly more time immobile in the water (Fig. 1b). During the SPT, CSDS mice showed a significantly decreased preference for sucrose water compared to control mice (Fig. 1c). These 3 tests validate that the CSDS treatment was successful in producing an MDD mouse model. The results are consistent with the MDD behavioral phenotype.

Figure Adapted from Tang et al. (2018). Neurochemical Research, 43:985-994 Fig. 1 Comparing performances of CSDS-treated and control mice during 3 behavioral tests: SIT (A), FST (B), and SPT (C). Tang et al. (2018) reported significant differences between the CSDS and control mice, as indicated with the asterisks.

CSDS mice show decreased hippocampal spine density and synaptic protein expression Along with behavioral tests, spine density and synaptic protein expression levels were also evaluated after CSDS treatment. Golgi staining in the hippocampus showed decreased spinal density in CSDS mice compared to control (Fig. 2a). Western blot data showed lower expression levels of PSD95 protein in CSDS mice compared to control (Fig. 2b). Immunofluorescence levels of PSD95 within neurons were also significantly lower in CSDS mice (Fig. 2c). The decreased spine density and PSD95 levels in CSDS mice provides further validation of a successful MDD mouse model.

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Figure Adapted from Tang et al. (2018). Neurochemical Research, 43:985-994

Figure Adapted from Tang et al. (2018). Neurochemical Research, 43:985-994.

Fig. 2 Pictures comparing spine density and synaptic protein expression in the hippocampus of CSDS and control mice. A Golgi staining of CSDS and control mice (the bar represents 10 m). B Western blot of PSD95 expression in CSDS and control mice C Immunofluorescence of PSD95 in CSDS and control mice (the bar represents 50m).

Fig. 3 Comparing RNA expression of M1 and M2 microglial markers iNOS, CD16, CD86, CXCL10, Arginase, and CD206 in CSDS and control mice. Tang et al. (2018) reported significant differences in the M1 microglial markers iNOS, CD16, CXCL10, and CD86 (as illustrated by the asterisks).

CSDS induces M1 microglial polarization This study used real-time PCR to analyze microglial polarization at the transcriptional level. Using this method, Tang et al. (2018) found that CSDS mice had significantly increased gene expression of iNOS, CD16, CD86, and CXCL10 (Fig. 3) compared to control mice. These genes are markers of M1 microglia, suggesting M1 polarization in CSDS mice. The change in iNOS expression in particular corresponds to a study done by Seneviratne et al. (2015). This study also found an increase in iNOS following stress. Tang et al. (2018) did not find a significant difference in Arg1 and CD206 expression between CSDS and control mice (Fig. 3). These are M2 microglial markers, suggesting little to no M2 polarization in mice after CSDS treatment. This is similar to a stress study by Xu et al. (2017), which also found an increase in iNOS with no change in Arg1. Double immunofluorescence and Western blot were used to assess microglial polarization at the protein level. The authors found a significant increase in iBa1 expression (a marker for active microglia) in the hippocampus of CSDS mice compared to control mice (Fig. 4a, b). There were also significantly more iNOS+ cells in CSDS mice (Fig. 4a, c). Moreover, immunofluorescence data showed significantly increased iBa1+ microglia merged with iNOS+ cells in CSDS mice (Fig. 4a). This suggests that the increased activated microglia found in CSDS mice are polarized to the M1 state. Furthermore, the amount of Arginase+ cells remained the same between CSDS and control mice (Fig. 4d). This provides further data that suggests unchanged M2 microglial polarization after CSDS treatment.

Figure Adapted from Tang et al. (2018). Neurochemical Research, 43:985-994. Fig. 4 Pictures comparing iNOS and iBa1 expression in CSDS and control mice. A Immunofluorescence of iNOS and iBa1 in hippocampus of CSDS versus control mice. B Immunofluorescence of Arginase in hippocampus of CSDS versus control mice. C Western blot of iNOS expression in hippocampus of CSDS versus control mice. D Comparing Western blot of hippocampal Arginase expression in CSDS and control mice.

Overall, these findings provide significant data which supports Tang et al.â&#x20AC;&#x2122;s (2018) suggestion that CSDS treatment induces M1 microglial polarization.

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by Tang et al. (2018) is the first to illustrate the state of M1 polarization after CSDS treatment.

Figure Adapted from Tang et al. (2018). Neurochemical Research, 43:985-994. Fig. 5 Pictures comparing activation of ER stress signaling pathways in CSDS and control mice. Western blot comparison of hippocampal p-IRE-1 (A), XBP1 (B), PERK (C), p-eIF2

(D), CHOP (E), and ATF6 (F) is between CSDS and control

mice. Tang et al. (2018) reported significant increases in p-eIF2 -1, and XBP1 in CSDS mice compared to control.

, CHOP, p-IRE

CSDS mice have increased activation of ER stress signaling pathways Certain stimuli (such as inflammation and oxidative stress) can activate the 3 ER stress signaling pathways. These include the ino-

The paper by Tang et al. (2018) also suggests that the activation of pro-inflammatory M1 microglia may have triggered the ER stress observed. Previous studies have also investigated ER stress in relation to MDD. Nevell et al. (2014) found a significant increase in ER stress signaling molecules CHOP and XBP1 in MDD patients, which were also increased in this review’s study. Although, Tang et al. (2018) are the first to study ER stress in CSDS-induced MDD models. Another study done by Timberlake & Dwivedi (2016) found that expression of the ER stress molecule ATF6 was upregulated in depression models. This contrasts the study by Tang et al. (2018), as they found no change in ATF6 levels between CSDS and control mice. The authors acknowledged this discrepancy and suggested that it might have been caused by the different models used. ER stress and M1-mediated neuroinflammation seem to be related, however further research into the directionality of their relationship is needed.

sitol-requiring protein-1 (IRE1)/XBP1 pathway, PERK/eIF2 /CHOP CRITICAL ANALYSIS pathway, and activating transcription factor 6 (ATF6) pathway Tang et al. (2018) concluded that CSDS-induced mice had higher (Schröder, 2008). In CSDS mice, the expression of both phosphorylevels of M1 polarization and ER stress signaling pathways comlated (p)-IRE1 (Fig. 5a) and XBP1 (Fig. 5b) was significantly higher pared to control mice. Their research found significant results illusthan control when examined by Western blot. The study’s data trating the increased expression of IRE1, XBP1, PERK, eIF2 and also showed that the expression of PERK (Fig. 5c), eIF2 (Fig. 5d), CHOP after CSDS treatment. However, RNA expression of ATF6, and CHOP (Fig. 5e) were also increased in CSDS mice, although a which is the third branch of the ER stress signaling pathway difference in ATF6 levels were not found (Fig. 5f). Nevell et al. (Bergmann & Molinari, 2018), was not found to be significantly (2014) found similar results, showing an increase in CHOP and different between control and CSDS mice. This result contradicts XBP1 in MDD patients. Overall, Tang et al.’s (2018) results indicate their initial prediction that ER stress signaling pathways would be the activation of the 2 ER stress signaling pathways, IRE1/XBP1 and upregulated following CSDS. It also contradicts the study by Timberlake & Dwivedi (2016), which found upregulated levels of ATF6 PERK/eIF2 /CHOP, in CSDS mice. after stress. The authors claimed that this discrepancy was a result of the different models used in each study (rats versus mice), howCONCLUSIONS/DISCUSSION ever this was not discussed any further. The researchers should The authors of this study concluded that following a successful look further into the reason of this inconsistency, as it contradicts CSDS treatment, microglia were polarized towards the M1 state. their idea that ER stress pathways are upregulated following Additionally, they proposed that ER stress signaling pathways were stress. Further investigation into the effects of CSDS on ER stress activated after CSDS treatment. These findings provide infor- mechanisms is needed to truly attribute this discrepancy to a mation which may be used for further difference in animal models. This is important because if ATF6 investigation into targeting M1 microglia and ER stress pathways in does in fact get upregulated following CSDS-induced stress, it may be a possible target for MDD treatment. Having support in the MDD patients. literature regarding changes to ATF6 is necessary to take further In this paper, they discuss how the significant increase in M1 steps into studying ATF6-based MDD treatments. microglia markers (iNOS, CD16, CD86, CXCL10) in Additionally, the authors suggest that M1-mediated neuroinCSDS mice compared to control illustrates M1 polarization in MDD. flammation might have triggered the ER stress in CSDS mice. HowThis study is not the first to investigate the effect of stress on mi- ever, they do not provide any previous research to supports this croglial polarization. A study done by Seneviratne et al. (2015) idea. In fact, Schröder (2008) suggests the opposite. This author illustrates the promotion of M1 microglial polarization after shear describes that the activation of ER stress signaling pathways actustress to the blood vessels of a mouse model. Similar to the study ally induces inflammatory signaling due to the UPR. Looking furdone by Tang et al. (2018), Seneviratne et al.’s (2015) findings indi- ther into the connection between ER stress and M1-mediated neucate an increase in the M1 marker iNOS after stress, along with roinflammation would allow for a better analysis of Tang et al.’s other M1 markers that this study did not analyze. Another study (2018) results. Understanding the direction of influence between by Xu et al. (2017) revealed the connection between M1 polariza- ER stress and neuroinflammation will open up the possibility of tion and spared nerve injury. Similar to Tang et al.’s (2018) article, integrating molecules such as PERK and IRE1 into the treatment of Xu et al. (2017) found an increase in M1 microglial marker iNOS, neurological disorders and diseases. with no change in the M2 marker Arg1. This study in particular also suggests that the polarization of M1 microglia provokes a depres- FUTURE DIRECTIONS sion-like phenotype. Although, unlike previous research, the study As mentioned in the previous section, Tang et al. (2018) explains

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that the discrepancy between their ATF6 expression results and previous research is due to the difference in animal species. However, the study by Timberlake & Dwivedi (2016) also induced MDD symptoms through learned helplessness, instead of CSDS. To understand the true direction of ATF6 expression change after CSDS treatment, a future experiment might involve examining its effect on ATF6 expression in both rats and mice. This experiment could even be taken a step further and compare ATF6 expression in MDD patients and control. These studies would reveal whether or not ATF6 is another ER stress signaling pathway implicated in MDD. Results from these experiments would allow for further study into creating treatments for MDD that target ER stress signaling pathways. Knowing which ER stress pathways are affected and in which direction guides studies towards which molecules/proteins should be targeted. Tang et al. (2018) found significant results showing increased expression of IRE1, XBP1, PERK, eIF2 and CHOP after CSDS treatment. Thus, further studies may investigate the effect of knocking out some of these factors in MDD models. The authors also found a significant increase in iNOS, CD16, CD86, and CXCL10, after CSDS treatment, illustrating M1 microglial polarization. Future experiments may look into creating a drug that affects M1 polarization. This may include knocking out certain factors released by M1 microglia (such as NO, IL-6 and TNF-Îą. As these molecules promote inflammation, it would be predicted that creating a knockout may reduce neuroinflammation in MDD models and possibly reverse certain depressive behaviors. If this hypothesis is correct, it would open up the possibility for investigating a new direction in MDD treatment. Overall, these experiments would provide more research into the effects of certain treatments on MDD and allow for exploration into new therapeutic targets.

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Overcoming Low-motivated Arousal after Early Life Stress using Chemogenetic Activation of the Lateral Hypothalamus Polina Rybitska

Lack of pleasure and low motivation towards normally rewarding stimuli are cardinal features of mood disorders such as depression, and early life stress (ELS) is a notable antecedent to the development of such disorders. The lateral hypothalamus (LH) is a key brain region that is implicated in motivated behaviour, so this study examined the effect of ELS on the LH circuitry and motivated behaviour of sucrose administration in rats. Furthermore, chemogenetic activation of this circuitry was performed to assess if sucrose responding could be modified in rats who experience early life stress and to see the impact on LH cells. This was done by separating rat pups from their mothers and then injecting designer receptors (hM3D(Gq)) that can be activated by designer receptors into the LH in adolescence. Later, in adulthood the ELS and no-ELS rates were trained to self-administer sucrose and tested under a progressive ratio schedule to assess their level of motivation, followed by an injection of either clozapine-N-oxide (CNO) or a vehicle drug. Also, cell population within the LH was assessed using fos -mapping. Results showed that ELS induced a state of low motivated behaviours but the administration of the designer drug (CNO) reversed these effects and increased fos-positive MCH and orexin cells in the LH. These results show that, LH circuits are plastic and the effects of early-life maltreatment can be reversed by using chemogenetic activation of these brain areas involved in motivated behaviours. This has important real life implications, as this knowledge may assist in the development of pharmacological treatments for mood disorders such as depression. Keywords: early life stress (ELS), lateral hypothalamus (LH), motivation, designer drugs activated by designer receptors (DREADD), orexin, MCH, fos-mapping, depression, clozapine-N-oxide (CNO)

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INTRODUCTION Early-life Stress (ELS) has been found to be an indicator of the development of neuropsychiatric diseases such as major depression in adulthood (Pace et. al., 2006). A notable symptom of depression is the lack of pleasure and low motivation towards stimuli that are normally rewarding, termed anhedonia (Sherdell et al., 2011). Studies in rodent animal models show that separation of rat pups from their mothers early in development (example of ELS) is correlated to the development of depression-like phenotypes in adulthood (Winslow and Insel, 1991). These results strongly mimic the abnormalities in behaviour seen in humans after experiencing childhood maltreatment. Early life stresses strongly impact the brain, particularly the neuroendocrine hypothalamus (paraventricular nucleus) mediated by the HPA axis (Meany et al., 2007). Depressed adults who have a history of childhood maltreatment were found to have an elevated neuroendocrine response to stress and impaired glucocorticoid signalling as well as a reduced hippocampal volume (responsible for stress response extinction regulation) (Danese et al., 2008). Besides the paraventricular nucleus of the hypothalamus, other hypothalamic regions are involved in the stress response such as the Lateral Hypothalamus (LH) (Winsky-Somerer et al., 2005). In stressful situations, the LH has been found to modulate stress-relevant adaptations such as changes in levels of arousal and motivational behaviours. Recent studies have shown the implication of lateral hypothalamic circuits and cells in the drive for motivated and rewarding behaviour (Stuber and Wise, 2015). The neuropeptide orexin is of particular interest as it regulates arousal, feeding and reward-motivated behaviours and targets the ventral tegmental area (VTA) which is also implicated in motivated behaviour. Studies have shown that the administration of Orx antagonist has shown a reduction in feeding behaviour (Haynes et al., 2002) but activation of Orx cells and administration of Orx into the VTA reinstates food-seeking behaviour (Harris et al., 2005). Another neuropeptide predominantly found in the LH is melanin-concentrating hormone (MCH) that also regulates feeding and the sleepwakefulness balance (Stuber and Wise, 2015). Studies have shown that injections of this peptide increases feeding in rodents (Qu et al., 1996) and overexpression result in obesity and hyperphagia (Ludwig et al., 2001). Overall, these studies show an increase in motivated behaviours from baseline levels, but not many have investigated manipulations to the LH and the effects when starting at lower than baseline levels due to early life stress. A previous study by James et al. (2014) observed the behavioural and neurological effects of ELS on rats. One effect of ELS was the reduction in socialization with other rats in an open field after experiencing stressful conditions, suggesting that ELS produces a state of lowmotivation (James et al., 2014). The other notable effect was in the LH circuit where orexin neurons were hypoactive in their response to restraint stress (James et al., 2014). This is consistent

with the literature where chronic stress in mice leads to the reduction in hypothalamic orexin neurons, and the restriction of orexin activity has been linked to symptoms of depression in humans (Nollet and Leman, 2013). To further assess the effects of ELS, rats were maternally separated and tested under the progressive ratio (PR) condition for sucrose administration by Campbell et al. (2017). Under ELS conditions, rats would experience reduce the motivational drive for attaining sucrose under the PR condition. Results showed to be consistent with this prediction. Compared to controls, the animals exposed to ELS displayed a decrease in motivated behaviour of lever pressing for reward reinforcement. In the second experiment, a chemogenetic technique using designer receptors exclusively activated by designer drugs (DREADD) to activate LH circuits was incorporated to see if the ELS effects on motivated behaviours could be reversed. DREADD is a technique that can precisely control major signalling pathways such as Gi or Gq where specifically human muscarinic receptors (ie. hM3D(Gq) or hM4D(Gi)) are genetically inserted into neuronal cells and can only be activated by binding of agonist clozapine-N-oxide (CNO) (Zhu & Roth, 2014). To understand the effects of DREADD, fos-mapping was used specifically to assess CNO-induced activity in the LH. Results showed that activation of the LH circuits using hM3D(Gq) DREADD increased motivated behaviour in ELS rats to the same levels as No-ELS rats. Therefore, pharmacological targeting of LH circuits can overcome deficits in motivated behaviour produced by ELS (Campbell et al., 2017). RESULTS Experiment one: effect of ELS on the motivational drive in adulthood The motivational drive was measured by sucrose selfadministration which first consisted of eight FR1 training sessions, where sucrose delivery was dependent on one lever press. Then came four FR3 sessions where delivery of sucrose was determined by three lever presses. The final training was on a PR schedule, where the number of lever presses needed to get the reward increased by one after receiving the sucrose (ie. first one lever press resulted in reward, the next time two lever presses were needed to receive the reward etc.). Analysis of the PR training showed a significant relationship between the PR day (day one-five) and the neonatal treatment. Also, there was a significant effect of neonatal treatment on active lever presses (ELS ratsâ&#x20AC;&#x2122; lever pressing was significantly reduced) (Fig. 2C). There was also a significant effect of neonatal treatment on the breaking point, where ELS rats had significantly lower breaking points than no-ELS rats (Fig 2D). Early studies done by Winslow and Insel (1991) show a similar relationship, where maternal separation is correlated to anxiety and depression-like behaviour in adults, which is closely related to observations in human adults (Winslow and Insel, 1991). Moreso, the persistent effects of maternal separation show lasting influence on brain regions associated with motivation, where disrupted early

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rearing lead to lasting changes in the mesocorticolimbic dopamine neuronsâ&#x20AC;&#x2122; responsiveness (Brake et al., 2004). The behavioural consequence of this is the lack of reward-seeking as seen in this first experiment. A forced swim test was also given to a subset of rats immediately prior to the final PR day to measure behavioural despair. This was done by placing them into a chamber filled with tepid water and assessing their responses. There was no significant effect of neonatal treatment on the time spent climbing, being in an immobile state or swimming (Fig 2E, F, G). Overall, under a PR schedule, ELS reduced responding for sucrose which is representative of motivated behaviour in adult rats.

er responding (Fig. 4A). Also, CNO had a significant effect on the breakpoint, as it increased the breaking points for sucrose administration in ELS-induced rats nearly-equivalent to no-ELS rats (Fig. 4C). Previous studies have shown that adverse early events tend to alter the function of pathways proximal to the hypothalamus, leading to notable alterations in adultsâ&#x20AC;&#x2122; responsiveness to psychological stresses (Ladd et al, 2000). This experimentation proved just that as a reversal of altered responsiveness due to early stresses was accomplished with the use of DREADD.

Figure 2: ELS effect on sucrose self-administration and forced swim test behaviours. Campbell, E.J., Mitchell, C.S., Adams, C.D., Yeoh, J.W., Hodgson, D.M., Graham, B.A., Dayas, C.V. (2017). Chemogenetic activation of the lateral hypothalamus reverses early life stress-induced deficits in motivational drive. European Journal of Neuroscience, 46(7), 2285-2296. Experiment 2: Effect of chemogenetic activation on the LH in ELSinduced rats with deficits in motivated behaviour Sucrose Administration Experiment 2 repeated the procedures of the first experiment but with the DREADD activation included. The chemogenetic activation of the LH was performed as it is a notable brain region also responsible for a stress response (Winsky-Somerer et al., 2005). It was done so prior to the final PR test to determine if it was possible to reverse the effects of ELS treatment on sucrose administration. The rats were either treated with vehicle (DMSO) drug or CNO drug. Data analysis shows that CNO significantly increased lever responding equivalent to no-ELS rats, while vehicle shows no significant effect with ELS rats experiencing reduced lev-

Figure 4: Effect of DREADD on sucrose self-administration responses. Campbell, E.J., Mitchell, C.S., Adams, C.D., Yeoh, J.W., Hodgson, D.M., Graham, B.A., Dayas, C.V. (2017). Chemogenetic activation of the lateral hypothalamus reverses early life stress-induced deficits in motivational drive. European Journal of Neuroscience, 46(7), 2285-2296. Assessing Fos-positive orexin cells and Fos-positive MCH cells Overall, CNO treatment resulted in significantly increased numbers of Fos-positive Orx neurons, as well as Fos-positive MCH neurons. A CNO bath was applied to cells in the LH and recordings were made to determine if the hM3D(q) cell transduction was capable of promoting neuronal activity. Most of the LH cells recorded that were bathed in CNO showed significant depolarization, which was enough to invoke an action potential spike. Studies have shown that Orx is a key modulator of reward, and its dysregulation is associated with neuropsychiatric disorders such as depression (Nollet & Leman, 2013). Furthermore, James et al. (2014) showed that ELS notably affected the LH circuit where Orx neurons were

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hypoactive in their response to restraint stress (James et al. 2014). Results of this experiment agree with these studies as the application of CNO to the LH helped increase motivated behaviour for the ELS condition and the overall number of Orx and MCH within the LH. DISCUSSION/CONCLUSION The primary conclusion made by the study by Campbell et al. (2017) is that exposure to ELS can have lasting effects on brain circuitry that controls motivated behaviour as animal models in the ELS condition showed deficits in motivated behaviour of sucrose administration under the PR schedule of reinforcement. These effects can be reversed by the activation of circuits in the LH using hM3D(Gq) DREADD using CNO which increased sucrose responding in the ELS condition to the no-ELS control levels. Furthermore, this was confirmed through recordings of neuronal cells bathed in CNO, which showed increased action potential spiking in the LH. Also, CNO increased the number of fos-positive cells such as MCH and orexin in the LH. Altogether, this shows that the behavioural deficits due to ELS can be overpowered by pharmacological activation of LH circuits. Earlier literature that studied ELSinduced changes of behaviour and mood focused on brain regions involved in the neuroendocrine system such as PVN and the hippocampus (Meany et al., 2007), but Campbell et al. (2017) focused on other hypothalamic areas such as the LH that are also vulnerable to ELS and produce a similar phenotype. Campbell et al.’s work builds on the work performed by James et al. (2014) that showed that ELS induced a state of low-motivated behaviour when exposed to other rats and induced hypoactivity of orexin neurons within the LH in adult rats (James et al. 2014). Campbell et al. (2017) looked further into the impact of ELS on motivated behaviour through the use of sucrose administration through a more sensitive measure of reinforcement training. They demonstrated a pharmacological approach to tackling this issue by using DREADD, which allows for selective activation of circuitry responsible for motivational states. Their results show that the LH circuits are plastic enough to overcome states of low motivation caused by ELS (Campbell et al., 2017). While there is more to discover about the LH circuits and their role in motivated behaviours, the knowledge developed through Campbell et al.’s (2017) work can be valuable in developing treatments for depression. CRITICAL ANALYSIS The authors of the paper demonstrate that ELS induces a state of low motivational arousal which can be overcome using chemogenetic activation of the LH circuits. In experiment one, the main outcome was that sucrose administration was decreased in the ELS rats in comparison to no-ELS rats, but both had similar behaviours during the forced swim tests (Campbell et al., 2017). Therefore, the maternal separation induced a state of specifically low motivated behaviours but not behavioural despair, implying that early life stresses produce a state of anhedonia as opposed to hopelessness which are two different characteristics of depression

(Sherdell et al, 2017). In assessing the effect of DREADD activation of LH circuits on responding after ELS, with CNO treatment active lever pressing increased for both conditions but both eventually converged and under vehicle conditions, differences were observed only in the first 15 minutes, with the responding similarly converging after. These results demonstrate there is an initial deficit in motivated behaviour that evens out between the groups with time. I suggest using a more extensive learning paradigm and test for motivated behaviours after longer periods of time to see if the effect of CNO has persistent effects or if it's just for a short period after experimentation. In this way the true nature of motivated behaviour can be assessed under both vehicle and CNO conditions. A discrepancy lies between the results of James et al.’s (2014) study, where ELS induced suppression of Orx cells in adulthood related to decreased interaction with other animals. This study showed that there was a decrease in motivated behaviour (in this case through sucrose administration), yet there was no corresponding decrease of Orx cells in ELS rats vs no-ELS. The known limitations in Fos-mapping may account for this inconsistency. In line with other studies, c-Fos activation may not capture the persistence of neural changes that occur due to stress induction after translation (Lin et al., 2018). Also, c-Fos is degraded quite quickly and has a half-life of about one hour (Lin et al., 2018). Therefore, the decrease in Orx cells in James et al.’s (2014) study may have been due to the issues mentioned with Fosmapping, providing faulty results that this study worked off of. I suggest that studies monitor Fos-positive cells more long term and use an additional technique such as electrophysiological recordings to get more precise results. FUTURE DIRECTIONS Overall, Campbell et al.’s (2017) work contributed knowledge about the LH circuits and how they mediate the effects of ELS, which can be incredibly useful in the development of pharmacological treatment for depressive illnesses. Considering the limitations of the study, the authors next steps should be using a more extensive learning paradigm to assess the effects of DREADD on LH circuits after ELS-induction as evidence showed that active lever pressing converged with time for both conditions after the application of CNO and vehicle. From this study, DREADD was seen to promote enhanced motivated behaviour produced by ELS, by transducing hypothalamic neurons involved in the regulation of LH output that targets the VTA (Campbell et al., 2017). Previous studies have shown that the persistent effects of maternal separation show lasting influence on brain regions associated with motivation, where disrupted early rearing lead to lasting changes in the mesocorticolimbic dopamine neurons’ responsiveness (Brake et al, 2004) and dopamine release is involved in consolidation of memory that adds motivational importance to generally neutral stimuli (Wise, 2004). Considering a large number of fos-positive cells were non-orexin and non-MCH after CNO, it is likely that the responsive neurons were other projection neurons.

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So while orexin and MCH are key neuropeptides involved in motivational behaviours, I would encourage the authors to further include dopamine in their assessments, especially if a learning paradigm is involved, since dopamine is implicated in reward-related incentive learning (Beninger and Gerjikov, 2005). To directly test these ideas, I am suggesting that the next experiment begins with a more extensive learning paradigm. This means that the training for sucrose administration would consist of FR1 training sessions, followed by FR3, then PR to test breaking points. After 1-2 days, this would be repeated but it would begin with an FR3, then proceed to FR5 and repeat the PR. This is important as so much of the results and conclusions depend on the animals properly learning the sucrose administration task. Also, the more they self administer the sucrose and get reinforced, the more they will become dependent on receiving the reward. Due to this, any decrease in motivation in ELS animals, will be clearly evident. After drug administration (CNO/vehicle), the sucrose administration test would be repeated under the PR ratio but the rats would be retested once a week for four weeks post drug injection to see how lasting the effects are. For the mapping of neuropeptides before and after drug administration, there would be an assessment of the Orx, MCH and dopamine between the LH and VTA. This would be done using Fos-mapping to stay consistent but also through the use of electrophysiological recordings to validate results. I predict that ELS rats would have a significantly reduced amount of Orx, MCH and dopamine compared to non-ELS rats, specifically between the LH and VTA, and CNO administration would reverse these effects similarly to the effects we saw with Orx and MCH cells. This would suggest that dopamine is strongly implicated in motivated behaviours and that the LH as well as VTA circuits are what pharmacological treatments for depressive illnesses should target next.

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Brake, W.G., Zhang, T.Y., Diorio, J., Meaney, M.J., Gratton, A. (2004). Influence of early postnatal rearing conditions on mesocorticolimbic dopamine and behavioural responses to psychostimulants and stressors in adult rats. European Journal of Neuroscience, 19(7), 1863-1874

Campbell, E.J., Mitchell, C.S., Adams, C.D., Yeoh, J.W., Hodgson, D.M., Graham, B.A., Dayas, C.V. (2017). Chemogenetic activation of the lateral hypothalamus reverses early life stress-induced deficits in motivational drive. European Journal of Neuroscience, 46(7), 2285-2296.

Cohen, M.M., Jing, D, Yang, R.R., Tottenham, N., Lee, F.S., Casey, B.J. (2013). Early-life stress has persistent effects on amygdala function and development in mice and humans. PNAS, 110(45), 18274-18278.

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The Role of Drp1 Mediated Mitochondrial Fission in Glial Activation and Neuronal Injury, in Mice Models of Neurodegenerative Disease. Pouria Saffaran

Models of neurodegeneration have revealed the role of glial activation, brought about by neurotoxic protein aggregates, in causing neural damage. Moreover, excessive dynamin-related protein 1 (Drp1) mediated mitochondrial fission, has been shown in these models. Joshi et al. (2019), examined whether Drp1 mediated mitochondrial dysfunction is related to glial activation, in models of neurodegenerative disease. Mice microglial cells, exhibiting various neurotoxic proteins, were used to determine the nature of microglial activation. Activated microglial cells were then transferred to astrocyte cultures, in order to examine astrocytic induction into the A1 state. Primary mice neurons were then subjugated to treatment with activated astrocyte cultures. It was found that microglial activation was dependent on Drp1 mediated mitochondrial fission. This was indicated by the attenuation of microglial activation by a heptapeptide (P110), which is a protein that blocks Drp1 signaling. Furthermore, astrocyte activation by microglial cells were dependent on Drp1 signaling as P110 treatment of astrocytes, or microglia, attenuated the induction of the A1 astrocytic state. Lastly, dysfunctional mitochondrial release from activated astrocytes led to neural damage, while the removal of dysfunctional extracellular mitochondria blunted these effects. Joshi et al. (2019) elucidate the nature of glial activation, in models of neurodegenerative disease. Furthermore, these findings reveal a mechanism by which glial activation may be inhibited, in turn reducing neural damage. However, Joshi et al. (2019) only focused on a unidirectional relationship between microglia and astrocytes. Future studies should investigate the role of astrocytes in activating microglia, and potentially leading to neural injury. Key Words: Microglia, NaĂŻve Astrocytes, A1 Astrocytes, Neurodegeneration, Drp1, Mitochondrial Fission, P110.

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c Introduction Various models of neurodegenerative disease have implicated the role of certain protein aggregates as a causal factor in neurodegeneration (Aguzzi and Oconnor, 2010). Neurotoxic protein accumulation has the potential to activate inflammatory responses within the central nervous system, which in turn may lead to neural dysfunction and apoptosis. The exact nature of this relationship has not yet been elucidated, although glial cells have been hypothesized to perform a mediatory role in this process (Bernhardi and Ramirez, 2001). Specifically, microglia and astrocytes have been shown to be activated by neurotoxic protein aggregates (Bernhardi and Ramirez, 2001) (Hou et al, 2011). Microglial cells perform various important functions within the central nervous system. These include neural surveillance, neuroprotection, and phagocytosis of redundant neurons. However, microglial cells can be induced to release proinflammatory markers, which can lead to neural apoptosis (Chen and Trapp, 2015). Similarly, astrocytes execute a multitude of essential functions within the brain, such as the maintenance of homeostasis and neuron health, alongside tissue repair. Conversely, astrocytes can be induced into a proinflammatory state, termed A1, which can engender neurodegeneration (Barreto et al, 2011). There exists a relationship between microglial and astrocytic functioning. These cells communicate through various signaling pathways. For instance, it has been shown that activation in microglial cells can lead to the activation of astrocytes (Liddelow et al, 2017), while the reverse effect may also exist (Lian et al, 2016). This becomes important in the context of neurodegenerative disease as activation of one glial cell line may lead to the proliferation of signaling cascades, activating other glial cell lines and accelerating neurodegeneration (Liu et al, 2011). The nature of the bidirectional communication between microglia and astrocytes has not been fully determined. Previous studies have also revealed increased mitochondrial dysfunction through the activation of the dynamin-related protein 1/mitochondrial fission 1 (Drp1-Fis1) pathway, in models of neurodegeneration brought about by neurotoxic protein accumulation (Guo et al, 2013) (Joshi et al, 2018) (Joshi et al, 2017). Specifically, it has been shown that increased neurotoxic protein accumulation leads to the increased recruitment of Drp1 leading to excessive mitochondrial fission through the activation of Fis-1 receptor. Increases in mitochondrial dysfunction, brought about by excessive Drp1-Fis1 signaling, induces neuronal death. Drp1-Fis1 signaling is attenuated by P110 treatment, which also reduces neurodegeneration and improves mitochondrial health. Furthermore, microglia and astrocyte activation have been reported in these models. Joshi et al. (2019) examined whether mitochondrial dys-

mitochondrial fission and mitochondrial dysfunction through Drp1

function, brought about by excessive Drp1 signaling, leads to glial

reveal a novel pathway in the relationship between neurotoxic pro-

activation and whether these changes mediate the relationship

tein accumulation and microglial activation, independent of LPS-

between neurotoxic protein accumulation and neural death. The

related pathways.

signaling. These in turn induce the transformation of astrocytes into the A1 state. The induction of the Astrocytic A1 state leads to increased mitochondrial fission and fragmentation in these cells and eventually leads to neuron death. These effects are attenuated by P110 treatment. Moreover, the effects of P110 treatment are specifically due to the blocking of glial activation by Drp1 recruitment. Results Microglial activation is induced through increased Drp1 signaling, in a lipopolysaccharide (LPS) independent manner. Using mice models of Huntington’s disease, it was shown that microglial activation can be induced by mitochondrial dysfunction. Mitochondrial dysfunction was partially indicated by increased reactive oxygen species in activated microglial cells genetically modified to express Huntington’s related proteins (Q73) (Figure 1A). Microglial activation was indicated by increased inflammatory markers such as tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β) (Figure 1C). P110 treatment attenuated mitochondrial dysfunction while also decreasing inflammatory markers (Figure 1A, C). Moreover, P110 treatment reduced Drp1 accumulation (Figure 1B). Decreased levels of mitochondrial dysfunction and Drp1 activity indicated that the Drp1-Fis1 pathway had been inhibited by P110, while decreased proinflammatory marker release indicated reductions in microglial activation through P110 treatment. These results suggest that neurotoxic protein expression leads to microglial activation through increased Drp1-Fis1 signaling and mitochondrial dysfunction. Similar results were observed in other models of neurodegenerative disease. TNF- α and IL-1β production were also increased by administration of LPS, often used in models of microglial activation (Figure 1 D). LPS administration exhibited an additive effect in R6/2 microglial cells (cell models for Huntington’s). In other words, inflammatory marker production was increased over and above the effect of protein aggregation, in R6/2 microglia treated with LPS. Moreover, inflammatory marker production was comparable in wild type microglial cells subjected to LPS treatment and R6/2 microglial cells treated with LPS and P110. These results indicated that LPS activation of microglial cells initiates signaling pathways independent of those initiated by neurotoxic protein aggregates. Previous studies have implicated the role of Drp1 mediated signaling in microglial activation. Increased Drp1 activity has been shown to lead to excessive mitochondrial fission and elevations in inflammatory signaling by microglial. Moreover, inhibition of Drp1 activity has been shown to decrease pro-inflammatory markers produced by microglia (Park et al, 2017). However, these effects had been induced by LPS administration. Joshi et al. (2019)

results suggest that microglia are activated as a result of excess

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by these cells (Figure 2B). Treatment of microglial cells with P110 reduced activation of astrocytes, suggesting that astrocytic activation was related to mitochondrial dysfunction in microglia (Figure 2A, B). Furthermore, the activation of astrocytes led to deficits in mitochondrial health in these glial cell lines (Figure 2A). In a separate trial, astrocytes were subjected to proinflammatory markers, TNF- α and IL-1β, resulting in the induction of astrocytes into the A1 state, which was indicated by increased expression of A1 related genes (Figure 2D) This effect was blunted by the direct treatment of astrocytes with P110 (Figure 2D), suggesting that Drp1 mediated mitochondrial fission is also important in astrocyte activation. Lastly, activation of astrocytes led to increased release of dysfunctional mitochondria by these glial cells. This was indicated by decreased cytochrome C concentration in the extracellular domain suggesting decreased mitochondrial membrane integrity (Figure 2C). Release of dysfunctional mitochondria was attenuated by P110 treatment of astrocytes (Figure 2C), providing evidence for the role of Drp1 in astrocytic mitochondrial fragmentation. These results are in line with previous research. It has

been shown that excessive mitochondrial fission in astrocytes leads to astrocytic activation, while the inhibition of mitochondrial fission has been shown to attenuate this effect (Li et al, 2016). Lastly, Figure1. Figures representing differential mitochondrial functioning and cytokine productions in tested microglial cells. A) microglial cells exhibiting the dysfunctional Huntington’s disease related protein (Q73) exhibited higher levels of mitochondrial reactive oxygen species (ROS), than microglial cells exhibiting a normal protein. Increases in mitochondrial ROS was reduced following P110 treatment. B) microglial cells exhibiting the Q73 protein express higher levels of Drp1 accumulation, when compared to a control protein (VDAC). This effect is blunted by P110 treatment. C) TNF- α and IL-1β levels are increased in microglial cells exhibiting the Q73 protein, compared to control microglial cells. These effects are blunted following P110 treatment. D) TNF- α and IL-1β production are increased in microglial cells modeling Huntington’s disease (R6/2 cells). LPS administration further increases TNF- α and IL1b production. P110 treatment of LPS administered R6/2 cells reduces proinflammatory marker production to levels comparable to those found in wild type cells treated with LPS. Figure 1. Adapted from “Fragmented mitochondria released from microglia trigger A1 astrocytic response and propagate inflammatory neurodegeneration.” by A. Joshi et al. (2019). Nature Neuroscience, 22(10), 1635–1648. Microglial activation leads to the induction of the A1 state in astrocytes, while also increasing mitochondrial fragmentation in these cells. Astrocytic activation was induced by activated microglial cells. Cell cultures containing activated microglial cells were transferred to astrocyte cultures. This led to the activation of astrocytes and their induction into the A1 state, which was partially indicated by increased release of proinflammatory cytokines TNF-α and IL-1α

microglia-astrocyte cross talk has been observed in previous studies. Specifically, it has been shown that microglia can induce astrocytic A1 state through the production of diffusible signals (Liddelow et al, 2017). Joshi et al. (2019) further previous research by revealing the importance Drp1 mediated mitochondrial fission in microglial activation of astrocytes.

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c Figure 2. figures representing changes in astrocytes and their induction to the A1 state, after being exposed to activated microglia. A) mitochondrial ROS levels increased in astrocytes after their treatment with microglial cells exhibiting the Q73 protein. This effect was attenuated after the treatment of microglial cells with P110. B) proinflammatory markers, TNF- α and IL-b were increased in astrocytes after being exposed to microglial cells expressing Q73 or G93A (an ALS model). These effects were attenuated after the treatment of microglial cells with P110. C) decreased cytochrome C concentration in mitochondria of astrocytes, following activation with LPS. This effect was attenuated by P110 treatment. D) astrocytes treated with TNF- α and IL-1α were induced into the A1 state, as indicated by increased transcription of A1 specific genes (shown in red). A1 induction was reduced following P110 treatment Figure 2. Adapted from “Fragmented mitochondria released from microglia trigger A1 astrocytic response and propagate inflammatory neurodegeneration.” by A. Joshi et al. (2019). Nature Neuroscience, 22(10), 1635–1648. Activated microglial and astrocyte cultures lead to neuronal death. The transference of microglia-activated astrocytic cultures to healthy neuronal cultures led to increased neuronal injury. This was indicated by increased lactate dehydrogenase (LDH) release from neurons (Figure 3). Since LDH is usually found in the intracellular space, increased concentrations of this molecule on the extracellular space indicate cell damage. The role of activated astrocytes in causing neural damage was mediated by increased extracellular concentrations of dysfunctional and fragmented mitochondria. The removal of dysfunctional mitochondria from the extracellular space decreased neuronal cell injury. Furthermore, treatment of microglial cells with P110 also decreased neuronal damage (Figure 3). These findings reveal the role of dysfunctional mitochondria in the relaying of signals from astrocytes and microglia to neurons, and eventually leading to neuronal degeneration. Previous studies support these findings. For instance, it has been shown that astrocytic mitochondrial health is related to neuronal functioning. Specifically, astrocytes release mitochondria into the extracellular space as a neuroprotective mechanism after injury, while decrements in this process worsen neuronal health (Hayakawa et al, 2016). The present study furthers these findings by revealing the injurious effects of dysfunctional mitochondria, released by glial cells into the extracellular space.

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Figure 3. Levels of LDH release as a result of neural treatment with naïve (ACM) or activated astrocyte cultures (aACM). Treatment of neurons with activated astrocyte cultures leads to increase LDH release by neurons, indicating injury. Activated astrocytic cultures treated with P110, or deprived of mitochondria (MitoΔ), did not cause neuronal injury. Figure 3. Adapted from “Fragmented mitochondria released from microglia trigger A1 astrocytic response and propagate inflammatory neurodegeneration.” by A. Joshi et al. (2019). Nature Neuroscience, 22(10), 1635–1648. Discussion/conclusion Joshi et al. (2019) reveals the importance of Drp1 mediated mitochondrial fission as a result of neurotoxic protein accumulation, in models of neurodegenerative disease. Specifically, it is shown that excessive mitochondrial fission leads to the activation of microglia, in turn inducing astrocytic A1 state, and eventually leading to neuronal cell death. These effects can be blunted by treatment of glial cells with P110, which inhibits Drp1 mediated mitochondrial fission. The results of Joshi et al. (2019) are mainly in line with the literature. Previous studies have shown glial activation in correlation with disease progression, in models of neurodegenerative disease caused by neurotoxic protein accumulation (Guo et al, 2013) (Joshi et al, 2018) (Joshi et al, 2017). Moreover, mitochondrial functioning has been implicated in neuronal maintenance (Hayakawa et al, 2016), while the role of Drp1 mediated mitochondrial fission in neurodegeneration has been indicated by previous research (Guo et al, 2013) (Joshi et al, 2018) (Joshi et al, 2017). The current findings reveal a mechanism by which mitochondrial dysfunction lead to neuronal death through the activation of microglia and astrocytes. Furthermore, these results reveal the therapeutic potential of P110, illustrating that this molecule can decrease neuronal apoptosis as a result of glial activation, in models of neurodegenerative disease. Moreover, Joshi et al. (2019) show that glial activation can be brought about by

pathways independent of LPS signaling. This is particularly important as previous studies use LPS treatment to induce glial activation (Park et al, 2017). However, protein aggregation may lead to glial activation through pathways independent of LPS signaling, indicating that research paradigms using LPS may not be ecologically valid. As noted by the authors, mitochondrial dysfunction in glial cells is but one mechanism through which neurotoxic protein accumulation may lead to neuronal apoptosis. Neurodegeneration can come about by the activation of blood derived immune cells and their entry into the central nervous system. Furthermore, neuronal death leads to the production of neuronal debris which further activate glial cells, independent of mitochondrial

dysfunction. Therefore, focusing on one possible causal factor, i.e.

Future studies

glial activation, can not provide a comprehensive theory of neurodegeneration.

Future studies need to consider the role of primary astrocytic activation as a result of neurotoxic protein accumulation. These models can make use of astrocytic cells which express proteins related to neurogenerative disease. Culturing of astrocytic

Critical analysis

cells with this genetic modification can reveal whether astrocytic Joshi et al. (2019) focus on the role of microglial activation in the propagation of inflammatory signals, through the induction of astrocytes into the A1 state, and culminating in neural damage. However, the nature of microglia-astrocyte cross talk need not be one directional. In fact, various studies point to the regulatory role of astrocytes on microglial activation. Astrocytes can activate microglial via the production of C3 molecules or chemokines (Kostianovsky et al, 2008) (Tanuma et al, 2006). Furthermore, astrocytes can regulate both nearby microglial cells and microglial cells in other regions of the brain, through indirect pathways (Liu et al, 2011). Moreover, astrocytic activation can be induced directly by neurotoxic protein accumulation, such as amyloid beta accumulation, in models of neurodegenerative disease (Hou et al, 2011). Therefore, glial activation may start with astrocytes rather than microglia. This notion seems to be supported by studies which reveal microglial activation by astrocytes, in models of neurodegenerative disease (Verkhratsky et al, 2010) (Tanuma et al, 2006). The present research also shows the release of dysfunctional mitochondria by astrocytic cells and reveals the importance of extracellular mitochondria in astrocytic activation and neuronal death. However, extracellular mitochondria can also activate microglial cells (Wilkins et al, 2016). Lastly, microglial activation can lead to neuronal death independent of astrocytic activation (Bate et al, 2004). Joshi et (2019) focuses on the effects of microglial activation on the induction of A1 state in astrocytes, in turn leading to neural death, but they neglects the direct effects of microglial activation, which may come about by activated astrocytes, on neuronal health.

activation, as a result of neurotoxic protein accumulation, is medi-

Previous research indicates the possibility of neural damage brought about by microglia activated by astrocytes. This is the case as astrocytes have been shown to activate microglial cells (Kostianovsky et al, 2008) (Tanuma et al, 2006) (Liu et al, 2011), a finding also observed in models of neurodegenerative disease. (Verkhratsky et al, 2010). Furthermore, Joshi et al. (2019) reveal that astrocytes release dysfunctional mitochondria which can lead to neural damage. However, dysfunctional extracellular mitochondria may also lead to microglial activation (Wilkins et al, 2016), while activated microglial cells can lead to neural damage directly (Bate et al, 2004). Since A1 astrocytes can release dysfunctional mitochondria (Joshi et al. 2019), it may be possible for astrocytes to activate microglia as a result of excessive Drp1 signaling, which may in turn lead to neural death. This notion is not examined by Joshi et al. (2019) as they simply focus on the one-way activation of astrocytes by microglia.

tions are possible. For instance, if the treatment of astrocytes with

ated by Drp1 signaling and mitochondrial dysfunction. Astrocytic activation would be indicated by increased inflammatory marker release, such as increased TNF-Îą or IL-1Î˛, or increased expression of A1 related genes. If P110 treatment of activated astrocytes attenuates proinflammatory marker release and A1 gene expression, it can be assumed that the activation of astrocytes is mediated by Drp1 signaling. This would confirm the results of Joshi et al. (2019). Next, activated astrocyte culture can be transferred to microglial cultures, in order to examine whether excessive mitochondrial fragmentation and release by astrocytes induces microglial activation. This can be deduced by monitoring the release of inflammatory markers from microglial cells. If P110 treatment of astrocytes cells inhibits microglial activation, it can be assumed that Drp1 signaling in astrocytes, leading to increased mitochondrial fragmentation and release, leads to the activation of microglia. Activated microglial cells can then be transferred to neuronal cell cultures to examine their effects on neural health. Monitoring the extracellular space in microglia-neuron cultures can reveal whether activated microglial cells lead to neuronal death through increased dysfunctional mitochondrial release. Increased dysfunctional mitochondria in the extracellular space would be the first indication of such a scenario. Moreover, if the clearance of extracellular mitochondrial fragments leads to the reduction of neuronal cell death, it can be assumed that activated microglial cells directly lead to neuronal cell death through excess mitochondrial fragment release. If these results do not occur, multiple interpretaP110 does not attenuate microglial activation, it could be that astrocytic activation of microglia is not mediated by Drp1 signaling. Furthermore, if the removal of extracellular mitochondria, produced by microglia, does not decrease neural damage, it could be that activated microglial cells lead to neural damage through other pathways. In any case, it is important that future studies consider the bi-directional nature of microglia-astrocyte cross talk as this would reveal a more comprehensive picture of neurodegeneration brought about by glial activation.

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Examination of Gut Microbial Dysbiosis Induced by ASDAssociated Environmental Risk Factors. Chanthie Sahota

The presence of autism spectrum disorder, abbreviated as ASD, is rapidly increasing among individuals worldwide. ASD includes an array of conditions that are identified by deficits in critical neurodevelopmental periods. Additional symptoms of ASD are heightened serum serotonin levels and gastrointestinal disorders, especially inflammatory bowel disease (IBD). However, the cause of these symptoms in relation to ASD is unknown. Another unique characteristic of ASD recently identified by researchers is the altered composition of gut microbiota. This alteration induces gut microbial dysbiosis that is likely responsible for the gastrointestinal comorbidities observed in ASD patients. As these complex interactions are currently unknown, research examining the causative agents of microbial dysbiosis may provide further insight on the onset of ASD. The research study conducted by Lim and colleagues (2017) analyzed the relationship between gut microbial dysbiosis and ASD by exposing mice (n = 10) to environmental risk factors of ASD to induce autism. The study found that mice prenatally exposed to ASD-related environmental risk factors VPA and poly I:C displayed gut microbial dysbiosis similar to that found in clinical reports of ASD. The researchers also observed a disruption in microbialassociated pathways that can influence the development of ASD. Greater amounts of serotoninproducing bacteria and serum serotonin levels were found in both experimental groups. These findings suggest that the environmental risk factors that contribute to ASD onset may also induce ASD-related gut microbial dysbiosis. This indicates that the gut microbiome may aid in the development of ASD. Keywords: autism spectrum disorder (ASD), gut microbiota, dysbiosis, gastrointestinal disorders, ASDassociated environmental risk factors

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c BACKGROUND & INTRODUCTION

prenatally exposed mice to ASD-related environmental risk factors valproic acid (VPA) and polyinosinic:polycytidylic acid (poly I:C) to Autism spectrum disorder (ASD) is considered to have a induce autistic and IBD symptoms (Lim et al., 2017). After 6 weeks, strong heritable component, where over 50% of ASD cases can be the microbial composition, behaviour, and serotonin levels were attributed to genetics (Betancur, 2011; Sandin et al., 2014). ASD is examined in the mice to identify the presence of any ASD-related defined by a range of conditions involving deficits in critical neurosymptoms (Lim et al., 2017). The study found that the mice disdevelopmental periods, such as repetitive behaviours and impaired played ASD-related behavioural deficits, decreased microbial diversocial communication (Lim et al., 2017). Additional comorbidities sity resulting in dysbiosis, increased bacteria associated with seroidentified in ASD patients are hyperserotonemia and gastrointestitonin production, and hyperserotonemia (Lim et al., 2017). These nal abnormalities, particularly inflammatory bowel disease (IBD), findings indicate that environmentally induced microbial dysbiosis with prevalence rates of 40% and 90%, respectively (McElhanon, may aid in the onset of ASD-related symptoms. McCracken, Karpen, & Sharp, 2014; Pagan et al., 2014). Despite the high prevalence rates of these conditions, their cause and relationship to ASD are currently unknown (Lim et al., 2017). MAJOR RESULTS ASD-related Behavioural Deficits in Treatment Mice Another feature recently observed by current research is As demonstrated in Figure 1, Lim and colleagues (2017) the alteration of gut microbiota in ASD patients (Hoban et al., prenatally injected environmental risk factors VPA (500 mg/kg) and 2016; Hsiao et al., 2013). Changes in the composition of the gut poly I:C (20 mg/kg) separately to model ASD in a total of 10 mice. microbiota can result in microbial dysbiosis (Lim et al., 2017). Gut VPA is a drug used to prevent epileptic pregnant women from exmicrobial dysbiosis is strongly correlated with gastrointestinal disperiencing seizure-induced miscarriages, but is associated with an orders as indicated by the findings of Vuong and Hsiao (2017). In increased risk of ASD (Heyer & Meredith, 2017). Therefore, VPA their review, Vuong and Hsiao (2017) analyzed 12 research studies serves as an excellent imitator of maternal VPA exposure to model and found greater dysbiosis severity to be associated with inenvironmentally induced ASD as seen in other environmental ASD creased gastrointestinal conditions in over 3500 ASD patients. Also, models of past literature (Heyer & Meredith, 2017; Vuong & Hsiao, microbial dysbiosis in the gut is correlated with elevated serum 2017). Also, poly I:C is an immunostimulant commonly used to serotonin levels (Hsiao et al., 2013). A study conducted by Liu and model ASD caused by maternal infection during pregnancy (Heyer colleagues (2017) found significantly higher plasma serotonin lev& Meredith, 2017). Prenatal exposure to these environmental risk els in obese and non-obese individuals with polycystic ovary synfactors resulted in the expected behavioural phenotypes of ASD drome in conjunction with microbial dysbiosis. (Lim et al., 2017). Using traditional behavioural tests, which were an open In addition to correlations with ASD-associated comorbidities, gut microbial dysbiosis is thought to be associated with a vari- field test and social interaction test, the VPA and poly I:C mice displayed lower social behaviour and greater anxiety at 6 weeks of ety of neurodevelopmental disorders, including ASD (Vuong & age (Lim et al., 2017). This is in line with previous literature to repHsiao, 2017). Despite the mechanism not being well understood, resent ASD in mice (Heyer & Meredith, 2017). For this reason, the many studies have found correlational evidence to support the VPA and poly I:C mice will be referred to as ASD mice due to their idea of a bidirectional interaction between the gut and brain in both mouse and human models (Hsiao et al., 2013; Vuong & Hsiao, display of ASD-associated deficits in behaviour (Lim et al., 2017). 2017). Demonstrating that dysbiosis induced by reduced gut bacterial diversity is linked to neurodevelopmental disorders, microbial dysbiosis is commonly seen in patients with schizophrenia (Severance, Prandovszky, Castiglione, & Yolken, 2015; Severance, Yolken, & Eaton, 2016). Decreased microbial diversity was also observed in 33 ADHD patients, who additionally reported gastrointestinal distress (Aarts et al., 2017; Prehn-Kristensen et al., 2018). As previously mentioned, current research examining ASD patients has seen improved gastrointestinal and ASD-related symptoms after fecal microbiota transplants of 8 weeks to enhance gut bacterial diversity, indicating a possible link between microbial dysbiosis and ASD (Kang et al., 2017; Yang, Tian, & Yang, 2018). Although ASD has a genetic basis, the rapid increase in the prevalence of the disorder worldwide indicates there are environmental factors that may be influencing its etiology (Lim et al., 2017). In addition to the onset of ASD, the co-occurrence of gastrointestinal abnormalities implies a relationship in which the shared underlying mechanism is currently unknown, but is hypothesized to be mediated by the gut microbiome (Lim et al., 2017). The present research paper by Lim and colleagues (2017) sought to investigate the relationship between gut microbial dysbiosis and the co-occurring disorders, ASD and IBD. The present Figure adapted from Lim et al. (2017). Molecular Brain, 10. study hypothesized that ASD-associated environmental risk factors Figure 1. Visual summary of the methods and results of the present induced microbial dysbiosis, which in turn would influence the study. onset of ASD-related symptoms (Lim et al., 2017). The researchers

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Lower Gut Microbial Diversity and Alteration in ASD Mice Gut microbial dysbiosis and distinct patterns of taxa associated with dysbiosis were observed in the ASD mice (Lim et al., 2017). Although the phylum microbial composition did not significantly differ between the ASD and control mice (Figure 2A), the ASD mice exhibited significantly lower diversity in microbial species as shown in Figure 2B (Lim et al., 2017). Findings of reduced microbial diversity are consistently reported in clinical studies of ASD children (Kang et al., 2017).

