Neuroscience Matters

Hippocampal Neurogenesis Forgetting and the Effects of Exercise, Aging, and Stress on Memory - PG77 The impact of

Social Defeat Stress on behavior and the Dopaminergic system PG224

Erasing Fear Memories - Is It Possible? PG178

Table of Contents Yasmine Abdelaal - The Potential Therapeutic Impact of an Enriched Environment on Animal Models of Post-Traumatic Stress Disorder - pg 2

Sonja Ing - A novel pharmacogenetic approach: Transient neuronal activation through TRPV1 and capsaicin - pg 116

Padmesh Ramanujam - Reviewing glutamate mediated excitotoxicity in miR-1000 Drosophila mutants - pg 219

Adeoluwa Adesina - Nanoparticle Drug Delivery and its many Benefits compared to Conventional Methods - pg 8

Sylvia Jennings - Differential Brain Activation in Sommeliers: Effects of expertise on flavour integration - pg 121

Joravir Riar - The impact of Social Defeat Stress on behavior and the Dopaminergic system - pg 224

Ashima Agarwal - Oxytocin enhances social bonding, an effect moderated by baseline individual differences in socio-emotional factors including empathy - pg 11

Nimara Dias - Modeling and Treatment of Familial Parkinson’s Disease Using iPSCs - pg 124

Ariba Alam - Increased Phosphorylation of CREB at Ser133 in the Dentate Gyrus Reverses Depressive Behavior in Rodents - pg 16 Samin Alikhanzadeh - The rate of increase in adult hippocampal neurogenesis and spatial learning in C57BL/6J mice is greater in response to voluntary exercise than in response to sensory stimuli manipulations - pg 21 Mie Andersen - subPCP induced alterations in gut microbiota associated with memory deficit in schizophrenia model - pg 26 Ami Baba - Environmental Enrichment: A Neurorehabiitation Method utilized to treat deficits conferred from Traumatic Brain Injury (TBI) - pg 30 Vanessa C. Bracaglia - Creatine Supplementation on brain performance suggestive of potential therapeutic agent - pg 34 Alana Brown - The Neural Mechanisms of SocioSexual Partner Preference - pg 37 Megan E. Cabral - Impact of KIBRA Polymorphism On The Hippocampus - pg 41 Sammy Cai - α5GABAA Receptors Mediate Inflammation-Induced Memory Deficits in the Hippocampus - pg 44 Qasid Chaudhry - Evaluating syanpto-protection in a three compartmented microfluidic chip model following a chemically induced axotomy. - pg 49 Chun-Chi Chu - Potential link between intestinal microbiota and anxiety - pg 54 Melissa Colaluca - Elevations in the Serum Levels of the Brain Derived Neurotrophic Factor during Aerobic Physical Activity - A Simple, yet Often Disregarded Remedy for Frontotemporal Dementia - pg 57 Erica Confreda - High fat diet intake is related to impaired hippocampal dependent memory in juvenile rats - pg 63 Akua Obeng-Dei - Caffeine prevents memory consolidation impairments associated with sleep deprivation - pg 66 Daniel Derkach - Conflicting or Corroborating Evidence? Interleukin-6 and the JAK-STAT Signaling Pathway in Neural Precursor Self-Renewal - pg 70 Rachel Duncan - Trans-Cranial Direct Stimulation: A device for out of the box thinking - pg 74 Saadia Esat - Hippocampal Neurogenesis, Forgetting and the Effects of Exercise, Aging, and Stress on Memory - pg 77 Vanessa Ferlaino - Deep brain stimulation and Alzheimer’s disease: Benefits, cost-effectiveness and feasibility of deep brain stimulation on Alzheimer’s disease and cognitive dysfunction. - pg 80 Floriana Ferri - The Efficacy of Neurofeedback Training as a Treatment for Attention-Deficit Hyperactivity Disorder - pg 85 Chantel George - Review of Intentional and Incidental forgetting - pg 89 Jessica Gosio - The Novel Role of mTOR-Dependent Macroautophagy in Autism Spectrum Disorder - pg 95 Man Lai Ho - The pivotal role of TNF-α in inducing cognitive dysfunction - pg 100 Patrick Hopper - AKAP150 Underlies Deficits Seen in Spatial Memory Following Short-Term Sleep Deprivation - pg 103 Patrick Hornlimann - Maternal Behavior Hormone Receptor might be a crucial player in the development of social and mood disorders - pg 107 Justin Huang - Consolidation of Memories Following Sleep is the Result of Synaptic Potentiation - pg 111

Xin Yue Kou - To accomplish more or loss less: the story of sleep deprivation and Alzheimer’s disease - pg 128 Alexandra Kubica - Reconsolidation and Extinction Are Dissociable and Mutually Exclusive Processes: Behavioral and Molecular Evidence The Importance of Specificity in Neuroscience - pg 131 Shikha Kuthiala - The Effects Of Kynurenic Acid On The Brain And Its Implications In Schizophrenia - pg 134 Soonji Kwon - Selectively Activating Endogenous A3 Receptors is The New Therapeutic Solution to Chronic Pain - pg 139 Shonali Lakhani - Working memory training is most effective in healthy young adults to improve cognitive skills - pg 142 Dong-Eun Lee - Neural Correlates of Artistic Imagination through the Visual Modality - pg 146 Victor Lee - Overcoming social difficulties with the help of medications - pg 151 Ella Lew - Role of mu-opioid receptors in stress affecting vulnerability to substance abuse - pg 155 Vivian Liu - Seeking Autism-Linked Performance Within the Synaptic World: Effect of Neurexins and Related Proteins - pg 158 Yi Xuan Li - Effects of systems consolidation, optogenetic inhibition, and adult neurogenesis in hippocampal memory traces - pg 163 Ziteng Li - Adult hippocampal neurogenesis and its role in Alzheimer’s disease in transgenic mice models - pg 166 Bernie Longange - Improved cognitive function through the elucidation of alcoholically induced changes in the brain - pg 170 Tong Mai - Down-Regulation of Amyloid-Beta Peptide Binding P75 in Basal Forebrain Cholinergic Neurons Rescued Neurodegeneration and Behavioral Deficits in AD Mouse Models - pg 173 Fazila Malek - Discovering Biomarkers to Detect Early Onset of Stroke - pg 175 Divya Mamootil - Erasing Fear Memories– Is it possible? - pg 178 Catherine B. Matolcsy1 - Bridging the Gap in Traumatic Brain Injury: The promise of the Collagen Matrix - pg 181 Lucy McPhee - The potential for epigenetic treatment of neuropsychological disorders. - pg 185 Amaara Mohammed - Distinguishing the neurobiological features of resilient cognition in Alzheimer’s Disease. - pg 188 Arinda Muntean - Musical experience enhances cognitive performance among the aging population - pg 191 Jena L. Niceforo - Visualizing anxiety through mGlu7 receptor immunocytochemistry - pg 195 Yuki Nishimura - The Next Step in Antidepressant Therapy: BDNF Oscillation Patterns as a Potential Early Predictor for Therapy Response - pg 198 Miranda Nong - Further Insight on Using Mean Diffusivity as a Potential Biomarker to Identify Mild Cognitive Impairment Converters to Alzheimer’s Disease - pg 201 Daria Pacurariu - Don’t Stress About it: 5HTT Genotype and Epigenetics - pg 204 Hyun Park - Chronic Sleep Deprivation is Enough Induce Neuronal Degeneration - pg 207 Hemish Patel - Can Neurogenesis Using Stem Cells Be the Key to Post-Stroke Functional Recovery? A Review of Neurogenesis and Stroke Recovery in Animal Models - pg 211 Maryna Pilkiw - Lateral entorhinal cortex encodes associations of past experience and location - pg 215

Ashkan Salehi - An investigation of the facilitative effects of exercise on learning and memory - pg 227 Husain Shakil - Brain Inflammation, A Link Between Obesity and Cognitive Deficits - pg 231 Arman Shekari - The role of cAMP in mediating hippocampal-dependent spatial memory loss following periods of acute sleep deprivation - pg 235 Jaclin Simonetta - Treating Alzheimer’s Disease with Magnetic Resonance Imaging-Guided Focused Ultrasound - pg 239 Olivia Singh - Novel Metabotropic Function of NMDARs in Alzheimer’s Disease - pg 243 Pranay Siriya - Dopamine D1/D5 Receptor mediated tLTP Pathway in the Dentate Gyrus and Implications in Spatially-Dependent Learning and Memory - pg 247 Stephanie Strug - Suppression of α-syn in Mice Model of Human Lewy Body Disorders Reverses Detrimental Effects of α-syn Accumulation - pg 251 Ola Taji - A reserve pool of glutamate receptors is required for LTP - pg 254 Daniel Takla - Demyelination: Prevention and Restoration - pg 258 Eugene C. Tang - Study Shows How Transcranial Magnetic Stimulation Changes Depressed Brains - pg 261 Lauren Tessier - PPARδ: New Target for Alzheimer’s Pharmacotherapy? - pg 264 Carmen Tu - Temporal-Spatial Disconnect of Tauopathy and Amyloidopathy in Alzheimer’s Disease - pg 268 Madli Vahtra - Neuregulin 1-ErbB4 Signaling and Reduced Activity in NMDA Receptors: A Molecular Pathway for the Development of Schizophrenia and a Potential Target for Future Antipsychotics - pg 272 Chuqi Sandy Wang1 - Amygdala Dependent Retroactive Consolidation of Episodic Memories - pg 275 Ting Ting Wang - Gene Down-Regulations and Neuronal Implications of Adderall Induction of the Developing Brain - pg 278 Vonny Wong - Chronic Coffee and Caffeine Ingestion Effects on the Cognitive Function and Antioxidant System of Rat Brains - pg 281 Jiawei Zhang - Engraftment of Stem Cell Derived Dopamine Neurons offers a Possible Regenerative Treatment for Parkinson’s disease - pg 285 Yidong Zhan - BDNF overexpression rescues symptoms of Huntington’s disease by ameliorating neuronal loss in the striatum. - pg 288

The Potential Therapeutic Impact of an Enriched Environment on Animal Models of Post-Traumatic Stress Disorder Yasmine Abdelaal

Environmental enrichment has been previously known to improve the cognitive functions, including learning and memory, as well as the physiology of animals in an experimental setting. However, little has been known on the impact of an enriched environment on mitigating anxiety-like behaviors in mood disorders such as depression and post-traumatic stress disorder (PTSD). This review paper shows that environmental enrichment in animal models of PTSD, such as avoidance escape task and time-dependent sensitization model, could ameliorate the numbing/avoidance behaviors as well as anxiety-like symptoms. At the molecular level, western blot analysis also revealed an increase in hippocampal neurotrophic factors, such as brain-derived neurotrophic factor (BDNF), as well as an increase in the expression levels of microtubule-associated protein light chain 3-II (LC3-II). These results suggest a possible critical role of autophagy signaling as well as neurotrophic factors in the process of neuronal plasticity and hence in the adoption of stress-resilient behaviour such as an improvement in numbing/avoidance behaviour during an EE procedure. However, the link between neurogenesis and autophagy signaling processes and the exact mechanism by which they could lead to amelioration of numbing/ avoidance behavior still remains unknown and needs to be further investigated. Since it has been shown that in previous experiments drugs such as paroxetine could ameliorate hypervigilant behaviour, this might indicate a therapeutic strategy that involves both pharmacological (paroxetine) as well as psychosocial approaches (enriched environment) to potentially treat patients with PTSD. Key words: environmental enrichment (EE); Post-traumatic stress disorder (PTSD); animal model; stress; autophagy; brain-derived neurotrophic factor (BDNF) Background There has been growing interest in the role of an enriched environment (EE) on the brain function, physiology and behavior. The use of an enriched environment as an experimental tool to study behavior has been documented since the 1970s and was defined as an environment that encompasses social interactions as well as inanimate objects such as a running wheel for physical activity, toys, tunnels and wooden blocks to stimulate an exploratory and motor behavior (Rosenzweig, Bennett, Hebert & Morimoto, 1978; Henriette, Gerd, & Fred, 2000). In general, the ‘enriched’ animals are kept in larger cages with larger groups to allow complex social interactions (Henriette, Gerd, & Fred, 2000; Takahashi, Shimizu, Shimazaki, Toda, & Nibuya, 2014).

Previous studies have shown that animals kept in an enriched environment have better overall learning and memory compared to those in standard conditions. However, recently there has been a focus on the effect of an enriched environment on emotionality such as reducing fearfulness (Fernández-Teruel et al., 2002), reducing anxiety-like behaviors (Roy, Belzung, Delarue, & Chapillon, 2001) and adaptation to stress (Benaroya-Milshtein et al., 2004). Therefore the therapeutic effect of an enriched environment in treating mood disorders such as depression or post-traumatic stress-disorder (PTSD) can be further elucidated.

Post-traumatic stress disorder (PTSD) is a longlasting maladaptive anxiety and stress response as a result of a severe traumatic event (DSM-IV). PTSD patients experience both avoidance as well as hypervigilant behaviors for long periods of time following the trauma. Although there have been several attempts to treat PTSD with antidepressant medications such

as escitalopram and paroxetine, however, many have been ineffective in treating patients and leading to replaces in some patients (Cohen, 2005; Davis, Frazier, Williford, & Newell, 2006; Baker, Nievergelt, & Risbrough, 2009; Stein, Ipser, & Sccdat, 2006).

Additionally, paroxetine was shown experimentally to ameliorate hypervigilant behavior, but not avoidance behavior (Sawamura et al., 2004). Therefore antidepressant medications alone are not sufficient for effective treatment of PTSD. In a recent study researchers were able to use a rat model of PTSD to investigate the impact of an enriched environment on the exhibition of both numbing/avoidance and hypervigilant behaviors (Henriette, Gerd, & Fred, 2000; Takahashi, Shimizu, Shimazaki, Toda, & Nibuya, 2014). Results of an avoidance escape task (AET) revealed amelioration in numbing/avoidance behavior but not in hyper-vigilant behavior. It was also found that there was an increase in hippocampal neurotrophic factors such as brain-derived neurotrophic factor (BDNF) as well a concurrent increase in hippocampal autophagy signaling (Henriette, Gerd, & Fred, 2000; Takahashi, Shimizu, Shimazaki, Toda, & Nibuya, 2014).

In another model of PTSD that used an inescapable foot-shock (IFS) paradigm, EE reduced anxiogenic behavior after IFS, which correlated with an increase in hippocampal cell proliferation (Hendriksen, Prins, Olivier, & Oosting, 2010). The above literature suggests that EE can reduce anxiety-like behavior via neurogenesis that involves the increase in BDNF levels. However, autophagy synaptic remodeling, which involves the degradation of unnecessary synaptic connections, was also shown to be involved in the process of neuronal plasticity and hence in 2

the adoption of stress-resilient behavior such as an improvement in numbing/avoidance behavior during an EE procedure (Henriette, Gerd, & Fred, 2000; Takahashi, Shimizu, Shimazaki, Toda, & Nibuya, 2014).

In order to further understand the effect of an EE on neuronal plasticity as a mechanism to adapt to stress, the link between increased levels of neurotrophic factors such as BDNF and the concurrent increase in autophagy signaling needs to be addressed in future studies. Additionally, we can investigate the degradation of AMPA receptors in the hippocampus following EE treatment, which was previously found to be as a consequence of the activation of autophagy signaling (Shehata, Matsumura, Okubo-Suzuki, Ohkawa, & Inokuchi, 2012).

Summary of Major Results

Environmental enrichment shows antianxiety effects in PTSD rat models

There have been consistent results in a number of studies showing reduced anxiety in rats housed under an enriched environment compared to a standard condition. In a recent study six-week-old male Wister rats were used as model organism for PTSD and the rats were divided up into the EE group (n=12) or the control group (n=16) for a period of two weeks (Takahashi, Shimizu, Shimazaki, Toda, & Nibuya, 2014).

To study the antianxiety effects of an enriched environment the researchers used an elevated plus maze test and an open field test as behavioral parameters.

The results revealed a significant increase in the time spent in the open arms, an increase in the number of entries to the open arms as well as a significant decrease in the distance traveled in the open field test (Figure 1) (Takahashi et al., 2014).

elevated plus maze. Additionally, the EE-treated mice were significantly more active by showing a higher rate of climbing in a staircase test.

Another behavioral parameter that was taken into account in one of the studies is the enhanced and faster habituation to an unfamiliar environment using a free exploration paradigm following environmental enrichment (Elliott & Grunberg, 2005). The combined results suggest that an enriched animal would also adapt faster to an unfamiliar environment compared to those left in a standard condition.

Animal models of PTSD

There have been many attempts to develop an effective animal model that would provide analogues to the specific symptoms of PTSD. In one of the major studies an avoidance escape task (AET) paradigm was used to study the effect of an enriched environment on both numbing/avoidance and hypervigilant behaviors (Takahashi et al., 2014). In this task a group of rats were given a series of initial inescapable shocks (ISs) with inter-trial intervals (ITIs), then they were placed in a shuttle box and given 5 mins to habituate to the environment. The number of crossings and the locomotor activities during adaptation were both found to be decreased after IS. However this decrease was ameliorated in the EE-treated group. In the second part of the AET, a conditioned stimulus was presented along with a foot shock and the numbers of crossings during the ITIs, as well as the avoidance of the CS were measured. Results revealed amelioration in the numbing/avoidance behaviors following EE-treatment, but not in the hypervigilant behavior, indicated by the number of crossings during the ITIs (Figure 2) (Takahashi et al., 2014).

Figure 1. Antianxiety effects shown in Wister rats housed in an EE for 2 weeks. A) Result of an open field test showing a significant increase in the time spent in the open arms in the EE group compared to the control. B) Result of an open field test showing a significant increase in the number of entries to the open arms in EE group compared to control group. C) Result of open field test showing a significant decrease in the distance traveled in the EE group compared to the control.

The same results were observed in another study that used ten-week-old male C3H mice housed in an EE for a period of 6 weeks (Benaroya-Milshtein et al., 2004). The EE-treated rats also showed a significant increase in the time spent in the open arms and in the total number of entries to the open arms of an 3

Figure 2. Results showing numbing/avoidance and hypervigilant behaviors in an avoidance escape task (AET) paradigm. (A and B) The decrease in the number of crossings and in locomotor activity during a 5 min adaptation period after IS was ameliorated following 2 weeks of EE-treatment. (C and D) No significant difference between the EE and control groups in the avoidance and number of crossings during intertrial interval (ITI).

In other studies a time-dependent sensitization model was used (Benaroya-Milshtein et al., 2004; Pynoos, Ritzmann, Steinberg, Goenjian, & Prisecaru, 1996). This animal model of PTSD involved repeated exposure to situational reminders of an initial stressful stimulus. This stressful stimulus, such as 1 mA shock for 10 seconds, was capable of allowing the animal to exhibit PTSD-like symptoms after situational reminders. Results revealed an increase in freezing time over a period of 5 weeks of situational reminders to the electric shocks. However, EE-treated mice had a significantly lower freezing time during the same time period (Figure 3) (Benaroya-Milshtein et al., 2004).

Figure 4. Expression levels of BDNF and LC3-II in EE-treated group and control after initial ISs in an avoidance escape task paradigm. Showing a significant increase in the expression levels of BDNF (A and B) and LC3-II (C and D) in the EE-treated mice after administration of ISs compared to the controls.

Conclusions and Discussion Figure 3. Results of time-dependent sensitization model Showing a significant decrease in freezing time in EE-treated mice after 5 situational reminders of an electric shock.

The effect of environmental enrichment on neurotrophic factors and autophagy signaling

It has been previously reported that following treatment of mood disorders like depression, there was an enhancement in neuronal plasticity via the upregulation of various neurotrophic factors including brain-derived neurotrophic factor (BDNF) (Duman & Monteggia, 2006). However, a number of studies also showed an increase in the expression levels of BDNF specifically in the hippocampus of environmentally enriched rats (Ickes et al., 2000; Fernández-Teruel, Escorihuela, Castellano, González, Toben˜a, 1997).

In one studies mentioned earlier that used the AET paradigm, the results of western blot analysis also revealed a significant increase of hippocampal BDNF expression as well as a concurrent increase in the expression levels of the active LC3-II, a biochemical marker of autophagy signaling, in inescapable stress (IS)-treated rats. This study was the first to show that an enriched environment could induce synaptic remodeling via autophagy signaling and in turn improve the numbing/avoidance behaviors in rat models of PTSD (Takahashi et al., 2014).

In another study exposure to an enriched environment following an inescapable food shock procedure (IFS) lead to complete recovery by reducing the anxiety of male Sprague Dawley rats (Hendriksen et al., 2010).

This behavioral recovery correlated with almost a 2 fold increase in cell proliferation in the dentate gyrus of the hippocampus (Hendriksen et al., 2010). The combined results suggest that an EE in animal models of PTSD might have an important role in neurogenesis via an increase in the levels of neurotrophic factors such as BDNF.

One of the key features of PTSD is the prolonged anxiety and stress response experienced by the patients and therefore researchers have been trying to develop an animal model that can exhibit those features and in turn use experimental techniques such as an enriched environment to decrease anxiety-like behaviors. From the experiments mentioned in the results section we can conclude that environmental enrichment can reduce anxiety by increasing the time spent in the open arms as well as increasing the number of entries in the open arms of an open field test (Benaroya-Milshtein et al., 2004; Takahashi et al., 2014; Elliott & Grunberg, 2005). Additionally, animals reared in an enriched environment were more active in the staircase test (Benaroya-Milshtein et al., 2004) and adapted quicker to novel environments by spending more time exploring freely the unfamiliar environment (Elliott & Grunberg, 2005).

However, the different strains and gender of the rats/mice used in the above experiments should also be taken into consideration. Although using different strains including C3H mice, Wister rats and Sprague– Dawley rats in these experiments all revealed similar results in exhibiting antianxiety behavior following EE treatment, on the other hand other studies found that anxious BALB/c strains were affected by EE treatment to a larger extent than C57BL/6 strains (Pynoos, Ritzmann, Steinberg, Goenjian & Prisecaru, 1996). Additionally, mostly male rats or mice were used however; in one of the studies EE had an overall greater effect in female Sprague–Dawley rats than males (Elliott & Grunberg, 2005). Thus, we can conclude that both the strain and gender of animal models contribute to the overall effectiveness of environmental enrichment in mitigating anxiety-like behavior.

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Animal models of PTSD

One of the animal models that were used to model PTSD in EE housing was the avoidance escape task (AET) paradigm (Takahashi et al., 2014). This model was effective since it allowed the researchers to investigate both numbing/avoidance and hyperarousal/hypervigilant behaviours at the same time by measuring the locomotor activity during the adaptation period and during inter-trial intervals following inescapable stress respectively (Takahashi et al., 2014; Sawamura et al., 2004; Wakizono et al., 2007; Kikuchi et al., 2008). Since it has been shown that in previous experiments drugs such as paroxetine could ameliorate hypervigilant behaviour, this might indicate a therapeutic strategy that involves a combination of pharmacological (paroxetine) as well as psychosocial approaches (enriched environment) to potentially treat patients with PTSD (Sawamura et al., 2004).

The time-dependent sensitization model was another stress paradigm that allowed the researchers to use repeated situational reminders to remind the animal of the initial traumatic or stressful stimulus (Benaroya-Milshtein et al., 2004). The results of this model indicated that EE could be used to decrease the freezing time for the animal throughout the five repeated situational reminders over a period of 5 weeks and was sustained (Benaroya-Milshtein et al., 2004). This concludes that using EE as a potential therapeutic technique in PTSD has to be achieved over a long-period of time in order to be effective.

Also, there is no single model that could feature all the symptoms of PTSD, therefore the time duration of the study as well as the type of traumatic stimulus has to be taken into consideration in future studies.

The effect of environmental enrichment on neurotrophic factors and autophagy signaling There have been consistent results in the literature revealing a significant increase in the levels of BDNF in the hippocampus of animals following EE treatment (Takahashi et al., 2014; Ickes et al., 2000; Fernรกndez-Teruel et al.,1997). However, the most recent paper that revealed a concurrent increase in the levels of BDNF and LC3-II reveals a new mechanism by which EE could ameliorate the numbing/avoidance behavior in PTSD animal models (Takahashi et al., 2014). Since BDNF is known to be a key regulator of neurogenesis, which involves the formation of new synaptic connections, and LC3-II is a key component of the autophagy signaling process, this suggests that both processes are involved in neuronal plasticity in the rat hippocampus to ameliorate the numbing/ avoidance behavior of PTSD animal models.

We also need to consider the results of another study that showed a correlation between the complete behavioral recovery of Sprague Dawley rats following IFS and the significant increase in the cell proliferation in their dentate gyrus (Hendriksen et al., 2010). This suggests that cell proliferation might play a role in the behavioral recovery following exposure to a traumatic or stressful stimulus, however from the experimental design we cannot conclude that cell proliferation is obligatory for complete behavioral recovery.

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Criticisms and Future Directions Although the results revealed a significant therapeutic impact of EE on animal models of PTSD, there still remains some limitations and further experiments need to be conducted to investigate the role of neurogenesis and autophagy signaling in the EE procedure.

One of the key strengths of one of the recent studies is that it involved both behavioural tests such as elevated plus maze and open field test to test for anxiety, as well as measuring the expression levels of molecular markers such as BDNF and LC3-II (Takahashi et al., 2014). However, a limitation of this study is that LC3-II was the only molecule used as biochemical marker for autophagy signaling. Another possibility is to use structural evidences of increased autophagosomes in hippocampal neurons following the EE procedure.

In the majority of the experiments conducted, the levels of neurotrophic factors or autophagy signaling molecules were measured only in the hippocampal region, however PTSD has been also shown to affect other regions of the brain such as the amygdala and the prefrontal cortex and therefore they also need to be taken into consideration in future studies (Bremner, Elzinga, Schmahl, & Vermetten, 2008; Yehuda & LeDoux, 2007).

Another limitation of the experiments discussed in this paper is that it was solely focused on environmental enrichments however, it was found in previous studies that physical activity alone using a running wheel, in the absence of an EE housing, could increase the hippocampal expression levels of neurotrophic factors such as BDNF as well as increasing cell proliferation and in turn could mitigate the anxiety symptoms experienced by rat models of PTSD (Bechara & Kelly, 2013). Therefore the effect of an enriched environment with and without physical activity needs to be further investigated.

Neurogenesis and autophagy signaling

As previously mentioned, one of the most recent studies revealed a concurrent increase in the levels of both hippocampal BDNF and autophagy signaling following EE treatment in the Wister rat model of PTSD (Takahashi et al., 2014). This was the first study to show a key involvement of autophagy signaling in the neuronal plasticity and behavioral recovery of PTSD rat models (Takahashi et al., 2014). However, the link between neurogenesis and autophagy signaling processes and the exact mechanism by which they could lead to amelioration of numbing/avoidance behavior still remains unknown.

Autophagy signaling was shown in previous studies to be enhanced via inactivation of mammalian target of rapamycin (mTOR), a serine/threonine protein kinase, and in turn could exert anti-anxiolytic effects (Cleary et al., 2008). On the other hand, another proposed mechanism for autophagy signaling activation was via an mTOR-independent pathway after the administration of mood-stabilizers that decrease the concentrations of inositol triphosphate (IP-3) (Sarkar,

Ravikumar, Floto, & Rubinsztein, 2009). Therefore a future experiment can be designed to examine the link between enhanced BDNF and autophagy signaling activation via mTOR-dependent or mTOR-independent mechanisms in a similar EE procedure. In such an experiment the numbing/avoidance behavior can be measured using the avoidance escape task (AET) after an initial period of inescapable stress. In the experiment rats can be divided up into two groups: with or without BDNF/mTOR activation plus attenuated autophagy and with or without mTOR-independent autophagy activation. Biochemical markers such as mTOR and IP3 can be used to differentiate between those two mechanisms of autophagy signaling activation. Finally, the behavioural differences such as numbing/avoidance behavior, distance traveled in the open field test or time spent in the open arms of an elevated maze test can be measured as well.

Finally, to further investigate the role of autophagy signaling in an EE procedure using rat model of PTSD, we can also look at the degradation of AMPA receptors in the hippocampus, which was previously found to be a consequence of activated autophagy processes (Shehata, Matsumura, Okubo-Suzuki, Ohkawa, & Inokuchi, 2012). Since the degradation of AMPA receptors requires protein phosphatase activity (PP1), PP1 can be used as a biochemical marker for this process. The effect of an enriched environment on the degradation of AMPA receptors and hence on autophagy signaling can then be determined.

In conclusion, environmental enrichment has a potential therapeutic effect on animal models of PTSD and can be a promising psychosocial approach that can be used for treating patients with PTSD after more studies are conducted.

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Physiology & Behavior, 90(4), 687-695. doi:10.1016/j. physbeh.2006.12.008 15. Kikuchi, A., Shimizu, K., Nibuya, M., Hiramoto, T., Kanda, Y., & Tanaka, T. et al. (2008). Relationship between post-traumatic stress disorder-like behavior and reduction of hippocampal 5-bromo-2′-deoxyuridine-positive cells after inescapable shock in rats. Psychiatry And Clinical Neurosciences, 62(6), 713-720. doi:10.1111/ j.1440-1819.2008.01875.x 16. Bechara, R., & Kelly, Á. (2013). Exercise improves object recognition memory and induces BDNF expression and cell proliferation in cognitively enriched rats. Behavioural Brain Research, 245, 96-100. doi:10.1016/j.bbr.2013.02.018 17. Cleary, C., Linde, J., Hiscock, K., Hadas, I., Belmaker, R., & Agam, G. et al. (2008). Antidepressive-like effects of rapamycin in animal models: Implications for mTOR inhibition as a new target for treatment of affective disorders. Brain Research Bulletin, 76(5), 469-473. doi:10.1016/j.brainresbull.2008.03.005

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18. Sarkar, S., Ravikumar, B., Floto, R., & Rubinsztein, D. (2009). Rapamycin and mTOR-independent autophagy inducers ameliorate toxicity of polyglutamine-expanded huntingtin and related proteinopathies. Cell Death And Differentiation, 16(1), 46-56. doi:10.1038/cdd.2008.110 19. Shehata, M., Matsumura, H., Okubo-Suzuki, R., Ohkawa, N., & Inokuchi, K. (2012). Neuronal Stimulation Induces Autophagy in Hippocampal Neurons That Is Involved in AMPA Receptor Degradation after Chemical Long-Term Depression. Journal Of Neuroscience, 32(30), 10413-10422. doi:10.1523/jneurosci.4533-11.2012 20. Elliott, B., & Grunberg, N. (2005). Effects of social and physical enrichment on open field activity differ in male and female Sprague–Dawley rats. Behavioural Brain Research, 165(2), 187-196. doi:10.1016/j.bbr.2005.06.025 21. Duman, R., & Monteggia, L. (2006). A Neurotrophic Model for Stress-Related Mood Disorders. Biological Psychiatry, 59(12), 1116-1127. doi:10.1016/j.biopsych.2006.02.013 22. Ickes, B., Pham, T., Sanders, L., Albeck, D., Mohammed, A., & Granholm, A. (2000). Long-Term Environmental Enrichment Leads to Regional Increases in Neurotrophin Levels in Rat Brain. Experimental Neurology, 164(1), 45-52. doi:10.1006/exnr.2000.7415 23. Fernández-Teruel, A., Escorihuela, R., Castellano, B., González, B., Toben˜a, A. (1997). Neonatal handling and environmental enrichment effects on emotionality, novelty/ reward seeking, and age-related cognitive and hippocampal impairments: focus on the Roman rat lines. Behav. Genet., 27, 513–526.

24. Bremner, J., Elzinga, B., Schmahl, C., Vermetten, E. (2008). Structural and functional plasticity of the human brain in posttraumatic stress disorder. Prog Brain Res., 167, 171–186.

25. Yehuda, R., & LeDoux, J. (2007). Response Variation following Trauma: A Translational Neuroscience Approach to Understanding PTSD. Neuron, 56(1), 19-32. doi:10.1016/j. neuron.2007.09.006

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Nanoparticle Drug Delivery and its many Benefits compared to Conventional Methods Adeoluwa Adesina

Overview: I discuss the history of nanoparticle use potentially important uses of nanoparticles as drug delivery aids and how the research on this topic has grown. I also mention the major results of a recent primary publication, the relevance of those results, and the implication they will have on future research in this field.

Background The idea of nanoparticles seemed extremely futuristic in the 1900s, and the thought that people would actually be able to use them effectively to help improve the delivery of drugs seemed even more so.

However, in the early-mid 1970s, some literature had already started to be published on nanoparticles and some of the things they can be used for. At this time, however, researchers weren’t exactly connecting the use of nanoparticles to drug delivery. Some were just injecting nanoparticles that they tagged with fluorescence to see where they would end up (1). One of the first few papers that used nanoparticles, or “liposomes”, as a drug delivery system encapsulated a form of insulin inside the liposomes. They then found that altering the liposomal surface properties could change the type of tissues the majority of the drug are eventually uptaken into (2). This was very promising, and led to more research being done on the use of nanoparticles for all sorts of things, including the controlled delivery of contraceptive steroids (3), the delivery of anesthetics (4), and the effective magnetic targeting of cancer treatment drugs in rats (5). Researchers were quickly starting to realize the potential implications these nanoparticles could have. Those earlier papers helped catapult the study of the use of nanoparticles for drug delivery to where it is today. By continuing down the drug delivery path of nanoparticles, we have learned more about what nanoparticles can do, such as their high permeability across the blood brain barrier (BBB)(6), and have expanded the range of diseases we attempt to come up with better treatments for through the use of nanoparticles and nanomedicine. For example, in the last few years, researchers have used nanoparticles to deliver drugs that help treat the symptoms of popular diseases, such as Alzheimer’s, with a lot of success (7, 8, 9).

Even though it seems that we know plenty about nanoparticles and their uses, there are still some things we do not know. One thing being how we’re going to apply our nanoparticle experiments to humans, since they’ve only been done in mice or other model organisms. The reasons for this could be many, but I believe uncertainty about certain doses of the nanoparticle/drug combination as well as other potentially serious issues or side effects that could occur because of the more complex human brain are some of the things that may be hindering this from being used in an human participants.

Summary of Major Findings Ying-mei Lui et al. (2014) followed in the footsteps of some of the previous papers and decided to choose the drug of a disease that has had difficulty being successfully and efficiently administered; a disease like brain ischemia. They wanted to test the effectiveness of nanoparticles on the delivery of ischemic drugs, like NBP, to the brain, since many of these drugs have limited permeability across the BBB. Their results showed that their PEGylated lipid nanoparticles (PLNs) containing NBP increased its permeability across the BBB. In conjunction with a Fas ligand antibody, which allows their PLN/NBP nanoparticle to have more accurate targeting, they were able to get more of the NBP to the ischemic brain region than using the NBP alone. Also, the PLN/NBP/Fas combination was more effective using far smaller dosages (5 mg/ kg) compared to NBP alone (10 mg/kg). Lastly, the amount of ischemic damage in the brain was greatly reduced using the PLN/NBP/Fas combination versus using NBP alone (10).

Figure 1. Shows the neurological scores of the mice after the treatment of the induced ischemia by NBP alone versus treatment by PLN/NBP/Fas (10).

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researchers were able to shed some light on other benefits of nanoparticle technology outside of simply allowing drugs easier access across the BBB. They showed that by adding specific antibodies on the surface of their nanoparticles, they can target specific tissues in the brain, which increases the effectiveness significantly as well as decreasing the usual required dose by at least half.

Criticisms and Future Directions

Figure 2. Shows the effect of NBP alone on improving ischemic tissue in the brain versus NBP/ PLN/Fas (10).

Consistency with the results of other papers

The results of this article are not just due to chance.

These results are accurate and precise, as there are many other researchers who used similar techniques and achieved the same result. This technique as well as the use of PLNs have been used years before this paper and have been shown to have similar results in drug delivery (11). The technique has also been replicated after this paper was published and, once again, similar results were documented (12) (though magnetic targeting was used instead of antibody targeting). It’s very clear that this technique, the use of drugs encapsulated by nanoparticles, and the results it produces are precise and consistent with the results of other papers which have used the same or a similar technique.

Discussion and Conclusion

These results are very significant; not just for this paper, but for nanomedicine in general. This is because the paper does more than just reiterate the effectiveness of encapsulated drugs crossing the BBB more easily, which has already been shown by other papers (6, 13). It shows how this increase in BBB permeability for drugs, as well as the highly accurate targeting effect of antibodies like Fas, can equate to less of the drug being required to have a more significant neuroprotective effect in the ischemic brain region. This maximizes the effectiveness of the drug, which also implies that any of the issues or side effects that are present at higher doses will be highly reduced using this technique, and at the same time, the individual will be experiencing a significantly stronger neuroprotective effect. Lastly, it’s one of the first studies to show a different pharmaceutical strategy that has the potential to better treat cerebral ischemia than conventional uses, as it has been difficult trying to find other non-conventional strategies over the past few years (14).

Conclusion

In conclusion, this article makes a big contribution to the study of nanoparticles and nanomedicine. These 9

This paper was well done and there were a lot of good things one could take from it. However, there were a few things that this paper could have improved on. The main thing I felt was lacking was the discussion of any future directions that their results could lead to. They just showed how encapsulating drugs in nanoparticles can maximize the effect of the drug itself, particularly drugs for brain diseases, and they didn’t give any specific examples of how the application of this important revelation can potentially be beneficial in future research. Even though this technique has been used many times before and it may have been difficult for them to come up with a unique or original idea, they could have at least mentioned a different way of doing an experiment that has already been done while incorporating their results to show they have actually given some thought to future applications of their research.

Future Directions

As I mentioned in the conclusion, one of the things they didn’t really add to their paper were any future directions their results could take research in the future. One questions they could have addressed is if there are other beneficial effects of using nanoparticles instead of conventional methods. Perhaps one could start off by seeing if there are other benefits of nanoparticle encapsulated NBP or other drugs. As was stated in the discussion, there are implications that because less of a dose than usual of the drug is required through the use of nanoparticles, using nanoparticles with a drug that is known to have many side effects may be able to reduce the incidence of those side effects. This is because less of the drug is needed to have a substantial effect. This could mean drugs that are very potent and effective, but aren’t usually recommended because of the serious side effects they may cause or their toxicity, may become more viable via nanoparticle administration (15).

Another future direction these results could lead to is nanoparticles being used as a gene delivery system rather than a drug delivery system, to combat the diseases that have a strong genetic component. One could create a genetic construct that will be able to cleave off the troublesome sequence in the genome permanently. Then, one could insert this construct into a nanoparticle to be transported into the body.

A popular technique used for genome editing called CRISPR/Cas9, which is thought to be the next big thing, works in a very similar way, and papers have already been published showing its effectiveness in successful gene therapy (16). For this technique, researchers generally use a virus to transport their

genetic construct into the body, reaping generally favourable results. However, using nanoparticles for transporting the genetic construct instead of a virus may be more beneficial (17).

References

1. Oppenheim, R.C., Marty, J.J. and Steward, N.F. The labelling of gelatin nanoparticles with g9m technetium and their in viva distribution after intravenous injection. Aust. J. Pharm. Sci., 7, 113-117 (1978).

2. Tanaka, T. et al. Application of Liposomes to the Pharmaceutical Modification of the Distribution characteristics of Drugs in the Rat. Chem. Pharm. Bull. 23 (12): 3069-3074 (1975).

3. Gao, Z. et al. Controlled Release of Contraceptive Steroids from Biodegradable and injectable gel formulations: In Vivo evaluation. Pharm. Research. 12(6): 864-868 (1995).

4. Wakiyama, N., Juni, K., and Nakano M. Preparation and Evaluation In Vitro of Polyactic acid Microspheres Containing Local Anesthetics. Chem. Pharm. Bull. 29(11): 3363-3368 (1981).

5. Witter, K. J. et al. Tumor Remission in Yoshida Sarcomabearing Rats by Selective Targeting of Magnetic Albumin Microspheres Containing Doxorubicin. Proc. Natl. Acad. Sci. 78(1): 579-581 (1981).

6. Ulbrich, K. et al. Transferrin- and transferrin-receptorantibody-modified nanoparticles enable drug delivery across the blood–brain barrier (BBB). Eur J. Pharm. Biopharm., 71, 251-256 (2009).

7. Song, Q. et al. Lipoprotein-Based Nanoparticles Rescue the Memory Loss of Mice with Alzheimer’s Disease by Accelerating the Clearance of Amyloid-Beta. Acs Nano. 8(3): 2345-2359 (2014).

8. Zhang, C. et al. Intranasal nanoparticles of basic fibroblast growth factor for brain delivery to treat Alzheimer’s disease.

Int. J. Pharm., 461, 192-202 (2014).

9. Zhang, C. et al. Dual-functional nanoparticles targeting amyloid plaques in the brains of Alzheimer’s disease mice.

Biomaterials, 35, 456-465, (2014).

10. Lu, Y. et al. Targeted therapy of brain ischaemia using Fas ligand antibody conjugated PEG-lipid nanoparticles.

Biomaterials, 35, 530-537 (2014).

11. Calvo, P. et al. PEGylated polycyanoacrylate nanoparticles as vector for drug delivery in prion diseases. J. Neurosci.

Methods, 111, 151-155 (2001).

12. Halupka-Bryl, M. et al. Doxorubicin loaded PEG-bpoly(4-vinylbenzylphosphonate) coated magnetic iron oxide nanoparticles for targeted drug delivery. J. Magn. Magn.

Mater., 384, 320-327 (2015).

13. Kreuter J., et al. Covalent attachment of apolipoprotein A-I and apolipoprotein B-100 to albumin nanoparticles enables drug transport into the brain. J. Controlled Release, 118, 54-58 (2007).

14. Kilic, U. et al. Evidence that membrane-bound G proteincoupled melatonin receptors MT1 and MT2 are not involved in the neuroprotective effects of melatonin in focal cerebral ischemia. J. Pineal. Res., 52, 228-235 (2012).

15. Ren, J. et al. The targeted delivery of anticancer drugs to brain glioma by PEGylated oxidized multi-walled carbon nanotubes modified with angiopep-2. Biomaterials, 33, 33243333 (2012).

16. Ding, Q. et al. Permanent Alteration of PCSK9 With In Vivo CRISPR-Cas9 Genome Editing. Circ. Res., 115, 488-497 (2014).

17. Harris, T. J. et al. Tissue-specific gene delivery via nanoparticle coating. Biomaterials, 31, 998-1006, (2010).

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Oxytocin enhances social bonding, an effect moderated by baseline individual differences in socio-emotional factors including empathy Ashima Agarwal

Melanie Feeser1,2,3, Yan Fan1,2,3, Anne Weigand1,2,3, Adam Hahn4, Matti Gärtner1,2,3, Heinz Böker5, Simone Grimm1,2,3,5, Malek Bajbouj1,2,3 1Cluster of Excellence “Languages of Emotion”, Freie Universität Berlin, Habelschwerdter Allee 45, 14195 Berlin, Germany; 2Department of Psychiatry, Campus Benjamin Franklin, Charité Berlin, Eschenallee 3, 14050 Berlin, Germany; 3Dahlem Institute for Neuroimaging of Emotion, Freie Universität Berlin, Habelschwerdter Allee 45, 14195 Berlin, Germany; 4 Social Cognition Center Cologne, University of Cologne, Richard-Strauss-Str. 2, 50931 Cologne, Germany; 5Department of Psychiatry, Psychotherapy and Psychosomatics, Hospital of Psychiatry, University of Zürich, 8032 Zürich, Switzerland

Social functioning and the development of social relationships rely on the ability to observe the behaviours and infer the mental states of others. Mentalizing abilities in particular have often been associated with social interaction, as they involve inferring the emotional states of others, and thereby contribute to the development and maintenance of relationships.

Oxytocin (OXT), a neuropeptide, has long been implicated in enhancing social bonding (Liberwirth & Wang, 2014). In support of this notion, literature concerning OXT has established its ability to enhancing mentalizing capabilities (Luminet et al., 2011), a relationship that has become well established.

However, previous research lacks in its ability to demonstrate the prevalence of a moderating influence between OXT and mentalizing.

There are many variables that have been implicated to have an affect on socio-emotional interactions. For example, empathy has been correlated with social functions, and similar to mentalizing, there is current evidence for the fact that OXT administration is related to improved empathetic abilities (Uzefovsky et al., 2014).

Concerning the ability of such socio-emotional traits to moderate the relationship between OXT and mentalizing abilities is important in our understanding of social relationships and interactions.

Key words: oxytocin; neuropeptide; mentalizing; empathy; social interactions; social bonding Background Social relationships are essential in maintaining societal coherence. They affect behavioural, physiological and psychological outcomes (Baumeister and Leary, 1995). Specifically, persistent attachments have impacts cognitive, emotional, social and physical well being. Social belonging protects against depression and anxiety (Lee and Robbins, 2000), and relationships have been shown tin increase life expectancy (Drefahl, 2012). Positive associations have been determined with immune functionality and cardiovascular health (Kiecolt-Glaser et al., 2010).

Past literature displays that the neuropeptide Oxytocin (OXT) is vital in the modulation of social-cognitive functions, promoting social memory, decreasing fear and anxiety, and stimulating social trust and approach (Feeser et al., 2015; Lieberwirth & Wang, 2014). If released in the hypothalamus, via the paraventricular and supraoptic nuclei, it acts as a neurotransmitter, which in turn has behavioural effects (Debiec, 2007).

Alternatively, it is shown to act as a hormone released by the pituitary gland.

In social interactions, it is important to perceive personal and others’ intentions. An operationalizing of this is mentalizing, which involves inferring mental and emotional states of other individuals, and is usually measured by the Reading the Mind in the Eye Test (RMET).

Mentalizing and OXT seem to follow a similar trajec11

tory; this relationship has long been established in literature as well. Specifically, OXT administration has been able to enhance mentalizing abilities as shown by increased accuracy on the difficult items of RMET (Luminet et al., 2011) with a comparatively lesser effect on the easier items of the test (Domes et al., 2007).

Simultaneously, Schultze et al. (2011) suggest that perhaps overly difficult items are too challenging, and that empathy operates best in moderation.

Regardless, in order to put this relationship into perspective, Bartz et al. (2011) have proposed an interactionist model concerning the social effects of oxytocin in humans. They highlight the importance of both stable individual differences and contextual or situational factors that moderate the effect of OXT. The model proposes that situation moderators may include stimuli effects, familiarity, perceived reliability as well as task difficulty. The individual differences are attachment anxiety, or psychiatric disorders. A similar model has been identified by Olff et al. (2013) who focus on the importance of perceiving a safe situation as well as inter-individual factors such as gender, childhood trauma, attachment style and the presence of psychiatric disorders.

However, little research has focused on the importance of individual traits within the soico-emotional domain, specifically. An example of this is empathy, or other baseline socio-cognitive and socio-emotional

Figure 1. Moderating factors of oxytocin. Situational cues and Interindividual factors predict the effect of oxytocin on social bonding and stress regulation. (Source: Olff et al., 2013).

traits that vary across individuals and that are still intrinsically biologically rooted, with nurturing elements.

One such trait is baseline emotional regulation abilities which, demonstrated by Quirin et al. (2011), affect OXT application and its resulting effect. Another trait was also supplied by Leknes et al (2013)’s experiment that showed that individuals with less emotional sensitivity displayed improved empathic accuracy following OXT administration. Similarly, Grimm et al. (2014) identified that early stress influenced the effect of OXT on cortisol and neural activity and finally, Feeser et al. (2015) demonstrated that OXT administration was influenced by baseline empathetic ability. In short, these studies deduced that the degree of OXT susceptibility varies depending on baseline socio-emotional skills.

Seemingly, these individual skills will help contribute to how OXT might affect an understanding of interpersonal interactions, social norms, different beliefs and different perspectives.

However, literature on this topic should further capitalize on possible brain mechanisms associated with this relationship. Additionally, it has been shown that there are innate genetic differences in the level of socio-emotional traits (i.e. empathy) that are based on the oxytocin receptor; this is a point that needs to be further investigated in light of mentalizing abilities and its interaction with the moderating effect of individual differences. Finally, it would be useful to expand studies to consider other socio-cognitive functions that could potentially moderate the effect of OXT on mentalizing.

Research Overview Summary of Major Results Feeser et al. (2015) recently conducted a study in which they investigated the effect of empathy on OXT administration. The researchers acknowledged the established relationship between empathy and mentalizing as well as the lack of consistency in OXT-administration effects that vary by baseline

socio-emotional traits. Given this, they hypothesized that OXT-induced results would enhance mentalizing, providing more distinct enhancements in the difficult items of the RMET as compared to easier items on the test. Furthermore, they suggested that OXT administration would enhance mentalizing abilities in individuals with low empathy as opposed to high empathy.