In the present study, Lim and colleagues (2017) identified Sporanaerobacter to be of the Clostridial class. The results of their study demonstrated significantly higher Sporanaerobacter levels in the ASD mice compared to the control in addition to elevated serotonin levels as seen in Figure 3 (Lim et al., 2017). These findings support the idea that spore-producing microbes indirectly enhance serotonin levels through increased production of SCFAs (Lim et al., 2017; Yano et al., 2015). The results also suggest a possible mechanism that links ASD-related environmental risk factors to ASDassociated symptoms via such dysbiosis (Lim et al., 2017).

Previous literature has also reported alterations in the microbiota at the species and genus levels in ASD and IBD patients (Balish & Warner, 2002; Golinska et al., 2013; Kang et al., 2017). Such studies have found reduced Oscillospira sp., Prevotella, and F. prausnitzii and greater Enterococcus, Desulfovibrio, and Sporanaerobacter levels in both ASD and IBD patients (Balish & Warner, 2002; Golinska et al., 2013; Kang et al., 2017). In the present study, Lim and colleagues (2017) used 16S rRNA survey of mice fecal DNA (Figure 1) to detect these similar patterns in the ASD mice. These findings indicate that environmentally induced prenatal events may provoke dysbiosis that likely progresses into ASD and its associated comorbidity IBD (Lim et al., 2017). Figure adapted from Lim et al. (2017). Molecular Brain, 10, 8. Figure 3. Potential metabolic alterations in ASD mice. A. Sporanaerobacter relative abundance in ASD mice and control. B. Serum serotonin levels analyzed via ELISA in mice groups. CONCLUSIONS/DISCUSSION

Figure adapted from Lim et al. (2017). Molecular Brain, 10, 4. Figure 2. Composition and diversity of gut microbiota in the mice groups. A. Phylum microbial composition of all mice groups. B. Alpha-diversity of all mice groups analyzed using Shannon index. Greater Serum Serotonin Levels in ASD Mice Through blood samples, Lim and colleagues (2017) observed significantly higher serum serotonin levels in the ASD mice compared to the control group. This particular finding has been commonly found among human ASD cases, but has not yet been reported in past literature using VPA- and poly I:C-induced ASD mice models (Lim et al., 2017). Despite this observation lacking supporting evidence, recent research papers have identified sporeforming microbes to enhance serotonin levels via greater shortchain fatty acid (SCFA) production (Reigstad et al., 2015; Yano et al., 2015). These particular publications examined germ-free and humanized (received microbiota from humans experiencing gastrointestinal issues) mice and found increased SCFA and serotonin levels in the humanized mice (Reigstad et al., 2015; Yano et al., 2015). Yano and colleagues (2015) further explored the role of various taxa and identified bacteria belonging to the spore-forming Clostridial class to restore SCFA and serotonin levels in specificpathogen free mice.

The main findings of the present study conducted by Lim and colleagues (2017) were that environmental ASD risk factors VPA and poly I:C resulted in mice demonstrating ASD-related behavioural deficits, decreased gut microbial diversity resulting in dysbiosis, increased SCFA-producing bacteria associated with serotonin production, and enhanced serum serotonin levels. These findings suggest a possible underlying mechanism where gut microbiota may promote the development of ASD by affecting various metabolic pathways associated with comorbidities of ASD (Lim et al., 2017). This would involve indirectly increasing serotonin levels to promote the development of hyperserotonemia and inducing gut microbial dysbiosis that in turn would lead to gastrointestinal abnormalities, such as IBD. Also, the results of the study comply with previous findings of gut microbial dysbiosis and associated patterns in taxa, reduced microbial diversity, and hyperserotonemia in ASD patients (Kang et al., 2017; Lim et al., 2017). However, Lim and colleagues (2017) are of the very first to speculate the underlying mechanism relating ASD-associated microbial dysbiosis to increased serotonin levels via correlational evidence. CRITICAL ANALYSIS The authors of the present paper imply that gut microbial dysbiosis may be an influential factor concerning the onset of ASD (Lim et al., 2017). Their research involved using environmental ASD risk factors VPA and poly I:C to suggest that the role of these agents in promoting ASD onset and its associated comorbidities, such as IBD, is by causing dysbiosis in the gut microbiome to alter particular taxa and produce the previously described findings (Lim et al., 2017). Despite demonstrating that the environmental ASD risk factors induced microbial dysbiosis, the other reported findings are correlational. The researchers could have measured SCFA

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levels in the ASD mice to further support the proposed idea that Sporanaerobacter indirectly enhanced serum serotonin levels through greater production of SCFAs. Although this link would have still been correlational, it would have strengthened their proposed mechanism and further supported previous findings concerning the role of spore-forming microbes and heightened serotonin levels (Reigstad et al., 2015; Yano et al., 2015). It also represents the need for additional research examining the role of SCFAs in relation to serotonin levels and ASD onset. Metatranscriptomic analysis could have also been used by the researchers to identify bacterial genes that were most active in the ASD mice (Franzosa et al., 2014). This would have been an accurate measure to assess whether genes involved in SCFA production of Sporanaerobacter were greatly expressed in relation to other genes within the microbiomes of the ASD mice, strengthening the proposed relationship between Sporanaerobacter and increased serotonin levels (Franzosa et al., 2014).

gest that another microbe might be influencing serotonin levels. Additionally, researchers can examine the gut microbiota of the mothers to assess whether they share similar dysbiosis induced patterns of taxa with their offspring. This would aid in reducing any confounding variables and possibly identify if the mothers’ microbial dysbiosis causes the dysbiosis in their offspring via transmission in milk. If findings of such a study indicate that the dysbiosis in the mothers are able to influence dysbiosis in the offspring, this information would identify the mothers as a potential target that treatments should be directed towards. If no findings suggest this, the role of the mothers can be excluded from further examination. Also, future studies can consider the use of genetic ASD models in addition to environmental ASD models. This may provide further insight on whether the microbial dysbiosis that involves greater abundance of Sporanaerobacter is also found in ASD patients whose onset was influenced by genetics. It can also lead to the investigation of whether these patients display greater SCFA levels in support of the previously described speculated relationship or In addition, the present paper did not examine the gut minot. By improving current knowledge of ASD induced by environcrobiota of the mothers (Lim et al., 2017). It is possible that the mental risk factors and its underlying mechanisms, this can aid in environmental ASD risk factors VPA and poly I:C induced gut mithe development of effective treatments for ASD patients. Specialcrobial dysbiosis in the mothers first, which in turn would cause ized treatments that target the gut microbiota, specifically sporedysbiosis of the gut microbiota of the offspring after birth (Lim et producing bacteria, may help to better alleviate ASD-related condial., 2017). This would occur through the consumption of the mothtions that impair the daily lives of those living with ASD. ers’ milk, which is known to consist of bacteria that aid in the maturing of the offspring’s gut microbiota (Lim et al., 2017). It is also possible that the mothers may have displayed elevated serum serotonin levels. However, measurements of the mothers were not taken and thus it is unclear what effects VPA and poly I:C may have directly had on the mothers before indirectly effecting the offspring after birth. Therefore, it is uncertain if the findings reported in the present study were of an indirect result of VPA and poly I:C via dysbiosis of the mothers’ gut microbiota rather than a direct relationship. This represents the need for research exploring how such factors may affect the mothers and how these effects are transferred to their offspring after birth. This information may aid in providing a novel treatment target it shown to incorporate the role of dysbiosis in the mothers.

FUTURE DIRECTIONS To better connect how environmental ASD risk factors VPA and poly I:C promote the development of ASD through gut microbial dysbiosis, future studies should examine the role of sporeforming Clostridial species on SCFA production and serotonin levels (Yano et al., 2015). Researchers can conduct an experiment that focuses on such bacteria, for example, Sporanaerobacter by using specific-pathogen free mice to explore the effects Sporanaerobacter may have on serotonin levels (Yano et al., 2015). A subset of germ-free mice could be given a gut microbiome that models a healthy gut while another subset receives a microbiome that lacks Sporanaerobacter, or another spore-producing microbe, to examine the effects Sporanaerobacter may have on serum serotonin levels and the onset of ASD-related behavioural symptoms (Yano et al., 2015). If serum serotonin levels are significantly lower in the mice lacking Sporanaerobacter compared to those with an intact microbiome, this would indicate that this particular microbe is likely responsible for elevated serotonin levels. It would also support the ideas of previous publications and of the present paper and aid in shifting from correlation to causation. However, if serotonin levels are unaffected in the treatment group, this would sug-

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Evaluating the efficacy of dance interventions on motor symptoms and quality of life aspects in Parkinson’s patients Rena Seeger

Parkinson’s disease (PD) is a neurodegenerative disorder that affects one’s physical and psychological functioning. The most common motor symptoms include resting tremor, rigidity, and bradykinesia, all of which negatively influence psychological health and can result in apathy and depression. There has been increasing evidence suggesting that rehabilitative exercise interventions, like dance, can protect the brain, allowing it to repair. However, few studies have evaluated the effects of dance therapy on both standard measures and qualitative perspectives of patients with PD. The present study by Westheimer et al. from 2015 investigated the effects of a dance intervention on motor and quality life aspects of PD following 16 dance sessions over the course of 8 weeks. The Hoehn and Yahr, UPDRS III, Berg Balance Scale, Beck Depression Inventory, and PDQ-39 were administered before and after the intervention, followed by individual interviews after the intervention. UPDRS III total scores and gait and tremor sub scores improved following treatment, and participants reported physical, emotional and social benefits during the interviews. This study demonstrates the importance of using both quantitative and qualitative data when evaluating the efficacy of a treatment since the interviews revealed benefits that were not seen from the quantitative measures. As dance therapy is relatively easy to administer and given its numerous benefits in alleviating motor symptoms and improving psychological health, it could be offered more frequently as one of the first lines of treatment for Parkinson’s patients. Key words: Parkinson’s disease, dance therapy, treatment

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BACKGROUND Parkinson’s disease is a neurodegenerative disorder characterized by motor impairments such as bradykinesia, rigidity, resting tremor and gait problems, as well as non-motor symptoms including sleep disorders, sensory alteration, cognitive impairment and depression (Nemes et al., 2019). Although often classified as a ‘motor disorder’, its effects on psychological, emotional, social and financial functions of life are often what make PD so debilitating (Sharp and Hewitt, 2014). As a result, there is a strong need for interventions that cater towards treating not just the motor symptoms, but other aspects of quality of life. There is accumulating research suggesting that regular physical activity is associated with a lower risk of developing PD, as well as can slow down progression of its physical manifestations (Romenets et al., 2015). Regular exercise has been shown to improve gait speed, strength, functional capacity, and has been shown to reduce falls in PD patients (Romenets et al., 2015). However, many exercise interventions are unappealing for patients with PD and have low compliance and participation (Heiberger et al., 2011a). Since less than half of PD patients meet recommended daily level of physical activity, there is a need to find interventions involving physical activity that can help patients overcome barriers to participating (Ellis et al., 2013). Dance, defined as a choreographed routine of movements usually performed to music (Hui et al., 2009) in particular is a promising intervention for PD patients as it offers auditory, visual and sensory stimulation, social interaction, motor learning and memory (Kattenstroth et al., 2010). Compared to other forms of exercise therapy, patients with PD have higher compliance rates when it comes to dance therapy and are more motivated to attend the classes even after the study period (Hackney and Earhart, 2009). This is particularly important since research shows that exercise interventions are only beneficial if performed regularly over a longer period of time (Goodwin et al., 2008). Evidence for the effects of dance on many non-motor symptoms of PD is limited (Romenets et al., 2015). Additionally, few studies have evaluated both standard measures and qualitative perspectives of patients who have undergone dance therapy (Westheimer, 2013). The current study by Westheimer et al. from 2015 employed both quantitative measures pre and post intervention to examine the intervention’s effects on motor function and quality of life. Comparing the post-intervention scores to pre-intervention, the total UPDRS III (motor) score improved by 10.4% for the group, and within the UPDRS III, gait and tremor both improved as well. Participants reported benefits related to quality of life and wellbeing that were not reflected in the quantitative measures; for example, participants reported improvement in social interactions and increased happiness. This study in particular not only demonstrates the benefits of dance treatment for Parkinson’s disease, but also establishes the importance of using qualitative as well as quantitative measures when evaluating the efficacy of any treatment.

interviews after the last class to examine the intervention’s effects on motor function and quality of life. The quantitative tests have all been used in Parkinson’s patients previously, and have been found to be related to outcomes by different forms of dance treatment (Brichetto et al,. 2005, Hashimoto et al., 2015, Nemes et al., 2019). There were 12 participants (6 male, 6 female), with a mean age of 66.2, that had their diagnosis of PD confirmed by a movement disorder specialist. Each participant was assessed while in their best on-medication state. Due to the large range in physical functioning pre-intervention, and to determine if there were differences based on preintervention level of functioning, the patients were divided into two groups – ‘Better’ and ‘Worse’ based on the PDQ-39SI scores at baseline. Patients that scored above the mean were part of the ‘Better’ group, and those that scored below the mean were part of the ‘Worse’ group. Motor symptoms An improvement of 10.4% in the total UPDRS III score was observed from baseline to post-intervention, as well as a significant change in the gait sub score, which improved by 26.7%. There was also an improvement in the rest tremor sub score, which improved by 18.5%, though this change was not significant. There was no change in HY, which makes sense since the intervention was not aimed at changing the stage of PD. There were no significant changes in BBS either, which is a measure of performance on different tasks testing balance.

Quality of life Quality of life was measured by the PDQ-39SI and BDI, and from the individual interviews conducted post-intervention. There were no significant changes from baseline to post-intervention in PDQ39S1 or BDI scores. The participants gave very similar responses in the interviews despite their differences on HY, UPDRS III, BBS, BDI, and PDQ-39. All participants said they would continue to attend classes if they were ongoing, and individuals from both groups reported physical, social and emotional benefits of the class. Multiple participants reported the sense of companionship they experienced from taking the class with other individuals with PD , feel-

ing happier and more energized, and being able to move more easily on a day to day basis. Figure 1: Visual summary outlining the methods and major results found in the study.

MAJOR RESULTS The current study employed both quantitative measures pre and post intervention, including the Hoehn and Yahr (HY – a scale for measuring the progress of PD), the Unified Parkinson’s Disease Rating Scale (UPDRS III), Berg Balance Scale (BBS), Beck Depression Table 1. Scores from the quantitative tests taken before (a) and Inventory (BDI), and the Parkinson’s Disease Questionnaire (PDQpost-intervention (b) are shown for both the ‘Better’ and ‘Worse’ 39 – a self-report measure of quality of life), as well as individual

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groups. Westheimer O., McRae C., Henchcliffe C., Fesharaki A., Glazman S., Ene H., Bodis-Wollner I. (2015). Dance for PD: a preliminary investigation of effects on motor function and quality of life among persons with Parkinson’s Disease (PD). J Neural Transm, 122, 1263-1270.

These dance forms also require working memory, attentional control, and multitasking to learn the dance steps and keep in time with the music (Foster et al., 2013).

CONCLUSIONS/DISCUSSION

CRITICAL ANALYSIS

Comparing the post-intervention scores to pre-intervention, the total UPDRS III score improved by 10.4% for the group, and within the UPDRS III, gait and tremor both improved as well. This is significant as problems with gait and balance are some of the most incapacitating symptoms of Parkinson’s disease, leading to the most falls (Boonstra et al., 2008; Bloem et al., 2004). The qualitative interviews revealed benefits related to quality of life and wellbeing that were not represented in the quantitative tests; for example, participants reported improvement in social interactions and increased happiness. This is significant as it demonstrates not only the effectiveness of dance as a form of treatment for Parkinson’s patients, but it also shows that quantitative measures alone cannot capture all of the effects of treatments. In this case, some of the results from the quantitative tests even contradicted the responses from the qualitative interviews. Some possible explanations will be discussed later on.

The trends found in this paper show that dance intervention leads to improvements in both motor symptoms and quality of life, however, there are many discrepancies between studies that will be further explored here. A likely reason is due to the differences in the administration of the interventions. The current study ran 16 dance sessions over the course of 8 weeks, while another study ran the study over a course of a year (Duncan and Earhart, 2012). The frequency of having dance sessions, as well as the duration of each one and length of the entire study all would influence the impact of the intervention. The authors of the paper discuss

In addition to changes in motor symptoms, the authors of this study were looking at the impacts of dance intervention on quality of life. While there were no significant differences in overall PDQ39 scores after intervention, four out of twelve subjects showed improvements on questions related to cutting up food, difficulty with speech, and feeling unpleasantly hot or cold. In the qualitative interviews, participants reported benefits related to quality of life and wellbeing not reflected in these quantitative tests. Contrary to what was found in this experiment, studies by Hackney and Earhart and Volpe et al. found improvements in PDQ-39 in Parkinson’s patients after dance intervention. The benefits measured from the qualitative interviews suggest that when possible, dance therapy should be offered more frequently as a form of treatment for PD patients.

that the period of 8 weeks may have been too short a period of time for participants to experience other measurable improvements. Looking to the literature, it appears that studies that had longer interventions showed more significant improvements in symptoms compared to These improvements in motor symptoms measured using UPDRS the current study. However, there is a lack of understandare in line with what other studies have found (Hackney and Ear- ing of whether longer intervention periods or longer seshart 2009a; Duncan and Earhart, 2012; Foster et al., 2013). There sions lead to more superior results (de Dreu et al., 2012). are variable results among studies looking at specific sub-scores or categories of motor symptoms. For example, studies by Hackney and Earhart, Duncan and Earhart, and Foster et al. found no differences in gait between participants who had undergone dance intervention and those had no intervention. A study by Brichetto et al. found that scores of Freezing of Gait Questionnaire, but not UPDRS scores significantly improved after treatment. This suggests that the type of quantitative measure being employed in these studies also affects which motor symptoms show improvement. The current study found that within UPDRS, the gait sub-score significantly improved. These changes in motor symptoms may be a result of increased activity in the basal ganglia, which is increasingly understood to play a role in the control of dance movements. Forms of physical activity also enhance the concentration of serotonin, which may explain why participants reported improvements in quality of life and well-being (Heiberger et al., 2011b). Many dance forms like tango and ballet require specific steps moving forward and backwards (ballet particularly require high motor control), so these may be particularly helpful for improving gait and preventing backwards falls (Brichetto et al., 2006; Hackney and Earhart, 2009).

Another important factor that likely influenced the results is that participants were tested while on medication. Although testing participants while on their best ON medication state can help provide insight to how they perform daily activities, some of the deficits of PD are not fully seen. As a result, the authors lack a full picture on the effects of dance intervention on symptoms, and whether the intervention is disease-modifying (Duncan and Earhart, 2012). Although testing participants while they are on medication appears to be common, as is shown in meta-analysis by Kathryn Sharp and Jonathan Hewitt, for future studies, testing off medication is warranted. A study by Duncan and Earhart found improvements in UPDRS scores at 3, 6, and 12 months in patients who were off medication. This allowed them to identify that participation in the dance program had a disease modifying effect.

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Although most dance training employs visual focus, rhythm, and reproducing distinct parts of dance sequences, it is possible that different types and regimes of dance would have different effects on motor symptoms. The current study employed activities such as tendus, port de bras, and plie, which are all ballet steps. Some modern, jazz, and tap moves were taught in addition to ballet. This is different from many other dance studies that employ tango dancing as an intervention for Parkinson’s patients (Romenets et al, 2015, Hackney et al., 2007a, Hackney et al, 2007b, Hackney and Earhart 2009, Duncan and Earhart, 2012). It would be interesting for future studies to compare different forms of dance on Parkinson’s patients to determine which forms of dance are the most effective, and if some forms improve motor symptoms more than others. It is likely that some forms of dance are more effective at treating specific motor symptoms, which has important applications when using dance therapy as treatment. Depending on what symptoms a patience is experiencing, and which are the most severe, a different kind of dance may be prescribed. Since the current study found improvements in motor symptoms and quality of life aspects reflected in the qualitative interviews but not the quantitative interview, it is likely that the type of tests used also influence whether improvements are seen. Further research in the field could determine standardized ways to test for the effects of dance interventions on Parkinson’s patients that include both quantitative and qualitative measures. FUTURE DIRECTIONS The current study only had twelve participants, which could have led to Type II errors in which statistically significant effects of dance intervention that happened were not found. Moving forward, studies could have a larger sample size if possible, having participants at different stages of Parkinson’s disease and with different lifestyles to see the effects of dance on a larger range of participants. Studies could also employ dance interventions to patients off their medication to gain a better understanding of the effects of dance on Parkinson’s symptoms. It would also be interesting to test the effects of not only different types of dance on PD, but also different types of dance compared to exercise. Previous studies that have compared dance to exercise interventions have found that dance showed significant improvements particularly in balance (Hackney et al, 2007b, Volpe et al., 2013). When evaluating the difference between these different interventions, it is important to take into account attrition rates as well as ease of delivering the intervention. When moving to applying these interventions in a clinical setting, it is important that patients are easily able to participate and enjoy them. Another possibility for a future study would be to separate participants into tremor predominant vs postural instability gait disorder forms of PD to examine potential differences in responsiveness of those motor symptoms to dance (Westheimer et al, 2015). The same quantitative measures employed in this study could be used, though the focus would be on the gait and tremor sub scores pre and post intervention. If participants with a primarily postural instability gait disorder form of PD show greater benefits to the dance treatment, that may demonstrate that gait, rather than tremor is more responsiveness to dance therapy.

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Bloem, B., Hausdorff, J., Visser, J. (2004). Falls and freezing of gait in Parkinson’s disease: a review of two interconnected episodic phenomena. Mov Disord, 19(8), 871-884.

Boonstra, T., Vdk, H., Munnenke, M., Bloem, B. (2008). Gait disorders and balance disturbances in Parkinson’s disease: clinical update and pathophysiology. Curr Opin Neurol, 21(4), 461-471.

Brichetto, G., Pelosin, E., Marchese, R., Abbruzzese, G. (2006). Evaluation of physical therapy in parkinsonian patients with freezing of gait: a pilot study. Clin Rehabil, 20, 31-35.

De Dreu, M.J., Vdw, A.S.D., Poppe, E., Kwakkel, G., van Wegen, E.E.H. (2012). Rehabilitation, exercise therapy and music in patients with Parkinson’s disease: a meta-analysis on the effects of music-based movement therapy on walking ability balance and quality of life. Parkinson Relat Disord 19(1), S114-S9.

Duncan, R.P., Earhart, G.M. (2012). Randomized Controlled Trial of Community-Based Dancing to Modify Disease Progression in Parkinson Disease. Neurorehabilitation and Neural Repair, 26(2),132-143.

Ellis, T., Boudreau, J.K., Deangelis, T.R., Brown, L.E., Cavanaugh, J.T., Earhart G.M., et al. (2013). Barriers to exercise in people with Parkinson disease. Phys Ther, 93, 628-636.

Foster, E., Golden, L., Ryan, P., Duncan, R., Earhart, M. (2013). A community-based argentine Tango dance program is associated with increased activity participation among individuals with Parkinson disease. Arch Phys Med Rehab, 94(2), 240-249.

Goodwin V., Richards S., Taylor R., Taylor A., Campbell J. (2008). The effectiveness of exercise interventions for people with Parkinson’s disease: a systematic review and meta-analysis, Mov Disord 23(5), 631-640.

Hackney, M.E., Earhart, G.M. (2009). Effects of dance on movement control in Parkinson’s disease: a comparison of Argentine tango and American ballroom. J Rehabil Med, 41, 475-81.

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Hui, E., Chui, B., Woo, J. (2009). Effects of dance on physical and psychological well being in older persons. Arch Gerontol Geriatr, 49(1), e45-d50.

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Kattenstroth, J.C., Kolankowska, L., Kailish, T., Dinse, H. (2010). Superior sensory, motor, and cognitive performance in elderly individuals with multi-year dancing activities. Front Aging Neurosci, 2(31), 1-9.

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Nemes, B.A., Pirlog, R., Tartamus, D., Capusan, C., Fodor, D.M. (2019). The role of dance therapy in the rehabilitation of Parkinson’s disease patients. Balneo Research Journal, 10(3), 300-304.

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Romenets, S.R., Anang, J., Fereshtehnejad, S.M., Pelletier, A., Postuma, R. (2015). Tango for treatment of motor and nonmotor manifestations in Parkinson’s disease: A randomized control study. Complementary Therapies in Medicine, 23, 175184.

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Westheimer, O. (2008). Why dance for Parkinson’s disease. Top Geriatr Rehabil, 24, 127-140.

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Westheimer O., McRae C., Henchcliffe C., Fesharaki A., Glazman S., Ene H., Bodis-Wollner I. (2015). Dance for PD: a preliminary investigation of effects on motor function and quality of life among persons with Parkinson’s Disease (PD). J Neural Transm, 122, 1263-1270.

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Volpe, D., Signorini, M., Marchetto, A., Lynch, T., Morris, M. (2013). A comparison of Irish set dancing and exercises for people with Parkinson’s disease: a phase II feasibility study. Biomed Cent Geriatr, 13(54), 1-6.

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Rifaximin Modulates Gut Microbiota in Rats with Induced Visceral Hyperalgesia, Leading to Reductions in Intestinal Permeability and Inflammation Amelia Semenak

Rifaximin is a broad-spectrum, non-absorbable antibiotic derived from Rifamycin that acts on gram positive and negative bacteria (Gao, Gillilland & Owyang, 2014). Rifaximin has anti-inflammatory properties, reducing both inflammatory cytokines and intestinal barriers when administered (Xu et al., 2014). The literature suggests these properties help positively modulate the gut microbiome, making Rifaximin a strong choice for treating a variety of gastrointestinal (GI) disorders (Gao et al., 2014). More research must be done to determine whether changes modulated by Rifaximin persist after treatment ends and the exact mechanism in which changes occur. An article published in 2014, by Xu et al. examined the role of Rifaximin in reducing GI symptoms associated with visceral hyperalgesia, a leading cause of GI disorders. The main findings of their study suggest that Rifaximin may aid in reducing GI symptoms by positively altering the bacterial composition of the gut, leading to an increase in lactobacilli (Xu et al, 2014). Furthermore, treatment with Rifaximin was found to reduce intestinal permeability resulting in normalization of tight junction integrity, and reductions in inflammatory factors. Researchers hypothesize that the normalization of GI symptoms can likely be attributed to the observed increase in lactobacilli following treatment with Rifaximin (Xu et al., 2014). The findings presented by Xu et al (2014) further reinforce the link between gut inflammation and visceral hyperalgesia, supporting its use as a model for GI dysfunction. These findings are significant as they provide a possible mechanism in which Rifaximin works to reduce visceral hyperalgesia and associated GI symptoms, supporting its use as a treatment. Keywords: rifaximin, antibiotic, lactobacilli, dysbiosis, intestinal permeability, mucosal inflammation, visceromotor response to colorectal distention

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c INTRODUCTION Humans are estimated to have around 1014 bacterial cells, many of which colonize the GI tract (DuPont, 2015). These cells, also known as the gut microbiota, play a number of critical roles to maintain proper health and prevent disease. Structurally, gut microbiota act as a barrier against pathogenic bacteria, maintaining the integrity of the mucosal barrier (Zhou, Zhang & Verne, 2009). Functionally, gut bacteria are important for influencing a number of metabolic processes and maintaining proper immune function, including recognizing harmful bacteria and inhibiting the expression of inflammatory factors (DuPont, 2015). Adverse changes in gut function are often caused by alterations in the composition of gut microbiota, known as dysbiosis. These changes are commonly associated with GI dysfunction, including increased intestinal permeability (Ahmad, Sorrell, Batra, Dhawan & Singh, 2017). A number of studies have shown that decreases in the integrity of the intestinal barrier, and changes in permeability can lead to mucosal inflammation and higher levels of inflammatory factors (Ahmad et al., 2017). One particular study conducted by Saitou et al (2000) found that mice with an induced mutation in the occludin gene showed increased inflammation compared to control mice. These findings highlight the role of occludin proteins as regulators of tight junction integrity, and more broadly reveal the relationship between intestinal permeability and inflammatory responses. One of the most common antibiotics used to treat GI dysfunction, including increased intestinal permeability is an antibiotic known as Neomycin (Xu et al., 2014). Neomycin has been shown to reduce bacterial overgrowth but is accompanied by harsh side effects and its efficacy is poor (Pimentel, Park, Mirocha, Kane & Kong, 2006). In light of this, another broad-spectrum antibiotic known as Rifaximin is commonly prescribed (Pimentel et al., 2006). While, a number of studies have shown the effectiveness of Rifaximin as a treatment for GI dysfunction, the exact therapeutic mechanism in which it functions was previously unknown (Xu et al., 2014). Xu et al. (2014) addressed this gap in the literature, studying the mechanism in which Rifaximin acts to reduce GI symptoms in rats with stress-induced visceral hyperalgesia. In order to test the effects of Rifaximin, two experimental conditions of rats were used. The first consisted of rats exposed to the chronic water avoidance stress (WAS) task, and the latter to repeat restraint stressors (RRS). Two different antibiotics were tested, Rifaximin and Neomycin, comparing changes to SHAM rats who did not undergo stress inducing tests and were treated with water injections. Researchers used qPCR, 454 pyrosequencing and 16s rRNA sequencing in order to analyze the ileal contents from the rats. Researchers also used reverse transcription, fluorescent traces, immunoblot, and histological analyses to examine levels of intestinal permeability, and inflammatory factors (Xu et al., 2014) Results showed that rat models induced with visceral hyperalgesia showed dysbiosis in the ileum, accompanied by increased permeability and mucosal inflammation (Xu et al., 2014). Treatment with Rifaximin was found to alleviate these symptoms, leading to an increase in lactobacilli and a reduction in gut permeability and inflammatory factors. Alternatively, treatment with Neomycin led to alterations in gut bacterial composition, however, its effects were much less profound (Xu et al., 2014). These results are relevant as they highlight a mechanism in which Rifaximin alters gut bacteria to reduce GI symptoms. Furthermore, these results provide evidence to support the use of Rifaximin as a treatment for GI disorders.

RESULTS Xu et al. (2014) used 2 rat models with induced visceral hyperalgesia after exposure to WAS and RRS protocols to study the effects visceral hyperalgesia on GI function. Researchers sought to determine whether symptoms were aided following treatment with Rifaximin and Neomycin. WAS and RRS Lead to Visceral Hyperalgesia A number of models to study visceral pain have been proposed, however, many are associated with significant limitations (Ness & Gebhart, 1988). Previously suggested methods such as measures of colonic slow wave frequency or gastrocolonic motor response have shown poor correlations with GI symptoms (Mertz, Naliboff, Munakata, Niazi & Mayer, 1995). One measure of visceral pain found to better correlate with the symptoms of GI dysfunction, measures changes in visceromotor response to colorectal distention. Visceromotor responses (VMR) to colorectal distension (CRD) exert a quantifiable pressure that can be measured to indicate visceral nociception and hyperalgesia (Ness & Gebhart, 1988). One study conducted in 2003 measured rectal perception thresholds to distention for 100 patients with irritable bowel syndrome (IBS). Researchers found rectal hyperalgesia was present in the majority of patients with IBS, supporting the use of VMR to CRD as a measure of GI dysfunction and visceral hyperalgesia (Miranda, Peles, Rudolph, Shaker & Sengupta, 2003). Similar to the findings discussed above, Xu et al (2014) found that after 7-10 days of exposure to stress-inducing tests, statistically significant increases in VMR to CRD were observed (Figure 1). These findings suggest the WAS and RRS tasks were sufficient to induce visceral nociception and hyperalgesia.

Figure 1: Changes in Visceromotor Response (VMR) to Colorectal distension (CRD) after Water Avoidance Stress Task: Black Squares: Experimental rats with exposure to WAS task. Black Circles: Control rats with no exposure to WAS task. Results show greater changes for WAS exposed rats. Figure Adapted from Xu et al. (2014). Gastroenterology, 146, 484-496. Rifaximin and Neomycin Reduce Stress-Induced Visceral Hyperalgesia: After inducing visceral hyperalgesia, Xu et al. (2014) tested the effects of Rifaximin and Neomycin on reducing the observed changes in VMR to CRD following exposure to WAS or RRS protocols. Researchers found both antibiotics led to a significant reduction in pressure changes, indicating a decrease in visceral hyperalgesia and nociception (Figure 2). These findings support the role of Rifaximin as a treatment for reducing GI symptoms associated with

visceral hyperalgesia.

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caused by stress (Eutamene et al., 2007). These findings will be discussed further in the discussion section but highlight a possible mechanism in which Rifaximin reduces GI symptoms by changing the abundance of Lactobacilli. WAS and RRS Protocols Lead to Mucosal Inflammation and Increased Intestinal Permeability Defects affecting intestinal barrier function have been associated with a range of GI issues, often leading to increases in intestinal permeability (Ahmad et al., 2017). Research has revealed Figure 2: Changes in VMR to CRD observed after WAS, Sham WAS and WAS + a number of causes of increased gut permeability, one of the most Treatment with Rifaximin: Black Circles: Sham WAS + water injection. Black prominent being stress (Camilleri, Madsen, Spiller, Meerveld & Triangles: WAS task with Rifaximin injection. Black Squares: WAS task + water Verne, 2012). One study, conducted in 2006, highlighted a mechainjection. Figure shows that changes in VMR to CRD from WAS task are reduced nism in which stress alters the intestinal barrier. Researchers following treatment with Rifaximin found that stress acts on mast cells to produce inflammatory facFigure Adapted from Xu et al. (2014). Gastroenterology, 146, 484-496. tors, resulting in increased permeability of the colon (Demaude, 2006). These findings are consistent with those of Zhou et al. Chronic Stress & Visceral Hyperalgesia Lead to Dysbiosis: (2009) whose study found an association between visceral hyperChronic stress has been associated with alterations in gut sensitivity (often caused by stress) and increased intestinal permebacterial composition and reductions in diversity, whereby even ability (Figure 4). The findings from both of these papers are analoshort-term exposure to stressors can greatly alter the contents of gous to that of Xu et al. (2014), whereby both stress-inducing WAS the gut (Galley et al., 2014). In a study conducted by Galley et al and RRS protocols led to increases in intestinal permeability re(2014), researchers examined the effects of short-term exposure vealed by heightened levels of plasma fluorescein-conjugated dexto a social stressor on the colonic bacterial profiles of mice. tran (Figure 5a). Through the use of pyrosequencing, Galley et al (2014) found a reduction in Lactobacilli and overall bacterial diversity following exposure to stress. These findings are analogous to that of Xu et al (2014) who similarly noted as reduction in bacterial diversity of the ileum following exposure to WAS and RRS protocols (Figure 3).

Figure 3: Changes in Diversity Following WAS Protocol: Graph shows the reduction in diversity for WAS exposed rats using two diversity indexes, Shannon evenness and Shannon diversity. Figure Adapted from Xu et al. (2014). Gastroenterology, 146, 484-496. Rifaximin and Neomycin Alter Bacterial Composition and Load: In order to test the effects of antibiotics on modulating the bacterial changes caused by visceral hyperalgesia, Xu et al. (2014) administered Rifaximin and Neomycin. Treatment with Neomycin led to an increase in proteobacteria, but an overall reduction in bacterial load. Similarly, Rifaximin treatment led to a reduction in bacterial load, however, this change was accompanied by an increase in lactobacilli (Xu et al., 2014). A number of studies have highlighted the ability of Lactobacilli to aid in reducing gut inflammation and promote proper intestinal barrier function (Eutamene et al., 2007). A study conducted in 2007, highlighted the ability of lactobacilli to reduce hypersensitivity to distension

Figure 4: Lactulose: Mannitol Ratio as a Measure of Intestinal Permeability: Black circles represent the control condition; black triangles show IBS patients with a lactulose: mannitol ratio of under 0.07 and white triangles show IBS patients with a ratio of over 0.07. Figure highlights the changes in intestinal permeability associated with IBS. Lactulose: Mannitol ratios of over 0.07 represent increased intestinal permeability, as seen in many patients with IBS. Figure Adapted from Zhou, Zhang and Verne (2009). PAIN, 146(12), 41-46.

Figure 5: Changes in Intestinal Permeability after Exposure to WAS Tests and Following Treatment with Rifaximin: Figure 5A) Highlights the increased intestinal permeability observed following exposure to WAS test. Figure 5B) Shows the reduction in intestinal permeability following treatment with Rifaximin. Figure Adapted from Xu et al. (2014). Gastroenterology, 146, 484-496.

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A number of recent studies have presented a relationship between increased gut permeability and mucosal inflammation (Fukui, 2016; Ahmad et al., 2017). This association was further confirmed by Xu et al. (2014) whereby in conjunction with increases in intestinal permeability, analyses using real-time quantitative reverse transcription PCR, showed higher levels of inflammatory factors including, IL-17, IL-6, and TNF-a in rats exposed to WAS and RRS protocols.

generalizability of their results, due to differences in rat models and humans. In order for Rifaximin to reduce inflammation, it must activate pregnane X receptors (PXR) (Xu et al., 2014). These receptors have diverged across evolution and are highly variable across species (Jones, 2000). As a result of this variability, it is difficult to determine whether these same anti-inflammatory effects would be seen in humans, and if they were, whether they could be attributed to the same mechanism of action.

Rifaximin and Neomycin Reduce Gut Permeability and Inflammation: Following these observations, Xu et al. (2014) treated rats with Neomycin and Rifaximin in order to observe changes in barrier function and inflammation. Treatment with both antibiotics led to significant decreases in intestinal permeability, however the effects of Rifaximin were much more profound (Figure 5b). Furthermore, Rifaximin was found to normalize levels of IL-17, IL-6 and TNF-a mRNA, reducing intestinal inflammation. Neomycin was not as effective in reducing inflammatory factors (Xu et al., 2014).

In terms of changes in gut microbiota, Xu et al. (2014) found hyperalgesia led to dysbiosis characterized by a reduction in diversity and overall bacterial load. Treatment with Rifaximin led to an even greater reduction in bacterial load, but a marked increase in the abundance of lactobacilli. Researchers suggest that changes in Lactobacilli could account for reductions in inflammation and permeability, however the effects of diversity loss are not discussed. A number of studies have presented a relationship between loss of microbial diversity and pathogenesis (Flanagan et al., 2007). Moreover, while it may be plausible that heightened levels of lactobacilli can reduce in GI dysfunction, studies warn about the Overall, treatment of Rifaximin to WAS and RRS induced lack of knowledge regarding the safety and stability of this bacteria rats led to changes in the composition of the intestinal ileum, (Cerbo, Palmieri, Aponte, Morales-Medina & Iannitti, 2015). One whereby lactobacilli became the most dominant bacteria (Xu et al., study conducted in 2004 found that lactobacilli was frequently 2014). Changes were accompanied by marked improvements in associated with endocarditis and bacteremia (Cannon, Lee, Bointestinal barrier function and reduced mucosal inflammation. All lanos & Danziger, 2004). Given that minimal studies have adof these changes led to a reduction in the levels of visceral hyperal- dressed the longitudinal effects of Rifaximin and the resulting elegesia and symptoms of GI dysfunction, supporting the role of Rifax- vated levels of lactobacilli, future research should be conducted to imin as an effective treatment. address these questions. CONCLUSIONS/DISCUSSION Rifaximin is a well-known antibiotic used to treat IBS, and other GI disorders, however, the mechanism in which it functioned was previously undefined. Xu et al. (2014) outlined a possible mechanism in which Rifaximin modulates gut bacteria in order to change the abundance of Lactobacilli. Rats with induced visceral hyperalgesia showed dysbiosis of the ileum accompanied by increased gut permeability, and elevated levels of inflammatory factors. Treatment with Rifaximin led to a reduction in bacterial diversity, but an increase in the abundance of lactobacilli. Researchers hypothesize this change led to normalization of intestinal permeability and associated inflammation (Xu et al, 2014). In order to compare the efficacy of Rifaximin to other antibiotics, changes were compared to Neomycin, which was much less effective (Xu et al., 2014). Overall, Rifaximin was found to be an effective treatment for visceral hyperalgesia, reducing the associated GI symptoms.

Lastly, as discussed in the results, Xu et al. (2014) found rats with induced visceral hyperalgesia showed increased intestinal permeability. While researchers attributed the loss of barrier function to visceral hyperalgesia, no other hypotheses were discussed. Recent literature suggests reductions in levels of glutamate synthetase in individuals with GI dysfunction can cause increased intestinal permeability (Zhou, Souba, Croce & Verne, 2009). This finding outlines future directions for new studies to assess a possible relationship between Rifaximin and glutamate. Studies should be conducted to determine whether treatment with this antibiotic increases the abundance of glutamate synthetase, or whether their mechanisms to reduce permeability are unrelated.

FUTURE DIRECTIONS Before researchers determine whether there are any consequences that can result from prolonged treatment with Rifaximin, more research must be done to determine whether the These results are important as they outline a possible antibiotic requires repeated use. Currently, there are discrepancies mechanism that was previously unknown to explain how Rifaximin in the literature, whereby a study by Pimentel et al. (2006) sugreduces GI dysfunction. They further the findings of the literature, gests IBS symptoms were improved 10-weeks following discontinuincluding that of Gao, Gillilland and Owyang (2014) who similarly ing treatment. Other studies, such as Saadi and McCallum (2013), suggest lactobacilli may be responsible for reducing inflammation found many patients required repeated treatments. In order to and visceral hyperalgesia in rat models with GI dysfunction. Lastly, reconcile this discrepancy, longitudinal analyses should be conthe findings of Xu et al. (2014) support the growing hypothesis that ducted to analyze the long-term efficacy of Rifaximin, and whether the onset of visceral hyperalgesia is facilitated by abnormal bacte- prolonged treatment and bacterial resistance is a cause for conrial interactions, leading to GI dysfunction. cern. This study would also enable researchers to address the longterm concerns (if any) of reducing bacterial load and diversity folCRITICAL ANALYSISlowing treatment with Rifaximin. In their study, Xu et al. (2014) highlighted a possible mechanism to explain how Rifaximin acts to improve GI function. As discussed in depth, increased intestinal permeability is While their findings support the use of Rifaximin as an antibiotic a common symptom of GI dysfunction, hypothesized to be related for treating GI disorders, questions should be raised about the to increases in inflammatory factors (Ahmad et al., 2017). Previous

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studies have found that glutamine deficiencies can lead to increases in intestinal permeability. Specifically, one study by Zhou et al, (2009) highlights a specific microRNA, miR-29a in IBS patients, which targets the GLUL gene, and can inhibit the expression of glutamine synthetase. These changes result in reduced glutamate and increased intestinal permeability. Given these findings, it would be interesting to determine whether administration of glutamate, or inhibition of miR-29a could be an effective treatment for GI symptoms.

To conduct this study, researchers could use rat models similar to those used by Xu et al. (2014). Researchers using either the 4-kDa fluorescent dextran marker, or the lactulose:mannitol excretion ratio could test for enhanced permeability (Zhou et al, 2009). Expression of miR-29a would be analyzed using quantitative real-time qPCR in order to determine whether itâ&#x20AC;&#x2122;s expression could account for the enhanced permeability. If miR-29a was indeed expressed, researchers could inhibit its expression, examining whether levels of glutamate synthetase would increase and as a result reduce GI symptoms. Furthermore, researchers could inject rats with glutamine synthetase, a known target of miR-29a. If intestinal permeability was reduced following the injection, it is hypothesized that inflammation and increased barrier function would accompany these changes. These findings would present a possible intervention for GI disorders, requiring no use of antibiotics or one that could be used to maintain the effects of Rifaximin after discontinuing treatment. If these effects were not observed, it is possible that the actions of miR-29a are synergistic with other molecules and simply inhibiting miR-29a would not be sufficient to prevent changes in intestinal permeability.

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Depression induced mice show increased interleukin-1β and NLRP3 inflammasome Jina Seok

Many studies have indicated the immune system to have a role in major-depressive disorder, however the mechanism by which this involvement occurs is still widely unknown. Major-depressive disorder consists of a variety of symptoms including anhedonia, lowered self-esteem and a disturbance in feeding, sleeping, and cognition. It has been found that the injection of lipopolysaccharide (LPS) or interleukin-1 (IL-1) causes depressive-like symptoms in animal subjects. The NLRP3 inflammasome is activated upon pathogen detection, and causes the creation of pro-inflammatory cytokines like IL-1β and IL-18. To detect whether the NLRP3 inflammasome has a role in depression, Zhang et. al (2013) performed a study using mice models which were injected with LPS to induce depressive-like symptoms. This is tested by using behavioural tests, like the forced swim test, and sucrose preference. Then the Bio‐Plex Mice Cytokine 6‐Plex panel immunoassay as well as a real-time PCR (RTPCR) was used to measure the levels of IL-1β and the NLRP3 inflammasome. It was found that mice with induced depression had higher levels of IL-1β as well as greater expression of the NLRP3 inflammasome compared to control mice. This was tested by injecting mice with a NLRP3 inflammasome inhibitor, Ac‐YVAD‐CMK, before administering LPS. This resulted in a significant decrease in depression symptoms which proves the involvement of the NLRP3 inflammasome in depression of LPSinduced mice. These findings suggest that depression and the immune system are more highly associated than previously thought, and more importantly, it serves as another target for the treatment of depression. Key words: depression, NLRP3 inflammasome, lipopolysaccharide, interleukin-1β, mice models

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INTRODUCTION

in mice models.

Major-depressive disorder, also called clinical depression, or major depression, is one of the most prevalent diseases worldwide, and is due to be the largest known disease-generated disability within the next few years. Depression has a range of symptoms which include; feeling hopeless, increased isolation, difficulty in making decisions or absorbing information, and a general loss of interest, leading to a significant decrease in the quality of life (Nystrom et al., 2015). The cause for this disorder is being widely studied, and as of now, there are many possible causes being considered, with both genetic and non-genetic factors having a contributional effect (Nestler et al., 2002). Although there is limited knowledge about the specific causes and little is known about its mechanism of action, there are many types of treatment which are successful to some degree, however there is no one cure for the disorder. The majority of people suffering from major-depressive disorder, show some reliviation of symptoms by using antidepressant medications or through cognitive and behavioural therapies which have shown to be an effective form of treatment. This tends to be the case especially when the two methods are used in conjunction (March et al., 2004). There has also been recent studies emerging about the use of physical activity in the prevention and treatment of depression, as can be seen in a study by Nystrom et. al (2015).

MAJOR RESULTS The study by Zhang et. al (2013) used a mouse model to induce depression by injecting LPS. Creating an animal model of depression is difficult due to the many different components of the disorder (Nestler, Gould & Manji, 2002). Using LPS to induce depressive-like symptoms is still a fairly new model with many postulations about its validity. However, LPS is known to induce immune activation and also causes depressive-like behaviours, especially anhedonia and decreased motor functions which accompany depression and therefore has been used as a model to study these components of depression (Kubera et al., 2013).

To ensure mice were exhibiting depressive-like symptoms, two different behavioural tests were performed; the sucrose preference test and the forced swim test. The sucrose preference test parallels anhedonia since normal mice will prefer water with more sucrose but depression induced mice will not show this preference (Figure 1A). In the forced swim test, mice are placed in a cylindrical tube of water and the time which they remain immobile is measured, this reflects levels of helplessness and despair. This test, shows depressive-symptoms in mice which are immobile for longer periods of time than the healthy, control mice (Figure 1B). Both the forced swim test as well as the sucrose preference test are widely used measures of depression in animal models (Paul et Despite the prevalence of depression, the pathology is still al., 2005). In the study, LPS injected mice showed significant differwidely unknown. It is hypothesized that many brain regions are ences compared to the control group for both tests, which indicatinvolved in depression, as shown in a study by Sheline (2011), ed that these mice had depressive-like symptoms where it was found that patients with major depression had smaller hippocampal volumes than those in the control. However, the direction of this relationship is still being studied. Furthermore, additional studies have shown the relationship between the immune system and depression, with the effect of neuroimmunological molecules causing inflammation in the brain (Miller & Raison, 2016). Generally pro-inflammatory cytokines like IL-1β and IL-16 cause systemic inflammation to aid in the generation of the inflammatory response, in comparison, the anti-inflammatory cytokines like IL-4 and IL-10 reduces inflammation and assists the healing process (Han et al., 2014). These cytokines are signal mediators for the immune cells, and pro-inflammatory cytokines have been found in elevated levels in depressed individuals. Cytokines may Figure Adapted from Zhang et. al (2013). CNS Neuroscialso be found to be produced in the brain by astrocytes, microglia, ence & Therapeutics, 20(2), 119-124. and certain endothelial cells. The role pro-inflammatory cytokines play in the brain as well as its impact on depression is still widely Figure 1. A. Results from mice sucrose preference test unknown (Han et al., 2014). The association between the immune system and majordepressive disorder is tested in the paper by Zhang et. al (2013). In this study, mice models were induced with depressive-like symptoms by injecting them with lipopolysaccharide(LPS). Depressed mice were tested using a Bio‐Plex Mice Cytokine 6‐Plex panel immunoassay for the levels of cytokine interleukin-1β. A real-time PCR (RT-PCR) was also executed to measure the levels of the NLRP3 inflammasome produced, which is a potent inflammatory modulator which gives rise to many pro-inflammatory cytokines. These tests showed an increase of both molecules in depression induced mice. The association between these inflammatory molecules and depression was proven by using a NLRP3 inhibitor, Ac‐ YVAD‐CMK, which caused a decrease in depressive-like symptoms in the mice injected with LPS. This proves the underlying connection between inflammation in the brain and depression symptoms

of control and LPS injected mice. B. Results from forced swim test comparing control and LPS injected mice. (***P < 0.001) Levels of IL-1β Protein and mRNA Interleukin-1β protein is a major proinflammatory cytokine which causes the recruitment of other immune regulatory cells. IL-1β has also been discovered to have a role in the development of acute depressive symptoms caused by stress factors (Zhang et al., 2013). Therefore, the levels of IL-1β was measured using a Bio‐Plex Mice Cytokine 6‐Plex panel immunoassay which uses fluorophore ratios to detect protein concentration, as well as a RT-PCR which allows for mRNA expression levels to be measured.

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These tests showed that IL-1β levels were higher in Figure 3. A. mRNA expression levels of the NLRP3 LPS injected mice than control mice (Figure 2). inflammasome receptor between control and LPS injected mice. B. mRNA expression levels of ASC component of the inflammasome between control and LPS injected mice. C. mRNA expression levels of the caspase-1 component of the inflammasome between control and LPS injected mice. (*P < 0.05 and **P < 0.01)

Figure Adapted from Zhang et. al (2013). CNS Neuroscience & Therapeutics, 20(2), 119-124

Figure 2. A. Levels of IL-1β protein in cells of control mice vs. LPS injected mice using Bio‐Plex Mice Cytokine 6‐Plex panel. B. Levels of IL-1β mRNA in cells of control mice vs. LPS injected mice using RT-PCR (*P < 0.05, **P < 0.01)

These findings suggest that the inflammasome is associated with depressive-like symptoms which is supported in another study by Pan et. al (2014), which suggests depression to be an inflammatory disorder. This paper reports an increase in the NLRP3 inflammasome as well as IL-1β in the prefrontal cortex of rats with depression symptoms. These results seem to agree with the majority of literature, as another study by Alcocer-Gomez et. al (2016), comes to similar conclusions that the NLRP3 inflammasome is required for the onset of stress-induced depressive-like behaviours which was shown through the deletion of the NLRP3 gene.

These results agree with a previous paper by Yu et. al (2003) which describes a study where the injection of IL-1β produced depressive-like symptoms in mice models. This may indicate the association be- NLRP3 Inflammasome Inhibitor, Ac‐YVAD‐CMK tween the pro-inflammatory cytokine, IL-1β, and major To examine the association between the NLRP3 -depressive disorder. inflammasome and depressive-like symptoms further in mice, a NLRP3 inflammasome inhibitor, Ac‐YVAD‐ CMK, was administered in mice prior to the LPS injecLevels of NLRP3 Expression tion. This showed a significant decrease in depressiveThe NLRP3 inflammasome is made of three main like symptoms compared to the mice which had been parts; the NLRP3 receptor, the adaptor protein ASC, injected with only LPS (Zhang et al., 2013)(Figure 4). and caspase-1 for effector functions. Using the receptor, the inflammasome is activated upon detection of damage-associated molecular pattern (DAMP) or pathogen-associated molecular pattern (PAMP) molecules. The activation of this molecule causes the caspase-1 molecule to cleave precursor IL-1β and IL-18 cytokine molecules into their active pro-inflammatory form for secretion (Zhang et al., 2013). The expression levels of the inflammasome was measured using a RT-PCR to show an increase in its expression (Figure 3).

Figure Adapted from Zhang et. al (2013). CNS Neuroscience & Therapeutics, 20(2), 119-124. Figure 4. A. Sucrose preference test performed on control, Ac‐YVAD‐CMK injected, LPS injected, and Ac‐ YVAD‐CMK/LPS injected mice. B. Forced swim test performed on control, Ac‐YVAD‐CMK injected, LPS inFigure Adapted from Zhang et. al (2013). CNS Neuroscijected, and Ac‐YVAD‐CMK/LPS injected mice. (*P < ence & Therapeutics, 20(2), 119-124. 0.05, **P < 0.01 and ***P < 0.001) 371

In a similar study by Li et. al (2016), it was shown that apigenin, a compound found in citrus fruits, had anti-depressive effects on rats. The mechanism behind apigenin was found to be a downregulation of the NLRP3 inflammasome and subsequently a decrease in IL-1β. Therefore, along with this study, it may be such that the NLRP3 inflammasome and the IL-1β cytokine may have a strong association with major-depressive disorder.

These types of depressive-like symptoms mimic anhedonia and despair symptoms in clinical depression, however many other symptoms exist as well. Other forms of animal depression models that may be utilized could be the learned helplessness model where animals with induced depression would show a decrease in escape behaviour. This model mimics the human symptom of helplessness and could be another way to measure depression in mice models (Krishnan et al., 2011). Using various symptoms will allow for greater generalizability since depression as a disorder encompasses a great variety of symptoms and ultimately the goal CONCLUSIONS/DISCUSSION is to be able to find a treatment that may be used for human subThe major findings in the paper by Zhang et. al (2013), jects. show that depression induced mice had higher levels of proinflammatory agents; IL-1β and the NLRP3 inflammasome expressed. The paper then shows that inhibiting the NLRP3 inflamFUTURE DIRECTIONS masome causes a decrease in depressive symptoms. These findings can conclude that the NLRP3 inflammasome which produces Future studies may be pointed in the direction of clinical activated IL-1β may have a role in the development of depressive- research. By testing anti-inflammatory agents and their effect on like symptoms. These results are crucial as they may indicate a depressive-like symptoms, it will show a clearer understanding of possible new target molecule for treatment of major-depressive the relationship between the immune system and major depresdisorder. Additionally, it has been stated that previous research sive disorder. A study done by Kohler et. al (2014) found using a has shown that different types of immune regulatory cytokines meta-analysis, that generally, using anti-inflammatory agents had have a role in the progression of depression and other mood disor- a decrease in depression symptoms. However studies taken into ders as can be seen in the study by Dunn et. al (2005). Unlike pre- consideration during this analysis were not consistent with the vious research which focused on the association between cyto- method or type of anti-inflammatory agents taken by trial pakines and depressive-like symptoms, the study by Zhang et. al tients. Therefore, researchers furthering the study by Zhang et. al (2013) incorporates the NLRP3 inflammasome and tests the effects (2013) may focus on finding the relationship between the inflamof the inflammasome by using the Ac‐YVAD‐CMK inhibitor. This masome NLRP3 and depressive symptoms. One way this could be approach goes one step further to confirm the effect that the in- done is by using an inhibitor similar to the one used on mice modflammasome has on depression symptoms shown in mice models. els in the study, like Bay 11-7082 which has been shown to have Previous research shown in the article by Song et. al (2010), re- inhibitory effects on the NLRP3 inflammasome (Juliana veals that the research was heavily about the relationship be- et al., 2010). Results of this study would show the eftween inflammation and depression rather than the mechanisms fects of the NLRP3 inflammasome on human subjects behind this relationship. The authors in the paper express that the with depression directly. Considering the results from study was based off the previously done research about the associthe NLRP3 inflammasome inhibitor on depressive-like ation between increased inflammation and increased cytokine molecules and the increased prevalence of depression symptoms. symptoms on mice models, it is possible to hypotheThis study was to explore the workings of specific cytokine mole- size a similar result in human subjects for a decrease in cules and the effect on depressive-like symptoms as was shown depression symptoms. Positive results would show through the NLRP3 inflammasome (Zhang et al., 2013). Further- another type of possible treatment that may be used to more, it contributes to our overall understanding of the relation- combat major-depressive disorder along with other ship between the immune system and major depression, and may lines of treatment. It may also be the case where there possibly have an effect on future treatment methods. is no positive correlation, and the NLRP3 inflamCRITICAL ANALYSIS In the study by Zhang et. al (2013), the involvement of the NLRP3 inflammasome in depressive-like symptoms is found, and the authors are hopeful that this may allow for more specific types of treatment for major-depressive disorder. Due to this new found discovery, many studies are now further exploring the effects of the inflammasome on depression induced animals (Iwata et al., 2013). However, there are yet to be many studies taking the opposite approach, by injecting NLRP3 inflammasome or proinflammatory cytokines and analyzing for any depressive-like symptoms. This type of study would allow for a causational relationship between inflammation and depression, which may be important in finding the possible causes of depression. Further research may also entail looking at other types of depressive symptoms since the ones studied were through the sucrose preference test as well as the forced swim test (Zhang et al., 2013).

masome inhibition does not show a decrease in depressive symptoms, which may also be valuable information as to determine that the mice model is not a sufficient model in understanding the complex relationship between immune regulatory molecules and depression symptoms. This would indicate the need for the development of different types of models which may mimic the human model in a more sufficient manner. Therefore, despite the conclusion of the results, a clinical study furthering the research by Zhang et. al (2013) would be beneficial in furthering the understanding of the involvement of the inflammasome in clinical depression.

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Is hyperserotonemia due to gut dysbiosis the driving force for autism spectrum disorder! Shirin Shamsaasef

Autism spectrum disorder, commonly abbreviated as ASD, is a neurodevelopmental disease that affects behavior and communication. It can be diagnosed at any age however its symptoms such as language deficits, repetitive pattern of behaviors and restricted social interaction and communication generally appear from 0 to 2 years of age. Although every individual with ASD is unique due to the severity and variation of symptoms, most of the kids diagnosed with ASD suffer from a wide range of systematic abnormalities such as GI distress, hyperserotonemia and inflammatory bowel disease (IBD). This highly suggests that the systematic aberrations and behavior abnormalities in children with ASD can be due to GI diseases and IBD disease. In a study by Lim et al. (2017), gut microbial dysbiosis and hyperserotonemia were proposed as a driving forces for systematic and behavioral abnormalities associated with people who have ASD. The results of the study indicated that the ASD mouse models had an increase in pathogenic bacterial abundance and a decrease in beneficial bacterial composition which closely resembles the clinical cases of ASD and IBD patients. An upregulation in lipopolysaccharides biosynthesis pathways and bacterial toxins was observed which initiated an immune response and caused the further release of immune factors that eventually lead to ASD like behavioral phenotypes. The authors observed increased serum serotonin levels due to changes in gut microbiota and considered it as a possible cause for ASD like behaviors. These results not only suggest gut microbiota plays multiple roles in systematic pathogenesis of hyperserotonemia and ASD, but it addresses a distinct pattern of alteration in metabolic pathways, serotonin pathway and the gut microbial composition which can be used to develop therapeutic methods to treat ASD and IBD.

Key words: Autism Spectrum Disorder, neurodevelopmental, gut dysbiosis, IBD, hyperserotonemia, environmental risk factors, gut microbiota, LPS biosynthesis, Short-Chain Fatty Acids (SCFA)

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INTRODUCTION: Autism spectrum disorder (ASD) is a neurodevelopmental disorder that found to have a major deficit in neurodevelopmental milestones. The main 3 features that are impaired in ASD are deficits are social interaction, language/communication, and repetitive pattern of behavior. While what causes ASD is unknown, primary research indicates that the etiology of ASD involves an interplay between multiple environmental risk factors, genetics, and both nervous system and systematic inflammation (Kong et al., 2019). The systematic and nervous system inflammation that occurs in individuals with ASD and contribute to the manifestation of ASD symptoms is thought to be due to gut microbial dysregulation (Ding, Taur, & Walkup, 2017; Vuong & Hsiao, 2017; Petersen & Round, 2014; Rossignol and Frye; 2014). This hypothesis is further supported by pieces of evidence that are gathered through ASD mice model studies. Scientists demonstrated maternal immune activation (MIA) in mouse models due to alteration in serum metabolites and gut microbiota results in ASD like behavioral phenotypes (Hsiao, McBride, Hsien, et al., 2013; Buffington, Di Prisco, Auchtung, et al., 2016). Given each case of ASD being unique, most of the individuals with ASD suffer from a wide range of systematic abnormalities such as GI distress, hyperserotonemia and inflammatory bowel disease (IBD) (Harrington et al., 2013; Pagan et al., 2014; Cook et al., 1996; McElhanon et al., 2014; Doshi-Velez et al., 2015; Lim et al., 2017). Both GI and IBD alter gut microbiota in a way that the abundance of pathogenic bacteria increases, while beneficial bacteria decreases. This microbial dysbiosis has severe negative consequences on the immune system and endangers the hostâ&#x20AC;&#x2122;s health status (Cryan and Dinan, 2012). While the co-occurrence of two etiologically different diseases is rare and there is a high prevalence of GI and IBD in most ASD kids, scientists hypothesized that common causes of abnormalities in behavior and systematic aberrations may be due to gut microbial dysbiosis. Gut bidirectionally communicates with the brain through the gut-brain-axis, vagus nerve, endocrine system, and immune system. Any imbalance in the composition and quantity of the gut microbiota may have negative consequences on the immunity, central nervous system and gut nervous system. It may also contain pathogenic risk factors for autism spectrum disorder. Moreover, epidemiological studies on prenatally induced autism rodents indicate an alteration in metabolic products and composition of the gut microbiome which is thought to contribute to the ASD behavioral phenotypes. Although evidence that supports the link between abnormalities in gut microbiota and Autism spectrum disorder has been accumulating recently, Lim et al. (2017) provide a distinct pattern of alteration in metabolic pathways, serotonin pathway and gut microbial composition. Authors examined gut microbiota in two prenatally ASD induced mouse models (Poly I:C (influenza virus) and Valproic acid (VPA)) and then measured anxiety-related behaviors, social interaction, and alteration in gut microbial abundance by gathering DNA from mouse feces. After analyzing DNA from feces, the authors found that the most prominent change in

Figure adapted from Lim et al. (2017). Molecular brain, 10(1), 14. Figure 1. illustrates the significant decrease in the decrease in the abundance of Prevotellaceae in both ASD induced mice models (Poly I:C and VPA) both ASD mice models and children with ASD was a decrease in the abundance of Prevotellaceae. Metabolic pathways in children with ASD were altered in the same manner as ASD mice models used for this study: 1) significant upregulation in lipopolysaccharide biosynthesis pathways and 2) increased serum serotonin (5-HT). This paper is significant because it can open new opportunities in disease management as it identifies hyperserotonemia due to gut dysbiosis as a causal factor for autism spectrum disorder. Unfortunately, there is no known treatment for ASD and drugs that are used are mainly for symptom management; thus, understanding and modulating the effects that gut microbiota has on the brain, through the microbial-gut-brain-axis, can aid scientists to come up with new effective and promising therapeutic ways to improve symptoms and maybe inhibit the ASD from happening. MAJOR RESULTS In Lim et al. (2017) study, ASD mouse models were created by prenatally injecting male mouse pops with two ASD environmental risk factors known as polyinosinic: polycytidylic acid (poly I:C) and valproic acid (VPA). After 6 weeks, the gut microbiota of the ASD mouse models was compared to gut microbial composition of the clinical cases of ASD and IBD to examine the effects of microbial dysbiosis on the hostâ&#x20AC;&#x2122;s metabolism and serum serotonin levels. The results of the study showed that ASD induced mouse models had significantly reduced in gut bacterial activity, beneficial bacterial abundance and exhibited ASD behavioral phenotypes such as anxiety and reduced social interaction. These ASD-like behaviors were measured through locomotor activity and 3-chamber assay. Alteration in gut microbial dysbiosis of ASD mice resembles the gut dysbiosis in the clinical cases of ASD and IBD Lim et al. (2017) studyâ&#x20AC;&#x2122;s results showed that specific microbial taxa in gut changes in response to prenatal injection of ASD

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community was comprised of Prevotellaceae while on the contrary, the ASD mice gut bacterial community had only ~2.5% with few members of the Prevotellaceae having zero abundance. These significant decrease in abundance of Prevotellaceae was also back up by other empirical data. In a study by Srikantha and Mohajeri (2019) where the link between gut microbiota and ASD was examined, authors found that class Prevotellaceae had significantly higher abundance in nonautistic children than ASD children. Compared to control heath children, Gonzalez and Martinez (2019) found that the abundance of Prevotella was significantly lower. Authors included that this change may be due to westernization and it may have consequences on the immune system and provoke an immune response. Similar metabolic pathways are altered in both ASD induced mice model and clinical cases of ASD and IBD. Given that ASD induced by environmental risk factors leads to microbial dysbiosis relevant to clinical cases, Lim et al. (2017) also investigate how these changes affect metabolic pathways in ASD mice. Authorsâ&#x20AC;&#x2122; investigations indicated that pathways such as steroid hormone biosynthesis and dioxin degradation that are altered in ASD individuals are also altered in the ASD induced mice in the same manner. Moreover, N-Glycan biosynthesis and sulfur metabolism were altered in ASD mice in the same manner as IBD patients. There was also an upregulation in lipopolysaccharide (LPS) synthesis and bacterial toxins. The elevated levels of bacterial toxins and LPS biosynthesis in circulation invoked an immune response which in return increased the number of immune factors being released and eventually result in ASD behavioral phenotypes. These results are backed up through a study by Srikantha and Mohajeri (2019). In this paper, a significant increase in LPS was observed. This increase would also provoke an inflammatory response in the host and carry negative health consequences for the host. Figures adapted from Lim et al. (2017). Molecular brain, 10(1), 14.

Hyperserotonemia and elevated Short-Chain Fatty Acids witnessed in ASD induced mice models

Figures 2, 3 and 4. illustrate the changes in the abundance of gut bacterial composition in control, ASD Poly I:C and VPA mouse models.

Lastly, Lim et al. (2017) showed that gut dysbiosis results in elevated serum serotonin levels. although protein digestion and absorption were not among pathways that implicated the progression of ASD and IBD. This pathway is however important due to its implication in the production of short-chain fatty acids (SCFAs) because SCFAs are known to stimulate the production of serotonin in enterochromaffin cells (Reigstad, Salmonson, Rainey, et al., 2014). Beside IBD and GI, elevated serum serotonin levels is another highly occurring comorbidity in individuals with ASD. Given that few recent studies mentioned spore-forming gut bacteria from Clostridia class are responsible for stimulating serotonin production in the host, Lim and colleges (2017) therefore analyze Clostridia class in ASD induced mice models. The analysis showed that Sporanaerobacter abundance was considerably increased in VPA and Poly I:C mice compared to control. Sporanaerobacter is a spore-forming and SCFA producing bacteria. Given the increase SCFAs pathways, as well as spore-forming and acetic acid-producing nature of this bacteria, scientists suspected that serotonin levels must be increased too. Lim and his colleges (2017) found that VPA and

environmental risk factors. Furthermore, this pattern of changes recapitulates those clinical cases of IBD and ASD. In both ASD induced mice model, there was a notable increase in the abundant of Desulfovibrio and Enterococcus. These findings are significantly important because it agrees with gut dysbiosis seen in clinical cases of ASD and IBD. In clinical cases, Enterococcus and Desulfovibrio have significantly higher abundance compared to other bacterial taxa. Enterococcus is also suspected as one of the key players in the progression of IBD. Moreover, 16rRNA gene sequencing indicated that the abundance of Prevotella, Oscillospira sp., and F. Prausnitzii was decreased in the ASD mice model. These bacterial taxa are also underrepresented in ASD and IBD clinical cases. Out of 259 microbial taxa that Lim et al. (2017) tested, Prevotella showed the most significant change compared to control mice. In healthy control mice, 7% of the gut bacterial

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Poly I:C mice models had a significant increase in their serum ed that autistic children have a significantly lower abundance of serotonin level, an event that had never been witnessed in ASD Prevotella (Srikantha and Mohajeri, 2019). These data suggest induced mice models before. that alteration in the microbial composition of gut in ASD children may have negative functional and biochemical conseIsraelyan and Margolis (2019), analyzed published research on quences on the host which eventually agrees with the findings the effects of gut dysbiosis on SCFA and serotonin production. of Lim et al. (2017). Compared to previous research, Lim et al., They concluded that short-chain fatty acids that are produced (2017) provided new insight into the relationship between the by pathogenic bacteria in gut eventually increase intestinal levdecreased abundance of Prevotella and ASD, that is exposure els of 5-HT which is a major compound for serotonin producto Poly I:C and VPA might cause the hostâ&#x20AC;&#x2122;s gut environment tion without changing the expression of SERT (serotonin transimpossible for the growth of Prevotella. Lastly and most importer). portantly, Lim et al., (2017) concluded that gut microbial dysbiosis due to environmental risk factors of ASD can negatively influence the hostâ&#x20AC;&#x2122;s health status through serotonin pathways and metabolic pathways. This result makes this paper very impactful and useful because the authors identified hyperserotonemia due to gut dysbiosis as a causal factor for Autism spectrum disorder. The reason the authors held gut microbial dysbiosis responsible for hyperserotonemia is due to the increased abundance of Sporanaerobacter. This bacterium is spore-forming bacteria and produces short-chain fatty acids (SCFA) which both are involved in the production of serotonin. The spore-forming bacteria in mouse (Sporanaerobacter) and SCFA in humans upregulates TPH1 transcription which promotes serotonin (5-HT) biosynthesis in colonic enterochromaffin cells (ECC) that provide serotonin in the colon (Yano et al., 2015). As mentioned by the authors, these findings may aid in developing medicine for ASD and IBD. Since there is no known medication for treating ASD, and there are pieces of evidence that suggests gut microbiota plays s role in behavioral symptoms of ASD, scientists can target multiple bacFigure adapted from Martin et al. (2018). Cellular teria taxa in the gut to develop a treatment for ASD. and molecular gastroenterology and hepatology, 6(2), 133-148

CRITICAL ANALYSIS

Figure 5. illustrates how spore-forming bacteria influence serotonin production and affects the brain through the Gut-Brain-Axis.

DISCUSSIONS Autism spectrum disease (ASD) is one of the leading neurodevelopment diseases in developed countries with approximately affecting 1 in 161 individuals worldwide. Even though children with ASD experience symptoms differently, most children with ASD are suffering from different GI disorders. Through the enormous empirical studies on humans with ASD and ASD mouse models, the link between gut dysbiosis and autism has been confirmed beyond doubt. In the paper by Lim et al., (2017) authors showed that prenatal injection of two widely used ASD environment risk factors induces gut microbial dysbiosis similar to clinical cases of IBD and children with ASD. Both Poly I:C and VPA ASD mice had a significant reduction in the abundance of Prevotella. Prevotella is fermenter of plant polysaccharides and its reduction in the gut might be related to many digestive disorders. This similar alteration has also been witnessed in clinical cases of ASD and IBD which further strengthens the hypothesis that gut microbiota may play a key role in ASD like behaviors. Empirical studies that compare the gut microbiome of autistic children to non-autistic also report-

The authors of the paper offer an alternative way to study Autism spectrum disorder. This approach allows scientists to know what pathways are affected, to what extent prenatal exposure to environmental toxins causes ASD and in what context gut dysbiosis causes similar changes in the gut bacterial population in both ASD mice models and clinical cases of SD and IBD. Although Lim et al. (2017) clearly discusses the findings and communicates why these data are important in the understanding of the disease, it does not include how these affected mice can benefit from microbial transfer therapy. Given that towards the end of the paper, authors suggest these findings can be used to develop therapeutic methods, authors could have applied the microbial transfer therapy on the ASD mice models to see if applying it would help with symptoms. Authors also used only male mice models for this study which inhibits it from applying data to the broader subjects. In addition, the ASD mice models are not an accurate resemblance of human anatomy and functions, thus it may cause difficulties for driving treatment strategies from this paper. To develop treatment methods, these studies must be done on humans or use models that accurately underline human anatomy and biological functions and pathways that take place in human body. This study, however, does a very great job at underlining how pathways are altered and what possibly causes the hyperserotonemia witnessed in autism spectrum disorder mice models. An issue with this howev-

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er is that Sporanaerobacter is only found to promotes serotonin production in mice and not in humans so this may not help find therapeutic methods for humans ASD clinical cases. FUTURE DIRECTION Although the results of this show that there is an increase in serotonin production and this plays a key role in connecting gut to the brain via gut-brain-axis and vagus nerve, many questions remain unanswered. Given serotonin can be found both in the brain and in the epithelial cells of the intestine and other organs that don’t even synthesize 5-HT – a major component to drive serotonin – further research are required to fully understand the importance of serotonin in this pathways and enhance our ability to drive therapeutic methods from it (Israelyan & Margolis, 2019; Martin et al., 2018). Considering that data that indicates gut dysbiosis increases pathogenic bacteria and decreases beneficial bacteria, future research should investigate the role gut microbiota in invoking immune response because one of the hypothetical causes of ASD is also the activation of the immune system. Moreover, the symptoms that people with ASD experience vary from one another and further research is required to fully investigate how gut microbiota affects ASD pathophysiology. Lastly, all the studies that are done up to this date suggest there is a connection between altered microbial composition in gut and ASD; thus, further studies must be done to possibly establish causation. One possible study design that can to some extent solve the issues raised for this paper could be the following. Prenatally inject both female and male mouse models with environmental risk factors of ASD to produce autism mice models so that data can be applied to a broader population. To see the alteration in Gut microbial composition I will collect feces from the control and ASD induced mice. To measure anxiety, social interaction and locomotor, I will put both the control and ASD mice models in a novel vs familiar chamber, expose them to a novel mouse. If the findings of Lim et al, (2017) are to be true, the results of this hypothetical study should show that Prevotella abundance in both female and male ASD mice model decreases while the abundance of pathogenic bacteria, Sporanaerobacter, increases. Furthermore, there should also be an upregulation in the LPS biosynthesis pathway and bacterial toxins. If spore-forming Sporanaerobacter is responsible for the increase in SCFA production and high serotonin serum levels, then this hypothetical study should show the same findings as Lim et al., (2017). To see if gut microbial dysbiosis is crucial for ASD like behaviors in ASD mice models, I will apply microbial transfer therapy to ASD mice to see if an increase in beneficial bacteria such as Prevotella can help with symptoms. If it does, microbial dysbiosis is responsible for ASD-like behaviors. Moreover, I will use antibiotics that reduce the abundance of Sporanaerobacter or reduce the uptake of serotonin produced to see if it will reduce the ASD symptoms. If it works, then hyperserotonemia due to gut dysbiosis is the causal factor for ASD and if not then the major conclusions of the study by Lim et al., (2017) are insignificant and there’s no connection between hyperserotonemia induced by gut microbial dysbiosis and ASD. 378

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Buffington, S. A., Di Prisco, G. V., Auchtung, T. A., Ajami, N. J., Petrosino, J. F., & Costa-Mattioli, M. (2016). Microbial reconstitution reverses maternal diet-induced social and synaptic deficits in offspring. Cell, 165(7), 1762-1775.

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The Lung-Brain Axis: Mechanisms of Ozone Exposure and Association with Alzheimer’s Disease Murdock Siegner

Ozone is a prevalent type of pollution common to urban areas and is recently found to be implicated in neurodegenerative disease such as Alzheimer’s. Although we have a good understanding of what happens to the brain after ozone exposure, it is not well understood the mechanisms by which this occurs. In this review, we examine a paper by Christen Mumaw et al. (2016) that begins to investigate the lung-brain axis as a possible mechanism for ozone effects on the brain. In the paper, Sprague-Dawley rats were exposed to 4 hours of ozone and tested for changes in microglial activation and changes in blood cytokine levels related to inflammation. In addition, the effects of age on increasing vulnerability to ozone was investigated. The authors found that ozone exposure lead to increases in inflammatory and neurotoxic molecule levels in the brain but not the blood, suggesting a different pathway for signalling molecules to affect the brain. In addition, aging populations had a reduced immune response and an enhanced proinflammatory response after mixed vehicle exhaust exposure indicating that aging populations are more susceptible to the neuroinflammatory responses found in Alzheimer’s when exposed to pollution. The results of the study implicated that inflammatory cytokines are not the signalling mechanism employed by ozone in the lung-brain axis and that age increases your susceptibility to microglial inflammatory responses to pollution. Key words: Ozone, O3, Alzheimer’s, lung-brain axis, inflammation, microglia, neurotoxic, mixed vehicle exhaust, TNFa, H2O2, MTT, neurodegeneration

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INTRODUCTION Pollution has become an increasingly important consequence of human action as we continue to burn fossil fuels for energy. In addition to global warming, increasing levels of pollution have been linked to major health problems including problems with respiratory and cardiovascular health as well as links to birth defects1,2. Pollution can affect these regions of the body through inhalation of particulate matter into the lungs where the air molecules can act on epithelial cells or enter the blood stream to reach various regions of the body3. In understanding that pollution can enter the body, it comes to question how pollution might affect the brain of individuals. Recent light has been shed on the potentially negative effects of pollution on the brain. Studies have begun to show how certain types of pollution might lead to microglial activation and the eventual death of neurons4,5. In this review we examined a study5 focusing on the effects of pollution as a basis to explore and expand on further research investigating this topic. Ground level ozone (O3) is a type of pollution that is not a direct result of fossil fuel burning; instead it is created by the reaction between volatile organic compounds (VOCs) and nitrogen oxides (NOx) with sunlight6. VOCs and NOx are released from fossil fuel burning and therefore indirectly contribute to increased levels of O3 in the lower atmosphere7. Increases in ozone levels alone can significantly heighten the chances of developing respiratory and cardiovascular diseases2,8. Only recently has research shed light on how the brain is affected. Recent findings have indicated that the activation of microglial cells of the brain plays an important role in the development of neurodegenerative diseases4,9. Microglial cells function as the immune system of the brain and work to maintain the neuronal environment10. In diseases such as Alzheimer’s disease (AD), high levels of microglial activation results in proinflammatory responses marked by the release of cytokines, the rounding of glial cell bodies, the shortening of axons and increased phagocytic activity 10. This immune-driven response contributes to the high levels of neuronal cell death we see in the brains of Alzheimer’s patients ultimately leading to the symptomatic consequences associated with the disease11. Understanding how microglial activity might negatively impact the development of the brain under certain circumstances continues to be essential to determining the causes of Alzheimer’s disease. In doing this, it has become increasingly important to develop an understanding of factors that can lead to the potentially harmful effects of microglial cells. We review a paper by Christen Mumaw et al. (2016)5 investigating pollution as one of those factors involved in microglial activation. Previous evidence has suggested a connection between ozone, Alzheimer’s disease12 and Parkinson’s disease13 in the brain, however, the mechanisms by which ozone has its effect is not well understood. Christen Mumaw et al. (2016)5 sought out to answer these questions by investigating how the ozone we breathe gets into our system, and the resulting effects on microglial activation. It is known that ozone cannot directly enter the body due to its reactivity, although it has been implicated that ozone reacts with lung surfactant to release secondary factors that enter the body 14. Mumaw et al. (2016)5 showed evidence to support that O3 has its effects through the lung-brain axis, that exposure to O3 increases microglial inflammatory response and Alzheimer’s-like neurotoxicity, and that aging brains are more vulnerable to exhibiting microglial inflammatory responses after O3 exposure. Using the study by Mumaw et al. (2016)5, this review hopes to come to conclusions about the major effects of ozone on the

brain and what steps need to be taken to improve our understanding of the mechanisms involved. MAJOR RESULTS In vivo rat models of microglial activation Mumaw et al. (2016)5 exposed rats to 4 hours of 1 part per million (PPM) of O3 which showed increased activation of microglial cells both 4 hours and 24 hours after initial exposure compared to control rats exposed to filtered air. O3 was not directly present in the brains of the rats after exposure, however, it was thought that interaction with lung cells mediated O3 signals to the brain even after 24 hours of exposure. The study points to this ongoing activation as evidence for a lung-brain axis implying that O3 continues to send signals from the lungs after exposure. These results support the work of other articles showing that oxidative stress as a result of O3 exposure results in activated microglia past the point of discontinued exposure15,16. O3 exposure was implicated with problems associated with hippocampal heath, which may explain the loss of memory commonly found in Alzheimer’s15,17.

Figure adopted from Christen L. Mumaw et al. (2016)5, FASEB J. 2016 May;30(5):1880-1891 Figure 1. Morphological differences between microglia in the brains of rats exposed to 4 hours of filtered air (left) and 4 hours of 1 PPM of O3 (right). Microglia in O3 exposed rats were more prominent with larger, round cell bodies and shorter axon projections. The red arrows point to activated microglia. Magnification was 40X. The scale bar is 50mm. Ex vivo rat models of microglial activation Mumaw et al. (2016)5 continued investigations into the effects of O3 on microglial cells in Ex vivo rat cell models. Morphological evidence showed changes in the shape of the microglial cells as seen by the rounding of the cell bodies and the shrinking of the cellular processes, indicating activation (Figure 1). Molecular investigation into the Ex vivo models showed that O3 exposed lipopolysaccharide (LPS)-induced cell lines demonstrated an increase in tumour necrosis factor alpha (TNFa), a decrease in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and an increase in hydrogen peroxide (H2O2) (Figure 2). Importantly, no significant changes were found in cultures that did not contain LPS. TNFa is a strong proinflammatory agent and an important component in the production of cytokines responsible for inflammation which might indicate its role in the inflammatory response seen in AD18. Decreased MTT is indicative of increased neurotoxicity found in O3 exposed LPS-induced microglial cells19 while increased H2O2 in LPS-induced cells might indicate higher levels of neural cell death20,21. In the case of all three measured molecules, O 3 appears to induce Alzheimer’s-like neuropathology only in cultures containing LPS. LPS is a saccharide molecule attached to lipids found on the surface of bacteria22. It is found in humans and has been shown to enhance inflammatory responses with O3 exposure23. Considering this, the results of this experiment appears consistent

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c with literature focused on LPS effects. Contrary to the study, Nery-Flores et al. (2019)24 found that there were increases in TNFa without the presence of LPS; however, this may have been because rats were exposed to 4 hours of 0.7PPM for 15 days and 60 days rather than a single exposure of 4 hours. It has also been shown that MTT assays decrease at higher levels of neurotoxicity25,26 providing confirmation that Mumaw et al. (2016)5 saw increased toxicity. Interestingly, one study concluded that glucose plays a key role in decreasing MTT levels after O3 exposure25, which was likely to be present in the fetal bovine serum used for the cell cultures. Lastly, studies have also shown the relationship between ozone exposure and increases in H2O2 as found in the Mumaw et al. (2016)5 study.

Testing aging effects on inflammatory response to MVE Mumaw et al. (2016)5 conducted experiments by determining whether Alzheimerâ&#x20AC;&#x2122;s, largely an age-related disorder, showed enhanced inflammatory responses in older populations as a result of pollution exposure. Younger (2 months) and older (10 months to 18 months) mice population were exposed to 6 hours per day of 50 days of mixed vehicle exhaust (MVE) composed of gasoline and diesel engine emission. Ozone was not tested as previous literature supported the effects of MVE on aging populations 28. They tested for inflammatory responses in the lungs and found that in young mouse populations, there was a significant increase in neutrophilic response after MVE exposure but there was no significant change in older mouse populations (Figure 3b). On the other hand, MVE exposure increased TNFa expression significantly in older populations but not in younger populations (Figure 3a). These findings suggest that while aging populations have a reduced immune response after MVE exposure they show enhanced proinflammatory responses. This might implicate that aging populations are more susceptible to the neuroinflammatory responses found in Alzheimerâ&#x20AC;&#x2122;s when exposed to pollution. Other research focusing on agerelated brain vulnerability to pollution is limited, but evidence suggests that exposure to higher levels of pollution predicts lower cognitive abilities in older populations28, perhaps owing to increased neuron death as a result of microglial activation.

Figure adopted from Christen L. Mumaw et al. (2016)5, FASEB J. 2016 May;30(5):1880-1891 Figure 2. Concentrations of TNFa, H2O2 and MTT in rat microglial cells exposed to ozone compared to filtered air, with comparisons to LPS-induced cell lines. A) TNFa was significantly increased in ozone exposed HAPI rat microglia containing LPS. B) TNFa was significantly increased in ozone exposed primary rat microglia containing LPS. C) Ozone increased H2O2 in LPS-induced primary rat microglial cells. D) Ozone significantly increased MTT in LPSinduced primary rat microglial cells.

Testing for changes in inflammatory cytokine levels in the blood After testing Ex vivo cell lines, Mumaw et al. (2016)5 used blood samples from O3 exposed rats to identify possible agents responsible for an inflammatory response through signalling from the lungs to the brain. They could not find such agents; there were no increases in blood CCL2, CCL11, TNFa, IL-6, and IL-1b levels which are associated with inflammatory signalling14. These findings indicate that these specific molecules are not involved in the inflammatory signalling used to activate microglia; the authors suggest that further investigation is required to determine the mechanism. According to previous research, increases in proinflammatory cytokines as a result of O3 exposure do occur, however, these findings were the result of tests on human lung epithelial cells 14. An important study published after the article by Mumaw et al. (2016)5, identified acute-phase protein serum amyloid A (A-SAA) as a possible signalling agent for O316. They hypothesized that A-SAA was linked to microglial inflammatory response after O3 exposure by the lung-brain axis16.

Figure adopted from Christen L. Mumaw et al. (2016)5, FASEB J. 2016 May;30(5):1880-1891 Figure 3. TNFa in the brain and total neutrophils present in the lungs after MVE exposure in 2-month-old and 18-month-old mice. A) Relative TNFa levels were increased in older mice population compared to younger populations indicating increases in neuroinflammation. B) Total neutrophils were increased in younger mice populations compared to older populations showing increased immune response.

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DISCUSSION AND CONCLUSION Indications that the activation of microglial cells plays an important role in the development of neurodegenerative diseases is emphasized by the role pollution plays in enhancing this effect. The paper by Mumaw et al. (2016)5 clarified this relationship; they found that microglia are activated by O3 in both In vivo and Ex vivo rat models. They also found increased TNFa, H2O2 and decreased MTT but only in models containing LPS, indicating higher levels of microglial inflammation, neurotoxicity and cell death. Results also indicated that there were no changes in inflammatory agents that might be responsible for signal transduction from the lungs to the brain. Lastly, the paper showed that young mice populations demonstrated an increase in lung neutrophilic response after MVE exposure while older mice populations demonstrated increased TNFa expression, indicating greater inflammatory responses in older populations. All points considered; the paper shows that their results support notions for a lung-brain mechanism by which pollution enhances neurodegenerative pathology. The findings of this study are significant as they begin to outline a mechanistic pathway that allows ozone to affect the brain. Investigation for a lung-brain axis in this study was backed by previous research indicating that there is an increased risk in developing AD in patients with lung related diseases, however, past works have not implicated an understanding behind the mechanisms29. There were indications that ozone does not directly infiltrate the body but rather impacts microglial responses to exposure and other organs of the human body30. These findings along with previously discussed knowledge provided a basis for Mumaw et al. (2016)5 to advance our understanding behind the mechanism of O3 action on the brain. Their results provide indications for a lung-brain axis; however, they explain that it is possible that other signalling factors may be more strongly involved in the pathway for mediating microglial activation. This indicated that more research needs to be focused towards understanding this pathway and the molecules involved. This study may pave the way for future studies, possibly resulting in better efforts directed towards reducing exposure to pollution and to developing a better understanding of the mechanisms involved in neurodegenerative diseases such as Alzheimerâ&#x20AC;&#x2122;s.

investigate the lung-brain axis, however, this topic appeared to be a small portion of what was focused on. The majority of the research was dedicated to morphological and molecular changes found within the brain rather than in peripheral tissue such as the blood or the lungs where previous research has indicated ozone releases its effect on the body14,16. In addition, the authors tested for the cytokines they believed would correspond to increased inflammation in the brain, rather than going through an exhaustive list of possible causes. As mentioned earlier, a study investigated molecular signalling of ozone-related interaction with human lung epithelial cells and found that lipid ozonation products (LOPs) are produced at the lungs after ozone exposure14. They hypothesized that LOPs then interacted with cells to induce the release of cell signals into the blood which lead to neuroinflammation14. Considering this, Mumaw et al. (2016)5 should have also focused on LOP increases in the blood and the resulting effects on cell signalling. The authors should have also focused more on studying peripheral attributions on a more thorough level in order to localize signalling to a specific region of the body such as the lungs. In the article discussion it would have benefitted the reader if the authors included an explanation for introducing LPS into Ex vivo models. It was briefly mentioned that LPS activates an immune response in microglial cells however it is never indicated what the significance of this is and how this is related to O3 exposure. It also comes into question how LPS might affect the results of the experiment considering that one study found that LPS alone is enough to induce neuroinflammation and increase some inflammatory cytokines such as TNFa31. The study by Mumaw et al. (2016)5 does not exclude LPS as a potential factor affecting the results of the experiment. The authors concluded that ozone increases microglial proinflammatory responses, however, it might have been more appropriate to conclude that LPS increases microglial inflammatory responses to ozone. In addition to LPS the authors lacked sufficient explanation for the reasoning behind testing only CCL2, CCL11, TNFa, IL-6, and IL -1b changes in blood. They mention the significance of these cytokines in transferring inflammation from the periphery to the brain, however, it is not clear why other molecules were not tested for.

One last criticism for the paper is that there was little connection between the results found in this study and the significance CRITICAL ANALYSIS in humans. Specifically, the authors should address if the results we The purpose of the Mumaw et al. (2016)5 article was to shed see in rats would be expected to translate to human populations, light on some potential mechanisms of O3 induced microglial activa- and if so, what might this mean for the future of preventative or tion and its relationship to Alzheimerâ&#x20AC;&#x2122;s. Considering previous works treatment options regarding pollution. on the topic of O3 induced microglial activation, the study takes the appropriate steps to test and explain the significance of their results. Methods were based off previous works, and tests were justi- FUTURE DIRECTIONS fied according to what has been done prior and what might be exAfter reviewing the Mumaw et al. (2016)5 study it bepected to occur. According to our research, other studies replicomes clear that further research is required to explore the mechacating or expanding on this study includes an article that identified nisms of O3 action on the brain. The focus article and a few articles a possible agent involved in the transmission of O3 in the lung-brain following it begin to explore this domain, however, our understand16 axis . Beyond this, expansion in this research area is mostly reing of how O3 has its effect on the brain is limited. Therefore, it is stricted to the outcomes of O3 interaction with lungs, rather than suggested that researchers focus more on testing for signalling molthe mechanisms involved. All points considered, the results of the ecules that might be responsible for O3 signalling to the brain. 5 Mumaw et al. (2016) study were consistent with previous research and the new findings of this study were intuitively explained by the A good model for studying molecular changes in the blood authors. after O3 exposure comes from a study by Michelle A. Erikson et al. (2017)16. The researchers of this study found increases in blood Some criticisms for the methods of the paper stem mostly acute-phase protein serum amyloid A (A-SAA) levels following 2 from the fact that the purpose of the research was to highlight and

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hours of O3 exposure in rats16. Based off this study, researchers should look for increases in signalling molecules first by exposing rats to 2 hours of 3 PPM of O3. A control group would be exposed to filtered air for basis of comparison. Blood samples should be collected after exposure and tested for quantities of various molecules including cytokines and signalling proteins. Although focus should be preferentially spent on testing an exhaustive list of molecules expected to be involved in neuroinflammation such as TNFa or LOPs, it would be wise to test other types of molecules as O3 signalling molecules might cause the release of other factors that are related to inflammation. After testing blood samples, organs that are suspected to be involved in the production of signalling molecules such as the lungs should be harvested and undergo protein extraction, immunoblotting, RNA extraction and quantitative reverse transcription polymerase chain reaction (qRT-PCR) to test for the upregulation of signalling molecules. After conducting these tests, we might expect to find that certain molecules will be significantly upregulated after O3 exposure. If so, pathway analysis can be conducted using Reactome software32 to determine whether or not the molecule is directly or indirectly related to brain inflammation. If the molecule is related to brain inflammation, further tests can be used to understand the specific relationship between O3, the molecule, and the brain to make conclusions about a lung-brain axis. On the other hand, if no molecules are found to be significantly upregulated then this might direct evidence away from a lung-brain axis and indicate other mechanisms that would require further investigation. Lastly, if some molecules are significantly upregulated but are not found to be related to pathways involving the brain, in-depth pathway investigation32 would be required to show the relationship between the molecule and O3 exposure. The importance of the proposed tests might provide evidence in favour of or against a lung-brain axis as a mechanism for O3 to act on the brain. Although current research favours the effects of O3 on the brain and its relationship to neurodegenerative disorders, it is equally important to investigate how this happens. Figuring out both sides of the story could be essential in understanding how to prevent or treat pollution-related diseases.

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The Exploration of GUO as a Potential Therapeutic Treatment for Focal Ischemic Stroke Vyshnave Sivasankar

Ischemic stroke is a medical condition where a part of the brain has temporarily or permanently reduced blood flow due to an embolic or thrombotic blockage of a major cerebral artery (Brouns & Deyn, 2009). This lack of blood flow results in an oxygen and glucose deficiency in the affected brain area causing energy failure and resulting in an ischemic cascade leading to oxidative and excitotoxic damage (Brouns & Deyn, 2009). The excessive release of glutamate by damaged cells in the penumbral zone causes the overstimulation of its excitatory receptors and the mass release of free radical species such as radical oxygen species (ROS) and radical nitrogen species (RNS) (Teixeira, et al., 2018). The severity of this post-stroke damage is dependent on the duration, location and size of the affected area. Guanosine (GUO) is a known neuroprotective agent against neurotoxicity induced by the over-activation of glutamatergic receptors as seen in the ischemic cascade (Petronilho, et al., 2012). In a study conducted by Hansel et. al (2014), GUO, an endogenous guanine-based purine, is explored as a therapeutic agent for rat stroke models. The main results of their study concluded that GUO administration following stroke induction, resulted in an overall reduction in stroke-based damage including reduced infarction area, increased forelimb function, decreased surge in ROS/RNS, and suppression of the glutamergic system (Hansel et al., 2014). Such results indicate that GUO is highly effective therapeutic agent for leading recovery from oxidative and excitotoxic damage induced by ischemic stroke. Keywords: cerebral ischemia, stroke, ischemic stroke, brain damage, oxidative stress, glutamate, lipid peroxidation, excitotoxicity, guanosine, ischemic cascade.

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INTRODUCTION Stroke is the worldâ&#x20AC;&#x2122;s second largest cause of disability and death; ischemic stroke makes up for about 80% of them (Teixeira, et al., 2018). Due to this many animal models for focal ischemic stroke have been developed, mainly utilizing rats since they have similar cerebrovascular and physiological components to humans (Durukan & Tatlisumak, 2007). The models are an integral part in analyzing how stroke affects the brain and testing methods to recover homeostasis and improve long-term clinical outcomes (Durukan & Tatlisumak, 2007). The affected area of the stroke is referred to as the infarct/ischemic zone and consists of necrotic tissue (Durukan & Tatlisumak, 2007). The tissue region surrounding the circumference of this zone, maintains structural integrity, and is referred to as the penumbral zone, consisting of mostly apoptotic tissue (Durukan & Tatlisumak, 2007). The two main treatment approaches for ischemic stroke are neuroprotection and recanalization. Recanalization works to lyse the clot stunting blood flow and recover at risk tissue, focusing on the infarct zone (Teixeira, et al., 2018). Conversely, neuroprotectants work to preserve neurons located in the penumbral region and prevent their death through working to fight the effects of the ischemic cascade (Minnerup, 2012). This includes reducing glutamate production and glutamergic receptor activity, apoptosis, excitotoxicity, release of free radicals, and inflammation (Minnerup, 2012). Currently, the only approved treatment for stroke is the use of recombinant tissue plasminogen activator (rt-PA) which works to recanalize the affected artery and restore brain function (Molina et al., 2004). In a study conducted by Molina et. al (2004), it was found that early recanalization using rt-PA allowed for better long-term recovery from stroke. However, in this same study, it was found that some stroke patients remained the same following treatment with rt-PA, regardless of early recanalization and did not show recovery following the administration of rt-PA (Molina, et al., 2004). This was consistently found to be associated with health factors including hypertension, hyperglycemia older age, and severity of stroke suffered (Molina, et al., 2004). Due to this, further research is heavily focused on discovering novel additive treatments that can be used alongside rt-PA to improve stroke treatment and optimize recovery. Many novel therapeutic agents work as neuroprotectants to offset damage of tissue within the penumbral zone. As previously mentioned, the viability of the cells within this area continually decreases as time progresses due to the effects of reperfusion, hypoxia, and the ischemic cascade (Xing et al., 2012). According to a review by Xing, Arai, Lo & Hommel (2012), the ischemic cascade is triggered through acidosis from the continual consumption of ATP following the cessation of its supply due to the lack of cerebral blood flow (CBF). The ionic imbalance stimulates the release of neurotransmitters without subsequent uptake resulting in a large uptake of water, sodium, and calcium. This results in cell swelling, degradation of essential cellular components and activation of catabolic processes (Xing et al., 2012). Additionally, the high sodium, calcium and adenosine diphosphate (ADP) levels stimulate the production of ROS, which damage lipids, proteins and other key macromolecules, at a higher rate than antioxidant enzymes, such as superoxide dismutase (SOD), and scavenging mechanisms, such as Vitamin C, that would regularly neutralize its effects (Xing et al., 2012). Such destructive processes ultimately lead to cell death in penumbral zone increasing the surface area of stroke damage, if neglected to be treated (Bruno et al., 2001).

Strong contributors of ischemic cascade neurotransmitter and receptor are the glutamergic receptor and glutamate. They fuel the excitotoxicity seen in ischemic stroke, due to this, they are common therapeutically targeted mechanisms. In a review conducted by Bruno et. al (2001), it was found that infarction volume was found to be reduced in models that blocked the activity of glutamate receptors (GluRs). The cessation of GluR function reduced calcium surge levels decreasing the activation catabolic processes, reducing cortical spreading depressions (CSD), and stimulating metabotropic GluRs (mGluRs), which control cell death and survival signaling (Bruno et al., 2001). In the 2014 study conducted by Hansel et. al, a novel neuroprotectant, GUO, is explored as a therapeutic agent for ischemic stroke and analyzed in the major contexts of stroke targets; the ischemic cascade and excitotoxicity through exploring effects on oxidative stress agents; ROS and RNS, and glutamergic effectors; glutamateaspartate transporter (GLAST) and glutamate transporter-1 (GLT1). The effects of ischemia were analyzed through inducing stroke in mice and comparing their behavioral and brain stressor outputs to sham mice. Behavioral comparisons were made using the cylinder test while, levels of ROS/RNS, antioxidant parameters (enzymatic and non-enzymatic), and glutamergic effectors indicated tissue stress levels. In mice treated with GUO post stroke induction, a lack of increase in ROS/RNS, increased GLAST, and increased antioxidant levels were observed when compared to the ischemic mice that were treated with saline. These comprehensive results suggest that GUO has a positive therapeutic effect on ischemic mice and can be used as a potential additive treatment in stroke patients. MAJOR RESULTS In the experiments conducted by Hansel et. al (2014), rat models were divided into 4 subgroups; sham rats treated with saline, sham rats treated with GUO, induced ischemic rats treated with saline, and induced ischemic rats treated with GUO. Impaired forelimb function observable through the decline in forelimb symmetry in the cylinder test and used as the hallmark of focal ischemic stroke within the motor cortex (Hansel et al., 2014). Following the induction of ischemic stroke, GUO treatments were administered immediately, 1 hour (h), 3 h, and 6 h following the treatment. A major finding by Hansel et. al (2014) is that the administration of GUO to ischemic rats partially recovered forelimb function. Their research also found that decreased ROS/RNS levels and increased glutamine synthetase (GS) levels were observed in ischemic mice treated with GUO indicating that it modulated the excitotoxic effect of ischemia through preventing lipid damage from oxidative stress and the upregulation of GluR activity, respectively (Hansel et al., 2014). Lastly, it was also found that the administration of GUO reduced the infarction volume at the stroke location as seen in Figure 1 from Hansel et. al (2014).

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Figure Adapted from Hansel et al. (2014). PLoS One, 9(2). Figure 1. Graph depicts an average of stroke infarction volumes in cubic millimeters for ischemic sham (IS) mice and ischemic GUO (IG) mice. IG mice had a significantly smaller infarction volume, about half of the infarction volume of IS mice. Restoration of Function GUO has been observed to be an agent for restoring bodily function impaired through various neurological disorders and injuries including ischemic stroke, parkinsonism and spinal cord injuries (Hansel et al., 2014; Jiang et al., 2007; Su et al., 2008). This is evident in the study by Hansel et al. (2014) where increasing forelimb symmetry scores were observed in the ischemic rats administered with GUO. Rats were administered different levels of GUO immediately, 1 h, 3 h, and 6 h post-surgery and forelimb symmetry was observed before surgery and up to 15 days following surgery (Hansel et al., 2014). Progressive increases in forelimb symmetry were observed as days progressed in ischemic rats administered with GUO, while ischemic rats given saline showed a significantly lower symmetry score post-surgery. Additionally, a higher increase in this score was observed with higher GUO dosage administrations (Figure 2) (Hansel et al., 2014).

Figure Adapted from Hansel et al. (2014). PLoS One, 9(2). Figure 2. Graph depicts forelimb symmetry score in percentage for the cylinder test. Day 0 depicts the day prior to surgery to induce ischemia and tests were repeated up to 15 days post-ischemia. Results depict the symmetry score of the control rats, sham saline and ischemia saline, and of ischemic rats with 30, 60, and 120 mg/ Kg of GUO This can be further seen in the study conducted by Su et al. (2008) where rat models for Parkinsonâ&#x20AC;&#x2122;s disease (PD) were administered GUO following the induction of mobility impairment. Rats were observed for rigidity and tremors during movement through and unrestricted areas (Su et al., 2008). Increases in mobility were observed in PD rats with GUO following 8 weeks of its injection (Su et al., 2008). An improvement in motor and sensory function with recovery of bladder function was observed in rats modelling spinal cord injury in a study conducted by Jiang et al. (2007). Rats were administered with GUO 4 h following cord injury continuing up to 14 days post-injury and tested for hind limb movement and sensitivity, forelimb movement, position maintenance on an inclined plane, urine expulsion (Jiang et al., 2007). Rats administered were found to have improvement across all motor test and recovered function of lower urinary tract when compared injured rats administered with saline (Jiang et al., 2007). Neuroprotective Measures and Effects As a neuroprotective agent, GUO has worked to prevent the effects of neurotoxicity or neurological damage in focal ischemic stroke (Hansel et al., 2014; Connell et al., 2013; Moretto et al.,

2005). This is evident in the study by Hansel et al. (2014) where ischemic rats administered with GUO did not have an increase in the levels of ROS when compared to ischemic rats given saline. The ROS levels are determined by dichlorofluorescin (DCF) fluorescence levels, since DFC is molecule that becomes fluorescent when oxidized (Hansel et al., 2014). DFC levels for ischemic rats with GUO were found to be similar to sham rats with saline and GUO (Figure 3a) (Hansel et al., 2014). This indicates that ROS levels remained at homeostatic levels in ischemic rats administered GUO. Through this study, it is also evident that RNS levels were decreased through the administration of GUO to ischemic rats (Hansel et al., 2014). This decrease in RNS levels are indicated through Nitric Oxide (NO) levels. NO levels are determined through the decrease in NO2-/NO3- levels, oxidation products of NO. Ischemic saline rats have significantly higher NO2-/NO3- levels when compared to other test groups (Figure 3b) (Hansel et al., 2014). When comparing NO2/NO3- levels between ischemic GUO rats and sham saline/ GUO rats (normal levels), ischemic GUO rats have a decreased level of NO2-/ NO3- (Figure 3b). In this same study, increased GS levels were observed in ischemic mice treated with GUO. This is visible in Figure 4, where there is a significantly higher level of GS measured in ischemic GUO rats compared to the control rats and ischemic rats with saline (Hansel et al., 2014). GS works to convert glutamate to glutamine within cells and its overactivation possibly prevents the overstimulation of glutamergic receptors. Additionally, the decrease in GLT1 activity by ischemia was reversed and GLT1 activity in ischemic GUO rats remained at levels comparable to the control rats (Hansel et al., 2014).

Figure Adapted from Hansel et al. (2014). PLoS One, 9(2). Figure 3. The graphs depict levels of oxidative stress measures for sham and ischemic mice administer with saline and GUO. (A) Depicts levels of ROS through the percentage of fluorescence in DFC molecules and show that ischemic GUO rats, maintained levels similar to control rats. (B) Depicts NO levels through NO2-/NO3levels and indicates that ischemic GUO rats had decreased levels when compared to control and ischemic saline rats.