Demographics and individual characteristics

Overall, demographic and individual characteristics were controlled for in the study, in order to ensure the matching of groups without inter-individual differences.

Results of the Mehrfach Wortschatz Intelligenztest (MWT), measuring verbal intelligence demonstrated that participant IQ was considered to be in the normal range or slightly above. Additionally, there were no significant personality differences assessed by the NEO Five-Factor Inventory (NEO-FFI), and no significant age differences were observed.

Furthermore, the Multidimensional Mood State Questionnaire (MDBF) was administered prior to OXT administration and after the RMET task, to ensure there were no differences in mood, calmness and wakefulness.

Mentalizing Accuracy

Overall, the results of the study echoed previous research that established the link between mentalizing ability and OXT, and presented it in form of a group-by task difficulty relationship. In the 2 (oxytocin or placebo) x 2 (difficult condition or easy condition) study, when a dose of 24 International Units (I.U.) of OXT was given to participants they showed significant improvement in mentalizing accuracy as compared to the placebo participants. This effect was pronounced for the difficult items of the RMET, with a comparatively subdued effect on the easier items of the test.

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Figure 2. Mean percentage of correct answers for item difficulty on the RMET (easy items vs difficult items) and experimental group (OXT vs placebo).

Bars are characteristic of the mean percentage accuracy on the test ± the standard error of mean (**p < 0.01). (Source: Feeser et al., 2015)

Additionally, when observing a group-by-EQ interaction, Feeser et al. (2015) found an effect for empathy mediating this interaction. Notably, regression analysis suggested the presence of a significant interaction between OXT or placebo and empathy on mentalizing abilities. Figure 3 demonstrates that participants that were rated to have low empathy showed greater in improvement in mentalizing abilities after administration of OXT as compared to individuals taking a placebo. Conversely, there were no significant group differences in individuals’ RMET performance of participants with high empathy, operationalized to have EQ values of 1 standard deviation above the mean.

Similarly, the results of both Hurlemann et al. (2010) and Uzefovsky et al. (2014) mirrored Feeser et al.’s (2015) results that empathy was shown to be privy to OXT stimulation. The neuropeptide enhanced empathic accuracy in those who were less socially proficient, by enhancing attentional selectivity towards relevant social stimuli (Hurlemann et al., 2010; Uzefovsky et al., 2014).

Figure 3. Mean percentage of correct answers determined by group (OXT vs placebo) and empathy scores assessed by Emotional Quotient. The data points are characteristic of mean percentage ± the standard error of mean (**p < 0.01). Low empathy was defined as participants with an EQ one standard deviation below the mean, while high empathy represents individuals with an EQ one standard deviation above the mean.

(Source: Feeser et al., 2015) 13

Another interesting result emerges in observing the relationship between mentalizing and empathy between OXT and placebo groups. The OXT group displayed a lack of a relationship between empathy and mentalizing abilities. However, for the placebo group, there was evidence of a significant positive relationship.

Higher empathy played a role in increasing mentalizing abilities in the placebo group, but failed to have an effect on individuals that were administered with OXT. This is in line with the results of a study conducted by Aoki et al. (2015). Aoki et al. (2015) determined that administration of 24 I.U. OXT increased the ability to infer the emotions of others, enhancing activity in the right anterior insula. Their results displayed OXT plays a role in understanding the emotions of others at both the behavioural and neurological level. As a result, similar results would have been seen to those of Feeser et al. (2015), as OXT improved empathy in the low empathy condition. Therefore, no difference would have been seen between the high empathy and the low empathy conditions in the OXT group.

In accordance with the research conducted by Domes et al., (2007) a three-way interaction should emerge between group, accuracy and empathy for difficult items on the RMET, but fail to show significance with the easy items. Yet, analyses determined that this was not significant in the study. Conducting separate analyses did however show this effect, suggesting the presence of some evidence of this effect, but remained insignificant in the analyses conducted. This echoes an idea by Schultze et al. (2011) who suggest that OXT’s effects may be accentuated from tasks that are challenging, but not extensively difficult.

Perhaps in accordance with this, significant moderating effects of OXT have been presented in other research in the field. More generally, Quirin et al.

(2011) provided substantial proof of the fact that the effects of intranasal OXT is dependent on the difference between high and low emotional regulation abilities (ERA). Additionally, Leknes et al. (2013) determined that OXT improved empathetic abilities selectively in individuals with less emotional sensitivity. Similarly, early life stress had an effect on OXT’s ability to attenuate cortisol stress responses and reduced neural activity during psychosocial stress (Grimm et al., 2014).

The findings of the studies demonstrate that OXT does have an effect in terms of improving mentalizing abilities, but that this relationship is significantly dependent on socio-emotional skills, one of which is empathy. In other words, such individual characteristics play a moderating role on the relationship between OXT and social function.

Conclusions and Discussion Interpersonal communication relies on the ability to infer emotional mental states of others. OXT has been demonstrated to increase this ability, and therefore presumably enhances the ability to develop and maintain social relationships.

Bartz et al. (2011)’s interactionist model concerning the social effects of OXT in humans, has previously defined the role of stable individual differences and

contextual factors in the moderation of OXT administration. This model has been determined to be appropriate as similar moderators have surfaced in the review conducted by Olff et al. (2013), who focused on similar situation and individual variables that presumably affected OXT. Despite research on individual differences such as gender and attachment levels present in these reviews, little research has focused on the importance of specific socio-emotional factors within this domain.

The review draws attention to the idea that OXT application is not uniform in its effect, and is shown to improve mentalizing in individuals who are generally less proficient at social interactions. In the limited studies that have been conducted on the topic, the effects of OXT application have been shown to be a function of baseline emotional regulation abilities (Quirin et al., 2011), emotional sensitivity (Leknes et al., 2013), early emotional stress (Grimm et al., 2014) and empathy (Feeser et al., 2015). In short, the degree of OXT susceptibility varies depending on baseline socio-emotional skills.

The significance of these findings points towards the notion that oxytocin is subtle in its effect on social cognitive and behavior, and strongly depends on baseline individual differences, specifically those that are within the socio-emotional.

Therefore, investigating the effects of socio-emotional individual differences on OXT application can provide insights into how potential emotional abnormalities can benefit from OXT administration. This would grant many individuals such as those inflicted with autism spectrum disorder, schizophrenia, drug addiction and other psychiatric communities the ability to normally interact in cases where they would not have been able to do so otherwise (Leknes et al. 2013).

As oxytocin receptors are found in the amygdala, striatum, hippocampus, nucleus accumbens and midbrain (Meyer-Lindenberg et al., 2011), the presence of this neuropeptide increases neural activity in these regions which have long been implicated with social bonding in neuroscience. The effects of OXT are clearly widespread with significant social, cognitive and behavioural effects.

These studies provide a new pathway of investigation into the wide array of socio-emotional individual differences that permit social interaction and the development of relationships.

Criticisms and Future Directions

The adaptation of oxytocin in motivating prosocial behaviour is crucial in determining our social interactions and relationships. The results of the review provided justification that this neuropeptide has a positive effect on social function, and that socio-emotional traits in particular are important in moderating the effects of oxytocin on increased awareness of socially relevant information, as well as an enhanced ability to mentalize. This is an incremental advancement to literature on the topic, as it includes the possibility of a third variable that moderates a previously established relationship. Nevertheless, it provides a facet by which

to improve and understand social interactions.

However, there exist some shortcomings in the methods employed. While the papers makes convincing arguments for the importance of oxytocin as well as its positive effect on mentalizing abilities (with regards to empathetic behavior), they fall short in being rather correlational, and are unable to determine exact causation.

Firstly, empathetic ability in this study was defined by self-report measures, which are privy to bias. In remedy to this, one solution would involve assessing saliva samples of the participants, as polymorphisms in oxytocin receptor genes have been implicated to have a relationship with empathetic related traits such as emotional processing (Laursen et al., 2014).

Alternatively, it would be helpful to implement performance-based computerized empathy tasks such as those proposed by Smith et al. (2014), or the Multifaceted Empathy Test (MET) that distinguishes between cognitive and emotional empathy (Hurlemann et al., 2010). Either of these proposed amendments would strengthen the evidence for the effect of empathy on the relationship between oxytocin and mentalizing abilities.

Secondly, the studies’ strengths lie in their ability to account for the relationship of a third variable with oxytocin, yet they could be improved by observing the mechanism by which oxytocin facilitates prosocial behavior. By using functional magnetic resonance imaging (fMRI), brain activity could be monitored to determine where, how and to what extent oxytocin acts (Hu et al., 2015) especially considering the different socio-emotional differences that were defined above. These two main areas for improvement will perhaps provide cause for future directions by which the experiments’ causal factor can be better isolated.

Additionally, it has been shown that there are innate genetic differences in the level of empathy that are based on the oxytocin receptor (Laursen et al., 2014); this is a point that needs to be further investigated in light of moderating effect of biological empathetic differences.

Finally, the studies did not isolate for presumable gender differences and cultural differences related to oxytocin dependent effects. Taking into account the fact that these individual differences may play a role will provide a more holistic view of individual traits being able to mediate roles in oxytocin’s effect on social interactions across a wide array of the population.

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14. Lieberwirth, C., & Wang, Z. (2014). Social bonding: regulation by neuropeptides. Frontiers in neuroscience, 8.

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Oxytocin increases amygdala reactivity to threatening scenes in females. Psychoneuroendocrinology, 37(9), 1431-1438.

16. Luminet, O., Grynberg, D., Ruzette, N., & Mikolajczak, M. (2011). Personality-dependent effects of oxytocin: greater social benefits for high alexithymia scorers. Biological psychology, 87(3), 401-406.

17. Meyer-Lindenberg, A., Domes, G., Kirsch, P., & Heinrichs, M. (2011). Oxytocin and vasopressin in the human brain: social neuropeptides for translational medicine. Nature Reviews Neuroscience, 12(9), 524-538.

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38(9), 1883-1894.

19. Quirin, M., Kuhl, J., & Düsing, R. (2011). Oxytocin buffers cortisol responses to stress in individuals with impaired emotion regulation abilities. Psychoneuroendocrinology, 36(6), 898-904.

20. Singer, T. (2006). The neuronal basis and ontogeny of empathy and mind reading: review of literature and implications for future research. Neuroscience & Biobehavioral Reviews, 30(6), 855-863.

21. Smith, M. J., Horan, W. P., Cobia, D. J., Karpouzian, T. M., Fox, J. M., Reilly, J. L., & Breiter, H. C. (2014). Performancebased empathy mediates the influence of working memory on social competence in schizophrenia. Schizophrenia bulletin, 40(4), 824-834.

22. Tabak, B. A., Meyer, M. L., Castle, E., Dutcher, J. M., Irwin, M. R., Han, J. H., ... & Eisenberger, N. I. (2015).

Vasopressin, but not oxytocin, increases empathic concern among individuals who received higher levels of paternal warmth: A randomized controlled trial. Psychoneuroendocrinology, 51, 253-261.

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Oxytocin receptor and vasopressin receptor 1a genes are respectively associated with emotional and cognitive empathy. Hormones and behavior, 67, 60-65 Received July, 7, 2014; revised December, 19, 2014; accepted December, 23, 2014.

This work was supposed by the Cluster of Excellence, “Languages of Emotion” (project number 88120057).

The authors thank Philipp Fuge, Karin Pestke and Emily Brandt for assistance with data collection.

Address correspondence to: “Languages of Emotion”, Freie schwerdter Allee 45, 14195 +49 30 838 50599; fax:

Cluster of Excellence Universität Berlin, HabelBerlin, Germany. Tel.: +49 30 838 52887.

Copyright © 2015 Elsevier Ltd. All rights reserved

Increased Phosphorylation of CREB at Ser133 in the Dentate Gyrus Reverses Depressive Behavior in Rodents Ariba Alam

Depression is a mental disorder that impair an individual’s ability to concentrate resulting in individuals feeling hopeless for short or long periods of time. According to the World Health Organization, depression by 2030 is hypothesized to be the second leading cause of disability in the world. In 2012, there have been 350 million cases reported globally of people that suffer from depression. Fortunately, there is research going on in this field and this disorder has both psychosocial treatment as well as treatment by medicine. Depression is studied in labs through use of behavioral paradigms such as unpredicted chronic mild stress (UCMS), tail suspension test (TST), sucrose preference test, or forced swim test (FST). These studies induce stress on the animal by a foot shock or social isolation to induce depression like behaviors on the rodent. These depressive symptoms are reversed by the administration of antidepressants that target the CREB-BDNF pathway which essentially lead to the overexpression of CREB in the hippocampus. Many studies have reported the dentate gyrus to be the most involved in behavioral symptoms that rodents experience, and CREB upregulation in this part of the hippocampus has reversed depressive behavior the most compared to other areas of the hippocampus such as CA3 and CA1 pyramidal cells. CREB is a protein that is integrated in a pathway of other factors involved such as BDNF, Trk receptor, kinases that must be functional in order for CREB to be expressed in the hippocampus.

Although the use of antidepressants that target upregulation of CREB-BDNF pathway, the causality of depression still has to be determined. These proteins are correlated with depression but the cause is still unknown.

There has to be further studies that explore the core role of these proteins that will lead to a better understanding of depression.

Key words: Depression; cAMP response element-binding protein (CREB); Brain Derived Neurotrophic Factor (BDNF); Dentate Gyrus; Hippocampus; Behavioral Test Paradigms; Antidepressent Drugs Background Studies have provided evidence that stress induces depression like behavior in mice. Depression is implicated to be due to an imbalance of CREB in the brain, particularly in the hippocampus. Depression was thought to be based on the monoamine hypothesis where deficiency in serotonin or noradrenaline caused depressive behavior (Mutlu et al., 2014; Chen, Shirayama, Shin, Neve & Duman, 2001; Gronli et al., 2006; Gass & Riva, 2007). However, this hypothesis does not explain all components of depression therefore is not the sufficient model that is followed (Mutlu et al., 2014). Alternatively, a recent hypothesis based on the CREB –BDNF pathway linked to depression specifically in the hippocampus is more commonly studied (Mutli et al., 2014; Chen, Shirayama, Shin, Neve & Duman, 2001; Gronli et al., 2006, Xiao et al., 2011; Gass & Riva, 2007). CREB is involved in neuronal plasticity and regulating transcription of genes that are associated with stress responses (Guan et al., 2013; Xiao et al., 2011; Gass & Riva, 2007). Stress prevents the phosphorylation of CREB (Ser133) in the dentate gyrus of the hippocampus.

CREB therefore cannot act on the Brain Derived Neurotrophic Factor (BDNF) gene downstream in the pathway therefore preventing the expression of this gene. BDNF has the capability to phosphorylate CREB Ser133,that is further responsible for activating genes that code for cyclic AMP (Gronli et al., 2006; Xiao et al., 2011). Studies have also shown that knocking out one allele for BDNF does not cause depression, but

if that is linked with an inhibitor for mitogen-activated protein kinase (MAPK/MEK) that also acts on CREB by phosphorylating it would cause depression in the animal (Duman, Schlesinger, Kodama, Russell & Duman, 2007). Therefore BDNF which aids neuron survival, synaptic transmission, and synaptic plasticity complements the functions of CREB. Post-mortem brains of depressed patients have showed decrease of CREB protein in the hippocampus (Chen, Shirayama, Shin, Neve & Duman, 2001; Gass & Riva, 2007).

This paper will focus on the expression of CREB protein in the hippocampus and it’s correlation with depression like behavior in rodents.

Primary Paper Introduction:

A study done by Guan L., Jia X., Zhao X., Zhang X., et al. (2013) analyzed the expression levels of CREB/ERK/Bcl-2 in rat brains and their immobility time through a swimming test after experiencing prenatal stress (PS). These levels were measured in the hippocampus, prefrontal cortex and striatum. The prenatal stress that was given was social isolation.

The motive of the article is to understand the role of the ERK-CREB pathway and its changes that occur after PS exposure and how that reflects in a swimming test. These proteins were studied due to their important functions. CREB is involved in neuronal plasticity and regulating transcription of genes that are associated with stress response. ERK responds to extracellular stimuli by regulating gene expression, 16

cellular growth, synthesis of new proteins in order to protect cells. Bcl-2 is regulated by CREB and is an anti-apoptotic factor. It is involved in regulating cell death, cell plasticity and growth of new neurons. ERK is activated by NMDA receptor excitation. MK-801 is a drug used in the study as it is been demonstrated to be affiliated with antidepressant properties. It is a non-competitive antagonist of NMDA receptor therefore blocks glutamate from binding to this receptor.

Saline was used alongside to MK-801.

Introduction to Additional Studies:

Many sorts of behavioral paradigms have been used in studies that test for depression. The more frequently used include a tail suspension test, forced swimming test, sucrose preference, and unpredicted chronic mild stress (UCMS) or chronic mild stress (CMS).

The unpredictable chronic mild stress model stresses out the animal that mimics natural stress which would parallel with human stress. These include food starvation, light-dark cycle altered, empty water bottle in their cage, etc. After exposing these mice to such stresses, they are given an antidepressant: agomelatine or melatonin for treatment (Mutlu et al., 2014).

Another study used foot shocks and then was given imipramine, fluoxetine, isofluran gas, amitriptyline (AMI) or saline to see the effects on the rodent (Chen, Shirayama, Shin, Neve & Duman, 2001; Gronli et al., 2006; Gourley et al., 2008). One study compared the results between antidepressants and a transgenic experiment where they injected Herpes Simplex Virus carrying CREB in the dentate gyrus. They wanted to see the effect of overexpressing CREB has on the animal (Chen, Shirayama, Shin, Neve & Duman, 2001). The animal can also be treated with MK-801, fluoxetine, desipramine, tranylcypromine, reboxetine that fixes CREB levels in the hippocampus after the animal experiences stress (Guan et al., 2013; Tardito et al., 2009). Acupuncture studies have also reversed depression behavior in rodents there is also used in many studies that study CREB-BDNF pathway related to stress (Yang et al., 2013; Sun et al., 2014). After the animals have been exposed to atleast one of these treatments, their CREB and BDNF and ERK levels are measured in the hippocampus and compared with the control group that was given no stress or the saline group. The saline group is given stress, but administering saline does not help them with their depressive behavior, therefore itâ&#x20AC;&#x2122;s represented as another reassurance of the experiment. Major results have indicated that these antidepressants indeed increase the levels of CREB, and BDNF, and ERK in the brain after these treatments and thus reversing depressive behavior.

Research Overview Summary of Major Results

Method/Experiment of Primary Paper:

The method that was utilized by Guan L., Jia X., Zhao X., Zhang X., et al. (2013) consisted of mating sexually active male rats to females over night within 17

a room temperature of 22-26 degrees, humidity 60% and with a 12 hour day/night cycle (control group).

Each pregnant rat was than separated into a control group, PS-Saline group, and PS-MK-801 group. The control group was not given any stress, but the other two groups were socially isolated 3 times a day for 45 minutes. The rats were given either saline or MK-801 on days 14-21 of pregnancy. The researchers than did a forced swimming test (FST) on the offspring to see the effects of PS on them. Also, RNA was extracted from the three brain areas (hippocampus, striatum and prefrontal cortex) and went through an RT-PCR to examine the intensity of ERK, CREB, and Bcl-2 mRNA expression in each.

Results from Primary Paper:

The results from Guan L., Jia X., Zhao X., Zhang X., et al. (2013) showed PS rats showed an increased immobility time. There was lower expression of ERK2 mRNA, CREB mRNA, Bcl-2 mRNA in the hippocampus, prefrontal cortex of PS rats with saline compared to control and MK-801 (modified the expression). The CREB levels were fixed by MK-801 as the levels of CREB were similar to that of the control even after experiencing stress prenatally. There was no significant difference seen in expression of mRNA expression among the three proteins in the striatum.

These changes were all reflected in the increased immobility timing correlated to depressive behaviour.

Results from Additional Studies:

The model size ranged from 9 (Chen, Shirayama, Shin, Neve & Duman, 2001; Gronli et al., 2006; Duman, Schlesinger, Kodama, Russell & Duman, 2007; Tardito et al., 2009) to 15 (Mutlu et al., 2014, Guan et al., 2013; Xiao et al., 2011). The largest experimental size used was 159 (Gourley et al., 2008). The studies showed a decrease in antidepressant behavior after administering an antidepressant such as Agomelatine (Mutlu et al., 2014). This antidepressant allowed the upregulation of BDNF-CREB in the hippocampus to decrease immobility time and also to increase memory of the animal (Mutlu et al., 2014). Another study injected a Herpes Simplex Virus (HSV)-CREB in the dentate gyrus of the hippocampus, which also resulted in less immobility time, and decrease in escape failures (Chen, Shirayama, Shin, Neve & Duman, 2001). This study compared the effect of antidepressants compared to a transgenic experiment where CREB was overexpressed. The results demonstrated that antidepressents decreased escape failures by 65% and CREB overexpression by 45% (Chen, Shirayama, Shin, Neve & Duman, 2001). However, in a swim test they demonstrated 35% reduced immobility time after administering imipramine in the dentate gyrus in comparison with 45% decrease in immobility time through CREB overexpression (Chen, Shirayama, Shin, Neve & Duman, 2001).

Therefore, depending on what stress the animal was given, different techniques to treat the depression would result in better outcomes. Phosphorylation of Ser-133 on CREB in one study also demonstrated a

higher preference for sucrose (Gronli et al., 2006).

One study used the chronic unpredictable stress model which produced depressive behavior in mice.

The results exhibited a 53% reduction of granule cell neurogenesis just after receiving seven days of stress, but this result was evident after a month of observation (Gronli et al., 2006). Although these studies used different methods, the end result was an increase in CREB expression that allowed the depressive behaviors to be reversed.

Figure 1. This figure from the paper by Gass & Riva (2007) show the pathway involved in depressant behavior. Protein CREB that binds to CRE on the DNA to activate BDNF is involved in depressive symptoms if down-regulated. CREB is phosphorylated by upstream factors that include Protein Kinase A, CaMKIV, and RSK proteins. The more these proteins are activated will allow this pathway to be expressed, and BDNF to get transcribed. Alterations in this pathway is correlated with depressive or antidepressive behavior.

Conclusions and Discussion After inducing different types of stresses in rodents, and then administering antidepressants or transgenically overexpression of CREB in the hippocampus resulted in expected results of increased sucrose preference, decreased immobility time during swimming tests and decreased escape failures (Mutlu et al., 2014; Chen, Shirayama, Shin, Neve & Duman, 2001; Gronli et al., 2006; Guan et al., 2013; Xiao et al., 2011; Tardito et al., 2009; Yang et al., 2013). One common factor in all of these studies is the CREB-BDNF pathway that is upregulated specifically in the dentate gyrus which results in antidepressant behavior in rodents (Gass & Riva, 2007). Despite, the drugs improving the performance of mice in these various experiments does not provide information on how long these drugs can be dependent upon. Would the mice ever become unresponsive to them or would a higher dose have to be given to sustain the antidepressant behavior?

Therefore, are these drugs only for short term use?

The protein CREB that is upregulated is influenced by many factors such as cAMP, BDNF, and kinases that allow CREB to be phosphorylated (Gass & Riva, 2007). Although, the involvement of CREB is evident in depression, but the role of this protein still has to be determined especially in all areas of the hippocampus (Gass & Riva, 2007).

The upregulation of CREB only in the dentate gyrus is seen to have antidepressant like effects, and not in the CA3 or CA1 pyramidal cells of the hippocampus or even the prefrontal cortex (Chen, Shirayama, Shin, Neve & Duman, 2001; Gronli et al., 2006; Xiao et al., 2011). Therefore, is it essential to know why CREB only acts in the dentate gyrus and how that influences

Figure 2. This figure from the paper by Chen, Shirayama, Shin, Neve & Duman (2001) show escape failures in rodents. In the control there are low chances of the rats to encounter escape failures. The saline treated rats that were given inescapable foot shock (IES) show higher levels of escape failures. Whereas, fluoxetine and imipramine treated rats after giving them inescapable foot shocks demonstrated a significant decrease in number of escape failures compared to ones treated with only saline (which is not an antidepressant). Figure 3. Rats exposed to inescapable foot shock (IES), and then microinjected with herpes simplex virus control (IES-HSV-LacZ) or HSV-CREB that allowed CREB to be overexpressed in the hippocampus and prefrontal cortex. Overexpression levels of CREB helped determine its involvement in decreasing escape failures or reversing depression symptoms. Different parts of the hippocampus such as dentate gyrus, CA1 pyramidal layer and prefrontal cortex were examined to determine any differences between the areas. The area that was the most significant in decreasing escape failures after overexpressing CREB using Herpes Simplex virus as a transgenic experiment was the dentate gyrus region of the hippocampus. However, the escape failures did not reduce at such a significant level in either the CA1 pyramidal layer or the prefrontal cortex

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the behavior of the rodent. Also, the role of this protein in the other regions of the hippocampus must also be determined to have a better understanding of depression. Knowing more about the projections that the dentate gyrus has would also help to determine where CREB gets transported to and if those areas have any involvement in altering behavior in mice.

A future approach can be taken on determining the role of CREB in CA1 and CA3 pyramidal cells of the hippocampus, and also the mechanism involved in depression.

Depressive behavior is examined when the CREBBDNF pathway is down regulated in the dentate gyrus. When the protein CREB is upregulated in the dentate gyrus reverses depressive behavior observed in many behavioral model paradigms. These behavioral paradigms include a sucrose preference test, tail suspension test (TST), or chronic mild stress model where the rodents are exposed to stressful scenarios that induces depressive behavior. After observing depression in rodents, an antidepressant such as MK-801 or imipramine is injected into the rodent in the hippocampus which reverses depression like behavior. These drugs are observed to act on upregulating CREB in the dentate gyrus.

Conclusions

Depression in people is becoming more frequent in all parts of the world according to the World Health Organization (Blendy, 2006). There is a 10-30% risk of women being affected by this, and 7-15% for men both of which are probable to occur (Blendy, 2006).

The primary approach taken to treat depression was through monoamines, or Selective Serotonin Reuptake Inhibitors (SSRI), however this approach is now not the ideal way of treating depression. The monoamine hypothesis is now replaced with a CREB-BDNF upregulation pathway being involved in depression (Blendy, 2006; Gass & Riva, 2007; Chen, Shirayama, Shin, Neve & Duman, 2001; Gronli et al., 2006; Schmidt, 2011). Through various behavioral models used such as sucrose preference test, inescapable foot shock, tail suspension test, immobility test in swimming that demonstrate depressed behavior are used in studies to study depression. The mechanism in all the studies presented are similar where the rodents are presented with stress initially and then treated with saline, or antidepressents. The pathway in the brain that is targeted is the CREB-BDNF pathway that is dependent on many transcriptional factors and upstream proteins such as TrkB receptors, CRE binding region, cAMP, and PKA (Gass & Riva, 2007). The protein that is essential for reversing depressive behavior is CREB protein, and the area that is involved in antidepressive behavior significantly is the dentate gyrus.

Criticisms and Future Directions

The validity of these behavioral paradigms are questionable. Although they demonstrate behavioral symptoms in rodents, it does not necessarily parallel with human depression. Depression in humans can last for long periods or short periods depending on what 19

the individual goes through in their life. Some humans are more susceptible to depression and some are not (Dzirasa & Covington, 2012). For instance, how does a tail suspension test (TST) or a sucrose preference test demonstrate anhedonia. Humans experience stress either early in life or later, but they are not in the form of foot shocks. Therefore, inducing foot shocks in rodents may alter different pathways in the animal compared to pathways that are altered by other forms of stress such as food starvation. Maybe the mechanism by which these mice are expressing depression symptoms is through a completely different pathway than what occurs in the brain. Therefore, mimicking the right type of stresses is essential. Moreover, upregulation of CREB has been helpful in reversing depressive symptoms, but this is not the cause of depression. CREB is a protein that is affected by another entity that yet has to be determined. The true function of this protein was not explored in all parts of the hippocampus, which is essential to understand the underlying cause of depression. Next, the temporal resolution or the amount of stress that is needed to start the production of these proteins was also not indicated in the study (Duman, Schlesinger, Kodama, Russell & Duman, 2007). It was not clearly identified to how long the rodent express antidepressant behavior after being administered the drug or overexpression of CREB in the dentate gyrus. For instance, after how much exposure to stress does the animal require to start upregulating these proteins or does the increased expression patterns of these proteins start at the same time the rat is exposed to even a little bit of stress? By looking at different approaches to resolve these questions will help gain a better understanding of the roles that these proteins play in depression and also contribute to a holistic understanding of this disease. The models used must also be easily transferred in clinical trials to improve the validity of the tests (Schmidt, 2011). Validity is conquered through integrating most essential features of depression in the animal model. Therefore, the model has to reflect practical scenarios and introduction to new models with predictive ability will aid in unveiling neurobiological mechanisms associated with depression.

Furthermore, measuring guilt, mood or suicidal feelings in rodents is not easily determined, but for humans is easily equitable through questionnaires. Therefore, equating animal models to humans is very difficult.

Another limitation in these studies is the effect of drugs on depressed individuals. In mice, acute administration of antidepressents has an effect on reversing depressive behavior. However, in humans, acute administration of drugs does not reverse depression in all individuals (Dzirasa & Covington, 2012). If the drug has an effect on an individual it would not be apparent until a couple of weeks of administration. Every individual is different with different responses to treatment therefore one form of model in rodents may not be enough to overcome this complex brain disorder.

References 1. Blendy, J. (2006). The Role of CREB in Depression and Antidepressant Treatment. Biological Psychiatry, 59(12), 1144-1150. doi:10.1016/j.biopsych.2005.11.003 2. Chen, A., Shirayama, Y., Shin, K., Neve, R., & Duman,

R. (2001). Expression of the cAMP response element binding protein (CREB) in hippocampus produces an antidepressant effect. Biological Psychiatry, 49(9), 753-762. doi:10.1016/ s0006-3223(00)01114-8 3. Duman, C., Schlesinger, L., Kodama, M., Russell, D., & Duman, R. (2007). A Role for MAP Kinase Signaling in Behavioral Models of Depression and Antidepressant Treatment. Biological Psychiatry, 61(5), 661-670. doi:10.1016/j. biopsych.2006.05.047 4. Dzirasa, K., & Covington, H. (2012). Increasing the validity of experimental models for depression. Annals Of The New York Academy Of Sciences, 1265(1), 36-45. doi:10.1111/j.1749-6632.2012.06669.x 5. Gass, P., & Riva, M. (2007). CREB, neurogenesis and depression. Bioessays, 29(10), 957-961. doi:10.1002/ bies.20658 6. Gourley, S., Wu, F., Kiraly, D., Ploski, J., Kedves, A., Duman, R., & Taylor, J. (2008). Regionally Specific Regulation of ERK MAP Kinase in a Model of AntidepressantSensitive Chronic Depression. Biological Psychiatry, 63(4), 353-359. doi:10.1016/j.biopsych.2007.07.016 7. Gronli, J., Bramham, C., Murison, R., Kanhema, T., Fiske, E., & Bjorvatn, B. et al. (2006). Chronic mild stress inhibits BDNF protein expression and CREB activation in the dentate gyrus but not in the hippocampus proper. Pharmacology Biochemistry And Behavior, 85(4), 842-849. doi:10.1016/j. pbb.2006.11.021 8. Guan, L., Jia, N., Zhao, X., Zhang, X., Tang, G., & Yang, L. et al. (2013). The involvement of ERK/CREB/Bcl-2 in depression-like behavior in prenatally stressed offspring rats.

Brain Research Bulletin, 99, 1-8. doi:10.1016/j.brainresbull.2013.08.003 9. Koch, J., Kell, S., Hinze-Selch, D., & Aldenhoff, J. (2002).

Changes in CREB-phosphorylation during recovery from major depression. Journal Of Psychiatric Research, 36(6), 369-375. doi:10.1016/s0022-3956(02)00056-0 10. Mutlu, Gumuslu, E., Sunnetci, D., Ulak, G., Komsuoglu Celikyurt, I., & Cine, N. et al. (2014). The Antidepressant Agomelatine Improves Memory Deterioration and Upregulates CREB and BDNF Gene Expression Levels in Unpredictable Chronic Mild Stress (UCMS)-Exposed Mice. Drug Target Insights, 11. doi:10.4137/dti.s13870 11. Schmidt, M. (2011). Animal models for depression and the mismatch hypothesis of disease. Psychoneuroendocrinology, 36(3), 330-338. doi:10.1016/j.psyneuen.2010.07.001 12. Sun, L., Liang, J., Guo, T., Guo, Z., Yang, X., & Wang, S. et al. (2014). Effect of Acupuncture on Expression of CREB and p-CREB in Hippocampus and Prefrontal Cortex of Depression Rats. The Journal Of Alternative And Complementary Medicine, 20(5), A84-A85. doi:10.1089/acm.2014.5222. abstract 13. Tardito, D., Musazzi, L., Tiraboschi, E., Mallei, A., Racagni, G., & Popoli, M. (2009). Early induction of CREB activation and CREB-regulating signalling by antidepressants.

The International Journal Of Neuropsychopharmacology, 12(10), 1367. doi:10.1017/s1461145709000376 14. Xiao, L., Shu, C., Tang, J., Wang, H., Liu, Z., & Wang, G. (2011). Effects of different CMS on behaviors, BDNF/

CREB/Bcl-2 expression in rat hippocampus. Biomedicine & Aging Pathology, 1(3), 138-146. doi:10.1016/j. biomag.2010.10.006 15. Yang, L., Yue, N., Zhu, X., Han, Q., Liu, Q., Yu, J., & Wu, G. (2013). Electroacupuncture upregulates ERK signaling pathways and promotes adult hippocampal neural progenitors proliferation in a rat model of depression. BMC Complement Altern Med, 13(1), 288. doi:10.1186/14726882-13-288 April, 6, 2015 This work was supported by The Association for the Development of Undergraduate Neuroscience Education (SRA & RLN), The Endowment for Science Education (EA), and The Synaptic State Faculty Research Foundation (EA).

The authors thank Mr. Spine L. Cord, Dr. Amy G. Dala, and the students in Neuroscience 101 for technical assistance, execution, and feedback on this lab exercise.

Address correspondence to: Ariba Alam, Human Biology Department, University of Toronto, Toronto, CA Email: rln@apc.edu Copyright Š 2015 Dr. Bill JU, Neurosciences, Human Biology Program

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The rate of increase in adult hippocampal neurogenesis and spatial learning in C57BL/6J mice is greater in response to voluntary exercise than in response to sensory stimuli manipulations Samin Alikhanzadeh

Adult hippocampal neurogenesis refers to the continual formation of new neurons from progenitor cells.

Previous studies on rodent models have shown that environmental enrichment possessing both sensory and motor stimuli increases neurotrophic levels which act to enhance neuritogenesis, cell survival, and neuronal differentiation. The aforementioned neurogenic benefits then are correlated with enhanced cognitive performance in test models. In later studies, the effect of the two components of enrichment were tested alone. The results indicated that enhanced adult hippocampal neurogenesis and spatial learning mice occurs solely following motor enrichment. This study conducted by Mustroph et al. (2012) tested the effect of voluntary wheel running and different sensory modalities including tactile, visual, dietary, auditory, and vestibular on neurogenesis and cognitive performance. Counting the number of co-labelled BrdU positive and NeuN positive cells per granule layer volume, it was shown that neurogenesis is greatest following motor enrichment. In the same study, testing for cognitive performance on a Morris water maze, it was shown that mice exposed to motor enrichment reduced path length to platform by 75% by the second day of the acquisition period while the learning period for mice in control and sensory enrichment group was 5 days. Consistent with previous findings, this study concludes that voluntary exercise confers neurogenic and cognitive benefit to rodents that are absent following sensory enrichment of different modalities. In many of the neurodegenerative and psychiatric disorders the rate of adult hippocampal neurogenesis is greatly reduced. Therefore, aerobic exercise can be used in conjunction with therapeutic interventions to enhance the proliferation of progenitor cells and survival of new neurons to reduce the burden of these conditions.

Key words: Adult Hippocampal Neurogenesis (AHN)), Spatial learning, Neurotrophic factors, Environmental enrichment, Aerobic exercise, Sensory stimulation.

Background Neurogenesis is the process of generating new neurons from the undifferentiated progenitor cells.

This multi-step process involves regulatory factors which induce newborn cells to proliferate, mature, differentiate, and ensure their survival. In an adult brain there are two regions in which neurogenesis occurs throughout oneâ&#x20AC;&#x2122;s lifetime: the subventricular zone (SVZ) and the subgranular layer of the dentate gyrus (DG). Adult hippocampal neurogenesis (AHN) declines with age and is disrupted in neurodegenerative diseases such as Alzheimerâ&#x20AC;&#x2122;s disease. Therefore enhancing neurogenesis may have the potential to rescue learning and memory dysfunction in these conditions as adult born neurons have enhanced excitability and synaptic plasticity.

Literature on AHN suggests that this process is influenced by external conditions which act to modulate the level of internal neurotrophic factors. For instance increasing VEGF and BDNF neurotrophic factors influence the proliferation of progenitor cells and survival of new born neurons respectively and thereby results in an enhanced rate of AHN. (Vithlani et al., 2013) Integration of these new neurons in existing neuronal circuitry has been suggested to improve spatial learning and memory. On the hand, research has shown that learning experiences can enhance AHN in rodent brains. In a study conducted

by Ambrogini et al. (2000) it was shown that spatial learning enhances survival of the new born neurons of 21

the dentate gyrus. Therefore, while neurogenesis can enhance hippocampal dependent learning, in a feed forward mechanism spatial learning can also enhance AHN. One of the external factors that reduces AHN through the reduction of neurotrophic factors is stress. Stress can also have detrimental effects on the health of the existing neurons by inducing the formation of reactive oxygen species (ROS). ROS are accumulated in the body as a result of aging; this can partly account for the reduced survival of the neurons and ultimately the reduction of AHN in older mice. (van Praag, Shubert, Zhao, & Gage, 2005) In a study by Lemon, Rollo, & Boreham (2008) it was shown that intake of dietary anti-oxidants -a component of enriched environmentreduces the burden of ROS and conferred neurogenic benefits.

Studying the effect of voluntary exercise â&#x20AC;&#x201C;a component of enriched environment- on aged mice, van Praag and colleagues showed that the decline in neurogenesis as a result of aging was ameliorated to half of the decline in an aged-match control group and behaviorally runner mice had improved learning and memory of the test maze. It was further suggested that the decline in synthesis of new neurons in major depressive disorders (MDD) and the associated depressive symptoms can be alleviated by exercise in a similar mechanism to the action of antidepressants by enhancing neurotrophic factors and increasing the number of new neurons.

In one study Kempermann, Kuhn, & Gage (1998)

tested for the effect of sensory and motor enrichment on AHN. In this study the animals were housed in groups with the opportunity for sensory exploration, social interactions, and exercise. The results indicated an increase in survival of new neurons in aged mice of enrichment group as compared to mice of the control group. In a follow up study by Kobilo et al. (2011), sensory and motor components of environmental enrichment were tested separately. The results showed that presence of motor stimuli alone is responsible and sufficient for neurogenic and procognitive benefits in mice.

By addressing the shortcoming of previous studies, Mustroph and colleagues aimed to analyze the impact of different novelty and modality of sensory stimulitactile, visual, dietary, auditory, and vestibular- and motor stimuli on AHN and cognitive abilities in singly housed male C57BL/6J mice. In this review we provide some details of their results and discus the implications of their findings in relation to other studies.

Furthermore, we provide the future directions that this study may take.

Research Overview Summary of Major Results

Average time spend interacting with sensory and motor stimuli

32 male C57BL/6J mice were singly housed in 4 different housing conditions as followed: Running wheel present in their cage (RUN), rotatable novel toys and diets presented in their cage (EE), RUN and EE, and standard housing conditions (Control). Video tracing of the animals were used to assess the time each animal spends doing physically activities and the time they spend interacting with toys. On average mice in RUN group spent 2.3 hours/day and mice in RUN+EE group1.8 hours/day on running wheels which exceeded the activity of mice in EE and Control group respectively. The difference between the activity of RUN and RUN+EE mice was not statistically significant. On the other hand, mice of the EE group spent 81% of their time in the enriched environment which exceeds the RUN+EE group by 13%. These data are used in assessment of the correlation of neurogenic and cognitive benefits to the sensory and motor stimuli in different housing conditions.

Motor stimuli enhances the rate of Adult Hippocampal Neurogenesis

The average granule layer volume in control animals was 0.46 μm3 ± 0.023. Running increased the volume of granule layer by 20% and environmental enrichment increased the volume of the granule layer by 12%. These effects were additive as the greatest increase was seen in EE+RUN group with granule layer volume of 0.65 μm3 ± 0.041.

The numerical count using confocal microscopy indicated that the greatest BrdU+/NeuN+ cell density was in the Run group with 7.6 ± 0.77 (new neurons per cubic mm × 103). This was followed by mice

of RUN+EE condition with 6.0 ± 0.45, mice of EE condition with 5.2 ± 0.34, and mice of control condition with 4.3 ± 0.39 new neurons respectively.

The immunohistochemical analysis indicated an approximate 80% overlap between BrdU+ cells and the NeuN+ neurons. (Fig. 1E) The co-expression of the two markers suggests that the dividing cells are differentiating into mature neurons in the hippocampus confirming enhancement of the AHN. Analysis of the co-variance showed significant correlation between the average distance traveled on the wheel and the BrdU+/NeuN+ cell density. (Fig.1G) However, posthoc pairwise analysis indicated no significant difference of co-labelled neurons between EE versus Control, and EE+RUN versus EE group.

Consistent with these findings, in a study by Kohl et al. (2002) it was shown that while postweaning enrichment-enhanced physical activity- possesses significant neurogenic benefits, preweaning enrichment-sensory nourishments- has no lasting effect on AHN. Moreover in a more thorough study by van Praag and collaugues analyzing different components of motor and sensort stimuli, it was shown that the greatest enhancement in AHN occurs in mice following voluntary wheel running as compared to mice following social interactions or swimming.

Figure 1.

Aerobic exercise is the critical component of environmental enrichment in inducing adult hippocampal neurogenesis. New neuron staining as an indication of neurogenesis in A) Control, B) EE, C)RUN, and D) EE+RUN mice.

E) NeuN+ neuron stained in green and BrdU+ neuron stained in red. Double labeled BrdU+/ NeuN+ neuron is shown by overlap of green and red. F) Average number of new neurons as measured by the number of BrdU+/NeuN+ neurons in the dentate gyrus of mice in different groups. G) Correlation of the distance traveled and the number of BrdU+/NeuN+ neurons.

(Mustroph et al., 2012)

Motor stimuli enhances spatial learning and memory Mice in all housing conditions spent more time on the quadrant with the hidden platform than other three quadrants of the Morris water maze.(Fig.2 B) Following the five days learning period all animals learned and retained memory of the location of the platform using outside environmental cues. Previous 22

work on exercise by van Praag and colleagues had shown that acquisition and retention of a Morris water maze in runner mice is better than the control nonrunner aged-match mice. Consistently in this study, there was a decrease in path length and latency to find the platform across the testing period; and mice in RUN group showed the fastest acquisition with a 75% decrease in length of the path by the second trial.

(Fig.2 A) There are significant posthoc differences elicited on day two between mice of the RUN versus mice of all other groups (p<0.05). Enhanced spatial learning and memory following enhanced AHN is the cognitive benefit observed as a result of exercise in many studies. (Kempermann et al., 1998; Rhodes et al., 2003)

Figure 2. Performance on Morris Water Maze. A) The path length to find the hidden platform in mice of different housing condition over 5 days of the testing period. B) Duration of time spent in the quadrant containing the hidden platform. (Mustroph et al. 2012)

Conclusions and Discussion Confirming previous study by Kobilo and colleagues, the results obtained in the study by Mustroph et al. indicates that in an enriched environment physical exercise is the critical component in increasing AHN and spatial learning in adult male C57BL/6J mice.

These results indicated that exercise can be used in conditions where the therapeutic aim is to enhance the proliferation and survival of neurons such as in MDD. Many research has been done to pinpoint the mechanism that accounts for enhanced AHN following environmental enrichment. It was previously thought that motor stimuli enhances the proliferation of progenitor cells and sensory stimuli enhances survival of the newly born neurons.(Olson, Eadie, Ernst, & Christie, 2006) Follow up studies by Fuss et al. (2010) showed that running enhances AHN by enhancing BDNF expression and increasing cell survival rather than proliferation. Therefore it can be concluded that 23

while environmental enrichment with sensory stimuli has no neurogenic benefits in C57BL/67 male mice, physical exercise enhances AHN by increasing the survival of new neurons.

Even though mice in EE+RUN group had equal access and spent similar amount of time on the running wheels as mice in RUN group, they failed to display similar cognitive benefits. This result may seem contradictory as the overall number of BrdU+/NeuN+ cells were also similar between the two groups. However, the results can be explain in two different ways: neuronal density and neuronal plasticity. Due to the additive effect of motor and sensory stimuli on the granule layer volume, mice in EE+RUN group displayed the greatest volume which make the area less dense with new neurons than the granule layer of mice in RUN group. The density of neurons in that sense can then impact the size of neurons, the dendritic and axonal processes, and the neuronal communications.

(Redila & Christie, 2006) Another possible explanation is that due to interaction with novel toys and for efficient processing of sensory stimuli, new neurons are recruited into existing neuronal circuitry. This integration of new neurons are absent in the RUN group which makes them more plastic and allows for enhanced spatial learning and memory. (Clark et al., 2012) The many modalities of environmental enrichment studied on singly housed mice in this experiment did not confer any of the neurogenic and cognitive benefits that were apparent following aerobic exercise. However, it cannot be concluded that sensory enrichment does not influence the brain. In a study by Schapiro (2002) it was argued that social interaction is an important component in an enriched environment.

Although this experiment does not account for social enrichment, previous studies by Kobilo and colleagues tested for the effects of social interaction on AHN with no improvement in female C57BL/6J mice.

Conclusions

Previous research has shown that an enriched sensory and motor environment enhances neurogenesis and cognitive performance in mice. Provided these results, researchers later aimed to segregate the two components of environmental enrichment; female mice exposed to motor stimuli showed enhanced neurogenic and cognitive benefits when compared to mice exposed to sensory stimuli. However, the study could not thoroughly discount the importance of sensory stimuli on brain function as they were solely conducted in female mice in group housing and did not account for different modalities of sensory stimuli.

By addressing these shortcomings this paper aimed to assess the impact of broad and novel sensory stimuli in single housed male mice. This study confirms the previous results of Kobilo and colleagues; showing that aerobic exercise is the essential component of environmental enrichment and that sensory stimuli of different modality and novelty do not confer any neurogenic and cognitive benefits in C57BL/67 male mice.

Criticisms and Future Directions

Previously Greenough, McDonald, Parnisari, & Camel, (1986) had shown differential brain changes including angiogenesis, dendritogenesis, and synaptogenesis within the visual cortex and the cerebellum following sensory stimuli. Therefore there are various brain changes that can occur in response to different experimental conditions. Structural changes such as angiogenesis, synaptogenesis, and astrogenesis confer cognitive benefits associated to the region in the brain in which they occur. Astrocytes in the tripartite synapses regulate development, maintenance, and plasticity of the cortex through the release of cytokines.

While it is known that sensory deprivation can lead to astrocytic dysfunction, the effect of sensory enrichment on astrogenesis is not well understood. (Bengoetxea, Ortuzar, Rico-Barrio, Lafuente, & Argandoña, 2013) Density of cortical astrocytes between mice in RUN and EE group can be analyzed using immunohistochemical staining for S-100 β protein.

Stress is a key modulator of AHN. Previous research on the hypothalamic-pituitary-adrenal (HPA) axis has shown that secretion of corticosterone is upregulated following stressful stimuli. Binding of this hormone to glucocorticoid and mineralocorticoid receptors can result in down regulation of AHN. Research has suggested that running and forced separation induces corticosterone release while complex environmental stimuli and social interactions reduce corticosterone levels. (Grégoire, Bonenfant, Le Nguyen, Aumont, & Fernandes, 2014) Using enzyme-linked immunoassay kits, the authors can measure the concentration of corticosterone in blood to assess the stress response of each animal to different sensory and motor stimuli.

Diet encompasses frequency, total intake, and content of food. Although one component of sensory enrichment in this study was diet, the dietary supplement provided were cashews and other nuts high in polyunsaturated fatty acids in a single dietary regimen.