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Figure Adapted from Hansel et al. (2014). PLoS One, 9(2). Figure 4. The graphs depict levels of glutamine synthetase (GS) in amounts of protein present in sham saline and GUO rats and ischemic saline and GUO rats. An increase in GS levels can be observed in ischemic GUO rats when compared to controls and ischemic saline rats. The neuroprotective effects of GUO in ischemic stroke can be further seen in a study conduct by Connell et al. (2013). In these experiments, test rats underwent oxygen-glucose deprivation (OGD) to mimic conditions of ischemia. It was found that the administration of GUO within 30 minutes of OGD initiation, decreased levels of Interleukin-8 (IL-8), a chemokine indicative of proinflammation (Connell et al., 2013). Subsequently, in a study conducted by Moretto et al. (2005), the effects of GUO administration on neonatal rats with induced hypoxic-ischemia (HI) were observed. It was found that, while HI greatly reduced glutamate uptake into the hippocampus 3-5 days after induction, GUO prevented this and maintained glutamate uptake at levels observed prior to HI induction (Moretto et al., 2005). Through this, it is evident that the research conducted by Hansel et al. (2014) on the effects of GUO on rats with induced ischemic stroke, shows results consistent with other studies conducted on ischemia and studies conducted on other neurological injuries and disorders. GUO is a neuroprotective agent that prevents the occurrence of neurotoxicity and aids in restorative functions following stroke (Hansel et al., 2014; Jiang et al., 2007; Su et al., 2008; Connell et al., 2013; Moretto et al., 2005). The observed restorative functions of GUO include recovery of forelimb symmetry, hindlimb movement and the recovery of function in the lower urinary tract seen in various neurological injuries and disorders (Hansel et al., 2014; Jiang et al., 2007; Su et al., 2008). Neuroprotective effects include decreasing levels of RNS and ROS, maintaining glutamate uptake, and reducing proinflammatory chemokines (Hansel et al., 2014; Connell et al., 2013; Moretto et al., 2005).

Hansel et al. (2014), was able to find conclusive results regarding the decrease in ROS/RNS pathway molecules following the administration of GUO to ischemic rats. This was not previously observed in studies conducted by Rathbone et al. in 2011 and Connell et al. in 2013, where ROS levels remained elevated even after GUO administration. Additionally, Hansel et al (2014) observed an increase in GS expression in ischemic GUO rats, an indicator of glutamate uptake and removal, preventing the overactivation of glutamatergic receptors. Such results are consistent with Moretto et al.’s (2005) observations of hippocampal glucose uptake in neonatal HI rats. Additionally, Hansel et al.’s (2014) findings on the effects of restorative function in forelimb symmetry are consistent with findings made by Jiang et al. in 2007 on limb and urinary tract function recovery in rats with spinal cord injury, and Su et al.’s findings on the reduction of tremors and limb function recovery in PD rats in 2008. Hansel et al. (2014) recognize that there has been previous research conducted regarding GUO and its role in reducing oxidative stress and glutamate overactivation in stroke models. Understanding this, they aimed to study the biochemical mechanisms that GUO targets to promote neuroprotection (Hansel et al., 2014). Through these conclusive findings by Hansel et al. (2014) and comparable literature, it is clear that GUO is a promising agent for the cessation of ischemia induced neurotoxicity, and effective in preventing the effects of the ischemic cascade. CRITICAL ANALYSIS Hansel et al. (2014) offer an additional therapeutic drug, GUO, to use in conjunction with rt-PA to act as a neuroprotective agent and prevent and recover tissue damage and death in the ischemic zone and penumbra. While this study shows some convincing results regarding the effectiveness of GUO administration to ischemic rats, more experiments regarding the use of GUO on other ischemic stroke rat models, such as MCAO, must be conducted to get a more thorough and widespread understanding on the therapeutic effectiveness of GUO (Hansel et al., 2014). Future research can be targeted towards better understanding the mechanisms by which GUO increases glutamergic uptake preventing overstimulation. This can give more clear results on the effect GUO has on the glutamergic pathway since only partial results supported this claim. In the study conducted by Hansel et al. (2014), GS was increased due to GUO, but GLAST and GLUT1 levels were not influenced by GUO. Due to this, the authors could have possibly added a tracer protein with GUO delivery to trace and identify areas effected by GUO and indicate whether the lack of response from the glutamergic receptors was due to the absence of GUO or lack of response to its effects. However, Hansel et al. (2014), was able to induce ischemia and obtain neuroprotective observations using an in vivo focal cerebral ischemic stroke rat. This model is a more effective and applicable model of study to humans since it is functional, and changes can be observed over time (Hansel et al., 2014). The depth of biochemical mechanisms observed and analyzed in this experiment allow for a deeper understanding of the effects of GUO on the glutamergic system and oxidative stress at a molecular level. This understanding of its function can than be applied to understand how it could work in human stroke patients.

CONCLUSIONS/DISCUSSION The overall conclusions that can be drawn from Hansel et al.’s (2014) research are that GUO promotes restorative function and works to prevent neurotoxic effects on ischemic stroke rats. These findings indicate that GUO is highly effective neuroprotective agent that can be implemented as a therapeutic drug and be used in conjunction with rt-PA for stroke recovery (Molina et al., 2003; Hansel et al., 2014). When comparing the results from the study conducted by Hansel et al. (2014) to the literature, the positive effects of GUO administration in ischemic conditions are mostly consistent with other studies and yield similar results. GUO works to maintain homeostatic activity in neurotoxic pathways and FUTURE DIRECTIONS limb function, that are comparable to measurements drawn prior To further apply current findings made by Hansel et al. to the induction of ischemia (Hansel et al., 2014; Jiang et al., 2007; (2014) and other literature, more application of therapeutic drugs Su et al., 2008; Connell et al., 2013; Moretto et al., 2005). that have been found to be successful in rat models should be ap-

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plied to human models in potential drug trials. GUO would be a great candidate for a trial drug, as there is extensive literature supporting its effectiveness as a neuroprotective agent in not just stroke but other neurological disorders and injuries (Hansel et al., 2014; Jiang et al., 2007; Su et al., 2008; Connell et al., 2013; Moretto et al., 2005). Additionally, GUO is a substance that is naturally occurring as a neuroprotective agent, because it is naturally occurring, the addition of it in these therapeutic trials shouldnâ&#x20AC;&#x2122;t have a harmful effect on patients making it an ethical trial drug. However, in this experiment, GUO is administered through intraperitoneal injections which may not be as effective in humans and a novel administration method should be observed. If GUO proves to be effective in human stroke patients as a neuroprotectant, it can provide better outcomes post-stroke for patients and allow for a better recovery and restoration of function. Another possible study can be conducted using GUO in conjunction with rt-PA to observe the therapeutic effects of both drugs since both prove to be therapeutic beneficial for stroke individually. This can also allow for observation of how both drugs work together and analyze potential positive or negative interactions between the drugs. If proven to be effective, this can provide better outcomes for stoke patients since rt-PA alone doesnâ&#x20AC;&#x2122;t always yield positive results and recovery in patients. Conducting such an experiment an provide novel results and therapeutic outcomes for stroke patients since there is only one drug, rt-PA, used of human patients. Further applying this drug combination in human trials can also provide novel insight on their effectiveness together. As previously mentioned, GUO is naturally occurring and neuroprotective making it an ethical sound drug to use. Additionally, rt-PA is already approved to use on human patients so using this drug cocktail in trials shouldnâ&#x20AC;&#x2122;t pose as an ethical conflict. Both proposed future steps can take current findings by Hansel et al. (2014) and other literature findings a step further towards finding more effective drug treatments for better recovery in human stroke patients. Regardless of the results these experiments yield, they can provide valuable insight on future research focus and initiatives. Since there is extensive research focusing on GUO in many stroke models, applying it to models more relevant to human stroke patients can allow for the discovery of novel therapeutic agent to treat stroke. ACKNOWLEDGMENTS Assignment for Dr. Bill Jeu.

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Connell, B. J., Iorio, P. D., Sayeed, I., Ballerini, P., Saleh, M. C., Giuliani, P., … Jiang, S. (2012). Guanosine protects against reperfusion injury in rat brains after ischemic stroke. Journal of Neuroscience Research, 91(2), 262–272.

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Hansel, G., Ramos, D. B., Delgado, C. A., Souza, D. G., Almeida, R. F., Portela, L. V., … Souza, D. O. (2014). The Potential Therapeutic Effect of Guanosine after Cortical Focal Ischemia in Rats. PLoS ONE, 9(2).

Jiang, S., Bendjelloul, F., Ballerini, P., D’Alimonte, I., Nargi, E., Jiang, C., … Rathbone, M. P. (2007). Guanosine reduces apoptosis and inflammation associated with restoration of function in rats with acute spinal cord injury. Purinergic Signalling, 3(4), 411–421.

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Molina, C. A., Alexandrov, A. V., Demchuk, A. M., Saqqur, M., Uchino, K., & Alvarez-Sabín José. (2004). Improving the Predictive Accuracy of Recanalization on Stroke Outcome in Patients Treated With Tissue Plasminogen Activator. Stroke, 35(1), 151–156.

Moretto, M., Arteni, N., Lavinsky, D., Netto, C., Rocha, J., Souza, D., & Wofchuk, S. (2005). Hypoxic-ischemic insult decreases glutamate uptake by hippocampal slices from neonatal rats: Prevention by guanosine. Experimental Neurology, 195(2), 400–406.

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Modeling Traumatic Brain Injury in Adult Zebrafish Shows Relevant Mammalian Injury Characteristics and Treatment Outcomes Isis So

Traumatic brain injury (TBI) is the global primary reason for death and disability. Mild TBI (mTBI) or concussion causes acute physical, behavioral, and cognitive impairments that typically resolve within 3 months. However, findings suggest that a single TBI can lead to progressive secondary injury, which consist of pathophysiological alterations. Rodent models of TBI â&#x20AC;&#x201C; although useful for understanding the underlying cellular and molecular mechanisms of TBI â&#x20AC;&#x201C; are expensive and time-consuming models for drug testing. As such, this study by MuCutcheon et al. (2017) investigated the validity and efficacy of modeling TBI with the adult zebrafish. The pulsed high-intensity focused ultrasound (pHIFU) technology was used to induce a closed-head TBI in zebrafish, which is equivalent to a mammalian mTBI caused by mechanical and/or stress forces. Behavioural outcomes measured with the novel tank test and shoaling test showed reduced locomotor activity and increased anxiety post-injury. Biochemical assays revealed increased expression of proteins that signal axonal injury and apoptosis. Injured fish showed better behavioural outcomes under hypothermic recovery conditions and neuroprotective MK801 drug injections, and increased survival rates under hypothermic recovery. These results are similar to observations made in mammalian models, which highlight the relevance of the adult zebrafish model of TBI, and suggests its potential application for further studying TBI pathophysiology, and for screening and evaluating TBI-related pharmacological agents. Key words: concussion, traumatic brain injury, zebrafish, high-intensity focused ultrasound, behaviour, hypothermia, drug response

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Traumatic brain injury (TBI) is the global primary cause of death and disability 1,2. Mild TBI (mTBI, or concussion) is the most common TBI subtype 3, and results in physical, behavioural, and cognitive impairments that typically resolve within 3 months, although 11-29% of patients continue to experience persisting concussion symptoms past this timepoint 4. Evidence shows that a single mTBI is sufficient to induce long-term pathophysiological brain changes, which constitute secondary injury 5. Changes such as glutamate excitotoxicity, calcium ion release, lactate buildup, and cerebral blood flow impairments can lead to altered brain metabolism, neurotransmission deficiencies, and cell death 5. However, the delayed onset of secondary injury presents a therapeutic time window at which intervention can be administered to slow or reduce injury 2, which highlights the importance of developing targeted drugs and/or therapies. Current models used to study the cellular and molecular mechanisms of mammalian TBI include the mouse, rat, and swine, but drug development and evaluation in these models is slow and difficult 2,6. Similarly, although in vitro brain organoids can be used for high throughput drug evaluation, they are unable to represent complex cellular and anatomical changes 7. Instead, the adult zebrafish – which has analogous brain anatomy to mammals 8 – is an ideal model for overcoming these limitations. Importantly, the adult zebrafish expresses receptors involved in mammalian neurotransmission and TBI-induced excitotoxicity (the α-amino-3hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) 9 and N-MethylD-aspartic acid (NMDA) receptors 10), and has cytoskeletal proteins that are susceptible to secondary injury post-TBI 11. Coupled with the ease of genetic manipulation and live imaging, and the ability to measure cognitive and behavioural outcomes, this organism has potential for modeling mammalian TBI and for evaluating TBIrelevant drugs. Ultrasound – a non-invasive technique that utilizes soundwaves – can be applied beyond clinical diagnostic and therapeutic purposes to induce TBI. High-intensity focused ultrasound (HIFU) is a minimally-invasive technique and can be used to emit targeted soundwaves to a tissue of interest 2. Pulsed HIFU (pHIFU) emits short pulses of HIFU waves, and can cause tissue damage equivalent to that caused by mechanical and/or stress forces 12. Past studies of zebrafish neurotrauma do not model closedhead TBI 13,14. Here, the study objective of McCutcheon et al. (2017) was to model closed-head TBI in the adult zebrafish using pHIFU and determine neurochemical and behavioral outcomes and drug response post-TBI. Biochemical and behavioural outcomes of injury were respectively identified using neuroprotective markers and behavioural tests, and response to pharmacological treatment was measured by injecting a neuroprotective drug and varying environmental temperatures post-TBI 2. To our knowledge, this study is the first that empirically evaluates the zebrafish for modeling closed-head mammalian TBI. MAJOR RESULTS Higher intensity and duration of pHIFU-induced injury required increased recovery time An image-guided 1-MHz pHIFU system was generated to emit high-pressure (1, 5, and 11MPa) spatially-focused soundwaves, which would cause damage on soft tissue and result in closed-head TBI (Fig. 1) 2. All experiments were performed on short-fin, wildtype zebrafish of both sexes aged 6-12 months 2. Recovery times of fish with pHIFU-induced injury increased when intensity and duration increased (data not shown here) 2.

Fig. 1: Setup and apparatus of the 1MHz image-guided pHIFU system and fish holder 2. Fish were temporarily anesthetized in 100ppm clove oil and positioned on its left side in the holder, which had minimal amounts of clove oil and water to maintain anesthesia 2. The holder was then positioned on the transducer such that the head was targeted for pHIFU emission from below 2. pHIFU amplitude and duration were set into the system’s function generator and activated to induce TBI 2. Control fish were also anesthetized and placed into the holder, but pFIHU was not delivered 2. Figure adapted from McCutcheon V et al. J Neurotrauma. 2017;34(7):1382-1393. pHIFU-Injured fish demonstrated less locomotor activity in novel tank test and more anxious behaviours in shoaling test Behavioural outcome measures included the novel tank test (NTT) and the shoaling test 2. In the NTT – a test of locomotor activity, stress, and anxiety – pHIFU-injured fish displayed trends of less locomotion as injury duration increased, with significance observed in overall distance travelled, velocity, meandering, and time spent freezing at various injury durations and hours post-TBI (Fig. 2) 2. In the shoaling test – which measures anxiety and social interaction – pHIFU-injured fish generally showed greater delays before engaging in vertical exploration, and displayed increased shoaling (grouping) behaviours (Fig. 3) 2.

Fig. 2: pHIFU-injured fish displayed less locomotor activity in the novel tank test compared to controls. The NTT is a 6 minute test of locomotion, stress, and anxiety in zebrafish 15, and was conducted with procedures based on previous studies (n=10 for each group) 16,17. Fish placed into a novel tank typically swim to the tank bottom and explore horizontally before gradually swimming upwards toward the surface 18. (E) Fish injured with 11MPa pHIFU travelled significantly less distances at 24 hours post-TBI 2. (F) Fish injured with 5 and 11MPa pHIFU moved with significantly lower velocities at 24 hours post-TBI 2. (G) Fish injured with 5 and 11MPa pHIFU meandered significantly more at 0 and 24 hours post-TBI, with significance maintained in the 11MPa-pHIFUinjured fish at 48 hours post-TBI 2. (H) Fish injured with 11MPa of pHIFU showed significantly more freezing behaviour at all timepoints, and fish injured

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with 5MPa of pHIFU showed significantly more freezing behaviour at 24 and 48 hours post-TBI 2. Figure adapted from McCutcheon V et al. J Neurotrauma. 2017;34(7):1382-1393.

Fig. 3: pHIFU-injured fish show more anxious behaviours during the 10 minute shoaling test 2. (A) Latency to enter the upper zone is a measure of anxiety 19. Fish injured with 11MPa pHIFU showed significant delay before vertical exploration (swimming towards the tank surface) at 0, 24, and 48 hours post-TBI. Fish injured at 5MPa pHIFU showed significant delay before entering the upper zone at 24, and 48 hours post-TBI 2. (B) All pHIFU-injured fish – regardless of injury duration – showed significantly less distance between subjects (more shoaling behaviour) at 0 hours post-TBI 2. This significance was maintained in more severely pHIFU-injured fish (5 and 11MPa) at 24 and 48 hours post-TBI. Fish were video-recorded, and software was used to analyze the average distance between fish (n=10 for each group; 5 fish per tank) 2. Figure adapted from McCutcheon V et al. J Neurotrauma. 2017;34(7):1382-1393.

Western blot analysis indicated increased expression of proteins Fig. 4: Western blot (A) gel and (B-D) quantification 20 of neurofilament 160 involved in axonal injury and apoptosis Levels of neurofilament 160 (NF-160), ß-III tubulin, activated caspase-3, and amyloid precursor protein (APP) were quantified to ascertain molecular characteristics of secondary injury post-TBI (Fig. 4) 2. NF-160 (an intermediate filament) and ß-III tubulin (a protein involved in microtubule formation) comprise neuronal cytoskeleton and axonal structure, which commonly become damaged post-TBI 21. Levels of these proteins showed significant increase at 12 hours post-TBI in 11MPa-pHIFU-injured fish (Fig. 4b & c) 2. APP is also a marker of post-TBI axonal damage 22. Caspase-3 is a cysteine protease that is activated by calcium ions and leads to apoptotic cell death, which has been observed post-TBI 23. Both APP and activated caspase-3 showed a trend towards significance from 6 to 12 hours post-TBI, with significant increase observed at 24 hours post-TBI (Fig. 4d & e) 2. Retro-orbital Injection of MK-801 at 1 hour pre-TBI improves behavioural outcomes post-TBI MK-801 is an NMDA receptor inhibitor that can prevent excitotoxicity, and has shown neuroprotective effects in TBI models 24. This drug was injected retro-orbitally, which directly targets the bloodstream while minimizing risk of injury to major organs and/or death 25. Injured fish showed dose-dependent improvement of behavioural outcomes on the NTT and shoaling tests with preinjury MK-801 injections, with higher drug dosage correlated to better behavioural outcomes (data not shown here) 2. Hypothermic recovery conditions improves survival chances and behavioural outcomes post-TBI All pHIFU-induced fish recovered from injury (and controls recovered from anesthesia) in tanks of 22°C (hypothermic) or 25 or 28°C (normothermic) to determine whether the therapeutic benefits of temperature intervention would be maintained in this model 2. Greater survival rates and locomotor activity, and fewer anxious behaviours, as measured by the NTT and shoaling test (data not shown here) were observed in fish that recovered at lower temperatures 2. All fish survived at hypothermic recovery conditions (Fig. 5) 2.

(NF-160), ß-III tubulin (ß-III tub), activated caspase-3, and APP – proteins involved in axonal injury and apoptotic cell death 21-23 – at 6, 12, and 24 hours post-TBI. Adult zebrafish whole brains were dissected and analyzed (n=4 per time observed) 2. (B) NF-160 and (C) ß-III tubulin are expressed at significantly increased levels 12 hours post-TBI compared to control, and (D) activated caspase-3 and (E) APP show trends of increased expression as hours post-TBI increased, with significantly more expression at 24 hours post-TBI compared to control 2. Figure adapted from McCutcheon V et al. J Neurotrauma. 2017;34 (7):1382-1393.

Fig. 5: Survival rates of 11MPa-pHIFU-injured fish are increased under hypothermic conditions compared to normothermic conditions 2. All injured fish survived when recovery occurred in hypothermic tanks (22°C), but injured fish that recovered in normothermic tanks (25 and 28°C) showed lower percentages of survival, with lower survival rates at higher normothermic temperature (n=10 per temperature group) 2. Figure adapted from McCutcheon V et al. J Neurotrauma. 2017;34(7):1382-1393.

DISCUSSION, CONCLUSIONS, & SIGNIFICANCE McCutcheon et al. (2017) demonstrated that the adult zebrafish is effective in modeling TBI, with injury characteristics and responses to hypothermic intervention and drug treatment similar to mammalian models. Specifically, closed-head injury was induced into fish using a pHIFU system, with increased injury severity – measured by ultrasound intensity and duration – correlated to increased recovery times required 2. Fish with greater severity of pHIFU-induced injuries displayed less locomotor activity on the novel tank test and more anxious swim behaviours on the shoaling test, as indicated by the reduced travel distance and increased latencies to zone 2. Biochemical analyses identified increased levels of NF-160 and ß-III tubulin at 12 hours post-TBI, and increased levels of activated caspase-3 and APP at 24 hours post-

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-TBI 2. These are all proteins that mark axonal injury and apoptotic moderate-severe TBI patients can likewise undergo acute recovery cell death, which are processes that occur post-TBI and have been and show behavioural deficits and biomarkers of injury post-TBI. It observed in mammalian models 21-23. may thus be useful to contemplate whether the quick recovery from injury was in part due to the regenerative capacity of the Additionally, it is known that moderate hypothermia can prozebrafish brain, which can start at 3 days post-injury 14. If regeneratect against various neurotrauma, including TBI, and has been suction had occurred, it may limit the observed injury severity and the cessfully utilized in clinic for TBI patients 26. This study validated applicability of the observed results to mTBI. the neuroprotective effect of hypothermia, where survival rates and behavioural outcomes improved in 11MPa-pHIFU-injured fish This study also only focused on acute recovery post-injury, and that recovered in hypothermic tanks compared to normothermic did not explore whether injury characteristics persisted and tanks 2. Similarly, a proof-of-concept test was conducted to evalu- whether the therapeutic benefits of hypothermic or drug intervenate the physiological response of the zebrafish TBI model to drugs tions could be maintained over months 2. Interestingly, despite relevant to TBI 2. Although MK-801 is not effective in the clinic, assessing behavioural and emotional outcomes, this study did not administration of this NMDA inhibitor is known to prevent exci- assess cognitive outcomes. Since cognitive deficits in processes like totoxicity 24. Here, a dose-dependent relationship between MK- attention, speed of processing, and memory are common post-TBI 801 and behavioural outcomes was observed, where increased MK and can persist past acute stages of recovery 4, it would be worth-801 dosage improved behavioural outcomes 2. while to examine whether the zebrafish TBI model experiences similar cognitive defects. The authors acknowledged that previous TBI models are sufficient for elucidating the cellular cascades and molecules involved in secondary injury, but also recognized that temporal and tech- FUTURE DIRECTIONS nical limitations exist in those models for high throughput assays Since impairments from mTBI typically take up to 3 months to of drugs 2. By characterizing the adult zebrafish in modeling mamrecuperate, with a small percentage of patients showing persisting malian TBI, findings from this study suggest a more efficient model concussion symptoms 1,4, it would be valuable to investigate the that can be used to further understand the cellular and molecular long-term pathophysiological, behavioural, and emotional effects mechanisms of TBI, and to test TBI-related drugs. of injury. In particular, future experiments can assess behavioural and emotional outcomes and quantify levels of the same biochemCRITICAL ANALYSIS ical markers at 2, 4, and 6 months post-injury via western blot to more clearly identify the window of time that is necessary for full This study was the first to date that characterized and validatacute recovery from mTBI impairments. Better delineating this ed the adult zebrafish as an effective model for closed-head TBI therapeutic time window would also provide basis for future studinduced with minimal human touch 2. Indeed, the model shows ies that aim to evaluate the efficacy of TBI-related drugs. I posit biochemical markers, behavioural outcomes, and treatment re2, that behavioural and emotional functions will decline up to 2 and sponses that are similar to those observed in mammalian models 4 months post-TBI, but gradually improve from 4 months post-TBI 21-23 . This high relevance to mammalian TBI indicates predictive and onwards. I also hypothesize that western blot results will show 2 validity , and offers potential use for delineating pathways behind increased expression of proteins involved in axonal injury and cell secondary injury and for drug testing. The adult zebrafish model death between 2 and 4 months post-TBI, with expression subsecan also overcome time and cost limitations that currently exist in quently reaching an asymptote without declining, given that these 2 drug assays using rodents . The fish is physically small, can be pathophysiological damages can be irreversible. If this did not occheaply stored, can breed quickly and more repeatedly than rocur, it may indicate that the expression of these proteins are only dents, and have behavioural measures that require minimal huacutely elevated (merely days, and not months, post injury) and man interference 2. Unlike rodents, tests and data can also be admay not be sufficient markers of long-term outcomes post-TBI. ministered and collected in batches as opposed to individually, Alternatively, the absence of my hypothesized results months post 2 which accelerates the process . -TBI may be due to the regenerative capacity of the zebrafish brain 14 However, McCutcheon et al. (2017) acknowledge that limita- , which would require further inquiry. tions exist in their study. One drawback is the small size of the fish It would also be interesting to explore cognitive functions posthead, which may result in artifacts that conceal the magnitude of TBI in fish, given that cognitive symptoms are a common occurprotein level changes, and undoubtedly prevents localized regionrence in mTBI patients. Emerging research has shown validity of an of-interest analysis of neuronal samples post-injury. Despite being object recognition test to assess discriminatory learning and visual able to induce closed-head injury, the team were unable to induce attention in zebrafish 27, a zebrafish version of the five-choice serilocalized injury due to the small size of the fish head and the limal reaction time task to assess speed of processing 28, and various ited capacity of current pHIFU wave technology 2. Furthermore, no tests to assess zebrafish learning and memory 29. Here, I hypothestandardizations of injury severity exist in animal TBI models, size that injured fish will experience cognitive defects including which renders judgment subjective compared to clinical severity worse visual attention, slower processing speeds, and deficits in scales used to diagnose TBI in humans 2. learning and memory. These tests would be ideal for examining Due to the non-specificity of injury and the imprecision of clini- how TBI affects cognition in zebrafish, and whether changes (if cal severity diagnosis, it would be worthwhile to interpret whether any) mirror those observed in mammalian models. If results show the behavioural deficits observed in pHIFU-induced fish were due that zebrafish do not experiences cognitive difficulties post-TBI, I to moderate-severe injury TBI instead of mild TBI. This study char- may question as to whether cognitive difficulties in the fish appear acterized TBI as mild by comparing post-injury behaviour, recov- with increasing injury severity. ery, and survival outcomes 2, and although acute recovery takes longer and survival chances are lower with greater TBI severity 1,

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Social Deficits in SHANK3 mutant mice caused by Anterior Cingulate Cortex Impairments Dhuvaraha Srikrishnaraj

Social deficits are cornerstone symptoms of Autism Spectrum Disorder (ASD). These include problems reciprocating interactions, inability to form relationships and impairments of non-verbal communication. Due to the complex nature of these symptoms, the mechanisms that underlie these characteristics remain mostly unknown. The current paper by Guo et al. (2019) investigated the structural and functional impairments associated with neurons of SHANK3 (an ASD associated gene) mutant mice in the anterior cingulate cortex (ACC) and its effects on social interaction. SHANK3 KO mice had reduced complexity in the pyramidal cells of the ACC, along with weaker synaptic transmission. Lowered activity was observed in the ACC during social interaction tasks of SHANK3 mutants and selective deletion of SHANK3 in the ACC was sufficient to induce social deficits. Optogenetic activation of the ACC, conditional knock-in of SHANK3 in ACC, and pharmacological enhancement of AMPA receptors in the ACC were all found to ameliorate the observed social deficits in SHANK3 mutant mice. These results suggest a causal relationship between impairments in ACC and social deficits observed in ASD. Since the treatment options for the core symptoms of ASD, such as social deficits, are lacking from a non-psychosocial angle, this study provides valuable insights into the molecular substrates involved in ASD that could be harnessed to develop potential pharmacological treatments targeting the ACC. Keywords: Autism spectrum disorder (ASD), SHANK3 KO (knock-out), Anterior cingulate cortex (ACC), social interaction deficits.

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c INTRODUCTION Autism spectrum disorder (ASD) is a neurodevelopmental disorder that can be characterized by deficits in social interaction, repetitive behaviors, emotional sensitivities, etc. Once diagnosed, the treatment options are mainly composed of psychosocial therapies, such as applied behavioural analysis, cognitive behavioural therapy and pivotal response treatment1. Pharmacological interventions have yet to be proven effective for treating the core symptoms, and are now only being used to treat associated symptoms such as anxiety, hyperactivity and insomnia1. Thus, more research on the underlying molecular mechanisms of ASD is required in order to develop pharmacological treatment options. From a molecular standpoint, mutations in synaptic proteins such as SHANK and NLGN3/4X have been found to be associated with autism2. SHANK3 is a crucial synaptic scaffolding protein that plays a role in excitatory synaptic function. In the post-synaptic neuron, it binds neuroligins and actin and influences cell growth, dendritic morphology and synapse function2. Studies have shown that SHANK mutant mice display autism-like characteristics, such as impaired social interactions and repetitive behaviours2 and examination of SHANK mutant synapses have also shown altered structure3. Brain imaging studies have found that individuals with ASD tend to have a reduced head size at the time of birth and then a sudden and abnormal brain growth in the following months, which was then followed by an abnormal slow growth 4. This has been observed in the frontal cortex, limbic system and cerebellum and these structures are known to be important in the development of abilities that seem to be impaired in those with ASD4.

SHANK3 mRNA and other neuronal growth proteins were found to be strongly co-localized in the pyramidal neurons of the ACC6. Given that SHANK3 affects the development of neurons, the effects of the deletion of the protein was examined on these specific neurons in the ACC. SHANK3 KO mice had reduced spine density and dendritic complexity compared to WT mice (Fig.1A, B)6. In line with previous studies indicating deficits in neuronal development due to SHANK3 deletion7, this shows the importance of SHANK3 in neuronal growth. Using the whole cell patch clamp recordings, it was also found that SHANK3 KO mice displayed a lowered frequency and amplitude of miniature ESPCs compared to wild type mice (Fig.1C)6. Western blot analysis showed that SHANK3 KO displayed lower levels of AMPAR subunit GluR1 compared to WT mice6. As AMPARs have been reported to facilitate LTP in pyramidal neurons of ACC8, the examination of LTP through paired training paradigm failed to produce LTP in SHANK KO mice6. Overall, it is evident that SHANK3 deletion weakens synaptic function in the pyramidal neurons of ACC. As expected from the impaired structural and functional development of SHANK3 deletion on the ACC, SHANK3 mutant mice required a larger stimulation to generate an action potential compared to WT mice6. In addition, with the use of real-time calcium dynamics observations, hypo-activity of the ACC during social contact was seen in SHANK3 KO mice6.

(A)

Since humans are a social species, the ability to interact with others and sustain relationships is crucial for survival. Pro(B) (C) cessing of social cues, emotions and critical thinking are important in survival and reproduction. Abnormalities in these can result in significant disruptions to daily life. The anterior cingulate cortex (ACC) in the frontal lobe is known to play a role in cognition as well as social behavior. Apps et al. report that the ACC is involved with various regions of social information processing and thus, is expected to play a role in social networking in the brain 5. To further the knowledge of the brain mechanisms involved in ASD, Guo et al. sought to identify the relationship between social deficit in SHANK3 Fig. 1. (A) Representative image of total spine density in WT mice vs. mutant mice and the ACC6. KO mice, (B) summary of total spine density between WT vs. KO mice By examining and manipulating the expression of SHANK3 in the ACC of SHANK3 mutant mice, the current study was able to establish a link between ACC dysfunction and social deficits associated with ASD. SHANK3 KO caused dendritic spine loss and impaired synaptic transmission in the ACC and selective SHANK3 KO in the ACC was sufficient to induce defects in social interaction and synaptic function that was comparable to the effects seen in global SHANK3 KO mice6. These deficits were alleviated by selective knock -in of SHANK3 in the ACC, as well as by optogenetic and pharmacological activation of the ACC6. This study provides concrete evidence for the role of the ACC in social interaction deficits experienced in ASD patients. With more research on the underlying mechanisms controlling these defects, tuning ACC activity can potentially be used for the treatment of social deficits caused by ASD.

(C) EPSC amplitude WT mice vs. SHANK3 KO mice. [Figure adapted from Guo, B., Chen, J., Chen, Q. et al. (2019). Anterior cingulate cortex dysfunction underlies social deficits in Shank3 mutant mice. Nat Neurosci (22):1223â&#x20AC;&#x201C;123.].

Social interaction deficits from selective SHANK3 KO in the ACC.

To establish a causal link between SHANK3 KO specifically in the ACC and social interaction deficits, virally mediated conditional knock-out (CKO) of SHANK3 was administered6. In line with this studyâ&#x20AC;&#x2122;s previous experiments, this resulted in structural and functional impairments highlighted above. To examine any social deficits in these mutant mice, the three-chamber test (to test response to social novelty) and the social interaction test (to test voluntary social interaction) were employed. It was found that CKO mice spent a significantly lower amount of time in cage with MAJOR RESULTS stranger mouse and with novel mouse in home cage (Fig.2)6. Changes in other behaviours, such as grooming, anxiety levels, etc. were SHANK3 deletion altered neuronal structure, impacted synaptic found to not be significant in comparison to WT mice6. This indifunction and reduced activity in pyramidal neurons of the ACC. cates that the knock-out of SHANK3 in only the ACC is sufficient to

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(B)

induce social deficits in mice.

(A)

(B) (C)

(D)

Fig. 2. (A) Summary of interaction time of KO and WT mice with novel mice (B) Social preference score of WT and CKO mice. [Figure adapted from Guo, B., Chen, J., Chen, Q. et al. (2019). Anterior cingulate cortex dysfunction underlies social deficits Fig.3. (A) Summary of optogenetic activation on interaction time with stranger mice of photo-activated vs. control (KO-EYFP) mice. (B)(C)(D) in Shank3 mutant mice. Nat Neurosci (22):1223â&#x20AC;&#x201C;123.]. Summary of optogenetic activation on anxiety using the plus maze test

Social deficits were rescued by optogenetic activation of ACC, of photo-activated vs. control mice. [Figure adapted from Guo, B., conditional knock-in of SHANK3 in ACC of mutant mice and phar- Chen, J., Chen, Q. et al. (2019). Anterior cingulate cortex dysfunction underlies social deficits in Shank3 mutant mice. Nat Neurosci (22):1223 macological enhancement of AMPAR receptors. â&#x20AC;&#x201C;123.].

When the activity of ACC was selectively enhanced optogenetically, SHANK3 KO mice displayed improvements in previously noted social interaction tasks (Fig.3A)6. They spent more time interacting with stranger and novel mouse in comparison to controls6. Anxiety-like behaviours were also found to diminish with this treatment (Fig.3B, C, D)6. The effects of optogenetic inhibition of the ACC was also examined in WT mice and it was found that this was sufficient to induce social impairments in WT mice6. Interestingly, optogenetic inhibition did not, however, increase anxietylike behaviours. This suggests that the ACC has bidirectional effects on social behaviours, but not on anxiety-like behaviours. Previous studies have shown that global re-expression of SHANK3 in mutant mice reversed structural and functional deficits of synapses as well as rescued behavioural abnormalities such as social deficits and repetitive behaviours9. Thus, Guo et al. examined whether a selective restoration of SHANK3 in only the ACC would result in the same amelioration. Selective knock-in of SHANK3 resulted in increased spine density and increased expression of AMPAR subunit GluR1 that was lowered in SHANK3 KO mice6. Behavioural tests showed a rescuing of the social impairments that were observed previously6. As AMPAR function was significantly reduced in SHANK3 KO mice in the ACC, the study sought to see the effects of the enhancement of AMPAR activity on social deficits. CX546, a positive allosteric modular of AMPAR, resulted in an increased amplitude and duration of AMPA current activity6. When different dosages of CX546 were administered subcutaneously in SHANK3 mutant mice, it was found that higher concentrations of the molecule improved social interaction6. Injecting only the ACC with CX546 also resulted in improved social deficits previously observed in the three-chamber and social interaction tests6.

(A)

Overall, these findings provide concrete causal evidence for social deficits in the ASD mouse model caused by impairments in the ACC and these new findings coupled with further research in this area could be harnessed for potential pharmacological treatments for ASD in humans. CONCLUSIONS/DISCUSSION Guo et al. demonstrate that loss of SHANK3 in the pyramidal neurons of the ACC is sufficient to cause neuronal structural and functional impairments such as reduced density of spines and dysfunction of AMPAR6. This loss was also found to cause the social impairments that are representative of SHANK3 mutant mice in the ASD mouse model. These deficits were rescued by enhancement of ACC activity in SHANK3 KO as well as conditional knock-in of SHANK3 in the ACC in global SHANK3 mutant mice6. Pharmacological enhancement of AMPAR was also sufficient to ameliorate these deficits6. The authors conclude that these findings outline a direct causal relationship between ACC function and social deficits in the SHANK3 KO mouse model. This evidence supports and enhances the current knowledge on neurobiological aspects involved in the impairments caused by ASD, and can be used to develop therapeutic techniques from a molecular perspective. Previous studies have reported structural abnormalities in the ACC neurons of ASD individuals. For instance, post-mortem studies of autism brains have shown decreased cell density and size in limbic regions such as the ACC10. However, clear evidence for why this occurs has been lacking. This study expands on this by demonstrating that a potential cause of these abnormalities is the lack of the functional SHANK3 protein in the ACC6. Rudebeck et al. found impairments in social functioning of rats with ACC lesions 11. Rats with lesions in the ACC showed decreased interest in other rats and memory of social stimuli and these rats maintained a high level of responsiveness to repeated exposure to the rats, unlike the control rats11. Although this illustrates that ACC impairments are associated with social dysfunction, the relationship between this dysfunction and ASD has not been explored in the past. The current study provides evidence for this relationship by demonstrating that specific deletion of an autism associated gene, SHANK3, in the

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ACC caused impairments in this brain region which in turn caused social deficits characteristic of ASD individuals6. In addition, Guo et al. provide evidence for the amelioration of these deficits by reversing the deletion of SHANK3, activating the ACC or enhancing the activity of receptors involved in excitatory signalling6. As evidence for reversing such social deficits has not been explored in the past, this study’s novel findings are extremely valuable for the progress in the field of ASD research. Overall, given the lack of knowledge surrounding the neuronal basis of ASD, these findings pave the way for more studies to elucidate the exact molecular substrates in the ACC that are affected by SHANK3 mutations. As mentioned previously, much of the treatment options available for those with ASD involve psychosocial therapies. However, with studies such as this, the development of new treatments for managing and treating this disorder can be tackled from a molecular standpoint. CRITICAL ANALYSIS The study puts forth evidence for a causal relationship between SHANK3 KO in the ACC and structural and social impairments observed in ASD. However, it is important to consider that the conditional knock-out and knock-in of SHANK3 in the ACC was done in adulthood, and not in germline. This fails to consider the important effects of neuronal mechanisms that would usually work in a germline SHANK3 KO to compensate for this loss throughout development. Adult KO and KI is not sufficient to observe the true effects of SHANK3 in an ASD model as ASD’s consequences arise from a very young age. As the brain is highly plastic, insufficient connections could potentially be replaced and adjusted for in this situation. In an adult CKO/CKI, such compensation will not likely be observed, and thus, may not be truly reflective of ASD changes. Guo et al. show that SHANK3 deletion causes structural and functional abnormalities in the ACC. Spine density and dendritic maturity were found to be reduced in SHANK3 KO mice compared to WT mice and the activity of pyramidal neurons in the ACC were also found to be reduced. The molecular substances and exact mechanisms through which this happens is, however, still remain unanswered. The specifics of the causes of SHANK3 deletion or mutations, and the downstream pathways that alter neuronal development is worth exploring. The authors also found that optogenetic activation of the ACC has anxiolytic effects in addition to improving social interaction in the three chamber and social interaction tests. This suggests a potential shared mechanism between two, which is an interesting avenue for more research. The ACC’s potential role in anxiety has been recorded in the past. Etkin et al. have demonstrated that patients with generalized anxiety disorder failed to engage the pregenual anterior cingulate and had reduced amygdala activity compared to normal individuals12. Kang et al. also found that inhibition of the ACC optogenetically did not affect anxiety levels in nociception models13. All in all, the relationship between the ACC and anxiety, as it is also a symptom of ASD, should be investigated. The tests that were administered to evaluate the social defects in the experimental mice was the three-chamber test and the social interaction tests. These are excellent tests for evaluating social behaviour and social memory. In addition to these, perhaps other tests such as the partition test, or the social transmission of food preference test (STFP) could be tested, as this would provide a more thorough characterization of the social deficits observed in models with ASD14. Finally, the current study examined the ACC as

a whole. But perhaps it may be worth considering the differences between the different sections/neurons of the ACC, such as the pregenual or sub-genual regions. For instance, von economo neurons in the ACC have been found to receive more input and connections than pyramidal neurons as well as play an adaptive role in social situations15. More research focusing such specific areas and neurons, perhaps in different animal models as well, could narrow down the exact substrates involved in ASD’s cause and symptoms. These present findings by Guo et al. are novel, with no evident contractions to existing literature. Overall, this study puts forth a basis for future studies in the field of ASD. FUTURE DIRECTIONS Given the current evidence for the relationship between the ACC and social impairments in the SHANK3 KO mouse model, the next step is to elucidate the specific mechanisms that underlie these changes. Understanding the specific proteins and potential molecular cascades involved in these would aid in streamlining potential drug targets to reverse these symptoms. For instance, given that SHANK3 is a scaffolding protein, perhaps a protein or molecule downstream of it may be important in downstream gene transcription that allows for neuronal development. Identifying such downstream proteins, and selectively inhibiting them would allow for the elucidation of their specific function or need in specific pathways. If such molecules/proteins are specified and interfered with, lack of neuronal function/growth would mean that SHANK3 deletion impacts the functioning of this protein, and restoring that protein may be important in rescuing effects caused by SHANK3 deletion. As mentioned in the previous section, the study failed to take into consideration compensatory events that would result from germline knock-out or knock-in of SHANK3 in the ACC by deleting SHANK3 only in adulthood. To remedy this, the authors should aim to selectively knock-out and knock-in SHANK3 in germline and then examine its effects on social behaviours. This would provide a better understanding of how ASD symptoms present from a realistic standpoint. Furthermore, different domains of the SHANK3 protein could be involved with different roles, such as the SH3 or Ankyrin domains. For instance, specific parts of SHANK3 have been found to connect the glutamate receptors to actin filaments16. The structural deficits that result from SHANK3 depletion can be attributed to the defect in this connection that prevents further neuronal development through actin polymerization. Understanding the role of the different regions of SHANK3 play in this could also be useful in developing targeted therapies. For example, CRISPR-CAS9 gene editing can be used to induce mutations in specific gene regions of the SHANK3 protein, and then observe the effects that result from that. The Ankyrin repeat domains have been found to be involved in protein to protein interactions 17. It can be expected that mutations in this region can affect actin polymerization or growth-cone motility that is crucial in neuronal development. If specific mutations in the different SHANK3 regions do not induce the same results observed in the current study, it may suggest that the entirety of SHANK3 is needed for proper neuronal development. Finally, as mentioned in the above section, different areas of the ACC could be examined separately, by selectively manipulating the expression of SHANK3 in those different areas. This would allow the authors to specify what region is mainly responsible for these changes, or if indeed they all work together.

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DeFilippis, M., & Wagner, K. D. (2016). Treatment of Autism Spectrum Disorder in Children and Adolescents. Psychopharmacology bulletin, 46(2), 18–41.

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Monteiro P., Feng G. (2017). SHANK proteins: roles at the synapse and in autism spectrum disorder. Nat Rev Neurosci. 18(3):147157.

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Amit Etkin, Katherine E. Prater, Fumiko Hoeft, Vinod Menon, and Alan F. Schatzberg. (2010). Failure of Anterior Cingulate Activation and Connectivity With the Amygdala During Implicit Regulation of Emotional Processing in Generalized Anxiety Disorder. American Journal of Psychiatry. 167:5, 545-554.

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Durand, C., Perroy, J., Loll, F. et al. (2012). SHANK3 mutations identified in autism lead to modification of dendritic spine morphology via an actin-dependent mechanism. Mol Psychiatry. 17: 71–84.

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Deciphering the role of the gut microbiome in the complementary relationship between stress and the development of depressive-type behaviors Mathusan Sritharan

Pearson-Leary et al. (2019) demonstrated the relationship between stress and the development of depressive-type behaviors in Sprague-Dawley rats. Depression is classified as a psychiatric disorder, with the causes and development of the illness undergoing research. However, vulnerability to stress may predispose one to develop depression, which is mediated by the gut microbiome. The gut microbiome is known to impact the brain plasticity through secretion of molecules that cause inflammation in the brain, as well as inducing the pro-inflammatory phenotype of microglia, which may thereby lead to depressive type behaviors. What remains to be elucidated involves the physiological mechanisms that underlie the ability of stress to mediate changes within the gut microbiome. Researches sought to determine the impact of the gut microbiome towards the development of depression by transplanting the gut microbiome of stress-vulnerable and stress-resilient rats into naive rats, respectively. It was determined that gut microbiome transplants from stress-vulnerable rats alone were enough to induce depressive-type behaviors in naive rats that have not been exposed to environmental stressors. The gut microbiota in stress-vulnerable rats were found to have an increased composition of Bacilli and Clostridia, which produce high concentrations of the pro-inflammatory cytokine, IL-1Î˛. This resulted in inflammation of the ventral hippocampus, increasing neural connections to stress-related regions of the brain such as the amygdala. Through this study, it was found that the gut microbiota impacts the vulnerability to stress, which invokes pathways that lead to the development of depressive-type behaviors through inflammation within the ventral hippocampus. This review seeks to shed light on proposed mechanisms towards the development of depression, as well as treatment options that may be designed which focuses on the intertwining relationship between stress, depression, and the gut microbiome. Key words: Cytokines, Ventral Hippocampus, Inflammation, Gut Microbiome, Stress, Dysbiosis

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BACKGROUND and INTRODUCTION Depression is described as a psychiatric disorder, associated with a large prevalence across the world, leading to high rates of mortality (Palazidou, 2012). A multitude of research experiments has been invested in discovering the underlying causes of depression, in hopes that effective treatment options would be designed (Monroe et al., 2009). Despite the vast amount of information related to depression being uncovered, the mechanisms surrounding the development of the disorder remain unclear (Bartolomucci et al., 2009). The development of effective treatment options remains elusive due to the lack of knowledge regarding the pathology of depression. Analyzing possible correlations with the development of depression results in a further understanding of the disorder, which allows for the determination of causational factors. Chronic stress is a factor that has been determined to be a link in the neurobiological basis and development of depression, as it also plays a key role in the development of other psychiatric disorders, such as anxiety and post-traumatic stress disorder (Davis et al., 2018). The concept of stress vulnerability has been elucidated in SpragueDawley rat models, where certain rats were shown to be resistant in developing stress-related mood disorders compared to other groups after exposure to chronic stress (Jankord et al., 2011). The mechanisms revolving around stress vulnerability are ambiguous however, the development of depression has been displayed in stress-vulnerable rats (Jankord et al., 2011). The vulnerability to stress and the development of stress-related mood disorders has been proposed to be the result of the increased production of inflammatory cytokines within the body, facilitating changes in neural structures (Raison et al., 2011). Individuals clinically diagnosed with depression have been found to have increased inflammatory cytokines within serum levels, with significantly higher levels of IL-1Î˛ and TNF-Îą compared to control groups (Zou et al., 2018). The neurobiological basis of inflammatory cytokines affecting stress vulnerability and the development of depression involves changes within the ventral hippocampus, inducing remodeling of the structure (Pearson-Leary et al., 2017). Remodeling within the ventral hippocampus caused by inflammatory cytokines results in increased neural connections to stress-related regions of the brain, such as the amygdala (Pearson-Leary et al., 2017). An elevated microglial density within the brain, as well as increased permeability to inflammatory cytokines in the blood-brain-barrier, affects the function and structure of the brain (Shigemoto-Mogami et al., 2018). The cause of the increased presence of inflammatory cytokines within the brain and serum in certain cases of depression has yet to be resolved. The presence of increased concentrations of cytokines affecting the vulnerability to stress, and consequently the development of depressive-type behaviors may be facilitated through the gut microbiome (Peirce et al., 2019). Dysbiosis within the bacteria making up the gut microbiome may result in the production of proinflammatory cytokines, ultimately affecting the brain (Schirmer et al., 2016). There is an observed difference between the gut microbiome of mice exhibiting depressive-type behaviors and the control group of mice, where different species of bacteria thrive and have a higher population depending on the group (Xie, 2017). The profound inter-communication between the gut and the brain is mediated through the vagus nerve, where both systems impact each other through this connection (Cheung et al., 2019). The

study conducted by Pearson-Leary et al. (2019) dwells upon whether the relationship between the gut microbiome and the development of psychiatric disorders such as depression is simply correlational. Pearson-Leary et al. (2019) studied the effects of the gut microbiome on the vulnerability to stress and the development of depressive-type behaviors. To determine whether the gut microbiome affects the development of depression, Pearson-Leary et al. proposed that stress vulnerability, characterized by the inflammation in the ventral hippocampus, is triggered by the effects of gut microbiome dysbiosis. The experiment was carried out by separating Sprague-Dawley rats into a control group without exposure to any stressors, and a group of rats undergoing stress to obtain a social defeat model. The rats that have undergone social defeat were separated into stress-vulnerable and stress-resilient groups. The stress-vulnerable phenotype was determined based on gut microbiome characterization using shotgun sequencing. Fecal transplantation of the gut microbiota from each group of rats was done on the control group of rats to screen for any changes in behavior and brain structures. The fecal transplant from the stress-vulnerable rats resulted in increased IL-1Î˛ concentrations within the serum, causing an inflamed ventral hippocampus. The presence of Clostridia dominated the gut microbiome, with depressive type behaviors being observed in the rats. Fecal transplantation from the stress-resilient rats resulted in no significant changes being observed, with no depressive-type behaviors being exhibited. The causes of the inflammation in the ventral hippocampus, as well as increased pro-inflammatory cytokine levels, remains controversial. The study conducted by Pearson-Leary et al. (2019) provides insight regarding the contribution of the gut microbiome to some of the major phenotypes observed in model organisms exhibiting depressive-type behaviors. It was hypothesized that the gut microbiota impacts vulnerability to stress, and therefore susceptibility to depression through changes in the ventral hypothalamus caused by inflammation (Pearson-Leary et al., 2019). There is increasing evidence suggesting that the gut microbiome plays a significant role in the development of psychiatric disorders such as depression. With increasing emphasis placed on dominant gut bacteria during dysbiosis and their effects on the brain structure, this can ultimately allow for the elucidation of the development of depressive-type behaviors (Pearson-Leary et al., 2019). MAJOR RESULTS Stress Vulnerability is Associated with Profound Differences in the Gut Microbiota of Rats Compared to Stress Resilient and NonStressed Groups In order to study the relationship between stress and the development of depressive-type behaviors, Pearson-Leary et al. (2019) examined the differences in the gut microbiota of rats that are stress-vulnerable, stress-resilient, and non-stressed control groups. The rats were exposed to stressors prior to distinguish between stress-vulnerable and stress-resilient rats. The mean latency to social defeat was observed to be significantly lower in stress-vulnerable rats, however, gut microbiota analysis was required to confirm the stress-vulnerable phenotype to control for any confounding factors. Following the social defeat of the two

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groups after 7 days, the alpha diversity observed within the stressvulnerable group was higher relative to the start of the study. The alpha diversity describes the mean species diversity along the intestinal tract within a specific group of rats. The alpha diversity within stress-resilient and control group rats did not display any significant changes within the 7-day span. The relevance of this finding suggests that increased alpha diversity within the gut microbiome may be associated with a greater vulnerability to stress. The induced proliferation of certain other species, otherwise found in low concentrations in control groups, may have contributed to the increase in diversity in response to stress. In comparison to a study conducted by Zhang et al., (2019), the alpha diversity within stress-vulnerable rats were observed to have decreased alpha diversity compared to the stress-resilient rats and control group. The mechanisms by which stress vulnerability associates with the alpha diversity in the gut microbiome are convoluted, however, there are distinct differences in the gut microbiota between stressvulnerable and stress-resilient rat groups.

technique on the various types of fecal matter from the different rat groups. Stress-vulnerable rats were observed to have significantly increased Bacilli and Clostridia, as well as decreased Bacteroidia within the gut microbiome. Consequently, stress-resilient rats were observed to have slightly increased Bacilli and Actinobacteria, but class differences were not significantly different from non-stressed control group rats. A study conducted on mice exposed to social stressors also displayed an increased abundance in Clostridia within the gut, although there were not any significant changes noted in the bacilli population (Bailey et al., 2011). Regarding the class-level changes within the gut microbiome, there was an observed reduction in Bacteroidetes Bacteroides and an increase in Firmicutes Lactobacillus in both stress-vulnerable and stress-resilient rat groups. The Firmicutes to Bacteroidetes ratio was also observed to be increased relative to the non-stressed control group. However, there was also an increase in the Bacilli to Clostridia ratio within the stress-resilient group. These findings suggest that exposure to stress can alter the gut microbial composition, however, the presence of certain types of bacteria may be an underlying factor in the stress-resilient and stress-vulnerable phenotype of the rats. Fecal Transplantation from the Stress-Vulnerable Group onto the Non-Stressed Control Group Results in Depressive Type Behaviours

Figure 1. Latency to social defeat by the stress-vulnerable (SL) and stress resilient (LL) rats depicted on the left. Rats were subject to stress for a period of 7 days, resulting in stress-resilient rats having a significantly higher latency to social defeat. Depicted on the right is a graph that displays the alpha diversity richness of the control, stress-resilient, and stress-vulnerable rat groups. Following exposure to stress, the SL and LL groups experienced increases in alpha diversity within the gut microbiome. Figures adapted from Pearson -Leary et al. (2019). The beta diversity within the gut microbiome was also analyzed to determine the differences between the microbiome communities among different groups (Pearson-Leary et al., 2019). Beta diversity refers to changes in species composition among different rat groups. On day 0, there were no differences in the beta diversity amongst the stress-vulnerable, stress-resilient, and control group rats. Following the 7-day timeframe of the study, the beta diversity in the stress-vulnerable and stress-resilient rats was found to have significantly shifted compared to the control group. This finding is consistent with the results of a study where an increase in beta diversity was observed amongst mice exposed to various types of stressors (Rocca et al., 2019). The significance of these findings implies that pre-existing differences among the rat groups are nonexistent before the exposure to stress. Stress as an environmental factor contributes a significant role in the development of the stress-vulnerable and stress-resilient phenotype. Social defeat is found to increase the beta diversity of the gut microbiome, regardless of the vulnerability to stress. Although a change in diversity was observed in the gut microbiome, the composition of the bacterial communities was analyzed to determine the implications of these findings.

The transplantation of the gut microbiome from the stressvulnerable rat group to the non-stressed rat group was administered through an oral gavage technique that contained fecal matter from the stress-vulnerable rats (Pearson-Leary et al., 2019). The same procedure was executed twice, with fecal matter originating from stress-resistant and naive groups transplanted to the non-stressed group of rats, which were subsequently screened for any deviations from the observed microbial communities recorded from the first half of the study. Over a 9-day span, naive rats that received gut microbiota from stress-vulnerable and stress-resilient rats had resulted in profound changes within the gut microbiome. The gut microbiome of the naive rats had started to resemble the gut microbiome of stress-vulnerable and stress-resilient rats, respectively, depending on which group the transplant was received from.

Following fecal transplantation, the naive rats were subjected to a series of tests to assess for depressive-type behaviors. Changes in body weight, as well as social interaction between other rats, were not affected. A forced swim test was performed, resulting in the naive rats with fecal transplantations originating from the stressvulnerable rats displaying a significantly lower latency to immobility compared to other groups. Naive rats treated with microbiota from the stress-vulnerable group also displayed significantly higher rates of total immobility, as well as significantly lower durations of time spent swimming compared to other groups of rats with transplanted gut microbiota. No differences between the time spent climbing were observed. A similar study focused on the contributions of stress towards gut microbiome dysbiosis, where mice exposed stressors exhibited higher percentages of immobility while undergoing a forced swim test (McGaughey et al., 2019). The relevance in these findings revolves around the gut microbiota. which plays a critical role in stress-vulnerability and the development of Pearson-Leary et al. (2019) analyzed the bacterial composition of depressive type behaviors. the gut microbiome, utilizing the shotgun metagenome sequencing

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Figure 2. Following fecal transplant, the naïve rats were subjected to exercises to test for depressive-type behaviors. Total immobility was observed in rats that received fecal transplants from stressvulnerable (SL) rats as depicted in graph D. A forced swim test was also conducted, which revealed that the percentage of time spent swimming in naïve rats that received fecal transplants from the SL rat group was significantly lower than that of the rats that received fecal transplants from the LL group. Figures adapted from PearsonLeary et al. (2019). Pearson-Leary et al. (2019) observed pro-inflammatory phenotypes within the ventral hippocampus of naive rats that received the gut microbiome transplantation from stress-vulnerable rats. Within this brain region, the expression of pro-inflammatory cytokine IL-1β was significantly increased in this group. The antiinflammatory cytokine IL-10 was also observed to be increased in both the rats that received fecal transplants from stress-vulnerable and stress-resilient rat groups. The blood-brain barrier was found to have increased permeability within rats with fecal transplants from the stress-vulnerable group, which was derived by utilizing the S100β permeability marker. These findings are consistent with a study analyzing the cytokine levels within individuals suffering from a depressive disorder, where increased levels of proinflammatory cytokines were observed, including IL-1β (Thomas et al., 2005). The major mechanisms by which the gut microbiota plays a role in the development of psychiatric disorders are through the production of cytokines that have various effects on the brain, including the increased permeability of the blood-brain barrier

Figure 3. The concentration of IL-1β was measured in naïve rats that received fecal transplants from stress-resilient (LL) and stressvulnerable (SL) rat groups (top graph). Naïve rats that received fecal transplants from SL rats had significantly higher concentrations of pro-inflammatory cytokine IL-1β in the ventral hippocampus, leading to inflammation. The bottom graph depicts the concentrations of IL-10 in the ventral hippocampus of antiinflammatory cytokine IL-10. Concentrations of IL-10 were found to be significantly higher in naïve rats that received fecal transplants from both SL and LL rat groups. Figures adapted from Pearson-Leary et al. (2019). CONCLUSIONS/DISCUSSION With the culmination of the study, Pearson-Leary et al. (2019) deduced the unequivocal role of the gut microbiota in the development of psychiatric disorders, namely depression. Dysbiosis in the gut microbiota of stress-vulnerable rats resulted in the induction of proinflammatory cytokines, which affects the brain by causing inflammation in regions such as the ventral hippocampus. The authors of this study concluded that the gut microbiome provides significant contributions to inflammatory processes observed in rats with depressive-type behaviors, further strengthening the relationship between stress and the development of depression. Chronic stress allows for the proliferation of bacteria that produce proinflammatory cytokines, which in turn led to depressive-type behaviors. This study provides an insight into the neurobiological mechanisms that underlie a form of depression, which can then be used as targets for the development of effective treatment options. A novel finding within the study revealed that the gut microbiome of stress-vulnerable and stress-resistant rats were identical before social defeat (Pearson-Leary et al., 2019). This suggests that vulnerability to stress may not be largely determined by genetics and that exposure to stress provides changes in physiological processes that allow for different microbial communities to thrive. Stress has been determined to result in structural changes to the brain which affect pathways involved in neuronal growth and proliferation, but the mechanisms behind these effects are still under research (Ross et al., 2017). The changes observed in the brain due to chronic stress may originate from dysbiosis within the gut microbiota and are mediated by proinflammatory cytokines, as was found in the study by Pearson-Leary et al. (2019). Rats that become stressvulnerable or stress-resilient are due to changes within the gut microbiome. The physiological processes that led to the stressvulnerable phenotype are unclear, however, this finding provides further insight as to how stress can induce depression. Rather than focusing on the phenotypes displayed in depression, this finding emphasizes the importance of the mechanisms underlying the development of this disorder, where the physiological processes that led to stress-vulnerability can provide further information towards this field of research.

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The highlight of this study that provided substantial support towards the gut microbiomeâ&#x20AC;&#x2122;s role in the development of depressive type behaviors involves the gut microbiome transplantation (Pearson-Leary et al., 2019). Transplantation of the gut microbiome from stress-vulnerable rats to the control group rats was enough to induce depressive type behaviors, without any external factors, such as exposure to stress at play. A similar study also arrived at the same conclusion, where the gut microbiome from patients suffering from major depressive disorder was transplanted onto healthy rats, which resulted in observed depressive-type behaviors (Kelly et al., 2016). Further research was required for this study to delve upon the mechanisms that produce depressive-type behaviors. Pearson-Leary et al. concluded that the microbiome of stress-vulnerable rats had higher compositions of Clostridia and Actinobacteria, which results in elevated production of inflammatory cytokine IL-1Î˛. It was interpreted that this resulted in the induction of the pro-inflammatory phenotype of microglia in the brain, leading to inflammation of the ventral hippocampus. This study elucidated a possible mechanism by which the gut microbiome causes depression through exposure to stress. With a possibility that this pathway towards the development of depression garners further support through future studies, methods to reverse depressive-type behaviors may be possible. Unlike other studies, Pearson-Leary et al. analyzed the effects of pro-inflammatory cytokines on brain regions such as the ventral hippocampus to explain the triggered depressive-type behaviors. They have formulated a possible pathway towards the development of depression, originating from the gut microbiome. A lot of studies involving the contribution of stress to the development of depression specifically involves observing changes in the phenotype of the brain, which has led to controversial debates surrounding the nature of this disorder (Richter-Levin et al., 2018). Not only does this study elucidate the mechanisms behind the development of depression, but it also provides insight into the relationship between the gut microbiome and the development of other psychiatric disorders such as anxiety. Pearson-Leary et al. demonstrated a clear link between the gut microbiome and the pathology of depression. CRITICAL ANALYSIS An element of this study that did not fit in line with previous studies involves the increased alpha diversity of the gut microbiome in the stress-vulnerable rats (Pearson-Leary et al., 2019). Several studies display a decrease in the alpha diversity following exposure to stress, through to the development of depression (Zhang et al., 2019; Winter et al., 2018). Although Pearson-Leary et al. concluded that stress-vulnerability is associated with increased alpha diversity, they also determined that stress increases the susceptibility towards developing depressive-type behaviors. Improved mental health is thought to be associated with increased gut microbiome diversity, which decreases the probability of developing psychiatric disorders such as depression (Bruce-Keller et al., 2018). It is possible that within the study by Pearson-Leary et al., exposure to stress may have led to the induction of physiological processes that allowed for more bacterial species to thrive, thereby increasing the alpha diversity. The thriving bacteria consequently would produce inflammatory cytokines that led to depressive-type behaviors, as determined by the analysis of the gut microbiome of stress-vulnerable rats (Pearson-Leary et al., 2019). This result challenges the idea that increased gut microbiome diversity may be beneficial towards mental health, however, the

physiological mechanisms that underlie the proliferation of certain bacterial species under stress remains to be elucidated. It is known that stress alters the gut microbiome composition, but how it does so require further studies to provide possible explanations. The authors of this study should determine how stress affects the communication between the brain and gut microbiome, mediated through the vagus nerve. This would allow for a possible explanation towards the increased alpha diversity observed in the stressvulnerable rats, also elucidating the development of this phenotype. Pearson-Leary et al. (2019) observed increased beta diversity and differences in microbiome compositions within stress-resilient rats. These were the proposed factors that led this phenotype, however, the physiological mechanisms that allow for stress-resilience were not elucidated. The cytokine IL-10 may contribute to antiinflammation within the ventral hypothalamus, but elevated levels were found in both stress-resilient and stress-vulnerable rats. It was proposed that the gut microbiome of stress-resilient rats produced more anti-inflammatory cytokines and little proinflammatory cytokines, which resulted in the phenotype. The conclusion derived revolves around the gut microbiome largely determining the stress-vulnerability of rats. However, a study conducted by Febbraro et al., (2017) determined that neuronal substrates such as c-fos are increased during the stress response, affecting certain brain regions and leads to increased stressvulnerability. To determine the magnitude of the gut microbiome on stress-vulnerability/resilience, Pearson-Leary et al. should study the effects of stress-resilient microbiota on stress-vulnerable rats. Future studies can also determine whether the ventral hippocampus is necessary for the gut microbiome to produce depressivetype behaviors and whether other brain regions should be analyzed. When the gut microbiome of stress-vulnerable and stress-resistant rats were transplanted to control group rats, both groups were observed to have increased concentrations of the antiinflammatory cytokine IL-10 (Pearson-Leary et al., 2019). This effect is a plausible outcome for stress-resilient rats, as it may help prevent inflammation induced by pro-inflammatory cytokines through stress. However, in a study conducted by Labaka et al. (2017), they observed significantly reduced Il-10 levels within the hippocampus of female mice expressing depressive-type behaviors, subjected to chronic stress. This finding contrasts the resulted received by Pearson-Leary et al., where the gut microbiome of stress vulnerable rats produces excess IL-10. Even with increased IL -10 levels, the rats that received gut microbiome transplants from the stress-vulnerable group developed depressive-type behaviors. A study conducted by Mesquita et al. (2008) determined that IL-10 modulates depressive type behavior, where they observed decreased depressive-type behaviors in mice that overexpressed this cytokine. In the study by Pearson-Leary et al., it is possible that IL10 is produced to reduce the amount of inflammation produced by the excess production of pro-inflammatory cytokines. Regardless, the levels of IL-10 were not sufficient to negate depressive-type behaviors in mice that received the gut microbiome from stressvulnerable rats. Rats subjected to stress have had their gut microbiome analyzed 7 days after, which garners the possibility that more time is required for the full effects of stress on a model organism to be elucidated. Further study is required to explore the induction of IL-10 through stress, and how it may lead to the reduction of depressive-type behaviors. The authors should also

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determine the effects of other factors that affect vulnerability to -Leary et al. (2019). It is predicted this microbiome would ultimatestress, and the development of depressive-type behaviors, such as ly allow for resilience to stress and the development of depressiveage and sex. type behaviors. The reverse experiment should also be conducted, where fecal transplants of stress-vulnerable rats are transferred to FUTURE DIRECTIONS stress-resilient rats. If the transplantation of the gut microbiome A possible continuation of the study conducted by Pearson-Leary from the stress-resilient rats to stress-vulnerable rats results in the et al. (2019) involves the physiological mechanisms surrounding retention of the stress vulnerable phenotype, it may be possible the proliferation of certain bacterial species as an organism is ex- that gut microbiota produces lasting changes to the brain. The periencing stress. An experiment that may elucidate this phenom- inflammation may be irreversible through the effects of gut microenon revolves around focusing on the vagus nerve of Sprague- biome transplant due to the presence of pro-inflammatory microDawley rats. The vagus nerve serves as a method of communica- glia. This study would further elucidate the contribution of the gut tion between the gut and brain, where inflammatory cytokines microbiota to the development of depression, as well as the develmay translocate into brain regions to induce psychiatric disorders opment of treatment options where gut microbiota transplants (Bonaz et al., 2018). To determine whether the brain mediates can allow for the elimination of depressive-type behaviors. changes to the gut during stress responses, the vagus nerve may be severed to prevent any molecules from reaching the gut micro- IL-10 is an anti-inflammatory cytokine that was observed to inbiome. Pearson-Leary et al. (2019) found that the gut microbiome crease due to stress, as observed in stress-vulnerable and stressof stress-vulnerable and stress-resilient rats were identical before resilient rats (Pearson-Leary et al. 2019). Although IL-10 levels exposure to stress. Various factors may contribute to the changes were increased in both groups, stress-vulnerable rats still exhibited in gut microbiota as an organism experiences stress. It is hypothe- inflammation in the ventral hippocampus as well as depressivesized that the brain may release hormones or chemicals that allow type behaviors. A study should be conducted to determine the for the proliferation of bacteria that produce pro-inflammatory effects of IL-10 in both phenotypes of the rats to allow for an excytokines in stress-vulnerable rats. By severing the vagus nerve, a planation of this observation. Higher levels of IL-10 should be inpossible result involves no change to the gut microbiota, due to jected into the ventral hippocampus of stress-vulnerable rats. It is the communication between the brain and gut being lost. Thus, a predicted that due to the anti-inflammatory effects of IL-10, the stress-vulnerable rat would most likely not develop any depressive inflammation within the ventral hippocampus should decrease. -type behaviors if the brain underlies the physiological changes to This would indicate that although stress-vulnerable rats have inthe gut microbiome. To determine the chemicals involved that the creased levels of IL-10, it does not exceed the threshold by which it brain releases under stress, the blood that flows within the vagus would mediate anti-inflammation of the ventral hippocampus. nerve may be sampled, which may help elucidate what causes Stress-vulnerable rats have increased permeability in their bloodchanges in the gut microbiome, This may include hormones that brain barrier, thus IL-10 may be injected into the bloodstream, or cause signaling cascades in the gut cells, leading to the transcrip- directly injected into the brain tissue of these rats. If increased ILtion of proteins that allow for the proliferation of certain bacterial 10 does not allow for decreased inflammation within stressspecies. An alternate outcome would result in the vagus nerve not vulnerable rats, this would indicate that these rats confer remediating changes in the gut microbiome during the stress resistance to IL-10. This result would also allow for the explanation sponse, which indicates that physiological mechanisms involving of increased IL-10 produced by stress-vulnerable microbiota, other organs may be at play. The brain may communicate with the where the rats simply do not respond to the effects mediated by gut through different pathways, such as the release of neurohor- this cytokine. A similar experiment should also be conducted, mones in the bloodstream that affect the gut and lead to the pro- where stress-resilient mice have the microbiota that produces ILliferation of certain microbiota. By determining the mechanisms 10 removed. This altered microbiome should be subsequently that involve changes within the gut microbiota, treatment options transplanted into naive rats, and exposed to stressors. The abmay be developed to counteract them and prevent the growth of sence of IL-10 would most likely result in lower resilience to stress, bacteria that produce proinflammatory cytokines. This would leading to the development of depression. Alternatively, this exthereby reduce the susceptibility of an organism to stress and re- periment would further elucidate the importance of IL-10 in stress duce the probability of developing depressive-type behaviors. resilience and can allow for the anti-inflammatory cytokine IL-10 to be key for the reversal of depressive-type behaviors. Although the effects of stress-vulnerable bacteria on the control group were carried out in the study by Pearson-Leary et al. (2019), As with many psychiatric disorders, many factors may be at play further studies on the stress-resilient gut microbiome can allow for towards its development, ranging from environmental to genetic a greater understanding of the relationship between the gut mi- factors (Nabeshima et al., 2013). The study conducted by Pearsoncrobiome and the brain. The gut microbiome of stress-vulnerable Leary et al. (2019) only looked at the effects of the gut microbiota rats should be eliminated with the use of antibiotics to reduce the in the development of depressive-type behaviors through geamount of pro-inflammatory cytokines. These rats should then netically identical rats. Factors such as sex and age were not acreceive fecal transplants from stress-resilient rats and should be counted for. Future studies may be directed at controlling for screened for any changes. A possible result of this study revolves these factors and observing any differences which may allow for a around the stress-resilient microbiota decreasing the vulnerability greater understanding of the relationship between stress and deto stressors of the stress-vulnerable rats, in addition to eliminating pression. If differences in gut microbiota and production of prodepressive-type behaviors. This may be due to stress-resilient rats inflammatory cytokines are observed between different groups containing microbiota that produce largely produce anti- such as sex and age, this may indicate a greater complexity toinflammatory bacteria through higher compositions of Clostridia wards the development of depression through stress. Studies on and Actinobacteria, as observed in the study conducted by Pearson psychiatric disorders are ultimately pursued to improve the under-

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standing behind the underlying mechanisms that lead to them, with the hopes that treatment options can be developed for individuals that suffer from them. Animal models of depression may not entirely reflect the physiological mechanisms that occur in humans. The gut microbiota of humans is different than that of rats due to differences in factors such as diet and environment. Studying the gut microbiota of human subjects that may be stressresilient can allow for the elucidation of factors that lead to this phenotype. Gut transplants may take place between a healthy, stress-resilient individual and an individual that is suffering from depression. Depressive-type behaviors may be reduced due to the phenotype being mediated by the gut microbiota. However, if the depressive-type behaviors persist, they may have arisen through other mechanisms that do not involve the gut microbiota. The research surrounding the relationship between the gut microbiome and the development of depression is relatively recent, thus further studies must be conducted on animal models to elucidate the physiological mechanisms behind this current phenomenon before tests can be conducted on human subjects.

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Autism Spectrum Disorder: Improvements Post Microbiota Transplant correlating with gastrointestinal symptoms Haushe Suganthan

Autism spectrum disorder (ASD) is a neurobiological disorder appearing early in life, impacting social interaction, and communication skills. This disorder is implicated to have an association with the gut microbiome, consisting of millions of bacteria. The gut-brain axis is considered a primary factor of ASD. ASD individuals have correlating gastrointestinal issues with ASD symptoms, leading many to believe that the gut microbiome heavily impacts ASD. In the research conducted by Kang, et al. (2017) a potential form of therapy for ASD targeting the gut microbiome alleviating ASD symptoms was found. This study conducted a modified fecal microbiota transplant (FMT), named microbiota transfer therapy (MTT). This treatment regimen consisted of a 2-week antibiotic treatment and bowel cleanse, and then MTT for 7-8 weeks initially with high doses, decreasing throughout treatment. The results showed significant improvement in gastrointestinal symptoms through the gastrointestinal symptom rating scale (GSRS) and ASD symptoms through clinical evaluations. In addition, increases in microbial diversity through bacterial sequencing analyses of fecal samples depicted increased relative abundances of bifidobacteria, prevotella, and desulfovibrio. The improvements and results lasted over 8-weeks post-treatment discontinuation, showing favourable long-term results for MTT as potential therapy for ASD. Therefore, targeting the microbiota via MTT depict great long-term improvement in symptoms for both gastrointestinal and ASD symptoms. Key words: Fecal Microbial Transplant (FMT), Microbiota Transfer Therapy (MTT), Autism Spectrum Disorder (ASD), Bifidobacteria, Prevotella, Desulfovibrio, Microbiota, gut-brain axis, neurodevelopmental disorder, Standardized Human Gut Microbiome (SHGM)

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BACKGROUND or INTRODUCTION Autism spectrum disorder (ASD) is a neurodevelopmental disorder, resulting in social communication deficits, and repetitive sensory-motor behaviours. It has been characterised neurobiologically that, those with ASD early in life have accelerated brain development, altering its connectivity. Depicting patterns of overall brain underconnectivity, additional to overconnectivity in certain regions, usually the frontal and occipital region, however this evidence needs further validation.1 The precise causation of ASD is unknown, however there is evidence of both genetic and environmental risk factors are involved.1,2 Gastrointestinal issues such as abdominal pain, diarrhea, and constipation is a common comorbidity in patients with ASD. These comorbidities have been linked to the gut microbiome.2-5 The human gut is comprised of millions of bacteria and microbiota which produce metabolites potentially involved in the pathophysiology of ASD.2-4 The brain-gut-microbiome axis is the interaction between central nervous system, gastrointestinal system, and microorganisms within the microbiota/gut.4-5 The gut microbiome influences the brain through neuroendocrine, neuroimmune, and autonomic nervous systems.3 Perturbations within the gut microbiome is associated with ASD, and this interaction through the gut-brain axis is seen to be altered in those with ASD compared to neurotypicals.4-5 Subjects with ASD were seen to have increased Zonulin levels, an enzyme associated with intestinal permeability regulation.5 Increased gut permeability, or “leaky gut”, allowing for bacterial metabolites to bypass the gut barrier, impacting neurodevelopment via the gut-brain axis as bacterial toxin/ products enter into the bloodstream.5,6,7 There is increasing evidence in ASD individuals that altered intestinal microbiome resulting in gastrointestinal distress and through the “leaky gut” these altered metabolites can enter systemic circulation and directly affect neurodevelopment.3,5,6,7 However, further validation is required into the potential treatment via the microbiome. Differences in the composition and variety of bacterial organisms is seen in studies between ASD and neurotypical populations, showing distinctive microbiomes.8,9 Through pyrosequencing of ASD gut microbiome compared to healthy/neurotypical subjects, there was evidence of decrease in diversity and distinct gut microbial compositions in ASD patients (Figure 4).8 However, amongst the literature there is little consensus on specific bacterial species that were similarly altered.8-10 Overall, individuals with ASD were seen to have a decrease in bacterial diversity, leading to dysbiosis within the microbiome contributing to the pathogenesis of ASD.5,8,9 Many studies targeting the microbiome as potential therapy for autism have been researched.8,9 A study found that vancomycin temporarily improved the behaviour and communication deficits in ASD patients, however once discontinued the behavioural symptoms relapsed.8,9 Showing only short-term improvement.6,8,9 Another study looked at administering probiotics such as lactobacillus and Bifidobacterium show improvement in gastrointestinal issues as a result of the exerted microbiota benefits provided through these bacteria, normalizing the gut microbiome. 6,8,9 This was seen to correlate with improved autistic core symptoms. 8 However, these studies were limiting as it only had a few bacterial species and the results were not always followed up with.8,9 However fecal microbial transplants (FMT) contain thousands of bacterial species native to the gastrointestinal system, being much more efficient in rebalancing the gut microbiome, and a more long-term solution.8,9

This study looks at altering the gut microbiome of ASD patients through FMT as potential therapy.9 A modified FMT treatment termed Microbiota Transfer Therapy (MTT) was conducted in 18 patients with ASD.9 This involved 14 days of oral vancomycin treatment, followed by 12-24 hours bowel cleansing, then a high initial dose of standardized human gut microbiota (SHGM), followed by daily lower doses for 7-8 weeks, with stomach acid suppressants.9 The results show about an 80% reduction in gastrointestinal symptoms at end the treatment, and improvements lasted 8-weeks post-treatment (Figure 1).9 Correlational to that behavioural ASD symptoms improved persisting also 8-weeks post treatment (Figure 2&3).9 Therefore, FMT/MTT proposes a long-term effect on ASD behaviour, gastrointestinal function, and microbiome enrichment.9 MAJOR RESULTS MTT treatment protocol consists of four main components: Oral vancomycin, MoviPrep, SHGM, and Prilosec.9 The treatment begins with 2 weeks of oral vancomycin, to ensure pathogenic bacteria is suppressed.9 Then an acid inhibitor Prilosec, is administered on day 12 and continued until end of low dose SHGM.8 Then on day 15 MoviPrep is administered to flush the bowel of remaining gut bacteria and vancomycin.9 Then on day 16 oral or fecal administration of SHGM, and then lower doses were given for the remaining 7-8 weeks.9 One major finding was gastrointestinal symptoms, when assessed by the gastrointestinal symptom rating scale (GSRS) showed significant improvement in abdominal pain, indigestion, diarrhea, and constipation (Figure 1).9 Another major finding was assessed and similarly found ASD core symptoms had decreased, showing improvement in ASD behaviour (Figure 2&3).9 Another major finding was changes in bacterial and phage diversity in gut microbiome overtime in response to the MTT (Fig.4&5).9 Gastrointestinal Evaluations The GSRS score dropped 82% throughout the treatment and remained 77% decreased 8-weeks post treatment. Two out of the 18 children were considered non-responders as they achieved less than 50% reduction in the average GSRS. Figure 1 clearly depicts the decrease in severity of gastrointestinal dysfunction overtime of treatment. Providing increasing evidence that with MTT, decrease in gastrointestinal issues, which may in part correlate to ASD severity.9 This is comparative to previous literature and studies done where alterations to the microbiome via probiotics resulted in decreased gastrointestinal symptoms.8,9

Figure 1. GSRS in 18 ASD patients post 8-week treatment, GSRS scores over time in weeks, scored on a scale rating 1 for no symptoms and 7 for very severe discomfort. Figure adapted from Kang, et al. (2017). Microbiome, 5(10), 1-16. ASD Evaluations

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Along with gastrointestinal symptoms, ASD- related behaviour also improved. Parent Global Impression -III (PGI-III) assessment evaluates 17 ASD-related symptoms. Post-treatment significant improvement was seen, and no reversion 8 weeks following treatment end. Evidence of negative correlation between change in GSRS and PGI-II is depicted when comparing Figure 1 and 2. Additionally, when core ASD symptoms were rated on child autism rating scale (CARS), they show 22% decreased throughout the treatment, and decreased 24% 8 weeks following end of treatment (Figure 3).9

. Figure 4. Microbial changes through stool analysis. Changes in Faiths PD in 18 children with ASD, orange line corresponds to median PD of donor, and dotted line indicates maintenance dose samples, and green line represents median PD of 20 neurotypical controls, at week 0. Figure adapted from Kang, et al. (2017). Microbiome, 5(10), 1-16.

Figure 2. ASD-related symptom rating of 18 ASD patients using PGI -III scores over course of treatment in weeks. -3 signifying much worse, -2 worse, -1 slightly worse, 0, no changed, 1 slightly better, 2 better, and 3 much better in comparison to baseline. Figure adapted from Kang, et al. (2017). Microbiome, 5(10), 1-16.

Figure 3. ASD-related symptom severity rating of 18 ASD patients using CARS assessment at pre-treatment, post-treatment, and 8weeks following end of treatment. Figure adapted from Kang, et al. (2017). Microbiome, 5(10), 1-16. Bacterial and Phage Diversity Changes after MTT After completion of the MTT treatment, there were changes in bacterial and phage diversity in gut samples. The phylogenetic diversity (PD) index, show gut bacteria were significantly less diverse in ASD children compared to neurotypical (Figure 4). During the treatment (week 3) bacterial diversity was not increased through initial SHGM. However, at the end of the treatment bacterial diversity increased in ASD children and remained high even after 8-weeks of treatment end. The donor bacterial community was partially engrafted in the recipient gut. Specific genera such as Bifidobacterium increased four-fold, and prevotella, and desulfovibrio also increased in relative abundance with treatment levels similar to neurotypical children. Thus, MTT successfully alters the ASD bacterial community towards that of healthy controls and donors.9

CONCLUSION/DISCUSSION The findings present that MTT depict significant improvements in gastrointestinal symptoms, ASD behaviour and core symptoms, and microbiome diversity.9 The improvements lasted for 8 weeks post-treatment end and showed promising results for ASD therapy.9 A follow-up study was conducted two years after this study, of the 18 participants, show persistent improvements in gastrointestinal and ASD symptoms, as well as remained increase in microbiota diversity and increased abundances in certain bacteria.10 Depicting long-term benefits in MTT as potential therapy for ASD.10 The findings of a gut-brain axis impacting ASD is consistent within literature.8 Literature has been consistent with interpreting a link between the gut microbiome and ASD, and these results further validate these interpretations.4,5 Other studies also looking into FMT and targeting the gut microbiome dysbiosis as potential therapy for ASD, have found beneficial effects.11Thus, results of our study, and similar others have found that just as authors interpreted altering the gut microbiome and modulating dysbiosis has beneficial effects in ASD, through increased microbial diversity, and communities.9,11 However, specific bacterial communities increasing in abundance relating to improvements in ASD, is controversial.8,9 This study is novel as it provides a long-term therapeutic approach, compared to vancomycin treatment, or probiotics, which have not shown significant long-term therapy in ASD.6,8 These results provide us with novel therapeutic approaches to ASD, as it shows potential significant long-term improvements and impacts in gastrointestinal and ASD symptoms in combination with microbial diversity.8,9 CRITICAL ANALYSIS The results of this study are consistent with literature, providing that there is a relationship between the gut microbiome and ASD.2 -9 Thus, treatment and therapy targeting the microbiome is efficient in improving ASD symptoms.8,9 This study used a combinatorial treatment of vancomycin, MoviPrep, SHGM, and Prilosec for the MTT.9 Further studies, implementing the effects of the individual treatment options should be studied individually to understand the importance of each step or factor, in comparison to the combinatorial method.9 Vancomycin and various antibiotics have been studied throughout literature previously alone as therapy for

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ASD.8,9 The results were effective but merely short-term, once vancomycin treatment ended, the ASD symptoms returned to prior levels.8,9,11 There are also inconsistencies in the benefits vancomycin/antibiotic treatment, as certain studies prove that vancomycin decrease microbial diversity which do not provide a benefit in ASD symptoms but cause it to regress.11, 12 In addition, literature is consistent in identifying decreased bifidobacterium and prevotella in ASD patients, however desulfovibrio is seen to be increased in some studies of ASD individuals.8,9 Probiotic treatment using these bifidobacteria, prevotella and lactobacilli further validate that these bacteria are beneficial to the microbiome, as ASD symptoms decreased post-probiotic treatment.8,9 However, desulfovibrio was seen to increase in ASD patients after post-MTT treatment contrary to literature.9 Specific desulfovibrio in ASD individuals have been retrieved and are considered aerotolerant, non-spore forming, propionic acid forming, and is antibacterial resistant, genotoxic, and produces hydrogen sulfide.13,14 Thus, desulfovibrio has been studied to be a key pathogen in regressive ASD and is increased in ASD compared to control subjects.13,14 Although, this is a limitation of this study as it is unknown how desulfovibrio is increased postMTT overall ASD core symptoms were improved.9 Authors should focus on studying further the interaction of the specific bacterial communities within the microbiome in respect to ASD. As certain bacterial species found to increase after treatment are considered adverse in ASD, contradicting literature.9,13,14 This is important to consider as future studies manipulating desulfovibrio alone could provide insight into potential ASD long-term treatment.

fovibrio and there impacts on ASD being beneficial or aversive pre and post-MTT is essential in better understanding its role. Also, studies looking at the effects of individual treatments with vancomycin, MoviPrep, Prilosec, SHGM alone and combined treatment is required to understand in depth if efficacy of MTT is contributed to a single factor or the combined effects of each.9 The results would most likely show increase efficacy in the combined treatment, as literature doing vancomycin treatment alone did not show longterm benefits in ASD symptoms.9 Thus, future studies are pivotal in further understanding and strengthening the results of the current study.

FUTURE DIRECTIONS Although this study had promising results in ASD therapy, it was not controlled for placebo effects, or randomized, making the data weak.9,15 By conducting a study with a placebo control, which is double-blinded, and contains randomization could potentially eliminate the confounding factors.9,15 This study will hopefully result in showing the placebo control arm, had no significant difference in gastrointestinal and ASD symptoms, when rated using the same scales by parents/guardians of ASD children in the study.9 However, if there were significant improvements in the result of placebo control arm group, then placebo may be a confounding factor in this study, making the results of this study inadequate. In addition to strengthen the results of this study, for future results looking at similar gastrointestinal issue, within similar age groups with similar ASD symptoms, making the population of study much more controlled and uniform can provide us with more tailored results, as inter-individual gut microbiome variation is high.9 We can make the population of study much more uniform through narrowing the inclusion criteria for age group, ASD severity, and gastrointestinal symptom severity. Using mouse models could also be a potential way to address the concern of homogeneity as these mouse models of study can have similar genetic variability and can be raised and nourished in similar environments giving them much more similar gut microbiomes.2 The results of these more controlled studies should provide us with varying microbiota changes per disease and varying outcomes per ASD symptoms, however if no difference between the homogenous groups are seen then perhaps MTT and gastrointestinal symptoms and ASD symptoms are much more generalized.9 Also, as per the critical analysis the literature on desulfovibrio is inconsistent, as it is heavily increased in ASD individuals, and considered aversive, and post-treatment of MTT it is increased compared to baseline.9,14 Future studies controlling for, regulating or looking only at desul-

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Lord C, Elsabbagh M, Baird G, Veenstra-Vanderweele J. Autism spectrum disorder. The Lancet. 2018;392(10146):508–520. doi:10.1016/s0140-6736(18)31129-2

Vuong HE, Hsiao EY. Emerging Roles for the Gut Microbiome in Autism Spectrum Disorder. Biological Psychiatry. 2017;81(5):411– 423. doi:10.1016/j.biopsych.2016.08.024

Li Q, Han Y, Dy ABC and Hagerman RJ. The Gut Microbiota and Autism Spectrum Disorders. Front. Cell. Neurosci. 2017; 11:120. doi: 10.3389/fncel.2017.00120

Luna RA, Savidge TC, Williams KC. The Brain-Gut-Microbiome Axis: What Role Does it Play in Autism Spectrum Disorder? Current Developmental Disorders Reports. 2016;3(1):75–81. doi:10.1007/s40474-016-0077-7

Fowlie G, Cohen N, Ming X. The Perturbance of Microbiome and Gut-Brain Axis in Autism Spectrum Disorders. International Journal of Molecular Sciences. 2018;19(8):2251. doi:10.3390/ijms19082251

Mayer EA, Padua D, Tillisch K. Altered brain-gut axis in autism: Comorbidity or causative mechanisms? BioEssays. 2014;36(10):933– 939. doi:10.1002/bies.201400075

Wasilewska JJ, Klukowski M. Gastrointestinal symptoms and autism spectrum disorder: links and risks – a possible new overlap syndrome. Pediatric Health, Medicine and Therapeutics. 2015:153. doi:10.2147/phmt.s85717

Yang Y, Tian J, Yang B. Targeting gut microbiome: A novel and potential therapy for autism. Life Sciences. 2018;194:111–119. doi:10.1016/j.lfs.2017.12.027

Kang D-W, Adams JB, Gregory AC, Borody T, Chittick L, Fasano A, Khoruts A, Geis E, Maldonado J, Mcdonough-Means S, et al. Microbiota Transfer Therapy alters gut ecosystem and improves gastrointestinal and autism symptoms: an open-label study. Microbiome. 2017;5(10). doi:10.1186/s40168-016-0225-7

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Kang D-W, Adams JB, Coleman DM, Pollard EL, Maldonado J, Mcdonough-Means S, Caporaso JG, Krajmalnik-Brown R. Long-term benefit of Microbiota Transfer Therapy on autism symptoms and gut microbiota. Scientific Reports. 2019;9(1). doi:10.1038/s41598 -019-42183-0

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Rosenfeld CS. Microbiome Disturbances and Autism Spectrum Disorders. Drug Metabolism and Disposition. 2015;43(10):1557– 1571. doi:10.1124/dmd.115.063826

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Vrieze A, Out C, Fuentes S, Jonker L, Reuling I, Kootte RS, Nood EV, Holleman F, Knaapen M, Romijn JA, et al. Impact of oral vancomycin on gut microbiota, bile acid metabolism, and insulin sensitivity. Journal of Hepatology. 2014;60(4):824–831. doi:10.1016/ j.jhep.2013.11.034

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Macfabe DF. Short-chain fatty acid fermentation products of the gut microbiome: implications in autism spectrum disorders. Microbial Ecology in Health & Disease. 2012;23(0). doi:10.3402/mehd.v23i0.19260

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Finegold SM. Desulfovibrio species are potentially important in regressive autism. Medical Hypotheses. 2011;77(2):270–274. doi:10.1016/j.mehy.2011.04.032

15.

Fattorusso A, Genova LD, Dell’Isola G, Mencaroni E, Esposito S. Autism Spectrum Disorders and the Gut Microbiota. Nutrients. 2019;11(3):521. doi:10.3390/nu11030521

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The Neuroprotective Potential of Physical Exercise Hiba Taha

Physical exercise has been implicated in improving mental health disorder symptoms, as seen in cases of depression and anxiety, cognitive health, as well as reducing risks of age-related chronic diseases such as cardiovascular disease (CVD) and Alzheimer’s disease (AD) (Hillman et al., 2008). Many studies show that exercise may improve symptoms as soon as following a single session of physical activity (Polska, 2004) However, the exact molecular or cellular mechanism outlining exercise and the resulting possible neuroprotective consequences is not well-understood. Jeong et al. (2018) highlights the neurological impact of treadmill exercise in High-Fat Diet (HFD) induced obese rats on a molecular level. Rats were divided into three main groups: control, HFD-control, and HFD-Treadmill Exercise (HFD-TE) all of which underwent learning and memory, biochemical and overall health assessments. Results revealed that the HFD-TE group had decreased body weight, insulin resistance and immunoreactivity. In addition, the HFD-TE group showed significant improvements in learning and memory, glucose tolerance, as well as brain-insulin signalling. These results support the neuroprotective role exercise plays on a molecular level that may have larger implications in therapeutic treatments for neurodegenerative diseases such as Alzheimer’s or Parkinson’s disease. Furthermore, the authors provide a direct link of how exercise may prevent deterioration of learning or memory. Through the prevention of Reactive Oxygen Species (ROS) formation and inhibition of specific protein hyperphosphorylation, it may be ultimately possible to prevent the pathology of many of the seemingly uncurable neurodegenerative or metabolic diseases. Key words: High-Fat Diet (HFD), High-Fat Diet Treadmill Exercise (HFD-TE), Reactive Oxygen Species (ROS), Tau hyperphosphorylation, NOX (NADPH Oxidase), Alzheimer’s Disease (AD), Parkinson’s Disease (PD), Obesity, Insulin Resistance

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BACKGROUND With increasing incidences of uncurable and untreatable neurodegenerative diseases, tremendous efforts are currently in place for understanding the mechanistic causes of Alzheimer’s (AD) and Parkinson’s disease (PD). More importantly, there is an urgent need for effective treatment and therapy options for affected individuals (Ang et al., 2010). Current research efforts uncovered common pathologies in both AD and PD in which specific protein aggregation results in the death of neurons, hence neurodegeneration (Reddy et al., 2019). In AD, genetic mutations in the Amyloid Precursor Protein (APP) causes the accumulation and aggregation of the toxic misfolded protein βamyloid (Lane et al., 2017). Post-mortem studies conducted in ADdiagnosed individuals reveal the resulting neuronal and synaptic loss in specific brain regions, in addition to the hyperphosphorylation of the micro-tubule associated protein Tau, involved in brain insulin signalling (Reddy et al., 2019; Gong and Iqbal, 2008; Jeong et al., 2018). Similarly, PD is characterized by the accumulation of the protein α-synuclein, formation of Lewy bodies and eventually leading to the loss of neurons in the Substantia Nigra SN), a region rich with dopaminergic neurons (Reddy et al., 2019). In both cases, this significant neuronal loss results in the classical neurodegenerative presenting symptoms such as considerable memory loss, cognitive impairments, as well as physical movement issues (Reddy et al., 2019). Interestingly enough, several studies propose a link between neurodegeneration and obesity. Spielman et al. (2014) suggests a possible mechanism involving excess adipose tissue administrating a pro-inflammatory response through the upregulation of proinflammatory cytokines. In addition, Spielman et al. (2014) provides evidence of obesity-induced insulin resistance resulting in reduced brain insulin signaling, weakening the brain’s neuroprotective potential. Some researchers have even considered neurodegenerative diseases such as AD to be ‘Brain Diabetes’ or ‘Type-III Diabetes’ (Duarte et al., 2012). On account of this thought-provoking link between metabolism, obesity and neuronal survival, several researchers such as Ang et al. (2010) began to implement and study the consequences of physical activity on neurodegeneration and the brain. A review article by Rolland et al. (2008) explored the repercussions of physical activity on patients diagnosed with AD. Results demonstrated 20 of the 24 studies showing a significant reduction in cognitive impairment following exercise (Rolland et al., 2008). Another study consisted of approximately 1700 subjects that were 65 years old and older (Paillard et al., 2015). A 13.0 dementia incident rate was observed per 1000 individuals who exercised three or more times per week in contrast to a 19.7 incident rate per 1000 less-exercising individuals (Paillard et al., 2015). Thus, researchers such as Jeong et al. (2018) decided to study the molecular link between exercise, obesity and neuronal survival or protection, in order to provide a possible mechanism for this phenomenon. Jeong et al. (2018) explored inflammation, oxidative stress, and insulin signalling in High-Fat Diet-induced obese rats on the basis that obesity affects how the body metabolizes and signals to the brain. Oxidative stress has been implicated in playing a major role in insulin resistance, diabetes mellitus, in addition to AD (Hurrle and Hsu, 2017; Yang et al., 2011). Oxidative stress occurs due to the formation of Reactive Oxygen Species (ROS) by NOX, NADPH Oxidase, as a result of endogenous or exogenous factors (Yang et al., 2011). Impaired insulin signalling can also result in Tau hyperphos-

phorylation which has been implicated in AD (Jeong et al. 2018). Following separation of three rat groups (control, HFD-control, and HFD-TE), several assessments and procedures were conducted to evaluate the following: learning and memory, overall health assessment, biochemical glucose and insulin analyses, in addition to protein analyses of Tau phosphorylation. The findings of Jeong et al. (2018) support the claim that exercise can reverse cognitive impairments, as seen in AD, by inhibiting oxidative stress. Moreover, brain insulin signaling is regulated, preventing Tau hyperphosphorylation. Thus, physical activity can possibly serve as a preventative and possibly therapeutic neuroprotective intervention in neurodegenerative cases. MAJOR RESULTS Results of the study conducted by Jeong et al. (2018) were divided into five major sections. To begin with, weight differences were first measured to ensure weight gain was successfully achieved in the HFD groups. A significant increase in weight, abdominal visceral fat (AVF), insulin resistance and a decreased glucose tolerance was reported in the HFD groups in comparison to the control Next, using immunohistochemistry, the presence of NAPDH subunits p22 phox, p47 phox and gp91 phox was assessed in the hippocampus and cerebral cortex of subjects in all three groups to understand the effects of exercise at the presence of oxidative stress. Results showed higher immunoreactivity of p22phox in the HFD-control group which was reduced following exercise in the HFD -TE group in the cortex and CA3 hippocampal regions (Jeong et al., 2018). Similar results were reported for immunoreactivity of p47 phox and gp91 subunits, suggesting that oxidative stress is increased as a result of the HFD-induced elevation of NOX (NADPH oxidase) which is reversed and inhibited by exercise (Jeong et al., 2018).

Fig. 1 Effect of exercise implementation on overall health and metabolism in control, HFD-control and HFD-TE experimental groups. (A) Both HFD-control and HFD-TE observe a significant increase in bodyweight. Following treadmill exercise, the HFD-TE showed a slight decrease in body weight. (B) Glucose was measured using the Oral Glucose

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Tolerance Test (OGTT) showing a large reduction in blood glucose in both control and HFD-TE groups, in comparison to the HFD-Con. (C) Abdominal Visceral Fat (AVF) was measured solely and (D) in ratio to body weight. Results show a reduction in AVF in the HFD group following exercise. Figures A, B, C and D were adapted from Jeong et al. (2018). Brain Research Bulletin. 142: 374-383 Next, using immunohistochemistry, the presence of NAPDH subunits p22 phox, p47 phox and gp91 phox was assessed in the hippocampus and cerebral cortex of subjects in all three groups to understand the effects of exercise at the presence of oxidative stress. Results showed higher immunoreactivity of p22phox in the HFD-control group which was reduced following exercise in the HFD-TE group in the cortex and CA3 hippocampal regions (Jeong et al., 2018). Similar results were reported for immunoreactivity of p47 phox and gp91 subunits, suggesting that oxidative stress is increased as a result of the HFD-induced elevation of NOX (NADPH oxidase) which is reversed and inhibited by exercise (Jeong et al., 2018).

Fig. 3 Assessing the presence of insulin signalling-related proteins in control, HFD-induced obese mice models, and after physical activity. (A) Results show a reduction in insulin signalling in mice under the HFD. A significant improvement in insulin signalling can be observed following implementation of exercise. Figure A was adapted from Jeong et al. (2018). Brain Research Bulletin. 142: 374-383 In addition, Jeong et al. (2018) immunohistochemically investigated Tau expression and activity by AT8 phospho-tau Ser202/ Thr205 protein. Following the trend of aforementioned results, Tau protein expression in the cerebral cortex, as well as Dentate Gyrus and CA3 hippocampal regions, was elevated in the HFDcontrol group and reduced in the HFD-TE exercise group (Jeong et al., 2018). This section of the results proposes that Tau hyperphosphorylation and aggregation caused by HFD can be inhibited by exercise (Jeong et al., 2018).

B Fig. 4 Phosphorylation levels of Tau proteins. (A) Results show a reduction in Tau phosphorylation following exercise in HFD groups. Figure A was adapted from Jeong et al. (2018). Brain Research Bulletin. 142: 374-383 Finally, learning and memory was assessed using the water maze and passive avoidance tasks across all three groups. In the water maze test, the HFD-TE group had a faster escape time and distance in comparison to the HFD-control group (Jeong et al., 2018). On the other hand, the HFD-Con group was unexpectedly reported to have a lower latency time than both the control and the HFD-TE exercise groups in the passive avoidance tasks (Jeong et al., 2018).

Fig. 2 Assessing the presence of NADPH Oxidase subunits in control, HFD-induced obese mice models, and after physical activity. (A) Immunoreactivity of NADPH p22 phox in the cerebral cortex and (B) CA3 hippocampal region. Results show a significant reduction in immunoreactivity following exercise in the HFD-induced models. (C) Expression levels of NADPH Oxidase subunits p47 phox and (D) gp91 phox subunits. Figures A, B, C and D were adapted from Jeong et al. (2018). Brain CONCLUSION/DISCUSSION Research Bulletin. 142: 374-383 Following assessment and verification of obesity induced by the Insulin signalling was assessed by measuring the expression of HFD program, Jeong et al. (2018) confirmed that rats in the HFD insulin-signalling associated proteins, such as insulin receptors (IR), group reported a significant increase of AVF, insulin resistance and in the hippocampus. It was found that IR and other insulinhyperglycemia in comparison to the control group. Furthermore, associated proteins were suppressed following HFD, interrupting an 8-week exercise regimen was implemented in the HFD-TE group insulin signalling but was increased following exercise rescuing in order to analyze the effects of exercise on brain insulin signalinsulin signaling (Jeong et al., 2018). ling, learning and memory, as well as Tau protein hyperphosphorylation or aggregation implicated in the pathology of neurodegenerative disorders (Reddy et al., 2019; Gong and Iqbal, 2008; Jeong et al. 2018). Reported results showed that physical activity suppressed HFD-induced weight gain, improved affected brain insulin signaling including Tau and NOX activity, and rescued learning and memory deficits (Jeong et al., 2018). Therefore, these results support the use of physical activity as a treatment plan to improve or reverse obesity-induced deficits in learning and memory, metabolism, and brain health (Jeong et al., 2018).

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It has been previously reported that exercise improves overall health, as well as insulin and glucose regulation (Bradley et al. 2008). In addition, inhibition of pro-inflammatory activity following exercise has been previously described in many diet-induced obese models (Kawanishi et al., 2010; Woods et al., 2009; Liu et al., 2015). However, results of this study by Jeong et al. (2018) provide a novel molecular perspective of how exercise may possibly act in a neuroprotective manner in diet-induced obese mice, in addition to providing supporting evidence that exercise improves overall health. A study conducted by Deng et al. (2009) found that Tau hyperphosphorylation occurs in association with insulin resistance and blunted insulin signaling. Jeong et al. (2018) proposes a mechanism by which exercise in obese mice can improve insulin resistance and signalling, thereby inhibiting Tau phosphorylation and aggregation, a crucial phenomenon of neurodegeneration observed in AD (Deng et al., 2009).

FUTURE DIRECTIONS

As acknowledged by Jeong et al. (2018), further studies need to be conducted to understand the exact mechanism by which physical activity improves learning and memory in both obese models and healthy models. Further studies outlining the exact type of diet would strengthen experiments such as these. In addition, studies on how different diets and exercise regimens affect the gut microbiome in healthy and obese models may be beneficial in understanding how exercise may impact our health. These studies could provide a potential mechanism of the neuroprotective potential of exercise. Furthermore, as Jeong et al. (2018) states, additional similar studies need to be conducted on humans. In this case, the exercise regimen can be manipulated in terms of intensity and duration which may have different effects on brain and overall health. Human studies can be conducted for a longer period of time which allows for a better understanding of the longAnother significant component of this study is the reduction of term consequences of exercising on diet-induced subjects. All in HFD-induced oxidative stress by inhibition of NOX activity found in all, these considerations in addition to the findings of this study the exercise group (Jeong et al., 2018). Some studies suggest that could create potential ways of preventing and targeting classical oxidative stress may play a role in regulating synaptic plasticity components of neurodegeneration. These options can then be (Jeong et al., 2018). Moreover, Jeong et al. (2018) suggests that implicated in future treatments and preventative measures of physical activity can improve cognition by inhibiting NOX activity. currently uncurable diseases such as AD and PD. This phenomenon is supported by many other studies that have shown that physical activity, such as running, improves cognition, learning and memory by upregulating neurogenesis and long-term potentiation (Praag et al., 1999). Overall, the relevance of this study lies in the fact that Jeong et al. (2018) provides direct evidence of exercising as an efficient treatment method with various targets to potentially prevent parallel cognitive impairments seen in neurodegenerative cases.

CRITICAL ANALYSIS Although Jeong et al. (2018) provides compelling evidence through cognitive, biochemical and immunological analysis of the benefits of treadmill exercises in obese mice, there are a few weak components of this study. To begin with, the authors did not disclose the exact nature of the HFD but rather simply stated that it was a 60% fat k-calorie diet (Jeong et al., 2018). The carbohydrate content of the diet was not acknowledged which may play a role in metabolic activity of the body. Many high fat diets such as the Keto Diet exist which have been reported to increase weight loss and improve metabolic activity and insulin resistance (Marvopoulous et al., 2005). Next, the authors did not acknowledge the results of the passive avoidance task in which the HFD-TE group had a higher latency time than the HFD-control group. In fact, it seems to have increased following implementation of the exercise regimen (Jeong et al., 2018). Finally, the HFD-TE underwent a 5-day exercise adaptation period prior to the official implementation of the exercise regimen (Jeong et al., 2018). This pre-adaptation may have influenced the results as the exercise regimen is then extended for a longer period of time than the period that was actually reported. In addition, both control groups were given water and food freely, which could also result in obesity although they were reported to be under normal diets (Jeong et. al, 2018). Overall, the conditions at which the control groups, both the control and the HFD-control, were kept in are slightly unclear and may have impacted the results of this study.