Therefore, while the study fails to fully account for the effect of diet it concludes that dietary manipulations as a part of environmental enrichment did not contribute to the rate of neurogenesis and cognitive functions in the animal models. In order to understand the relative contribution of diet, the authors should measure the rate of neurogenesis and spatial memory in response to various mode of calorie restriction, intake of polyphenols which have neuro-protective effects, and intake of polyunsaturated fatty acids which are known to decrease BDNF and neurogenesis. (Murphy, Dias, & Thuret, 2014) References 1. Ambrogini, P., Cuppini, R., Cuppini, C., Ciaroni, S., Cecchini, T., Ferri, P., … Del Grande, P. (2000). Spatial learning affects immature granule cell survival in adult rat dentate gyrus. Neuroscience Letters, 286(1), 21–24.

2. Bengoetxea, H., Ortuzar, N., Rico-Barrio, I., Lafuente, J. V., & Argandoña, E. G. (2013). Increased physical activity is not enough to recover astrocytic population from dark-rearing. Synergy with multisensory enrichment is required. Frontiers in Cellular Neuroscience, 7(October), 170. http://doi.org/10.3389/fncel.2013.00170

Clark, P. J., Bhattacharya, T. K., Miller, D. S., Kohman, R. A., Deyoung, E. K., & Rhodes, J. S. (2012). New neurons generated from running are broadly recruited into neuronal activation associated with three different hippocampusinvolved tasks. Hippocampus, 22(9), 1860–1867. http:// doi.org/10.1002/hipo.22020 3. Fuss, J., Ben Abdallah, N. M. B., Vogt, M. A., Touma, C., Pacifici, P. G., Palme, R., … Gass, P. (2010). Voluntary exercise induces anxiety-like behavior in adult C57BL/6J mice correlating with hippocampal neurogenesis. Hippocampus, 20(3), 364–376. http://doi.org/10.1002/hipo.20634 4. Greenough, W. T., McDonald, J. W., Parnisari, R. M., & Camel, J. E. (1986). Environmental conditions modulate degeneration and new dendrite growth in cerebellum of senescent rats. Brain Research, 380(1), 136–143. http:// doi.org/10.1016/0006-8993(86)91437-X 5. Grégoire, C. A., Bonenfant, D., Le Nguyen, A., Aumont, A., & Fernandes, K. J. L. (2014). Untangling the influences of voluntary running, environmental complexity, social housing and stress on adult hippocampal neurogenesis. PLoS ONE, 9(1). http://doi.org/10.1371/journal.pone.0086237 6. Kempermann, G., Kuhn, H. G., & Gage, F. H. (1998).

Experience-induced neurogenesis in the senescent dentate gyrus. The Journal of Neuroscience : The Official Journal of the Society for Neuroscience, 18(9), 3206–3212. http:// doi.org/10.1016/S0149-7634(97)00008-0 7. Kobilo, T., Liu, Q.-R., Gandhi, K., Mughal, M., Shaham, Y., & van Praag, H. (2011). Running is the neurogenic and neurotrophic stimulus in environmental enrichment. Learning & Memory (Cold Spring Harbor, N.Y.), 18(9), 605–609. http://doi.org/10.1101/lm.2283011 8. Kohl, Z., Kuhn, H. G., Cooper-Kuhn, C. M., Winkler, J., Aigner, L., & Kempermann, G. (2002). Preweaning enrichment has no lasting effects on adult hippocampal neurogenesis in four-month-old mice. Genes, Brain and Behavior, 1(1), 46–54. http://doi.org/10.1046/j.16011848.2001.00009.x 9. Lemon, J. A., Rollo, C. D., & Boreham, D. R. (2008).

Elevated DNA damage in a mouse model of oxidative stress: Impacts of ionizing radiation and a protective dietary supplement. Mutagenesis, 23(6), 473–482. http://doi. org/10.1093/mutage/gen036 10. Murphy, T., Dias, G. P., & Thuret, S. (2014). Effects of diet on brain plasticity in animal and human studies: Mind the gap.

Neural Plasticity. http://doi.org/10.1155/2014/563160 11. Mustroph, M. L., Chen, S., Desai, S. C., Cay, E. B., DeYoung, E. K., & Rhodes, J. S. (2012). Aerobic exercise is the critical variable in an enriched environment that increases hippocampal neurogenesis and water maze learning in male C57BL/6J mice. Neuroscience, 219, 62–71. http://doi. org/10.1016/j.neuroscience.2012.06.007 12. Olson, A. K., Eadie, B. D., Ernst, C., & Christie, B. R. (2006). Environmental enrichment and voluntary exercise massively increase neurogenesis in the adult hippocampus via dissociable pathways. Hippocampus. http://doi. org/10.1002/hipo.20157 13. Redila, V. A., & Christie, B. R. (2006). Exercise-induced changes in dendritic structure and complexity in the adult hippo24

campal dentate gyrus. Neuroscience, 137(4), 1299–1307. http://doi.org/10.1016/j.neuroscience.2005.10.050 14. Rhodes, J. S., van Praag, H., Jeffrey, S., Girard, I., Mitchell, G. S., Garland, T., & Gage, F. H. (2003). Exercise increases hippocampal neurogenesis to high levels but does not improve spatial learning in mice bred for increased voluntary wheel running. Behavioral Neuroscience, 117(5), 1006–1016. http://doi.org/10.1037/0735-7044.117.5.1006 15. Schapiro, S. J. (2002). Effects of social manipulations and environmental enrichment on behavior and cell-mediated immune responses in rhesus macaques. Pharmacology Biochemistry and Behavior, 73(1), 271–278. http://doi. org/10.1016/S0091-3057(02)00779-7 16. Van Praag, H., Shubert, T., Zhao, C., & Gage, F. H. (2005). Exercise enhances learning and hippocampal neurogenesis in aged mice. The Journal of Neuroscience : The Official Journal of the Society for Neuroscience, 25(38), 8680–8685. http://doi.org/10.1523/JNEUROSCI.1731-05.2005 17. Vithlani, M., Hines, R. M., Zhong, P., Terunuma, M., Hines, D. J., Revilla-Sanchez, R., … Moss, S. J. (2013).

The ability of BDNF to modify neurogenesis and depressivelike behaviors is dependent upon phosphorylation of tyrosine residues 365/367 in the GABA(A)-receptor γ2 subunit.

The Journal of Neuroscience : The Official Journal of the Society for Neuroscience, 33(39), 15567–77. http://doi. org/10.1523/JNEUROSCI.1845-13.2013

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subPCP induced alterations in gut microbiota associated with memory deficit in schizophrenia model Mie Andersen

Over the years, Schizophrenia has been intensely studied to find the underlying cause for the disorder. Many studies have focused on the genetic factors involved, but an ac-cumulation of data is now pointing toward a possible im-mune system mediated pathogenesis for the disorder. In line with this, there is an increasing interest in the gut-brain axis and the gut microbiota as possible players in the pathogenesis of schizophrenia. This was addressed by Jørgensen et al. Using the subchronic PCP induced schizophrenia rat model, they discovered a correlation between altered GM and cognitive deficits in the model. In this paper, their research will be reviewed with emphasis on the findings implying the GM in schizophrenia, and furthermore how they comply with other data. Furthermore arguments for and against the role of gut microbiota in the pathogenesis of schizophrenia will be discussed in this article.

Key words: Acute phencyclidine (aPCP); Gut-brain axis; Gut microbiota (GM); Locomotor activity assay (MOTR); Novel object recognition (NOR); Schizophrenia; Subchronic phencyclidine (subPCP) Background Schizophrenia is a complex disorder characterized by a multitude of symptoms including positive and negative symptoms. The underlying mechanism for its pathology is not well understood, and both environmental and genetic factors seem to be responsible for the phenotype.

In recent years, a bidirectional pathway between the gut and the brain â&#x20AC;&#x201C; termed the gut-brain axis â&#x20AC;&#x201C; has become more evident. Furthermore, the importance of the gut mi-crobiota (GM) with its regulatory role in the gut-brain axis is of increasing interest in disease and as possible therapeu-tic tool. Thus, variations in the GM have been implied in various CNS related disorders such as autism and multiple sclerosis2,3.

For instance, the gut microbiota has been shown to trigger an autoimmune response leading to multi-ple sclerosis, thus presenting a possible target for treatment3. Hence, the research, reviewed in this article, focus on the possible involvement of the gut-brain axis in schizo-phrenia4.

The gut microbiota plays an important role in regulation of the immune system, and thus alterations in the GM can lead to dysregulation of the immune system and cause inflammatory responses5.

In search of genetic factors underlying schizophrenia numerous genome wide studies have been conducted, and interestingly genes commonly linked to schizophrenia are related to the immune system6,7, suggesting that alterations in the immune system is contributing to the pathogenesis of schizophrenia. In addition, autoimmune diseases has also been linked with schizophrenia8, further implicating the immune system in the disorder.

Gastrointestinal dysregulation and inflammation has al-so been associated with schizophrenia. Namely, a there is considerable gastrointestinal comorbidity in schizophrenia, such as irritable bowel syndrome and inflammatory bowel disease observed in9.

In this fashion the GM and the gut brain axis is able to explain both genetic and environmental factors contributing to schizophrenia. However, despite this abundance of evidence linking the immune system and the GM with schizophrenia, it is not clear, whether the immune system constitutes a causative factor in the schizophrenic phenotype, or if the two are otherwise associated.

Figure 1 The gut brain axis. Abnormal CNS function leads to dysregulation of the gastrointestinal dynamics and alterations of gut microbiota, which in turn affect the brain.

Source: Cryan and Dinan1

26

Figure 2 NOR performance by vehicle- and subPCP-treated rats, respectively, at 0 weeks (T0), 3 weeks, (T3) and 6 weeks (T6) after washout. DI: discrimination index (Source: Jørgensen et al13)

Research Overview Summary of Major Results

Duration of subPCP induced effects

The subPCP induced schizophrenia model is a widely used model for studying the effect of new therapeutic candidates in treating schizophrenia. The model mimics both positive and negative symptoms of schizophrenia.

Thus, subPCP has been shown to increase locomotor sensitivity and to impair working memory. However, the duration of the subPCP-induced effect on memory had not previously been described, why Jørgensen et al wanted to examine this.

After subjecting rats to subPCP-treatment followed by one week of washout, locomotor activity and memory were evaluated by acute PCP (aPCP) induced locomotor activity assay (MOTR) and novel object recognition (NOR), respectively. In agreement with previous findings10, the group found subPCP to increase locomotor activity in response to aPCP and to impair the memory performance. To study the duration of the memory deficit, the rats were tested NOR either immediately after, three weeks, or six weeks after washout, respectively. The group found that the decreased NOR performance resulting from subPCP was evident up until three weeks after washout (Figure 2), and that the increased locomotor activity was still evident at six weeks after washout – the latter serving as a control that subPCP were able to induce changes in all groups of rats tested.

These results show that the subPCP-induced cognitive effects in this model last for 3 weeks after washout and are reversed after 6 weeks.

Roseburia and Dorea of the Lachnospiraceae family and the genus Odoriabacter tended to be elevated in subPCP treated rats immediately after washout. Likewise, an un-known genus belonging to the S24-7 family tended to be elevated at three weeks after washout.

These results show a correlation between the GM and the behavioural changes observed in the subPCP-induced model.

A causative relation between GM and NOR

Jørgensen et al wanted to explore the nature of the dynam-ics between the GM changes and the impaired NOR in subPCP-treated rats to find a possibly causative relation between the two.

In order to address this, the group repeated the experi-ments in rats that were administered ampicillin. It was found that NOR performance was similar between the subPCP treated group and the vehicle treated group (Figure 3) when the GM was reduced – thus, antibiotic reduction was able to rescue the cognitive deficits induced by subPCP. The ampicillin had no effect in MOTR. This suggest that the subPCP-induced changes in GM is upstream of the memory deficits seen in the model.

Changes in GM after subPCP

The authors hypothesize that variance within the subPCP induced model can be caused by variations in the GM. Thus, they investigated the effect of subPCP on GM and the association between GM and behaviour.

In order to examine the influence of subPCP on the gut flora, samples of the GM were collected from the rats at 0 and three weeks after washout, and the samples were sequenced. Comparing the samples from subPCP treated and vehicle treated rats, the Jørgensen et al found a weak but significant difference between the groups. The genera 27

Figure 3 NOR performance by vehicle- and subPCPtreated rats that were either administered antibiotics or not.

DI: dis-crimination index (Source: Jørgensen et al13)

Conclusions and Discussion The results suggest that the subPCP induced rat model can be used for studying pharmaceuticals against schizo-phrenia up to three weeks after washout, which is relevant as it permits testing for a longer period using the same animals, and this in turn means that fewer animals are needed in evaluations of new drugs. While the authors make a somewhat general statement that this is useful in utilizing the subPCP-induced animal model, it is important to keep in mind that they did only show this in the rat mod-el, and that other numbers may apply to the mouse model.

The fact that the ampicillin treatment seemed to abolish the cognitive effects in the subPCP treated rats suggests that the GM is operational in causing the cognitive changes. Prior studies by the group showed similar changes in GM in two different stress models – elevated levels of Roseburia and Dorea in social disruption, and elevated Odoribacter in the trippletest11,12. The authors reason that this supports that changes in GM in the present is due to subPCP-induced stress.

At three week after washout, an unknown genus be-longing to the S24-7 family tended to be elevated in com-parison to the vehicle treated rats. Another unknown genus of the S24-7 family has previously been associated with enhanced spatial memory13. As pointed out by the authors this could indicate that the rise in the unknown genus could be the initiation in restoring the memory, as the NOR per-formance is back to normal at 6 weeks. However, this would need further investigation before a conclusion can be made.

Sequencing GM samples from rats at six weeks after washout might be useful in testing this theory. In addition, this may also show whether the GM has been completely normalized thus supporting the correlation between the GM and the memory deficit.

In conclusion Jørgensen et al made two major findings: (1) that the effects of subPCP on NOR is evident in the rat model for three weeks following washout; and (2) that sub-PCP induced changes in GM correlated with decreased NOR performance, and that antibiotic reduction was able to rescue this memory deficit .

Criticisms and Future Directions

The authors argue that the findings are useful for future studies on schizophrenia using this model, as they de-scribed the period for which it can be used following wash-out and account for some variance within the model. How-ever, it is worth considering whether their findings in fact suggest that the model is not applicable in studying schiz-ophrenia. That is, if the cognitive deficits in actual schizo-phrenic individual is not caused by changes in the GM, then the pharmaceuticals that shows a positive impact on cognition in the model will not necessarily show the same effect in schizophrenic patients. Therefore, it is important to research the GM composition in other schizophrenia models that exhibit similar cognitive deficits. Even more relevant would be to examine the dynamics of GM in schizophrenic individuals.

In fact, a recent study showed that minocycline, an anti-biotic compound, was able to reduce the cognitive

symp-toms in schizophrenic patients14, supporting the theory that these can actually be caused by dysregulation in the GM. Furthermore, studies have shown that thioridazine – a first generation antipsychotic drug used for treating schizophre-nia – has antibiotic abilities15. However, this drug was used to reduce the positive symptoms in schizophrenic patients and not the cognitive ones. On the contrary, a meta-analysis indicated a negative impact of thioridazine on working memory in schizophrenia16. These effects could in turn be due to other pathways than inflammation and thus not connected to the antibiotic effect of the drug.

However, this would need further investigation.

As mentioned by Jørgensen et al it is necessary to eliminate the possibility of a confounding factor between the ampicillin treatment and the rescue of memory impairment. This could be done by transplanting microbiota from the subPCP treated rats into a control group of germ-free rat, as done by others17,18, in order to see if the phenotypes change correspondingly. This could reveal whether ampicillin treatment has a direct impact on the brain. Additionally it would show if the GM alone could induce a memory deficit without any residual PCP. Furthermore, using specific antibiotics19 targeting each of the microbial species elevated by subPCP may lead to finding the microbe accountable for the phenotype. This could be the first step in elucidating the possible molecular pathway from GM to brain function observed in the subPCP induced schizophrenia model by Jørgensen et al. If in fact the genera Roseburia, Dorea and Odoribacter are contributing to the memory deficits in the rat model, it would be interesting to examine the NOR performance in the stress models that were previously shown to have ele-vated levels of those genera11,12. This might help under-stand the necessity or sufficiency of the microbiota in causing the phenotype.

It is important to keep in mind that the study conducted

by Jørgensen et al show that GM may be accountable for some but not all phenotypic traits of schizophrenia in the model. Nevertheless, these findings may lead to a better understanding of the disorder and maybe eventually a GM based treatment of said symptoms.

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14. Liu, F. et al. Minocycline supplementation for treatment of negative symptoms in early-phase schizophrenia: a double blind, randomized, controlled trial. Schizophrenia research 153, 169-176, doi:10.1016/j.schres.2014.01.011 (2014).

15. Thorsing, M. et al. Thioridazine induces major changes in global gene expression and cell wall composition in methicillin-resistant Staphylococcus aureus USA300. PloS one 8, e64518, doi:10.1371/journal.pone.0064518 (2013).

16. Fenton, M., Rathbone, J., Reilly, J. & Sultana, A. Thioridazine for schizophrenia. The Cochrane database of systematic reviews, CD001944, doi:10.1002/14651858.CD001944. pub2 (2007).

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Current issues in molecular biology 10, 17-24 (2008).

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19. Citorik, R. J., Mimee, M. & Lu, T. K. Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases. Nature biotechnology 32, 1141-1145, doi:10.1038/ nbt.3011 (2014).

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Environmental Enrichment: A Neurorehabiitation Method utilized to treat deficits conferred from Traumatic Brain Injury (TBI) Ami Baba

Traumatic brain injury (TBI) is a debilitating injury that is prevalent in the general population, and often results in various impairments in speech, memory, attention, mobility, and executive functions. In order to regain deficits that have been acquired after sustaining a TBI, environmental enrichment (EE) has been used as a neurorehabilitation model that exposes individuals with TBI to a social, interactive, and complex space. EE has found to be effective in improving deficits in cognitive and motor functioning in rodents that have sustained a TBI. One study investigated the efficacy of EE in rodents that have sustained a TBI, and compared if the benefits conferred from short-term and long-term EE exposure differed. The results of the study revealed that EE is an effective method for regaining back spatial memory and mobility; in addition, results revealed that short-term EE was equally as effective as long-term EE. As the study demonstrated that a long duration of EE is not necessary in order to recover cognitive and motor functioning, EE is a promising therapy model that has the potential to reach out to patients that cannot receive sufficient treatment due to various constraints. Further investigation into the effectiveness and optimum distribution of EE can change the landscape of TBI rehabilitation.

Key words: traumatic brain injury (TBI), environmental enrichment (EE), neurorehabilitation, cognitive rehabilitation, motor recovery Background Traumatic brain injury (TBI) is an injury that results from both open-headed injuries and closed-headed injuries, resulting in mild TBI (concussions), or moderate-severe TBI1-3. TBI is pervasive and has a high incidence rate in the general population, and in the United States alone, around 1.7 million people are affected by TBI annually1. TBI affects a wide array of individuals, as the leading causes of TBI includes motor vehicle accidents, athletics, falls, and violent assaults, and those who sustain a TBI often experience a multitude of deficits that affect their quality of living and daily functioning, preventing individuals from pursuing an active lifestyle that would assist with their recovery1-4. Individuals with TBI experience deficits in cognitive, emotional, and motor functioning1-6. In addition, TBI has been observed to cause anatomical changes in the brain4.

In order to regain functioning of impaired cognitive and motor abilities, a variety of therapy methods are used, including invasive methods such as pharmacotherapies: 5-HT1A receptor agonists 8-OH-DPAT, or dopamine receptor agonists bromocriptine and methylphenidate, and neurotrophic support3,7. However, non-invasive therapy methods are also utilized and are starting to be implemented as part of neurorehabilitation for TBI patients, such as voluntary exercise and environmental enrichment (EE)2,4-6,8,9. EE is a method that has been identified to be a promising therapy model that been successful in improving cognitive and motor functions that were impaired in those who have sustained a TBI2,4-11. EE is a therapy method that exposes a subject affected with TBI to a stimulating, social, complex space that allow for interaction and engagement with novel, interactive objects and activities2-5,7-13. Studies have found that animals

and humans that have cognitive, emotional, and motor impairments as a result of a TBI have conferred long-term benefits in their memory, mobility, executive functions, and attention after exposure to EE3-5,7-9,12.

In addition to improvements in behaviour, anatomical changes in the brain has also been observed due to EE exposure â&#x20AC;&#x201C; the brain has exhibited enhanced synaptogenesis, spine remodeling, hippocampal neurogenesis, and maintenance of tissue integrity â&#x20AC;&#x201C; which has been correlated with improved behavioural functions conferred from EE2-7,9,10,13-15. While EE has shown to be a promising neurorehabilitation method, there are still some issues that are unclear. Studies have shown that the benefits conferred from EE are relatively the same regardless of the length of time that subjects were exposed to EE; therefore, it is unclear as to whether a longer EE duration is more beneficial7,8.

In addition, most studies that have been conducted utilizing EE have investigated benefits conferred from EE in animal models. Since there are few studies that have examined the use and benefits of EE in human patients, the transferability of the benefits acquired from EE that have been observed in animal models has not been fully investigated.

Research Overview Summary of Major Results Cheng et al. was interested in investigating the efficacy of EE in TBI rehabilitation, in addition to examining whether the cognitive and motor benefits conferred from EE exposure would be relatively equal in subjects that were exposed to the EE paradigm for varying durations9. In order to study this, the researchers utilized a rat model with experimental TBI, which was induced through controlled cortical impact (CCI). Two different housing environments were used for the duration of the experiment; rats were housed 30

Figure 1. Graphical representations of mean time (seconds) that rats remained on the balance beam9. (A) Phase 1 results. TBI+EE performed better on beam balance tasks than the TBI+STD group (*p<0.0117). The sham group (control) performed significantly better on the task compared to both TBI+STD and TBI+EE. (**p<0.0001)9. (B) Phase 2 results. TBI+EE, TBI+EE+STD group performed significantly better than TBI+STD group (*p<0.0021). No significant difference was found in performance between TBI+EE and TBI+EE+STD group (p>0.05)9.

in either a standard steel-wire mesh (STD) cage, or a multi-level environmental enrichment (EE) cage.

Inside the EE cage, the rats had access to a variety of interactive, novel objects which created a complex, stimulating environment for the rats to live in. Both the TBI and control group rats were divided into groups randomly, and placed into either STD or EE housing.

For phase 1 of the experiment, which lasted for 3 weeks, the rats stayed in their assigned housing; in phase 2, half of the rats in the EE cage stayed in their original cage, while the other half were moved to STD housing for the next 6 months. Therefore, researchers investigated three experimental groups: 31

Figure 2. Graphical representations of mean time (sec) spent finding the platform in the Morris Water Maze (MWM)9. (A) Phase 1 results. Control outperformed both TBI+EE and TBI+STD (**p<0.0001). The TBI+EE group was able to locate the platform faster than TBI+STD group (*p<0.0001)9. (B) Phase 2 results. Performance of TBI+EE and TBI+EE+STD group had no significant difference (p=0.53), but outperformed the TBI+STD group (*p<0.0001)9.

rats placed in the EE cage for 6 months and 3 weeks (TBI+EE), rats placed in EE housing for 3 weeks and STD housing for 6 months (TBI+EE+STD), and rats placed in STD housing for the entire duration of the experiment (TBI+STD).

In order to study the cognitive and motor improvements after exposure to EE, the Morris Water Maze (MWM) and beam balance tasks were used9. In phase 1 of the experiment, the rats took part in the MWM and beam balance tasks on days 1-5 and 14-18, and in phase 2, tests were run once a month.

Within the rats that have sustained a TBI, the results for both phase 1 and 2 indicated that the rats that

were exposed to EE for both short and long durations (TBI+EE, TBI+EE+STD) showed significantly better results on the Morris Water Maze than rats that were not exposed to EE at all (p<0.0001). In phase 2, all groups exposed to EE (TBI+EE, TBI+EE+STD) performed markedly better on the beam balance (p<0.0021) and MWM task (p<0.0001) than the TBI+STD group, but the performance between the TBI+EE and TBI+EE+STD group were equally comparable and did not show a significant difference (p>0.05). These results indicate that EE does result in better recovery in cognitive and motor function, as all groups that were exposed to EE showed significantly better performance on both tasks compared to the TBI group that was not exposed to EE at all9.

Similar results have been reported in other studies – TBI rats that were exposed to early or continuous EE demonstrated better performance on beam-walking compared to rats with no EE exposure; in addition, better cognitive training was observed in rats with continuous EE exposure5,7. The results also show that there is no difference between the benefits conferred between TBI+EE and TBI+EE+STD. Conclusions and Discussion Cheng et al.’s study suggest that longer exposure to EE is not necessarily beneficial compared to a shorter exposure to EE, because the performance on both the MWM and beam balance tasks did not have a significant difference9. Research suggests that there is a minimum duration (1-4 weeks) of EE exposure that is necessary to observe behavioural benefits8, which supports Cheng et al.’s results by demonstrating that short exposure to EE is sufficient in acquiring improvements in cognitive and motor deficits6,7,9.

Another study determined that the length of EE in terms of conferring behavioural benefits was not the most important factor for improvements in cognitive and motor function, as long as there was exposure to EE7. Rats that received exposure to EE for only 1-week directly after sustaining a TBI, then moved to STD (early EE) and rats that were exposed to the EE paradigm for three weeks continuously (continuous EE) both showed enhanced performance on beamwalking tasks7. In addition, rats that were exposed to EE for 21 days demonstrated similar learning rates to rats that received EE for 14 days, so long as there was a continuous exposure to EE for at least 2 weeks11 – strengthening the notion that there is a minimum necessary duration. Therefore, the results all indicate that a longer duration of EE exposure is not necessary to acquire benefits, as long as there is a short exposure to EE. The results suggest that there may be an upper-limit to the benefits that can be acquired from long-term EE, as these results indicate that longer durations of EE does not result in more cognitive and motor benefits9.

While behavioural benefits may require a minimum of 1-4 weeks of exposure to an EE paradigm, plasticity changes in the brain were observed from as little as 1 hour of EE exposure per day6. Rats that sustained a TBI through fluid percussion (FP) were exposed to EE for 1 hour/day for 20 days, and researchers

observed that in their ipsilesional dentate gyrus, the survival of the neural progenitors that are endogenous to this region were protected, in addition to increased neurogenesis in the granule cell layer6. These results strengthen that EE benefits arise from short durations in the brain in terms of plasticity, even if it is not apparent behaviourally. Furthermore, plasticity changes in the brain seem to benefit from a continuous exposure to EE11. EE has demonstrated potential in improving behavioural functions, but also in maintaining and improving neuroplasticity and anatomy in the brain; EE had an effect on preventing atrophy in the hippocampus, slowing down of CA3 cell loss in the dentate gyrus, decreased size in site of lesion in the cerebral cortex, and an upward increase in spine density and dendritic growth4,7,9,10.

Criticisms and Future Directions

However, there is conflicting evidence regarding the effectiveness of longer durations of EE. Amaral et al. reported that rats exposed to the EE paradigm for a longer duration (8-weeks) gained behavioural improvements that were more persistent compared to rats that were exposed to EE for a shorter duration (4-weeks) of time, which suggests that longer durations of EE does have a benefit in making more permanent, fixated improvements. These results suggest that while the benefits that are conferred from a shorter duration of EE is equivalent to the benefits acquired from a longer duration of EE, the difference is in how long these benefits last for after EE treatment is over. Furthermore, longer periods of EE seem to allow more time for anatomical and neurochemical alterations in the brain to occur, which contributed to the persistence of the benefits8,10,11. Cheng et al.’s research did not investigate the post-EE long-term effects and persistence of the benefits acquired from the EE paradigm; therefore, it would be of value to conduct a study investigating the potential differences in persistence of cognitive and motor benefits acquired from short and long EE exposure. This could potentially be done by exposing groups of TBI rats to EE cages for a select duration that vary from 1-week to around 6-weeks, and then testing their performance on cognitive and behavioural tasks after 6-months from EE exposure. The benefits of long-term EE could then be elucidated in terms of the persistence of the benefits acquired, along with the anatomical changes through histological analysis. Further investigation will give insight into the optimum duration of EE that should be distributed during treatment.

Furthermore, Cheng et al. mentions that the results obtained from the study would benefit TBI treatment.

However, because the research that focuses on EE is predominantly conducted on animal models that have sustained an experimental TBI, the transferability of the benefits that have been observed to be associated with EE exposure was not studied. However, past research has shown that EE also does help humans recover from brain injuries. One study found that stroke patients that were in a facility that allowed for patients to partake in more interaction and activities experienced faster recovery of lost motor abilities, 32

while those who spent more time alone took longer to recover12. In addition, TBI patients that have identified to be involved in EE activities on the Lifestyle Activities Questionnaire (LAQ) also showed less deterioration in their hippocampus4. These results indicate that behaviour, brain anatomy, and plasticity recovery are also evident in humans as well; as such, EE is a promising neurorehabilitation method that could be incorporated in patientâ&#x20AC;&#x2122;s daily lifestyles for rehabilitation. To further investigate the benefits of EE in patients that have sustained TBI, a group of patients receiving a novel therapy model that exposes patients to an environment that facilitates cognitive, social, and physical aspects can be compared to another group of patients receiving standard care. In the EE paradigm, mind stimulating games such as crosswords and chess can be incorporated with physical activity and socialization opportunities.

Conclusion

The results from Cheng et al.â&#x20AC;&#x2122;s experiment demonstrated that rats that sustained a TBI conferred cognitive and motor improvements from EE exposure.

Similar studies that were interested in looking into the effects of EE have also shown similar results, and express that EE is a beneficial recovery and therapy paradigm for regaining back impairments that resulted from TBI2-15. These findings validate that EE is a relevant rehabilitation model that indicate that exposure to a social, complex, and interactive environment facilitate recovery of important skills such as memory, attention, and mobility2-14.The results of these studies demonstrate that in treating TBI patients, rehabilitation and therapy should integrate activities and environments that focus on cognitive, physical, and social aspects2,4,12. With a better understanding of how EE should be distributed, along with how effective the model is for human patients, EE is a neurorehabilitation model that could potentially alter the current standard care used for TBI patients.

References 1. Faul, M., Xu, L., Wald, M. M. & Coronado, V. Traumatic Brain Injury in the United States. Atlanta, GA: Centers for Disease Control and Prevention, National Center for Injury Prevention and Control (2010) 2. Frasca, D., Tomaszczyk, J., McFadyen, B. J. & Green, R. E. Traumatic brain injury and post-acute decline: what role does environmental enrichment play? A scoping review.

Front. Hum. Neurosci. 7 (2013).

3. Sozda, C. N. et al. Empirical comparison of typical and atypical environmental enrichment paradigms on functional and histological outcome after experimental traumatic brain injury. J. Neurotrauma 27, 1047-1057 (2010).

4. Miller, L. S., Colella, B., Mikulis, D., Maller, J. & Green, R. E. Environmental enrichment may protect against hippocampal atrophy in the chronic stages of traumatic brain injury.

Front. Hum. Neurosci. 7 (2013).

33

5. Passineau, M. J., Green, E. J. & Dietrich, W. D. Therapeutic effects of environmental enrichment on cognitive function and tissue integrity following severe traumatic brain injury in rats. Exp. Neurol. 168, 373-384 (2001).

6. Gaulke, L. J., Horner, P. J., Fink, A. J., McNamara, C. L. & Hicks, R. R. Environmental enrichment increases progenitor cell survival in the dentate gyrus following lateral fluid percussion injury. Mol. Brain Res. 141, 138-150 (2005).

7. Hoffman, A. N. et al. Environmental enrichment-mediated functional improvement after experimental traumatic brain injury is contingent on task-specific neurobehavioral experience. Neurosci. Lett. 431, 226-230 (2008).

8. Amaral, O. B., Vargas, R. S., Hansel, G., Izquierdo, I. & Souza, D. O. Duration of environmental enrichment influences the magnitude and persistence of its behavioral effects on mice. Physiol. Behav. 93, 388-394 (2008).

9. Cheng, J. P. et al. A relatively brief exposure to environmental enrichment after experimental traumatic brain injury confers long-term cognitive benefits. J. Neurotrauma. 29, 2684-2688 (2012).

10. Leggio, M. G. et al. Environmental enrichment promotes improved spatial abilities and enhanced dendritic growth in the rat.Behav. Brain Res. 163, 78-90 (2005).

11. Matter, A. M., Folweiler, K. A., Curatolo, L. M. & Kline, A. E. Temporal effects of environmental enrichment-mediated functional improvement after experimental traumatic brain injury in rats. Neurorehabil. Neural Repair. 25, 558-564 (2011).

12. De Wit, L. et al. Motor and functional recovery after stroke: a comparison of 4 European rehabilitation centers. Stroke.

38, 2101-2107 (2007).

13. Lambert, T. J., Fernandez, S. M. & Frick, K. M. Different types of environmental enrichment have discrepant effects on spatial memory and synaptophysin levels in female mice.

Neurobiol. Learn. Mem. 83, 206-216 (2005).

14. Bennett, J. C., McRae, P. A., Levy, L. J. & Frick, K. M. Long-term continuous, but not daily, environmental enrichment reduces spatial memory decline in aged male mice. Neurobiol.

Learn. Mem. 85, 139-152 (2006).

15. Bruel-Jungerman, E., Laroche, S. & Rampon, C. New neurons in the dentate gyrus are involved in the expression of enhanced long-term memory following environmental enrichment. Eur. J. Neurosci. 21, 513-521 (2005).

Creatine Supplementation on brain performance suggestive of potential therapeutic agent Vanessa C. Bracaglia

Background Creatine is a naturally synthesized molecule in vertebrates that plays an important role in energy metabolism.

Catalyzed by creatine kinase, phosphorylated creatine (PCr) has the ability to transfer it’s high energy phosphate group to ADP in order to produce ATP. This process of ATP synthesis is quicker than production via oxidative phosphorylation or other de novo pathways (Rae et al., 2003). Creatine is used by cells both in skeletal muscle and in the brain where it has been advocated to provide a neuroprotective effect. One way it achieves this is through its buffer for energy homeostasis and ability to maintain the integrity of the membrane potential during stressful circumstances. Elevated levels of brain creatine have been observed as an outcome of mental training (Rae et al., 2003). Studies have indicated oral creatine supplementation is sufficient to increase total creatine levels in skeletal muscle and is associated with a greater rate of ATP regeneration most likely attributable to having a more readily available pool of PCr to begin due to the creatine supplementation and thus delaying muscular energy depletion (Greenhaff et al., 1994). In another study, increases in muscular phosphocreatine was observed after 16 weeks of creatine supplementation in patients with fibromyalgia. Although there was not any significant changes on the general symptoms of the disease, there were improvements with the upper and lower body muscle function demonstrating the potential of creatine as a therapeutic agent (Alves, 2013). Oral creatine supplementation does not only increase creatine stores within the muscles, but studies have also confirmed its ability to significantly increase neural creatine stores within the brain. Increases in total creatine levels were observed in brain regions such as the cerebellum, gray matter, and more amply within the thalamus and white matter (Dechent et al. 1999) Another study has associated neural creatine levels with brain and more specifically, memory performance. Using cognitive tests such as the Raven’s Advanced Progressive Matrices and Wechsler Auditory backward digit span (BDS) task, individuals undergoing a double-blind, placebo controlled, cross-over trial were able to demonstrate that creatine supplementation leads to increased brain creatine which was thus associated with improved working memory function and storage (Rae et al., 2003).

Research Overview

Summary of Major Results

This was the first study performed in vivo examining the effects of Creatine monohydrate (CrM) supplementation on cognition and corticomotor excitability

during a state of hypoxia. Previous findings have suggested creatine to have neuroprotective aspects in vitro thus it is important that this study was able replicate consistent results in vivo (Shen and Goldberg, 2012). The current study used a double-blind placebo controlled design to assess the influence of creatine supplementation on neural creatine levels and how that impacts neuropsychological and neurophysiological features. Before the intervention trials, participants underwent baseline testing for neuropsychological data as well as were exposed to the hypoxia treatment. The state of hypoxia was created by inhaling a mixture of gas containing only 10% oxygen through a one-way face mask for 90 minutes.

Using Magnetic Resonance Spectroscopy(MRS) imaging, it was observed that supplementing with CrM for 7 days was enough to increase neural creatine levels therefore individuals who were supplementing had greater amounts of creatine in the brain than those who given the placebo(Turner et al., 2015).

Neuropsychological adequacy was tested using a standardized series of seven computerized tests assessing multiple factors such as complex attention, executive function, processing speed, visual memory, overall neurocognitive index score and more. Neurophysiological data was determined using peripheral nerve stimulation, transcranial magnetic stimulation and measuring motor evoked potential (MEP) amplitudes. Both neuropsychological and neurophysiological data was collected from participants at baseline as well as during the hypoxic state. CrM supplementation was able to prevent or at least reduce the cognitive deficits associated with being in an oxygen-deprived environment (Turner et al., 2015). Hypoxia causes a deterioration in mental functioning as observed by the 12% reduction in overall neurocognitive index score.

Notably, complex attention appeared to be preserved the most when coupled with creatine supplementation as there was a 21% difference between treatment and placebo groups (Turner et al., 2015). Along with improved cognitive functions, creatine supplementation increased corticomotor excitability when in an oxygen-deprived environment. Interestingly, creatine supplementation alone did not increase excitability in normal conditions—it is not until an oxygen-deficient state is induced that creatine then demonstrates its ability to increase neuronal excitability by 70%(Turner et al., 2015). Due to it’s energy-buffering capacity, creatine supplementation prior to energy depletion has exhibited the ability to maintain the integrity of the cell’s membrane potential in vitro thus preventing axonal degradation (Shen and Goldberg, 2012). The creatine is suggested to have stimulated ATP-synthesis needed to drive the Na+/K+ ATP-ase which main34

tains the ion gradient that control neuron excitability (Shen and Goldberg, 2012). Previous studies have demonstrated in rat hippocampal samples that creatine is a potential modulator of Na+/K+ ATP-ase activity through the NMDA–calcineurin pathway (Rambo et al., 2012).

Figure 1. Creatine pretreatment reduced or prevented the cognitive deficits experienced during hypoxia. (Turner et al., 2015).

Figure 2: During an oxygen deprivation state, corticomotor excitability increased significantly in individuals who supplemented with creatine(black bar) compared to the placebo group. (Turner et al., 2015).

Discussion/Conclusions/Future Directives Supplementation or pre-treatment of creatine seems to be imperative in order to achieve its full neuroprotective benefits. Supplementation of creatine prior to energy depletion decreased axonal degradation by 50% and relieving damages related to ATP loss and depolarization (Shen and Goldberg, 2012). When creatine is added to neurons after inducing energy depletion, regular axonal deterioration was observed (Shen and Goldberg, 2012). Energy depletion compromises the structural and functional integrity of axons by a means of accumulation of calcium ions 35

producing adverse effects by activating downstream pathways and through excess Na+ ions(hence why tetrodotoxin—a Na+ channel blocker—reduced axonal damaged when applied and coupled with energy depletion) (Shen and Goldberg, 2012). Disruption of membrane potential and neuronal dysfunction is observed in in vitro stroke models and more commonly among many neurodegenerative disorders which is why the neuroprotective effects of creatine treatment is being explored for its potential as a therapeutic agent.

It has been established that oral creatine supplementation is sufficient to increase creatine storage in multiple brain regions. This enriched creatine availability has been associated with increased brain performance and reduces cognitive defects related to conditions such as oxygen deprivation and energy depletion. A 16% reduction in fRMI Blood Oxygen Level Dependant was observed and associated with an increase in memory span after another study using creatine supplementation where these results were not seen in the placebo group (Hammett et al., 2010). These results suggest creatine as a potential therapeutic agent for treating Alzheimer ’s disease as individuals carrying the ApoE E4 allele (and therefore predisposed to a higher risk of developing AD) exhibit increases in BOLD response and decreases in memory function (Hammett et al., 2010). The authors of this study hypothesize the reduction in BOLD due to an increase in oxidative glycolysis; However, in the present study, oxidative glycolysis becomes compromised as a result of the hypoxia and yet increased creatine stores were still able to contribute to phosphocreatine-mediated ATP-synthesis leading to enhanced cognitive performance. Further investigations are warranted to see if reductions in BOLD response are observed during hypoxia treatment and if that response is associated with preventing cognitive function. Mutations within the mitochondria leading to disrupted energy homeostasis in the brain appear to be a commonality among neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s Disease, Huntington’s Disease and ALS. An oxidative form of cytosolic-brain creatine kinase (frequently seen in AD patients among other oxidative protein modifications) results in drastically less activity levels than its wildtype counterpart leading to altered energy metabolism within the brain (Burklen et al., 2006). Another study observed less brain phosphocreatine levels within AD patients compared to healthy individuals which further sanctions the importance of the creatine kinase/phosphocreatine shuttle in maintaining energy homeostasis (Burklen et al., 2006). Since neurodegeneration and aging appear to utilize similar pathways, one study investigated the role of creatine supplementation in regular aging mice compared to mice who were not given creatine pre-treatment. An average life span increase of 9% was seen accompanied with improved neurobehavioral test scores as well as less reactive oxygen species within the brain revealing its potential antioxidant properties (Klopstock et al., 2011).

Immunohistochemical methods have also revealed the expression level of the creatine transporter to be widespread within the brain, considerably in large projection neurons, whereas less expression is seen in cells that are known to be involved in neurodegeneration—

medium the spiny neurons of the striatum (implicated in Huntington`s Disease), and the dopaminergic neurons in the substantia nigra region (Parkinson`s) (Lowe et al., 2015). It is evident that creatine-related mechanisms plays a role in neurodegeneration, and since creatine has exhibited neuroprotective features, it is not surprising that it is being considered as a possible therapeutic agent. However, further studies are needed to elucidate these neuroprotective characteristics and how creatine supplementation influences the phosphocreatine-creatine kinase shuttle and creatine transporter expression—both of which impact energy metabolism and brain plasticity.

References

Received Month, ##, ##, 200#; accepted

200#; Month,

revised ##,

Month, 2013.

This work was supported by The Association for the Development of Undergraduate Neuroscience Education (SRA & RLN), The Endowment for Science Education (EA), and The Synaptic State Faculty Research Foundation (EA). The authors thank Mr. Spine L. Cord, Dr. Amy G. Dala, and the students in Neuroscience 101 for technical assistance, execution, and feedback on this lab exercise.

Address correspondence to: Dr. Rita L. Neurotrophin, Biology Department, 123 Growth Cone Avenue, Action Potential College, Hillock, IL 60101 Email: rln@apc.edu Copyright © 2015 Dr. Bill JU, Neurosciences, Human Biology Program

1. Alves C.R.R. (2013) Creatine Supplementation in Fibromyalgia: A Randomized, Double-Blind, Placebo-Controlled Trial. Arthritis Care and Research. 65:1449-1459.

2. Burklen TS, et al. (2006) The Creatine Kinase/Creatine Connection to Alzheimer’s Disease: CK Inactivation, APP-CK Complexes, and Focal Creatine Deposits. Journal of Biomedicine and Biotechnology. 2006:1-11.

3. Dechent P, Pouwels PJ, Wilken B, Hanefeld F, Frahm J (1999) Increase of total creatine in human brain after oral supplementation of creatine monohydrate. Am J Physiol 277:R698 –R704 4. Greenhaff P.L., Bodin K, Soderlund K, Hultman E (1994) Effect of oral creatine supplementation on skeletal muscle phosphocreatine resynthesis. American Physiological Society.

Am J Physiol. 266: E725–E730.

5. Hammett ST., Wall MB., Edwards TC., Smith A.T. (2010) Dietary supplementation of creatine monohydrate reduces the human fMRI BOLD signal. Neuroscience Letters. 479:201205.

6. Klopstock T, Elstner M, Bender A (2011) Creatine in mouse models of neurodegeneration and aging. Springe.

40:1297–1303 7. Lowe M, Faull R, Christie D, Waldvogel H . (2015) Distribution of the Creatine Transporter Throughout the Human Brain Reveals a Spectrum of Creatine Transporter Immunoreactivity.

The Journal of Comparative Neurology. 523:699–725 8. Rae C, Digney A, McEwan S, Bates T.C. (2003) Oral creatine monohydrate supplementation improves brain performance: a double-blind, placebo-controlled, cross-over trial.

Proc. R. Soc. Lond. 270: 2147–2150.

9. Rambo LM, et al. (2012) Creatine increases hippocampal Na+,K+-ATPase activity via NMDA–calcineurin pathway.

Elsevier 88: 553–559 10. Shen H, and Goldberg MP (2012) Creatine pretreatment protects cortical axons from energy depletion in vitro Neurobiol Dis. 47:184-193 11. Turner CE., Byblow WD, Gant N (2015) Creatine Supplementation Enhances Corticomotor Excitability and Cognitive Performance during Oxygen Deprivation. The Journal of Neuroscience. 35:1773-1780.

36

The Neural Mechanisms of Socio-Sexual Partner Preference Alana Brown

This review focuses mainly upon a study involving the induction of same-sex socio-sexual partner preference conditioning in male rats that experience cohabitation with other male rats under the enhanced influence of D2-type receptor and/or oxytocin activity (Triana-Del Rio et al., 2015). This review examines the history behind sexual preference research, the results of this particular experiment, a critical evaluation of its conclusions and suggestions for further research. Using this experiment, the significance of the D2 receptor in partner preference formation is explored, and the novel implication of oxytocin in this complex system in the nucleus accumbens (as it pertains to mate selection) is considered in more detail. The sizes of the sexually dimorphic nucleus of the medial preoptic area (SDN-POA) and supraoptic nucleus of the hypothalamus (SON) are not seen to be correlated with same-sex partner preference conditioning; this relevance is questioned and the nucleus accumbens is discussed as a brain region more likely to be involved with partner preference alteration.

Study shortcomings and propositions are described and an experiment involving a MC4 receptor agonist in the nucleus accumbens is suggested to hone understanding of the interrelationship between oxytocin and dopamine as it relates to partner preference and social cognition.

Key words: conditioning, social cognition, partner preference, homosexual, sex, oxytocin, dopamine, nucleus accumbens, sexually dimorphic nuclei Background The mechanism underlying what causes a male animal to seek a female mating partner remains a complex issue of considerable dispute, for partner preference is the result of a multifarious relationship between genetics, hormones and learning (Kamiya et al., 2014). It is known that in nature there are varying types of social attachments. These attachments are acquired as a result of cohabitation and are often measured in terms of increases in social recognition, motivation and reward. The interaction between oxytocin and D2 receptors in the nucleus accumbens seems to facilitate partner preference and attachment, while D1 receptors have been seen to facilitate parental attachment (Coria-Avila et al., 2014). Dopamine in the nucleus accumbens is considered critical for pair-bond formation in male prairie voles (Aragona, Liu, Curtis, Stephan, & Wang, 2003). Dopamine, particularly in the rostral shell of the nucleus accumbens, seems to be important to pair-bond formation in male prairie voles, with enhanced D2-type receptors facilitating pair-bond formation and enhanced D1-type receptors preventing it (Aragona et al., 2005). Furthermore, it has been made clear that an individual’s experience with reward (e.g. dopamine) can overturn presumably “innate” mate choices (e.g. male rats experiencing female partner preference) by means of Pavlovian conditioning. Prior studies have shown that pleasure seems to be a more powerful predictor of social behavior than genetics or reproductive fitness (CoriaAvila, 2012).

The Triana-Del Rio et al. (2015) study of focus reiterates previous conclusions suggesting that cohabitation can affect partner preference via conditioning while also recapitulating the finding that sexually naïve male rats injected with the D2 agonist 37

quinpirole show same-sex partner preference for scented sexually receptive male rats that they have cohabitated with over sexually receptive female rats (Cibrian-Llanderal et al., 2012). The current study is noteworthy for implicating both D2-type receptors and oxytocin in same-sex partner preference development.

It demonstrates that enhanced oxytocin alone (without quinpirole) can induce same-sex partner preference in male rats who cohabitate with other male rats.

Implicating oxytocin directly with this sexual preference behavior is important for furthering research that looks at oxytocin as a mediator for prosocial behavior, particularly in terms of how it interacts with dopamine to promote partner preference formation (Modi et al., 2015). Oxytocin has been seen to lessen aggression levels and enhance social exploration; therefore it may be important to look more precisely at the behavior patterns in rats to experimentally tease apart those more influenced specifically by oxytocin or dopamine (Calcagnoli, Kreutzmann, de Boer, Althaus & Koolhaas, 2015).

The Triana-Del Rio et al. (2015) study also investigates associations between reductions in size of the sexually dimorphic medial preoptic area (SDMPOA) and supraoptic nucleus of the hypothalamus (SON), as past research has suggested that lesions of SDM-POA induce same-sex partnership in male ferrets (Alekseyenko, Waters, Zhou, & Baum, 2006).