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Bradley, R., Jeon, J., Liu, F., Maratos-Flier, E. 2008. Voluntary exercise improves insulin sensitivity and adipose tissue inflammation in diet-induced obese mice. Am J Physiol Endocrinol Metab. 295(3): E586-E594

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Rapid eye movement sleep modulated by melaninconcentrating hormone neurons via the ventrolateral periaqueductal gray Jeffrey Wang

Melanin-concentrating hormone (MCH) containing neurons in the lateral hypothalamus (LH) have been implicated in the promotion of rapid eye movement (REM) sleep. However, the exact pathway through which these neurons modulate REM sleep is unclear. A novel experimental model is created that involves chemogenetic activation at the soma of MCH neurons in the LH and optogenetic inhibition at the axon terminals that innervate the ventrolateral periaqueductal gray (vlPAG). In this method, MCH neuron projections to the vlPAG can be evaluated on their role in REM sleep regulation via chemoactivation and photoinhibition, alone and in combination. It is demonstrated that MCH neurons in the LH densely innervate the vlPAG, a key REM-suppressing region while moderately innervating the sublaterodorsal nucleus (SLD), a primary REM-promoting region. Chemoactivation and photoinhibition demonstrated that MCH neurons inhibit the vlPAG thereby increasing REM sleep by increasing transitions from NREM to REM without effects to other aspects of arousal states. This elucidates the role that MCH neurons play in the REM sleep circuit by confirming that MCH neurons promote REM sleep primarily through inhibition of the vlPAG. This provides a prominent expansion to the model of REM sleep regulation as a distributed network of circuits Key words: REM sleep, chemogenetics, optogenetics, ventrolateral periaqueductal gray, sublaterodorsal nucleus, REM sleep transitions, melanin-concentrating hormone

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INTRODUCTION Rapid eye movement (REM) sleep has been studied extensively for its biological function, including memory consolidation, since being first characterized over 50 years ago (Peever & Fuller, 2017). However, the neural circuitry that regulates the onset, maintenance, and termination of REM sleep is not fully understood. The current, widely-accepted model for REM sleep regulation involves a ‘flip-flop switch’ of mutually inhibitory REM-on and REM-off neuronal populations in the brain (Lu et al., 2006). The sublaterodorsal nucleus (SLD) has subsequently been identified as a primary REM-on region (Lu et al., 2006). The SLD is seen to be REM sleep promoting, with pharmacological activation inducing REM sleep like motor atonia and lesion experiments demonstrating a reduction in REM sleep and prevention of muscle atonia (Fraigne et al., 2015). SLD neurons induce motor atonia via glutamatergic activation of inhibitory neurons in the ventromedial medulla (vmM) which subsequently inhibit skeletal motor neurons (Lu et al., 2006). The ventrolateral periaqueductal gray and lateral pontine tegmentum (vlPAG/LPT) was also identified to contain a key REM-off population of GABAergic neurons that suppress the SLD. Various methods of inhibition of the vlPAG lead to promotion of REM sleep via disinhibition of the SLD neurons (Peever & Fuller, 2017; Lu et al., 2006; Weber et al., 2015). Combined, this provides evidence to demonstrate the vlPAG/SLD circuit as a key regulator of REM sleep (Lu et al, 2006). However, research indicates that there are other inputs into this principal circuit. The dorsal raphe nucleus (DR) is seen to inhibit the SLD and promote wakefulness, with the DR inhibited by GABAergic neurons in the dorsal paragigantocellular reticular nucleus (DPGi) and the vlPAG (Luppi et al., 2007). The vmM is also seen to influence REM sleep promotion via GABAergic inhibition of the vlPAG (Weber et al., 2015). The lateral hypothalamus (LH) also sees significant input into the vlPAG/SLD circuit. Orexin containing neurons in the LH have been seen to influence REM sleep with activation of these neurons significantly increasing time spent in wakefulness at the expense of non-REM (NREM) sleep and REM sleep (Sasaki et al., 2011). Inhibition of orexin neurons result in narcolepsy with inappropriate activation of SLD during wakefulness resulting in cataplexy (Chen et al., 2013). Orexin neurons appear to innervate the vlPAG, thus facilitating the vlPAG’s REM sleep suppressing effects (Chen et al., 2013). In addition to orexin, melanin-concentrating hormone (MCH) neurons in the LH have also been associated with REM sleep generation (Jego et al., 2013; Vetrivelan et al., 2016).

MCH neurons have been determined to be associated with REM sleep regulation, with maximal excitation during REM sleep (Hassani et al., 2009). Brief optogenetic stimulation of MCH neurons results in increased NREM-REM transitions when initiated during NREM sleep and increased REM duration when initiated during REM sleep (Jego et al., 2013). Long-term optogenetic stimulation of MCH neurons show variable results, increasing REM sleep but sometimes increasing or decreasing NREM sleep (Konadhode et al., 2013; Tsunematsu et al., 2014). Chemogenetic activation of MCH neurons also increases REM sleep via only NREM-REM transitions without significant effect to REM sleep duration and NREM sleep (Vetrivelan et al., 2016). This suggests MCH neurons have a REM sleep specific role in modulating sleep regulation, with particular indication towards REM sleep generation as opposed to maintenance. However, the question remains as to where do MCH neurons project their influences onto the REM sleep circuit. MCH neurons may promote REM sleep either by modulating the primary SLD/ vlPAG switch or by inhibiting wakefulness-promoting regions. In inhibiting wakefulness-promoting regions of the brain, MCH neurons would delay REM sleep termination and thereby increase REM sleep duration. MCH neurons are known to project to the wake-active serotonergic neurons in the DR and pharmacological injections of MCH into the DR results in an increase in REM sleep episodes (Lagos et al., 2009). However, it was later determined that such activation would also entail depressive effects (Devera et al., 2015). Thus, while MCH neurons may input onto the DR and modulate sleep, this does not appear to be the principal mechanism through which they regulate REM sleep in otherwise healthy individuals. Instead, REM sleep may be promoted via projections directly to the SLD/vlPAG switch. In this regard, MCH neurons may inhibit REM sleep supressing neurons in the vlPAG or activate REM sleep promoting neurons in the SLD. Through a novel combination of chemogenetic activation and optogenetic inhibition, Kroeger et al. set out to confirm that MCH neurons regulate REM sleep primarily via the vlPAG. To confirm MCH innervation in the vlPAG, MCH-Cre mice were stereotaxically injected with an adeno-associated virus (AAV) carrying Archaerhodopsin T (ArchT) and a green fluorescent protein (GFP), AAV8-CAGFlex-ArchT-GFP, into the LH unilaterally. This allowed for confirmation that MCH neurons did indeed innervate vlPAG. Furthermore, MCH terminals exist at a 10-fold higher density in the vlPAG in comparison to the SLD, supporting a hypothesis that MCH primarily acts via the vlPAG. In another cohort of MCH-Cre mice, AAV8CAG-Flex-ArchT-GFP and AAV-DIO-hM3Dq-mCherry were injected bilaterally into the LH. In addition, bilateral optic fibers were implanted at the vlPAG region. Here, clozapine N-oxide (CNO) can be injected at the LH to chemically activate MCH neurons while photoinhibition (593.5nm) can occur specifically at MCH projections to the vlPAG. By studying the effects of chemoactivation and photoinhibition, separately and simultaneously, the changes to REM sleep can be studied. Kroeger et al. determined that MCH neurons innervating vlPAG were sufficient to induce the REM sleep changes that Figure Adapted from Peever & Fuller, 2017. are observed when MCH neurons are activated, providing a confirFig. 1. Illustration of putative REM sleep circuit. The sublaterodor- mation of the pathways through which MCH neurons modulate sal nucleus (SLD) promotes REM sleep and motor atonia. The ven- REM sleep regulation. trolateral periaqueductal gray (vlPAG) and SLD reciprocally inhibit each other to create a flip-flop switch of REM suppression and MAJOR RESULTS REM promotion, respectively. Various other inputs modulate this Histological processing of mice with ArchT-GFP selectively exprincipal switch including the ventromedial medulla (vmM), dorsal pressed in MCH neurons originating from the LH clearly demonraphe nucleus (DR), and the lateral hypothalamus (LH) via orexin and melanin-concentrating hormone. strated projections to both the vlPAG and the SLD. After 4 weeks of

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of expression, the vlPAG was densely innervated (171.4 ± 65.2 boutons/mm2 ; n = 3 mice) while the SLD was moderately innervated (19.7 ± 6.1 boutons/mm2 ; n = 3 mice). This showed preference for MCH neurons towards modulating vlPAG activity as opposed to SLD activity. To further elucidate the modulatory effects of MCH neurons to the vlPAG, Kroeger et al. created a mouse model capable of soma chemoactivation via Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) and terminal photoinhibition at the vlPAG via ArchT and a wavelength of 593.5nm. Sleep-wake analysis of mice subjected to either saline or CNO demonstrated clear increases in REM sleep episodes in CNO-exposed mice with no other appreciable effects (Fig. 3). This is in agreement with past MCH neuron activation studies exhibiting increases in REM sleep due to number of episodes as opposed to duration.

Figure Adapted from Kroeger et al., 2019. Fig. 2. Tracing via GFP (black) of MCH neurons from the LH (A) densely innervating the vlPAG (B) and moderately innervating the SLD (C). A’, B’, C’ are magnifications of the black squares in A, B, C. B” C” are magnifications of the white square in B’ C’. Red arrows indicate axon terminals. Scale bars: (A) 1 mm; (B, C) 0.5 mm; (A′) 200 μm; (B′, C′) 100 μm; (B′′, C′′) 20 μm. 3V: 3rd ventricle; 4V: 4th ventricle; Aq: aqueduct; f: fornix.

Figure Adapted from Kroeger et al., 2019. Fig. 3. Chemoactivation of MCH neurons in the LH specifically increases REM sleep via an increase in REM sleep episodes. CNO (in black) or saline (in white) was injected before a 3h recording period in MCH-Cre mice (n=9). Significant increases in REM sleep bouts and REM sleep proportions without significant changes to duration of REM sleep or affecting other arousal states. A, B, C show percentages of time spent in wakefulness, NREM sleep, and REM

sleep; D, E, F depict mean number of episodes per hour for each stage; G, H, I depict mean episode duration in seconds. Next, a combination of CNO and photoinhibition was used to examine the properties of MCH neurons projecting to the vlPAG. Photoinhibition occurred after 30 seconds of stable NREM sleep and continued until the next waking period regardless of whether NREM sleep transitioned into REM sleep or wakefulness. Each mouse was subjected to (a) saline and sham inhibition, (b) saline and photoinhibition, (c) CNO and sham inhibition, or (d) CNO and photoinhibition on 4 separate days with 3 days in between and in a randomized crossover design. Saline or CNO was injected and recording with sham or laser light occurred an hour later for 3 hours.

Figure Adapted from Kroeger et al., 2019. Fig. 4. Comparisons of effects from various combinations of chemoactivation and photoinhibition. Mice were exposed to either saline or CNO and to sham inhibition or photoinhibition at 593.5nm, in some combination (n=9). A shows percentage of NREM sleep episodes that transitioned into REM sleep. CNO + sham inhibition significantly increased these transitions, while photoinhibition with or without CNO significantly decreased such transitions. B and C depict mean REM sleep episode duration and REM sleep onset latency, respectively. D shows mean NREM sleep episode duration.

Chemoactivation of MCH neurons resulted in a significant increase in NREM-REM sleep transitions (41.5 ± 3.1% over 29.6 ± 3.4% in baseline saline and sham inhibition, Fig. 4A). Chemoactivation furthermore did not affect any other feature of REM sleep, including latency of onset and average duration of REM sleep episodes (Fig. 4B, 4C). This demonstrates that MCH neurons increase REM sleep by promoting transitions to REM over wakefulness rather than decreasing NREM sleep (decreasing REM sleep latency). Photoinhibition of MCH terminals at the vlPAG resulted in significantly fewer NREM-REM sleep transitions (11.1 ± 2.2%, Fig), without altering REM sleep duration (Fig. 4A, 4B). Without change to NREM sleep duration at the expense of NREM-REM sleep transitions (Fig. 4A, 4D), this indicates that inhibition of MCH neurons results in disfacilitation of NREM-REM sleep transitions, allowing more NREM-wake transitions. Combined chemoactivation and photoinhibition completely negated the increase in NREM-REM sleep transitions observed in chemoactivation alone, and was not significantly different from photoinhibition alone (Fig. 4A). While NREM sleep duration significantly increased over baseline, REM sleep duration did not, indicating that MCH neurons projecting to the vlPAG do not influence REM sleep maintenance but rather regulate REM sleep generation (Fig. 4B, 4D). These revelations demonstrate a clear connection between MCH neurons and the vlPAG in modulating REM sleep.

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c DISCUSSION The results of these experiments demonstrate that inhibition of MCH neuron terminals in the vlPAG during simultaneous soma activation of MCH neurons in the LH is sufficient to reproduce the effects of nonspecific MCH neuron activation. In this regard, MCH neuron projections into the vlPAG are sufficient to cause the increase in NREM-REM transitions without other effects. This indicates that MCH neurons promote REM sleep primarily through the inhibition of the REM suppressive vlPAG. Histology experiments also demonstrated this circuitry with the presence of a significantly higher density of MCH terminals in the vlPAG compared to the SLD (Fig. 2). This alleviates the concern that inhibition at the vlPAG may cause inhibition in downstream targets of MCH axons – these axons terminate at the vlPAG with no further downstream targets. However, MCH neurons in the LH also innervate other regions related to REM sleep regulation (Jego et al., 2013; Lagos et al., 2009). Despite this, activation of these regions, while do increase REM sleep overall, do not lead to an increase in NREM-REM sleep transitions that is exhibited in MCH neuron activation (Jego et al., 2013; Vetrivelan et al., 2016). This suggests that while these regions may extend REM sleep, the principal means through which MCH neurons promote REM sleep is via REM sleep generation. This generation is accomplished by the vlPAG facilitating transitions into REM sleep. The chemoactivation and subsequent photoinhibition of MCH neuron projections to the vlPAG demonstrate that these projections play the principal role in driving the REM sleep generation properties of MCH neurons. This is in support of previous studies of MCH neurons that point towards the vlPAG. Possibilities for innervation to the SLD are contradictory to evidence showing MCH application to the SLD decreases REM sleep (Monti et al., 2016). Orexin may be theorized to be an intermediate transmitter with MCH neurons inhibiting orexin pathways. However, MCH neurons have been shown to promote REM sleep even in orexin knock -out mice (Naganuma et al., 2018), demonstrating that MCH neurons likely act directly onto the REM sleep circuit. This study uses a novel experimental method in sleep research by combining optogenetics with chemogenetics to identify the nature of the MCH neuron pathway and its impact on the REM sleep circuit. Kroeger et al. confirm a body of evidence that points to MCH neurons directly innervating the vlPAG to promote REM sleep by inhibiting the REM-off GABAergic neurons in the vlPAG and allowing a bias towards NREM-REM sleep transitions. This expands the model of REM sleep regulation currently held and further demonstrating the diffuse nature of the networks involved in REM sleep. CRITICAL ANALYSIS In their examination to confirm the MCH pathway into the REM sleep circuit, Kroeger et al. make a significant preference in their examination of previous research. Optogenetic methods have shown variable results with respect to MCH neurons and their influences on REM sleep as well as NREM sleep. In particular, there is variance in the exact manner through which MCH neurons increase REM sleep, either by duration or by generation. Kroeger et al. make heavy reliance on the shortcomings of optogenetics and a single study of their own via chemogenetics. This leaves Kroeger et al. vulnerable in their assumption that the effects of MCH neurons are indeed due to MCH neurons and not due to differences in stimulation paradigms.

Further flaws weaken their experimental model. In their chemogenetic activation of MCH neurons, CNO exposure induces a significant increase in REM sleep via increasing the number of episodes without change to any other aspect of REM sleep or any other arousal state (Fig. 3). This stands at odds as an increase in REM sleep necessitates a decrease in one of or both of NREM sleep and wakefulness. Furthermore, this increase in REM sleep is not reconfirmed in their second experiment – CNO and sham inhibition compared to saline and sham inhibition should also be shown to increase REM sleep. This would have helped consolidate that the authors’ experimental model does not cause undesirable and confounding effects. Furthermore, the over-negation demonstrated by simultaneous chemoactivation and photoinhibition may point to the importance of MCH neuron projections to the vlPAG but may also reveal potential issues with the methodology, where photoinhibition dominates over chemoactivation and skews the results. Particularly, CNO with photoinhibition apparently exhibited stronger effects than just photoinhibition alone (Fig. 4D), which would not be expected given CNO’s stimulatory effects. Therefore, further study into the validity of this novel method is needed to ensure that it is indeed examining the effects of MCH neurons in isolation. What the method does provide is a distinct advantage over photoactivation which is seen to cause overheating and monotonous artificial firing which present a number of confounds (Cardin et al., 2010). By using photoinhibition, this is neatly avoided. By adding chemoactivation, the authors are still able to test if activation of MCH neurons is necessary to create its modulatory effects. In this regard, Kroeger et al. provide a technique that may allow further research that is capable of both stimulation and inhibition with minimal confounding variables. A major point that Kroeger et al. fail to address is an explanation as to the functional role of MCH neuron projections into the SLD as well as into other brain regions that modulate REM sleep. While they clearly demonstrate that projections to the vlPAG are sufficient to reproduce the effects of MCH neuron activation, Kroeger et al. do not explore this pathway in relation to other pathways to consolidate the nature of the role that MCH neurons play in REM sleep regulation as a whole. FUTURE DIRECTIONS Critically, further verification of this novel chemoactivation and photoinhibition experimental method is needed. This is the first study to use this method in sleep research and so further studies examining its validity are needed, particularly due to the issues presented already. Chemoactivation at the soma with terminal photoinhibition does not isolate this pathway, as the activation may cause intermediate targets to be affected and influence the effects observed. In particular, the increase in NREM sleep duration and decrease in NREM-REM sleep transitions under simultaneous chemoactivation and photoinhibition indicate that photoinhibition dominates over the chemoactivation. This may be interpreted as the vlPAG as the principal relay for MCH effects and that MCH neurons partially inhibit the vlPAG tonically, as Kroeger et al. have done. However, it may also be an artefact due to stronger effects seen by photoinhibtion. To remove this confound, refinement of the experimental model is needed by confirming that photoinhibition itself is equal in magnitude of effect to chemoactivation. That is, all else being equal, the two methods counterbalance each other. In vitro experiments with a simple neural circuit can work to confirm if this is the case by isolating the synapse and examining if

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chemoactivation and photoinhibtion equally cancel each other out. If this is not the case, then some of the dominant effects seen in the simultaneous condition as well as in the photoinhibition only condition may be a result of the experimental paradigm, confounding the findings presented by Kroeger et al. Beyond the validity of the experimental model presented by Kroeger et al., future studies should look towards identifying the specific neurotransmitter that MCH neurons rely on to modulate the REM sleep circuit. This is as of yet unknown with some key neurotransmitters under scepticism. MCH neurons in the LH are primarily inhibitory and contain GAD67, necessary for GABA synthesis, suggesting they may act through GABA signalling. However, these neurons also lack VGAT, necessary for GABA transport and release, casting this theory into doubt. Glutamate may be involved through feed-forward inhibition, however the REM sleep effects seen by MCH neuron activation can be seen without glutamate involved. To determine the signalling mechanisms, studies will need to examine each candidate neurotransmitter to isolate which is necessary to induce the effects seen by MCH neuron activation, including knockouts of various transmitters until one removes the exhibited effects. With the complexity of the brain, it will be likely that various signalling molecules will be involved in the REM sleep modulatory effects of the MCH neurons â&#x20AC;&#x201C; MCH neurons may innervate the SLD through one neurotransmitter and the vlPAG through another.

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Cardin, J., Carlen, M., Meletis, K., Knoblich, U., Zhang, F., Deisseroth, K., … Moore, C. (2010). Targeted optogenetic stimulation and recording of neurons in vivo using cell-type-specific expression of Channelrhodopsin-2. Nature Protocols, 5(2), 247-254.

Chen, L., McKenna, J., Bolortuya, Y., Brown, R., & McCarley, R. (2013). Knockdown of orexin type 2 receptor in the lateral pontomesencephalic tegmentum of rats increases REM sleep. European Journal of Neuroscience, 37, 957-963.

Devera, A., Pascovich, C., Lagos, P., Falconi, A., Sampogna, S., Chase, M., * Torterolo, P. (2015). Melanin-concentrating hormone (MCH) modulates the activity of dorsal raphe neurons. Brain Research, 1598, 114-128.

Fraigne, J., Torontali, Z., Snow, M., & Peever, J. (2015). REM sleep at its core – circuits, neurotransmitters, and pathophysiology. Frontiers in Neurology, 6(3).

Hassani, O., Lee, M., & Jones, B. (2009). Melanin-concentrating hormone neurons discharge in a reciprocal manner to orexin neurons across the sleep–wake cycle. Proceedings of the National Academy of Sciences, 106(7), 2418-2422.

Jego, S., Glasgow, S., Herrera, C., Ekstrand, M., Reed, S., Boyce, R., … Adamantidis, A. (2013). Optogenetic identification of a rapideye movement (REM) sleep modulatory circuit in the hypothalamus. Nature Neuroscience, 16(11), 1637-1643.

Konadhode, R., Pelluru, D., Blanco-Centurion, C., Zayachkivsky, A., Liu, M., Uhde, T., … Shiromani, P. (2013). Optogenetic Stimulation of MCH Neurons Increases Sleep. Journal of Neuroscience, 33(25), 10257-10263.

Kroeger, D., Bandaru, S., Madara, J., & Vetrivelan, R. (2019). Neuroscience, 406, 314-324.

Lagos, P., Torterolo, P., Jantos, H., Chase, M., & Monti, J. (2009). Effects on sleep of melanin-concentrating hormone (MCH) microinjections into the dorsal raphe nucleus. Brain Research, 1265, 103-110.

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The Use of Gold Nanoparticles to Treat Alzheimer’s Disease Ziru Wang

Alzheimer’s disease (AD) is a neurodegenerative condition that is associated with a decline in cognitive ability and memory loss that interferes with the daily life of an individual. Current hypotheses regarding the cause of AD include the accumulation of plaques composed of amyloid-B and aggregations of hyperphosphorylated tau protein. The rationale behind current treatments is to treat the symptoms of the disease as oppose to changing the course of the disease progression. Additionally, current treatments that are available are associated with some adverse side effects. Recent research has been done to examine the possibility of using gold nanoparticles (AuNPs) to treat AD due to its ability to reduce inflammation, and its biocompatibility. A study in 2019 done by Tramontin & colleagues used okadaic acid (OA) to create a rat model for AD. They then used AuNPs to examine its effects as a potential treatment option for AD. The results showed that rats treated with OA alone showed higher levels of phosphorylated tau protein. Furthermore, the Barnes maze test revealed that OA treated rats showed a decline in spatial memory whereas the OA+AuNPs treated rats prevented this decline and showed similar results to the sham control. Lastly, the authors showed a significant decrease in superoxide dismutase (SOD) activity in the OA rats and that treatment with AuNPs prevented this decline. Overall the relevance of these results suggest that AuNPs can be used to treat AD. Key words: Alzheimer’s disease (AD), gold nanoparticles (AuNPs), okadaic acid (OA)

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Introduction Alzheimer’s disease (AD) is one of the most common forms of dementia and is rapidly becoming a worldwide epidemic1. Alzheimer’s disease refers to a condition where there is a significant decline in cognitive ability and memory loss that interferes with the ability of an individual to continue with daily activities1,2. It is also characterized by the accumulation of plaques composed of amyloid-B (Aβ) and aggregations of hyperphosphorylated tau protein1. One of the leading theories regarding the progression of pathology for AD includes the amyloid cascade hypothesis3. The amyloid cascade hypothesis is centered around the abnormal cleavage of amyloid precursor protein resulting in abnormal Aβ production3. The aggregation of Aβ can lead to neuronal damage and loss3,4. Furthermore, this production ultimately leads to the accumulation of amyloid plaques and neurofibrillary tangles composed of hyperphosphorylated tau protein3. Another emerging mechanism that is thought to contribute to the progression of AD is neuroinflammation3,5. Neuroinflammation involves the activation of microglial cells in the brain and it has been shown to contribute to neuronal injury and memory loss3,5. Furthermore, this neuroinflammation has been implicated with an increase in reactive oxygen species (ROS) ; which is another feature that is commonly observed in patients with AD1,6. Some studies have even suggested that antiinflammatory drugs may have protective effects on AD7.

ment alone was sufficient to cause a significant increase in phosphorylation of tau. This finding is consistent with existing literature which states that a defining feature of Alzheimer’s disease is the phosphorylated tau protein1. Together, these results suggest OA rats are a good model for AD.

Figure 1: A comparison of the levels of tau phosphorylation in the hippocampus between treatment groups. Rats that received OA injection exhibited higher levels of tau phosphorylation while rats that received both OA and the AuNPs treatment had similar phosphorylation levels as the sham controls1. (P<0.05, n=4)

AuNPs Treatment Prevents a Decline in Spatial Memory

To determine the cognitive effects that AuNPs treatment had on the AD rat model, researchers compared results from the Barnes maze spatial memory task one day after completing the treatments and at 5 days following the last treatment1. This To date, pharmacological treatments that exist are those that treat task involved getting the rats to remember one specific hole the symptoms associated with AD itself, rather than being able to that contained an exit out of a total of 19 holes that were 3 alter the progression of the disease . Furthermore, existing drugs placed on a platform1,10. Figure 2 shows that OA rats treated also have several unwanted side effects such as fatigue, muscle with the AuNPs significantly decreased the amount of time the cramps, nausea and have an overall small effect on AD3. Recently, animal took to find the target hole when compared to the OA more research was conducted to investigate the potential use of 1 show AD gold nanoparticles for AD treatement . Gold nanoparticles (AuNPs) rat. This result is similar to several other studies that 9,11 animal models having a decline in spatial memory . are of particular interest in developing new forms of treatment because they have been shown to have anti inflammatory properties by inhibiting the expression of NF-κB8. Additionally, gold nanoparticles are already used on occasion as a method of drug delivery to the brain because of its biocompatibility9. An article written by Tramontin & colleagues explored the potential to use these gold nanoparticles to treat Alzheimer’s disease1. To do this, they created an AD rat model by intracerebroventricularly injecting okadaic acid (OA) into the brains. The authors then compared how the AuNPs treatment affected levels of phosphorylation of tau protein, any changes in spatial memory and activity levels of superoxide dismutase (SOD)1. The major results of this study determined that OA injection increased the levels of phosphorylated tau, while treatment with AuNPs prevented it1. Furthermore, OA rats treated with AuNPs prevented a loss in spatial memory as well as restored levels of superoxide dismutase activity1. Overall their research has presented evidence to suggest that AuNPs is a promising treatment for AD.

Figure 2: Comparison of the time spent to find the exit in a Barnes maze task1. (P<0.05, n=12) AuNPs Treatment Restores Superoxide Dismutase Activity

Researchers quantified the level of superoxide dismutase (SOD) activity by measuring the inhibition of adrenalin oxidation1. As seen in figure 3, treatment of OA on rats significantly decreased the level of SOD activity relative to the sham controls in both the hippocampus and the cortex. Furthermore, the AuNPs Major Results treatment on OA rats restored the levels of SOD activity. The AuNPs Treatment Prevents Hyperphosphorylation of Tau relevance of this result suggests that AuNPs treatment can reTo examine the effects of gold nanoparticles (AuNPs), Tramontin & store the activity of SOD and therefore, reduce neuroinflammacolleagues first created an animal model for Alzheimer’s disease tion, which is a common feature of patients experiencing AD12. (AD) by intracerebroventricularly injecting okadaic acid (OA) into Critical Analysis male wistar rat models1,3. When OA induced rats were treated with the AuNPs, hyperphoshorylation of tau protein was prevented in By examining how the AuNPs treatment affected the levels of the hippocampus as seen in figure 1. Furthermore, the OA treatphosphorylation of tau, spatial memory and levels of superox430

ide dismutase activity, the study was able address several aspects of Alzheimer’s disease that could be treated with this new therapy1. The results seen in the Barnes maze test as well as the SOD activity were consistent to the ones performed in another study done by Muller & colleagues9. With the results obtained from the study, the authors concluded that AuNPs prevented hyperphosphorylation of tau, a decline in cognition, and improved the function of superoxide dismutase in response to OA1. The results presented in the paper were aligned with this conclusion. However, one of the results showing the analysis of inflammatory cytokines demonstrated that the hippocampus had higher levels of the pro-inflammatory cytokine IL-1β in both the OA and OA+AuNPs groups when compared to the sham group1. This specific result does not align with the literature that suggests AuNPs have anti-inflammatory properties8. Despite the inconsistency, the authors provided supporting evidence to suggest that IL-1β may be an important component for plasticity and spatial memory formation, which could potentially explain the increase seen in the results 1, 14. Another limitation of this study was the lack of experiments done to show the biological mechanism by which AuNPs work to protect the brain from AD pathology. Without a concrete mechanistic explanation, it is more difficult to associate the results solely due to the AuNPs treatment. Confounding variables such as the age and only the use of male rats in the study lowers the level of external validity of the study.

recognition test multiple times over time. This test involves recording the amount of time a rat spends exploring new objects after being habituated with older objects in their surrounding9. Rats with no memory and recognition impairments are expected to spend more time exploring new objects because they recognize and remember the old objects9. It is expected that rats who receive the OA treatment will perform worse on both the Barnes maze test and object recognition test. More specifically, these rats are expected to spend more time finding the exit hole in the Barnes maze test and spend more time exploring old objects in the object recognition test. Furthermore, the OA and AuNPs treated rats should exhibit faster times in the Barnes maze test and spend more time exploring new objects in the object recognition test. If these results are seen, then it may be concluded that AuNPs treatment has long lasting effects. If the expected results are not observed, the next step would be to follow up and perform studies to examine the mechanism by which these AuNPs work. The purpose for this is to better understand how they provide neuroprotective effects as seen in the results that were reviewed in the study.

Finally, Alzheimer’s disease is a progressive neurodegenerative disease and thus the effect of its treatments should be analyzed over time. The authors sacrificed the rats immediately after administering the Barnes maze test which was 5 days after the last treatment was given1. By sacrificing the rats in a short period of time, the authors were not able to determine how long the effects of AuNPs as a treatment would last as well as any potential long term side effects that may be associated with it.

Future Directions The study that was examined in this review looked at the potential to use AuNPs for the treatment of Alzheimer’s disease1. To further support the results demonstrated in that study, a more longitudinal study can be done to examine the longevity of this treatment option as well as any potential long term side effects. To do this, rats can be grouped into 4 categories that will receive 4 different treatments. One group will be the sham control and receive no OA or AuNPs, the second will receive only AuNPs, the third group will receive only OA and the fourth group will receive both OA and AuNPs treatment. Since rats reach sexual maturity at 6 weeks and can live on average 3 years, a future study can start the treatment in the rats at 6 weeks time because it can allow researchers to also determine if the AuNPs treatment can also act as a preventative treatment for AD15. Following a protocol from previous studies, each rat will be administered their given treatment every 48 hours for 21 days1. Once the rats finish treatments, they will all be subject to perform the Barnes maze test immediately after the last treatment as well as every subsequent week. Each rat will be administered Barnes maze test over a long period of time. Furthermore, to assess recognition memory, the rats can also be administered the object

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1. Tramontin, N. D. S., Silva, S. D., Arruda, R., Ugioni, K. S., Canteiro, P. B., Silveira, G. D. B., … Muller, A. P. (2019). Gold Nanoparticles Treatment Reverses Brain Damage in Alzheimer’s Disease Model. Molecular Neurobiology. doi: 10.1007/s12035-019-01780-w

Du, X., Wang, X., & Geng, M. (2018). Alzheimer's disease hypothesis and related therapies. Translational neurodegeneration, 7, 2. doi:10.1186/s40035-018-0107-y

Briggs, R., Kennelly, S. P., & O'Neill, D. (2016). Drug treatments in Alzheimer's disease. Clinical medicine (London, England), 16(3), 247–253. doi:10.7861/clinmedicine.16-3-247

Morales, I., Guzmán-Martínez, L., Cerda-Troncoso, C., Farías, G. A., & Maccioni, R. B. (2014). Neuroinflammation in the pathogenesis of Alzheimer's disease. A rational framework for the search of novel therapeutic approaches. Frontiers in cellular neuroscience, 8, 112. doi:10.3389/fncel.2014.00112

Cai, Q., & Tammineni, P. (2017). Mitochondrial Aspects of Synaptic Dysfunction in Alzheimer’s Disease. Journal of Alzheimers Disease, 57(4), 1087–1103. doi: 10.3233/jad-160726

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Youn, H. S., Lee, J. Y., Saitoh, S. I., Miyake, K., & Hwang, D. H. (2006). Auranofin, as an anti-rheumatic gold compound, suppresses LPS-induced homodimerization of TLR4. Biochemical and Biophysical Research Communications, 350(4), 866–871. doi: 10.1016/ j.bbrc.2006.09.097

Muller, A. P., Ferreira, G. K., Pires, A. J., Silveira, G. D. B., Souza, D. L. D., Brandolfi, J. D. A., … Silveira, P. C. L. (2017). Gold nanoparticles prevent cognitive deficits, oxidative stress and inflammation in a rat model of sporadic dementia of Alzheimers type. Materials Science and Engineering: C, 77, 476–483. doi: 10.1016/j.msec.2017.03.283

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Gawel, K., Gibula, E., Marszalek-Grabska, M., Filarowska, J., & Kotlinska, J. H. (2019). Assessment of spatial learning and memory in the Barnes maze task in rodents-methodological consideration. Naunyn-Schmiedeberg's archives of pharmacology, 392(1), 1–18. doi:10.1007/s00210-018-1589-y

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The role of gut microbiome in contributing to the development of schizophrenia via kynurenine metabolism? Wenjuan Wang

The tryptophan metabolism pathway has been found to be dysregulated in patients with different mental disorders including schizophrenia and bipolar disorder. The degradation of tryptophan mainly depends on two major pathways: the 5-hydroxytryptamine (5-HT) pathway and the Kyn pathway. In addition, people with mental disorder like schizophrenia are often shown to have a disturbed gut microbiome. Since that bacteria from the gut microbiome catabolize tryptophan and contribute to the formation of a variety of catabolites such as kynurenine (Kyn), kynurenic acid (Kyna) and quinolone acid (Quia), it would be important to find out whether there is a correlation between disturbed microbiome and the dysregulated kynurenine pathway in order to further understand the underlying pathology of schizophrenia. In 2019, Zhu et al transplanted fecal microbiota from schizophrenia (SCZ) patients and healthy control to specific-pathogen-free mice and compare the effects on these two recipient groups in terms of behavior and brain activities. The results showed that there was a significant increase in the kynurenine-kynurenic acid (Kyn-Kyna) pathway resulting in an elevation in the production of Kyna in the group that received fecal transplantation from SCZ patients, whereas such change was not seen in the healthy control group, suggesting a correlation between gut microbiome and disrupted Kyna level observed in SCZ patients. Furthermore, there was also changes in the behaviors of the SCZ-recipient mice, such as hyper-locomotion, increased reciprocal social interaction as well as impaired spatial learning and memory, which high assembles abnormal behaviors shown in SCZ patients. All of these findings suggest disturbance in gut microbiome might cause the imbalance of neuroactive metabolites which then leads to abnormal brain functions. Key words: Schizophrenia, gut microbiome, tryptophan, fecal microbiota transplantation (FMT)

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c INTRODUCTION Schizophrenia is a brain disorder that has drawn an increasing number of peopleâ&#x20AC;&#x2122;s attention for the past decades. This disorder is often associated with both positive and negative symptoms such as delusions and hallucinations, as well as impaired cognitive functions and low motivations (Saha et al, 2005). Though Schizophrenia only affects around 1% of the entire population, it is still considered as one of the major economic burden of disease, costing up to $176.9 billion per year (Martin et al, 2019). This is mainly due to the fact that Schizophrenia typically onset from early adulthood and persists its symptoms regardless of advanced treatments (McCutcheon, 2019). Therefore, such feature as global burden of disease urges the need to find out the underlying pathology of schizophrenia and the development of more efficient treatment measures. Kyna is the one of the end products of kyn pathway in tryptophan metabolism. Tryptophan is first degraded into either 5-HT through the 5-HT pathway or into Kyn through the Kyn pathway, after which Kyn can then be processed through two different processes: Kynrenic acid (Kyna) and 3-hydroxykynurenine (3-HK). Many researches in the last decades have found an elevated concentration of kynurenic acid (Kyna) in SCZ patients compared to healthy individuals (Erhardt, 2016). Additionally, accumulation of Kyna was proved to be associated with impaired cognitive performance (Plangar et al, 2012), suggesting a possible correlation between Kyna imbalance and SCZ development. Since that gut bacteria are known to have functions in modulating catabolism of food-derived tryptophan and affect the concentration of final metabolite product like Kyn and Kyna, changes in the gut microbiome might disrupt the tryptophan metabolism process.

like abnormal behaviors observed in the recipient mice suggests that altered gut microbiome does indeed has some role in inducing SCZ-like symptoms in animal models.

Figure Adapted from Zhu et al. (2019). Molecular Psychiatry. Figure 1. The graph shows the total amount of distance travelled within 30 min where the blue line represents SCZ mice and the black line shows the movement pattern of the healthy control group. Overall, SCZ mice travel more distance compared to HC mice and its hyper locomotion did not reduce. Figure 2. This figure compares the spatial learning ability of the two groups where the filled dot represents the SCZ mice group and the hollow dot is the healthy mice group. The primary latency for SCZ mice and healthy control differs significantly; overall SCZ mice took much longer period of time to locate things.

Enhanced Tryptophan-Kyn-Kyna pathway in SCZ mice

To further explore the underlying mechanism of how an altered gut microbiota contributes to the induction of SCZ-like behaviors in animal model, mice tissues including gut, spleen, liver as well as serum were examined throughout the experiments in terms of changes in the concentrations of metabolites and meIn previous studies, both the gut microbiome and the tryptophan tabolism enzymes. The researchers found that tryptophan level metabolism were found to be similarly disturbed in SCZ patients decreased significantly in SCZ mice right after the transplanta(Zhang et al, 2019). This leads to the assumption of their correlation and such pattern continued throughout the experiment tion and whether this interaction contributes to the development (Zhu et al, 2019). Furthermore, there was an opposite trend in of SCZ. Kyn and 5-HT level observed in SCZ mice. While Kyn increased significantly increased after FMT, 5-HT level decreased (Zhu et MAJOR RESULTS al, 2019). This change returned back to its normal state within 10 days which was in line with the fact that human microbiota The gut microbiome has some function in regulating the tryptophan metabolism and is a possible contributor to the imbalance of readily diminishes in mice and only survive up to two weeks. end metabolite products that was observed in SCZ patients. To test Additionally, the level of Kyna elevated continuously in SCZ out the effects of a disturbed microbiome, Zhu et al performed mice as shown in figure 3 whereas the 3-HK concentration refecal microbiota transplantation (FMT) on specific-pathogen-free mained unchanged (Zhu et al, 2019). This shows that only one (SPF) mice in which one group of mice received microbiota from of the Kyn metabolism pathways was being affected by the SCZ patients and the other group was the healthy control. foreign SCZ microbiome. Altered microbiome induced SCZ-like behaviors In order to investigate the effects of altered microbiome on the The microbiota of the recipient mice was depleted using antibiotics and were checked that most microbiota were eliminated before FMT (Zhu et al, 2019). After the transplantation, a series of behavior tests were performed on SCZ and healthy control mice. During the first 10 min of the tests, both groups displayed similar level of activity; however, as shown in figure 1, the SCZ mice showed hyperlocomotion after the habituation period and continued this increased activity pattern without showing any sign of decline (Zhu et al, 2019). The same results were also observed in level of their exploratory behaviors and reciprocal social interactions. In addition to this, the SCZ group also found to have impaired spatial learning and memory ability as shown in figure 2. All of these SCZ-

activity of metabolism enzymes that are important in tryptophan degradation process, Zhu et al performed assays to quantify the mRNA level of enzymes in tryptophan-Kyn-Kyna pathway. It was found that the mRNA levels of indoleamine 2,3dioxygenase (IDO-1), tryptophan-2,3-dioxygenase (TDO-2) as well as kynurenine aminotransferase II (KAT II) were markedly increased in SCZ mice, which was in line with the findings of enhanced tryptophan-kyn-kyna pathway because they are the main enzymes responsible for this process. Along with the suppression of 5-HT pathway observed in SCZ mice, tryptophan hydroxylase 1 (TPH-1), an important enzyme in the pathway was also found to be lower than normal in concentration.

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Overall, these results suggest that the altered microbiome from SCZ patients can indeed disrupt Tryptophan metabolism, specifically via the tryptophan-Kyn-Kyna pathway, leading to changes in the behaviors and cognitive functions in the recipient mice. As the transplanted microbes diminished in animal model, the level of several metabolites also returned back to their normal status.

microbiota and pathogenesis of SCZ provide additional new evidence supporting the role of disturbed microbiome in SCZ. Compared with previous studies in the field, zhu et al chose SPF mice as the animal model instead of germ-free mice used in another research done by Zhang et al. Though the survival rate of transplanted microbes in germ-free mice is higher than other types of mice models(Zhu et al, 2019), previous research have shown that germfree mice do not have normal neural development due to the lack of some microbiota needed for the brain development (Neufeld et al, 2010). This feature of germ-free mice might cause the whole experiment to have misleading results. SPF mice would be the better animal model for this research and therefore might answer some of the questions in the previous research done by Zhang in which GF mice were used. Though the finding of enhanced tryptophan-Kyn-Kyna pathway and an elevated level of Kyna level observed in the microbiotainduced SCZ mice were consistent with the results of previous Figure Adapted from Zhu et al. (2019). Molecular Psychiatry. studies on SCZ patients, some researches showed completely opFigure 3. The blue bar represents the Kyna level of SCZ mice group posite results. In a research focusing on the imbalance in and the black bar shows the serum Kyna level of the healthy con- kynurenine metabolism and its correlation with SCZ pathogenesis, trol group (HC). Overall, Serum level of Kyna elevated significant- it was found that the Kyna level significantly reduced while 3-HK ly in SCZ mice immediately after FMT; however, such increase level increased in SCZ patients (Myint et al, 2011). They also found returned back to normal state after 10 days. that this shift to the 3-HK pathway is probably caused by the elevated production of inflammatory cytokines which was observed in some SCZ patients. In contrast, the levels of 3-HK as well as the CONCLUSIONS cytokine production remain unaffected in the zhuâ&#x20AC;&#x2122;s SCZ models. As the concept of microbiota-gut-brain-axis become increasingly This difference suggests two different mechanisms that might lead popular in the science filed, researchers start to focus more on the to the development of SCZ. Further researches are needed to unimpact of disturbed microbiome on human brain and whether or derstand the role of these two pathways in order to come up with not it plays a functional role in inducing some types of brain disor- a more efficient treatment target. ders. So far, there have not been many studies on the effects of FUTURE DIRECTIONS disturbed microbiota in the case of SCZ; however, current limited experiments have shown that there is an significant difference in This paper provides the first evidence of strong correlation bethe diversity of gut microbiome between SCZ patients and healthy tween altered gut microbiome and SCZ-like symptoms, in which individuals, as well as a high similarity in the microbiome diversity transplantation of microbiota from SCZ patients into SPF mice was in the patients with SCZ. A research paper published last year sug- enough to disrupt Kyna production and the expression of metabogests a higher abundance of Proteobacteria and Firmicutes in the lism enzyme KAT II. This remarkable finding again emphasizes the gut of SCZ patients (Shen, 2018). In this research done by zhu et al, importance of microbiome in the pathogenesis of SCZ; however, they focused on the impact of gut microbiota in the case of SCZ. the underlying mechanism in which how altered microbiota upregThe researchers collected focal microbiota from Treatment-free ulates enzymes like KAT II thereby favoring Kyna production is still SCZ patients and healthy individuals and transplanted it into SPF unknown. Future researches should look more deeper into the mice. The overall results showed that changing the diversity of gut mechanism behind for things like a specific product of gut microbimicrobiome through introducing foreign SCZ microbiota was ome metabolism affects the production of enzyme KAT II. By doing enough to disrupt the tryptophan metabolism pathway and induce this, we can then come up with a possible efficient treatment tarSCZ-like behaviors in the recipient mice. get to downregulate KAT II thereby fixing the disrupted Kyna proIn SCZ mice, the tryptophan metabolism was disrupted immediate- duction. ly after the FMT compared to the healthy control, resulting in a Furthermore, previous experiment showed that cognitive function remarkable increase in Kyna level (Zhu et al, 2019). This finding is of SCZ mice can be improved through a KAT II inhibitor PFin accordance with previous studies in which there was a signifi04849989 in which Kyna production was downregulated (Kozac et cant elevation in the concentration of kyna in most SCZ patients al, 2014), suggesting a possible treatment target. Future studies (Schwarcz et al, 2001). The fact that the introduction of foreign SCZ can investigate the role of such inhibitor and its efficiency in regut microbiota immediately led to a disruption in the Kyn pathway versing the impaired cognitive performance through downreguand such disruption diminished progressively as the number of SCZ lating Kyna in SCZ models. microbes in mice decreased suggest a strong correlation between Lastly, since that nowadays it is hard to find volunteers with SCZ gut microbiome and SCZ symptoms. Furthermore, the SCZ mice that are medication-free, this experiment done by zhu et al is also displayed abnormal behaviors that assembles SCZ behaviors based on the gut microbiome from a relatively small number of (Zhu et al, 2019). This again implies that gut microbiome from SCZ SCZ patients and cannot represent all cases of SCZ, thus further patients has the ability to disrupt brain functions and induce SCZ. researches should probably re-do this type of experiment many times in order to further understand the role of microbiome in SCZ CRITICAL ANALYSIS development. The authors of this paper focusing on the relationship between gut

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Saha, S., Chant, D., Welham, J., & Mcgrath, J. (2005). A Systematic Review of the Prevalence of Schizophrenia. PLoS Medicine, 2(5). doi: 10.1371/journal.pmed.002014

Martin, A., Bessonova, L., Osullivan, A., Weiden, P., Hughes, R., Berger, S., & Harvey, P. (2019). Pmh21 The Economic Burden Of Illness Of Schizophrenia In The Us. Value in Health, 22. doi: 10.1016/j.jval.2019.04.1066

Mccutcheon, R. A., Marques, T. R., & Howes, O. D. (2019). Schizophrenia—An Overview. JAMA Psychiatry, 1. doi: 10.1001/ jamapsychiatry.2019.3360

Erhardt, S., Schwieler, L., Imbeault, S., & Engberg, G. (2017). The kynurenine pathway in schizophrenia and bipolar disorder. Neuropharmacology, 112, 297–306. doi: 10.1016/j.neuropharm.2016.05.020

Zheng, P., Zeng, B., Liu, M., Chen, J., Pan, J., Han, Y., … Xie, P. (2019). The gut microbiome from patients with schizophrenia modulates the glutamate-glutamine-GABA cycle and schizophrenia-relevant behaviors in mice. Science Advances, 5(2). doi: 10.1126/ sciadv.aau8317

Zhu, F., Guo, R., Wang, W., Ju, Y., Wang, Q., Ma, Q., … Ma, X. (2019). Transplantation of microbiota from drug-free patients with schizophrenia causes schizophrenia-like abnormal behaviors and dysregulated kynurenine metabolism in mice. Molecular Psychiatry. doi: 10.1038/s41380-019-0475-4

Shen, Y., Xu, J., Li, Z., Huang, Y., Yuan, Y., Wang, J., … Liang, Y. (2018). Analysis of gut microbiota diversity and auxiliary diagnosis as a biomarker in patients with schizophrenia: A cross-sectional study. Schizophrenia Research, 197, 470–477. doi: 10.1016/ j.schres.2018.01.002

Schwarcz, R., Rassoulpour, A., Wu, H.-Q., Medoff, D., Tamminga, C. A., & Roberts, R. C. (2001). Increased cortical kynurenate content in schizophrenia. Biological Psychiatry, 50(7), 521–530. doi: 10.1016/s0006-3223(01)01078-2

Neufeld, K. M., Kang, N., Bienenstock, J., & Foster, J. A. (2010). Reduced anxiety-like behavior and central neurochemical change in germ-free mice. Neurogastroenterology & Motility, 23(3). doi: 10.1111/j.1365-2982.2010.01620.x

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Myint, A., Schwarz, M., Verkerk, R., Mueller, H., Zach, J., Scharpé, S., Kim, Y. (2011). Reversal of imbalance between kynurenic acid and 3-hydroxykynurenine by antipsychotics in medication-naïve and medication-free schizophrenic patients. Brain, Behavior, and Immunity, 25(8), 1576–1581. doi: 10.1016/j.bbi.2011.05.005

11.

Kozak, R., Campbell, B. M., Strick, C. A., Horner, W., Hoffmann, W. E., Kiss, T., … Castner, S. A. (2014). Reduction of Brain Kynurenic Acid Improves Cognitive Function. Journal of Neuroscience, 34(32), 10592–10602. doi: 10.1523/jneurosci.1107-14.2014

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Antioxidant-like protein 1 loss of function in streptozotocininduced diabetes rat model, indicates a mechanism for chronic hyperglycemia and cognitive impairment. Sebastian Warma

It is well documented that diabetes and subsequent hyperglycemia have long term effects on brain function and memory. Hyperglycemia increases oxidative stress and cell damage. Previous studies have shown the effects of hyperglycemia on numerous brain antioxidants and memory. The antioxidants examined are critical scavengers of reactive oxygen species. However, the effect of hyperglycemia on Antioxidant-like protein 1 (AOP-1) expression is unknown and this is the main focus of this paper. The researchers in this study induced diabetes in rats using streptozotocin, an agent that is known to be particularly toxic to the insulin-producing beta cells of the pancreas. Rats were examined over the course of 4 weeks and compared to a control. At each time point, an analysis of the expression of AOP-1 in neurons in the CA1, CA3, and dentate gyrus regions of the hippocampus was conducted. Results indicated that levels of AOP-1 were significantly decreased in the CA1 and CA3 regions but unchanged in the dentate gyrus, versus control. Additionally, blood glucose levels were significantly elevated in the STZ treated group relative to the control, thus, indicating a potential mechanism for neuron degeneration via increased oxidative stress damage. Moreover, as biopsies of hippocampal tissue were examined in this research, it provided a possible mechanism linking a reduction in antioxidant activity and increased oxidative stress to cognitive impairment such as memory loss. These findings are interesting and support the connection between diabetes and increased rates of neurodegenerative diseases such as Alzheimerâ&#x20AC;&#x2122;s disease. Keywords: Streptozotocin (STZ), diabetes, oxidative stress, CA1, CA3, dentate gyrus, Antioxidant like protein-1(AOP-1)

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c INTRODUCTION. Diabetes is a disease that is estimated to affect roughly 370 million people by the year 2030 (Wild et al. 2004). As the global incidence of this disease increases, more research will be needed into potential treatment options (Harding et al. 2019). One of the most effective methods of inducing diabetes for experimental models is through the injection of the drug Streptozotocin (STZ) (Akbarzadeh et al. 2007). STZ induces diabetes by entering pancreatic beta cells via the GLUT2 transporter, resulting in downstream effects and ultimately the death of the beta cells (Akbarzadeh et al. 2007). A consequence of the pancreatic beta-cell death is hyperglycemia, this is also a consequence in the in vivo disease state (Vatanen et al. 2018). This elevated blood glucose from Diabetes has numerous consequences in the brain. It has been shown to increase cognitive ageing such as memory loss and learning problems, and potentially aid in the development of Alzheimer’s disease in older age (Kapogiannis et al. 2011). Current research points to increased permeability of the blood-brain barrier (BBB) and a resulting increase in vascular inflammation as a mechanism for memory loss and cognitive impairment (Salva et al. 2019). This has been indicated by the upregulation of genes promoting BBB disruption and inflammation and leading to neurodegeneration (Salva et al. 2019, Han et al. 2010). Lee et al. 2015 examined a mechanism for this neurodegeneration. The mechanism studied was a reduced capacity of hippocampal cells to manage metabolic and oxidative stress (Kapogiannis et al. 2011). They aimed to address a gap in the understanding of how certain species that combat the damage caused by oxidative stress are affected under Streptozotocininduced type 1 diabetes. Chronically elevated glucose levels generate an increase in the number of free radicals or reactive oxygen species (ROS) in the brain (Jaduan et al. 2018). This results in increased oxidative stress (Ceriello 2000). Oxidative stress deteriorates neurons and other cells, impairing cell function (Muriach et al. 2014). Damage from this stress includes increased lipid peroxidation, as well as DNA damage and protein damage (Sies 1997). However, the body has numerous evolutionary adaptations to mitigate the effects of these reactive oxygen species. One example is an antioxidant, a compound that neutralizes the free radicals (Sies 1997). The study analyzed in this review studied one protein antioxidant in particular; Antioxidantlike protein 1 (AOP-1). AOP-1 is found in the cytoplasm and mitochondria (Hwang et al. 2005). It plays a critically important role in neutralizing reactive oxygen species (Hwang et al. 2005). It has also been linked to promoting cell proliferation and differentiation (Fujii et al. 2013). This study addressed a significant gap in research on Streptozotocininduced type-1 diabetes and its effect on AOP-1 expression in hippocampal neurons. The researchers are addressing whether there is a change in AOP-1 expression as a result of STZ treatment. This is being examined alongside analysis for evidence of the harmful effects of ROS species in the brain, such as lipid peroxidation and protein damage. The downstream effect of this damage is memory loss, which as stated, is associated with long-term diabetes. In the study analyzed, rats were separated into either an STZ treated or control group (Lee et al. 2015). Additionally, these primary groups were divided into subgroups for 2,3, and 4 weeks post-STZ treatment or control. Blood glucose levels were analyzed for each group and time period. At the conclusion of the time periods for each respective group, biopsies of the hippocampus were taken, from the dentate gyrus, CA1 and CA3. The presence of AOP-1 immunoreactivity was visualized using 3-3’ diaminobenzidine tetra-

chloride, in each of these regions. Signs of lipid peroxidation were also measured for alongside testing for differences in AOP-1 expression.