No such associations were found in the study being evaluated here. Researchers might consider exploring further by prolonging the experiment (to see if there are any changes in size that take longer to develop) or looking more closely at the interaction between dopamine and oxytocin in the nucleus accumbens, an area that may be more relevant to partner preference and copulatory behavior (Liu & Wang, 2003).

Research Overview

Summary of Major Results

Male rats received an injection of saline, quinpirole, oxytocin, both quinpirole and oxytocin or no injection at all during either cohabitation with a male rat or alone. Rats in the cohabitation groups experienced three 24-hour trials of cohabitation with an almondscented male (the conditioned stimulus). Social and sexual preference was measured after the last trial, drug-free (to ensure results were due to learning).

Subjects later chose between their familiar scented male and an unscented, sexually receptive female (see Figure 1). Preference was measured in terms of mounts, contacts, genital investigations, intromissions, ejaculations, non-contact erections and female-like solicitations. After the first experiment, subject brains were processed for Nissl dye (stained with cresyl violet) to aid in measuring the sexually dimorphic nucleus of the medial preoptic area (SDN-POA) and supraoptic nucleus of the hypothalamus (SON).

Digital photographs enabled software to calculate nuclei areas (Triana-Del Rio et al., 2015).

Same-Sex Partner Preference Induction Male rats who received saline, quinpirole, oxytocin or no injection without cohabitation showed preference for females in terms of higher visit incidence, more body contact, mounting, non-contact erections and olfactory investigations. Rats who received quinpirole, oxytocin and quinpirole and oxytocin together, all with cohabitation, displayed same-sex preference for their male partner in terms of increased visit frequency and duration, shorter contact latency, more body contact, olfactory investigation, female-like solicitation, and noncontact erection with the male partner (see Figure 1).

This research validates previous findings that same-sex socio-sexual preference can be induced in male rats injected with quinpirole that cohabitate with other male rats, while also providing support for the novel conclusion that oxytocin injections with cohabitation can also induce same-sex socio-sexual preference (Triana-Del Rio et al., 2015). The most significant findings suggest that with increased dopamine receptor and oxytocin activity, conditioned same-sex social and sexual partner preference can develop when accompanied by cohabitation. Dopamine and oxytocin injections without cohabitation were not seen to result in same-partner social and sexual preference.

Brain dimorphism Areas of the SDN-POA and SON were not seen to correspond to same-sex socio-sexual preference development (see Table 1). Males who received oxytocin had smaller SDN-POA sizes, no matter if they showed female or male socio-sexual preference. Males that cohabitated with other males and received quinpirole and both quinpirole and oxytocin injections had the largest SON nuclei. Same-sex partner preference does not seem to be correlated with changes in the sizes of the SDN-POA or SON. These findings do not validate other research that shows lesions of the SDN-POA can induce same-sex partner preference in male ferrets (Alekseyenko, Waters, Zhou, & Baum, 2006).

Figure 1. Mean total time of visit demonstrated by experimental male rats toward stimuli (male vs. sexual receptive female) (Triana-Del Rio et al., 2015).

Conclusions and Discussion Triana-Del Rio et al. (2015) conclude that Pavlovian associations under the influence of cohabitation and enhanced D2 and oxytocin can facilitate the development of same-sex socio-sexual partner preference in male rats. Consistent with past research, D2 receptors are implicated in the induction of same-sex partner preference, although a D1-type receptor agonist was not experimented with in this particular study. This was an appropriate decision. The conclusion that oxytocin injections with cohabitation can induce same-sex partner preference is novel. There is also immense strength in the observation that the males influenced by enhanced quinpirole and oxytocin (separately) exhibit similar preference behaviors after cohabitation conditions. Researchers acknowledge that there is a significant relationship between oxytocin and D2 activity in the nucleus accumbens as it pertains to partner preference development, and they confirm that this has little correlation with hypothalamic areas like SON (Triana-Del Rio et al., 2015).

The results of this study fail to support the notion that same-sex partner preference in males is associated to a smaller, more female-like SDN-POA. Researchers suggest that this may be due to the dimorphic structures in question being organized during a perinatal period, making them less modifiable during the conditioning of same-sex preference. It is possible that after same-sex preference conditioning the nucleus accumbens takes over control of preference motivation (Triana-Del Rio et al., 2015). It is important that this research is able to take the SDN-POA and SON out of the equation, in a sense, in order to apply a more solid foundation for future studies of the nucleus accumbens.

This validation that there is an interrelationship between dopamine and oxytocin is perhaps the most significant component of this study. Authors note that dopamine release in the nucleus accumbens increases quickly in male rats just before ejaculation. After ejaculation, dopamine in the nucleus accumbens seems to decrease rapidly and substantially. Researchers posit 38

that during this decrease, oxytocin plays a significant role in crystalizing new social attachments, as it may be involved with enhancing trustfulness and calmness (Fiorino, Coury, & Phillips, 1997). It is likely that dopamine works to enhance attention and arousal during a sexual encounter, while oxytocin may be involved with contact, consummatory behavior and bonding (Pfaus, 2009). This does not necessarily explain why separate oxytocin and quinpirole injection cohabitation conditions result in similar patterns of behavior for the male rats; however, it is relevant to future discussion involving the precise pathways of oxytocin and D2 receptors as they affect partner preference, for it is clear that they are interrelated.

Table 1. Main results: partner preference and brain dimorphism after conditioning (groups are intact- (involving no injection and no cohabitation), saline (SAL) with (+) and without (-) cohabitation, quinpirole (QNP) with and without cohabitation and oxytocin with and without cohabitation) (Triana-Del Rio et al., 2015).

Criticisms and Future Directions

Critical Analysis

The impact of this study is moderate, as it connects oxytocin distinctively to the process of partner preference induction involving dopamine, but it does not necessarily elucidate the connection. Researchers note that blocking either oxytocin or D2-type receptors prevents heterosexual partner preference induced by injections of oxytocin or D2 agonists in prairie voles and therefore opt to utilize only quinpirole and no D1-type receptor agonist (Liu & Wang, 2003). This enhances the experimentâ&#x20AC;&#x2122;s specificity and acknowledgment of inducing partner preference as opposed to the parental attachment associated with D1-type receptors.

Triana-Del Rio et al. (2015) also recognize the importance of delving more deeply into the interrelationship between D2-type receptors and oxytocin, although they make few suggestions regarding how to do so. They investigate many different socio-sexual behaviors, but do not attempt to experimentally separate the ones associated specifically with oxytocin or D2-type receptors. Increased attempt to investigate the roles of oxytocin and dopamine in the nucleus accumbens would have likely been more insightful.

Future Directions In terms of furthering this research, it may be worth considering the addition of a vasopressin injection condition (with and without cohabitation), as vasopressin has previously been shown to facilitate social attachment and may also contribute to the develop39

ment of same-sex preference (Bielsky & Young, 2004). This additional condition might be important for dissociating the effects of vasopressin on attachment from those of dopamine and oxytocin. It might also be interesting to prolong this experiment (e.g. for months, as opposed to weeks). Drug-free preference tests could take place intermittently to measure how long-lasting the conditioned preference is. Authors show that this conditioned partner preference may last only a few weeks, but posit that experiments involving extending the time to longer periods may be beneficial.

It might also be advantageous to conduct an experiment during which oxytocin receptor activity is blocked by injecting oxytocin antagonist bilaterally into the nucleus accumbens of male rats to see if cohabitation will still have an effect when increased levels of oxytocin or quinpirole are injected as well (Liu & Wang, 2003). This might help to differentiate the socio-sexual behaviors induced by oxytocin and quinpirole (e.g. dopamine may be more relevant to formation of the preference behavior and oxytocin more relevant to expression of partner preference), and perhaps show whether one is dependent on the other to facilitate same-sex preference (Young et al., 2001).

Triana-Del Rio et al. (2015) also suggest that future research might involve attempting to induce samesex socio-sexual preference in male rats that are not sexually naĂŻve (that have had sexual experiences with a receptive female prior to any conditioning). This experimentation would be important for demonstrating the effects of conditioning strength on previous learning and reward, particularly because this sort of situation

is more pertinent to humans, who are consistently exposed to sexual preference conditioning.

It would also be immensely beneficial to further this research at a more specific molecular level. The MC4 receptor (MC4R) has been shown to interact with neurochemical systems involving social and emotional behaviors in the nucleus accumbens, particularly those associated with oxytocin and dopamine (Modi et al., 2015). Agonists of MC4R have been seen to enhance partner preference formation in the prairie vole, while co-administration of an oxytocin receptor antagonist has the capacity to prevent this partner preference, demonstrating that MC4R and oxytocin interact and impact partner preference (Modi et al., 2015). An experiment similar to that of Triana-Del Rio et al. (2015) involving a MC4R agonist would be valuable. One might consider injecting the MC4R agonist in a male mouse during cohabitation to see if same-sex socio-sexual preference could be induced through mere engagement of the oxytocin system in the nucleus accumbens (rather than through increased levels of oxytocin itself). This may also aid in implicating dopamine in the nucleus accumbens.

Infusing the agonist in other brain areas (such as the ventral tegmental area or the amygdala) could also be informative.

On a larger scale, partner preference induction in rats may be illustrative of a strategy to enhance social cognition in humans who have deficiencies in social functioning (e.g. in people with autism or schizophrenia) (Modi et al., 2015). MC4R agonists may be the key to implicating the involvement of both oxytocin and dopamine in partner preference formation and perhaps other significant clinical facets of social functioning.

References 1. Alekseyenko, O. V., Waters, P., Zhou, H., & Baum, M. J. (2007). Bilateral damage to the sexually dimorphic medial preoptic area/anterior hypothalamus of male ferrets causes a female-typical preference for and a hypothalamic fos response to male body odors. Physiology & Behavior, 90(2-3), 438-449.

2. Aragona, B. J., Liu, Y., Curtis, J. T., Stephan, F. K., & Wang, Z. (2003). A critical role for nucleus accumbens dopamine in partner-preference formation in male prairie voles. The Journal of Neuroscience, 23(8), 3483-3490.

3. Aragona, B. J., Liu, Y., Yu, Y. J., Curtis, J. T., Detwiler, J. M., Insel, T. R., & Wang, Z. (2005). Nucleus accumbens dopamine differentially mediates the formation and maintenance of monogamous pair bonds. Nature Neuroscience, 9(1), 133-139.

4. Bielsky, I. F., & Young, L. J. (2004). Oxytocin, vasopressin, and social recognition in mammals. Peptides, 25(9), 1565-1574.

5. Calcagnoli, F., Kreutzmann, J. C., de Boer, S. F., Althaus, M., & Koolhaas, J. M. (2015). Acute and repeated intranasal oxytocin administration exerts anti-aggressive and pro affiliative effects in male rats. Psychoneuroendocrinology, 51, 112-121.

6. Cibrian-Llanderal, T., Rosas-Aguilar, V., Triana-Del Rio, R., Perez, C. A., Manzo, J., Garcia, L. I., & Coria-Avila, G. (2012). Enhaced D2-type receptor activity facilitates the development of conditioned same-sex partner preference in male rats. Pharmacology, Biochemistry and Behavior, 102(2), 177-183.

7. Coria-Avila, G. A., Manzo, J., Garcia, L. I., Carillo, P., Miquel, M., Pfaus, J. G. (2014). Neurobiology of social attachments. Neuroscience & Behavioral Reviews, 43, 173-182.

8. Coria-Avila, G. A. (2012). The role of conditioning on heterosexual and homosexual partner preference in rats.

Socioaffective Neuroscience & Psychology, 2, 1-12.

9. Fiorino, D. F., Coury, A., & Phillips, A. G. (1997).

Dynamic changes in nucleus accumbens dopamine efflux during the Coolidge effect in male rats. The Journal of Neuroscience, 17, 4849-4855.

10. Kamiya, T., Oâ&#x20AC;&#x2122;Dwyer, K., Westerdahl, H., Senior, A., & Nakagawa, S. (2014). A quantitative review of MHC-based mating preference: the role of diversity and dissimilarity.

Molecular Ecology, 23, 5151-5163.

11. Liu, Y. & Wang, Z. X. (2003). Nucleus accumbens oxytocin and dopamine interact to regulate pair bond formation in female prairie voles. Neuroscience, 121(3), 537-544.

12. Modi, M. E., Inoue, K., Barrett, C. E., Kittelberger, K. A., Smith, D. G., Landgraf, R., & Young, L. J. (2015). Melanocortin receptor agonists facilitate oxytocin-dependent partner preference formation in the prairie vole. Neuropsychopharmacology,1-10.

13. Pfaus, J. G. (2009). Pathways of sexual desire. Journal of Sexual Medicine, 6(6), 1506-1533.

14. Triana-Del Rio, R., Tecamachaltzi-Silvaran, M. B., DiazEstrada, V. X., Herrera-Covarrubias, D., Corona Morales, A. A., Pfaus, J. G., & Coria-Avila, G. A. (2015). Conditioned same-sex partner preference in male rats is facilitated by oxytocin and dopamine: Effect on sexually dimorphic brain nuclei, Behavioural Brain Research, 283, 1-9.

15. Young, L. J., Lim, M. M., Gingrich, B., & Insel, T. R. (2001). Cellular mechanisms of social attachment. Hormones and Behavior, 40(2), 133-138.

Received Month, ##, ##, 200#; accepted

200#; Month,

revised ##,

Month, 2013.

This work was supported by The Association for the Development of Undergraduate Neuroscience Education (SRA & RLN), The Endowment for Science Education (EA), and The Synaptic State Faculty Research Foundation (EA). The authors thank Mr. Spine L. Cord, Dr. Amy G. Dala, and the students in Neuroscience 101 for technical assistance, execution, and feedback on this lab exercise.

Address correspondence to: Dr. Rita L. Neurotrophin, Biology Department, 123 Growth Cone Avenue, Action Potential College, Hillock, IL 60101 Email: rln@apc.edu Copyright Š 2015 Dr. Bill JU, Neurosciences, Human Biology Program

40

Impact of KIBRA Polymorphism On The Hippocampus Megan E. Cabral

The KIBRA gene is found throughout both the brain and kidney in humans and has recently been linked to memory and cognition by a number of studies. As interest in the gene grows more researchers are seeking answers to how a single nucleotide polymorphism in the gene (Cď&#x192; T) affects both the brain and behaviour.

CC- homozygotes were found to preform worse on average on memory and cognition tasks than their T-allele carrying counterparts (Wersching et al, 2011). The KIBRA protein is found in the highest concentrations in the hippocampus making this structure an area of high interest in relation to the genes function and effects.

In a study conducted by Palombo et al. (2013) it was found that T-allele carriers of the gene have significant increases in volume in both the Cornu Ammonis and Dentate Gyrus of the hippocampus providing preliminary evidence that the KIBRA genotype does affect the volume of hippocampal sub-regions.

Key words: KIBRA; Cognition; memory; Hippocampus; Cornu Ammonis (CA); Dentate gyrus (DG); Structural MRI Background KIBRA was first identified in a study by Kremerskothen et al in 2003 as a protein of the WWC family, found in both the brain and kidney that interacts with the postsynaptic protein dendrin. A study by Papassotiropoulos et al in 2006 later expanded on this finding making it a gene of interest to all researchers in the scientific community studying memory, cognition and related diseases. The KIBRA protein was found to exist in higher concentrations in the Cornu Ammonis (CA) and the Dentate gyrus (DG) of the hippocampus. KIBRA was found to be encoded for by the WWC1 gene and has been identified as a regulator of the hippocampal signaling pathway as well as being involved in cell polarity, membrane and vesicular trafficking, mitosis and cell migration (Zhang et al, 2014).

In 2006, evidence was found suggesting a polymorphism in the KIBRA gene was involved in memory and cognition performance (Papassotiropoulos et al, 2006). A single nucleotide polymorphism (SNP) in this gene from cytosine to thymine on the ninth intron of the gene has been linked to significant increases in performance on episodic memory tasks, especially those involving consolidation or delayed retention, than c-homozygotes. The same study found that brain activity recorded via fMRI, in areas related to memory retrieval were higher in people who did not carry the t-allele (Papassotiropoulos et al, 2006). Since these original findings the KIBRA gene has been an area of interest in many labs studying memory and cognition, and the KIBRA gene has also been implicated as a possible risk factor for developing late onset Alzheimerâ&#x20AC;&#x2122;s disease as well as dementia (Schneider, 2010). Being a CC homozygote has been associated with decreased memory performance and less efficient MTL activation (Schwab et al, 2014). Carriers of the T allele were recently found have increased hippocampal volume, especially in two sub regions of the hippocampus: the DG and CA (Palombo et al, 2013).

This finding is contrary to a prior study, which had 41

found no volumetric difference between carriers and non-carriers using automated segmentation (Papassotiropoulos et al, 2006) Conclusions and Discussion

Summary Of Major Results

The purpose of the study conducted by Palombo et al in 2013, is to look for any volumetric differences associated with the SNP of the KIBRA gene in the hippocampus and associated MTL structures. The study hypothesizes that carriers of the T-allele will have volumetric differences significantly different than those of non-carriers in these areas. Carriers and noncarriers of the t-allele were separated into groups and matched on performance on behavioral tasks as well as other genes which have been implicated in episodic memory, including APOE, BDNF and COMT. Highresolution T2 weighted images, taken perpendicular to the long axis of the hippocampus were obtained and analyzed using Region of Interest (ROI) segmentation (see figure 1 for an example). Areas analyzed were separated into the CA1, DG/CA, subiculum, medial temporal lobe (further subdivide into perirhinal cortex, entorhinal cortex, and parahippocampal cortex) and the head and tail of hippocampus.

The study used three-mixed design ANCOVAs in order to to test for significant differences in the hippocampus, segmented hippocampus and MTL cortices between t-allele carriers and cc homozygotes. These two areas were further subdivided into the CA1, DG/ CA, subiculum, entorhinal cortec (ERC), perirhinal cortex (PRC) and parahippocampal cortex (PHC).

Their hypothesis was validated when they found that T-carriers had larger overall hippocampal volume than CC-homozygotes as well as significantly larger segmented hippocampi than non- carriers. Using post-hoc analysis the CA1 and DG/CA were found to be significantly larger in T-carriers. The subiculum

volume. Prior research studies on both animals and older populations have found a functional differentiation between these two hippocampal subregions. The CA has been shown to be involved in late retrieval and memory consolidation while the DG has been associated with encoding and early retrieval (Milnik et al, 2012).

These differences were observed in the absence of any behavioral differences due to the matched subject groups which allows us to separate and describe the independent effects of KIBRA.

Table 1.

In parentheses the standard error is shown.

Segmentation of hippocampus is as follows: CA, DG/CA, and Sub (subiculum). Segmentation of MTL cortices is as follows: ERC, PRC, and PHC. Columns with * contain significant differences between groups (p<0.05) and ** contain significant differences of (p<0.10). (Palombo et al, 2013).

Conclusions

In conclusion, the KIBRA polymorphism of a CT was found to be associated with increased Figure 1. Depicted on the left is a 3D representation of the hippocampus. hippocampal volume. This finding was found to The yellow lines mark coronal slices which are shown to the right in be strongest in two sub regions of the hippoT2 weighted slices. The middle column depicts and un-segmented view of MTL scans while the furthest right is an example of a segmentedcampus: the DG and CA. This finding is one step scan. The colors denote different structures green=CA1, dark blue=DG/towards identifying one of KIBRA’s many differential CA, red=Sub, orange= head of the caudate, brown=tail of the caudate,effects on the brain and memory. By identifying the yellow= PHC, pink=PRC, light blue= ERC.(Palombo et al, 2013). effects of the KIBRA polymorphism we may be able to develop ways of applying this knowledge towards clinical treatments to aid patients with damage to the was found to be relatively larger in non-carriers than memory and cognition centers affected by KIBRA, carriers of the T-allele. There were no significant like patients suffering from Alzheimer’s disease and group interactions and KIBRA was found to have dementia. no significant effect on most of the MTL structures.

Using post-hoc analysis, T-carriers were found to Criticisms and Future Directions have a small but significant increase in volume of the parahippocampal cortex indicating some small effect The hippocampus and surrounding MTL structures of KIBRA here. The mean volumes observed in both carriers and non-carriers in these sub-regions of the have been researched previously in their entirety to hippocampus and MTL cortices can be found in Table look for any volumetric differences associated with the 1: A & B below and are further divided by hemisphere. SNP of the KIBRA gene and results have been null (Papassotiropoulos et al., 2006). Within the same study it was also found that expression of KIBRA in Conclusions and Discussion the brain varies and is higher in the hippocampus and more specifically two of its sub-regions: the Cornu The study used structural imaging to investigate Ammonis (CA) and the Dentate Gyrus (DG). These the effects of KIBRA on both hippocampal and MTL variances in concentration may suggest affects of the volume. They were ultimately able to show evidence KIBRA gene also vary regionally. supporting the association between the t-allele SNP The study conducted by Palombo et al. (2013) is of the KIBRA gene and increased CA and DG/CA unique in its approach by regionally segmenting 42

and analyzing the hippocampus as well as looking at overall hippocampus and MTL structure volumes.

Using this method they found significant changes in regional substructures associated with higher KIBRA expression: CA1 and the DG. These differences may not have been found in the earlier study discussed due to differences in segmentation and demarcation strategies between researchers which is a common challenge in the field. Past studies also did not match participants between groups on behavioral performance to allow KIBRA related differences to be shown independently, which may also account for some of the differences in findings (see Papassotiropoulos et a., 2006). The finding of regional hippocampal volumetric differences between carriers and non-carriers provides a possible mechanism for differences observed in episodic memory performance found in multiple other studies (Milnik et al, 2012; Corneveaux et al, 2010; Papenberg et al, 2015). By working to identify the mechanism of the KIBRA SNP the current study contributes to the creation of new targets for possible development of clinical in associated diseases like dementia and AD. Wersching et al (2011) found a complex interaction of KIBRA on cognitive function that is modified by both gender and arterial hypertension. The current study did not take effects of gender into account and the majority of its participants were female. This inequality in gender ratio may have skewed the findings because of possible effects of gender, leading to different effects on volume gene interactions. One simple possible future study that could be preformed would look to see if sex alters the effects of KIBRA on regional hippocampal volumes by separating the two genders and looking for any significant differences between the two, using the same methodology as the current study.

A case has also been made for the differing effect of KIBRA with age, suggesting that the effects of the gene may accumulate, as differences in cognitive abilities between the T and C allele carriers tend to both appear and/or become larger with age (Papenberg et al, 2015). A longitudinal study is needed in which individuals are first given a genotypic screening to determine allele type and a preliminary cognitive performance assessment. After being matched for performance and placed into T and C carrier groups they would then be given episodic memory tasks as well as a structural MRI’s on intervals (~ every 1-2 years) over a period of 10-15 years. The data would then be analyzed for any significant accumulation effects of allele type in episodic memory, regional hippocampal volumes, as well as interactions and correlations between the two. Findings from this study or a similar longitudinal study would shed light on mechanisms behind the increasing gap in cognitive performance tasks we see between the two variant KIBRA allele groups. With data from both gender and longitudinal studies we would e able to make more concrete conclusions about the total effect of the KIBRA polymorphism on hippocampal structures volumes.

43

References 1. Corneveaux, J. J., Liang, W. S., Reiman, E. M., Webster, J. A., Myers, A. J., Zismann, V. L., … Huentelman, M. J. (2010). Evidence for an association between KIBRA and late-onset Alzheimer’s disease. Neurobiology of aging, 31(6), 901–9. doi:10.1016/j.neurobiolaging.2008.07.014 2. Kremerskothen, J., Plaas, C., Büther, K., Finger, I., Veltel, S., Matanis, T., … Barnekow, A. (2003). Characterization of KIBRA, a novel WW domain-containing protein. Biochemical and biophysical research communications, 300(4), 862–7.

3. Milnik, A., Heck, A., Vogler, C., Heinze, H.-J. J., de Quervain, D. J., & Papassotiropoulos, A. (2012). Association of KIBRA with episodic and working memory: a meta-analysis.

American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics, 159B(8), 958–69. doi:10.1002/ ajmg.b.32101 4. Palombo, D. J., Amaral, R. S., Olsen, R. K., Müller, D. J., Todd, R. M., Anderson, A. K., & Levine, B. (2013). KIBRA polymorphism is associated with individual differences in hippocampal subregions: evidence from anatomical segmentation using high-resolution MRI. The Journal of neuroscience : the official journal of the Society for Neuroscience, 33(32), 13088–93. doi:10.1523/JNEUROSCI.1406-13.2013 5. Papassotiropoulos, A., Stephan, D. A., Huentelman, M. J., Hoerndli, F. J., Craig, D. W., Pearson, J. V., … de Quervain, D. J. (2006). Common Kibra alleles are associated with human memory performance. Science (New York, N.Y.), 314(5798), 475–8. doi:10.1126/science.1129837 6. Papenberg, G., Salami, A., Persson, J., Lindenberger, U., & Bäckman, L. (2015). Genetics and Functional Imaging: Effects of APOE, BDNF, COMT, and KIBRA in Aging. Neuropsychology review. doi:10.1007/s11065-015-9279-8 7. Schneider, A., Huentelman, M. J., Kremerskothen, J., Duning, K., Spoelgen, R., & Nikolich, K. (2010). KIBRA: a new gateway to learning and memory? Frontiers in aging neuroscience, 2. doi:10.3389/neuro.24.004.2010 8. Schwab, L. C., Luo, V., Clarke, C. L., & Nathan, P. J. (2014). Effects of the KIBRA Single Nucleotide Polymorphism on Synaptic Plasticity and Memory: A Review of the Literature.

Current neuropharmacology, 12(3), 281–8. doi:10.2174/157 0159X11666140104001553 9. Wersching, H., Guske, K., Hasenkamp, S., Hagedorn, C., Schiwek, S., Jansen, S., … Floel, A. (2011). Impact of common KIBRA allele on human cognitive functions. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology, 36(6), 1296–304. doi:10.1038/npp.2011.16 10. Zhang, L., Yang, S., Wennmann, D. O., Chen, Y., Kremerskothen, J., & Dong, J. (2014). KIBRA: In the brain and beyond. Cellular signalling, 26(7), 1392–9. doi:10.1016/j.cellsig.2014.02.023

Received Month, 04, 2015; revised Month, 04, 2015; accepted Month, 04, 2015.

α5GABAA Receptors Mediate Inflammation-Induced Memory Deficits in the Hippocampus Sammy Cai

The mechanism by which systemic inflammation induces learning and memory deficits still remains poorly understood. This study investigates the pathogenesis of inflammation-induced memory deficits using a combination of behavioral, electrophysiological, and biochemical methods combined with genetic and pharmacological approaches. The results suggest that acute systemic inflammation leads to contextual fear memory deficits in wild-type mice primarily due to the reduction in amplitude of long-term potentiation in CA1 hippocampal slices.

It was determined that inflammation-induced memory deficits were reversible upon pharmacological inhibition or gene deletion of α5-subunit-containing γ-aminobutyric acid type A (α5GABAA) receptors. Inflammatory cytokines, primarily interleukin-1β (IL-1β), increase the inhibitory tonic current generated by α5GABAA receptors via the p38 mitogen-activated protein kinase signaling (MAPK) cascade. Activation of this pathway resulted in an increase in surface expression of α5GABAA receptors. IL-1β-mediated upregulation of α5GABAA receptors was attenuated via pharmacological inhibition of p38 MAPK. Collectively, these results show that α5GABAA receptors are upregulated by IL-1β and can mediate memory deficits during acute systemic inflammation.

Key words: inflammation; hippocampus; α5GABAA receptors; electrophysiology Background Learning and memory deficits can be caused by acute systemic inflammation, which can be induced by autoimmune disease, infection, and stroke. In humans, acute systemic inflammation results in impaired explicit recall whereas it impairs acquisition of fear-associated memories in mice1. Chronic inflammation can contribute to neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, and multiple sclerosis1.

Systemic inflammation results in increased production of cytokines in the brain, including interleukin-1β (IL-1β), IL-6, and tumour necrosis factor-α (TNFα). It is known that there is a correlation between increased IL-1β plasma levels with memory deficits in patients. In the laboratory, mice that underwent orthopedic surgery had elevated levels of IL-1β in the hippocampus, which was accompanied with memory deficits2. In addition, the effects of IL-1β on memory consolidation only affected hippocampal dependent memories, whereas hippocampal independent memories were unchanged3.

The binding of the neurotransmitter γ-aminobutyric acid (GABA) to GABA Type A (GABAA) receptors modulates the inhibitory tone found throughout the CNS3. GABAA receptors are known to generate two forms of inhibitory currents: a phasic, fast inhibitory postsynaptic current and tonic, which is mediated by extrasynaptic GABAA receptors. The inhibitory tone found in the CA1 region of the hippocampus is primarily generated by tonic currents mediated by the α5-subunit-containing GABAA (α5GABAA) receptors4. Studies have shown that drugs that increase α5GABAA receptor activity can result in memory deficits5. Alternate studies have shown that reducing α5GABAA receptor function or expression in mice can result in improved performance in trace fear-conditioning as opposed to contextual fear-conditioning6.

The binding of IL-1β to IL-1 receptors during inflammation activates a variety of signalling pathways in neurons, such as p38 mitogen-activating protein kinase (MAPK), c-Jun N-terminal kinases (JNKs), and phosphatidylinositol 3-kinases (PI3Ks)7. The mechanism of GABAA receptor trafficking is currently under debate as findings from various studies have been contradictory. For example, one study demonstrates that activation of p38 MAPK and PI3K via TNF-α downregulates cell-surface expression of GABAA receptors within the hippocampus8. Conversely, another study showed that TNF-α had no effect on the tonic inhibitory current9. It is hypothesized that α5GABAA receptors are upregulated in inflammationinduced memory deficits.

Despite the efforts made to elucidate the link between inflammation and memory deficits, no treatments are available to effectively prevent or reverse the memory deficits associated with neuroinflammation. General inhibition of IL-1β binding to IL-1 receptors are impractical treatments as that would delay wound healing and increase the risk of infection10. More studies are required to identify additional downstream mediators of neuroinflammation in order to develop more effective treatments.

Research Overview

α5GABAA Receptors Regulate InflammationInduced Contextual Fear Memory Deficits

Both wild-type (WT) and α5-subunit null mutant (Gabra5-/-) mice were treated with IL-1β to mimic acute systemic inflammation. Three hours after the treatment, the mice were trained to pair an electric foot shock (unconditioned stimulus) with an auditory tone (conditioned stimulus) for contextual fear learning, which is hippocampal dependent. To test for cued fear 44

mice were reversed after treatment with L-655,708 or MRK-016. Both WT and Gabra5-/- mice treated with either IL-1β or LPS exhibited no deficits in cued fear memory (Figure 1D; n = 14-16). Collectively, these results show that inflammation only impairs hippocampal-dependent memories3.

α5GABAA Receptors Reduce the Amplitude of Long-Term Potentiation During Inflammation

Figure 1. Inflammation-Induced Contextual Fear MemoryDeficits Is Absent in Gabra5-/- Mice and Can Be Prevented in WT Mice With Pharmacological Inhibition of α5GABAA Receptor3 A. IL-1β lowered freezing scores for contextual fear in WT mice.

B. L-655,708 or MRK-016 restored freezing scores of IL-1βtreated WT mice to control values.

C. LPS reduced freezing scores in WT mice only. Treatment with LPS then L-655,708 restored freezing scores to control values.

D. IL-1β did not affect freezing scores in cued memory response to tone in WT and Gabra5-/- mice.

memory, which is hippocampal independent, the mice were reintroduced to the tone after training11.

It was found that mice treated with IL-1β had impaired contextual fear memory, as seen in the lower freezing scores compared to controls (Figure 1A; n = 14-16, p < 0.05). This is consistent with previous studies1.

Gabra5-/- mice treated with IL-1β exhibited no memory deficits (Figure 1A; n = 14-16; p > 0.05).

Inhibition of the α5GABAA receptor with L-655,708 or MRK-016 did not modify contextual memory in either WT or Gabra5-/- mice under control conditions (Figure 1B; n = 16). Treatment with L-655,708 or MRK-016 in IL-1β-treated WT mice exhibited attenuation of impaired contextual fear memory3.

Systemic inflammation was induced in WT and Gabra5-/- via injection of lipopolysaccharide (LPS).

After three hours, WT, but not Gabra5-/- mice exhibited impairment of contextual fear memory (Figure 1C; n = 10-15). The deficits found in WT 45

Brain slices were prepared three hours after mice were injected with LPS. Long term potentiation (LTP) was induced via theta burst stimulation of the Schaffer collateral pathway in the CA1 region of the hippocampus (Figure 2A, inset). The field postsynaptic potentials (fPSPs) were measured after stimulation.

In vehicle-treated WT mice, stimulation increased the slope of fPSPs to 136.1% ± 5.6% of baseline (n = 9). In contrast, LPS-treated mice had only a 113.1% ± 2.5% increase of fPSP from baseline (Figures 2A and 2B; n = 10; p < 0.05). Pharmacological inhibition of α5GABAA receptors of the LPS-treated brain slice using L-655,708 eliminated the reduction of LTP induced by LPS (Figure 2B). LPS failed to reduce LTP in brain slices from Gabra5-/- mice, having an fPSP slope of 133.1% ± 4.3% (n = 15; p > 0.05 compared with control) for the LPS treatment compared to 135.4% ± 5.9% (n = 13) for the vehicle treatment (Figure 2B)3. This suggests that α5GABAA receptors are required for impairing LTP via inflammation.

IL-1 receptor antagonists (IL-1ra) were applied to LPS-treated mice brain slices. IL-1ra) restored LTP to 126.2% ± 3.9% of baseline (Figure 2C; n = 10; p < 0.05 compared with LPS). These results confirm the findings from previous studies, suggesting that reduction in LTP is largely mediated by increased IL-1β activity12.

IL-1β Increases α5GABAA Receptor Activity in Neuronal Cultures

Whole-cell currents were recorded from hippocampal neurons to determine whether IL-1β directly enhances tonic inhibitory conductance. Neurons treated with IL-1β had an increase in tonic current by 45% (IL-1β 1.6 ± 0.1 pA pF-1, n = 22, versus control 1.1 ± 0.1 pA pF-1, n = 21; p < 0.001 compared with control; Figure 3A). Increasing concentrations of IL-1β resulted in an increase in current amplitude. After 12-15 hours, no further increase in current amplitude was observed3.

The effects of TNF- α and IL-6 were also examined, as they are known to increase along with IL-1β during acute inflammation1. When neurons were treated with TNF- α or IL-6, no changes were observed (1.0 ± 0.1 pA pF-1; n = 10-14, and 0.9 ± 0.05 pA pF-1 respectively; n = 5, vs control 1.0 ± 0.06 pA, n = 5). To examine whether IL-1β enhanced tonic currents were mediated by α5GABAA receptors, α5GABAA receptor antagonists and genetic approaches were used. Using L-655,708, it was found that it decreased tonic current in WT neurons by 56.6% ± 9.2%3. No observable changes for tonic current was recorded in Gabra5-/- mice injected with

IL-1β (Figure 3B). This suggests that IL-1β enhances the tonic current generated by α5GABAA receptor. To see the effects of endogenous IL-1β, neurons cocultured with microglia were treated with LPS. Treatment with LPS resulted in an increase in tonic current, which was reversible when treated with IL-1ra. When testing neurons alone, LPS did not elicit any changes (Figure 3C). This suggests that IL-1β was released from microglia.

IL-1β Increases α5GABAA Receptor Activity via the p38 Mitogen-Activated Protein KinaseDependent Pathway

Hippocampal neurons were treated with selective kinase inhibitors to identify the predominant signalling pathway of IL-1β.

Using a p38 MAPK inhibitor, SB203,580, blocked the increase in tonic current induced by IL-1β, whereas, SB202,580, the inactive analog, had no effect (Figure 4A; n = 10-14; p < 0.05 compared with control). This suggests that p38 MAPK mediates IL-1β-induced increase in tonic current. No change in IL-1β-induced tonic current was reported when using selective inhibitors for JNKs and PI3Ks (Figures 4B and 4C)3.

To determine whether IL-1β increased mobilization of α5GABAA receptors, hippocampal slices were treated with IL-1β and then analyzed using a quantitative western blot analysis. It was found that surface expression of the α5-subunit protein increased to 157.4% ± 17.6% of baseline in vehicle-treated control slices (Figure 4D; n = 6)3. This suggests that IL-1β increases surface expression of α5GABAA receptors.

Discussion These findings suggest that neuroinflammation can cause memory loss by increasing tonic inhibitory conductance via α5GABAA receptors. Pharmacological or genetic inhibition of α5GABAA receptors was capable of preventing contextual fear memory deficits

induced by IL-1β or LPS. Increased tonic inhibitory conductance and reduction in LTP was due to an increase in surface expression of α5GABAA receptors, which was mediated by the p38 MAPK pathway.

These results are consistent with findings from other studies13. Although other signalling pathways are activated, such as JNKs and PI3Ks, they do not affect expression of α5GABAA receptors.

There are two proposed mechanisms of the p38 MAPK pathway. Phosphorylation of radixin, an actin-binding protein that anchors α5 subunits to cytoskeletal elements, increases the stability of α5GABAA receptors14. Alternatively, activation of p38 MAPK can increase the activity of cAMP response element-binding proteins, which can also enhance surface expression of α5GABAA receptors9.

Figure 3. IL-1β Increases Tonic Currents Generated by α5GABAA Receptors3 A. IL-1β increases tonic currents generated by α5GABAA receptors in a dose-dependent manner.

B. α5GABAA receptors are required for generation of the tonic current.

C. Tonic currents were increased in neuron and microglia cocultures, but not neurons alone. Changes were reversible upon treatment with IL-1ra.

Figure 2. Inflammation Reduces LTP in CA1 Region in WT Mice and This Can Be Prevented by Pharmacological Inhibition of α5GABAA Receptors3 A. LPS impaired LTP induced by theta burst stimulation only in brain slices of WT mice.

B. LPS-mediated reduction of LTP was mediated by IL-1β.

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Figure 4. p38 MAPK Pathway Mediates Inflammation-Induced Enhancement of α5GABAA Receptor Tonic Current3 A. SB203,580, a p38 MAPK inhibitor, abolished the effects of IL-1β whereas SB202,580, its inactive analog, did not block the tonic current (right).

B. Treatment with a JNK antagonist did not block the IL-1βinduced tonic current.

C. Treatment with a PI3K antagonist did not block the IL-1βinduced tonic current.

D. IL-1β increased surface expression of the α5 subunit in hippocampal slices compared to vehicle-treated control slices.

There are currently no treatments that are available to reduce memory deficits induced by neuroinflammation.

Potential treatment strategies for inflammation-induced memory loss can include administration of inverse agonists for α5GABAA receptors. Compared to nonselective GABAA receptor antagonists, the CNS has a higher tolerance for inverse agonists because they lack anxiogenic effects15. GABAA receptor expression is not restricted only to the CNS16. Because of this, acute systemic inflammation may also upregulate GABAA receptors in the periphery. It may be of interest for future studies to investigate the effects of GABAA receptors on organ dysfunction after acute or chronic systemic inflammation.

Criticisms and Future Directions Despite finding the pathway by which IL-1β mediates α5GABAA receptor activity to impair hippocampaldependent memories, there are several limitations associated with this study.

While this study looked at the effects of IL-1β on α5GABAA receptor activity, it is possible that there is an increase in GABA concentration in the extracellular space of the CNS during acute systemic inflammation. It is known that events such as stress, stroke, or surgery can increase extracellular concentrations of GABA2. Rather than increased vesicular release 47

of GABA, it is believed that there is a reduction in GABA transporter activity, thus reducing GABA reuptake17. Future studies can look at the effects of IL-1β on GABA transporters to determine whether acute systemic inflammation affects GABA reuptake.

Electrophysiological recordings can be performed to analyze the activity of GABA transporters, specifically GABA transporter-1, after application of IL-1β to cultured neurons.

Although the findings suggests that inflammation induces increased α5GABAA receptor activity within the hippocampus, the use of Gabra5-/- results in global deletion of α5GABAA receptors within the CNS. Global deletion of α5GABAA receptors may result in effects that are mediated by other structures within the CNS since α5GABAA receptors are also expressed outside the hippocampus3. To restrict loss of α5GABAA receptors to the CA1 region of the hippocampus, future studies can explore the use of Cre-Lox recombination.

Previous studies have shown that the use of a CaMKII promoter can restrict gene modifications specifically in the CA1 region of the hippocampus18. After deletion of the α5-subunit containing gene, behavioural studies, such as Pavlovian fear conditioning, can be performed to determine whether memory deficits induced by inflammation are specific to α5GABAA receptors within the hippocampus.

Another area of interest would be to restrict inflammation to the hippocampus. In the original study, intraperitoneal injections of IL-1β or LPS was used to induce acute systemic inflammation. While this provides a model that is similar to acute systemic inflammation in humans, it is not restricted within the hippocampus3. Potential studies can explore the use of mice expressing IL-1β excisional activation transgene (IL-1βXAT) within the hippocampus.

Hippocampal-restricted overexpression of IL-1β can be achieved via intrahippocampal injections of retroviral Cre into IL-1βXAT mice19. Memory deficits caused by hippocampal inflammation can then be tested for by using Pavlovian fear conditioning.

No treatments were available for inflammationinduced memory deficits prior to 20123. A recent study has determined that a salt extracted from the Glycyrrhiza glabra root, glycyrrhizin (GRZ), was able to attenuate inflammation-induced memory deficits in a dose dependent manner. GRZ has been found to reduce IL-1β mRNA levels within brain tissue, thus potentially reducing the effects of the p38 signaling cascade20. Future studies could examine the effects of GRZ on α5GABAA receptor surface expression by inducing inflammation via intraperitoneal injection of LPS, followed by oral administration of GRZ. This can be used to determine whether reduction of IL-1β is an effective treatment strategy for inflammation-induced memory deficits.

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3. Wang, D.-S. S. et al. Memory deficits induced by inflammation are regulated by α5-subunit-containing GABAA receptors.Cell Rep 2, 488–96 (2012).

4. Caraiscos, V. B. et al. Tonic inhibition in mouse hippocampal CA1 pyramidal neurons is mediated by alpha5 subunit-containing gamma-aminobutyric acid type A receptors. Proc. Natl. Acad. Sci. U.S.A.101, 3662–7 (2004).

5. Cheng, V.Y. et al. a5GABAA receptors mediate the amnestic but not sedative-hypnotic effects of the general anesthetic etomidate. J. Neurosci. 26, 3713–3720 (2006) 6. Collinson, N. et al. Enhanced learning and memory and altered GABAergic synaptic transmission in mice lacking the alpha 5 subunit of the GABAA receptor. J. Neurosci. 22, 5572–80 (2002).

7. O’Neill, L.A. Signal transduction pathways activated by the IL-1 receptor/toll-like receptor superfamily. Curr. Top. Microbiol. Immunol. 270, 47–61 (2002).

8. Pribiag, H. & Stellwagen, D. TNF-α Downregulates Inhibitory Neurotransmission through Protein Phosphatase 1-Dependent Trafficking of GABAA Receptors. Journal of Neuroscience 33, 15879–15893 (2013).

9. Srinivasan, D., Yen, J.-H. H., Joseph, D. J. & Friedman, W. Cell type-specific interleukin-1beta signaling in the CNS. J. Neurosci. 24, 6482–8 (2004).

10. Fleischmann, R. M. et al. Safety of extended treatment with anakinra in patients with rheumatoid arthritis. Ann. Rheum. Dis. 65, 1006–12 (2006).

11. Fanselow, M. S. & Poulos, A. M. The neuroscience of mammalian associative learning. Annu. Rev. Psychol. 56, 207–34 (2005).

12. Lynch, M.A. Long-term potentiation and memory. Physiol.

Rev. 84, 87–136 (2004).

13. Coogan, A. N., O’Neill, L. A. & O’Connor, J. J. The P38 mitogen-activated protein kinase inhibitor SB203580 antagonizes the inhibitory effects of interleukin-1beta on long-term potentiation in the rat dentate gyrus in vitro. Neuroscience 93, 57–69 (1999).

14. Koss, M. et al. Ezrin/radixin/moesin proteins are phosphorylated by TNF-alpha and modulate permeability increases in human pulmonary microvascular endothelial cells. J. Immunol. 176, 1218–27 (2006).

15. Atack, J. R. Preclinical and clinical pharmacology of the GABAA receptor alpha5 subtype-selective inverse agonist alpha5IA. Pharmacol. Ther. 125, 11–26 (2010).

16. Watanabe, M., Maemura, K., Kanbara, K., Tamayama, T., and Hayasaki, H. GABA and GABA receptors in the central nervous system and other organs. Int. Rev. Cytol. 213, 1–47 (2002).

17. Wu, Y., Wang, W., Díez-Sampedro, A. & Richerson, G. B. Nonvesicular inhibitory neurotransmission via reversal of the GABA transporter GAT-1. Neuron 56,851–65 (2007).

18. Tsien, J., Huerta, P. & Tonegawa, S. The Essential Role of Hippocampal CA1 NMDA Receptor–Dependent Synaptic Plasticity in Spatial Memory. Cell 87, 1327-1338 (2000).

19. Hein, A. et al. Behavioral, structural and molecular changes following long-term hippocampal IL-1β overexpression in transgenic mice. J. Neuroimmune Pharmacol. 7, 145–55 (2012).

20. Song, J.H. et al. Glycyrrhizin Alleviates Neuroinflammation and Memory Deficit Induced by Systemic Lipopolysaccharide Treatment in Mice. Molecules 18, 15788-15803 (2013).

Received Month, ##, ##, 200#; accepted

200#; Month,

revised ##,

Month, 2013.

This work was supported by The Association for the Development of Undergraduate Neuroscience Education (SRA & RLN), The Endowment for Science Education (EA), and The Synaptic State Faculty Research Foundation (EA). The authors thank Mr. Spine L. Cord, Dr. Amy G. Dala, and the students in Neuroscience 101 for technical assistance, execution, and feedback on this lab exercise.

Address correspondence to: Dr. Rita L. Neurotrophin, Biology Department, 123 Growth Cone Avenue, Action Potential College, Hillock, IL 60101 Email: rln@apc.edu Copyright © 2015 Dr. Bill JU, Neurosciences, Human Biology Program

48

Evaluating syanpto-protection in a three compartmented microfluidic chip model following a chemically induced axotomy.

Qasid Chaudhry

Microfluidic chips allow for the manipulation, at small quantities of neuronal cells. Further, these chips allow for precise temporal and spatial control, allowing for a model that can be useful in the framework of neurodegeneration. Deleglise et al (2013) fabricated a 3 chamber microfluidic chip. The chambers held one of: coritical neuron soma and dendrites, cortical axons, or striatal neurons. The chip was designed to replicate an oriented neuronal network, with connections going from the coritcal to the striatal neurons. Through the use of a chemically induced axotomy, by adding fluid to the central chamber (housing the cortical axon), Deleglise et al (2013) showed that their chip could be used to simulate a lesion in the neuronal network. An emphasis was placed on the protection of synapses, an event that in axotomy studies, traumatic injury and in many neurodegenerative disorders precedes the loss of neuronal cell soma and axons. They then showed that zVAD-fmk, a caspase inhibitor and resveratrol did not show synaptic protection, while NAD+ and Y27632, a Rho Kinase inhibitor showed significant synaptic protection, despite the mechanism not being clear. NAD+ and rho kinase inhibition point at potential therapeutic targets for neurodegeneration. This can also be further looked at from the functionality of the chip - it is a useful tool in the evaluation of drugs in the axotomy model was presented in the study.

Key words: Microfluidic Chip; synpato-protectoin; axotomy; neural network Background When studying neurodegenerative diseases or other brain injuries that result in neurodegeneration, we are lacking in a good in vitro model. Currently, commonly used models consist of either the use of whole brains or dissociated cell culture systems. While both do have their benefits, the use of entire brains limits the ability to interact with and manipulate individual cells, while cell cultures do not have the intricate networks found within the brain. A solution would be the use of a microfluidic chip, which would allow for the manipulation of single cells, while also allowing for the growth of an in vitro neural network.

Microfluidic chips have been used as early as the early 1990s (Chin et al, 2006). Microfluid chips have been used to study a variety of different processes and systems such as genetic analysis (Liu et al, 2009), drug screening (Caplin et al, 2015) and cancer diagnosis (Ying et al, 2013). In neurons, microfluidic chips have been used to study developmental processes such as axonal guidance (Huang et al, 2014) and neurodegeneration (Deleglise et al, 2013; Kilinc et al, 2010) among others. The diversity of microfluidic chips shows how potentially powerful this technique is. The strength of microfluidic chips, especially when looking at neuronal systems is in the simplification of vast, complex networks into easily manipulable, both temporally and spatially, chips.

When investigating neurodegeneration, the death of the soma occurs after synapses are lost (Deleglise et al, 2013). This is evident in axotomy and in early stages of neurodegenerative diseases and results in a further dying of downstream neurons. Yet little has been done in terms of investigating potential therapies in regards to synapto-protection.

Both the need of synaptic protection and a model simulating neural networks are important when considering the effect of neurodegeneration and testing the

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efficacy of drugs to prevent it from occurring. Deleglise et al (2013) look at the effect of several drugs in respect to dealing with chemically induced damaged resulting in axotomy. More interestingly, the model that they used to evaluate the drugs of interest was a threechambered microfluidic chip. This study spans two important fields within neuroscience â&#x20AC;&#x201C; neurodegeneration and the need for good in vitro models. This study, further used a microfluidic chip, but rather than simply use it to separate the axon from the somatodendritic portion, it also added a third chamber which held a full neuron (soma, dendrite and axon) and had one way synaptic connections. This microfluidic chip method is a step forward in creating complex in vitro models, which at the same time provide good spatial and temporal control (Siddique et al, 2014) â&#x20AC;&#x201C; something that could be used to study a variety of neuronal phenomena, including neruonal development, neurodegenerative disease, and in the case of the study, traumatic injuries.

Research Overview

Summary of Major Results and Discussion

Deleglise et al (2013) created a three chambered microfluidic chip in order to study the effects of synaptoprotective drugs following axotomy. The chip used was composed of 3 separate compartments; one for the cortical cells, one for striatal cells and one for the axons of the cortical cells. The compartments allowed, through micro channels, the axons from the cortical cells to send axons to the striatal cells. Each compartment could be manipulated separately through fluids, due to 2 reservoirs on each compartment (Fig. 1a).

The ability of this chip to manipulate specific parts of the neuron and study the effects on connected neurons is one that could lead to many different uses.

For one, due to the simplicity of the model, mecha-

nistic pathways of neurodegeneration and of therapies could more readily be assessed. The efficacy of drug therapies involved in treating neuronal conditions could also be screened in a spatially and temporally controlled environment. The inherent nature of the chip allows for the use of fewer neuronal cells and allows for results to be obtained quicker. The applications of the chip, because of the capacity for different designs is almost limitless and extends to beyond the brain (Liu et al, 2009; Caplin et al, 2015; Ying et al, 2013).

The cells were seeded onto the chip and the network was allowed to mature for 14 days prior to axotomy

Figure 1. a: A schematic for the 3 compartment microfluidic chip is shown, the reservoirs are labelled R. b: A phase contrast image of the microfluidic chip. The left side shows the cortical neuron somas, while the left portion shows the striatal neurons. The two are connected by microchannels. The decreasing diameter of the microchannels when going from the cortical to striatal cells can be seen.

Source: Deleglise B, Lassus B, Soubeyre V, Alleaume-Butaux A, Hjorth J, Vignes M, ... Peyrin J (2013) Synapto-protective drugs evaluation in reconstructed neuronal network. PLoS ONE 8(8), e71103

Neural Network

Once 15 days had passed from the seeding, Deleglise et al (2013) showed that the neural network had grown as predicted. They found that 80% of the cortical axons that made it to the central compartment made connections and synapsed on the dendrites of the striatal neurons. This was evidenced by the association of VGLUT1 positive presynaptic terminals from the cortical axons and MAP-2 positive dendrites of the striatal cells in the receiving compartment.

The decreasing diameter of the microchannel proved effective in the prevention of allowing striatal axons from projecting into the microchannel (Fig. 1b).

This all points to the formation of a neural network, with unidirectional projections as found by Peyrin et al (2011) as well under a similar design. The study

by Deleglise et al (2013) showed that the addition of reservoirs and a compartment to the microchannels didn’t alter the functional or morphological aspects of

the neurons in the network.

Further, these results can be further extended - due to the polarizing of the axons, a further receiving chamber, downstream of the striatal neurons could be added to allow for a more complex network.

Axotomy

Prior to assessing the efficacy of the synaptoprotective drugs, the axons needed to be lesioned. This was carried out through chemical axotomy, through the use of a 0.1% Triton in DMEM solution, along with a control DMEM only solution treatment. The solution was added to the reservoirs of the middle compartment, housing the cortical axons. The Triton solution was shown to result in a small lesion, while not affecting other portions of the axons (Fig 2).

Figure 2. Following axotomy, the lesion seemed to be contained to the central channel, while the surrounding axonal segments seemed unharmed. The axons were stained for α-tubulin.

Source: Deleglise B, Lassus B, Soubeyre V, Alleaume-Butaux A, Hjorth J, Vignes M, ... Peyrin J (2013) Synapto-protective drugs evaluation in reconstructed neuronal network. PLoS ONE 8(8), e71103

Following the axotomy, the cortical neurons showed no differences in either their dendrites or soma, as shown through staining using MAP-2 and α-tubulin, and the portion of the axon still attached to the cortical soma could be regrown.

The portion of the cortical axon that was no longer attached to the cortical soma began to show some degeneration after four hours, and was fully fragmented after six. Interesting to note is that there was no difference in the structure of the axons of the control (DMEM) and the 0.1% Triton treated axons.

The striatal post synaptic neurons did not show any architectural changes over the six hours, however, even just two hours, there was a significant decrease in the presynaptic clusters (60% decrease), and within six hours, the synapses seemed to have disappeared 50

(v-GLUT1 staining, indicative of presynaptic clusters disappeared) (Fig 3). In addition, the postsynaptic striatal neurons did not show any degeneration over the six hour time period of the, which the authors took to suggest that the structure of the striatal cells could be preserved and rescued for at least that time period.

The six hour period points to a critical window in which the striatal neurons could potentially be rescued with some of the synapses still being maintained that long. The 60% decrease in synaptic clustering within the first two hours is alarming and points further to the importance of immediate treatment following any sort of axonal lesion.

The time period over which the post synaptic striatal cells are shown to still be viable could be used in future assays of neuroprotection.

resveratrol pre-treatment did not show any significant synapto-protection. Y27632 pre-treatment of 1 hour prior to axotomy showed a retention of 75% of the synapses (Fig 4).

Y27632 and NAD+ provide interesting potential future therapeutic targets for dealing with trauma in the brain.

Figure 4. The relative efficacy of all of the drugs evaluated in the study in protecting from synaptic degeneration 3 hours following the initial axotomy is shown. Resveratrol and z-VAD-fmk show no significant difference from the untreated axotomized neurons, while NAD+ and Y27632 treated neurons do.

Source: Deleglise B, Lassus B, Soubeyre V, Alleaume-Butaux A, Hjorth J, Vignes M, ... Peyrin J (2013) Synapto-protective drugs evaluation in reconstructed neuronal network. PLoS ONE 8(8), e71103 Figure 3. Through staining with MAP-2 (blue) and v-GLUT1 (red) staining, loss of synaptic association of the axons can be seen, as decreased v-GLUT1 around the MAP-2 stained dendrites points to a loss of axonal projections in c: control condition, and after the axotomy at d: 2 hours e: 4 hours f: 6 hours.

Source: Deleglise B, Lassus B, Soubeyre V, Alleaume-Butaux A, Hjorth J, Vignes M, ... Peyrin J (2013) Synapto-protective drugs evaluation in reconstructed neuronal network. PLoS ONE 8(8), e71103

Synaptoprotective Effects

The cell cultures were also exposed to NAD+, z-VADfmk (caspase inhibitor), resveratrol (NAD+ dependant histone deacetylase activator), and Y27632 (Rho kinase inhibitor) prior to axotomy. Only Y27632 and NAD+ showed any significant protective effects on synapses.

3 hours after axotomy, cells not treated with any of the drugs showed a decrease to 25% of the initial number of synapses. Treating with NAD+ for 24 hours prior to axotomy showed a retention of 65% of their synapses. Pre-treatment with z-VAD-fmk for 1 hour did not show any significant synapto-protection compared to the non-treatment group. Similarly,

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Conclusions The study by Deleglise et al (2013) provides a look at a modification to the use of microfluidic chips in studying neuronal cultures to the chip used by Peyrin et al (2011). The addition of the reservoir to the microchannels allows for the manipulation of the axons and provides more control when manipulating neurons.

The use of microfluidic chips themselves allows for the study of small populations of neuronal cells and allows for the control of multiple sections of neurons, such as the soma or axons of individual populations.

Further, due to the flexibility of the design of the chip, microfluidic chips can be used to model multiple degenerative and developmental pathways. Peyrin et al (2011) also put forward that modifying the size of the microchannels, the size and amount of axons that could pass through could be controlled and a neural network that would mimic in vitro growth conditions could be obtained. The orientation of the network could easily be manipulated by modifying the size of microchannels, and through the use of different fluids in the chambers multiple conditions could be mimicked (Deleglise et al, 2013).

Deleglise et al (2013) further showed that Rho

kinase inhibitor Y27632 and NAD+ are both effective in protecting synapses from degeneration, while caspase inhibitors do not. Treating cells with Y27632 or NAD+ prior to the axotomy resulted in a lower loss of synapses three hours after the axotomy. Despite the pathway being unclear, the effects seem to be significant when compared to zVAD-fmk, a caspase inhibitor, and resveratrol. One of the earliest changes observed in neuronal degeneration is the loss of synaptic and axonal integrity (Shahidullah et al, 2013). Protecting against these effects could provide direction for further targets that could be used to prevent the degeneration of neurons in neurodegenerative diseases.

Criticisms and Future Directions As previously mentioned, one of the greatest strengths of using a microfluidic chip when looking at neurons is the amount of control that it provides, both temporally and spatially. Due to it being an in vitro method, visualizing the cells become easier. The different compartments allow for very specific targeting of chemicals to axons or the cell soma – something that would be difficult in in vivo models. The chip also provides a good temporal resolution. The chip, being clear allows for fluorescence to be observed outside it with no issues, and can thus easily be recorded.

Another strength is that microfluidic chips increase the viability of neurons (Huang et al, 2012).

The level of control however could also be seen as a flaw when considering translating the knowledge obtained in these systems to in vivo systems. As mentioned before, chemicals, through the 3 compartments could be specifically targeted to a specific area of the neuron, this would not be the case in in vivo environment. While the control helps to create the initial in vivo environment, the fact that the compartments are distinct is a reason for caution, and would have to be addressed.

Another potential flaw in the use of microfluidic chips was the use of polydimethylsiloxane. Despite its benefits of being clear, flexible and biocompatible, polydimethylsiloxane also has problematic properties, such as its ability to take up small proteins (Huang et al, 2012). The intake and release of proteins can be random, and as such could be a source of error.

This points to the need to be able to make disposable chips.

The effects of the drugs (NAD+ and Y27632) in the study were only evaluated for three hours after the axotomy – a very limited time scope. Further studies should look at extended time periods to determine the length of time that they are effective for.

In addition, Deleglise et al (2013) pretreated the chips with the drugs of interest up to 24 hours before.

Though there was an increase in synaptic viability using NAD+ and Y27632, the results should be taken with some doubt. NAD+ as Deleglise et al (2013) state, is involved in a wide manner of cellular including nuclear signalling and metabolism. Due to the addition of these potential therapeutic agents significantly before the axotomy, there will be multiple downstream pathways that would need to be further investigated.

Further, resveratrol indirectly stimulates NAD+ produc-

tion, yet does not induce the same synaptic protection as NAD+ does in the axon. Again, as Deleglise et al (2013) mention, the effects of resveratrol may be due to long distance signalling, as resveratrol has been shown to have effect in delaying Wallerian degeneration (Calliari et al, 2014). Another caveat to introducing these chemicals before the axotomy is that it is not something that would be observed in the real world – pre-exposing neurons with these chemicals to prevent synaptic degeneration is not a realistic treatment. But, this does warrant further investigation in a in vivo model and in respect to their effectiveness after axotomy. These pathways could also be useful in determining other agents to protect against synaptic degeneration.

The use of microfluidic chips is an interesting approach in neuroscience. Despite the use in observing single and even two neuronal populations, there is still significant amounts of further use that could potentially be gained.

Microfluidic chips are very versatile in that they can be used to study almost any neurodegenerative process in the brain. For example, this study looked at trauma, however, it used a very simple model of two neural populations. We know that in vivo, neural networks extend over significantly more neurons, which is one of the possible ways in which microfluidic chips can be further used. This study used a single synaptic connection, but in future studies, this could be extended to larger pathways. This combined with a 3-dimensional brain-on-a-chip (Park et al, 2015) could lead to the recreation of very complex networks, which, could even more closely resemble the in vivo environment in the cell. As in the study by Park et al., this could be applied to Alzheimer’s disease.

Using the microchannels and separated chambers from the Deleglise et al. study in conjunction with the neurospheroids of the Park et al. study, a very convincing model for Alzheimer’s disease in the brain could be produced. This could be used to further test therapies for Alzheimer’s and to further understand the mechanism in a much simpler, but still complex and manipulable model. Due to the diversity of the microfluidic chip, this could be further used for other forms of neurodegenerative diseases such as Parkinson’s. Another level of complexity could be added by introducing glial cells into the chip as well, to further emulate the in vivo environment (Soe et al, 2012).

In essence, microfluidic models could become very similar to mimicking in vivo models, despite being in vitro.

As it is with most in vitro studies, there needs to be some translatability to in vivo models when evaluating the drug therapies. Synaptoprotective therapies have been shown to be beneficial in the treatment of Alzheimer’s disease. The use of magnesium-L-threonate in late stages of Alzheimer’s was also shown to beneficial (Li et al, 2014). Considering Y27632 and the possible therapeutic synaptoprotective properties, the next step would be to either use an in vivo mouse model, to see the global and long term effects of the two in the brain (NAD+ has already been shown to be effective in vivo (Deleglise et al, 2013)). The second option would be to test the two using an in vitro model 52

of human cells. Both of these would be necessary before testing in in vivo human models, as again, with using in vitro studies, there are numerous other complicating factors in vivo. Furthermore, despite being a good model, the mouse and human brains are inherently different, and this would also be needed to be taken into consideration when looking at the applicability to therapies in humans. In essence, it would be of interest to almost repeat the experiment, but using human neurons and to also using the same controls and treatments, observe longer term effects on mice (the study used a three hour period after the axotomy).

References 1. Calliari A, Bobba N, Escande C, Chini EN (2014) Resveratrol delays Wallerian degeneration in a NAD(+) and DBC1 dependent manner. Exp Neurol 251:91-100 2. Caplin JD, Granados NG, James MR, Montazami R, Hashemi N (2015) Microfluidic organ-on-a-chip technology for advancement of drug development and toxicology. Adv Healthc Mater doi:10.1002/adhm.201500040 3. Chin CD, Linder V, Sia SK (2007) Lab-on-a-chip devices for global health: past studies and future opportunities. Lab Chip 7:41–57 4. Deleglise B, Lassus B, Soubeyre V, Alleaume-Butaux A, Hjorth J, Vignes M, ... Peyrin J (2013) Synapto-protective drugs evaluation in reconstructed neuronal network. PLoS ONE 8(8), e71103 5. Huang H, Jiang L, Li S, Deng J, Li Y, Yao J, Li B, Zheng J (2014) Using microfluidic chip to form brain derived neurotrophic factor concentration gradient for studying neuron axon guidance. Biomicrofluidics 8(1) doi: 10.1063/1.4864235 6. Huang Y, Williams J, Johnson S (2012) Brain slice on a chip: Opportunities and challenges of applying microfluidic technology to intact tissues. Lab Chip 12:2103-2117.

7. Kilinc D, Peyrin JM, Soubeyre V, Magnifico S, Saias L, Viovy JL, Brugg B (2011) Wallerian-like degeneration of central neurons after synchronized and geometrically registered mass axotomy in a three-compartmental microfluidic chip. Neurotox Res 19(1):149-61 8. Li W, Yu J, Liu Y, Huang X, Abumaria N, Zhu Y, ... Liu G (2013) Elevation of brain magnesium prevents synaptic loss and reverses cognitive deficits in Alzheimer’s disease mouse model. J Neurosci 33(19):8423-41 9. Liu P, Mathies RA (2009) Integrated microfluidic systems for high-performance genetic analysis. Trends Biotechnol 27(10):572-81 10. Park J, Lee BK, Hyun J, Jeong GS, Lee CJ, Lee SH (2015) Three-dimensional brain-on-a-chip with an interstitial level of flow and its application as an in vitro model of Alzheimer’s disease. Lab Chip 15(1):141-150.

11. Peyrin JM, Deleglise B, Saias L, Vignes M, Gougis P, Magnifico S, … Brugg B (2011) Axon diodes for the reconstruction of oriented neuronal networks in microfluidic chambers. Lab Chip 11:3663-73 12. Shahidullah M, Le Marchand SJ, Fei H, Zhang J, Pandey UB, Dalva MB, Pasinelli P, Levitan IB (2013) Defects in synapse structure and function precede motor neuron degeneration in Drosophila models of FUS-related ALS. J Neurosci 33(50): 19590-98 53

13. Siddique R, Thakor N (2014) Investigation of nerve injury through microfluidic devices. J R Soc Interface 11(90) doi: 0.1098/rsif.2013.0676 14. Soe A, Nahavandi S, & Khoshmanesh K (2012) Neuroscience goes on a chip. Biosens Bioelectron 35:1-13.

15. Ying L, Wang Q (2013) Microfluidic chip-based technologies: emerging platforms for cancer diagnosis. BMC Biotechnol 13:76

Received Month, ##, 200#; revised ##, 200#; accepted Month, ##,

Month, 2013.

This work was supported by The Association for the Development of Undergraduate Neuroscience Education (SRA & RLN), The Endowment for Science Education (EA), and The Synaptic State Faculty Research Foundation (EA). The authors thank Mr. Spine L. Cord, Dr. Amy G. Dala, and the students in Neuroscience 101 for technical assistance, execution, and feedback on this lab exercise.

Address correspondence to: Dr. Rita L. Neurotrophin, Biology Department, 123 Growth Cone Avenue, Action Potential College, Hillock, IL 60101 Email: rln@apc.edu Copyright © 2015 Dr. Bill JU, Neurosciences, Human Biology Program

Potential link between intestinal microbiota and anxiety Chun-Chi Chu

The importance of the intestinal microbiota that is developed in our first few days of life has been established.

However, the relationship between the intestinal microbiota and the central nervous system is still poorly understood. This literature review examines an experiment conducted by Neufeld et al. and derives potential future researches to further explore this area. Neufeld et al. collected both behavioural testing and measured anxiety-related brain mRNA expression level from adult germ free (GF) and specific pathogen free (SPF) Swiss Webser female mice. They found that GF mice presented less anxiety-related behaviours and more exploratory behaviours compared to SPF mice. GF mice also increased in the mRNA expression of brainderived neurotrophic factor (BDNF) and decreased in the mRNA expression of both 5HT1A serotonin receptor and N-methyl-D-aspartate (NMDA) NR2B subunit.

Key words: anxiety; intestinal microbiota; elevated plus maze; central nervous system; BDNF; NMDA; 5HT1A Background Previous studies have shown and established the importance of intestinal microbiota, which is formed in the first few days after birth, on the development and function of gut, immune, and endocrine systems. It helps the body to maintain homeostasis and regulate inflammation.1 Although it has not been fully investigated, several studies have suggest the connection between gut microbacteria and the central nervous system. In a cohort study, researchers found that the prevalence for anxiety, mood, and other psychological disorders tend to be higher in those who have disturbed intestinal microbiota, or bowel diseases.2 Diet, which changes intestinal microbiota, also alters learning and memory.3 The direct interaction between microbiota and the brain/behaviour is, however, poorly understood.

A study conducted by Neufeld et al.4 attempts to close this gap. They elucidated and demonstrated the effect of intestinal microbiota on behaviour and on the central nervous. Neufeld et al. measured mice’s anxiety behaviours using the elevated plus maze and mRNA expression of brain-derived neurotrophic factor (BDNF), 5HT1A serotonin receptor, and N-methylD-aspartate (NMDA) subunit. These proteins have been found to be associated with anxiety behaviors.

They found that germ free (GF) mice with no intestinal microbiota have lower anxiety and higher exploratory behaviours compared to mice raised in standard conditions, or specific pathogen free (SPF) mice.

Research Overview

Summary of Major Results

Eight-week-old GF and SPF female Swiss Webster mice were used. Fifty-one hours after the mice arrived, their anxiety-related behaviours were tested using elevated plus maze. Blood and brain samples were also collected from a separate group of mice to test for corticosterone, BDNF, 5HT1A serotonin receptor, and NMDA subunit levels.4

Elevated Plus Maze

Using elevated plus maze, the mice’s number of arm entries and duration in each arms were recorded. Figure (A) and (B) traces SPF’s and GF’s movements in the maze. GF mice spent more time in the open arms, less time in the closed arms, and entered open arms more frequently compared to SPF mice.4

Corticosterone Levels

A standard radioimmunoassay kit was used to measure the corticosterone levels. GF mice’s plasma corticosterone level was higher than SPF mice’s.4

In situ Hybridization

And, in situ hybridization was used to identify and measure the mRNA expression levels of BDNF, 5HT1A receptor and NMDA subunit. Then, statistical programs were used to compare and determine the statistical significance of the data.

GF mice increased in BDNF mRNA gene expression, specifically in the dentate gyrus of the hippocampus, while the expressions in other regions, CA1 and CA3, did not differ compared to SPF mice (Figure (C)). On the other hand, compared to SPF mice, GF mice’s 5HT1A receptor (Figure (D)) and NMDA mRNA expression decreased in the dentate gyrus and central amygdala, respectively. The expression of 5HT1A mRNA expression did not differ between GF mice and SPF mice in the CA1 region. The NMDA subunit that showed significant decrease was the NR2B subunit (Figure (E)).4

Conclusions and Discussion

The presence or the absence of intestinal microbiota influences the development of behaviour and central nervous system in mice. GF mice with no microbiota decreased in anxiety-like behaviours and increased in exploratory behaviours. They also expressed more BDNF and expressed less 5HT1A and NMDA subunit NR2B in the brain.4 Altered gene expression in these 54

proteins is consistent with the behavioural findings and contributes to the exploratory behaviour observed.

Previous studies have shown that low BDNF levels in the dentate gyrus increase anxiety-like behaviours5; the decrease in serotonin level from 5HT1A receptor

increases exploratory behaviour6; and NR2B antagonist blocks amygdala synaptic plasticity and fear learning7. Thus, higher BDNF levels and lower 5HT1A and NR2B levels in GF mice are related to reduced anxiety-like behaviour observed.

Figure (A) presents the movement of specific pathogen free (SPF) mice in the elevated plus maze (EPM). (B) presents the movement of germ free (GF) mice in the EPM. (C), (D), and (E) show the mRNA expression of BDNF, 5HT1A, and NR2B, respectively.

55

Criticisms and Future Directions The first few days of life after birth define the lifelong mutual relation we have with the microbacteria in our gut. This relation is not only essential for the development and function of gut, immune, and endocrine system but also for the development and function of the brain/behavior. Neufeld et al.4 found that GF mice with no intestinal microbiota have lower anxiety and higher exploratory behaviours compared to mice reared in SPF environments.4 This study ties the gap between intestinal microbiota and the brain/behavior interaction. It also provides new insights to therapeutic approaches in mental health. Innovating methods to treat and prevent psychiatric illnesses, specifically, anxiety-related disorders, can be derived.

Using the elevated plus maze model and measuring anxiety and fear related protein mRNA levels, Neufeld et al. showed that GF mice spent less time in the open arms and expressed higher BDNF levels and lower 5HT1A and NR2B levels. A group of mice were used for behavioral testing, and a separate group of mice were used for sample testing.4 This study, however, failed to account for the effect of menstrual cycles, the unexpected increase in corticosterone levels, and the effect of restoring microbiota.

Researchers did not take mice’s reproductive cycles into consideration and used eight-week-old adult female Swiss Webster mice for the experiment. These mice underwent hormone level changes that could have resulted in behavioural alterations. A study has shown that rats in the proestrus phase with high levels of progesterone and estrogen of the cycle reduced in anxiety-like behaviours.8 This means GF mice’s exploratory behaviours may be due to their proestrus phase rather than their lack of gut microbiota. To rule out this confounding factor, one should replicate the experiment using male mice. If the same results are found, the correlation between gut microbiota and behavior can be established and confirmed.

Neufeld et al. also failed to explain the unexpected high levels of corticosterone in GF mice that showed reduced anxiety-like behavior. High corticosterone levels tend to be accompanied by increased anxiety behaviours and low corticosterone levels by decreased anxiety behaviours.9 In this study, however, an inverse relation was observed. Mice with elevated corticosterone levels surprisingly showed less anxiety behaviours.4 To follow up on this finding, one measure the corticosterone samples from the group of mice that underwent behavioural testing instead of measuring from a separate group of mice. The blood samples should be collected immediately after testing.

The possibility of replenishing the gut microbiota and its effect on behaviour was not explored. When GF mice’s microbiota is re-established by introducing Bifidobacterium infantis, GF mice’s anxiety-like behaviours increase.10 For future experiments, one could administer pro-biotic supplements to facilitate the growth of microbiota in GF mice and antibiotics to suppress the growth in SPG mice and observe the behaviour/brain changes.

These manipulations should be done at different periods after birth to discover the time frame in which the interaction between the brain and the microbiota is still plastic.

Further experiments on male mice, on corticosterone levels, and on pro-biotic and antibiotic should be conducted to fill in the gaps in this study and understand more on anxiety disorders.

References 1. Backhed, F., Ley, R.E., Sonnenburg, J.L., Peterson, D.A., & Gordon, J.I. (2005). Host-bacterial mutualism in the human intestine. Science, 307, 1915–1920.

2. Walker, J.R., Ediger, J.P., Graff, L.A., Greenfeld, J.M., Clara, I., Lix, L. et al. (2008). The Manitoba IBD cohort study: a population-based study of the prevalence of lifetime and 12-month anxiety and mood disorders. Am J Gastroenterol, 103, 1989–1997.

3. Li, W., Dowd, S.E., Scurlock, B., Acosta-Martinez, V., Lyte, M. (2009). Memory and learning behavior in mice is temporally associated with diet-induced alterations in gut bacteria. Physiol Behav, 96, 557–567.

4. Neufeld, K.M., Kang, N., Bienenstock, J., & Foster, J.A. (2011). Reduced anxiety-like behaviour and central neurochemical change in germ-free mice. Neurogastroent Motil 23, 255-e119.

5. Chen ZY, Jing D, Bath KG et al. (2006). Genetic variant BDNF (Val66Met) polymorphism alters anxiety-related behavior. Science, 314(5796), 140–143.

6. Holmes A, Yang RJ, Lesch KP, Crawley JN, Murphy DL.(2003). Mice lacking the serotonin transporter exhibit 5-HT1A receptor-mediated abnormalities in tests for anxiety-like behavior. Neuropsychopharmacology, 28(12), 2077–2088.

7. Rodrigues SM, Schafe GE, LeDoux JE. (2001). Intraamygdala blockade of the NR2B subunit of the NMDA receptor disrupts the acquisition but not the expression of fear conditioning. J Neurosci, 21(17), 6889–68896.

8. Sayin, A., Derinoz, O., Yuksel, N., Sahin, S., & Bolay.

H. (2014). The effects of the estrus cycle and citalopram on anxiety-like behaviors and co-fos expression in rats.

Pharmacol Biochem Behav 124, 180-187.

9. Conboy, L., & Sandi, Carmen. (2010). Stress at learning facilitates memory formation by regulating AMPA receptor trafficking through a glucocorticoid action. Neuropsychopharmachology 35, 674-685.

10. Sudo, N. et al. (2004). Postnatal microbial colonization programs the hypothalamic-pituitary-adrenal system for stress response in mice. J Physiol 558.1, 263-275.

Received Month, 03, 04, 2015; Accepted

2015; Revised Month, 04,

Month, 2015.

This work was supported by The Association for the Development of Undergraduate Neuroscience Education (SRA & RLN), The Endowment for Science Education (EA), and The Synaptic State Faculty Research Foundation (EA). The authors thank Mr. Spine L. Cord, Dr. Amy G. Dala, and the students in Neuroscience 101 for technical assistance, execution, and feedback on this lab exercise.

Address correspondence to: Chun-Chi Chu, Human Biology Department, 300 Huron Street, Wetmore Hall Rm105, Toronto, ON M5S 3J6 Email: Catherine.chu@mail.utoronto.ca Copyright © 2015 Dr. Bill JU, Neurosciences, Human Biology Program 56

Elevations in the Serum Levels of the Brain Derived Neurotrophic Factor during Aerobic Physical Activity - A Simple, yet Often Disregarded Remedy for Frontotemporal Dementia Melissa Colaluca

Abstract: Within the last few decades, researchers in the field of neuroscience have taken an interest in exercise-meditated cognitive enhancement, with the intent of identifying plausible techniques and therapies to successfully reduce or delay cognitive decline in the elderly. A multitude of observational studies have found improved cognitive performance on a series of executive function assessments subsequent to aerobic activity, whereas stretching and toning training has been shown to have little to no effect on a subjects reasoning and rationalizing skills. More recently, researchers have identified surges in serum brain derived neurotrophic factor (BDNF) levels subsequent to aerobic activity and thus, have come to deduce that this protein mediates improved executive control subsequent to exercise. Leckie et al. (2014) sought to demonstrate the effect of BDNF on executive function in an elderly subject pool by having them complete a 1-year aerobic activity or stretching and toning intervention. Findings revealed that only subjects completing daily aerobic activity showed elevations in serum BDNF levels and improved performance on a test of executive function, the task-switch paradigm. Such is the case due to increased blood flow to cortical sites during aerobic activity, which increased the expression of BDNF, up-regulated neuronal outgrowth and inhibited apoptotic factors. Such enabled neural structures involved in executive function - the prefrontal cortex (PFC) and the anterior cingulate cortex (ACC) to perform cognitive processing at an optimal level.

Key words: brain derived growth factor (BDNF); insulin-like growth factor (IGF-1); Vascular endothelial growth (VEGF), prefrontal cortex (PFC); anterior cingulate cortex (ACC); task-switch paradigm, frontotemporal dementia; long term potentiation (LTP); grey matter volume (GMV); white matter volume (WMV); Event related potential (ERP) Background For centuries, several experts in the field of science have urged the elderly to participate in daily physical exercise to hinder the onset of debilitating physical diseases. Though several scientists agree that exercise regulates vascular disease symptoms - there is currently heated debate on its ability to initiate the cellular and molecular mechanisms that delay the onset of various neurodegenerative diseases. Exercise in popular media has been portrayed as the under the radar simple remedy to many of modern societies psychical ailments. Unfortunately, exercise is but an arduous and unpreferred way the elderly wish to spend their leisure time. Though several individuals are fit enough to walk around the block or jog on the spot in the comfort of their own household, few take the initiative to practice this behavior. Some use the excuse that their jam-packed busy schedules impede them from exercising or on the contrary, that their lazy disposition in a technologically dependent culture decreases their drive to stay fit. Often sedentary, older adults in Western States appear to have a high incidence of cardiovascular disease, but a more intriguing trend is that many have been diagnosed with one or more cognitive deficits.

Though humans anticipate a loss of memory in old age, they fail to realize that this outcome can indeed be reversed given that neurogenesis and synaptogenesis bare no age limits. Many recent studies have linked lower BDNF levels - the brain derived neurotrophic factor - with inactivity in old age. Increased expression 57

of this protein at cortical sites functions to enhance neuronal survival and outgrowth and so, if up regulated during exercise, neurons that would otherwise undergo apoptosis, thrive. After decades of observational and behavioral research on physical activities ability to prevent executive decline, scientistâ&#x20AC;&#x2122;s prime interest is to now pinpoint the neuronal proteins, diffusion patterns of pleasure inducing neurotransmitters and/or classes of endorphins involved in preventing cognitive decline. Of the studies conducted, it is evident that neurotrophic factors during and subsequent to physical activity show increased expression and transmittance to optimize and improve brain functioning in elderly subjects. Due to innovations in neuroimaging techniques and the rapid advancements in immunofluroescent technologies, neuroscientists have been able to demonstrate that a strict exercise plan leads to increased performance on tests of executive function as BDNF expression and the many downstream molecules it stimulates, increase.

Unfortunately, the visualization of proteins in the human cortex has not been perfected and so many preliminary studies on executive function augmentation as mediated by BDNF during exercise were conducted on rodents. One such immunofluroescent study found that as BDNF levels increased subsequent to a 20-minute exercise condition, CAMKII levels elevated, a protein known to initiate and increase long-term potentiation (LTP) via trafficking of AMPA receptors to the post-synaptic membrane of neurons. An increase of this protein at the frontal cortex correlated with improvements on tasks of executive function. Larson

et al. (2006) investigated the molecular mechanisms that mediate the effect of exercise on executive function by at protein expression at the frontal cortex of rodents, a model organism that holds several parallels in terms of neuronal structure and function to humans. Rats completed a 5-day treadmill exercise intervention for 15 or 30 minutes. Western blotting revealed elevations in BDNF expression in subjects completing 15 minutes of exercise. However, rats in the 30-minute condition showed diminutions in BDNF. Thus, it seemed as though this duration of exercise induced a stress response rather than having neuroprotective value and so moderate rather than vigorous exercise increases BDNF levels and improves cognitive functioning. The same study identified elevations in PI3K, CREB and MAPK proteins involved in the downstream signaling pathway of BDNF that work to inhibit apoptosis.

Psychologists have also studied the influence of aerobics on cognition via longitudinal observational studies such as the CAIDE investigation, which found that exercise for a duration of one year enhanced cognitive performance by 52% in subjects over the age of 65 (Cotman, Berchtold & Christie, 2007).

These subjects demonstrated near perfect scores on a series of executive function tasks. Kluding, Tseng and Billinger (2011) conducted a similar study whereby they had older adults complete a 6-month aerobic exercise program. Subsequently, subjects completed a flanker task - where they were to respond to target stimuli and disregard distractors - prior and subsequent to the intervention. Subjects demonstrated an enhanced ability to discriminate between distractors and target stimuli as a function of increased activity at the frontal sites.

Erickson et al. (2012) - one of the leading neuroscientists in a novel field of research investigating the effects of neurotrophic factors such as IGF-1 and BDNF on cognition - carried out an MRI study on elderly subjects looking at their grey matter volume (GMV) in the PFC and ACC during a 6-month aerobic exercise intervention. Subjects demonstrated increased volume at the PFC and ACC demonstrating there to be an association between physical activity and the integrity of grey matter microstructure. This was correlated with the improved performance on a Stroop task where individuals were to report the colour and not the semantics pertaining to a word, a difficult assignment considering that humans are more inclined to verbalize words rather than font colour. Thus, the following task requires that the elderly subject inhibit a reflexive response, a task that requires executive function.

Furthermore, Vaynman, Ying and Gomez-Pinilla (2007) reported elevations in cerebral blood volume as visualized using MRI in the hippocampus of 11 elderly subjects after a three-month aerobic exercise program. This was correlated with elevations in oxygenation levels at the cortex and improvements on tasks of executive function and memory. Thus, CBI may be a biomarker for neurogenesis in humans.

Colcombe et al. (2006) took to using MRI on a subject pool of older adults subsequent to an aerobic or toning intervention. Those in the aerobic inter-

vention - required to walk for 1 hour 2 days a week - showed elevations in white matter volume (WMV) at the frontal and temporal cortex. Volumetric increases in the nonaerobic control group as well as in a condition of middle-aged participants showed no changes in WMV. Similarly, Benedict et al. (2012) demonstrated increased synaptic connectivity via DTI in elderly subjects subsequent to a 12-week aerobic exercise intervention. The long fiber white matter tracts at the frontal cortex of these subjects displayed decreased functional anisotropy (sheering) and increased diffusion (Figure 1). Thus, neuronal coherence decreases in older subjects permitted that they remain active.

Figure 1: The following image illustrates a negative correlation between elderly subjects reaction time during a task of executive function and fractional anisotropy at the frontal cortex, subsequent to aerobic activity. Contrary to findings in the following study, the following demonstrates younger adults as showing greater improvements in cognitive functioning than the elderly (Benedict et al., 2012).

Event related potential (ERP) studies have also revealed that chronic, prolonged, rather than acute aerobic exercise in elderly subjects is correlated with increased amplitude in the P300 component at 300-800ms. This brain wave activity is associated with motivational attention, an updating of memory and decision-making. Thus, as performance on a task-switch paradigm increased so too did the P300 amplitude in elderly neurotypical subjects that exercised more than 4 times a week (Savikko, Timo, & Kaisu, 2009).

A less supported hypothesis has proposed that during exercise IGF-1 along with BDNF transmission is up regulated. Blocking BDNF signaling using antibodies to TrkB - that is the receptor for this neurotrophic factor - thwarted improvements in cognitive function, specifically improvements in executive function (Figure 2) (Trejo et al., 2007). In the study, elevations in neurospheres â&#x20AC;&#x201C; that is neural stem cells - at the prefrontal cortex, evidence of neuronal outgrowth (Figure 3).

Blocking IGF-1 using antibodies that act on its receptor also produced deficits in cognitive performance of subjects. Subsequent studies have shown that low levels of IGF-1 leads to an attenuation of Synapsin I and CAMKII, proteins involved in synaptic plasticity.

When IGF-1 was infused into rodents or its receptors 58

were overexpressed, an antidepressant effect, that is improvements in executive function, preceded. These, subjects showed diminished hypofrontality due to increased neurogenesis at prefrontal site.

Figure 2: When Trk-B receptors are blocked via antibodies (TrkB-Fe), the magnitude of LTP measured using theta burst stimulation is reduced, evidence that BDNF is involved in synaptic plasticity and strengthening (Trejo et al., 2007).

ventions: a daily aerobic activity condition or a daily stretching and toning activity condition. Prior to intervention, subjects had blood drawn so as to quantify their preliminary serum BDNF levels and to rule out any health conditions that may limit their participation in the study. The aerobic exercise condition had subjects complete 5 minutes of cardiovascular activity daily, that being a light jog or power walking. Subjects were to increase the duration of this activity by 5 minutes until they reached a maximum of 40 minutes of daily aerobic activity. Individuals in the stretching and toning exercise condition completed 10 minutes of daily weight training, yoga and one exercise of their choosing. Intermittently, a participant was required to complete a task-switch paradigm, a test used by psychologists to assess an individual’s executive function. Completed in isolation, these elderly subjects were to make very rudimentary judgments, that being, whether a number was even or odd, or higher or lower than the number five. However, the paradigm was divided into two conditions, a single task condition wherein the participants were to make only higher/ lower or even/odd judgments. Whereas, in a mixed condition, subjects were to make higher/lower judgments intermixed with even/odd judgments. When the computer screen background was blue, participants were to press the X key when the number presented was higher than five or the Z key when the number was lower then 5. Conversely, when the background was pink, if the number was odd they were to press the N key and when even, the M key. Thus, this task demanded appropriate discriminatory behavior, the ability to keep track of stimuli and inhibit inappropriate responses. Accuracy, as measured via percent correct responses, was extrapolated to quantify the subject’s executive skills. The subject’s serum BDNF levels were measured at the end of the intervention.

Results and Discussion Figure 3: The following demonstartes and increase in neural stem cell outgrwoth subsueqnt to the increased expression of BDNF as mediatted by aerobic activit (Trejo et al., 2007).

Thus, by old age, due to frequent mild blows to the skull, stress or other pre-existing psychiatric illnesses leads to ventricle enlargement, hemorrhaging of the underlying white matter structures beneath sites of focal contact, as well as hypoxia and cerebral perfusion often at the frontal cortex. These symptoms can be reversed via aerobic exercise, which increases the delivery of oxygenated blood to the brain by inducing vasodilation of capillaries. Increased oxygen delivery leads to increased metabolic activity and thus, greater BDNF transmission.

Research Overview Methods In the following investigation, Leckie et al. (2014) sought to deduce whether elevations in serum BDNF in elderly subjects arbitrates the effect exercise has on improving executive function. 92 elderly participants were randomly assigned to one of two exercise inter59

Participants assigned to the 1-year aerobic exercise condition showed striking elevations in post-serum BDNF levels as performance on the task-switch paradigm improved drastically. Even more remarkable and unexpected to Leckie et al. (2014) was that levels of BDNF increased as a function of age. Thus, subjects that were older showed marked improvements on the task-switch paradigm raising a not yet proposed question, “does age moderate the effect of aerobic exercise on serum BDNF levels?” Global accuracy, that is the measurement used to quantify whether a subject showed improvements in executive functioning during the cognitive task, looked at the difference in accuracy scores form the mixed trial and single trial conditions. It was evident that subjects older than 71 years of age in comparison to younger subjects showed increased global accuracy as a function of increased serum BDNF levels. Thus, BDNF seemed to improve some aspect of cognitive functioning during exercise by increasing neuronal outgrowth and cell survival.

Participants in the stretching and toning condition did not show elevations in BDNF levels but rather marked decreases in post-serum levels as performance on the task-switch paradigm declined. Subjects were unable to shift between making one judgment and switching

to another. Evidently, the stretching and toning condition exerted excessive stress not only on the subjectâ&#x20AC;&#x2122;s muscles, but on their mental health and thus, likely increased cortisol levels.

Therefore, Leckie et al. suggests that elderly subjects should be encouraged by general practitioners to participate in daily aerobic exercises, rather than flexibility training, which has more consequences than benefits.

Conclusions

The following study by Leckie et al. (2014) clearly demonstrated that the age of elderly subjects moderates changes in BDNF serum levels as demonstrated by significantly higher amounts of neurotrophic factor in subjects over the age of 65. Subjects in aerobic exercise condition, over the age of 71 displayed better task-switch performance, with higher accuracy in mixed trials that demanded executive control. Thus, elevated serum BDNF levels due to aerobic exercise mediate the effect of physical activity on task-switch performance. However, stretching and toning did not elevate BDNF levels and thus performance on the task-switch paradigm declined. Thus, elderly individuals should be encouraged to partake in daily aerobic exercise, rather than flexibility exercise to prevent the waning of executive function. Leckie et al. (2014) demonstrated that an increase in serum BDNF levels mitigates neurodegeneration, most likely - though not demonstrated - in regions involved in executive control.

Future Directions

Clearly, however, the benefits of aerobic exercise are far greater than that of flexibility training in terms of hindering executive dysfunction. However, though exercise does improve mental health, the exact mechanism in which it does so was not effectively demonstrated in the following study. Further studies should be conducted so as to identify a means of preventing frontotemporal dementia onset or diminishing executive disruption in individuals with pre-existing cognitive impairments. This is necessary as the proportion of elderly individuals with dementia on a global scale continues to surge. One major fallout and criticism of the following study, rests in the fact that no neuroimaging techniques were correlated with serum BDNF levels and the behavioral measures acquired from the task-switch trial, so as to elucidate whether elevations in BDNF occur at the frontal cortex during exercise to improve cognitive processing. The particular mechanism or neural substrates targeted

by BDNF are not explicated in this study. It is not known whether elevations in BDNF improved cognition, for there is no way to illustrate that the PFC and ACC involved in executive functioning show increased GMV, elevated activity or more importantly, increased BDNF transmission.

Furthermore, findings from many studies on exercisemeditated cognitive enhancement have demonstrated that aerobic interventions are far more effective in middle aged subjects rather than the elderly. Thus, Leckie et al. (2014) should take to deducing whether

BDNF will better mediate the effect of exercise on middle-aged subjects in comparison to elderly individuals. This may be due to the fact that middle-aged adults for one are typically more active and so the oxygenated blood flow to the cortex is far greater during exercise and so BDNF expression is increased.

Furthermore, they are less likely to have had several mild traumatic brain injuries and so the amount of atrophy, sheering of white matter tracks and inflammation of cortical structures is diminutive and so apoptotic factors are scarcely activated. They are also less likely to have a preexisting medical condition that impedes production of BDNF such as hypertension, cardiovascular disease and bouts of major depression or anxiety. Thus, Leckie et al. (2014) should look at whether physical activity provides greater neuroprotective benefits in middle age, contrary to the finding in the following study.

Furthermore, the findings in several studies suggest that women respond more to exercise interventions than males, quite possibly due to elevations in estrogen levels. Higher bouts of estrogen secretion during menstrual cycles are believed to increase the secretion of BDNF at cortical sites as well as the hippocampus. One observational study found that the risk of Alzheimerâ&#x20AC;&#x2122;s Disease and cognitive decline in woman participants over 65 years of age was annulled by 50% for those who exercised more than 4 times a week compared to those less active.

However, there was no such difference in cognitive function for men that were more active (Fratiglioni, Viitanen, & Von Strauss, 1997). A smaller study demonstrated a reduction in cognitive impairment by a whopping 88% in females who partook in a 5-year exercise intervention compared to controls that did not participate in daily exercise activity (Lautenschlager, Cox, & Cyarto, 2012). These less active woman had a five times greater risk of developing a cognitive impairment however, in an identical study with male subjects, there was little difference between active and inactive participants. Thus, Leckie et al. (2014) should take to comparing the enhancement in cognitive performance between male and female subjects.

Other such studies have demonstrated that cognitive impairments are more greatly ameliorated when subjects partake in meditative exercise and stretching activities where as those that partake in cardiovascular activity show higher levels of stress and discomfort and so exhibit little improvements in cognitive function, as quantified by longer reaction times and reduced performance on the IOWA gambling task, task-switch paradigm and the N-back test. It would be interesting to tag BDNF and IGF-1 and then deduce whether stretching and toning or aerobic activity more greatly increases their expression at the frontal cortex. The following study did not clearly elucidate whether physical exercise need be moderate or vigorous, chronic or acute, in order for physical activity to take its effect on cognitive centers in the brain to improve metabolic activity. A recent study, however, found that Tai Chi - a multimodal physical activity incorporating aerobic and flexibility exercises - successfully improved cognitive performance on a shift card test, digit span and Stroop test in a large sample of 2553 elderly adults (Wayne et al., 2014).

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Additionally, Leckie et al. (2014) did not look at the effect of aerobic activity on elderly individuals with pre-existing executive function impairments such as psychiatric disorders, including bipolar disorder and schizophrenia with characteristic hypofrontality.

Subjects with bipolar disorder showed improvements in performance on tasks of executive function subsequent to aerobic activity (Figure 4) (Sylvia, Ametrano, & Nierenberg, 2008). What was not demonstrated in this study was an elevation in BDNF serum levels, thus it is possible that a dissimilar protein acts to increase activity at the frontal cortex to improve the subjects ability to make accurate and appropriate discriminatory judgments and to inhibit inappropriate responses.

Another such study found that in comparison to neurotypical controls, patients clinically diagnosed with panic disorder, characteristic of poor executive function due to frequent displays of impulsive behavior and uncontrolled aggression. Subsequent to a 30-minute aerobic exercise intervention, exercise was shown to increase BDNF levels in panic disorder patients.

Figure 4: BDNF levels in panic disorder patients showed significant elevations subsequent to exercise (Sylvia, Ametrano, & Nierenberg, 2008).

Leckie et al. (2014) should also take to looking at the increased expression of the neurotransmitter dopamine subsequent to aerobic activity, which has also been proposed as contributing to the up-regulation of BDNF. For quite some time, dopamine has been known to act on receptors in the prefrontal cortex wherein it improves executive function. Thus, subsequent to physical activity, many individuals report feelings of ease, content and elation. Once such study demonstrated that exercise increases serum calcium levels and dopamine levels after a daily 15-minute aerobic activity (Hogan, Mata, & Carstensen, 2013). Controls that did not partake in this intervention showed no differences in serum calcium and dopamine levels.

Thus, it can be inferred that aerobic activity works to increase calcium influx into various neurons, which in turn increases dopamine and BDNF levels. Thus, Leckie et al. (2014) should employ PET imaging to correlate a subjectâ&#x20AC;&#x2122;s dopamine transmittance at the prefrontal cortex with increased serum BDNF levels to demonstrate that neurotransmitters play a role in cognitive improvement.

Given the large body of evidence in support of the role of insulin growth factor-1 (IGF-1) in executive function, Leckie et al. (2014) should also take to 61

measuring serum levels of this protein. Though this growth factor acts peripherally, it has been shown to enhance cognitive performance. Subjects in an aerobic activity condition demonstrated significantly higher levels of serum IGF-1 as well as increased LTP at various cortical neurons as measured via thetaburst stimulation, evidence of synaptic strengthening.

(Wieczorek-Baranowska et al., 2011).

The researchers should also genotype the subjects to deduce whether they have the Val66Met isoform of the BDNF gene or the Val66Val polymorphism. Those with the heterogeneous isoform are likely to show less responsiveness to the exercise intervention due to a decrease expression and transmission of BDNF and thus reduced neuronal outgrowth at frontal sites. Elderly with the Val66Met polymorphism are likely to show declinations in cognitive function despite the duration, length and type of intervention they are assigned to (Figure 5) (Swathi et al., 2014).