MAJOR RESULTS The treatment of rats with STZ induced pancreatic beta-cell death and thus hyperglycemia. Blood samples were then collected via a tail nick to confirm these symptoms.

Figure 1: Graph of Blood Glucose levels. Blood glucose levels were measured for the control group and 2,3, and 4 weeks post-STZ treatment groups. The blood glucose levels collected are with fasting, so there is no confounding from meals/diet. *the mean blood glucose levels were found to be significantly different (n=15, p<.05). Bars indicate standard error. Streptozotocin (STZ), Weeks (W). Figure Adapted from Lee et al. (2015). Neural Regenerative Research, 10(3), 451-456.

There was an almost 4-fold increase in blood glucose levels starting 2 weeks after STZ treatment (See Figure 1). Thus, indicating the successful induction of type-1 diabetes and hyperglycemia. This allowed for the analysis of AOP-1 immunoreactivity via 33’ diaminobenzidine tetrachloride markers. Tissue from the CA3, CA1 and dentate gyrus were analyzed. Immunoreactivity was noted in non-pyramidal cells of the hippocampus.

Figure 2: Number of AOP-1 Immunoreactive cells in hippocampus CA1 samples. Reactive cells were visually detected with 3-3’ diaminobenzidine tetrachloride. *significant decrease in AOP-1 expressing cells (n=5 per group, p<.05). Bars indicate standard error. Streptozotocin (STZ), Weeks (W). Images of AOP-1 reactivity, no magnification given. 2A: AOP -1 Reactivity Control. 2B: AOP-1 reactivity 2 weeks post-STZ treatment 2C: AOP-1 reactivity 3 weeks post-STZ treatment 2D: AOP-1 reactivity 4 weeks post-STZ treatment. Stratum oriens (SO), Stratum pyramidal (SP),

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decrease in AOP-1 immunoreactivity. Additionally, direct evidence of increased oxidative stress was demonstrated by an increased presence of MDA and protein carbonyl levels.

Stratum radiatum (SR). Figure Adapted from Lee et al. (2015). Neural Regenerative Research, 10(3), 451-456.

Figure 4: Number of AOP-1 immunoreactive cells in hippocampus dentate gyrus samples. Reactive cells were visually detected with 3-3â&#x20AC;&#x2122; diaminobenzidine tetrachloride. No significant decrease in AOP-1 expressing cells. Streptozotocin (STZ), Weeks (W). Images of AOP-1 reactivity, no magnification given 2A: AOP-1 Reactivity Control. 2B: AOP-1 reactivity 2 weeks post-STZ treatment 2C: AOP-1 reactivity 3 weeks post-STZ treatment 2D: AOP-1 reactivity 4 weeks post-STZ treatment. Layers of the dentate gyrus: Polymorphic Layer (PoL), Molecular Layer (ML), Granule Cell Layer (GCL). Figure Adapted from Lee et al. (2015). Neural Regenerative Research, 10(3), 451-456.

The reduction of AOP-1 levels in the STZ treated groups provides a possible mechanism and rationale for increased cell damage. Additionally, this research indicated that neurons in the dentate gyrus were not susceptible to STZ treatment, while those in the CA1 and CA3 regions were significantly affected, albeit differently. The effects observed by the increase in oxidative stress in the CA1 and CA3 regions appear to be associated to those neurons being highly vulnerable to oxidative stress (Kesner 2013). Previous studies have also indicated an increase in susceptibility of these regions to oxidative stress, specifically in the CA1 area (Wang et al. 2005, Rehman et al. 2017). This is likely due to an increased need for oxygen and glucose in cells in that region, relative to others. The dentate gyrus is critical for the integration of sensory inputs as well as pattern separation (Kesner 2013). This study is the first to analyze changes in AOP-1 expression alongside changes in diabetic states. However, previous research has shown significant neurodegeneration in the hippocampus due to oxidative stress and its possible link to depression (Rehman et al. 2017). Numerous studies have examined oxidative stress response in the hippocampus, however, this is the first to examine changes in AOP-1 expression. In addition, the authors examined the long-term effects of neuron degradation in diabetics rats and its relationship to the development of cognitive impairment and memory loss.

CRITICAL ANALYSIS

The data in the experiment provide strong evidence for changes in AOP-1 expression in the CA1, CA3 and dentate gyrus regions of the hippocampus. Previous experiments have also shown increased oxidative stress damage in the CA1 and the CA3 regions, consistent with Lee et al. (Wang et al. 2005). However, previous studies have indicated high levels of oxidative stress-related damage in the denThere was a significant decrease in AOP-1 immunoreactive cells tate gyrus (Huang et al. 2015), which Lee et al. do not observe. It in the CA1 and CA3 regions of the hippocampus, but not in the den- should be noted, however, Huang used different measurements tate gyrus (See Figure 2, 3, 4). In the CA1 the 2 weeks after STZ other than AOP-1 expression to show the damaging results of oxidatreatment group AOP-1 expressing neurons decreased to 24% of tive stress. It would have been helpful for the authors to comment the control. This was maintained in weeks 3 and 4. In the CA3, the on these somewhat contradictory results. Interestingly, Lee et al number of AOP-1 immunoreactive neurons decreased to 78.3% at showed there was a noted difference in loss patterns of AOP-1 exthe 2-week mark and then roughly 40% for the 3- and 4-week mark. pression between the CA1 and CA3 regions of the hippocampus. This indicates the potential for a significantly higher risk of damage The CA3 appeared to have a modest reduction in AOP-1 expression from oxidative stress in these regions of the hippocampus. The im- at the 2-week mark (78.3% of control),with a further reduction at portance of the CA1 and CA3 for long-term potentiation has been both the 3rd and 4th week post-STZ treatment (41.4% and 39.4% well documented in literature (Zakharenko et al. 2003). respectively). The AOP-1 expression levels in the CA1 neurons, howAdditionally, Lee et al. tested for signs of oxidative stress by ever, indicated a rapid drop to 24.4% of control at 2-weeks and looking for an elevated presence of malondialdehyde (MDA) and maintained those levels for weeks 3 and 4. The authors did not protein carbonyl levels, in the tissue samples. Both are indicators of comment on any potential reasoning for these differences in the oxidative stress damage from ROS. Levels of these two indicators loss of AOP-1 expression between these otherwise similar areas of were greatly increased in the week 3 STZ-treatment group comthe hippocampus. The paper surprisingly, only comments on the pared to the control, indicating increased oxidative stress. This indata observed in the CA1 region, failing to mention the intercreased cellular damage indicates a potential consequence for the variability between CA1 and CA3. loss of AOP-1 immunoreactive neurons. Additionally, the authors provide no hypothesis as to why the DISCUSSION neurons in the dentate gyrus show no reduction in AOP-1 expression levels and why they remain similar to controls. It is known that Lee et al. demonstrated that hyperglycemia, resulting from the the dentate gyrus is a separate structure to the CA regions of the onset of type-1 diabetes, often leads to a significant increase in oxidative stress. Interestingly, they have demonstrated that this can hippocampus, comprised of a tightly packed layer of small granule have damaging effects on certain areas of the hippocampus but not cells (Giap et al. 2000). Anatomically the CA differs from the dentate others. The effects of oxidative stress were primarily measured by a gyrus. They do make some reference to the difference in structure

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stating that the AOP-1 immunoreactivity was detected in the interneurons in the polymorphic layer of the dentate gyrus, and not in the pyramidal cells. They also confirm that in the CA1 and CA3 regions, AOP-1 immunoreactivity was detected in the pyramidal cells. This might suggest that AOP-1 expression on pyramidal cells is somehow more vulnerable to oxidative stress than in the interneurons of the dentate gyrus. It would have been interesting for the authors to expand on this. Lastly, analyzing different areas of the brain associated with cognitive impairment in old age such as the frontal cortex would provide more comprehensive results in relation to the development of dementia and Alzheimerâ&#x20AC;&#x2122;s as a complication of diabetes. Additionally, analysis of other evolved mechanisms and molecules which fight oxidative stress, other than AOP-1 would provide more robust data for this field. Moreover, increasing the sample size per group from n=5 in future iterations of this study would yield more robust data. However, through the analysis of AOP-1 expression changes, Lee et al. have provided a strong analysis of this subject, filling a gap in current research.

FUTURE DIRECTIONS This study provided a comprehensive analysis of changes in AOP1 expression in the hippocampus. Further research should examine why AOP-1 levels in the dentate gyrus are not impacted by oxidative stress, as suggested by these authors. Perhaps AOP-1 expression is somehow protected by this tightly packed layer of cells in the dentate gyrus and, therefore, the effects of oxidative stress have less impact. A potential mechanism that allows for the dentate gyrus to remain unaffected could provide insight into a potential drug that might be able to offer similar protection to the more vulnerable CA1 and CA3 regions, and thus, limiting the effects of oxidative stress in diabetes patients. Additionally, another interesting direction for future study would be the analysis of tissues outside the hippocampus. Specifically, areas of the brain linked to mental illness and depression. As links between diabetes and mental illness are being brought to light, analysis of AOP-1 expression in areas of the brain such as the frontal cortex or amygdala would highlight a potential mechanism for cognitive deficit (Harding et al. 2019). Additionally, analysis of reward pathways, in areas of the brain such as the nucleus accumbens could give insight on whether oxidative stress causes increased cell-damage. Thus, providing a possible mechanism for reward pathway impairment in mental illness. Testing of AOP-1 levels could be done using similar methods to the Lee et al 2015 study. It could be hypothesized that oxidative stress as a result of STZ induced diabetes, would lead to a decrease in AOP-1 immunoreactivity in the nucleus accumbens and frontal cortex. Some may hypothesize that the amygdala would not be affected as it is hyperactive in animals suffering from anxiety and depression (Grace 2016). Should this be the case it would provide interesting groundwork as no previous studies have examined the relationship between diabetes, mental illness, and AOP-1.

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Running to the Rescue â&#x20AC;&#x201C; How Exercise Induces Hippocampal Neurogenesis through Platelet Activation Isabella Watson

With the many known health benefits of exercise, a major physiological benefit is the effects of exercise on the brain. A link has been found between physical activity and an increase in neurogenesis in regions of the hippocampus; however, the mechanism behind it is still not known. Countless researchers have tried to find the answer behind this phenomenon. In a recent study by Leiter et al., a pathway has been explored through the activation of platelets and release of plasma factor 4. The plasma was isolated from adult mice that had either exercised for 4 days or remained sedentary. Plasma factor 4 and other blood-borne proteins were seen to be upregulated in the mice that exercise for 4 days when proteomic analysis was performed. The study observed an increased amount of activated platelets in mice after physical activity. These activated platelets, along with the release of plasma factor 4, were found to induce differentiation of neuronal precursor cells. Additionally, in ex vivo, cells cultured in platelet rich plasma showed increases in cell maturation and proliferation. Since it was established that activated platelets induced neurogenesis, and their numbers increased during and after exercise, this can likely play a role in the mechanism of exercise-induce neurogenesis in the dentate gyrus of the hippocampus. Key words: exercise; neurogenesis; dentate gyrus; hippocampus; platelets; adult mouse; neural precursor cells

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Introduction Physical activity plays an important role in keeping our bodies healthy and functioning. Many physiological systems benefit from exercise including the cardiovascular, skeletal, motor and nervous systems (Leal-Galicia et al. 2015). In the brain, specifically in the hippocampus, exercise can increase neurogenesis and synaptic plasticity as well as significant delay of cognitive impairment and neurodegeneration (Oâ&#x20AC;&#x2122;Callaghan et al., 2009). Exercise induces a change in blood composition by releasing several systemic compounds and factors. Factors like vascular epithelial growth factor (VEGF), Insulin-like growth factor-1 (IGF-1) and serotonin affects proliferation of neuronal precursor cells (NPCs) (Niu et al. 2019). Platelets are a component of blood that are known to play a role in clotting after tissue or blood vessel damage. However, platelets may play another role, in which they can induce neurogenesis. Neurogenesis is the process by which new NPCs are generated in the brain; this process occurs continuously throughout adulthood in very few regions of the brain including the dentate gyrus (DG), striatum and subventricular zone to olfactory bulb (Iismaa et al. 2018). These regions have been confirmed in rodent brains, however, in humans, only the dentate gyrus and striatum have strong evidence showing neurogenesis. The evidence behind exercise causing neurogenesis is strong, however, how this is done is yet to be discovered. One hypothesis is that exercise alters the length of the cell cycle, allowing the cells to remain in the proliferative state for a longer amount of time, in order to produce more NPCs. (Overall et al. 2016). Another hypothesis, is that after exercise, a systemic component in the blood is released and induces NPC proliferation in the DG. This was observed by Leiter et al. through analyzing mouse plasma after running on a wheel through proteomic screening to determine its composition. Plasma factor 4 (PF4) was one of the proteins found at much higher levels after exercise. Ex vivo DG cell cultures were then treated with activated platelets and neurosphere formation was tracked. The study investigated the role of platelets in neurogenesis and its activation through physical activity to determine the mechanism behind activity-induced NPC proliferation. The results showed that platelets may have a broader function than originally thought and play a key role in adult hippocampal neurogenesis.

CD62P+ was measured to indicate the number of activated platelets and CD61+ was measured to see the total number of platelets in the blood. Through this the proportion of platelet activation can be found and compared between groups. After only 1 day of acute exercise, significant increases in activated platelets were seen and continued to increase by day 4 of exercise (Figure.1B).

Figure 1. Blood composition changes due to exercise cause cell proliferation. A. Shows neurosphere cultures (n=3) injected with 0.01% of serum isolated from 4dRUN mice have an increase in neurosphere numbers relative to control culture. Results are compared to normalized control value (dashed line). * indicates statistically significant results as p < 0.05. B. Indicate amount of platelet activation of C57BL/6 mice after running as percent increase of standardized STD platelets numbers. **p < 0.01, significant increase of activated platelets in 4d RUN mice. Figure Adapted from Leiter et al. (2019). Stem Cell Reports, 12(4), 667-678.

Exercise-induced activated platelets act on hippocampal cells to promotes neurogenesis Ex Vivo As previously stated, the DG is a region of the hippocampus known to be involved in neurogenesis. Therefore, DG cells were cultured with either platelet-rich plasma (PRP) or platelet-poor plasma (PPP) from STD and RUN mice, then compared to untreated control cells. Results showed, the cultures with PRP from RUN mice had a significant increase in number and size of neurospheres while PPP treatments from both STD and RUN mice showed no difference in cell proliferation compared to control (Figure.2A). Lysophosphatidic acid receptor 1 (LPA1) tagged with green fluorescent protein (GFP) was used as a marker for NPCs and neural stem cells. Cells with LPA1-GFP were cultured with PRP or PPP and the results show a significant increase in neurospheres in PRP treated culture Major Results Exercise promotes platelet activation, changes in protein expression, (Figure.2B). This indicated activated platelets act on NPCs and progenitor cells of the DG. and release of systemic compounds in the blood To test for platelet activation after exercise, Leiter et al. isolated plasma from mice that were either housed in standard conditions, remaining sedentary (STD mice) or with a running wheel where the mouse would exercise for short periods over 4 days (4d RUN mice). The Proteomic screen done through mass spectrometry found 38 upregulated proteins in the 4d RUN plasma, 16 of which were significant. Reactome pathway analysis found a large majority of the proteins were of the 14-3-3 family and were involved in platelet activation. 14-3-3 proteins are highly expressed in the brain and are involved with neural signalling, development and protection (Foote & Zhou, 2012). It is thought that these proteins also play a role in neural differentiation, migration and survival. A neurosphere assay was performed with isolated serum from STD and 4d RUN mice and when placed with primary cells of the DG showed a significant increase in the number of neurospheres (Figure.1A). This demonstrates that there are also additional components to the plasma that may induce NPC proliferation.

Figure 2. Neurogenesis of hippocampal NPCs through exerciseinduce platelet activation. A. Neurosphere assay of DG cells treated with platelet poor and platelet rich plasma from STD and RUN mice. n = 11. *p < 0.05. B. STD cultures with LPA1-GFP stained cells. Increase in neurosphere numbers in STD PRP cultures compared to untreated (control) cultures. n = 7. **p < 0.01. Figure Adapted from

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Leiter et al. (2019). Stem Cell Reports, 12(4), 667-678. aid with neurogenesis. Although Leiter et al. discovered a beneficial role of platelets in the brain, conflicting research has shown the activated platelets are a determinant for ischemic stroke, demonstrating that platelets may also have negative effects. (Schmalbach et al., 2015). Additionally, a study found that activated platelets may play a role in Alzheimerâ&#x20AC;&#x2122;s disease (AD) through stimulating microglial cells, inflammation and damaging cortical blood vessels (Kniewaller et al., 2018). Even though some studies show a negative effect of platelets, Leiter et al. found a protective effect which may be important for ageing. Ageing leads to reduced cognitive function and neurogenesis, as well as changes in blood composition, but with exercise and activated platelets, this decline Figure 3. Increase in neurogenesis in the hippocampus caused by may be prolonged (Villeda et al., 2011). The role platelets play on plasma factor 4. A. Levels of PF4 in plasma in STD and 4dRUN mice. the brain is very complex and it has not yet been determined *p < 0.05, significantly more PF4 release in RUN mice. n = 5 per whether, overall, activated platelets do more help or harm to neugroup. B. Varied concentrations of PF4 added to DG neurosphere ral cells. Leiter et al. showed strong evidence that activated platecultures. n = 12. Significant (*p < 0.05) change in neurosphere lets and PF4 induced neurogenesis Ex vivo, however, when tested number when 10ng/ml and 100ng/ml of PF4 is added. Figure Adapted from Leiter et al. (2019). Stem Cell Reports, 12(4), 667- in vivo, although it showed that PF4 aided in neuronal cell survival, there was no indication of neurogenesis. Additionally, the authors 678. Plasma factor 4 release in the dentate gyrus increases neurogene- only administered PF4 directly into the hippocampus and no systemic tests with PF4 were done. Further research should look into sis Leiter et al. determined that PF4 is released into the blood the systemic administration of PF4 and if the reason no neurogenstream by activated platelets and was found to induce neurogene- esis was seen after the hippocampal injection could be potentially sis in the DG. Through proteomic analysis, it was found that a sig- due to multiple blood-borne components acting on the DG. The authors did look at the relative amount of platelet nificantly greater amount of PF4 was released after 4 days of exeractivation over a longer period of time (i.e. >4 days) and found cise in RUN mice compared to STD mice (figure.3A). Additionally, in there was a decrease in activated platelets. However, Leiter et al. the neurosphere assay, a greater number of neurospheres were should further tract the effects of other systemic blood compoobserved when DG cells were treated with PF4 (figure.3B). nents over weeks or months of exercise as it has been found that long-term exercise also affects cell proliferation and health of the Discussion & Conclusion The primary conclusion in the study by Leiter et al. is that hippocampus (Oâ&#x20AC;&#x2122;Callaghan et al., 2009). As there are many systemexercise-induced platelet activation increases neurogenesis in the ic blood compounds released as a result of physical activity, other adult hippocampus. Furthermore, the activated platelets released upregulated factors can be analyzed to determine their involvePF4, which was greatly upregulated in RUN mice. This factor, when ment in the exercise-induced neurogenesis mechanism. released by activated platelets after exercise, generates proliferation and differentiation of NPCs and neural stem cells of the DG. Future Directions With age comes a decline in hippocampal neurogenesis This was determined by isolating plasma from sedentary and active mice, culturing the plasma with cells from the dentate gyrus and changes in blood composition (Villeda et al. 2011). Platelets and observing significant increases in cell growth after just one day have been seen to clearly show age-related changes as they dein the RUN plasma culture. This mechanism may be the missing crease in numbers and increase in reactivity (Jones, 2016). It is still part that explained how exercise is linked to increased neurogene- unclear whether these age-related changes in blood could coincide sis and provides us with a better functional understanding of neu- with potential cognitive impairment and decline in hippocampal function and neurogenesis. rogenesis. Future research can look into whether PF4 levels also Their findings are consistent with other research focusing decline with age through isolating the plasma of young and old on blood biomolecules interacting with the blood-brain barrier. mice who have either exercised for acute periods of time or have Other studies have found that the compounds released from exerremained sedentary. By preforming in vivo neurosphere assays cise may also improve cognitive function and play a role in neuroprotection, reducing risk of neurodegenerative diseases (Tari et al. with cells from the DG and the different plasma, it can be deter2019). Additionally, other blood-borne factors were also found mined through number of neurospheres whether PF4 levels in that influenced hippocampal cell differentiation such as chemo- blood remained the same in young and old mice. If PF4 levels dekines (eotaxin) which increases with age and when present in high crease with age then there should be no significant difference between RUN and STD old mice, as well as noticeably less neurolevel in blood reduces neurogenesis (Villeda et al., 2011). The authors determined that exercise induces acute, transi- spheres in old RUN mice compared to young RUN mice. However, ent platelet activation and that PF4 acts as a peripheral regulator if no changes in PF4 levels occur with age then old mice should for hippocampal NPC proliferation (Schroer et al., 2019). Overall, show similar result as young mice in the STD and RUN neurosphere this provides further evidence into understanding how and why assays. This would then indicate this factor may not be the primary compound that induced neurogenesis in the brain. Additionally, a exercise is so beneficial for the brain. proteomic screen can be done on the young and old RUN mice to Critical Analysis Previously mentioned, Leiter et al. found a new systemic observe any changes in protein levels and platelet responsiveness. Past experiments have used heterochronic parabiosis as component in the blood released by activated platelets that may

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well as blood transfusions to determine that blood borne factor induce neurogenesis (Villeda et al. 2011). Intravenous injections of young plasma to old mice has been found to have regenerative capabilities on cells, tissues and organs including the brain (Hofmann, 2018). To further test PF4 in vivo, researchers can inject PF4 and young mouse serum systemically or locally into old mice to determine if it can induce neurogenesis and slow down cognitive decline and/or enhance hippocampal function. To measure learning and memory in mice the novel object recognition test, the Morris water maze and the Y-maze test may be used (Holter et al., 2015). Additionally, MRI brain scans can be done to study hippocampal volume as an indicator for neurogenesis (Erickson et al., 2011). If neurogenesis is observed in the hippocampus or any learning or memory improvements are seen when injected with only PF4, then PF4 can very likely be a key component in the exercise-induced neurogenesis mechanism. However, if no changes occur with only PF4 administration but intravenous injections of young plasma still cause neurogenesis then PF4 either does not induce neurogenesis in vivo, or some other systemic compounds may be playing a role in this pathway such as VEGF, IGF-1, Irisin or Cathepsin B (Rendeiro & Rhodes, 2018). It also may show that PF4 is only a small part of a larger mechanism involving multiple bloodborne compounds interacting, all of which are needed for proliferation of NPCs in the DG. Further research must be performed to better understand the role of exercise-induced platelet activation on hippocampal NPCs. A better understanding of this pathway and the biomolecules involved may allow for potential therapeutic targets to induce hippocampal neurogenesis and increase hippocampal function such as with learning and memory.

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Brain-Selective Estrogen improves α-Synuclein homeostasis, neuropathology, and motor phenotype in Parkinson’s Disease model Claire Wilson

Parkinson’s Disease (PD) is a neurodegenerative disorder characterized by the accumulation of a misfolded protein called α-synuclein. This protein aggregates in inclusions called ‘Lewy bodies,’ which are associated with neuronal cell death. Damage occurs especially to dopaminergic (DAergic) neurons in the substantia nigra, which are responsible for controlling movement via dopamine signalling. Motor deficits ensue, including tremor, rigidity, bradykinesia, poor balance, swallowing difficulties, facial masking, and slurred speech. It is unknown what causes Parkinson’s disease, or what causes α-synuclein to aggregate into Lewy bodies. The disorder seems to result from a complex interplay between genes and the environment, and the progression of each case is unique. However, it has been welldocumented that risk increases with age, and that men are more likely to develop the disease than women. In the study conducted by Rajsombath et al. (2019), the authors seek to elaborate on the effect of female sex and estrogen on the progression of Parkinson’s. They discovered that female sex and treatment with brain-selective estrogen significantly reduced α-synuclein aggregates, PD neuropathology, and motor deficits in vivo. These findings illuminate the role of sex differences in α-synuclein aggregation and neuropathology. The authors provide evidence for a new therapy that could revolutionize the treatment of a disease whose progression has not been slowed thus far. Keywords: Parkinson’s disease α-synuclein, dopamine, neurons, neuropathology, female sex, estrogen, treatment

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BACKGROUND or INTRODUCTION. Although Parkinson’s is often branded as a movement disorder, it may trigger autonomic dysfunction, emotion dysregulation, and cognitive impairment, including dementia and psychosis (Kalia & Lang, 2015). There is no cure, and no treatment that can stop or slow the progression of the disease. So far, treatments are focused on symptom management, and even these can have devastating side effects and lose their effectiveness over time (Kalia & Lang, 2015). So, there is an urgent need for treatment that can curtail the progression of the disease. Parkinson’s disease (PD) is incredibly complex, but two main epidemiological findings have emerged – risk increases with age, and according to sex. Men may have as much as 1.5 times the risk of developing PD, and often experience a younger age of onset (Wooten et al., 2004; Taylor, Cook, & Counsell, 2007). Men also experience more severe motor deficits than women (Brann et al., 2007). The disparity between sexes may be partially attributable to differences in exposure or genetic factors, but it is more likely due to female sex hormones. The neuroprotective effects of estrogen (17β-estradiol) are wellestablished (Brann et al., 2007). It can shield the brain from environmental toxins and can also enhance the survival and growth of dopamine neurons (Sawada et al., 1998; Brann et al., 2007). Studies have shown that women who experienced decreased estrogen stimulation during life had higher PD risk, while women who experienced increased estrogen stimulation had reduced PD risk (Liu & Dluzen, 2007; Rocca, Grossardt, & Maraganore, 2008, Gillies et al., 2014). Furthermore, low-dose estrogen has been shown to improve motor symptoms in postmenopausal PD patients (Tsang, Ho, & Lo, 2000). However, there are still reports which debate the effect of estrogen on PD risk, and estrogen therapy is controversial due to the hormone’s role in breast cancer (Wang et al., 2015; Gillies et al., 2014; Yager & Davidson, 2006). Two recent developments in the field have inspired the present study by Rajsombath et al. (2019). Firstly, a new form of brainselective estrogen called DHED (10β,17β-dihydroxyestra-1,4-dien3-one) was discovered in 2015 (Prokai et al., 2015). DHED is an inactive estrogen precursor that is converted to active estrogen (17β-estradiol) in the brain. Since DHED remains inactivated in the rest of the body, it can be safely used to treat neurological disorders without causing detrimental peripheral side effects. DHED was recently successful in treating a mouse model of Alzheimer’s disease, which has a misfolded protein pathology similar to Parkinson’s (Tschiffely et al., 2018). However, the effect of DHED on αsynuclein, the protein which becomes misfolded in Parkinson’s disease, is unclear. The second recent development is the discovery that native α-synuclein exists not only as an unfolded monomer, but also as an aggregation-resistant tetramer (Bartels, Choi, & Selkoe, 2011). The α-synuclein E46K mutation (3K) disrupts the tetramer:monomer (T:M) ratio and causes neurotoxicity and αsynuclein cytoplasmic inclusions (Dettmer et al., 2015). Thus, the α -synuclein tetramer:monomer ratio is increasingly recognized as a relevant measure of α-synuclein PD pathology (Tschiffely et al., 2018). In light of these new developments, Rajsombath et al. (2019) tested the effect of female sex and brain-selective estrogen (DHED) on α-synuclein neuropathology and motor symptoms in an α-synuclein tetramer-abrogating (3K) mouse model of PD. They measured α-synuclein tetramerization & solubility, dopaminergic & cortical fibre integrity, and associated motor deficits. They found that female 3K mice had a significant delay in onset and reduced

severity of pathology in comparison to male mice. Furthermore, DHED-treated male mice exhibited improved pathology on par with female levels (Rajsombath et al. 2019). This study reveals that α-synuclein homeostasis may be an important new mechanism for understanding sex differences in PD prevalence. The article also presents evidence for a new treatment, brain-selective estrogen, which may finally be the drug that slows down Parkinson’s disease. MAJOR RESULTS The authors first observed earlier onset of motor symptoms with greater deficits overall in 3K (PD) males compared to 3K females. The PD motor phenotype first presented at ten weeks of age and worsened throughout the 6-month observation period. At 3-4 months of age, male mice demonstrated significantly increased hindlimb clasping compared to females, who did not succumb to clasping until 6 months of age. By 6 months of age, all male mice demonstrated stiff, uncoordinated gait. 3K males had significantly increased pole test times in comparison to 3K females, who were actually on par with wild-type (WT) females in that test (Figure 1). While 3K females had decreased wire test (Figure 1) and rotarod endurance, 3K males had even less endurance (Rajsombath et al. 2019). These results support the hypothesis that males have increased motor deficits, and that these deficits are due to sex hormones since the model controlled for genetic and environmental factors (Brann et al., 2007; Gillies et al., 2014).

Figure 1. Sex differences in pole test time and wire test endurance at age 6 months in 3K (PD) mice versus age-matched controls. Bars represent mean +/- standard error margin (SEM). *p<0.05, ** p<0.01, ***p<0.001; two-way ANOVA post Turkey. (Figure adapted from Rajsombath et al. 2019). Female sex increases α-synuclein T:M ratio, solubility, and neurite complexity The mechanism behind these sex differences in motor symptoms is elucidated by Rajsombath et al. (2019) in their study of αsynuclein homeostasis and neurite complexity. Quantitative Western Blots of cortical extracts revealed significantly increased aggregate-resistant tetramers and decreased aggregate-prone monomers in 3K females v. 3K males. Thus, females have a higher αsynuclein tetramer:monomer ratio (Figure 2). Furthermore, 3K females have greater prevalence of soluble α-synuclein, and less insoluble (Lewy body) α-synuclein than 3K males (Figure 2). So, female sex hormones are not just protective of neurons, but are also associated with improvement of the earliest known PD pathology. Female 3K mice also demonstrated an associated increase in cortical and dopaminergic neurite fibre length and branching in cortical sections (Figure 3). This increase corresponded with augmented striatal dopamine levels measured by HPLC assay. This

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finding provides further evidence for estrogen’s neuroprotective and trophic effects on dopaminergic neurons (Brann et al., 2007; Sawada et al., 1998). Although, this protection may be occurring via α-synuclein homeostasis.

This finding confirms the estrogen-responsive effects of DHED in the brain. The authors also performed motor testing and found that 3K males had significant improvement in pole test performance and rotarod endurance, and even surpassed WT males in motor skills. 3K DHED-treated females also surpassed their WT counterparts. This evidence suggests that DHED ameliorates Parkinson’s pathology via healthy regulation of the α-synuclein T:M ratio.

Figure 2. Sex differences in α-synuclein tetramer:monomer (αS60:αS14) ratio and solubility. Syn1 antibody detects increased T:M ratio in female 3K mice using quantitative Western Blot (WB) (n-3 mice per genotype). WBs of TBS-soluble, Triton X-100-soluble, and Triton X-100-insoluble extractions in 3K females v. males. *p<0.05 (unpaired two-tailed t-test). (Figure adapted from Rajsombath et al. 2019). Figure 4. DHED treatment improves aS-60 tetramer: aS-14 monomer ratio in a stepwise manner. Bars represent mean +/- SEM. *p<0.05; two-way ANOVA post-Turkey. (Figure adapted from Rajsombath et al. 2019).

Figure 3. Sex differences in cortical and dopaminergic neuron length and complexity in non-transgenic (Ntg) and 3K mice. Estimated by NeuronStudio software (n=10 traced neurons or cortical sections). Bars represent mean +/- SEM. *p<0.05, ** p<0.01, ***p<0.001; two-way ANOVA post Turkey. (Figure adapted from Rajsombath et al. 2019). DHED treatment improves T:M ratio by autophagy clearance, DAergic fibre density motor symptoms The authors Rajsombath et al. (2019) first assessed α-synuclein T:M ratio and observed that DHED treatment significantly increased T:M ratio in 3K males & females in a stepwise manner (Figure 4). Treatment in 3K males recovered tetramerization to female levels, and treatment boosted 3K female tetramerization even closer to the wild type. The authors also observed a substantial decrease in vesicle-rich α-synuclein aggregates and suspected increased autophagy turnover as a mechanism. Electron microscopy established the presence of highly abnormal lysosomes in untreated 3K males. They investigated LC3 (autophagic protein) overlapping with p-SER-LAMP-1 inclusions. Confocal microscopy staining revealed increased LC3 puncta within pSer129 aggregates in 3K -DHED-males and 3K females. Additionally, overall LC3 protein levels increased significantly along with colocalized LC3: aggregate ratio (Figure 4) These data substantiate the hypothesis that increased autophagy turnover of aggregate-prone monomers is responsible for increasing the T:M ratio. Next, Rajsombath et al. (2019) measured levels of the dopamine precursor, tyrosine hydroxylase (TH), and found that DHED treatment in 3K males recovered TH to wild-type levels. TH staining of caudate putamen sections also depicted increased dopaminergic fibre densities in 3K-DHED males on par with 3K females (Figure 5).

Figure 5. DHED treatment improves dopaminergic fibre density (represented by TH+ staining). Images from dorsal caudate putamen sections of 3K males, 3K-DHED males, and 3K females (n=4 per group). (Figure adapted from Rajsombath et al. 2019). CONCLUSIONS/DISCUSSION Rajsombath et al. (2019) conducted the first study which observed the effects of female sex and brain-selective estrogen on a transgenic mouse model of Parkinson’s disease. The authors expanded the body of literature by using a newly-discovered α-synuclein tetramer-abrogating mouse model (3K) of PD, whereas previous studies commonly used toxin-based models, or other transgenic mutants. They found that in comparison to male 3K mice, female 3K mice experienced ameliorated PD neuropathology and delayed onset of motor symptoms. Treatment with brain-selective estrogen (DHED) had beneficial effects on both male and female 3K mice by improving T:M ratio, autophagy turnover, dopaminergic fibre density, and motor symptoms. These findings provide evidence that the sex differences in PD are due to female sex hormones, rather than genetic or environmental factors. Furthermore, the improved neurite fibre densities are supportive of a neuroprotective role for estrogen. Estrogen’s neuroprotective effects have been previously documented, but Rajsombath et al. (2019) provide further evidence for estrogen’s ability to ameliorate PD symptoms once the disease has already set in, a finding that has been replicated only a few times. Importantly, the authors elucidate a new mechanism for the effect of estrogen on Parkinson’s disease. They found evidence that estrogen most likely improves the α-synuclein tetramer:monomer ratio via increased autophagy turnover of aggregate-prone monomers (Rajsombath et al., 2019). The authors also observed strong

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correlations between α-synuclein solubility and fibre complexity, and between α-synuclein tetramerization and improved motor symptoms (Rajsombath et al., 2019). These combined results point strongly towards an upstream role for α-synuclein tetramerization in maintaining fibre integrity and delaying subsequent PD motor pathology. The success of treatment with brain-selective estrogen paves the way for developing a new therapy to treat men and postmenopausal women with Parkinson’s disease. CRITICAL ANALYSIS Although the authors (Rajsombath et al., 2019) have proposed a mechanism for α-synuclein homeostasis, more research must be done to determine if they are correct in assuming that estrogen is increasing autophagy turnover of aggregate-prone monomers. So far, they have only pointed toward a single association between an autophagic protein (LC3) and colocalization with vesicle-rich aggregates. Colocalization does not confirm causation. As opposed to looking at a snapshot of aggregation and protein colocalization, future research should take many cortical sections throughout the course of DHED treatment. This would clarify the association between autophagic proteins and α-synuclein solubility. Then, DHED should be administered at different doses in order to determine if autophagic proteins are expressed in a dose-dependent manner.

relationship between T:M ratio and fibre densities. Although, a delay in effect on fibre densities would not rule out an upstream role for α-synuclein aggregation, since it may act cumulatively. If there was no relationship between T:M ratio and fibre densities, then T:M ratio could be affecting motor symptoms by regulating a different pathway. Furthermore, researchers should test the effects of DHED on various genetic and toxin-based animal models of Parkinson’s disease to see if DHED continues to ameliorate α-synuclein aggregation, dopaminergic fibre complexities, and PD motor symptoms. If DHED reduces aggregation, then this supports a role for estrogen in autophagy turnover. If aggregation is not ameliorated, but fibre complexities and motor symptoms improve, then the positive effects of estrogen may be attributable instead to its neurotrophic effects or unknown neuroprotective capabilities. To date, the effects of estrogen are still under debate. This may be due to the prevalence of retrospective studies that cannot control for variation in estrogen dosage and timing. Given the paucity of research on the role of estrogen in Parkinson’s disease, scientists should immediately undertake a large observational and prospective human study to study the effects of estrogen and hormone therapy on Parkinson’s disease occurrence and pathology.

Moreover, it is unclear how estrogen is able to increase vesicle fusion for autophagy. If estrogen does indeed regulate autophagy turnover, it remains to be seen whether this is its only mechanism for modulating α-synuclein tetramerization. In addition, the authors indicate that α-synuclein has a potential upstream role in ameliorating PD neuropathology and motor symptoms. While an upstream role is possible, they overstate their case, which relies on associations instead of causations. Despite observing an increased α-synuclein T:M ratio, neurite complexity, and motor symptoms after DHED treatment, it is quite possible that estrogen is modulating each of these outcomes independently. Previous research has shown that estrogen has neurotrophic effects on dopamine fibres. Also, it is becoming clear that men and women experience Parkinson’s disease differently, and women naturally have less motor symptoms. So, DHED could be amplifying an existing pathway for female motor resilience. The authors should study the effect of DHED on α-synuclein and neuropathology in the many genetic and environmental models of Parkinson’s disease so that they are not limited to a tetramer-abrogating model, which would already be more sensitive to T:M ratio. FUTURE DIRECTIONS Future research should be directed at replicating the effects of DHED treatment on Parkinson’s disease pathology. In order to validate the proposed mechanism suggested by Rajsombath et al. (2019), researchers should administer DHED at low, medium, and high doses. If T:M ratio increases in a dose-dependent manner, it would verify estrogen’s modulation of tetramerization. It is also necessary to confirm that α-synuclein tetramerization is indeed a modulator of PD neuropathology. Otherwise, estrogen’s effect on tetramerization is irrelevant, and sex differences may not be due to α-synuclein homeostasis. To corroborate an upstream role of tetramerization in PD neuropathology, researchers should sample cortical sections throughout the disease course to observe T:M ratio and fibre densities. Researchers should expect to find a direct

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Estrogen Matters: The Impact of Neuron-Derived Estradiol Katrina Wugalter

While estrogen is necessary for female sexual development, recent research has found that it may also impact certain areas of the brain. Neuroimaging research and neuropsychological tests have revealed that the hippocampus may be an important region of estrogen function. Additionally, studies with ovariectomized animals or post-menopausal women have shown associations between estrogen loss, cognitive decline, and reductions in hippocampal volumes. Researchers have proposed that the reduction of circulating estrogens post-menopause may explain the high prevalence of Alzheimer’s Disease in older women. An interesting finding that complicates estrogen’s effects is the neural production of 17B-estradiol by the aromatase enzyme, found in animals and humans. The recent paper by Lu et al. (2019) utilized a forebrain-specific aromatase knockout mouse model to investigate the impact of neuron-derived estrogen on learning, memory, neural transmission, kinase and neurotrophic signalling, and synaptic plasticity. Their results indicated that loss of neuron-derived estradiol led to reductions in hippocampal-dependent cognitive performance, excitatory transmission, and long-term potentiation induction. The knockout mice also had lower concentrations of phosphorylated kinases and brain-derived neurotrophic factor. Exogenous estradiol treatment in vivo “rescued” the deficits in kinase and neurotrophic signalling, and spatial learning and memory, while in vitro addition of estradiol to hippocampal slices recovered long-term potentiation induction. The researchers conclude that neuron-derived estradiol is a key component of synaptic plasticity and memory and may act through kinases and neurotrophic factors. The limitations of this paper and future directions for the field are discussed.

Key words: estrogen, estradiol, synaptic plasticity, hippocampus, menopause, aromatase, estrogen receptors, hormone replacement therapy

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INTRODUCTION Recent research in endocrinology, psychology, and neuroscience has discovered unexpected effects of estrogen on neuroplasticity and cognition. While estrogen’s role in sexual development is well-established, its impact on the brain is a growing topic as more researchers are investigating women’s health issues. The higher prevalence of Alzheimer’s Disease among women compared to men suggests that there are unaddressed sex differences in the nervous system (Chambers, Bancej, & Mcdowell, 2016). Research regarding the interplay between estrogen and the brain may provide important insight to develop prevention and therapeutic strategies for women affected by neurological conditions. The connection between estrogen and cognition has motivated many studies investigating older women due to the decline in estrogen concentrations caused by menopause. A fundamental study by Sherwin (1988) found that women who underwent total abdominal hysterectomy or bilateral salpingo-oophorectomy had higher scores on working memory tasks after taking estrogen hormone replacement therapy than those taking a placebo. Another experiment found that women using a gonadotropin releasing hormone antagonist had worse performance on working memory and executive function tasks and reported worsened mood over time (Grigorova, Sherwin, & Tulandi, 2006). Additionally, a longitudinal study found that a longer reproductive window and the use of hormone therapy were positively associated with better cognitive status later (Matyi, Rattinger, Schwartz, Buhusi, & Tschanz, 2019). Neuroimaging has revealed that women with higher estrogen concentrations have higher gray matter volume and more functional connectivity in the hippocampal region than women with minimal estrogen (Lisofsky et al., 2015; Lord, Buss, Lupien, & Pruessner, 2008). Together, these findings support the hypothesis that estrogen plays a role in cognition and therefore, postmenopausal women may be at risk of cognitive decline. While research with human subjects has its benefits, confounding factors may impact the results. Oophorectomized women represent an interesting group in this field because their estrogen production stops abruptly; however, many women who undergo oophorectomies and hysterectomies have been diagnosed with other conditions such as endometriosis or cancer which may have unprecedented cognitive effects. Additionally, researchers believe surgical menopause is more detrimental than natural menopause for bone, cardiovascular, and cognitive health and therefore may not accurately depict the effects of natural estrogen loss on women’s health (Bove et al., 2014). Due to the limitations of human research, a significant amount of animal research has been conducted. Woolley and her colleagues are responsible for foundational studies establishing a positive correlation between estrogen levels and spine density in the cornu ammonis (CA1) pyramidal cells of the rat hippocampus (Gould, Woolley, Frankfurt, & McEwen, 1990; Woolley, 1998). Multiple studies with rodents have found similar results to human research regarding estrogen’s effects on cognition, with estrogen treatment eliciting better performance on spatial and working memory tasks (Talboom, Williams, Baxley, West, & Bimonte-Nelson, 2008; Tuscher et al., 2016). Both human and animal research currently shows a significant effect of estrogen on the brain, inducing structural and functional changes. Several different mechanisms contribute to estrogen’s effect in the nervous system. First, previous research has highlighted β subtype estrogen receptors (ERβ) as more important than α subtype receptors (ERα) for enhancing plasticity, synaptic develop-

ment, and cognition (Liu et al., 2008; Zhao, Woody, & Chhibber, 2015). Second, estrogen signaling may rely on kinase networks because the excitatory effects of estradiol (E2) have been shown to be inhibited when kinases were blocked (Hasegawa et al., 2015). Lastly, and most importantly for this review, estrogen is not only synthesized in the ovaries but is produced in the brain by the aromatase enzyme which converts testosterone to estradiol (E2), and is expressed in the temporal and hippocampal regions (Azcoitia, Yague, & Garcia-Segura, 2011; Stoffel-Wagner, Watzka, Schramm, Bidlingmaier, & Klingmueller, 1999 ). The multi-experiment study by Lu and colleagues (Lu et al., 2019) sought to investigate multiple different proposed mechanisms regarding the connection between estrogen and neural activity. The inconsistency in past studies due to “off-target” effects of inhibition methods and lack of precision in knocking out aromatase only in neurons encouraged the authors to create a novel forebrain-neuron-specific-aromatase-knockout (FBN-ARO-KO) mouse model to further investigate the impact of neuron-derived estrogen on synaptic growth, synaptic transmission, spine density, kinase activity, spatial memory, fear conditioning, and depression and anxiety symptoms (Lu et al., 2019). The first part of the study demonstrated that structural and functional deficits elicited by lack of neuron-derived estrogen in the FBN-ARO-KO mouse in comparison to the control (FLOX) mouse may be explained by abnormal kinase and neurotrophic signaling pathways. The second and third parts of the study used hippocampal slices and in vivo treatment, respectively, to reveal that neuron-derived E2 is necessary for long -term potentiation (LTP) and exogenous E2 may reverse the effects of estrogen loss. Lu et al. (2019) found significant structural and cognitive deficits in mice lacking neuron-derived estradiol, indicating estrogen’s role in plasticity and cognition. MAJOR RESULTS In Lu et al.’s (2019) study, immunohistochemistry and enzyme-linked immunosorbent assay revealed a reduction in neuron-derived E2 levels in the CA1 and cortex of male and female ovariectomized (ovx) FBN-ARO-KO mice in comparison to FLOX mice. The overall E2 levels between FBN-KO-ARO mice and FLOX mice were not significantly different, suggesting the knockout procedure successfully disrupted neural E2 but not circulatory E2 (Lu et al., 2019). FBN-ARO-KO mouse behavioral and structural differences The researchers compared behavioral and neurostructural differences between FBN-ARO-KO mice and FLOX mice. Results revealed that hippocampal-dependent learning and memory performance was significantly reduced in the FBN-ARO-KO mice compared to the FLOX mice because escape latency on the Barnes maze task was significantly lower, preference for a new object in the novel object recognition task was decreased, and contextual fear conditioning was disrupted (Lu et al., 2019). Using the forced swim test and open field test, Lu et al. (2019) found no differences in depressive or anxious behaviors between FBO-ARO-KO mice and FLOX mice. Lu et al. (2019) also observed a decrease in mean spine density (Figure 1.) and reduced concentrations of synaptic markers synaptophysin and PSD95 in the cortex and the CA1 region of the hippocampus in FBN-ARO-KO mice compared to FLOX mice. Additionally, they found an increase in ERβ and a decrease in ERα in FBN-ARO-KO mice, suggesting a compensatory upregulation (Lu et al., 2019).

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Figure 1. Spine density in the CA1 and cortex of female FLOX and female FBN-ARO-KO mice. FLOX mice have significantly more spines than FBN-ARO-KO mice. Figure adapted from Lu et al. (2019). Neuron-derived estrogen regulates synaptic plasticity and memory. Journal of Neuroscience, 39(15), 2792–2809. https:// doi.org/10.1523/jneurosci.1970-18.2019

Results revealed that BDNF levels, synaptophysin and PSD95 levels, and phosphorylation of AKT, ERK, and CREB were rescued by administration of exogenous E2 to female ovx FBN-ARO-KO mice (Lu et al., 2019). The authors highlight that this is a “true rescue” because E2 treatment had no enhancing effect on FLOX mice (Lu et al., 2019). According to Figure 3., the exogenous E2 administration also normalized ERβ and ERα levels in the FBN-ARO-KO mouse (Lu et al., 2019). Lastly, the deficits in spatial learning and memory observed on the Barnes maze and novel object recognition tasks were attenuated by E2 treatment in FBN-ARO-KO mice (Lu et al., 2019).

Synaptic transmission and cellular signaling mechanisms The findings revealed reduced excitatory transmission in the FBN-ARO-KO mice compared to FLOX mice (Lu et al., 2019). Application of E2 to hippocampal slices from FBN-ARO-KO mice was shown to attenuate these reductions (Lu et al., 2019). Using high frequency stimulation, Lu et al. (2019) measured LTP induction in the mice, demonstrating significant impairments in LTP amplitude in the FBN-ARO-KO mice, which was rescued in vitro by adding E2 to brain slices. U0126 was added to inhibit extracellular signal-regulated kinase (ERK) signalling which prevented the “rescue effects” of exogenous E2 in both excitatory transmission and LTP induction, suggesting the importance of kinase pathways in estrogen’s effects (Lu et al., 2019). As demonstrated in Figure 2., Lu et al. (2019) also found a decrease in brain-derived neurotrophic factor (BDNF) and phosphorylated protein kinase B (AKT), ERK, and cyclic adenosine monophosphate response element binding protein (CREB) concentrations in the hippocampus and cortex of FBN-ARO-KO mice compared to the FLOX mice.

Figure 3. Levels of estrogen receptors in ovx female FBN-ARO-KO mice relative to FLOX mice. A. Levels of ERβ in the FBN-ARO-KO placebo group are significantly different from FLOX mice and the E2 treatment group in the CA1 and the cortex B. Levels of ERα in the FBN-ARO-KO placebo group are significantly different from the FLOX mice and the FBNARO-KO E2 treatment group in the CA1 and the cortex. *p<0.05 versus FLOX + PL group, #p<0.05 versus FBN-ARO-KO + PL group. Figure adapted from Lu et al. (2019). Neuronderived estrogen regulates synaptic plasticity and memory. Journal of Neuroscience, 39 (15), 2792–2809. https://doi.org/10.1523/jneurosci.1970-18.2019

CONCLUSIONS The most important conclusion drawn from the Lu et al. (2019) study is that neuron-derived estrogen impacts cognition and synaptic plasticity in mice. Using an aromatase knockout model, the researchers investigated the effects of neuron-derived estrogen loss on neural structure, synaptic transmission, LTP induction, and hippocampal-dependent cognitive performance (Lu et al., 2019).

Figure 2. Levels of phosphorylated AKT, ERK, and CREB, and BDNF in ovx female FBN-ARO-KO mice relative to FLOX levels. A. Levels of p-AKT, p-ERK, p-CREB, and BDNF in the CA1. B. Levels of p-AKT, p-ERK, p-CREB, and BDNF in the cortex. Figure adapted from Lu et al. (2019). Neuron-derived estrogen regulates synaptic plasticity and memory. Journal of Neuroscience, 39(15), 2792– 2809. https://doi.org/10.1523/jneurosci.1970-18.2019

Exogenous estradiol treatment in vivo

The results demonstrated significant reductions in spine density and synaptic markers, indicating that estrogen is important for the growth and maintenance of synapses (Lu et al., 2019). These findings are consistent with past research that discovered a positive correlation between estradiol levels and dendritic spine density in CA1 pyramidal neurons (Frankfurt & Luine, 2015; Woolley, 1998). LTP was inhibited in the FBN-ARO-KO mice but could be rescued by exogenous E2 treatment (Lu et al., 2019). The researchers believe the rescue effects demonstrate that LTP inhibi-

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tion is not caused by the decrease in spine density because the effects of exogenous E2 were immediate. Lu et al. (2019) also found a significant decrease in BDNF and phosphorylated kinases in the FBN-ARO-KO mice in comparison to the FLOX mice, indicating a potential rapid intracellular pathway that may underlie the observed changes in synaptic plasticity. BDNF is an important protein for neuron survival which suggests that low BDNF levels could be modulating the structural and functional deficits in the FBNARO-KO mice (Lu et al., 2019). Another significant finding from this paper is that estrogen loss disrupted hippocampal-dependent cognitive performance, while exogenous E2 treatment rescued the learning and memory deficits (Lu et al., 2019). These findings are consistent with prior research demonstrating a significant relationship between estrogen and memory in rodents (Talboom et al., 2008; Tuscher et al., 2016 ). By employing a variety of measures, the authors were able to capture a detailed explanation of the function of neuron-derived estrogen that is supported by previous research.

that NMDA antagonism inhibited the effects of estradiol on spine density, while AMPA and muscarinic receptor antagonism had no effect (Woolley & McEwen, 1994). Lu et al. (2019) did not investigate NMDA receptor activity but it is possible that LTP is blocked in the FBN-ARO-KO mice due to decreased sensitivity of NMDA receptors upon estrogen loss. Considering the importance of neuronderived estrogen highlighted by Lu et al. (2019), a future experiment should investigate the sensitivity of NMDA receptors in the presence of estrogen by treating the FBN-ARO-KO mouse with an NMDA agonist, antagonist, and placebo. If the effects on LTP are elicited by neuron-derived estrogen’s effect on NMDA receptors, the addition of an agonist should rescue the deficits in the FBNARO-KO mouse, while an antagonist and placebo should both have no effect because LTP is already reduced in the mice due to lack of estrogen. If the NMDA agonist has no effect in the FBN-ARO-KO mouse, it would suggest that the impact of neuron-derived estrogen on LTP is not mediated by NMDA receptors. The proposed study would provide insight into the mechanisms underlying the connection between neuron-derived estrogen and memory.