Figure 5: Individuals with the Val66Met polymorphism show lower cortical thickness at frontal sites and thus deficits in executive function (Swathi et al., 2014).

Cortisol levels in subjects should also be measured to rule out whether increased stress impedes the effect of exercise on neurogenesis and synaptogenesis.

Chronic activation of the HPA axis due to increased anxiety in old age (as a result of decreased mobility and ailments) may impede the effects of BDNF on executive function (Figure 6) (Strohle et al., 2010).

Figure 6: As serum BDNF levels in the elderly decreased, arousal increased. Subjects lower than normal BDNF levels displayed severe executive function deficits (Strohle et al., 2010).

References 1. Baker, L. D., Frank, L. L., Foster-Schubert, K., Green, P. S., Wilkinson, C. W., McTiernan, A., … Craft, S. (2010).

Effects of Aerobic Exercise on Mild Cognitive Impairment: A Controlled Trial. Archives of Neurology, 67(1), 71-79.

2. Benedict, C., Brooks, S.J., Jullberg, J., Nordenskjold, R., Burgos, J., Le Greves, M., Kilander, L., Karsson, L., Ahlstrom, H., Lind, L., & Schioth, H.B. (2012). Association between physical activity and brain health in older adults. Neurobiol of Aging. 34(1), 83-90.

3. Colcombe, J.C., Erickson, K.I., Scalf, P.E., Kim, J.S., Prakash, R., McAuley E., Elavsky, S., Marquez, D.X., Hu, L., & Kramer, A.F. (2006). Aerobic Exercise Training Increases Brain Volume in Aging Humans. Journal of Gerontology, 58(2),176-180.

4. Cotman, C.W., Berchtold, N.C, & Christie, L.A. (2007).

Exercise builds brain health: key roles of growth factor cascades and inflammation. Trends Neurosci, 30(9), 464-472.

5. Dahl, A., Hassing, L.B., & Fransson, E. (2009). Being overweight in midlife is associated with lower cognitive ability and steeper cognitive decline in late life. J. Gerontol. A Biol.

Sci. Med. Sci, 65(1), 57-62.

6. Erickson, K. I., Weinstein, A. M., Sutton, B. P., Prakash, R.S., Voss, M. W., Chaddock, L., … Kramer, A. F. (2012).

Beyond vascularization: aerobic fitness is associated with N-acetylaspartate and working memory. Brain and Behavior, 2(1), 32–41.

7. Fratiglioni, L., Viitanen, M., & Von Strauss, M. (1997).

Very old women at highest risk of dementia and Alzheimer’’s disease: incidence data from the Kungsholmen project, Stockholm. Neurology, 48(1), 132-138.

8. Gold, B. T., Powell, D. K., Xuan, L., Jicha, G. A., & Smith, C. D. (2010). Age-related slowing of task switching is associated with decreased integrity of frontoparietal white matter. Neurobiology of Aging, 31(3), 512.

9. Gomez-Pinilla, F. (2008). Brain derived neurotrophic factor functions as a metabotrophin to mediate the effects of exercise on cognition. Eur J Neurosci, 28(22), 2278-2287.

10. Hogan, C. L., Mata, J., & Carstensen, L. L. (2013).

Exercise Holds Immediate Benefits for Affect and Cognition in Younger and Older Adults. Psychology and Aging, 28(2), 587-594.

11. Kluding, P. M., Tseng, B. Y., & Billinger, S. A. (2011).

Exercise and Executive Function in Individuals with Chronic Stroke: A Pilot Study. Journal of Neurologic Physical Therapy : JNPT, 35(1), 11-17.

12. Larson, E.B., Wang, L., Bowen, J.D., McCormick, W.C., Teri, L., Crane, P., & Kukull, W. (2006).Exercise is associated with reduced risk for incident dementia among persons 65 years of age or older. Ann Intern Med, 144(30), 73-81.

13. Lautenschlager, N.T., Cox, K., & Cyarto, E.V.(2012).The influence of exercise on brain aging and dementia.Imaging Brain Aging and Neurodegenerative Disease,1822(3), 474-481.

14. Leckie, R. L., Oberlin, L. E., Voss, M. W., Parkas, R. S., Szabo-Reed, A., Chaddock-Heyman, L., …Erickson, K. I. (2014). BDNF mediates improvements in executive function

following a 1-year exercise intervention. Front. Hum. Neurosci, (8) 985, 1-10.

15. Lopez-Lopez, C., LeRoith, T., & Torres-Aleman, I. (2004). Insulin-like growth factor I is required for vessel remodeling in the adult brain. Proceedings of the National Academy of Sciences of the United States of America, 101, 9833-9838.

16. Raji, C. A., Ho, A. J., Parikshak, N., Becker, J. T., Lopez, O. L., Kuller, L. H., … Thompson, P. M. (2010). Brain Structure and Obesity. Human Brain Mapping, 31(3), 353-364.

17. Savikko, N., Timo, E.S., & Kaisu, H.P. (2009). Effect of physical exercise on cognitive performance in older adults with mild cognitive impairment or dementia. Dementia and Geriatric Cognitive Disorders, 38 (5). 347-365.

18. Strohle, A., Stoy, M., Graetz, B., Michael, S., Wittmann, A., Gallinat, J., Lang, U.E., Dimeo, F., & Hellweg, R. Acute exercise ameliorates reduced brain-derived neurotrophic factor in patients with panic disorder. (2010). Psychoneuroendocrinology, 35(3), 364-368.

19. Swathi, G., Manuck, S.B., Ferrell, R.E., Flory,J.D., & Erickson, K.I.(2014).The BDNF Val66Met polymorphism does not moderate the effect of self-reported physical activity on depressive symptoms in midlife. Psychiatry Research 218(2),93–97.

20. Sylvia, L.G., Ametrano, R.M., & Nierenberg, A.A. (2008). Exercise treatment for bipolar disorder: potential mechanisms of action mediated through increased neurogenesis and decreased allostatic load. Eur J Neurosci, 20(10), 2580-2590.

21. Trejo, J.L., Piriz, J., Llorens-Martin, M. V., Fernandez, A.M., & Bolós, M. (2007). Central actions of liver-derived insulin-like growth factor I underlying its pro-cognitive effects, Molecular psychiatry, 12(12), 1118-1128.

22. Vaynman, S., Ying, Z., Gomez-Pinilla, F. (2007). Hippocampal BDNF mediates the efficacy of exercise on synaptic plasticity and cognition. Neurochem Research, 33(1), 51-58.

23. Wayne, P. M., Walsh, J. N., Taylor-Piliae, R. E., Wells, R. E., Papp, K. V., Donovan, N. J., & Yeh, G. Y. (2014). The Impact of Tai Chi on Cognitive Performance in Older Adults, Journal of the American Geriatrics Society, 62(1), 25-39.

24. Wieczorek-Baranowska, A., Nowak, A., Michalak, E., Karolkiewicz, J., Pospieszna, B., Rutkowski, R., Laurentowska, M., & Pilaczyńska-Szcześniak, L. (2011). Effect of aerobic exercise on insulin, insulin-like growth factor-1 and insulin-like growth factor binding protein-3 in overweight and obese postmenopausal women. J Sports Med Phys Fitness, 51(3), 525-32.

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High fat diet intake is related to impaired hippocampal dependent memory in juvenile rats Erica Confreda

Recent studies in the literature have indicated that there is a link between obesity and cognitive dysfunction.

This is a prevalent issue in today’s society and raises a lot of concern due to the dramatic increase of obesity among children and adolescents. This study conducted by Boitard et al. investigated whether adolescence is a life stage that is particularly vulnerable to the negative effects of a high fat diet. The authors hypothesized that when juvenile rats are exposed to a high fat diet, the levels of pro-inflammatory cytokines in the hippocampus increase and as a result hippocampal dependent memory is impaired.

Results showed that juvenile high fat diet exposure significantly impaired long-term memory and spatial flexibility but not short-term memory. Furthermore, the levels of interleukin-1β and tumor necrosis α were significantly increased within the hippocampus of these juvenile rats after administration of LPS. Interestingly, the same composition and duration of the high fat diet did not affect long-term memory, spatial flexibility and short-term memory nor cytokine expression in adult rats.

These results indicate that adolescents are specifically vulnerable to the negative effects a high fat diet has on the hippocampus and that elevated levels of pro-inflammatory cytokines impair spatial memory.

Key words: Spatial memory; obesity; cytokines; hippocampus; interleukin-1β; tumor necrosis α

Background Obesity is a prevalent issue in today’s society since it has risen dramatically among children and adolescents. For instance, In the United States the occurrence of obesity is 31%8. Obesity is an important health issue that needs to be addressed since it is associated with other health concerns such as cardiovascular disease, type II diabetes and certain cancers. Moreover, recent studies show that there is a relationship between obesity and adverse neurocognitive functioning8. However, although research shows there is a relationship between obesity and neurocognitive functioning, little is known on exactly how they are connected. In the literature, inflammation is one proposed mechanism to explain this relationship9. The hormone leptin, through its signalling pathway, produces pro-inflammatory cytokines, reactive oxygen species and increase macrophage phagocytosis6. As a result, these cytokines promote insulin resistance by preventing phosphorylation of the insulin receptor6. In particular, elevated levels of the cytokine interleukin- 1 beta (IL-1β) for a long period of time impairs hippocampal dependent memories13.

This may be because of the higher concentration of microglia within the hippocampus and therefore this part of the brain might be more vulnerable to inflammation13. In some studies, the authors administered lipopolysaccharide (LPS) to exaggerate the immune response of animals. Some examples of cytokines that are increased after administration are: interleukin 6, Interleukin-1β and tumor necrosis factor α (TNFα)5. Some research also indicates that obesity may play a role in neurodegenerative diseases such as Parkinson’s and Alzheimers4. The authors Boitard et al. conducted this study on juvenile rats in order to see whether a high fat diet influences hippocampal 63

dependent spatial memory and if increased levels of cytokines play a role3. They also wanted to address the age that is more vulnerable to high fat diets. The authors hypothesized that the levels of inflammatory cytokines in the hippocampus increase when juvenile rats are exposed to a high fat diet, and as a result spatial memory is altered.

Research Overview

Summary of Major Results

The authors used male rats that were either 3 weeks old (juvenile group) or 12 weeks old (adult group) and had no weight differences. Animals of both age groups were subdivided into two treatment conditions.

One received a control diet containing 2.5% lipids and the other received a high fat diet containing 24% lipids. The rats were exposed to these diets for two months and then behavioural tests were performed.

One behavioural test used was the Morris water maze in order to assess spatial memory and the other was a reversal-learning test to assess spatial flexibility. After these behavioural tests were performed, the rats were either injected with saline, with lipopolysaccharide (LPS) or were not injected at all. The rats were then killed and the levels of insulin, leptin and cytokines were measured.

The authors assessed short-term memory two hours after the acquisition phase and assessed long-term memory four days after the acquisition phase. Results showed that long-term memory and spatial flexibility were significantly impaired in the juvenile high fat diet group whereas there was no significant difference in short- term memory (figure 1). In addition, there were increased levels of interleukin- 1β and tumor necrosis

factor α (TNF- α) within the hippocampus after LPS was administered (figure 2). In contrast, long-term memory, short-term memory and spatial flexibility were not impaired in the adult high fat diet group and there were no significant changes in the levels of cytokines (figure 1, figure 2).

Figure 1. The left diagram demonstrates there is no significant difference in short-term memory for both control and high fat juvenile diet groups. However long-term memory is significantly reduced for the juvenile high fat diet group. The right diagram demonstrates there is no significant change in short-term and long-term memory for both adult diet groups. Picture from: Boitard C. et al (2014) Impairment of hippocampal-dependent memory induced by juvenile high-fat diet intake is associated with enhanced hippocampal inflammation in rats. Brain, Behavior, and Immunity 40:9-17

Conclusions and Discussion The results illustrate that juvenile rats are more vulnerable to the effects a high fat diet has on the hippocampus. Long-term memory and spatial flexibility were altered and there was a higher inflammatory immune response when LPS was administered. In contrast, the same duration and percentage of high fat diet did not have the same significant affect in the adult diet groups. The result of the current study is in agreement with the findings of Acebes et al. such that a high fat diet has a more serious consequence on memory performance in adolescent mice rather than young adults mice1. In addition, their results indicated that it is not caloric intake but rather food composition that affects memory1. These two studies illustrate that adolescence is a life stage that is critical for the maturation of the hippocampus to control memory. In contrast, other studies showed that adult rats do in fact experience hippocampal dependent memory impairments when exposed to a high fat diet. Heyward et al. revealed that when adult rats are exposed to a higher percentage of fat for a longer duration they experience memory impairment7. Therefore, duration and fat composition act as potential confounders. Additionally, Pistell et al. noted that when 12-month-old male mice were exposed to a diet consisting of 60% lipids for 16 weeks, they experienced significant behavioural changes and had increased levels of TNF- α10. The discrepancy in data between these two studies may be due to the different composition of fat administered, the duration of the diet and the different behavioural

tests that were used.

The results from the current paper show that interleukin-1β is a significant cytokine that when elevated at high levels for long durations impair spatial memory. In another study conducted by Sobesky et al. inflammation is reduced when an antagonist blocks the interleukin-1β receptor and as a result eliminated the effect of the high fat diet by increasing the freezing times of rats13. This result indicates that inflammation plays a key role in memory decline. Sobesky et al. noted that obesity may prime the cells in the brain such that when a secondary challenge is administered there is an exaggerated inflammatory response13. This is in agreement with the current study since at basal levels there was no difference in cytokine production, but after administration of LPS there were elevated levels of interleukin- 1β and tumor necrosis factor α.

However it is important to note the stressfulness of the diet being administered.

The current study has several strengths and limitations that should be considered when interpreting the data. This is one of the first studies to investigate how a high fat diet affects hippocampal dependent memory in juvenile rats. In addition, the study eliminated weight gain as a potential confounder and the diet was not stressful enough to cause insulin resistance thus also eliminating that as a potential confounder. A limitation of the study is that the rats were only exposed to the high fat diet for a short length of time. Another limitation is that the authors only used the Morris water maze and reversal learning as behavioural tests. Other tests should be used to see if similar results are obtained.

Figure 2. Demonstrates the effect of diet on cytokine expression in juvenile and adult treatment groups. Interleukin- 1β and Tumor Necrosis Factor α are significantly increased within the hippocampus of the juvenile high fat diet group receiving LPS. In contrast, these cytokines did not change in any of the adult diet groups. Picture from: Boitard C. et al (2014) Impairment of hippocampal-dependent memory induced by juvenile high-fat diet intake is associated with enhanced hippocampal inflammation in rats. Brain, Behavior, and Immunity 40:9-17.

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Conclusions In conclusion, the data from Boitard et al. suggest that exposure to a high fat diet containing 24% lipids impairs spatial flexibility and long-term memory in juvenile rats but not adult rats. Additionally, a high fat diet leads to increases in pro-inflammatory cytokines, specifically interleukin-1β, within the hippocampus of juvenile rats. Although the exact mechanism on how elevated cytokines impair hippocampal dependent memories is poorly understood, adolescents are specifically vulnerable to the deleterious effects of a high fat diet. This study addresses an important issue since obesity is increasing dramatically among children and adolescents.

Criticisms and Future Directions

The results from this study indicate that the levels of interleukin-1β increases within the hippocampus of juvenile rats after they were exposed to a high fat diet.

In order to see if this cytokine is critical for impairing hippocampal dependent memories, this study should be repeated with injecting interleukin-1β antagonists into the group of rats consuming a high fat diet. The rats would then undergo a behavioural test such as the Morris water maze, and if preference for target annulus increases then interleukin-1β is impairing spatial memory. An additional experiment would be to expose both age groups to a diet containing higher percentage of lipids for a longer period of time. This would allow the authors to see if spatial memory of adult rats is altered depending on the severity of the diet. Moreover, less stressful behavioural tests can be used in order to eliminate the amygdala as a possible confounder. One example would be to use novel location recognition behavioural task since it is less stressful than the Morris water maze but still hippocampal dependent1. Future studies should conduct a similar experiment on female rats to see if a high fat diet has similar negative consequences. Also, future studies should test to see if it is possible to reverse the effects of high fat diet, either through diet or exercise, and see if spatial performance is improved. Future experiments that illustrate why obesity increases the levels of pro-inflammatory cytokines within the hippocampus and how they influence spatial memory, are needed to describe the link between obesity and cognitive dysfunction.

References 1. Acebes I. et al (2013) Spatial memory impairment and changes in hippocampal morphology are triggered by high-fat diets in adolescent mice. Is there a role of leptin? Neurobiology of Learning and Memory 106:18-25.

2. Arnold SE. et al (2014) High fat diet produces brain insulin resistance, synaptodendritic abnormalities and altered behavior in mice. Neurobiology of Disease 67:79-87.

3. Boitard C. et al (2014) Impairment of hippocampaldependent memory induced by juvenile high-fat diet intake is associated with enhanced hippocampal inflammation in rats.

Brain, Behavior, and Immunity 40:9-17.

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4. Cai D (2013) Neuroinflammation and neurodegeneration in overnutrition-induced diseases. Trends in Endocrinology and Metabolism 24:40- 47.

5. Chen J. et al (2008) Neuroinflammation and disruption in working memory in aged mice after acute stimulation of the peripheral innate immune system. Brain Behavior and Immunity 22:301-311.

6. Gil A. et al (2007) Altered signalling and gene expression associated with the immune system and the inflammatory response in obesity. British Journal of Nutrition 98: 121-126.

7. Heyward FD. et al (2012) Adult Mice Maintained on a High-Fat Diet Exhibit Object Location Memory Deficits and Reduced Hippocampal SIRT1 Gene Expression. Neurobiology Learning Memory 98: 25-32.

8. Liang J (2014) Neurocognitive correlates of obesity and obesity-related behaviors in children and adolescents. Nature 38:494-506.

9. Miller AA, Spencer SJ (2014) Obesity and neuroinflammation: A pathway to cognitive impairment. Brain, Behavioural, and Immunity 42:10-21.

10. Pistell P. et al (2010) Cognitive Impairment Following High Fat Diet Consumption is Associated with Brain Inflammation. J Neuroimmunol 219:25-32.

11. Prada PO, Areias M (2015) Mechanisms of insulin resistance in the amygdala: Influences on food intake. Behavioural Brain Research 282: 209-217.

12. Sainz N. et al (2015) Leptin resistance and diet-induced obesity: central and peripheral actions of leptin. Metabolism Clinical And Experimental 64:35-46.

13. Sobesky JL. et al (2014) High-fat diet consumption disrupts memory and primes elevations in hippocampal IL-1b, an effect that can be prevented with dietary reversal or IL-1 receptor antagonism. Diet, Inflammation and the Brain 42:2232.

14. Wang D. et al (2015) Cardiotrophin-1 (CT-1) Improves High Fat Diet-Induced Cognitive Deficits in Mice. Neurochem Res 40:843-853.

15. Winer AD. et al (2014) B Lymphocytes in obesity-related adipose tissue inflammation and insulin resistance. Cell. Mol. Life. Sci 71:1033-1043.

Caffeine prevents memory consolidation impairments associated with sleep deprivation Akua Obeng-Dei

Numerous of studies have been able to report strong correlations between sleep and the positive contribution it makes to memory consolidation. Studies have demonstrated that sleep deprivation negatively contributes to memory consolidation that impairs learning and memory. The induction of late phase long-term potentiation (L-LTP) is required for the consolidation of short-term memory to long-term memory through the strengthening of synapses. Caffeine has been shown to have cognitive protective properties in rat models of neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease preventing some of the cognitive decline that is associated with these aliments. Chronic caffeine treatments were given to the rats over the course of 4 weeks before the sleep deprivation period, rats in this treatment group committed fewer errors when compared to the control during in the RAWM task. Furthermore, Sleep deprived rats treated with caffeine showed L-LTP induction that had an amplitude and slope similar to that of the control. Additional, P-CREB levels increase within the control rats and those treated with caffeine, but the rats that were sleep deprived showed no increase in P-CREB levels.

When sleep deprived rats were treated with caffeine, the P-CREB levels were similar to levels exhibited in the control group. The significance of these present results illustrate a possible mechanism in how sleep deprivation may impair the process of consolidation and but provides evidence that caffeine is able to act as a protective against cognitive impairments associated with hippocampal-dependent spatial long-term memory.

Key words: sleep deprivation; caffeine; radial arm water maze (RAWM); late long-term potentiation (L-LTP); cAMP response element binding protein (CREB); brain-derived neurotrophic factor (BDNF) Background Previous studies have drawn strong correlations between sleep and the positive contribution it makes to memory consolidation. Two pivotal hypotheses that elaborate on the phenomenon of sleep and memory consolidation are synaptic homeostasis and the active system consolidation1. Synaptic homeostasis hypothesis proclaims that during REM sleep there is a synaptic downscaling, on the global scale, permitting consolidation to occur in the brain1. Active system consolidation hypothesis states that during REM sleep there is selectively re-activating of the memories allowing for memory consolidation to occur1.

Numerous of studies have demonstrated that sleep deprivation negatively contributes to memory consolidation that impairs learning and memory. Spatial learning tasks performed by human participants have shown that sleep after learning enhances performance while the opposite is associated with sleep deprivation, where consolidation of memories is hindered, thereby participants perform poorly2. Sleep deprivation leads to reduction AMPA receptor phosphorylation occurring at GluR1-S845 site and twelve hours after rats performed novel arm recognition task3. Sleep deprivation before learning impairs the ability for learning to occur and decreases the magnitude of L-LTP in the CA1 region of the hippocampus4, 5.

The induction of late phase long-term potentiation (L-LTP) is required for the consolidation of short –term memory to long-term memory through the strengthening of synapses. The mechanism by which L-LTP allows for the strengthen of synapses involves an electrical stimulation to the Schaffer collaterals in the CA1 region of the hippocampus, glutamate will be release from the presynaptic membrane and will bind to NMDA receptors on the

post-synaptic membrane. Subsequently, this lead to the influx of calcium into the post synaptic cell allowing for the activation of CaMKIV, which will go on to phosphorylate CREB leading to the activation of genes that is required for the induction of L-LTP6, 7. Normally, after the induction of L-LTP once learning has occurred there is an increase in the expression of CREB and BDNF in the hippocampus, in sleep deprived rats there is a decrease in the expression P-CREB and BDNF levels exhibited in hippocampus8.

Caffeine has been shown to have cognitive protective properties in rat models of neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease permitting the prevention of the cognitive decline that is associated with these aliments9, 10. The implications associated with the cognitive protective effect of caffeine on impairments of hippocampal-dependent spatial long-term memory due to sleep deprivations have not been fully studied. This review will highlight the findings of Alhaider et al paper and elaborate on concepts that could be further elucidated in future research.

Research Overview

Summary of Major Results

Chronic caffeine treatments prevents impairments to spatial long-term memory exhibited due to sleep deprivation

The impact of caffeine was investigated in sleep-deprived rats to see whether or not caffeine has a positive effect in the spatial long-term memory defects associated with sleep deprivation. The experimental behavioral approach that was implication was the radial arm water maze (RAWM), longterm memory is measured by comparing the number of errors done by the various treatment groups to the control.

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The rats that were sleep deprived committed more mistakes after the learning phase than the control groups. Chronic caffeine treatments were given to the rats over the course of 4 weeks before the sleep deprivation period, the rats in this treatment group committed fewer mistakes when compared to the control group. (Refer to figure 1)

Chronic caffeine treatments prevents the decrease of L-LTP magnitude in sleep deprived rat

Multiple high frequency stimulation (MHFS) was applied to the Schaffer collaterals in the CA3 region of the hippocampus to induce L-LTP, the slope of fEPSP and the amplitude of pSpike were measured. Sleep deprivation in the rats was still able to evoke L-LTP, but even though there was a significant increase in the amplitude of pSpike and the slope of fEPSP, the increase was smaller when compared to the control rats. Sleep deprived rats treated with caffeine showed a stronger overall induction of L-LTP that was similar to that of the control.

Levels of P-CREB, total CREB, and BDNF proteins after the induction of L-LTP

Five hours after the induction of L-LTP, the hippocampus was removed and the protein levels of P-CREB, total-CREB, and BDNF were measured using immunoblotting techniques. The P-CREB levels increase within the control rats and those treated with caffeine, but the rats that were sleep deprived showed no increase in P-CREB levels. When the sleep deprived rats were treated with caffeine the P-CREB levels were similar to levels exhibited in the control group. However, the total levels of CREB were increased in all of the experimental groups. BDNF levels in sleep-deprived rats exhibit no increase after the induction of L-LTP unlike the control or rats that were only treated with caffeine. (Refer to figure 2)

Conclusions and Discussion

Alhaider et al.11 were able to show that chronic treat-

ment of caffeine was able to prevent the impairments of hippocampal-dependent learning and memory associated with sleep deprivation in rats. The findings from the hippocampal-dependent spatial memory task, RAWM, suggest that sleep deprivation is the cause of long-term memory impairments this is marked by the increase number of errors compared to the control. Caffeine treatments have been shown to alleviate the impairments induced by sleep deprivation associated with memory consolidation. When normal rats that were not sleep deprived but were treated with caffeine, there was not further reduction in the errors made, suggesting that caffeine acts as a neuroprotective rather than an enhancer consolidation.

These results correlate with numerous of studies that have shown a positive correlation between impairments during hippocampal-dependent spatial memory tasks and sleep deprivation, in rats and well as humans2, 3, 4. Additional, there are studies that have shown that sleep deprivation has no correlation at all with impairment memory12; this could be due to the different experimental conditions such as the behavioral model or the method by which sleep deprivation was induced in the rats.

The findings from the electrophysiological induction of L-LTP suggest that the reduction in L-LTP coincide with the memory impairments seen during the RAWM task.

Caffeine will prevent the sleep deprivation association of L-LTP impairment in the CA1. Previous studies have demonstrated LTP impairments associated with sleep deprivation that was reported in both vivo and vitro models5.

The protein levels of P-CREB in sleep-deprived rats suggest that the reduction in the phosphorylation of CREB could be a possible mechanism to explain the impairments associated with sleep deprivation. Furthermore, the decrease in BDNF in sleep-deprived rats also seems to contribute to the impairments associated with memory consolidation. Caffeine treatments seem to prevent the decline that is seen in the sleep deprived rats pertaining to the levels of P-CREB and BDNF protein expression, suggesting that caffeine acts prevents the negative impairments of sleep deprivation.

Figure 1. The impact of caffeine on sleep deprivation mediated spatial long-term memory impairments was investigated using the radical arm water maze (RAWM). All of the groups underwent a learning phase and then there was a 24-hour break where some of the groups were sleep deprived. Then the researcher measured the number of errors made by all of the rat groups when trying to find the hidden platform and compared it to the control. Rats that were sleep deprived made more mistakes after the learning phase compared to the control rats and S-caffeine/sleep deprivation created similar number of error to the control rats. Source: Alhaider et al (2011) 11 67

Figure 2. They investigated the amounts of the signaling molecules P-CREB within hippocampal extracts using immunoblot assay. Five hours after the induction of L-LTP, there was a significant increase in phosphorylated CREB in S-control, S-caffeine, and S-caffeine/sleep deprivation. Source: Alhaider et al (2011) 11

Conclusions Through the use of three different experimental approaches, the researcher were able to conclude that without the increase in the phosphorylation of CREB and BDNF is important for L-LTP to occur therefore impaired learning and memory. The reduction in the proteins essential for L-LTP illustrates a possible mechanism to the spatial long-term memory impairments associated with sleep deprivation in the rats. Additional, caffeine was shown to prevent the cognitive impairment permitting a stronger induction of L-LTP in sleep-deprived rats thereby allowing the levels of P-CREB and BDNF to match levels in the control mice. The significance of the results presented is that not only does it provide for a mechanism into how sleep deprivation may impair the process consolidation but also illustrates that caffeine has protective properties against these cognitive process associated with hippocampal-dependent spatial memory.

Criticisms and Future Directions Alhaider et al. failed to provide a reasonable explanation for the results that showed that caffeine treatments only partially improved the impairments to long-term memory induced by sleep deprivation while L-LTP impairments fully recovered in the sleepdeprived rats. Microdialysis-HPLC assay can be conducted to measure the levels of Glutamate, GABA and Histidine, which are associated with wakefulness in the hippocampus of sleep-deprived rats before and after caffeine treatments13. In previous studies it has been shown that caffeine is able to induce the release of glutamate in the nucleus accumbens14, providing rational that this can be also occurring in the hippocampus. It has been proposed that caffeine act through the adenosine receptor A2 to increase glutamate release in the glutamatergic neurons, which will then allow NMDA receptor activation for LTP to occur13, explaining the inconsistent results. Furthermore, why the caffeine treatments in the control rats

that were not sleep deprived did not exhibit further improvements to learning and memory was also not accurately discussed. To address the results that showed no further increase in learning and memory in the control group of rats, conduct EEG while the control rats are either injected with caffeine or saline as they conduce various neurocognitive tasks allowing for real-time information that can be record as learning and memory occurs15. Previously is has been shown that caffeine is able to improve cognitive function15, suggesting that in order for the control rats to show an improvement in cognitive function the researchers will need to administrate a higher dose of caffeine. In order to test for whether the behavioral task, RAWM, is creating stress within the rats leading to misleading results need to physically measure for corticosterone levels before and after the RAWM task to rule out stress has a factor3. The two different experiments outlined will be able to give a broader picture and new understanding on the mechanism by which caffeine is able to act as a neuroprotective to improve hippocampal-dependent spatial learning and memory in sleep deprived rats.

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2. Ferrara, M. et al. Sleep to find your way: the role of sleep in the consolidation of memory for navigation in humans.

Hippocampus. 18, 844-851 (2008).

3. Hagewoud, R., Havekes, R., Novati, A., Keijser, J.N., Van Der Zee E.D., & Meerlo, P. Sleep deprivation impairs spatial working memory and reduces hippocampal AMPA receptor phosphorylation. J. Sleep Res. 19, 280-288 (2010).

4. Li, S., Tian, Y., Ding, Y., Jin, X., Yan, C., & Shen, X. The effects of rapid eye movement sleep deprivation and recovery on spatial reference memory of young rats. Learning & Behaviour. 37, 246-253. (2009).

5. Kim, EY., Mahmoud, GS., & Grover, LM. REM sleep deprivation inhibits LTP in vivo in area CA1 of rat hippocampus.

Neuroscience Letters. 388, 163-167 (2005).

6. Bito, H., Deisseroth, K., & Tsien, RW. CREB phosphorylation and dephosphorylation: a Ca2+ - and stimulus duration-dependent switch for hippocampal gene expression.

Cell. 87, 1203-1214 (1996).

7. Tokuda et al. Involvement of calmodulin-dependent protein kinase-I and –IV in long-term potentiation. Brain Research.

755, 162-166 (1997).

8. Guzman-Marin, R et al. Suppression of hippocampal plasticity-related gene expression by sleep deprivation in rats.

J. Physiol. 573, 807-819 (2006).

9. Gevaerd, M.S., Takahashi, R.N., Silveira, R., & Da Cunha, C. Caffeine reverses the memory disruption induced by intra-nigral MPTP-injection in rats. Brain Research Bulletin.

55, 101-106 (2001).

10. Arendash, GW et al. Caffeine protects alzheimer’s mice against cognitive impairment and reduces brain β-amyloid production. Neuroscience. 142, 941-952 (2006).

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11. Alhaider, I. A., Aleisa A.M., Tran T.T., & Alkadhi K. A. Molecular and Cellular Neuroscience. 46, 742-751 (2011).

12. Samkoff, J.S., Jacques, C.H., A review of studies concerning effects of sleep deprivation and fatigue on residents’ performance. Acad. Med. 66, 687-693 (1991).

13. ohn, W., Kodama, T,. & Siegel, JM,. Caffeine promotes glutamate and histamine release in the posterior hypothalamus. Am J Physiol Regul Integr Comp Physiol. 307, 704-710 (2014).

14. Solinas, M., Ferré, S,. You, ZB,. Karcz-Kubicha, M,.

Popoli, P,. & Goldberg, SR. Caffeine induces dopamine and glutamate release in the shell of the nucleus accumbens. J Neurosci. 22, 6321-6324 (2002).

15. Bruce, SE., Werner, KB., Preston, FP., & Baker, LM. Improvements in concentration, working memory and sustained attention following consumption of a natural citicoline-caffeine beverage. Int J Food Sci Nutr. 8, 1003-1007 (2014).

Received Month, ##, ##, 200#; accepted

200#; Month,

revised ##,

Month, 2013.

This work was supported by The Association for the Development of Undergraduate Neuroscience Education (SRA & RLN), The Endowment for Science Education (EA), and The Synaptic State Faculty Research Foundation (EA). The authors thank Mr. Spine L. Cord, Dr. Amy G. Dala, and the students in Neuroscience 101 for technical assistance, execution, and feedback on this lab exercise.

Address correspondence to: Dr. Rita L. Neurotrophin, Biology Department, 123 Growth Cone Avenue, Action Potential College, Hillock, IL 60101 Email: rln@apc.edu Copyright © 2015 Dr. Bill JU, Neurosciences, Human Biology Program

69

Conflicting or Corroborating Evidence? Interleukin-6 and the JAK-STAT Signaling Pathway in Neural Precursor Self-Renewal Daniel Derkach

Maternal cytokine surges have been implicated in long-lasting neurological consequences in progeny, such as autism spectrum disorder and schizophrenia-like behavior. However, the precise mechanism(s) underlying these effects are yet to be fully understood. This review analyzes and evaluates the findings of Gallagher et al., which support the notion that a maternal IL-6 surge, which can be caused by maternal infections or distresses, deregulates the proliferation and self-renewal of neural precursor pools in the mammalian embryonic forebrain.

The authors report that a single maternal IL-6 injection may result in increased adult forebrain precursor proliferation, increased precursor self-renewal, and sustained precursor pluripotency long into adulthood. These findings contribute to the current archetype of neural precursor regulation by suggesting and investigating a novel signaling pathway, which has yet to be thoroughly clarified. However, these findings are only an incremental advance in this field of research, where several questions remain to be answered. In addition, similar studies exhibit findings that may appear to be conflicting with the article of focus, but further research is necessary to resolve any apparent discrepancies.

Key words: neural precursor cells (NPCs); neural stem cells (NSCs); cytokines; interleukin-6 (IL-6); JAK-STAT; neurogenesis; cognitive disorders; schizophrenia Background Abnormalities associated with stem cells can result in long-lasting impairment of tissue maintenance and repair, as well as functionality (Simons & Clevers, 2011).

However, the mechanisms underlying the regulation of adult stem cell pool growth and composition are still not understood. Investigations seeking to uncover these mechanisms are significant because it has been shown that human cognitive function and adult neurogenesis are directly affected by variations in adult neural precursor cells (NPCs) (Ming & Song, 2011). In order to link NPC variation with cognitive function, Gallagher et al. (2013) investigate a maternal cytokine surge, which may occur during prenatal perturbations such as maternal infection or stress exposure. In humans, this cytokine surge is implemented in long-term cognitive outcomes such as schizophrenia and autism spectrum disorder (ASD) (Patterson, 2007, 2011).

Cytokines such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α chemically mediate inflammation, and may cross the blood-brain barrier, thereby facilitating maternal to fetal transmission, but the mechanisms underlying their effects on fetal brain development are unclear (Boksa, 2008). Gallagher et al. focus on a specific cytokine, IL-6, which is thoroughly studied in relation to NPC pool size, composition, and self-renewal.

Some regulatory factors of embryonic NPCs have previously been identified, such as epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2), but IL-6 is a relatively new candidate. It is currently understood that IL-6 is associated with stem cell proliferation in muscles (Muñoz-Cánoves, 2013) and the spinal cord (Kang, 2008), but research involving its role in the mammalian brain is limited. The current investigation by Gallagher et al. provides an incremental advance for cytokine and NPC research because it expands upon the pre-established functions of IL-6, specifically examining its association with NPC regulation in the mammalian forebrain, whereas it has

conventionally been assumed to be an immune regulator.

It is currently understood that IL-6 activates the Janus kinase-signal transducer and activator of transcription (JAKSTAT) pathway, but requires the formation and activation of a glycoprotein 130 (gp130) and IL-6 receptor (IL6R) complex. IL-6 binds its receptor (IL6R), which forms a complex with gp130, thereby activating JAK. In turn, JAK phosphorylates tyrosine residues on the cytoplasmic aspect gp130, which act as docking sites for STAT. JAK subsequently phosphorylates STAT, which forms homodimers before localizing to the nucleus where STAT acts as a transcriptional activator (Dittrich et al., 2012).

As previously identified by Barnabé-Heider et al. (2005), gp130 expression is exhibited by NPCs in the embryonic forebrain. These factors have the potential to contribute to more expansive research involving the downstream events resulting from JAK-STAT signal activation and deregulation.

Research Overview

Summary of Major Results

To investigate the potential effects of IL-6 on NPC proliferation, Gallagher et al. initially administered a single intraperitoneal injection of IL-6 into pregnant mice on gestational day 13.5 (G13.5) and subsequently immunostained and analyzed the subventricular zone (SVZ) of 2-month-old adult mice. Compared to wild type, maternally exposed IL-6 embryos showed 2-fold more proliferating BrdU-positive SVZ cells and significantly increased levels of Sox2-positive precursor cells. Additionally, an almost 1.5-fold increase was seen in neurosphere-initiation SVZ cells compared to wild type.

As elucidated by previous research, NPCs in the adult SVZ originate from the dorsal cortex and ventral ganglionic eminence (GE) (Ventura & Goldman, 2007; Young et al., 2007). To determine if cortical contribution of NPCs to the SVZ is modified by maternal IL-6 upregulation, Gallagher 70

et al. used an electroporation experiment to induce green fluorescence protein (GFP) expression for lineage tracing.

Compared to wild type, a maternal IL-6 surge resulted in a greater contribution from the dorsal cortex, which led to a 2-fold increase in GFP-expressing NPCs in the adult SVZ. Concurrently, additional in vivo analysis showed that maternal IL-6 injection resulted in a 2-fold increase in neurosphere generation at clonal density in the embryonic cortex and ventral GE. Since it was previously demonstrated that gp130 receptors are expressed by NPCs in the embryonic cortex, the authors continued their investigation by examining if IL6Rs are also expressed by these NPCs. By isolating Sox2-positive cortical NPCs in embryos, RT-PCR and immunostaining revealed the presence of IL6R mRNA at embryonic day 13 (E13) in the cortex and SVZ. Since both IL6R and gp130 were found to be expressed by cortical NPCs, the authors investigated if IL-6 injection led to activation of the IL6R/gp130-mediated JAK-STAT pathway. Western blot analyses revealed almost 2-fold increases in phosphorylated STAT3 levels in embryonic cortices at E12.5, both in vivo and in vitro.

The authors then went on to see if this downstream signaling promoted proliferation of cortical precursors. In vitro cultural analyses following immunostaining for BrdU, Ki67, phospho-histone H3, and Pax6 each demonstrated increased percentages of proliferation in radial precursors treated with IL-6 compared to wild type.

After showing that IL-6 mediates proliferation of NPCs, the authors explored the possibility of IL-6-mediated enhancement of forebrain NPC self-renewal. Via clonal analysis, it was demonstrated that IL-6-treated cortical precursors formed clones with significantly greater multicellularity, as well as more diverse clonal bodies. Compared to wild type, IL-6 treatment resulted in a slight decrease in clones containing neurons only, and nearly doubled the amount of mixed clones containing a mixture of BIII-tubulin-positive neurons, glial fibrillary acidic protein (GFAP)-expressing glia, and undifferentiated neural precursors.

Cytokine antibody dot blot experiments revealed that IL-6 was also synthesized and secreted by cortical precursors in a precursor-conditioned medium, whereas other cytokines, such as IL-4/5/9/10 were not. Moreover, immunostaining and cultural analysis of IL-6-/- and IL-6+/+ cortical precursors revealed similar rates of cell death, but significantly decreased proliferation of IL-6-/- precursors.

Finally, immunostaining and siRNA-mediated knock-

down of IL6R resulted in significantly decreased levels of Sox2-positive precursors and increased levels of Satb2-positive neurons. Likewise, immunostaining revealed parallel results in IL-6-/- embryos relative to wild type embryos (Fig. 1).

Discussions and Conclusions

Although the evidence provided by Gallagher et al. strongly support their hypothesis, claiming that a maternal IL-6 surge upregulates NPC proliferation and self-renewal, several questions remain unanswered and controversy is still present among research in this area.

Multiple studies report that gestational perturbations have long-lasting effects on adult hippocampal neurogenesis and cell proliferation, but such effects are also shown to be independent of maternal IL-6 (Coe et al., 2003; Uban et al., 2010). These reports suggest that hippocampal and SVZ precursor populations are inherently distinct. Similarly, Li et al. (2013) report that hippocampal and SVZ precursor populations derive from discrete populations of embryonic progenitors.

A study by Bowen et al. (2010) supports the findings of Gallagher et al., showing that a dearth of IL-6 results in decreased proliferation of adult SVZ precursors. However, the findings of Bowen et al. contradict those of Coe et al., Uban et al., and Li et al., who all report differences between hippocampal and SVZ precursor cell populations. On the other hand, Bowen et al. report that IL-6-/- mice exhibited significantly decreased NPC survival in both the dentate gyrus and the SVZ, relative to wild type mice. This suggests similarities between hippocampal and SVZ progenitor populations, which conflicts with several other studies.

The findings of Gallagher et al. also raise the question of how JAK-STAT signaling maintains NPC pluripotency.

Although the authors show an increase in phosphorylated STAT3 in the E12.5 progeny of maternally exposed IL-6 mice in vivo and in NPCs directly exposed to IL-6 in vitro, no direct association is illustrated between STAT3 and sustainment of NPC pluripotency. Additionally, Gallagher et al. face an array of opposition concerning their results that show IL-6-mediated downstream signaling to preserve pluripotency and defer differentiation. Several studies support the claim that IL-6-mediated downstream JAK-STAT signaling promotes astrocytic differentiation.

Using Western blot and immunocytochemical analysis, Gu et al. (2005) show that STAT3 suppression directly

Figure 1 (Gallagher et al., 2013). BrdU (red) proliferation marker and Sox2 (green; left) precursor marker or Satb2 (green; right) neuron marker.

Left: Analysis of proportion of precursors in IL-6 knockout mice relative to wild type (control).

Right: Analysis of proportion of differentiated neurons in IL-6 knockout mice relative to wild type (control).

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inhibited astrogliogenesis and induced neurogenesis.

Further-more, Nakanishi et al. (2007) report that microglia-derived IL-6 did not increase NPC proliferation. Instead, it was shown that microglia-derived IL-6 activated the JAK-STAT pathway, leading to astrocytic differentiation of E16 NPCs. This was affirmed by inhibiting the JAK-STAT pathway, leading to reduced astrocytic differentiation.

However, a more thorough analysis creates the possibility that both views may be valid, but at different stages of embryogenesis. Fan et al. (2005) perform an experiment involving a DNA methyl-transferase 1 deletion (Dnmt1-/-), resulting in accelerated demethylation of the GFAP promoter and subsequent JAK-STATmediated astrogliogenesis. Interestingly, this concept is expanded upon by Takizawa et al. (2001), reporting that the STAT3 binding element in the wild type GFAP promoter is methylated at E11.5, but demethylated at E14.5 (see Fig. 2). Since Nakanishi et al. examined the JAK-STAT pathway and astrogliogenesis at E16, it is likely that the STAT3 binding element in the GFAP promoter was demethylated, thereby allowing GFAP transcription and astrocytic differentiation. On the other hand, Gallagher et al. examined the JAK-STAT signaling pathway at E12.5, when the STAT3 binding element in the GFAP promoter was likely still methylated.

Even if these are valid conditions and JAK-STAT signaling results in astrocytic differentiation only after E14.5, but not before, the question remains: How does JAK-STAT signaling maintain NPC pluripotency prior to E14.5? If JAK-STAT signaling prior to E14.5 causes sustainment of NPC pluripotency, it then also raises the question of whether or not STAT3 binds targets other than the GFAP promoter.

Boksa (2008), in conjunction with Khan & Brown (2002), give reason to hypothesize that it may not necessarily be the IL-6-mediated JAK-STAT signaling pathway that is responsible for behavioral disorders in the progeny of infected mothers, but may potentially be caused by fever-induced apoptosis of neocortical cells, instigated by a maternal IL-6 surge. Khan & Brown affirm this by demonstrating elevated activation of caspase 3 in E17 cortical cells 10 hours after a heat shock.

Figure 2 (Taga & Fukuda, 2005). Mechanism of IL-6-mediated JAK-STAT signaling and GFAP promoter activation in neural precursors at E11.5 (methylated) versus E14.5 (demethylated).

Conclusions The experimental findings of Gallagher et al. provide strong evidence in support of their hypothesis, claiming that a maternal IL-6 surge, which may be caused by maternal infection in mammals, has long-lasting implications on NPC pools in progeny.

Based on the authorsâ&#x20AC;&#x2122; findings of cytokine antibody dot blot experiments, it can be concluded that IL-6 is endogenously synthesized and secreted by embryonic forebrain NPCs in wild type mice. Moreover, RT-PCR and immunostaining support the hypothesis that embryonic forebrain NPCs also express IL6Rs, suggesting that IL-6 signaling functions in an autocrine/paracrine manner.

Mechanistically, as shown in Western blots depicting upregulated STAT3 levels by Gallagher et al. and demonstrated in several other reports, it can be concluded that IL-6 has downstream effects in the mammalian forebrain via JAK-STAT-mediated signaling. However it cannot be firmly concluded that JAK-STAT signaling directly and specifically promotes either sustained pluripotency of NPCs or astrocytic differentiation in the embryonic forebrain of mice. It may, however, be a possibility that astrocytic/glial differentiation is observed due to the specific inhibition of neuronal differentiation, or may be due to a different mechanism altogether.

Although not explicitly investigated by Gallagher et al., but relevant to the topic of embryonic NPC variation nonetheless, discrepancies between studies comparing the nature of hippocampal and SVZ precursor populations give reason to take caution when deducing a conclusion, and supplementary experimentation is necessary. Finally, although one may naturally assume that a deficiency in NPCs would result in cognitive deficits, Gallagher et al. demonstrate that IL-6 may, in fact, play a role in prompting harmful functional consequences by promoting the over-accumulation of neural precursors.

Criticisms and Future Directions

As research in neural stem cells has widespread potential in modern biomedical applications, there is a plethora of additional research to be carried out, especially concerning proliferation and self-renewal of NPCs. Although Gallagher et al. perform a broad range of experiments to identify the IL-6-medited effects on embryonic NPCs, there are several questions left unanswered.

The authors determine that IL6R is expressed by NPCs, allowing autocrine/paracrine IL-6 signaling. However, it is not elucidated as to which type of IL6R is predominantly incorporated in the activation of the JAK-STAT signaling pathway. Since classic membrane-bound IL6R signaling and soluble IL6R (sIL6R) trans-signaling have antagonistic inflammatory effects, it would be beneficial to further investigate the specificity of IL-6 signal transduction in the mammalian embryonic forebrain. It is understood that classic signaling has anti-inflammatory effects, transsignaling has pro-inflammatory effects, and that sIL6R in the trans-signaling pathway can activate the membranebound gp130 on any cell, unlike membrane-bound IL6R, which can only activate gp130 in an autocrine manner (Rabe et al., 2008). In order to further investigate this concept, experiments incorporating inhibition of sIL6R can be performed by transgenically overexpressing soluble 72

gp130 (sgp130), which exclusively inhibits sIL6R, allowing predominant anti-inflammatory membranebound gp130 signaling. Coupling such an experiment with immuno-cytochemical analysis of NPC proliferation and pluripotency would contribute to the clarification of the mechanism responsible for IL-6 mediated regulation of NPCs. This research could also help to elucidate the anti-inflammatory effects of NPC deregulation in respect to previously reported IL-6-associated behavioral disorders (Smith et al., 2007).

Additional clarification of STAT3 targeting is also needed to resolve the controversial observations reported by various authors (Gu et al., 2005; Nakanishi et al., 2007). By evaluating STAT3-mediated GFAP expression in embryonic neural precursor pools, future experiments should be performed to investigate differences in two related aspects.

Firstly, phosphorylated STAT3 levels following IL-6 administration should be quantified at varying times or stages in embryonic development, both before E11.5, after E14.5, and in between. Secondly, such quantifications should be made in a spatially comparative manner, for example, the hippocampus versus the SVZ precursor pools. In addition to these analyses, ChIP experiments could potentially be utilized to determine if promoters or regulator regions in genes other than the GFAP gene are implemented in IL-6mediated JAK-STAT signaling in the embryonic forebrain.

Finally, Western blot analyses should be implemented in order to discern whether other (non-STAT3) mammalian STAT family members are activated by JAK during a maternal IL-6 surge. Coupling these aforementioned experiments with immunocytochemical analyses of NPC proliferation and pluripotency may result in valuable findings in the growing field of neural stem cell research.

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22. Uban, K.A., Sliwowska, J.H., Lieblich, S., Ellis, L.A., Yu, W.K., Weinberg, J., & Galea, L.A.M. (2010). Prenatal alcohol exposure reduces the proportion of newly produced neurons and glia in the dentate gyrus of the hippocampus in female rats. Hormones and Behavior. 58, 835-843.

23. Ventura, R.E., & Goldman, J.E. (2007). Dorsal radial glia generate olfactory bulb interneurons in the postnatal murine brain. The Journal of Neuroscience. 27, 4297-4302.

24. Young, K.M., Fogarty, M., Kessaris, N., & Richardson, W.D. (2007). Subventricular zone stem cells are heterogeneous with respect to their embryonic origins and neurogenic fates in the adult olfactory bulb. The Journal of Neuroscience. 27, 8286-8296.

Rachel Duncan

Trans-Cranial Direct Stimulation: A device for out of the box thinking

Newell and Simon proposed that to solve a problem, steps need to be taken from the current state to the goal state within relevant constrains such as time (Newell and Simon, 1972). However, what makes insight problems particularly challenging is that unlike well defined problems which rely on past experience and relevant knowledge, insight problems are often ill-defined (DyYoung, Flanders, Peterson, 2008).

Previous knowledge, expectation and goals may bias how entering information is processed through “top down” cognitive processing which may lead to impasses and cognitive rigidity when solving insight problems (Corbetta and Shulman, 2002). In noninsight problems, the operations required to get to the goal state as well as how the current/goal states themselves are perceived is unpredictable (DyYoung, Flanders and Peterson, 2008). They often require the subject to reconstruct their perspective on the situation including inappropriate constrains in order to overcome the impasse. Reliance on a routine frame to solve a problem has also been called the “mental-set effect”, a phenomena associated with left hemisphere dominance (Chi and Snyder, 2011). While top down cognitive processes involve a flow of information from higher to lower cortical areas, bottom up cognitive processes have minimal influence from higher order cognition on how sensory stimuli are salient, allowing novel and unexpected sensory information to direct the content of attention (Corbetta and Shulman, 2002).

Learned or habitual thought patterns fuel top down processes can cause blindness to obvious solutions.

One area of research that has been getting attention because of it’s effects on the quality of attention is mindfulness meditation. Mindfulness practices cultivate an orientation to experience through “fresh eyes” as though an individual is experiencing the stimuli for the first time (Bishop et al, 2004). It practices an objective, empirically based orientation rather than one clouded by expectation, emotion and subjective filter (Bishop et al, 2004). Researchers have been studying the relationship between mindfulness practice and mental set effects- a reliance on previous knowledge or experience. Greenberg, Reiner and Meiran (2012) found that compared to unexperienced meditators, experienced meditators were better able to identify simple or novel solutions to the Einstellung water task and were less blinded by rigid, experienced based problem solving strategies. Other researchers have found support for trait mindfulness and state induced mindfulness on increased insightful problem solving ability and creating thinking even when controlling for positive affect on the prisoner’s rope, antique coin and steel pyramid insight tasks (Ostafinand Kassman, 2012). Moore and Malinowski (2009) found that measures of attention and cognitive flexibility were higher for experienced meditators who showed less Stroop interference on the Stroop

task; a measure of the participant’s ability to detect novelty among visual stimuli. In these studies, heuristically or top down driven processes of thinking had less influence, as the mindfulness groups were better at overcoming habitual problem solving strategies in order to solve the novel insight tasks.

Chi and Snyder (2011) investigated the effects of temporarily reducing cognitive rigidity using noninvasive trans-cranial direct current stimulation (tDCS). Their hypothesized was drawn from research of “paradoxical facilitation”; a cognitive style that is less influenced by prior knowledge and experience in individuals with left temporal lobe impairments.

Interestingly, these individuals can have an enhanced ability for solving insight problems and creating thinking. Cognitive processes facilitated by creativity and novel meaning making draw from a similar vein as the mindfulness research. Experienced meditators may be less prone to mental-set effects having practiced a fresh eyed awareness that relies less on habitual patterns of thinking than their unexperienced counterparts. However, unlike the mindfulness manipulation, Chi and Snyder used tDCS on to influence an insight prone cognitive style by stimulating the right and left anterior temporal lobe areas of healthy right handed, left hemisphere dominant individuals. The researchers amplified and muted the left and right anterior temporal lobes (ATL) to reduce heuristically driven mental set effects on a novel insight problem: matchstick arithmetic. Likewise, other studies have tried to reversed mental set effects by stimulating the dorsolateral prefrontal gyrus and left angular gyrus using tDCS and included neurophysiological imaging (Dandan et al. 2013).

Image 1: Example of three types of problems and their accompanied solutions atypical of matchstick arithmetic. To overcome the mental impasse in the first two problems, participant must turn a 3D display into a 2D display. (Figure source: Chi and Snyder, 2011)

Methods Sixty undergraduate participants without prior experience with the matchstick arithmetic paradigm were recruited ands randomly assigned to one of three groups: L- R+, L+ R-, or control. All three groups were asked to solve 27 type 1 problems (see figure 1) to induce a mental 74

set effect whereby each of the 27 questions required the same ‘X’ to ‘V’ maneuver to reach the solution, during the mental set phase participants who did not reach a solution after two minutes per problem were given the solution. Following the mental set phase, participants were hooked up to one of three tDCS manipulations.

Participants in the left cathodal/right anodal ATL (L-R+) group and L+ R- group experienced two minutes of active 1.6mA stimulation for the duration of the test.

Meanwhile, participants in the sham group were hooked up to a device with an ‘on’ display setting at minimal stimulation, to imply active simulation at minimum sensation of 1.6 mA for 30s followed by 5 minutes of waiting for the minimal effect to wear off. Following the mental set phase, groups were asked to solve one of each type 2 and type 3 problem with 6 minutes per puzzle. The researchers predicted that reliance on the first strategy ‘X’ to’ V’ to would hinder their performance when solving type 2 and 3 problems which require a ‘+’ to ‘=’ sign change. Participants from the three groups were measured based on time to ‘event’; number of seconds until they reached the solution. Statistical analysis was performed using a two tailed Fisher’s exact test for performance between stimulation groups and a logrank test to compare time to event.

Results Neither age, gender, nor time required to complete the mental set phase was a predictor for solving the type two problem. The majority of participants (95%) were able to solve the 27 type 1 problems in the mental set phase. However, at the end of the 6 minutes given for the type 2 problem, 60% of participants in the L- R+ stimulation group reached the correct solution compared to only 20% of participants in the L+ Rand sham groups. This result supported the author’s hypothesis. These findings were not observed in the type 3 problem, as both L+ R- and L- R+ stimulation groups showed smaller differences between group but both outperform participants in the sham group by 40%. However, this doesn’t disprove the author’s hypothesis because they were expecting to see improvement for the L- R+ for type 2 problems only, a finding in line with previous research on individuals with brain lesions ability to solve type 2 but not type 3 problems.

Discussion Although the results of this investigation were in line with the author’s hypothesis, the mechanism by which tDCS influenced participants cognition and behavior is unclear. They suggested but were not able to prove that the enhanced performance of L- R+ group may have been facilitated by increased excitability to the right ATL rather than decreased excitability in the left ATL. Chi and Snyder (2011) suggested that a single or combination of the following mechanisms could have been at work behind their results: reduced topdown processing (paradoxical facilitation), facilitated insight (increased right hemispheric firing), diminished mental set effect (decreased left hemispheric firing), and switching hypotheses. They also considered the 75

Figure 2 (above left) shows group comparisons for solving the Type 2 insight problem while Figure 3 (above right) indicated groups comparisons for solving the Type 3 insight problem. Figure source: (Chi and Snyder, 2011).

possibility of a ceiling effect whereby tDCS wouldn’t be able to induce further mental set effects. This paper particularity has been met with controversy in the media; some suggest that the limitations of replicability and the possibility of confounding factors may outweigh it’s significance. Considering target area of electrodes ranged 35cm^2, the researchers could included neurophysiological imaging techniques like an FMRI to determine if the tDCS stimulation was influencing the right/left ATL independently of other cortical areas or if it’s activation/inhibition influence had spread (Dandan et al, 2013). It is worthwhile to be aware that other studies have shown that as little as 5% of drift in electrode position may have significant influence on current intensity for particular cortical regions (Wood, et al. 2014). Also, they could have also tested for state-dependent contextual information (including arousal levels, affect valence, amount of sleep) anything that could have account for behavioral or cognitive priming that may inflate the effects of tDCS (Smith, Vartanian and Goel, 2014; Horvath, Forte and Carter, 2015) Limitations of this study include the demographics of the sample. Only twenty participants were included in each strimulation group and the average age was 22. However, taken

with a grain of salt, the article presents a small cue of the use of tDCS which in itself is a safe, non-invasive, non-pharmacological procedure.

Future Directions It would be interesting for future studies to explore an older age group. Considering the brains of young people may be working at optimal plasticity levels, perhaps even greater effects would be seen in older adults (65+) who may have life long experience with habitual heuristic processing and declines in working memory and problem solving abilities. Additionally, they could recruit left-hand dominant participants to see if (L-R+) stimulation helped them reach the solution more efficiently than in the sham control group, or if any mental set effects would occur in (L+R-) stimulation. Left hand dominant participants may be less susceptible to mental set effects. Although most studies shy away from left hand dominant participants it would be interesting to include their results to the data set.

As well, they could have included other insight problems like the nine dot problem to further investigate inter hemispheric rivalry in tDCS (Chi and Snyder, 2012). However, the nine dot problem and matchstick arithmetic are not a typical problems of everyday life.

To make their experiment more relevant to everyday life, they could present participants with cognitive tasks that are more realistic and require breakdown of existing heuristics such as the prisoner’s rope, the antique coin, steel pyramid or stroop tast in conjunction with tDCS and FMRI imaging (Dandan et al, 2013; Ostafin and Kassman, 2012; Moore and Malinowski 2009).

tions from single-session transcranial direct current stimulation (tDCS). Brain Stimulation, doi:10.1016/j.brs.2015.01.400 8. Moore, A., & Malinowski, P. (2009). Meditation, mindfulness and cognitive flexibility. Consciousness and Cognition, 18(1), 176-186. doi:10.1016/j.concog.2008.12.008 9. Nelson, T. O., Kershaw, T. C., & Ohlsson, S. (2004).

Multiple causes of difficulty in insight: The case of the nine-dot problem. Journal of Experimental Psychology: Learning, Memory, and Cognition, 30(1), 3-13. doi:10.1037/0278-7393.30.1.3 10. Newell, A., & Simon, H. A. (1972). Human problem solving.

Englewood Cliffs, NJ: Prentice-Hall.

11. Ostafin, B. D., & Kassman, K. T. (2012). Stepping out of history: Mindfulness improves insight problem solving. Consciousness and Cognition, 21(2), 1031-1036. doi:10.1016/j. concog.2012.02.014 12. Smith, K. W., Vartanian, O., & Goel, V. (2014). Dissociable Neural Systems Underwrite Logical Reasoning in the Context of Induced Emotions with Positive and Negative Valence.

Frontiers in Human Neuroscience, 8, 736. doi:10.3389/ fnhum.2014.00736 13. Woods, A., Bryant, V., Sacchetti, D., Gervits, F., & Hamilton, R. (2014). Effects of electrode drift in transcranial direct current stimulation. Brain Stimulation, 8(2), 320-321.

References 1. Bishop, S. R. (2004). Mindfulness: A proposed operational definition. Clinical Psychology: Science and Practice, 11(3), 230-241. doi:10.1093/clipsy/bph077 2. Corbetta, M., & Shulman, G. L. (2002). Control of goaldirected and stimulus-driven attention in the brain. Nature Reviews Neuroscience,3, 201-215.

3. Chi RP, Snyder AW (2011) Facilitate Insight by Non-Invasive Brain Stimulation. PLoS ONE 6(2):e16655 doi:10.1371/ journal.pone.0016655 4. Dandan, T., Haixue, Z., Wenfu, L., Wenjing, Y., Jiang, Q., and Qinglin, Z. (2013). Brain activity in using heuristic prototype to solve insightful problems. Behav. Brain Res. 253, 139–144. doi:10.1016/j.bbr.2013.07.017 5. DeYoung, C. G., Flanders, J. L., & Peterson, J. B. (2008).

Cognitive abilities involved in insight problem solving: An individual differences model. Creativity Research Journal, 20(3), 278-290. doi:10.1080/10400410802278719 6. Greenberg, J., Reiner, K., & Meiran, N. (2012). “Mind the trap”: Mindfulness practice reduces cognitive rigidity. PLoS One, 7(5) doi:http://dx.doi.org/10.1371/journal.pone.0036206 7. Horvath, J. C., Forte, J. D., & Carter, O. (2015). Quantitative review finds no evidence of cognitive effects in healthy popula-

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Hippocampal Neurogenesis, Forgetting and the Effects of Exercise, Aging, and Stress on Memory Saadia Esat

The following review discusses the role of neurogenesis in forgetting, and the substantial correlation that appears to exist. Additionally, exercise has been shown to increase the survival of newborn neurons and therefore improve neurogenesis, speeding up the forgetting process. Aging, however, has the opposite effect as it slows neurogenesis and allows the existing memories to be recalled. Looking at stress, it can have positive and negative effects in relation to memory depending on the extent and for what treatment it is being used. Studying the correlation between neurogenesis and forgetting is helpful in understanding how to maximize learning and memory in terms of hippocampal-dependent memories, as well as improving the mental health of those who are suffering from chronic stress.

Key words: hippocampus; neurogenesis; forgetting; fear conditioning; exercise; memory Background The hippocampus has been vastly studied as it is essential to the formation and retrieval of contextual memories, a process important for many cognitive activities including learning, prediction-making, problem solving, and decision making. Studies have been done in various scenarios to see the effects of different variables on memory retrieval, specific to those memories stored in the hippocampus. Variables such as exercise, stress, post-traumatic stress disorder (PTSD), and contextual and non-contextual dependent memories demonstrate an effect on one key process in the hippocampus â&#x20AC;&#x201C; that is neurogenesis. As these studies are discussed, this review will cover the process of neurogenesis, how it is affected, and what that means for neurogenesis and learning.

Learning, overall, is greatly affected by how much an individual can remember. Specific to the hippocampus, learning in the use of spatial memory and context dependency will be used. In the study by Winocur et al. (2012), neurogenesis was disrupted in adult rats. It was then demonstrated through a learned discrimination that rats placed in a high-interference environment had an impaired performance on the task, where as those in a low-interference environment did not. This is because the rats in the high-interference environment relied heavily on the contextual cues for performance, which were now unavailable to them due to the disruption in the hippocampus. From this, it is concluded that the hippocampus is context-dependent and that the following discussion of memory will be based on this (Winocur, 2012).

Neurogenesis has been shown to be affected by other internal factors including exercise and stress.

These factors, as they affect how much is information is remembered, in turn affect learning. However, the interesting aspect of the process of remembering is that it appears to come hand in hand with forgetting.

Akers et al. (2014) experiment on the effects of hippocampal neurogenesis and forgetting to determine the extent of the correlation between neurogenesis and forgetting, and how that is further interfered with by 77

exercise. From studying this, it may lead to developments in maximizing learning and memory by being able to balance the tradeoffs. In addition, it is important in order to study the effects of stress on both the processes of remembering and forgetting, and where it can help with those with PTSD. Research Overview SUMMARY OF MAJOR RESULTS & DISCUSSION Akers et al. (2014) experimented with infant and adult mice, to look at the difference between the various amount of neurogenesis occurring and how it affected memory of a fear-conditioned contextdependent stimulus. In infants, neurogenesis is rapid and occurs to a larger extent than it does in adult mice. Additionally, the effects of having access to an enriched environment (a running wheel) were also used as a variable. Finally, this was tested on TK+ mice vs. wildtype mice, in which the modified mice were impaired for the process of neurogenesis.

The major results here indicated that with increased neurogenesis, there was also increased amount of forgetting. This was true with the adult vs. infant condition, in which after the fear condition, both groups tested a day later showed the same amount of freezing time, whereas when both were tested 28 days later, the adults showed more freezing than the infants. In addition, the above was true in the case of enriched vs. non-enriched rats, in which those who voluntarily accessed a running wheel showed more neurogenesis with GFP tracking in the dentate gyrus, as well as a reduced response the conditioned stimulus context. Interestingly, the transgenic TK+ mice that were impaired in the neurogenesis process were unable to forget the conditioned stimulus and did not reduce their reaction time during the tested context 28 days later (Akers, 2014).

The finding that enriched environment does promote forgetting, is in parallel with other evidence suggesting that in fact volunteer running promotes neurogenesis by increasing the survival chances of 1-3 week old

neurons (Zhao, 2008). By promoting the growth of these neurons, memory on spatial tasks and trace conditioning may be improved, though other memories are forgotten in the process (Frankland, 2013).

The evidence for this lies in both the experiment

by Akers et al. and how over a delayed time period, the memory of the fear conditioning was forgotten.

Additionally, Winocur et al. (2006) performed experiments with spatial tasks and trace conditioning to show that in fact impairing the adult neurogenesis will cause disruption for the rat to recalls memories over a long time delay, as well as in contextual circumstances.

Hence, hippocampus is important in these areas, and that the survival of these new neurons is important for an extended recall of memories, though it affects the memory of other retrieval patterns.

Memories formed in the hippocampus are proven to be formed on the idea of pattern separation (Mongiat 2014). Pattern separation is the idea that in circumstances where conditions are similar, little contextual cues, indicated by the hippocampus, allow for the appropriate response. In this case, a memory would be considered a specific retrieval pattern, and forgetting would occur when one is unable to recall that exact retrieval pattern. Putting together the two above ideas, this indicates that when neurogenesis occurs, the new neurons disrupt the old pathways and cause forgetting of the old retrieval pathways. Interestingly, however, neurogenesis is very helpful in cases where new information learned conflicts with old information (Winocur 2006).

This leads to another point, in that there may be tradeoffs for how much one can learn and how much they will remember, based on the process of neurogenesis.

If there is too much neurogenesis, then it appears one will forget a lot more; if there is too little then one will not be able to learn anything new (make new retrieval patterns). This is illustrated in the following diagram:

Conclusions and Discussion As mentioned, it appears there is a tradeoff level between how much can be learned and remembered.

As Akers et al. proved, there is a correlation between neurogenesis and forgetting. This brings to light an interesting discussion on where the tradeoff is and how can one maximize this learning and retention when in life. One aspect of this tradeoff is affecting by the aging process.

Many studies have shown that with age comes impaired performance in spatial memory, or tasks that involve the hippocampus. Not surprisingly, neurogenesis also reduces with age, as well as synaptic plasticity (Barak, 2014). However, Barak et al. (2014) showed that with fitness and aerobic exercise, one can reduce the effects of age-related impairment in memory. This is very much in line with Akers et al. experiment in that there conditions with adult vs. infant rats showed the difference between neurogenesis with the age gap and that in the adult condition, some rats had access to a running wheel and some did not.

Conclusions

In exploring the relationship of the tradeoff between learning and forgetting, as well as factoring age and exercise, it can begins the process of studying how to maximize memory and learn efficiently. Akers et al. provided a novel insight to just how much correlation there is between learning and forgetting, as much of the literature on forgetting, in this aspect, is just theory.

Being able to know when and how much certain aspects of learning, in relation to the hippocampus, should occur is essential to maximizing human interaction with the world. Additionally this novel experiment has shown how much forgetting is related to learning, and can be evidence for the processes of both LTP and LTD. The study provided confirmation to certain aspects of hippocampal memory, as discussed above, but also allowed for new insight of forgetting. Additionally, Akers et al. checked to see if their hypothesis was transfer appropriate, by also testing on guinea pigs. The correlation carried through, hence there is a substantial correlation between forgetting and hippocampal neurogenesis.

Criticisms and Future Directions

http://www.sciencemag.org.myaccess.library.utoronto.ca/ content/344/6184/594/F1.expansion.html Figure 1.

The rats respond to learning what is announced by the radio. When too little neurogenesis occurs, there is a limitation on how much can be learned, as well as remembered. When too much neurogenesis occurs, then a lot can be learned, but very little can be remembered. It is then suggested, a tradeoff level where neurogenesis balances with forgetting and so learning and retention is maximised (Mongiat 2014).

In future, in addition to studying the tradeoff relationship, it would worthwhile to study the effects of stress and neurogenesis. Stress is a part of everyday lives, and in small doses, is said to be helpful for learning, memory, and performance, but chronically it can impair all three.

Chronic stress is shown to alter the hippocampus by affecting the NMDA-dependent excitatory pathway, and thereby inhibiting neurogenesis in the dentate gyrus (Gould 1999). This is demonstrated by individuals with PTSD, who have proven to have a smaller hippocampus than those without (McNamara 2006). Studying these effects can help alleviate consequences from stress by being able to counteract the effects of stress on the hippocampus. For example, with the administration of Ziprasidone, anxietylike behaviours in rats decreased and neurogenesis was then up-regulated (Zhengqu 2013).

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However, in another experiment where a categorized list was learned, and then the individual was exposed to a stressor or control environment, those under stress showed an improvement in memory in that they did not suffer from retrieval-induced forgetting (Koessler 2009). Additionally, veterans with PTSD showed that selective retrieval of trauma related stimuli leads to enhancement of induced forgetting, meaning that it helped to alleviate the effects of PTSD (Brown 2012).

Looking at both sides of the spectrum, it appears that the balance of memory and forgetting can be regulated by stress and anti-depressants/stress treatments (Dranovsky 2006). Being able to do this, is key to the mental health of individuals and finding alternate methods of dealing with chronic stress and further maximising learning and memory in the hippocampus.

Another part of the brain that also influences memories in the hippocampus is the amygdala. Young rats exposed to chronic childhood stress, were more succumbed to stress in adulthood and showed a more activated amygdala during times of stress (Tsoory 2007). To put these three aspects: stress, the amygdala, and hippocampal neurogenesis together would be a crucial aspect of studying mental health and being able to create environments that are appeasing to all three. Additionally, helping those with chronic stress and the long term effects of it (like the amygdala response) would greatly be effected by studies on neurogenesis and forgetting. Potentially, the same experiment from the Akers et al. study could be replicated but over an extended period of time. Stressors and learning can be alternated throughout growth to see what the effects of neurogenesis end up being, and an MRI scan can be used to look at the size of both the hippocampus and the amygdala. Akers et al. experiment has provided the first stepping stone to looking further into the correlation between forgetting and memory and moving toward the most efficient treatments for stress, as well as the most effective methods for learning and memory.

References 1. Akers K, et al. (2014). Hippocampal Neurogenesis Regulates Forgetting in Adulthood and Infancy. Science 344(6184), 598-602.

2. Barak B, et al. (2014). Cardiovascular Fitness and Cognitive Spatial Learning in Rodents and in Humans. J Gerontol A Biol Sci Med Sci. 3. Brown A, et al. (2011). Forgetting Trauma: Socially Shared Retrieval-induced Forgetting and Post-traumatic Stress Disorder. Appl. Cognit. Psychol. 26:24-34.

4. Dranovsky A, Hen R (2006). Hippocampal Neurogenesis: Regulation by Stress and Antidepressants. Biol Psychiatry 59: 1136-1143.

5. Frankland P, et al. (2013). Hippocampal neurogenesis and forgetting. Trends in Neurosciences, 36(9): 497-503.

6. Gould E, Tanapat P (1999). Stress and Hippocampal Neurogenesis. Biol Psychiatry, 46: 1472-1479.

7. Koessler S, et al. (2009). No Retrieval-Induced Forgetting Under Stress. Psychological Science, 20(11): 1356 79

â&#x20AC;&#x201C; 1363.

8. McNamara D (2006). Chronic PTSD linked to smaller hippocampus. Clinical Psychiatry News, 34(5), 19.

9. Mongiat L, Schnider F (2014) A Price to Pay for Adult Neurogenesis, Science 344(6184): 594-595.

10. Tsoory M, et al. (2007). Amygdala modulation of memory-related processes in the hippocampus: potential relevance to PTSD. Progress in Brain Research, 167: 35-51.

11. Winocur G, et al. (2012). Adult hippocampal neurogenesis and memory interference. Behavioural Brain Research, 227: 464-469.

12. Winocur G, et al. (2006). Inhibition of Neurogenesis Interferes with Hippocampus-Dependent Memory Function.

Hippocampus, 16: 296-304 13. Zhao C, et al. (2008). Mechanisms and Functional Implication of Adult Neurogenesis. Cell, 132(4): 645-660.

14. Zhengqu P, et al. (2013). Ziprasidone ameliorates anxiety-like behaviours in rat model of PTSD and up-regulates neurogenesis in the hippocampus and hippocampus-derived neural stem cells. Behavioural Brain Research, 244: 1-8.

Received Month, ##, 200#; revised ##, 200#; accepted Month, ##,

Month, 2013.

This work was supported by The Association for the Development of Undergraduate Neuroscience Education (SRA & RLN), The Endowment for Science Education (EA), and The Synaptic State Faculty Research Foundation (EA). The authors thank Mr. Spine L. Cord, Dr. Amy G. Dala, and the students in Neuroscience 101 for technical assistance, execution, and feedback on this lab exercise.

Address correspondence to: Dr. Rita L. Neurotrophin, Biology Department, 123 Growth Cone Avenue, Action Potential College, Hillock, IL 60101 Email: rln@apc.edu Copyright Š 2015 Dr. Bill JU, Neurosciences, Human Biology Program

Deep brain stimulation and Alzheimer’s disease: Benefits, cost-effectiveness and feasibility of deep brain stimulation on Alzheimer’s disease and cognitive dysfunction.

Vanessa Ferlaino

Many neurodegenerative diseases are associated with common neurological and psychiatric conditions and are often the result of disruptions to neurological circuits. Due to the complexity of neurological circuits involved in neurodegenerative diseases that result in a vast array of symptoms, as identified in Alzheimer’s disease (AD), there are very limited effective treatments available for AD patients. Deep brain stimulation (DBS) is a new technique that has shown great efficacy and benefits in other neurodegenerative diseases, including Parkinson’s diseases (PD), Obsessive Compulsive Disorder (OCD), and depression. It has been recently used to stimulate various targets of the brain , including the fornix, anterior thalamic neucli and the nucleus basilis of meynert to improve cognitive function and memory in AD patients. Additionally, DBS has been coupled with imaging techniques to enhance understanding of the neural circuitry underlying AD, suggesting glucose cerebral metabolism plays a role in AD symptoms. However, there are many drawbacks of DBS to AD, including its use being limited to patients with mild AD, its cost-effectiveness and its feasibility as very few patients qualify for DBS treatment. As a result, this review assesses the use of DBS as a treatment for AD and includes an analysis of its cost-effectiveness and feasibility in the healthcare field. We conclude that DBS in conjunction with imaging techniques is very valuable to understanding neurological circuits of AD, and will be truly effective as a treatment if its feasibility can be increased to include treating patients at higher stages of AD or if combined with imaging techniques that can screen patients earlier in the AD development process.

Key words: Deep brain stimulation (DBS); Alzheimer’s disease (AD); fornix; anterior thalamic nuclei (ATN); nucleus basilis of Meynert (NBM);midline thalamic nuclei (MTN); feasibility; cost-effectiveness; PET; FDG-PET. Background Lozano and Lipsman stated that many neurodegenerative diseases are associated with common neurological and psychiatric conditions that are often the result of changes, damage, and death of neurons in neurological systems. As a result, because numerous multiple neurological systems tend to be involved a variety of symptoms emerge, including cognitive and motor function deficits, leading to a large gap in terms of treatments for these patients as these neurological systems are multi-factorial and difficult to study.

Lyketsos et al. describe Alzheimer’s disease (AD) as a neurodegenerative disease characterized by progressive cognitive dysfunction. Hirao, Pontine and Smith state the most common symptoms include depression, apathy and irritability. Pathologically, the disease appears to be due to accumulation and deposition of Beta-amyloid protein (Aβ) resulting in loss of neuronal function and neuronal death. Again, the neural circuits involved in AD are not completely understood making the development of a drug or other treatment quite difficult. However, a recent method known as Deep Brain Stimulation (DBS) has proved to be a beneficial treatment for AD patients as shown by Smith et al.

DBS is best described by Lozano and Lipsman as a technique that involves the neurosurgical implantation of an electrode into a target brain region, ex. fornix.

It is connected to a pulse generator, implanted under the skin below the collarbone, and settings such as stimulation type, frequency, amplitude, and pulse width can be controlled externally allowing it to be used to the effects of DBS as a potential treatment for

numerous neurodegenerative diseases. It has been successful for treating Parkinson’s disease, obsessive compulsive disorder (OCD), and depression (Pereira et al.), and has been incredibly useful as a technique to study the neural circuits of these diseases. However, DBS has many drawbacks, such as its beneficial effects being more prevalent in those with early AD thus limiting its feasibility as a treatment. Additionally, as the health care field prepares to shift to patientcentered care of chronic diseases, the feasibility of innovative techniques is important to assess. This review assesses the use of DBS as a treatment for AD and includes an analysis of its cost-effectiveness and feasibility in the healthcare field. We conclude that DBS in conjunction with imaging techniques is very valuable to understanding neurological circuits of AD, and will be truly effective as a treatment if its feasibility can be increased to include treating patients at higher stages of AD or if combined with imaging techniques that can screen patients earlier in the AD. Research Overview

DBS of the fornix is critical for controlling spatial memory in mild AD patients as it may be related to cerebral glucose metabolism

DBS of the fornix has been especially popular for treatment of AD. Initially, Hamani et al. had used DBS of the fornix to treat an obese patient, though it unexpectedly resulted in the upbringing of autobiographical

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memory events in this patient. As a result, the authors began assessing the hypothalamic mechanism behind this memory. The authors concluded that the patient had enhanced cognition after hypothalamic stimulation as well as increased activity in the hippocampus, showing that electrical stimulation of the hypothalamus modulates limbic activity and improves hippocampusdependent memory. They hypothesized the effects of this hypothalamic stimulation on memory were due to activation of the fornix. Recognizing the role of the fornix in memory enhancement, Smith et al. investigated stimulation of the fornix and its role in memory enhancement in AD in a breakthrough study that involved DBS of the fornix for one year in 5 patients. Figure 1 shows PET scans that identified a frontal-temporal-parietal-striatal-thalamic network, important for memory and other cognitive functions, and a frontal-temporal-parietal-occipital-hippocampal network, important for the default mode network, after 1 year of DBS at constant electrode settings.

High baseline metabolism in brain regions commonly affected by AD indicated less global cognitive decline and increased quality of life, denoted by a decline in ADAS-cog and QOL-AD scores, and correlated with increased cerebral metabolism after 1 year of DBS. Low baseline metabolism in brain regions less commonly affected by AD indicated less global cognitive decline and correlated with decreased metabolism after 1 year of DBS. However, they concluded that patients with mild AD benefitted more from DBS of the fornix as they showed more memory improvement. Their results also suggested that glucose metabolism plays a role in memory enhancement in AD, as indicated by PET imaging. Gao et al. investigated this relationship by using FDG-MicroPET to quantify baseline and postDBS treatment cerebral glucose levels in the brain of mice to show that bilateral anterior thalamic nucleus (ATN) stimulation increased glucose uptake in the thalamus and hippocampus. DBS in mice with lesions in the ATN did not show any increased glucose uptake post-treatment, thus glucose metabolism is regulated by the ATN. This is consistent with Smith et al. as the ATN is present in the frontal-temporal-parietalstriatal-thalamic network, important for memory and other cognitive functions, therefore cerebral glucose plays an important role in memory and further studies may reveal its importance in AD as well as its potential use as a screening technique. Additionally, it is clear that DBS is an exceptional tool for studying neurological systems especially when compared with imaging techniques.

Hescham et al. also used DBS to stimulate the fornix at 200 µA, 100 µA, and 50 µA in mice treated with scopolamine, a muscarinic acetylcholine receptor antagonist that induces memory dysfunction. Their main goal was to identify the stimulation parameters that allowed optimal improved memory in experimental dementia. Although this paper did not exclusively look at AD, Pereira et al. state dementia is the result of progressive AD. Object location task and open field (OF) tests were used to assess memory recognition and anxiety. They concluded that 200 µA at 10 Hz as well as 100 µA at 10 Hz and 100 HZ enabled the best enhanced memory performance in the object location task. The OF results showed no significant 81

difference between sham rats and experimental rats, indicating no side-effects on locomotor and anxietyrelated behaviours. In conclusion, DBS of the fornix in humans and mice models has shown that the fornix appears to be critical for controlling spatial memory function and can be shown to improve this memory function in patients with mild AD and has clear benefits as a therapeutic treatment.

Figure 1. Frontal-temporal-parietal-striatal-thalamic network and (red) frontal-temporal-parietal-occipital-hippocampal network (blue) associated with increased cerebral metabolism (Smith et al.).

DBS of the midline thalamic nuclei and nucleus basilis of meynert may help improve cognitive dysfunction and memory

Despite the clear benefits of DBS of the fornix, other targets have been found to be beneficial for AD, including the midline thalamic nuclei (MTN) and the nucleus basalis of Meynert (NBM). Arrieta-Cruz, Pavlides and Pasinetti initiated DBS first in the Schaffer Collateral (SC) of TgCRND8 hippocampal slices to activate CA1 neurons and found that TgCRND8 mice did not undergo short-term potentiation until after high frequency stimulation (HFS) at 200 Hz in contrast to wild type slices. They also used c-Fos to show increased activity of α-secretase in TgCRND8 mice after HFS at 50, 100 and 200 Hz but there was no effect on β-secretase activity. They then performed HFS of midline thalamic nuclei in TgCRND8 mice followed by an object recognition task immediately after stimulation. TgCRND8 mice showed enhanced short-term memory acquisition after DBS in this task, which is again only associated with early AD as this is the first type of memory affected in AD (Tramatula et al.). However, Chen et al. replicated these results in long term memory by injecting mice with Aβ1-40 and

then performing ANT-DBS. These mice had increased platform-traversing time and time spent in the target quadrant in the Morris Water Maze compared to AD mice injected with Aβ1-40 but did not undergo ANT-DBS. Therefore, cognitive dysfunction and memory of AD can be treated by ANT-DBS in mice.

Kuhn et al. performed bilateral stimulation for one year of the Ch4 region in the NBM, as it is known to be most affected by AD, resulting in increased acetylcholine release in this area, correlating with increased cognitive performance. Figure 2 shows ADAS-cog scores showing improved memory, though authors could not make any conclusions as to whether or not the hippocampus or other memory-dependent circuits were affected. Consistent with Smith et al., these authors also found an increase in glucose metabolism in the entire cerebrum in three out of four patients that underwent PET post-DBS after 1 year of treatment.

Therefore DBS of both the fornix and the NBM result in increased cerebral glucose metabolism, again suggesting the importance of glucose metabolism in AD and proving that DBS is an excellent tool for understanding neural circuitry when used in conjunction with imaging techniques.

DBS is a safe and feasible treatment, but targets very few patients with mild AD

Kuhn et al. also assessed the safety of DBS technique and concluded there were no dangerous side effects.

They also noted that out of six patients, quality of life appeared to only improve in two. Two others indicated a decrease and another two patients indicated

no change at all, while the quality of life of relatives did not show a significant increase. In regards to the popularity of the use of DBS, one patient and one relative of another patient indicated they would not choose to use the treatment again, suggesting that although DBS has promising results its feasibility may not be realistic for treatment if its interest is not perceived well by the public.

As a result, feasibility of DBS was investigated by Fontaine et al. After a one year period of inclusion, 9/110 patients fit the inclusion criteria, including having episodic memory impairments. 4/9 originally accepted the idea, though only 2/4 gave consent.

In the end, only 1 patient received bilateral DBS of the fornix for one year as the other patient withdrew consent. Fontaine et al. reported the surgery was well-received but many global cognitive functions did not improve until after 6 months of treatment and were then stabilized. Nonetheless, they were able to conclude that DBS is a safe procedure that should not result in any adverse complications and does not appear to impair daily living activities. However, they were only able to operate on one patient suggesting their restriction criteria, which required AD patients with impairments in episodic memory often associated with later stages of AD, was too specific. As this coincides with Smith et al. in terms of AD being most effective for patients with mild AD, this also indicates that a small percentage of patients are capable of receiving DBS. They also list factors such as lack of awareness of DBS, its invasiveness, as well as patient denial in regards to their cognitive decline leading to an underestimation of the benefit/risk ratio as leading

Figure 2. Mean scores reflecting long term effects of DBS on memory, cognition and global functioning in 6 AD patients (Kuhn et al.).

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to this low acceptance level. That being said, the authors contrast the high acceptance rate of DBS with its low feasibility rate and question whether DBS is a treatment to invest in when so little can be treated or accept to be treated with it.

Mirsaeedi-Farahani et al. also conducted a costeffectiveness analysis of DBS for AD treatment, looking at the clinical and economic thresholds especially as the cost of healthcare increases. They performed a literature review and determined that in order for DBS to be successful, its success rate must be at 3% to overcome effects of possible surgical complications on quality of life. Cost-effectiveness of DBS depends on society’s willingness to pay DBS. The authors concluded that DBS is cost-effective if its success rate is 20% or 74% for mild AD. With a success rate of 80%, DBS is more clinically effective and more cost-effective. In conclusion, DBS is highly effective in comparison to standard treatment of AD and that it is cost-effective if society is willing to pay. That being said, DBS is an expensive treatment; at a 20% success rate Mirsaeedi-Farahani et al. reported a cost of $200K and $50K for a 74% success rate. Kuhn et al., Fontaine et al. and Smith et al. showed recruitment of patients with mild AD was difficult as most patients suffer from progressive AD and also confirmed that DBS was mostly beneficial for mild-AD patients. Thus as so little will benefit from AD, it may not be entirely cost-effective at the healthcare level which seeks to provide treatments that will serve a greater proportion of the general public. Additionally, the success rate needs to be determined experimentally which may deviate greatly from this mathematical model as only so little patients benefit from AD. Thus, perhaps another way to tackle this issue is to investigate the use of better scanning techniques that will recognize neurodegenerative diseases like AD at earlier stages, such as imaging techniques described by Shivamurthy et al., Nasrallah and Wolk, and Laske et al.

Conclusions As reported in this review, there are many benefits from the use of DBS of the fornix, ATN and NBM as a sole treatment for treating memory and cognitive function associated with AD. DBS of the ATN and NBM showed enhanced memory and cognitive function in mice as shown by Kuhn et al., Chen et al., as well as Arrieta-Cruz, Pavlides and Pasinetti. Additionally, Smith et al., Gao et al and Kuhn et al. have shown that pairing DBS with imaging techniques can help in establishing and understanding neural circuits underlying AD, including the role of glucose cerebral metabolism. DBS of the fornix is proven to be critical for controlling spatial memory in both AD patients and mice models, as indicated by Hescham et al. However, Smith et al. showed memory in humans is better enhanced in mild AD patients.

As a result, despite DBS being effective in improving cognition, Fontaine et al. and Mirsaeedi-Farahani et al. proved DBS to be feasible and cost-effective but unrealistic as it has been best proven for mild AD patients in many cases and thus there may not be a 83

market for DBS as a treatment among AD patients.

Thus, perhaps the real potential of using DBS lies within its ability to aid in the understanding of neural circuitry in AD and the effects of glucose metabolism effects in conjunction with imaging techniques to help understand the disease and aid in the development of treatments and medications that are more feasible and acceptable by society. That being said, with the advance of neuroimaging techniques as screening tools for diagnosis at earlier stages of disease we may see the efficacy of DBS increase as DBS will be able to target more patients. Nonetheless, whether DBS is used as a treatment alone or in conjunction with imaging techniques to enhance understanding of the disease, it certainly is beneficial for AD.

Future Directions

As indicated in this review, plenty of evidence exists in supporting DBS as a therapeutic treatment for mild AD symptoms. Thus, in the future, the study of the effects of cerebral glucose metabolism as well as the effects of DBS on neurogenesis, BDNF transport and Aβ pathology will be useful to further assess the benefits of DBS as a sole treatment and maybe even a potential cure for AD. Cell and molecular biology techniques can be used to study the effects of increased cerebral glucose metabolism and neurogenesis on the brain, as previously shown

by Gao et al as well as Arrieta-Cruz, Pavlides and Pasinetti . Additionally, Stone et al. showed DBS of the entorhinal cortex (EC) in mice participating in a water maze task resulted in activated neurons in the Dentante Gyrus (DG) with increased cFos and IdU expression, indicating new DG neurons integrated into hippocampal memory circuits. To assess the effect of DBS on Aβ metabolism, Aliaga et al. determined a correlation between BDNF levels and Aβ levels to use BDNF levels as a marker for Aβ presence. They used real-time PCR to show that sublethal concentrations of Aβ led to decreased BDNF mRNA levels and trkB mRNA levels in neurons. p75 mRNA levels did not change, but protein expression decreased as a result of internalization. Protein expression of trkB did not significantly change. Finally, Mark et al. showed that Phloretin, increased Aβ concentrations and FeSO4 presence resulted in decreased glucose uptake.

Glucose transport impairment occurs quicker when exposed to HNE, a cytotoxic product of lipid peroxidation due to Aβ production and also occurred before decreased cell ATP; in conjunction with the observation that phloretin resulted in neurotoxicity led to the conclusion that Aβ-induced impairment of glucose transport is a result of Aβ-induced neurotoxicity. In conclusion, combining DBS, PET, FDG-MicroPET and cell and molecular biology techniques to study cerebral glucose metabolism and its effects on neurogenesis, BDNF transport and Aβ pathology in mouse models may be useful to enhance understanding of AD circuitry in the brain to aid in development of medications and other treatments for AD. Additionally, these studies may also increase the feasibility of DBS as a treatment to assist hospitals in their transition to treating chronic diseases, including debilitating neurodegenerative diseases.

References 1. Aliega E, Silhol M, Bonneau , Maurice T, Arancibia S, Tapia-Arancibia L (2010). Dual response of BDNF to sublethal concentrations of beta-amyloid peptides in cultured cortical neurons. Neurobiol Dis 37:208-217.

2. Arrieta-Cruz I, Pavlides C, Pasinetti GM (2010). Deep brain stimulation in midline thalamic region facilitates synaptic transmission and short-term memory in a mouse model of Alzheimer’s disease. Translational Neuroscience 3:188-194.

3. Chen N, Dong S, Yan T, Ma Y, Yu C (2014). Highfrequency stimulation of anterior nucleus thalamus improves impaired cognitive function induced by intra-hippocampal injection of Aβ1-40 in rats. Chin Med J 1:129-139.

4. Fontaine D, Deudon A, Lemaire JJ, Razzouk M, Viau P, Darcourt J, Robert P (2013). Symptomatic treatment of memory decline in Alzheimer’s disease by deep brain stimulation: A feastibility study. Journal of Alzheimer’s disease 34:315-323.

5. Gao F, Guo Y, Zhang H, Wang S, Wang J, Wu JM (2009). Anterior thalamic nucleus stimulation modulates regional cerebral metabolism: An FDG-MicroPET study in rats. Neuorbiology of Disease 34:477-483.

6. Hamani C, McAndrews MP, Cohn M, Oh M, Zumsteg D, Shapiro CM, Wennberg RA, Lozano AM (2008). Memory enhancement induced by hypothalamic/fornix deep brain stimulation. Ann Neurol 63:119-123.

7. Hescham S, Lim LW, Jahanshahi A, Steinbusch HWM, Prickaerts J, Blokland A, Temel Y (2013). Deep brain stimulation of the forniceal area enhances memory functions in experimental dementia: The role of stimulation parameters.

Brain stimulation 6:72-77.

8. Hirao K, Pontone GM, Smith GW (2014). Molecular imaging of neuropsychiatric symptoms in Alzheimer’s and Parkinson’s disease. Neuroscience and Biobehavioural Reviews 49:157-170.

9. Kuhn J, Hardenacke K, Lenartz D, Gruendler T, Ullsperger M, Bartsch C, Mai JK, Zilles K, Bauer A, Matusch A, Schulz RJ, Noreik M, Buhrle P, Maintz D, Woopen C, Haussermann P, Hellmich M, Klosterkotter J, Wiltfang J, Maarouf M, Freund HJ, Sturm V (2015). Deep brain stimulation of the nucleus basalis of Meynert in Alzheimer’s dementia. Molecular Psychiatry 20:353-360.

10. Laske C, Sohrabi HR, Frost SM, López-de-Ipiña K, Garrard P, Buscema M, Dauwels J, Soekadar SR, Mueller S, Linnemann C, Bridenbaugh SA, Kanagasingam Y, Martins RN, O’Bryant SE (2014). Innovative diagnostic tools for early detection of Alzheimer’s disease. Alzheimers Dement 14:2463-24637.

11. Lyketsos CG, TargumSD, Pendergrass JC, Lozano AM (2012). Deep brain stimulation: A novel strategy for treating Alzheimer’s disease. Innovations in clinical neuroscience 9:10-17.

12. Lozano AM, Lipsman N (2013). Probing and regulating dysfunctional circuits using Deep Brain Stimulation. Neuron Review 77:406-424.

13. Mark RJ, Pang Z, Geddes JW, Uchida K, Mattson MP (1997). Amyloid beta-peptide impairs glucose transport in

hippocampal and cortical neurons: involvement of membrane lipid peroxidation. J Neurosci 17:1046-1054.

14. Mirsaeedi-Farahani K, Halpern CH, Baltuch GH, Wolk DA, Stein SC (2015). Deep brain stimulation for Alzheimer disease: a decision and cost-effectiveness analysis. Advanced online publication. DOI 10.1007/s00415-015-7688-5 15. Nasralla I, Wolk DA (2014). Multimodality imaging of Alzheimer disease and other neurodegenerative dementias. J Nucl Med. 12:2003-2011.

16. Pereira JL, Downes A, Gorgulho A, Patel V, Malkasian D, De Salles A (2014). Alzheimer’s disease: The role for neurosurgery. Surg Neurol Int. 5:385-390.

17. Sankar T, Chakravarty MM, Bescos A, Lara M, Obuchi T, Laxton AW, McAndrews MP, Tang-Wai DF, Workman CI, Smith GS, Lozano AM. Deep Brain Stimulation Influences Brain Structure in Alzheimer’s Disease. Brain Stimulation (2015), doi:10.1016/j.brs.2014.11.020.

18. Shivamurthy VK, Tahari AK, Marcus S, Subramaniam RM (2015). Brain FDG PET and the diagnosis of dementia. AJR Am J Roentgenol 204:76-85.

19. Smith GS, Laxton AW, Tang-Wai DF, McAndrews MP, Diaconescu AO, Workman CI, Lozano AM (2012). Increased cerebral metabolism after 1 year of deep brain stimulation in Alzheimer’s disease. Archives of Neurology 69:1141-1148.

20. Stone SS, Teixeira CM, Devito LM, Zaslavsky K, Josselyn SA, Lozano AM, Frankland PW (2011). Stimulation of entorhinal cortex promotes adult neurogenesis and facilitates spatial memory. J Neurosci 38: 13469-13484.

21. Tramutola A, Triplett C, Di Domenico F, Niedowicz DM, Purphy MP, Coccia R, Perluigi M, Butterfield DA (2015).

Alteration of mTOR signalling occurs early in the progression of Alzheimer disease (AD): analysis of brain from subjects with pre-clinical AD, amnestic mild cognitive impairment and late-stage AD. Journal of Neurochemistry 10:1-11.

Received April, 01, 01, 2015; accepted

2015; April,

revised 06,

April, 2015.

This work was supported by The Association for the Development of Undergraduate Neuroscience Education (SRA & RLN), The Endowment for Science Education (EA), and The Synaptic State Faculty Research Foundation (EA). The authors thank Mr. Spine L. Cord, Dr. Amy G. Dala, and the students in Neuroscience 101 for technical assistance, execution, and feedback on this lab exercise.

Address correspondence to: Dr. Rita L. Neurotrophin, Biology Department, 123 Growth Cone Avenue, Action Potential College, Hillock, IL 60101 Email: rln@apc.edu Copyright © 2015 Dr. Bill JU, Neurosciences, Human Biology Program

84

The Efficacy of Neurofeedback Training as a Treatment for Attention-Deficit Hyperactivity Disorder Floriana Ferri

The present study examined the efficacy of neurofeedback training in treating attention deficit hyperactivity disorder (ADHD) in children and adolescents by comparing it to the standard pharmacological intervention. Of the 63 recruited participants, 23 participants, 11 boys and 12 girls, were selected and completed the study.