CRITICAL ANALYSIS The paper by Lu et al. (2019) had several advantages beyond previous studies. As mentioned in the paper, the use of a forebrain specific knockout model ensured precision and accuracy because there was no “off-target” effects of inhibitory methods and estrogen production in the ovaries remained, allowing the researchers to investigate neuron-derived estrogen (Lu et al., 2019). Additionally, comparing male, female intact, and female ovx mice allowed Lu et al. (2019) to draw conclusions regarding which effects are different between the sexes, which are driven by circulatory estrogen, and which are influenced by neuron-derived estrogen. While Lu et al. (2019) presented significant findings and interesting conclusions regarding neuron-derived estrogens, it may be difficult to generalize many of the findings to humans. The mouse is a reliable model organism; however, the human body is much more complex. First, the results from Lu et al. (2019) cannot explain why the reduction in circulatory estrogen leads to cognitive defects in post-menopausal women (Matyi et al., 2019). Additionally, while exogenous estradiol treatment reversed the deficits in the FBN-ARO-KO mice in the present study, estrogen therapy is not an ideal treatment for all women affected by estrogen-related neural changes because certain cancers are estrogen-sensitive and therefore, their growth is exacerbated by high estrogen concentrations (Chen et al., 2004). Chen et al. (2004) demonstrated that hormone replacement therapy increased the risk of estrogenreceptor-positive carcinoma; therefore, it would be ideal to find an alternative treatment rather than additional exogenous E2. The authors should further investigate underlying mechanisms to understand if a potential treatment could act on intracellular systems rather than stimulating the estrogen receptors themselves.

A unique aspect of exogenous estrogen treatment that was not mentioned by Lu et al. (2019) is the theory of a critical period for estrogen’s rehabilitative effects. Research in rodents and humans suggests that the benefits of exogenous estrogens may be limited to a certain post-menopausal time period (Daniel, Hulst, & Berbling, 2006; Maki, 2013). Researchers should employ a longitudinal study comparing the effects of exogenous estradiol on control mice and FBN-ARO-KO mice after ovariectomy to visualize whether neural changes over time are mediated by circulating or neuron-derived estrogens. An abrupt reduction in cognitive performance after ovariectomy in both groups would suggest that circulating estrogens are crucial for brain functioning, while no decline after oophorectomy in the control mice but a sharp decline in FBNARO-KO mice would suggest that neuron-derived estradiol is important. They could then observe changes in memory performance after treating control and FBN-ARO-KO mice with exogenous E2 at different timepoints post-ovariectomy to determine the critical period for estrogen’s effect on cognition. This would help create a timeline for the efficacy of post-menopausal estrogen treatment to develop adequate treatment plans to prevent cognitive decline. Although further research is required, the paper by Lu et al. (2019) confirms that neuron-derived estradiol is important for cognition and plasticity, and therefore plays a role in brain health.

FUTURE DIRECTIONS The findings and limitations from Lu et al. (2019) suggest potential future research in this field. First, the finding that LTP induction is reduced by estrogen loss should be studied further because it might explain the observed memory deficits. Previous research has highlighted an interaction between estrogen and NMDA receptors that may mediate changes in memory performance and cognitive structure (Smith & McMahon, 2005; Vedder, Smith, Flannigan, & McMahon, 2013). An older experiment found

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Azcoitia, I., Yague, J.G., & Garcia-Segura, L.M. (2011). Estradiol synthesis within the human brain. Neuroscience, 191, 139-147. Bove, R., Secor, E., Chibnik, L. B., Barnes, L. L., Schneider, J. A., Bennett, D. A., & de Jager, P. L. (2014). Age at surgical menopause influences cognitive decline and Alzheimer pathology in older women. Neurology, 82(3), 222–229. https://doi.org/10.1212/ WNL.0000000000000033 Chambers, L. W., Bancej, C., & Mcdowell, I. (2016). The Prevalence and Monetary Costs of Dementia in Canada. Retrieved from www.alzheimer.ca Chen, W. Y., Hankinson, S. E., Schnitt, S. J., Rosner, B. A., Holmes, M. D., & Colditz, G. A. (2004). Association of hormone replacement therapy to estrogen and progesterone receptor status in invasive breast carcinoma. Cancer, 101(7), 1490–1500. https:// doi.org/10.1002/cncr.20499 Daniel, J.M., Hulst, J.L., & Berbling, J.L. (2006). Estradiol replacement enhances working memory in middle-aged rats when initiated immediately after ovariectomy but not after a long-term period of ovarian hormone deprivation. Endocrinology, 147, 607-614. Frankfurt, M. & Luine, V. (2015). The evolving role of dendritic spines and memory: Interaction(s) with estradiol. Hormones and Behaviour, 74, 28-35. Gould, E., Woolley, C. S., Frankfurt, M., & McEwen, B. S. (1990). Gonadal steroids regulate dendritic spine density in hippocampal pyramidal cells in adulthood. The Journal of Neuroscience, 10(4), 1286-1291. Grigorova, M., Sherwin, B. B., & Tulandi, T. (2006). Effects of treatment with leuprolide acetate depot on working memory and executive functions in young premenopausal women. Psychoneuroendocrinology, 31(8), 935–947. https://doi.org/10.1016/ j.psyneuen.2006.05.004 Hasegawa, Y., Hojo, Y., Kojima, H., Ikeda, M., Hotta, K., Sato, R., … Kawato, S. (2015). Estradiol rapidly modulates synaptic plasticity of hippocampal neurons: Involvement of kinase networks. Brain Research, 1621, 147–161. https://doi.org/10.1016/ j.brainres.2014.12.056 Lisofsky, N., Mårtensson, J., Eckert, A., Lindenberger, U., Gallinat, J., & Kühn, S. (2015). Hippocampal volume and functional connectivity changes during the female menstrual cycle. NeuroImage, 118, 154–162. https://doi.org/10.1016/ j.neuroimage.2015.06.012 Liu, F., Day, M., Muñiz, L. C., Bitran, D., Arias, R., Revilla-Sanchez, R., … Brandon, N. J. (2008). Activation of estrogen receptor-β regulates hippocampal synaptic plasticity and improves memory. Nature Neuroscience, 11(3), 334–343. https://doi.org/10.1038/ nn2057 Lord, C., Buss, C., Lupien, S. J., & Pruessner, J. C. (2008). Hippocampal volumes are larger in postmenopausal women using estrogen therapy compared to past users, never users and men: A possible window of opportunity effect. Neurobiology of Aging, 29(1), 95–101. https://doi.org/10.1016/j.neurobiolaging.2006.09.001 Lu, Y., Sareddy, G. R., Wang, J., Wang, R., Li, Y., Dong, Y., … Brann, D. W. (2019). Neuron-derived estrogen regulates synaptic plasticity and memory. The Journal of Neuroscience, 39(15), 2792–2809. https://doi.org/10.1523/jneurosci.1970-18.2019 Maki, P.M. (2013). Critical window hypothesis of hormone therapy and cognition: a scientific update on clinical studies. The Journal of The North American Menopause Society, 20(6), 695-709. Matyi, J. M., Rattinger, G. B., Schwartz, S., Buhusi, M., & Tschanz, J. T. (2019). Lifetime estrogen exposure and cognition in late life: the Cache Country Study Menopause, 26(12) https://doi.org/10.1097/GME.0000000000001405 Sherwin, B.B. (1988). Estrogen and/or androgen replacement therapy and cognitive functioning in surgically menopausal women. Psychoneuroendocrinology, 13(4), 345-357. Smith, C.C. & McMahon, L.L. (2005). Estrogen-induced increase in the magnitude of long-term potentiation occurs only when the ratio of NMDA transmission to AMPA transmission is increased. Journal of Neuroscience, 25(34), 7780-7791. Stoffel-Wagner, B., Watzka, M., Schramm, J., Bidlingmaier, F., & Klingmueller, D. (1999). Expression of CYP19 (aromatase) mRNA in different areas of the human brain. Journal of Steroid Biochemistry and Molecular Biology, 70(4-6), 237-241. Talboom, J.S., Williams, B.J., Baxley, E.R., West, S.G., & Bimonte-Nelson, H.A. (2008). Higher levels of estradiol replacement correlate with better spatial memory in surgically menopausal young and middle-aged rats. Neurobiology of Learning and Memory, 90 (1), 155-163. Tuscher, J.J., Szinte, J.S., Starrett, J.R., Krentzel, A.A., Fortress, A.M., Remage-Healey, L., & Frick, K.A. (2016). Inhibition of local estrogen synthesis in the hippocampus impairs hippocampal memory consolidation in ovariectomized female mice. Hormones and Behaviour, 83, 60-67. Vedder, L.C., Smith, C.C., Flannigan, A.E., & McMahon, L.L. (2013). Estradiol-induced increase in novel object recognition requires hippocampal NR2B-containing NMDA receptors. Hippocampus, 23(1), 108-115. Woolley, C. S. (1998). Estrogen-mediated structural and functional synaptic plasticity in the female rat hippocampus. Hormones and Behaviour, 34(2), 140-148. Woolley, C. S., & McEwen, B. S. (1994). Estradiol regulates hippocampal dendritic spine density via an N-Methy-D-Aspartate receptor-dependent mechanism. The Journal of Neuroscience, 14(12), 7680-7687. Zhao, L., Woody, S.K., & Chhibber, A. (2015). Estrogen receptor β in Alzheimer’s disease: From mechanisms to therapeutics. Ageing Research Reviews, 24, 178-190.

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Bisphenol A Upregulates the Expression of Neuropeptide Y Through Disruptions of the Regulation of Circadian Rhythm Jia Xi Xu

Long recognized as an obesogen and endocrine disruptor, bisphenol A (BPA) has been linked to disrupted energy balance. However, BPA’s direct effects on hypothalamic neurons that regulate energy homeostasis have not been thoroughly elucidated. The arcuate nucleus (ARC) of the hypothalamus contains neurons that secret various neuropeptides, including the orexigenic neuropeptide Y (NPY) that stimulates feeding behaviours. The suprachiasmatic nucleus (SCN), on the other hand, is the master regulator of circadian rhythm. SCN regulates circadian rhythm through the molecular clock, which involves a gene known as brain and muscle ARNT-like 1 (Bmal1). It is known that one of the ways that BPA acts to disrupt cellular functions is through altering gene expressions. In the study conducted by Loganathan et. al (2019), it was proposed that BPA upregulates NPY’s expression in the hypothalamus through the disruption of circadian rhythm, namely, through a Bmal1-dependent mechanism. Their findings indicated that the expressions of several clock genes, including Bmal1, were altered by BPA treatment. They also found that NPY expression levels were upregulated in wild-type (WT) cells following BPA treatment, however, the upregulation was abolished in Bmal1-knockout (KO) cells. To further support their findings, the authors conducted in silico analysis of the NPY promoter sequence, and potential binding sites for Bmal1 were identified. These results elucidated the mechanisms behind BPA’s orexigenic effects on a molecular level. The results of this study may help to generate a better understanding of the control of energy homeostasis in the hypothalamus and to direct future efforts in attempting to block BPA’s disruptions of energy balance. Key words: bisphenol A (BPA), circadian rhythm, neuropeptide Y (NPY), brain and muscle ARNT-like 1 (Bmal1), energy homeostasis, feeding behaviours, obesity

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INTRODUCTION Bisphenol A (BPA) is an organic synthetic compound. It is one of the most common chemicals in polycarbonic plastics and is found in receipts, canned food linings, and has been ubiquitously detected in drinking water.1 BPA exposure has been linked to adverse effects in humans including increased body mass index (BMI) and obesity, which can lead to elevated risks for type II diabetes mellitus, osteoarthritis, and some cancers.2 As such, it is of value to investigate BPA’s effects on the regulation of energy homeostasis and the molecular mechanisms behind any disruptions. The regulation of energy homeostasis is largely controlled by the arcuate nucleus (ARC) of the hypothalamus. A heterogeneous population of neurons in the ARC secret several types of neuropeptides that directly affect food intake and energy expenditure.3 Disruptions in the balance between orexigenic versus anorexigenic neuropeptides secreted by these neurons can drastically alter feeding behaviours and can potentially lead to obesity. One of the most potent orexigenic neuropeptides is neuropeptide Y (NPY).4 With both food intake and energy expenditure showing circadian patterns, the regulation of energy homeostasis is tightly associated with the regulation of circadian rhythm, which is predominantly controlled by the molecular clock in the suprachiasmatic nucleus (SCN).5 The molecular clock consists of several proteins, including the ones coded by circadian locomotor output cycles kaput (Clock) and brain and muscle ARNT-like 1 (Bmal1). Clock and Bmal1 are transcription factors that heterodimerize to form the Clock:Bmal1 complex.6 The Clock:Bmal1 complex, in turn, activates the expressions of other clock genes.7 Although the association between the regulation of energy homeostasis and circadian rhythms has long been established,8 molecular mechanisms behind this connection as well as how environmental chemicals such as BPA may disrupt the function of this system have not been thoroughly explored. In the study conducted by Loganathan et. al, the authors utilized immortalized hypothalamic cell lines to investigate BPA’s effects on the expression of the orexigenic neuropeptide NPY and clock genes, as well as the connections between the regulations of energy homeostasis and circadian rhythms.9 In addition to several previously generated cell lines, the authors also generated a Bmal1 -knockout (KO) cell line specifically for this study. Their major experimental approach included quantitative reverse transcription polymerase chain reaction (qRT-PCR), which detects changes in gene expression on the RNA level and Western blotting, which detects the presence of specific proteins. In addition, the group performed in silico analysis of the regulatory region of the NPY gene. Their findings indicated that BPA upregulates the expression of NPY in a Bmal1-dependent manner in that in Bmal1-KO lines, the upregulation of NPY by BPA seen in wild-type (WT) cell lines was abolished. They also identified potential binding sites for the Clock:Bmal1 complex on the NPY promoter. These findings offered crucial insights into how BPA disrupts energy homeostasis and revealed the interdependent nature between the energy balance regulators in the ARC and the circadian rhythm regulators in the SCN. The newly generated Bmal1-KO cell line can also be used in future studies interested in the cellular and molecular functions of Bmal1 in relation to circadian rhythm regulations.

of three clock genes, including Bmal1, were altered by BPA treatment at different time points in a 24-hour time course, as shown in Figure 1.9 These results indicate that BPA can disrupt the regulation of circadian rhythm though altering levels of gene expression. Most importantly, Bmal1 expression level was seen to be elevated at the 4-hour timepoint in all four cell lines when compared to the control. Two other clock genes, Per2 and Rev-Erbα also showed altered expression levels following BPA treatment.

Figure 1. The expression levels of three genes involved in circadian rhythm regulation, including Bmal1, were altered by BPA treatment at different timepoints in four mouse hypothalamic cell lines (two embryonic and two adult). Gene expression levels in BPA treated cells (shown in black squares) were compared to vehicle controls (shown in white circles). Figure Adapted from Loganathan et al. (2019). Endocrinology, 160(1), 181-192.

BPA Upregulates NPY Expression in Bmal1-WT Cells In two immortalized Bmal1-WT cell lines (one male and one female) newly generated by Loganathan et. al from Bmal1-WT mice, significant upregulations of NPY expression were found following an 8-hour BPA treatment, as identified by qRT-PCR (shown in Figure 2). As NPY directly stimulates food intake, these results can be interpreted as NPY mediating BPA’s long-established orexigenic effects.11

MAJOR RESULTS BPA Disrupts Clock Gene Expressions Using previously generated embryonic and adult hypothaFigure 2. The expression levels of NPY was upregulated following an 8lamic cell lines,10 Loganathan et. al found that the expression levels hour treatment in the Bmal-WT cells and the upregulation was abol458

ished in both the male and the female Bmal-KO lines. CRITICAL ANALYSIS Figure Adapted from Loganathan et al. (2019). Endocrinology, 160(1), Although the article corroborated that Bmal1 is the key reg181-192. ulator in BPA’s upregulation of NPY, the evidence was mostly indi-

Upregulation of NPY Expression by BPA Abolished in Bmal1-KO Cells Loganathan et. al generated two immortalized Bmal1-KO cell lines from Bmal1-KO mice (one male and one female) as well as a Bmal-WT cell line to act as a control. The absence of the Bmal1 protein was attested by Western blotting, as shown in Figure 3. It was found that the upregulations of NPY expression seen in the WT cells were abolished in the Bmal1-KO cell lines, as seen in Figure 2. These results suggest that the upregulation of the orexigenic neuropeptide NPY was mediated by the Bmal1 protein. Thus, a direct association between the energy balance regulatory system and the circadian rhythms regulatory system was established.

rect. For example, the binding sites identified by the authors were only hypothetical, the actual binding of Bmal1 was not determined. Granted, the authors showed that in Bmal1-KO cells, the upregulation of NPY by BPA was abolished, which is the strongest evidence for Bmal1’s direct involvement in the upregulation, the abolishment of the upregulation could be due to confounding factors introduced during the generation of the novel KO cell lines. It is possible that expression levels of other genes were altered in the KO cell lines, which actually mediated the upregulation of NPY by BPA. To rule out this possibility, the authors need to further characterize their newly generated immortalized cell lines by screening for the expression levels of genes involved in circadian regulations and energy homeostasis, especially the ones with expression regulatory functions, and compare the expression profiles of the KO cells to the WT cells. As well, the study was only conducted in vitro with cell cultures acutely exposed to BPA.16 To improve the generalizability of their results, the authors may want to follow up with an in vivo study focusing on the effects of long-term BPA exposure on energy homeostasis and circadian rhythm. In addition, potential roles of other cells present in the hypothalamus, such as microglial cells, Figure 3. Western blotting results confirm that Bmal1was not present in were neglected in the study, which may require further investigathe Bmal1-KO cell lines. tion. Figure Adapted from Loganathan et al. (2019). Endocrinology, 160(1), 181-192. FUTURE DIRECTIONS

Potential Binding Sites for the Clock:Bmal1 Complex Identified on the NPY Promoter Using sequences 2500 bp upstream of the transcription starting site of NPY, Loganathan et. al identified several potential binding site for the heterodimer Clock:Bmal1, known to have DNA binding activities.9,12 These putative binding sites for the Clock:Bmal1 complex were identified with reference to previously determined sequences.13 The in silico analysis further supports the authors’ hypothesis that Bmal1 can directly alter NPY’s expression levels through its DNA binding activities.

CONCLUSIONS/DISCUSSION The results of the study conducted by Loganathan et. al showed a clear connection between circadian rhythm regulation and energy homeostasis.9 They demonstrated the mechanisms behind the effects of BPA on the expression of NPY, an orexigenic neuropeptide. Bmal1, a part of the circadian rhythm regulatory system, was identified to be the key transcription factor mediating BPA’s upregulation of NPY expressions in the neurons regulating energy homeostasis.9 Three novel immortalized cell lines were generated for this study – a Bmal1-WT cell line containing a heterogeneous population of neurons and two Bmal-KO cell lines. The study elucidated the previously unknown pathway behind BPA’s actions on neurons involved in energy homeostasis regulation. As a ubiquitously present environmental chemical, the elucidation of the mechanisms behind BPA’s orexigenic effects may eventually lead to the discovery of potential ways of blocking its actions on the molecular level. The results also offered further evidence that the circadian rhythm regulatory system and the energy homeostasis regulatory system are closely linked, especially with regards to the Clock:Bmal1 complex since it has been repeatedly shown to play a role in energy balance.14,15 As well, the newly generated immortalized cell lines can be used in future studies interested in Bmal1 and its functions as well as for studying other functions of hypothalamic neurons.

To rigorously test if Bmal1 directly regulates NPY’s expression, the authors should perform a chromatin immunoprecipitation sequencing (ChIP-Seq) procedure to verify Bmal1’s binding to NPY’s regulatory region.17 ChIP-Seq aims both to verify the putative binding of Bmal1 to the promotor of NPY and to identify the sequence of DNA that Bmal1 binds to. Another technique that could be used to study the binding of transcription factors to DNA is electrophoretic mobility shift assay (EMSA), however, EMSA is not capable of identifying the specific sequence of DNA that the transcription factor binds to. Since several different sequences of potential binding sites, each around 6-bp long, were identified by the authors, ChIPSeq’s ability of pinpointing the specific sequence of the binding site becomes valuable. If Bmal1 indeed binds to NPY’s promotor, the DNA sequence identified by ChIP-Seq can be used to clarify which, if any, of the proposed binding sequences is consistent with the experimental results. If the hypothesized binding of Bmal1 cannot be verified by ChIP-Seq, then it is possible that Bmal1 is not the direct regulator of NPY expression, but an upstream regulator. If this is the case, then the authors may proceed to investigate potential downstream targets of Bmal1 that may be directly binding to NPY’s promotor to alter its expression. The authors should also look into conducting in vivo studies aiming at investigating effects of chronic BPA exposure using Bmal-WT and Bmal-KO mice from which their cell lines were generated to determine if BPA’s disruptions of gene expressions are maintained over a longer period of time and if Bmal1 is also involved in the chronic effects of BPA. BPA is known to induce neuroinflammation, which may be mediated by microglial cells in the hypothalamus. Therefore, the author may also want to investigate BPA’s effects on hypothalamic microglial cells, for example, they might see an elevated production of proinflammatory cytokines following exposure to BPA, which may interact with the neurons involved in circadian and energy homeostasis regulations.

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Physical Exercise as an Effective Therapy for Delaying Alzheimer’s Disease Progression Beatrix Yu

Physical exercise is increasingly being recognized as an effective therapy for neurodegenerative diseases. However, the underlying mechanisms involved in the therapeutic efficacy of exercise for Alzheimer’s disease (AD) is still not fully understood. Previous studies have implicated brain energy metabolic impairment as one of the main features of AD and may be an underlying factor involved in cognitive impairment. Glucose is the main source of energy for neurons and require glucose transporters (GLUTs) to cross the blood-brain barrier into the extracellular space of the brain, as well as into neurons. A large body of evidence suggest that GLUT1 and GLUT3 deficiencies coupled with impairment in glycolysis correlate with the severity of AD pathology. Recently, the study by Pang et al. (2019) demonstrated that regular exercise ameliorated AD pathological hallmarks in AD model mice by improving brain glucose metabolism. Results from the study showed that regular exercise significantly increased mitochondrial integrity and ATP production levels as well as GLUT1 and GLUT3 expression in the brain of AD model mice. Regular exercise also significantly decreased amyloid-beta (Aβ) and phosphorylated tau (P-tau) expression in the brain of AD model mice, with subsequent improvements in cognitive function. The findings by Pang et al. (2019) suggest that physical exercise can be a potent non-pharmacological intervention in delaying AD progression and have important implications for future studies targeting glucose metabolic impairment in the brain to investigate promising treatments for AD. Key words: Alzheimer’s disease, glucose metabolism, energy metabolism, regular exercise, glucose transporters, mitochondria, amyloid-beta, phosphorylated tau, cognitive function

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INTRODUCTION. Alzheimer’s disease (AD) is the most common neurodegenerative disorder and form of dementia world-wide. Up to 95% of cases of AD are sporadic and late-onset, whereas only up to 5% of AD is genetic (Bali, Gheinani, Zurbriggen, & Rajendran, 2012). AD pathology is characterized by the accumulation of beta amyloid (Aβ) and neurofibrillary tangles (NFTs) composed of abnormally phosphorylated tau (P-Tau) (Grundke-Iqbal, et al., 1987). To date, the exact mechanisms involved in the therapeutic efficacy of exercise for AD is still not fully understood or characterized completely. However, it has been implicated that brain energy metabolic impairment is considered an underlying factor involved in cognitive impairment (Hunt, et al., 2007). Glucose, the primary energy source for neurons, cannot be synthesized or stored by neurons and cannot cross the blood-brain barrier (BBB) freely. Thus, glucose transporters (GLUTs) are essential in facilitating the transport of glucose from the peripheral circulation across the BBB and into brain tissue, as well as mediating cerebral energy metabolism homeostasis (Liu, Liu, Iqbal, Grundke-Iqbal, & Gong, 2008). While GLUT1 is highly expressed in the endothelial cells of the BBB and is responsible for the transport of glucose from the blood into the extracellular space of the brain, GLUT3 is mainly expressed on neurons and helps transport glucose from the extracellular space into the neuron (Liu, et al., 2008). Since neurons have limited glycolytic capacity, mitochondrial energy production is crucial and accountable for approximately 90% of cerebral adenosine triphosphate (ATP) production (Rolfe & Brown, 1997). There is a large body of evidence suggesting that glucose metabolism and mitochondrial dysfunction is an underlying cause of cognitive impairments in neurodegenerative diseases, including AD. Previous studies have demonstrated a reduction in GLUT1 and GLUT3 expression in the brain of AD patients (Simpson, Chundu, Davies-Hill, Honer, & Davies, 1994). The pathogenic link between glycolysis and the development of Aβ has also been implicated by studies as it was found that the distribution of regional glucose metabolism via glycolysis correlates spatially with Aβ deposition in both healthy young adults and individuals with AD (Vlassenko, et al., 2010). In fact, the production of ATP from glucose metabolism in sporadic AD was observed to decline by 50% (Hoyer, 2002), and continued to decline throughout disease progression. Moreover, it has been demonstrated that the downregulation of GLUT3 in brain regions vulnerable to AD pathology were due to low rates of glycolysis and high glucose concentrations in brain tissue (An et al., 2018). Currently, research in non-pharmacological interventions for AD has become increasingly important as there is no treatment or approved drug available for the disease. With AD being a major healthcare burden and the number of AD patients expected to reach 106 million by 2050 (Norton, Matthews, Barnes, Yaffe, & Brayne, 2014), effective and safe therapies for AD are urgently needed. Thus, emerging evidence supporting physical exercise as an effective therapy for delaying AD progression by means of ameliorating brain glucose metabolism has become a topic of interest and importance. In the study by Pang et al. (2019), amyloid beta precursor protein and presenilin 1 (APP/PS1) double-transgenic AD model mice were used to investigate the effects of regular exercise on AD pathological symptoms including cognitive function. Mice were divided into four groups; wild-type mice (WT-NT) and AD model mice with no treatment (AD-NT) were used as control groups and did not have access to exercise; wild type mice (WT-T) and AD

model mice with treatment (AD-T) were used as treatment groups and received regular swimming exercises for 1 hour, six days per week over the course of 4 weeks. After 4 weeks, the cognitive function of mice was tested using the Morris water maze apparatus. Aβ and P-tau expression in the hippocampus and cortex of mice was analyzed via western blotting. GLUT1 and GLUT3 expression was detected using double immunofluorescence labelling assays. Lastly, electron microscopy was used to detect mitochondrial integrity in the brain of mice and ATP levels were measured using an ATP Assay kit. Results of this study demonstrated that regular exercise significantly increased GLUT1 and GLUT3 expression, with subsequent decrease in Aβ and P-tau expression. Regular exercise also significantly improved mitochondrial integrity and ATP levels in the brain of AD model mice, as well as improving cognitive impairments. These findings suggest that physical exercise can be an effective therapy for delaying AD progression and have important implications for future studies that could target brain glucose dysregulation in AD to investigate therapeutic interventions. MAJOR RESULTS GLUT1 and GLUT3 expression Through immunofluorescence labelling, the authors observed a significant reduction of GLUT1 and GLUT3 expression in AD-NT mice (Figure 1), aligning with the observation of decreased GLUT1 and GLUT3 levels in the brains of AD patients (Simpson, et al., 1994). The decrease in GLUT3 expression in AD-NT mice is also consistent with the study by An et al. (2018) which proposed brain glucose dysregulation and the subsequent downregulation of GLUT3 as a feature of AD. In addition, results indicate that AD-T mice showed significantly higher GLUT1 and GLUT3 expression in both the hippocampus and cortex compared to AD-NT mice (Figure 1). More notably, GLUT3 expression in the cortex of AD-T mice after 4 weeks of regular exercise was almost on par with WT-NT mice (Figure 1C). These findings validate the existing hypothesis of brain glucose dysregulation as a feature of AD and demonstrate that regular exercise can ameliorate GLUT1 and GLUT3 expression in AD model mice.

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Figure 1. GLUT1 and GLUT3 expression in the cortex and hippocampus of mice. A, B. immunoblots of GLUT1. C, D. immunoblots of GLUT3. #p<0.05, ##p<0.01 compared with the AD-NT group. n=5 for each group. AD-T mice showed significantly higher GLUT1 and GLUT3 expression than AD-NT mice in both the cortex and hippocampus after regular exercise. Figure adapted from Pang et al. (2019). Mitochondrial integrity and ATP production levels Mitochondrial integrity in the brain of mice was observed with electron microscopy and ATP levels were measured using an ATP Assay kit. While mitochondria in the AD-NT group showed low integrity, with blurred edges and broken cristae, the AD-T group showed improved mitochondrial integrity after 4 weeks of regular exercise, with clear mitochondrial edges and dense cristae (Figure 2B-F). Subsequently, ATP levels in the AD-T group was significantly improved, and much higher than the AD-NT group in both the cortex and hippocampus (Figure 2G, H). The results corroborate with studies that have found a fragmented mitochondrial phenotype that disturbed mitochondrial activity in AD patients' brains (Bernardo, et al., 2016). The results also verify mitochondrial dysfunction as a feature in AD pathology by demonstrating lower levels of ATP production in the brain of AD model mice, which further validates the existing hypothesis of brain energy metabolism dysfunction as a feature of AD. Overall, the results demonstrate that regular exercise restored mitochondrial integrity and ATP levels in the brain of AD model mice and can ameliorate brain energy metabolism dysfunction.

Figure 2. Mitochondrial integrity and ATP levels in the brain of mice. A-F. Mitochondrial integrity in the cortex. G. ATP levels in the hippocampus H. ATP levels in the cortex. ##p<0.01 compared with AD-NT group. n=5 for each group. AD-T mice showed significantly higher ATP levels in the cortex and hippocampus compared to AD-NT mice after regular exercise. Figure adapted from Pang et al. (2019).

to reduce Aβ deposition by strengthening the activity of an Aβ degrading enzyme known as neprilysin. Moreover, the results also align with previous studies that have found a negative correlation between GLUT1 and GLUT3 expression with levels of tau phosphorylation (Liu, et al., 2008). Overall, the results demonstrate that regular exercise significantly reduced Aβ and P-Tau expression in AD model mice.

Figure 3. Aβ and P-Tau expression. A-C. Immunoblots of Aβ and PTau in the cortex. D-F. Immunoblots of Aβ and P-Tau in the hippocampus. #p<0.05, #p<0.01 compared with the AD-NT group. n=5 for each group. AD-T mice showed significantly lower Aβ and P-Tau expression compared to AD-NT mice after regular exercise. Figure adapted from Pang et al. (2019). Cognitive ability Regular exercise improved cognitive abilities in AD model mice. Spatial learning and memory ability evaluated by the Morris water maze test showed that AD-T mice had a higher frequency of passing the hidden platform position compared to AD-NT mice (Figure 4B). In fact, the AD-T group showed almost similar learning ability to the WT-NT group. These results provide evidence that regular exercise was able reduced cognitive impairments in AD model mice by increasing spatial learning and memory ability. These findings are also consistent with an existing study by Lin et al. (2015) which demonstrated that exercise was able to increase neurogenesis in the hippocampus and subsequently enhance hippocampus-associated memory in addition to Aβ clearance in AD model mice.

Aβ and P-Tau expression Western blotting indicated that regular exercise reduced Aβ and P-Tau expression in the cortex and hippocampus of AD model mice. While the AD-NT mice showed a significant increase in Aβ and P-Tau expression in both the hippocampus and cortex, AD-T mice showed significantly decreased expression in both areas after 4 weeks of regular exercise (Figure 3). The observed decrease in Aβ expression levels after regular exercise aligns with the study by Maesako et al. (2012) which demonstrated that exercise was able

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Figure 4. Learning and memory ability tested by the Morris water maze apparatus. A. Navigation traces for the Morris water maze test. B. Number of times mice cross the hidden platform position. #p<0.05 compared with the AD-NT group. n=25 for each group. AD -T mice cross the hidden platform at a higher frequency than ADNT mice, demonstrating better learning and memory ability. Figure adapted from Pang et al. (2019). DISCUSSION/CONCLUSIONS Overall, Pang et. al (2019) demonstrated that regular exercise ameliorated AD symptoms by increasing brain glucose metabolism. The results indicate that increased mitochondrial ATP production and expression of GLUTs correlate with lower Aβ and P -Tau expression in AD model mice along with subsequent cognitive improvements. These findings support the hypothesis of brain energy metabolic impairment being an underlying factor of cognitive impairment in AD and suggest that physical exercise can be a potent non-pharmacological therapeutic to delay AD progression. The findings by Pang et a. (2019) has important implications for future research in the controversial topic regarding physical exercise as an effective therapy for delaying AD progression. In another study by Tarumi et al. (2019), cognitive function and brain volume between two groups of adults with Aβ-positive amnestic mild cognitive impairment were compared. Over 12 months, one group did regular aerobic exercise, while the other group only did stretching exercises. Results after 12 months showed that although the aerobic exercise group showed less volume reduction in the hippocampus than the stretching group, Aβ accumulation and cognitive abilities did not differ between the two groups. The current study by Pang et al. (2019) has demonstrated a novel finding that regular exercise was able to reduce both Aβ levels and cognitive impairments in AD model mice.

methodology. Considering that AD-T mice were treated with regular swimming exercise over 4 weeks, it may have put them in favor to better performance during the Morris water maze test compared to AD-NT mice. The performance of both AD-T and AD-NT mice during the test could have been affected by mere expertise or competence in swimming, more so than cognitive ability such a spatial learning and memory alone. Other forms of exercise, such as wheel running, could be used as treatment to account for this confound. In addition, other factors known to contribute to mitochondrial dysfunction and glucose metabolism such as reactive oxygen species (ROS) and brain derived neurotrophic factor (BDNF) should be studied further. Leuner et al. (2012) demonstrated that mitochondrial dysfunction involved the increased production of reactive oxygen species (ROS) which was found to enhance Aβ production. BDNF release from the human brain was demonstrated to increase two-to three-fold during exercise (Rasmussen, et al., 2009), and its plasma levels in AD patients have found to be positively correlated with their physical activity level (Coelho, et al., 2014). BDNF has also been linked with synaptic mitochondrial health by improving glucose transport and respiratory coupling efficiency, as well as mediating mitochondrial biogenesis (Marosi & Mattson, 2014). Furthermore, BDNF is demonstrated to play a neuroprotective role against toxic effects induced by Aβ peptides (Arancibia, et al., 2008). More importantly, BDNF was found to decrease over the course of AD progression with negatively correlated serum levels and severity of dementia (Laske, et al., 2005). As BDNF is demonstrated to contribute to synaptic mitochondrial health, glucose transport and resistance to Aβ toxicity, BDNF can be another potential candidate marker for AD therapy.

CRITICAL ANALYSIS Pang et. al (2019) demonstrated that regular exercise was able to reduce a range of AD pathological hallmarks and could be an effective non-pharmacological therapy to delay AD progression. However, there are discrepancies between the authors’ results and literature, and there are elements of the paper that requires further study. The efficacy of exercise as a therapy for AD progression remains controversial as multiple studies have found no correlation between exercise and the modification of Aβ deposition. Frederiksen et al. (2019) demonstrated that moderate to high intensity exercise does not modify cortical Aβ levels in AD patients. In the work by Hatashita & Wakebe (2019) it was also found that Aβ deposition is independent of glucose metabolism and that glucose metabolism does not contribute to AD progression in preclinical AD subjects. Although the results from Pang et al. (2019) provided evidence for the brain energy metabolic dysfunction hypothesis behind AD pathology and demonstrated that regular exercise can ameliorate AD symptoms, the underlying mechanisms behind such observations still remain unknown and current findings in the study as well as literature are still largely correlational. Therefore, even though Pang et al. (2019) have concluded that the failure of glucose utilization is a fundamental feature of AD, further studies are needed to verify the directionality of this relationship as it is not clear whether glucose hypometabolism is a cause or an effect of AD. The study by Pang et al. (2019) also had limitations in

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Linking the Hypotheses of Depression: Dopamine D3 Receptor Knockout Mice Exhibit Both Depressive Symptoms and Microglia Activation Cindy M. Zhou

Depression has been extensively studied in recent years, yet the etiology behind it remains complex and difficult to pinpoint. Research has generated many theories of depression, but two hypotheses remain the leading motivators for current research. One being the gliocentric hypothesis, which suggests that glial cells—in particular microglia—are involved in depression by inducing the release of pro -inflammatory cytokines such as tumour necrosis factor alpha (TNF-a), interleukin-1beta (IL-1b), and IL-6. The second being the monoamine imbalance hypothesis, which suggests that imbalances in serotonin, norepinephrine—and most recently dopamine—are responsible for the depressive symptoms. A deficiency in the D3 receptor in particular has been shown to be linked to depression. Yet, the mechanism through which it induces depressive symptoms is unclear. A study conducted by J. Wang et al (2019) using D3 receptor knockout (D3RKO) mice proposed that depressive symptoms of D3RKO may be mediated by microglia activation. Namely, D3RKO mice exhibited neuroinflammation in mesolimbic regions of the brain in addition to depressive symptoms. These effects were localized to microglia and were reversible via administration of microglial inhibitors like minocycline and PLX3397. Their findings are particularly relevant in the etiology of depression due to the fact that i) it provides a possible answer to the question of how D3 deficiency induces depression ii) it links the gliocentric and monoamine hypotheses of depression and iii) it has implications for improving the selectivity of depression treatments. Key words: depression, D3 receptor knockout (D3RKO), minocycline, PLX3397, microglia activation, neuroinflammation, pro-inflammatory cytokines

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INTRODUCTION Depression remains a debilitating disease with increasing prevalence rates (Hasin et al., 2018). As a result of this, research in recent years has focused on elucidating the etiology behind depression (Czeh & Nagy, 2018). One major hypothesis of depression is the “gliocentric hypothesis”, which suggests that all three types of glial cells are involved in depression (Czeh & Nagy, 2018). However, majority of research seems to focus on the role of microglia. In particular, there is an abundant amount of evidence suggesting that activated microglia induce neuroinflammation via the release of pro-inflammatory cytokines such as tumour necrosis factor alpha (TNF-a), interleukin-1beta (IL-1b), and IL-6, all of which map onto depressive symptoms (Czeh & Nagy, 2018; Feltes et al., 2017). This is true in both human and rat models of depression (TorresPlatas, Cruceanu, Chen, Turecki & Mechawar, 2014; Setiawan et al., 2015; Tynan et al., 2010; Fagundes, Glaser, Hwang, Malarkey & Kiecolt-Glaser, 2013, Hodes et al., 2014). In depressed human and rat models, there is either an increase in the amount of activated microglia observed via Iba1 staining (Torres-Platas et al., 2014), or a direct increase in the number of inflammatory cytokines like IL-6 (Hodes et al., 2014). Thus, it is well-established that microglia activation induced neuroinflammation mediates depressive symptoms. An older hypothesis of depression that is still being extensively studied is the monoamine imbalance hypothesis (Leggio et al., 2013). This hypothesis focuses mainly on the contribution of serotonin and norepinephrine, but recent findings have shifted the focus onto dopamine (Leggio et al., 2013; Lemke, Brecht, Koester, Kraus & Reichmann, 2005). Specifically, imbalances in the mesolimbic dopaminergic system seem to be implicated in feelings of anhedonia and a loss in motivation (Lemke et. al, 2005). Within this system, the D3 receptor in particular has been shown to correlate with depression in a number of ways (Leggio et al., 2013; Moraga-Amaro, Gonzalez, Pacheco, & Stehberg, 2014; Y. Li et al., 2015; Rogóz & Skuza, 2006; Penciña et al., 2017). For example, D3 receptor knockout (D3RKO) mice have been shown to exhibit depressive symptoms that are not attributable to motor dysfunctions (Moraga-Amaro et al., 2014). As well, many antidepressants such as ketamine and pramipexole are known to target the D3 receptor (Y. Li et al., 2015; Rogóz & Skuza, 2006). Although a D3 receptor deficiency is known to be involved in depression, the exact biological mechanism through which it induces depressive symptoms is unclear (Moraga-Amaro et al., 2014; J. Wang et al., 2018). Recently, J. Wang et al. (2018) were able to show that administration of lipopolysaccharides (LPS)—which induce depressive symptoms—was able to lower D3 receptor levels and increase proinflammatory cytokines in mesolimbic regions of the brain; such as the medial prefrontal cortex (mPFC), nucleus accumbens (NAc), and ventral tegmental area (VTA). This finding in addition to the existing literature suggests that both microglia activation and D3RKO can induce depression via the release of pro-inflammatory cytokines (Hodes et al., 2014; J. Wang et al., 2018). Based on this observation, Wang and colleagues hypothesized in their current study that the mechanism through which D3RKO induces depression may in fact, be due to microglia activation. In order to test this hypothesis, the authors examined whether or not depressive symptoms in D3RKO mice can be ameliorated by microglia inhibitors of minocycline and PLX3397 (J. Wang et al., 2019). It was observed that both minocycline and PLX3397 were able to reverse the depressive symptoms in D3RKO mice, suggesting that microglia activation is the mediator of D3RKO induced depression. Their

research provides a preliminary answer to the question of how D3 inhibition induces depression, whilst also introducing an interesting link between the two major hypotheses of depression.

MAJOR RESULTS The results of J. Wang et al. (2019) were able to confirm the presence of depressive behaviour and neuroinflammation in D3RKO mice, whilst localizing D3RKO’s effects to microglia. The major finding of their study was that treatment using microglia inhibitors minocycline and PLX3397 was able to differentially improve performance on behavioural measures of depression, whilst also reducing the number of pro-inflammatory cytokines in certain mesolimbic regions. Their results overall suggest that D3RKO induced depression is mediated by microglia activation. Evidence of depression and neuroinflammation in D3RKO mice D3RKO mice were shown to have significantly worse performance in a battery of behavioural measures of depression compared to wild-type (WT) mice (J. Wang et al., 2019). D3RKO mice showed increased immobility in both the tail suspension test (TST) and the forced swimming test (FST), as well as a decreased sucrose preference on the sucrose preference test (SPT) (Figure 1).

Figure 1: Reduced performance on behavioural measures of depression in D3RKO mice. (C, D) D3RKO mice exhibited greater immobility on TST, and a decreased preference for sucrose. FST results not shown. Figure adapted from J. Wang et al. (2019). Brain, Behaviour, and Immunity, S0889-1591(19):30622-1.

Compared to WT mice, D3RKO mice had significantly more proinflammatory cytokines like TNF-a, IL-1b and IL-6 in the mPFC, NAc, and VTA (Figure 2). Both behavioural and biochemical data suggest that D3RKO mice show depressive symptoms. These findings are consistent with the authors’ past study (J. Wang et al., 2018), as well as an earlier study showing that D3RKO mice exhibit chronic depressive symptoms (Moraga-Amaro et al., 2014). D3RKO’s effects on glia cells are localized to microglia Although the D3 receptor was shown via western blot analysis to be present in both microglia and astrocytes of the mPFC, NAc, and VTA, the effects of D3RKO was only visible in microglia (J. Wang et al., 2019). Namely, only microglia showed differences in the number and density of Iba1 labelled cells between WT and D3RKO mice (Figure 3N); with Iba1 being a marker for activated microglia (J. Wang et al., 2019). On the other hand, GFAP was used to measure the number of activated astrocytes, but there was no significant difference in the number of GFAP labelled cells between WT and D3RKO mice. This observation broadly supports the gliocentric hypothesis, but more importantly supports the abundance of literature showing that microglia as opposed to astrocytes are the main contributors to neuroinflammation-induced depression (Feltes et al., 2017). The localization of D3RKO in microglia supports the authors’ hypothesis that D3RKO induced depression is microglia mediated.

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In terms of neuroinflammation, both minocycline and PLX3397 were able to lower the level of pro-inflammatory cytokines, but in differing mesolimbic regions (J. Wang et al. 2019). For example, minocycline lowered TNF-a only in the NAc and VTA, whilst PLX3397 lowered TNF-a in all three brain regions (Figure 5). Selective reduction was also seen for the other pro-inflammatory factors but are not reported in the present review due to space conFigure 2: Upregulation of pro-inflammatory cytokines in the mPFC, straints. NAc, and VTA of D3RKO mice compared to WT mice. (E, F) TNF-a, and IL-1b levels are significantly different between D3RKO and WT mice. IL6 levels are not shown, but also had increased expression in all three brain regions of D3RKO mice. Enzyme-linked immunosorbent assay (ELISA) was used to measure pro-inflammatory cytokine levels. Figure adapted from J. Wang et al. (2019). Brain, Behaviour, and Immunity, S0889-1591(19):30622-1. Figure 5: Minocycline and PLX3397’s effects on TNF-a levels in three mesolimbic regions. (A) Minocycline reduced TNF-a in NAc and VTA, but not mPFC. (E) PLX3397 reduced TNF-a in all three brain regions. Figure adapted from J. Wang et al. (2019). Brain, Behaviour, and Immunity, S0889-1591(19):30622-1.

CONCLUSIONS/DISCUSSION Figure 3: Immunofluorescence staining of activated microglia and astrocytes in WT and D3RKO mice. D3RKO induced glial activation is restricted to microglia. (N) Microglia were stained using Iba1 antibody and were observed to have more stained cells in D3RKO mice. (G) Astrocytes were stained with GFAP but did not show differences in the number of activated cells between WT and D3RKO mice. Figure adapted from J. Wang et al. (2019). Brain, Behaviour, and Immunity, S0889-1591(19):30622-1.

Microglia inhibitors minocycline and PLX3397 improve depressive symptoms in D3RKO mice Microglia inhibition via minocycline and PLX3397 were able to improve depressive symptoms and lower pro-inflammatory cytokines in selective brain regions. Mice that were given minocycline intraperitoneally at 25 mg/kg and 50 mg/kg showed improvements on all three behavioural tests (J. Wang et al., 2019, data only shown for TST). Mice that were given PLX3397 intragastrically at 40mg/kg showed improvement on TST, but not FST (not shown). Both drugs were able to improve sucrose preference, but minocycline in particular, was shown to have a dose-dependent effect on SPT (Figure 4).

The study by J. Wang et al. (2019) was able to conclude that D3RKO mice experience depressive symptoms likely due to an increase in pro-inflammatory cytokines in the mesolimbic system. This increase seems to be microglia mediated and can be reduced via the administration of microglia inhibitors like minocycline and PLX3397. The authors highlight how their findings support the role of D3 receptors in depression (J. Wang et al., 2019), and does in fact validate previously reviewed literature showing the relationship between D3 receptor deficiency and depression (MoragaAmaro et al., 2014; J. Wang et al., 2018). Previous research was able to show that a D3 receptor deficiency leads to depressive symptoms (Leggio et al., 2013; Lemke et al., 2005; Moraga-Amaro et al., 2014) whilst D3 receptor agonists are effective antidepressants (Leggio et al., 2013; Y. Li et al., 2015; Rogóz & Skuza 2006; Lammers et al., 2000). Yet, they were unable to elucidate the mechanism behind how D3 receptor deficiency leads to depression. Although the findings of the present study do not provide a definitive answer to this question, it does suggest that microglia induced neuroinflammation is involved. Thus, by proposing a preliminary link between the gliocentric and monoamine deficiency hypotheses of depression, J. Wang et al.’s (2019) research reveals a novel way of approaching the etiology of depression. If microglia induced neuroinflammation is the mechanism behind D3RKO depression, then microglia inhibitors may become more popularized as a selective, first-line treatment of depression. CRITICAL ANALYSIS

Figure 4: Minocycline’s effects on behavioural measures of depression. (C) TST immobility decreased significantly after both doses of minocycline treatment. (D) Minocycline had a dose-dependent effect on sucrose preference, where the higher dose yielded a greater preference than the lower dose. Figure adapted from J. Wang et al. (2019). Brain, Behaviour, and Immunity, S0889-1591(19):30622-1.

J. Wang et al. (2019) were able to conduct a meticulous experiment, but it was not without flaws. Although their results demonstrate that D3RKO only affects microglia and not astrocytes, there exists literature showing that astrocytes—in addition to having lower numbers in depressive patients (Si et al., 2014)—also play a role in neuroinflammation; and thus a possible role in D3RKO mediated depression (Kang et al., 2010; Ma et al., 2010). Furthermore, there is evidence that the neuroinflammatory factors secreted by activated microglia can trigger subsequent activation of astrocytes, promoting them into an A1 state akin to the M1 state of

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microglia (Liddelow et al., 2017). If this is the case, then the cause of D3RKO-induced depression cannot be attributed to microglial activation alone. A possible reason as to why astrocyte activation was not observed in J. Wang et al’s (2019) study is because the GFAP marker used may not be entirely selective for activated astrocytes (Liddelow & Barres, 2017). Studies have shown that high levels of GFAP labelled cells may indicate cortical atrophy as opposed to astrocytic activation or proliferation (Serrano-Pozo, Gómez-Isla, Growdon, Frosch, & Hyman, 2013). If GFAP does not accurately stain for astrocyte activation, it is possible that astrocytes were in fact activated in the study by J. Wang et al (2019). If this is true, then the authors cannot conclude that D3RKO induced depression is exclusively caused by microglia.

al., 2017), and would thus provide a better measure of astrocyte activation. Iba1 and C3 staining results can be used to infer whether microglia or astrocyte activation is responsible for the depressive symptoms in D3R-. For example, if neither are stained in D3R   mice, this suggests that although D3 inhibition can induce depressive symptoms, it is not enough to induce microglia OR astrocyte activation. This also suggests that the microglia activation seen in genetic D3RKO mice may therefore be due to compensatory mechanisms during development (J. Wang et al., 2019). If only Iba1 staining occurs, then it is possible that microglia activation is the sole contributor. If microglia activation led to astrocyte activation as previously observed (Liddelow et al., 2017), then there should be evidence of both Iba1 and C3 staining. If only C3 staining occurs, this would mean that an increase in neuroinflammation is Furthermore, the authors recognize that a limitation of their study solely due to astrocytes. However, this would be unlikely given the is the fact that mice were born D3 receptor deficient (J. Wang et well-characterized role of microglia in neuroinflammation (Czeh & al., 2019). This is problematic because it becomes unclear if micro- Nagy, 2018). glia activation is the result of D3 deficiency, or if it is the result of a compensatory mechanism (J. Wang et al., 2019). If a D3 deficiency The contributions of microglia and astrocytes to D3R- depressive post-birth cannot trigger microglia activation, then microglia may symptoms can be further elucidated via administering microglia not be the mediators of D3RKO induced depression. The authors and astrocyte inhibitors separately and concurrently. M1 selective should investigate further by inducing a loss of D3 receptors after minocycline (Kobayashi et al., 2013) should be used for microglia, birth, as opposed to starting with a genetically deficient model. and fluorocitrate (FC) (Y. Wang et al., 2019) should be used for This way, the causality of microglia activation can be determined. astrocytes. The goal of this research should be to test whether one inhibitor alone is more or less effective at reversing inflammatory Finally, while the antidepressant role of minocycline is well- and depressive symptoms compared to both. If either microglia or established (Zhang et al., 2019; A. Wang et al., 2005), the role of astrocytes alone are responsible for the D3R- induced depression, PLX3397 is less clear. Conflicting research has shown that PLX3397 then minocycline and FC treatment respectively should be able to can both reduce (M. Li et al., 2017), as well as increase (Jin et al., reduce neuroinflammatory and depressive symptoms. However, if 2017) the number of pro-inflammatory cytokines in the brain. This both microglia and astrocytes contribute, then the effect of giving is likely due to the fact that unlike minocycline which selectively both inhibitors should be greater than simply giving one. In other inhibits M1 microglia (Kobayashi et al., 2013), PLX3397 inhibits the words, there should be an even greater decrease in inflammatory entire CSF1R pathway which is needed for microglial survival (Li et and depressive behaviours when both minocycline and FC are gival., 2017; Jin et al., 2017). The use of non-selective GFAP and en compared to when only one is given. PLX3397 in J. Wang et al’s (2019) study demands further research to be conducted before the conclusion that microglia activation By conducting the experiments outlined above, researchers should mediates D3RKO depression can be validated. be able to gain a better understanding of i) what the true mechanism behind D3 deficiency mediated depression is ii) whether or FUTURE DIRECTIONS not this depression involves microglia, astrocytes, or both, and iii) how to improve selectivity of depression treatments, regardless if In order to rule out the effects of compensatory mechanisms on they are caused by neuroinflammation or monoamine deficiency. D3RKO-induced depression, researchers should test whether D3 receptor inhibition in WT mice will have the same effect on microglia activation and neuroinflammation. To do this, future studies should administer D3 receptor antagonists such as NGB 2904 (J. Wang et al., 2018) in WT mice as opposed to starting with mice that are genetically D3 deficient (D3RKO). Behavioural measures of depression such as FST, TST and SPT should then be taken, and compared between WT and D3 antagonist (D3R-) mice. Levels of pro-inflammatory cytokines should also be measured using quantitative real-time PCR (J. Wang et al., 2019). Both depressive symptoms and an increase in pro-inflammatory cytokines are expected given the well-characterized role of D3 deficiency in depression (Moraga-Amaro et al., 2014; Leggio et al., 2013; Y. Li et al., 2015). However, in order to fully understand whether or not these symptoms are due to microglia activation, immunofluorescence staining of microglia using Iba1 (J. Wang et al., 2019), and astrocytes using C3 (Liddelow et al., 2017) should be performed in the mesolimbic regions of the brain. Unlike GFAP, C3 has been shown to be a marker that is selective for A1 but not A2 astrocytes (Liddelow et

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