Through random selection, half of the participants (N=11) began taking daily doses of methylphenidate (standard medication), while the other half (N=12) completed 40 sessions of neurofeedback (NF) training. According to results in the behavioral rating scales, which were completed by parents and teachers, the participants that received NF training showed significant improvements in inattention, functional impairment, academic performance and overall ADHD symptoms. The improvements were parallel to those receiving methylphenidate, however, only the NF group showed significant improvement in academic performance. These results indicate that NF has the potential to be used as an alternative treatment for ADHD as it leads no side effects and it has an additional advantage of increasing academic performance.

Key words: neurofeedback; attention deficit/hyperactivity disorder; children; randomized-control trial; two and six-month follow-up.

Background Attention deficit hyperactivity disorder (ADHD) is a neurodevelopmental disorder that affects close to 5% of children worldwide (Polanczyk, de Lima, Horta, Biederman & Rohde, 2007) making it one of the most common childhood psychiatric disorders (Campbell, 2000). ADHD is also common among adults but remains highly undiagnosed in this segment of the population (Schweitzer, Cummins & Kant, 2001). Some of the core symptoms of ADHD are hyperactivity, impulsivity, inattention and often, but not always, increased executive functioning impairments (Biederman et al., 2004).

As a result of these symptoms, children with ADHD often have reduced academic outcomes (Mautone, DuPaul & Jitendra, 2005) and express higher rates of physical and verbal aggression, attention-seeking behavior and non-compliance in comparison with their peers (Junod, DuPaul, Jitendra, Volpe & Cleary, 2006). In addition, children with ADHD are also more likely to be part of the special education program at their school, have to repeat a grade or be expelled or suspended (Klein & Abikoff, 1997). Of the cases that persist into adolescence and adulthood, which is 40-60%, problems such as poor socialization, unsatisfactory academic performance and increased chance of traffic accidents are common (Faraone, Biederman & Mick, 2006).

The present-day method for treating ADHD is primarily through pharmaceutical treatments; more specifically, through psychostimulant medications. Psychostimulants are drugs that increase psychomotor activity in the central nervous system and they have shown to be efficacious in reducing symptom severity of ADHD (Schachter, Pham, King, Langford, & Moher, 2001), however, they have also shown to cause adverse side effects (Steiner, Frenette, Rene, Brennan, & Perrin, 2014). According to recent studies, psychostimulant medications are associated with growth suppression (Germinario et al., 2013), decrease of appetite 85

and development of insomnia (Wigal et al, 1997); these effects have been reported to reverse only after discontinuation of treatment (Germinario et al., 2013; Wigal et al., 1997). Additional issues regarding ADHD pharmaceutical treatments include low adherence rates to the medication, as only 13%-64% of the children continue long-term use of the medication (Van de Loo-Neus, Rommelse & Buitelaar, 2011), and incompetency to cure symptoms after long-term use, as symptoms return after treatment discontinuation (Steiner et al., 2014). Additionally, 20-30% of children suffering with ADHD are irresponsive to the available medications, reporting lack of progress during use of treatment (Wigal et al, 1997). These finding raise the importance of finding new efficacious methods of treating ADHD. One such new treatment that is gaining more attention in the research field is neurofeedback. Neurofeedback is an operant conditioning technique that trains people to self-regulate their brain activity through real-time feedback of their neurophysiological signals (Arns, de Ridder, Strehl, Breteler, & Coenen, 2009). The neurophysiological signals are measured using EEG and are presented to the participant in a form of visual or auditory match (game), such as a race or a puzzle.

Since previous studies have shown that ADHD is associated with abnormalities in electrophysiology, specifically increased frontal theta wave activity in the brain and decreased beta activity, neurofeedback games are designed to make the participant suppress theta activity and increase beta activity, resulting in reduced inattention and hyperactivity and improved cognitive functioning (Arns et al., 2009).

The literature on the efficacy of neurofeedback training to treat ADHD has yet to expand seeing as neurofeedback is a moderately new approach. Some of the primary studies that have examined the efficacy of neurofeedback were weak in that they lacked randomization, adequate blinding of participants,

control treatments, and reliable outcome measures consequently resulting in inconsistent and divisive study outcomes (Lofthouse, Arnold, Hersch, Hurt, & DeBeus, 2011). Taking that into consideration, the studies from the past five years have attempted to control for previous limitations and have found much more statistically significant results.

Duric, Assmus, Gundersen and Elgen (2012), performed a randomized and controlled study to evaluate neurofeedback as a treatment for ADHD in children, where 91 children, randomized into three groups, 1/3 receiving methylphenidate (MF) treatment, 1/3 sessions of neurofeedback training and 1/3 receiving both. After completion of the study, as reported by parents, no significant differences between treatment groups were observed, meaning that NF was shown to be as effective as methylphenidate in treating ADHD. In 2014, Duric, Abmus and Elgen, used self-reports to examine the efficacy of NF in children and similarly found that NF was as effective as methylphenidate in improving attention and hyperactivity, however, only NF lead to significant improvement in school performance, which is important to examine further in future studies.

Overall, recent studies are strong indicators that NF is a efficacious method of treating ADHD as they have moderate sample sizes, include treatment control conditions and randomization, use self and observation reports to measure outcomes, and control for co-morbidity and concomitant treatments.

In spite of these positive effects, these studies lack a long-term post-treatment follow-up assessment, which is why this study aims to evaluate the efficacy of NF in comparison to standard medication, using a randomized and controlled trial with two and sixmonth follow-up.

Research Overview

Summary of Major Results

Pre-assessment of participants was conducted one week prior to the study. The participants were selected based on age, sex, IQ and ADHD symptoms and did not differ significantly based on these criteria.

Behavioral changes were assessed at pre-treatment (PRE), post-treatment (POST), two-month followup (FU1) and six month follow-up (FU2) using the following standardized measures: ADHD rating scale, Weiss Functional Impairment Rating and Oppositional defiant disorder rating scale.

Based on parent reports, the NF group showed significant improvement in ADHD symptoms, functional impairment, inattention and academic performance; the teachers reported moderate and insignificant improvements in those categories. Regarding hyperactivity and oppositional defiant behavior, mother and teachers reported less than moderate improvements.

The MPH group, on the other hand, showed significant improvements in ADHD symptoms, including hyperactivity, and inattention and the teachers also reported a significant reduction in oppositional defiant behavior. The MPH group, however, did not show

significant improvement in academic performance by neither parent nor teacher reports. As measured by statistical tests, the most significant difference between the two groups was the change in academic performance. Although assessments showed that the NF group maintained the achieved improvements at FU1 and FU2, after the completion of the study, the parents were free to start medicating their children, and at FU1, 6 of the participants were medicated and at FU2, 8 were medicated.

Fig 1. Changes in Academic Performance scores at pre-treatment (PRE), post-treatment (POST), two-month follow-up (FU1) and six-month follow-up (FU2) for the neurofeedback (NF) versus methylphenidate (MPH) participants

Discussion and Critical Analysis

From the results of this study, neurofeedback training shows to be equally capable of treating primary symptoms and associated functional impairments of ADHD as the standard pharmacological medication.

The strength of this study relies on its randomizedcontrolled trial design, in which the efficacy of NF is evaluated through comparison with the current standard treatment for ADHD. As previous studies have simply used placebo-control interventions, this study overcame that limitation by comparing NF to a standard/highly effective treatment, which offers more compelling support for the efficacy of NF as a treatment for ADHD. Importance of this study also includes two naturalistic follow-up assessments, at two and six months following the intervention, use of two groups of evaluators (parents and teachers) and the use of a varying and standardized\behavioral rating scales.

These assessments were useful to demonstrate that the progression resulting from NF can be maintained over a period of time as the majority of the NF participants maintained their cognitive improvement and even continued to progress after discontinuation of NF training. Above all, three of the children maintained their progression even six months after the treatment, being able to cope without the use of pharmacological interventions; this suggests that training the brain through an operant-conditioning method can have 86

lasting benefits, in contrast to pharmaceutical interventions, where discontinuation causes full return of symptoms (Steiner et al., 2014).

Although this study overcame previous limitations, it also presented a few limitations of its own. The primary limitation of the current study is the small sample size of 27 participants, which decreases the feasibility of the conclusions drawn from this study. As only 12 participants completed the NF training and only 4 remained medication-free at six-month follow-up, it cannot be concluded that the effects of NF training are long-lasting until these effects are shown in larger number of participants.

Secondly, this study lacked blinding of participants and assessors in its design, which could indicate that findings of this study are partially a result of performance bias and outcome assessment bias. A 2013 study, for example, found that placebo-neurofeedback was equally effective in treating ADHD symptoms as EEG-neurofeedback (van Dongen-Boomsma, Vollebregt, Slaats-Willemse, & Buitelaar, 2013), showing that motivation and performance bias are capable of affecting the progression acquired from NF training.

Thus, it is important for future studies to control for such biases. More importantly, a feasibility study, using a double-blind placebo-controlled design showed that, regarding NF, double-blind studies are not feasible as the reward thresholds have to be manually adjusted, however, blinding of the parents and the children is valuable and should be incorporated into future study designs (Lansbergen, van Dongen-Boomsma, Buitelaar, & Slaats-Willemse, 2011).

Thirdly, parent and teacher reports were used as outcome measure, which can be biased and inconclusive; another study justified using parent reports, as they were able to correctly determine the clinical range of the children’s ADHD symptoms (Steiner et al, 2014). In this study, however, the fathers’ reports differed from those of mothers’ and teachers’ in that they detected less behavioral improvements, which shows inadequacy in either observation reports or in the behavioral rating scales. Generally, using parents as proxy respondents has shown to not be very efficacious as parent reports correlate poorly with those of their children (Duric et al. 2014). As other studies such as that of Duric, Aßmus, and Elgen (2014) have used only self-reports to evaluate changes in ADHD symptoms, to increase the validity of the results, it would wise to use of both parent/teacher and selfreports in future studies.

Lastly, a critical weakness in this study is the onset of medication use by the participants in the NF group at two and six-month follow-up. Although the gained benefits of NF were maintained in the NF participants according to the behavioral reports, regardless of medication use, it is not clear whether medication helped maintain the progress and whether NF benefits can be maintained after discontinuation of treatment.

This does not, however, indicate that NF is less proficient than medication in treating ADHD long-term, as during the follow-up period, the children in the medication group continued their regular pharmaceutical treatment, while those in the NF terminated their treatment. In future studies, the children in the medication 87

group should also cease to continue their treatment during the follow-up period in order to determine whether progression is preserved equally after both interventions.

Conclusions and Future Directions

In conclusion, this study suggests that NF is an effective long-term alternative treatment and also a complementary treatment to medication for treating ADHD, as it enhances cognitive functioning and improves academic performance. Due to the small sample size and medication use during follow-up period, the results from this study should be interpreted carefully until the results are replicated in future studies. A study design necessary to overcome some of the limitation in this study would have to be a randomized single-blind study with a large sample size, and three groups: neurofeedback-placebo group, a pharmacological intervention group and a neurofeedback intervention group. The length of the study would be at least one year, as regulation of brain waves can be time-dependent and attrition rates would be used to indicate lack of efficacy of the interventions. The outcomes would be measured through tests that evaluate attention and impulsivity specifically in the children and not general reports of behavior, while proxy reports by teacher and parents would only be used to support the findings. Factors such as feedback animations and reward thresholds would be maintained in the design as they have shown to be important in encouraging the participants to strive towards achievement of cognitive regulation (Gevensleben et al., 2012) and implementation of active learning strategies to supplement the NF training would be added as recent studies have indicated that learning strategies are important for enhancing the efficacy of NF (Sherlin et al., 2011). Follow-up assessments would be non-naturalistic, where both groups would have the choice of continuing NF training and/or using medication.

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5. Duric, N. S., Assmus, J., Gundersen, D., & Elgen, I.

B. (2012) Neurofeedback for the treatment of children and adolescents with ADHD: a randomized and controlled clinical trial using parental reports. BMC psychiatry, 12(1), 107.

6. Faraone, S. V., Biederman, J., & Mick, E. (2006).

The age-dependent decline of attention deficit hyperactivity disorder: a meta-analysis of follow-up studies.Psychological medicine, 36(02), 159-165.

7. Gevensleben, H., Rothenberger, A., Moll, G. H., & Heinrich, H. (2012). Neurofeedback in children with ADHD: Validation and challenges. Expert Review of Neurotherapeutics, 12, 447–460.

8. Germinario, E. A., Arcieri, R., Bonati, M., Zuddas, A., Masi, G., Vella, S., ... & Panei, and the Italian ADHD Regional Reference Centers, P. (2013). Attention-Deficit/ Hyperactivity Disorder Drugs and Growth: An Italian Prospective Observational Study. Journal of child and adolescent psychopharmacology,23(7), 440-447.

9. Junod, R. E. V., DuPaul, G. J., Jitendra, A. K., Volpe, R. J., & Cleary, K. S. (2006). Classroom observations of students with and without ADHD: Differences across types of engagement. Journal of School Psychology, 44(2), 87-104.

10. Klein, R. G., & Abikoff, H. (1997). Behavior therapy and methylphenidate in the treatment of children with ADHD. Journal of Attention Disorders, 2(2), 89-114.

11. Lansbergen, M. M., van Dongen-Boomsma, M., Buitelaar, J. K., & Slaats-Willemse, D. (2011) ADHD and EEGneurofeedback: a double-blind randomized placebo-controlled feasibility study. Journal of Neural Transmission, 118(2), 275-284.

12. Lofthouse, N., Arnold, L. E., Hersch, S., Hurt, E., & DeBeus, R. (2011). A review of neurofeedback treatment for pediatric ADHD. Journal of attention disorders, 1087054711427530.

13. Mautone, J. A., DuPaul, G. J., & Jitendra, A. K. (2005).

The effects of computer-assisted instruction on the mathematics performance and classroom behavior of children with ADHD. Journal of Attention Disorders, 9(1), 301-312.

14. Polanczyk, G., de Lima, M. S., Horta, B. L., Biederman, J.,& Rohde, L. A. (2007). The worldwide prevalence of ADHD: a systematic review and metaregression analysis. The American journal of psychiatry, 164(6), 942-948.

15. Schachter, H. M., Pham, B., King, J., Langford, S., & Moher, D. (2001). How efficacious and safe is short-acting methylphenidate for the treatment of attention-deficit disorder in children and adolescents? A meta-analysis. CMAJ: Canadian Medical Association Journal, 165(11), 1475–1488.

16. Schweitzer, J. B., Cummins, T. K., & Kant, C. A. (2001).

Attention-deficit/hyperactivity disorder. Medical Clinics of North America, 85(3), 757-777.

17. Sherlin, L. H., Arns, M., Lubar, J., Heinrich, H., Kerson, C., Strehl, U.,et al. (2011). Neurofeedback and basic learning theory: Implications for research and practice. Journal of Neurotherapy, 15, 292–304.

18. Steiner, N. J., Frenette, E. C., Rene, K. M., Brennan, R. T.,& Perrin, E. C. (2014). Neurofeedback and cognitive attention training for children with attention-deficit hyperactivity disorder in schools. Journal of Developmental & Behavioral Pediatrics, 35(1), 18-27.v

19. an de Loo-Neus, G. H., Rommelse, N., & Buitelaar, J. K. (2011). To stop or not to stop? How long should medication treatment of attention-deficit hyperactivity disorder be extended?. European Neuropsychopharmacology, 21(8), 584-599.

20. van Dongen-Boomsma, M., Vollebregt, M. A., SlaatsWillemse, D., & Buitelaar, J. K. (2013). A randomized placebo-controlled trial of electroencephalographic (EEG) neurofeedback in children with attention-deficit/hyperactivity disorder. J. Clin. Psychiatry, 74, 821-827.

21. Wigal, T., Swanson, J. M., Regino, R., Lerner, M. A., Soliman, I., Steinhoff, K., ... & Wigal, S. B. (1999).

Stimulant medications for the treatment of ADHD: Efficacy and limitations. Mental Retardation and Developmental Disabilities Research Reviews, 5(3), 215-224.

Copyright © 2015 Dr. Bill JU, Neurosciences, Human Biology Program

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Review of Intentional and Incidental forgetting Chantel George

Rizio & Dennis (2013) studies cognitive processes involved in encoding and maintaining information. Specifically, the neural correlates and cognitive processes existing for incidental and intentional forgetting are under question. It is hypothesized that incidental forgetting is involved in encoding attempts whereas intentional forgetting is involved in inhibitory processes and as a result, have different brain activity. A directed forgetting paradigm is used to acquire intentional and incidental forgetting in participants. .An MRI is also used to visualize the brain regions that show activity during incidental and intentional forgetting. Results indicate that intentional forgetting in participants is involved with right parietal cortex activity, prefrontal cortex activity and less medial temporal lobe activity. Furthermore, incidental forgetting is involved with more activity in the left inferior frontal gyrus, left superior frontal gyrus, early visual cortex, and left superior parietal lobe.Therefore, intentional and incidental forgetting are two separate processes with different neural correlates. Further research is discussed in order to advance knowledge of forgetting and ultimately the control of cognitive processes and memory.

Background Rizio & Dennis (2013) studies neural activity and processes occurring during remembering and forgetting. The neural activity expressed during forgetting is essential to understanding cognitive processes involved in the encoding and maintenance of information in the brain. The two types of forgetting are incidental and intentional forgetting. The difference between intentional and incidental regarding neural activity. Concrete evidence of activity in certain parts of the brain for intentional forgetting and different activity for incidental forgetting is needed. It is questionable whether incidental forgetting is related to intentional forgetting in the sense that both follow the same neural processes or if both adopt different neural activity. Rizio & Dennis (2013) proposes that if they have different neural activity, incidental forgetting should adopt neural activity involved in the inadequacy of encoding information and intentional forgetting should adopt neural activity involved in control and suppression. Many studies have shown that the prefrontal cortex (PFC) is associated with inhibitory cognitive processes and control. Depue et al., (2007) shows that there is increased PFC activity and decreased hippocampus activity when analyzing participants trying to subdue emotional memories. Anderson et al., (2004) also found that when analyzing neural correlates of participants trying to suppress undesired memories, there was more prefrontal cortex activity and less hippocampus activity. In addition, Benoit & Anderson (2012) used FMRI imaging to investigate neural correlates underlying intentional forgetting and found that there was an increase in PFC as well. Berkman et al., (2009) study investigates the intentional forgetting of emotional stimuli and found more prefrontal cortex activity and less amygdala activity. Regarding the encoding of information, Dougal et al., (2007) illustrates that the medial temporal lobe (MTL) plays an important role. When using an FMRI to measure brain activity in participants encoding information, the MTL activity was shown significantly.. Although neural correlates underlying cognitive encoding and inhibition is observed, the connection to 89

incidental and intentional forgetting is uncertain. Rizio & Dennis (2013) seeks more concrete evidence of the PFC activity and MTL suppression through imaging.

Therefore if incidental and intentional forgetting stimulate different cognitive processes, incidental forgetting should have more prefrontal cortex and MTL activity.

Alternatively, intentional forgetting should have more prefrontal cortex and parietal lobe activity. Dennis et al explores the neural correlates underlying intentional forgetting and incidental forgetting. As a result, the role of MTL, parietal lobe and prefrontal cortex in the context of memory is investigated.

Research Overview SUMMARY OF MAJOR RESULTS Neural activity in intentional and incidental forgetting To prompt intentional and incidental forgetting, Rizio & Dennis (2013) use a directed forgetting paradigm was executed with participants from18-26 years of age.

In the first stage, participants were instructed to either forget or remember words that were presented to them.

Then, there was a distraction task. Lastly, there was a retrieval stage in which participants informed whether they recalled words that were presented to them. The words included old words from the first stage and new words that they were not shown before. The retrieval stage indicated whether words were intentionally forgotten or incidentally forgotten. A word was deemed intentionally forgotten if it was directed to be forgotten in the first stage and was not recalled in the third stage.

A word was deemed incidentally forgotten if participant was told to remember it in the first stage but they ended up forgetting it in the third stage. An MRI scanner was utilised in order to show what brain regions were active during incidental and intentional forgetting. The right parietal cortex and prefrontal cortex is active during intentional forgetting. During incidental forgetting, the left inferior frontal gyrus, left superior frontal gyrus, early visual cortex, and left superior parietal lobe are shown to have more activity. In addition, there is an increase

in prefrontal cortex activity and a decrease in medial temporal lobe activity during intentional forgetting suggesting inhibition by the prefrontal cortex. On the contrary, there is no such inhibition by the prefrontal cortex activity suppressing medial temporal lobe activity during incidental forgetting.

Image from: Dennis, N. A., Rizio, A. A. (2013). The Neural Correlates of Cognitive Control: Successful Remembering and Intentional Forgetting. Journal of Cognitive Neuroscience, 25(2), pp. 297-312.

Figure 1. shows neural correlates in incidental and intentional forgetting

Conclusions and Discussion Rizio & Dennis (2013) et al reveals that memory inhibition and memory loss entail different neural pathways depending on whether memories are incidentally or intentionally forgotten. The hypothesis that incidental and intentional forgetting are separate and have different underlying neural activity is evident. The right parietal cortex and prefrontal cortex activity are shown to be involved in cognitive control and therefore are seen to be active during processes that allow one to control memory such as intentional forgetting. Since there a decrease in MTL activity and an increase in PFC activity during intentional forgetting, it suggests that an inhibitory processes may be in place in which PFC has an inhibitory role of suppressing MTL activity.

In contrast, results indicate that incidental forgetting is the consequence of the aim but lack of success in encoding information.

Criticisms and Future Directions

Although Rizio & Dennis (2013)) provides substantial evidence of incidental and intentional forgetting being two different processes, further considerations can be made to improve research on the cognitive manipulation of memory. Rizio & Dennis (2013) studied participants between the ages of 18 and 26.

Studying other age groups aids in the understanding of the control of memory regarding intentional and incidental forgetting. Rizio & Dennis (2013) observes intentional forgetting for young and middle age adults as well as the elderly. Results show that younger adults have more prefrontal cortex activity during intentional forgetting. Hence, age makes a difference

when observing neural correlates in forgetting and should be considered in future research.

Another important variable that may affect results is the amount of attention allocated to the memory task.

Chiu (2014) uses probes to serve as stimuli during a memory test in order to observe its affect on oneâ&#x20AC;&#x2122;s ability to control memory. It is done in order to assess any trade offs for cognitive resources. In another study, Fawcett(2011) uses a directed forgetting and remembering task and then a task involving the recognition of colours to observe how fast participants were able to respond. Results show that after participants were directed to voluntarily forget a word, they were slower at recognising colours during the second task.

This suggests that there intentional forgetting may use cognitive resources in attempt to control working memory. Regarding the directed forgetting paradigm initiated in Rizio & Dennis (2013), it gives rise to the question of cognitive resources and if there are trade offs between attention to oneâ&#x20AC;&#x2122;s surroundings and forgetting.

Rizio & Dennis (2013) did not test for differences in gender for intentional and incidental forgetting. Yang (2013) observes the affect of female and male voice on directed forgetting and memory. Thus, listening to words being verbally spoken instead of just being shown may have an affect on incidental and intentional forgetting and should be included in further studies. In addition, a test for gender differences in forgetting is beneficial.

Rizio & Dennis (2013) did not explain the affect of timing on intentional and incidental forgetting.

Mcgregor (2014) studies the recollection of memories over a 24 hour period. This brings into question the role of timing of recognition in the current study. By testing different time periods participants are given to make a decision as to whether they recall or do not recall a word will aid with observing the role of timing in recollection.

For the directed forgetting paradigm, Rizio & Dennis (2013) chose words randomly from a medical database. Things brings into question the types of words chosen for the directed forgetting paradigm. Sahakyan (2008) tests the strength of words in intentional forgetting. Specifically, word strength was primed by associating words with a positive emotion in comparison to other words, allotting more time to process some words but not others, and allowing some words to be repeated over some time in comparison to some words being repeated consecutively. The current study can be improved by incorporating the strength of words and the affect strong and weak words it has on the ability to intentionally and incidentally forget.

Festini(2013) tests for the familiarity of words and the ability to intentionally forget. Festini(2013) notes that information learned in the past(familiar information) can interfere with the learning of new material. Therefore, further studies should be initiated in order to test the affect of familiarity on intentional and incidental forgetting by using a memory test inducing familiar words and a test to recall them.

The affect of emotion on intentional and incidental forgetting is not addressed in the Rizio(2013) study.

Maddock (2009) reveals the importance of emotion 90

in memory as results show that words associated with positivity are increased in spatial and temporal memory and are recalled more in comparison to words associated with negativity. In addition, Padovani (2011) studies memory by presenting words that encourage emotional responses and feelings. Therefore, along with studying neutral words, an experiment using emotional words can aid in elucidating the affect of emotion on incidental and intentional forgetting.

Learning about memory control helps with one’s understanding of memory control. However, the next step can to be to learn more and apply the knowledge in issues today. One way would be to apply knowledge about forgetting to post traumatic disorders so that memory control involved in traumatic experiences can be understood. Trauma involves occurrences that result in negative and shocking emotions. Bailey (2012) study reveals that participants have more difficulty with forgetting emotionally driven words because emotions hinder ones cognitive ability to control memories. Zwissler (2012) investigates intentional forgetting in Post traumatic stress disorder patients.

Findings show that patients exhibit less intentional forgetting. Therefore, more research on intentional and incidental forgetting may facilitate a better understanding of memory and existing issues such as post traumatic stress disorder.

References 1. Bailey (2012). When can we choose to forget? An ERP study into item-method directed forgetting of emotional words 2. Chiu, C., Egner, T. (2014). Inhibition-Induced Forgetting: When More Control Leads to Less Memory. Psychological science, 26(1), pp.27-38.

3. Padovani, T., Koenig, T., Brandeis, D., Perrig, W. T. (2011). Different Brain Activities Predict Retrieval Success during Emotional and Semantic Encoding. Journal of Cognitive Neuroscience, 23(12), pp. 4008–4021.

4. Rizio, A. A., Dennis, N. (2013). The Neural Correlates of Cognitive Control: Successful Remembering and Intentional Forgetting. Journal of Cognitive Neuroscience, 25(2), pp.

297-312.

5. Dennis, N. A., Rizio, A. A. (2013). The Neural Correlates of Cognitive Control: Successful Remembering and Intentional Forgetting. Journal of Cognitive Neuroscience, 25(2), pp.

297-312.

6. Depue, B. E., Curran, T., & Banich, M.T. (2007).

Prefrontal regions orchestrate suppression of emotional memories via a two-phase process. Science, 317, 215-219.

7. Fawcett & Taylor(2012). The control of working memory resources in intentional forgetting: Evidence from incidental probe word recognition 8. Festini (2013)Cognitive control of familiarity: Directed forgetting reduces proactive interference in working memory.

9. Maddock (2009). Reduced memory for the spatial and temporal context of unpleasant words 10. Mcgregor (2014). What a Difference a Day Makes: Change in Memory for Newly Learned Word Forms Over 24 Hours 91

11. Anderson, M. C., Ochsner, K. N., Kuhl, B., Cooper, J., Robertson, E., Gabrieli, S.W., et al (2004). Neural systems underlying the suppression of unwanted memories. Science, 303, 232-235.

12. Benoit, R. G., & Anderson, M. C., (2012). Opposing Mechanisms support the voluntary forgetting of unwanted memories. Neuron 76, 450–460.

13. Berkman, E. T., Burkland L., Lieberman M. D. (2009).

Inhibitory spillover: Intentional motor inhibition produces incidental limbic inhibition via right inferior frontal cortex.

NeuroImage 47, 705–712.

14. Dougal S., Phelps E. A., & Davachi, L. (2007)/ The role of medial temporal lobe in item recognition and source recollection of emotional stimuli . Cognitive, Affective, & Behavioral Neuroscience 7 (3), 233-242.

15. Sahayakan (2008). Intentional Forgetting Is Easier After Two “Shots” Than One. 16. Rizio, A., & Dennis, N. (2014). The Cognitive Control of Memory: Age Differences in the Neural Correlates of Successful Remembering and Intentional Forgetting. PLoS ONE, 9(1), pp. 1-12.

17. Yang, Sujin & Gih0 (2013). Her Voice Lingers on and Her Memory Is Strategic: Effects of Gender on Directed Forgetting 18. Zwissler (2012). Memory control in post-traumatic stress disorder: evidence from item method directed forgetting in civil war victims in Northern Uganda

Received Month, ##, 200#; revised ##, 200#; accepted Month, ##,

Month, 2013.

This work was supported by The Association for the Development of Undergraduate Neuroscience Education (SRA & RLN), The Endowment for Science Education (EA), and The Synaptic State Faculty Research Foundation (EA). The authors thank Mr. Spine L. Cord, Dr. Amy G. Dala, and the students in Neuroscience 101 for technical assistance, execution, and feedback on this lab exercise.

Address correspondence to: Dr. Rita L. Neurotrophin, Biology Department, 123 Growth Cone Avenue, Action Potential College, Hillock, IL 60101 Email: rln@apc.edu Copyright © 2015 Dr. Bill JU, Neurosciences, Human Biology Program

Narrowing It Down to One Locus, to One Chromatin Remodeling by G9a David Giang

Chronic stress and drug addiction have been known to modify the transcription factors of both rodents and humans. The process of histone post-translational modification achieves this, which will either allow for up-regulation or down-regulation of a gene expression of specific transcription factors. However, specificity of locus causality to the observed behaviour plasticity was poorly understood, due to the nature of genome-wide epigenetic modifications and chromatin remodeling. With the uses of engineered transcription factor and the novelty of using them in vivo, the ΔFosB transcription factor was one of the many candidates that can be investigated in detail of their direct contribution and their possible molecular mechanism. It is suggested that the ΔFosB may play a role in a feed-forward loop that stabilizes the ΔFosB levels by phosphorylation by CaMKII. Other proposed mechanism involves the levels of CREB being phosphorylated by the downstream action of the ΔFosB. In hopes to be applied in a clinical setting, the use of engineered transcription factors shows promising future in neuropsychiatric treatment, as well as generalizing to possible gene therapy of other neurological disorders by epigenetic approach.

Background Drug addiction and stress have been recognized to cause changes within the human genomes, which have been correlated with the change in baseline levels of transcription factors. The regulations of these transcription factors consist of either an increase levels from activation or decrease levels from repressive mechanisms. One fundamental way to regulate the transcription factors responsible for the behaviour plasticity we observed is due to epigenetic changes at the sites of crucial gene loci.1 Drug abuse, such as repeated cocaine administration, and stress are environmental factors that can result the respective behaviours of addiction and stress-evoked depression.1,2 The main determinant of the penetrance of causing these behaviours is the amount of histone modifications associated on the gene of interest from drug or stress induction.1,2 As a result, one main focus for epigenetic for addiction and depression is the role of chromatin remodeling in their corresponding pathways and which genes and enzymes are involved.2,4 The focus of this study is the action of the main enzyme is the histone methyltransferase is the G9a enzyme, which dimethylize on histone 3, lysine 9 (H2K9me2) and the FosB gene.3,4 In particular, the FosB and its splice variants ΔFosB transcription factor have been found to play an important in the behaviour plasticity in cocaine addiction. When cocaine is administrated, the ΔFosB transcription factor is induced and overaccumulation of ΔFosB in the nucleus accumbens (NAc) leads to increase locomotor sensitivity, consistent with cocaine addiction.1,4,5 The molecular mechanism proposed was that CaMKIIalpha was stabilizing the ΔFosB after the induction of CaMKIIalpha gene expression by ΔFosB.2 This feed-forward loop allows for the stabilization of ΔFosB and the addictive behaviour observed. Additionally, stress-evoked depression and chronic stress were both shown to exhibit reduced Fosb/ ΔFosB levels in humans and rodents1,2 Ultimately, ΔFosB plays a key role in both of these reward behaviours.

However, these inferences were made under the limitation that the epigenetic modifications were done on a global genome level.5 Many loci are affected simultaneously by epigenetic changes, so a causal relationship of a certain histone modification on a specific gene cannot be translated into the observed behaviours of humans and rodents. In order to bypass this problem, the authors of this article plan to utilize zinc-finger proteins (ZFP) to act on specific site on the genome when fused with the G9a catalytic subunits.3,6 ZFP are transcription factors that can provide a means to regulate certain loci in the genome. When fused with a catalytic subunit, the preferential histone modification may occur at the locus of interest.

In addition, transcription activator-like effectors (TALEs) were also used in order to allow for increased level of gene expression by histone acetylation. ZFPs and TALEs, even though are engineered transcription factors to enhance specificity and proof on epigenetic changes, were not used,in an intact biological context in retrospect.1 Therefore, the goal of this study is to obtain this specificity in an in vivo setting for locusspecific epigenetic causality, using these bidirectional transcription factors.

Research Overview

Summary of Major Results

The experiment conducted for this article consisted of the rodent model of mice, injecting the viral vectors at the site of the NAc. A control virus was used, as well as saline to control for the cocaine injections. Two series of models were used for each behaviour plasticity. For addiction, repeated cocaine treatment was given with the intention of a sensitizing treatment or a subthreshold treatment. The difference between the two is that the mice with the subthreshold treatment will not have the locomotor sensitivity that the sensitizing treatment will have, but are more susceptible 92

than the control mice without either dosing. The mice were then transfected with a herpes simplex virus (HSV) that have either a ZFP or TALE transcription factors that binds to the Fosb gene, as well the G9a methyltransferase catalytic subunit or the p65 catalytic subunit, which is responsible of activating transcription by histone acetylation. A non-functional domain (NFD) was also incorporated in order to control for the catalytic domain’s presence being sufficient to cause ΔFosB-dependant behaviours.

TALEs and underwent a chronic social defeat stress, leaving the mice in a subthreshold social stress. It was observed that the mice infected with HSV-FosbZFP35-G9a have a reduction in social interaction, as well as less time in the open arms of the elevated plus maze. These indicate increased anxiety and depression-like behaviour responses in comparison to the control virus.

These results observed from this study were consistent on other publications’ result on the association that ΔFosB has in the roles of these reward behaviours.

The authors were able to obtain the expected result from H3K9me2 on the Fosb gene, where overexpression of this histone modification leads to blocking locomotor sensitivity or stress-evoked depression. On the other hand, transcriptional activation of VP64 and p65 subunits overexpressed ΔFosB and Fosb when the analyzing mRNA levels by quantitative reverse transcription PCR. Conclusions and Discussion

(Heller E.A. et al, 2014) Figure A & B– Schematic setup for the experiment of mice that were infected with the ZFP-p64 (“On”), as well as ZFP-TALE1v64 (“On”) or ZFP-G9a (“Off”). NFD strain is representing a ZFP with no functional domain.

Figure C – The locomotion shown in the rats, where elevated locomotion were found a trend between days and the high cocaine dose for figure C. Figure D - The low dose of cocaine that is subthreshold (cause no locomotion for normal mice with this cocaine dose) It was observed that enhanced locomotion from day 4 to day 16, and indication of a trend of dose and days. Both high dose and low dose cocaine treatment can be blocked by ZFP-G9a injection.

Figure E – Control ZFP with NFD strain Figure G to J – Shows the depression behaviour of the mice that were injected with H3K9me2 enrichment. Decrease in social interaction and exploration of open arm maze.

When the mice were injected with an HSV-FosbZFP35-G9a, it was found that its expression was proficient to block cocaine locomotor sensitivity. On the other hand, HSV-fosb-ZFP35-p65 and HSVTALE1-VP64, another activator catalytic subunit were able to increase the sensitivity of the locomotion, indicating a more heightened response and susceptibility to cocaine addiction. The findings in this study indicate that an increase in G9a activity can prevent locomotion sensitivity, while activating the ΔFosB gene will augment the sensitization of cocaine. Heat maps and the amount of beam break counts measured the results observed above.

For measuring stress-evoked depression in the mice that were transfected with the same sets of ZFP and 93

The authors were able to confidently display how a locus-specific interaction was able to regulate the behaviour plasticity of the mice that would normally show either resilience to depression or increased locomotor activity that is consistent with repeated cocaine administration.1,7,8 In order to establish the efficacy of engineered transcription factors, ZFP and TALE were used to target the ΔFosB gene. Since the function of ΔFosB in addiction and depression is well understood, it is clear that the epigenetic changes on a single locus is causing the behaviour changes observed and not any other factors, such as a confound phenomenon controlled from using the FosB gene or any technical confounds. These confounds, such as the administration of an injection, a catalytic subunit, or the use of HSV lead to the results that are observed. In addition, 28 off-target genes were also observed to control for ΔFosB expression in the NAc having adverse effects. Other related histone modifications were investigated, such as other methylations within the same histone, in order to establish the mechanism of action by the G9a subunit. The authors believe that the results they have received can only be explained from the G9a subunit activities acting on the H3K9 site of the Fosb gene promoter, where it is only this one kind of histone modification and one locus of the genome.

It was a fairly clean experiment, where all confounds that can arise in an in vivo setting can skew the results were controlled for, from the mechanism of action by the enzyme to the techniques of transfection. Overall, this ruled out any data obtained stochastically.

A possible molecular mechanism that may result from the H3K9me2 that the authors presented is that it involves the molecule CREB.1,9 H3K9me2 is believed to prevent the phosphorylation of the CREB at the Fosb gene, preventing further expression of ΔFosB and Fosb proteins alike. This was observed in the qChIP analysis, where cocainetreated mice had a 3.9-fold increase in phosphor-CREB levels when compare to the saline injection and did not affect total number of CREB or increased CREB binding.

This suggest that the M3K9me2 is only specific to halting phosphorylation of CREB, in turn leading to ΔFosB levels to be blocked and causing the observed reward behav-

iours that are expected with this gene.

The significance of this paper was one of the first innovators in the field to use engineered transcription factors as a way to alter the behavioural plasticity in depression and addiction. Epigenetic have been a field for how gene regulation can impact human health from a neuropsychiatric stand point, but were not able to obtain any possible causality due to the limits of global chromatin remodelling that can occur. By utilizing ZFP and TALE, the ΔFosB gene was well regulated

by H3K9me2 or activation.1,5,7 This suggests that the novel use of single locus regulation can not only help study the epigenetic basis of addiction and depression better, but also be able to impact the treatment for these neuropsychiatric disorders. The impact this paper has shown is the possibility that the future of gene therapy is possible and that may have hopes of generalizing this approach to other diseases with a epigenetic basis.

Criticisms and Future Directions

As mentioned earlier, the paper was able to successfully present that a single locus that is regulated by epigenetic mechanisms play a role in addiction and depression behaviour. However, being a novel field in using engineered transcription factor in an intact biological context, more insight will be required to determine whether or not they will be sufficient enough to be used in a clinical setting.

Also, the data were based off of repeated cocaine administration in the mice but any indications for acute drug administration were not investigated. ΔFosB have been recognized by other literatures as the molecule switch for addiction. To appropriate evaluate the role of ΔFosB requires fine regulation of the gene promotion, which have became possible from the revolution of this method. The use of injecting HSV vector virus may not be the only virus that should be used, as it has its own limitations. Therefore AAV and other virus to use as a vector should be considered.

In addition to the experiment, mice were put under the chronic stress model that involved an aggressor. That is a fear-based response and is too simple compare to the kind of depression a human may face from losing a loved one or undergoing meaninglessness in their everyday life. This may involve different mechanisms since the response to anger and tragedies do have variable differences. In addition, elevated plus maze was the only model to measure the mice for depression based off of their will to explore, but did not cover any other aspects of depression such as their lost of appetite or possible cortisol measurement to determine if it was truly stress-dependent. Overall, stress-evoked depression was not covered as explicitly as cocaine-addiction and further works to conduct in chronic stress could lead to a better understanding.

Other future directions this paper should consider would be to investigate other gene targets in the genome in this locus-specific manner in order to see its relevance in other fields of neuropsychiatric treatment. For example, HDAC2 has been found to be affecting the responses in antipsychotic treatment. By being able to regulate the expression of the mGluR2 promoter activity, this may lead to increase efficacy of antipsychotic drug treatment,

yielding better response and aspiring for a bright future in clinical treatment for schizophrenia.10,11 In addition to HDAC2 as an epigenetic approach to target gene, stress and depression can also be regulated from another pathway involving the epigenetic changes in RAC1.11,12, Though the evidence for this is recent, this publication was carried out under the limitation of multiple genome-wide epigenetic.13 By applying the same use of engineered transcription factors, it may bring more insight into the synaptic remodelling that occurs in this other gene involved in stress evoked depression.14,15 References 1. Heller, E.A. et al. Locus-specific epigenetic remodeling controls addiction- and depression-related behaviours. Nat. Neurosci. 17, 1720-1727 (2014).

2. Robison, A.J. et al. Behavioural and structural responses to chronic cocaine requires a feedforward loop involving ΔFosB and calcium calmodulin-dependent protein kinase II in nucleus accumbens shell. J. Neurosci. 33, 4295-4307 (2013).

3. McNamara, A.R. et al. Zinc finger protein targeted epigenetic gene regulation toward direct long-term gene control. Mol. Ther. 9, S123-s124 4. Maze, I. et al. Essential role of the histone methyltransferase in cocaine-induced plasticity. Science. 327,213-216 (2010).

5. Shinkai, Y. & Tachibana, M. H3K9 methyltransferase G9a and the related molecule GLP. Genes Dev. 25, 781-788. (2011) 6. Kelly, M.A. et al. Locomotor activity in D2 dopamine receptor-deficient mice is determine by gene dosage, genetic background and developmental adaptation. J. Neurosci. 18, 3470-3479. (1998) 7. Oh S-T. et al. H3K9 histone methyltransferase G9a-mediated transcriptional activation of p21. FEBS Letters.

588, 685-691. (2014) 8. Rice, C.J. et al. Histone methyltransferase direct different degrees of methylation to define distinct chromatin domains.

Mol. Cell 12, 1591-1598. (2003) 9. An essential role for ΔFosb in the nucleus accumbens in morphine action. Zachariou, V. et al. Nat. Neurosci. 9, 205-211. (2006).

10. HDAC2 regulates atypical antipsychotic responses through the modulation of mGlu2 promotor activity. Kurita, M. et al.

Nat. Neurosci. 17, 1245-1254. (2012).

11. Epigenetic regulation of RAC1 induces synaptic remodeling in stress disorders and depression. Golden, S.A. et al.

Nat. Med. 19, 337-344. (2013).

12. Hyman, S.E. Target practice: HDAC inhibitors for schizophrenia. Nat Neurosci. 15, 1180-1181. (2012).

13. Damez-Werno, D. et al. Drug experience epigenetically primes Fosb gene inducibility in rat nucleus. J. Neurosci. 32, 10267-10272. (2012).

14. Nestler, E.J. et al. FosB: a sustained molecular switch for addiction. PNAS. 98, 11042-11052. (2001) 15. Garcia-Perez et al. Glucocorticoids regulation of Fosb/ ΔFosb expression induced by chronic opiate exposure in the brain stress system. PLoS ONE. 9, 1-13. (2012) 94

The Novel Role of mTOR-Dependent Macroautophagy in Autism Spectrum Disorder Jessica Gosio

Autism spectrum disorders (ASD) is characterized by deficits in cognitive abilities including communication skills, social interactions, and emotional control, and yet is poorly understood at a biological level. One common characteristic of the disorder is aberrant excessive cortical dendritic spine growth and reduced pruning of postsynaptic glutamatergic synapses. Tang et al. hypothesize mutations in genes that inhibit the mammalian target of rapamycin (mTOR) kinase to be linked to ASD, as overactive mTOR is thought to lead to excessive synaptic protein synthesis. mTOR also acts downstream to inhibit macroautophagy (autophagy) – a process involved in neuronal pruning. They found that the hyperactivity of mTOR lead to reduced autophagy and loss of autophagy caused increased spine densities in late postnatal development in ASD-model mice. The findings by Tang et al. enhance the understanding of ASD at a cellular level thereby providing molecular targets for novel therapeutics, as well as establish a foundation upon which future ASD research may be conducted.

Key words: Autism Spectrum Disorder (ASD); mammalian target of rapamycin kinase (mTOR); rapamycin; autophagy; TSC2; synaptic pruning; dendritic spines Background Autism spectrum disorders (ASDs) are characterized by cognitive and social deficits, as well as aberrant changes in cortical size. Structural differences in the cortices of ASD patients have been hypothesized to play a role in the behavioral phenotypes observed.

In non-ASD subjects, synapse formation and high dendritic spine densities are found early in human development and are balanced with protein degradation creating a reduction in spine density and increased synaptic pruning during childhood and adulthood (Penzes, Cahill, Jones, VanLeeuwen, & Woolfrey, 2011) in order to maintain homeostasis (Purves & Lichtman, 1980). In ASD patients, these processes are disrupted, resulting in increased spine density in excitatory pyramidal cells of the frontal, parietal, and particularly strong increase found in cells of the temporal lobe (Hutsler & Zhang, 2010), thought to be due to decreased synaptic pruning (Zhan et al., 2014).

These changes create the differences in cortical size reported in ASD subjects, as well as provide structural evidence that may explain earlier reported anomalous changes in brain circuitry connections(Belmonte et al., 2004), as synaptic pruning and spine density reductions are critical for correct formation of synaptic circuits in a developing brain. Further investigation is required to understand the importance of spines and pruning in the onset and progression of ASD. Although only a small proportion of ASDs are due to known genetic mutations, some cases with genetic origin are caused by mutations in Tsc1/Tsc2 - genes that normally act to inhibit the mammalian target of rapamycin (mTOR) kinase (Bourgeron, 2009). mTOR is a regulator of cell growth and activates protein synthesis at the synapse (S. J. Tang et al., 2002). It has been found to be associated with proteins found in neuronal synapses and dendritic spines, such as Shank (Peça & Feng, 2012), a protein that has recently been reported to share a protein binding partner with Tsc1/Tsc2 at neuronal 95

synapses (Sakai et al., 2011), connecting two molecular pathways related to ASD (Peça & Feng, 2012).

It is predicted that overactive mTOR signaling due to described mutations in mTOR-inhibitory genes results in excessive synaptic protein synthesis observed in ASD. ASD, though, is also associated with decreased synaptic pruning – a process involving synaptic protein degradation and therefore macroautophagy (also referred to as autophagy) – the removal of damaged or degraded proteins in a cell. mTOR lies upstream of autophagy and acts to inhibit its processes (Kim, Kundu, Viollet, & Guan, 2011). It can be inferred then that loss of mTOR inhibition also leads to strong inhibition of autophagy. Tang et al. therefore predicted that mTOR-dependent autophagy coupled with mTOR over-activity is responsible for the reduction in synaptic pruning and increase in dendritic spine production observed in ASD subjects (G. Tang et al., 2014).

Evidence supporting this new theory of ASD stems from previous studies of tuberous sclerosis model mice where heterogeneous inactivation of their Tsc2 gene (Tsc2+/-) showed deficits of learning and memory related to mTOR hyperactivity, and rapamycin administration (an inhibitor of mTOR kinase) reversed learning and behavioral deficits observed in the mice (Ehninger et al., 2008). ASD behavioralphenotypes have not been assessed in this paradigm, though autism and tuberous sclerosis are both functionally related to mutations in the mTOR pathway (García-Peñas & Carreras-Sááez, 2013). Few studies have assessed autophagy in relation to ASD, but a recent study identified ASD-related mutations in genes encoding proteins for autophagy processes (Poultney et al., 2013). Autophagy, however, has been assessed in synaptic remodeling paradigms involving C. elegans (Rowland, Richmond, Olsen, Hall, & Bamber, 2006) and Drosophila (Shen & Ganetzky, 2009), but never in a developing mammalian model system. Tang et al. therefore tested their hypothesis, and found mTOR-dependent autophagy to be required for correct synaptic pruning and spine

growth in the brains of developing mice, and mTOR over-expression to underlie ASD behavioral and physical pathologies.

Research Overview

Summary of Major Results

Dendritic spine pruning deficits in temporal lobe of human ASD patients compared to controls

Tang et al. began by confirming previously reported increased dendritic spine density in layer V pyramidal neurons of the superior middle temporal lobe from the brains of post-mortem ASD patients, an area known for its participation in social and communication networks in the brain. They also found ASD patients displayed greater dendritic spine density throughout later stages of life, in-line with predicted reduced spine reduction throughout development (Fig 1a, b).

Figure 1. a) Representative Golgi images of human temporal lobe for two control subjects (C = aged 8 years and 18 years) and 2 ASD subjects (A = aged 7 years and 15 years). Images depict a greater density of basal dendritic spines found in layer V of the human temporal lobe. b) Linear regression displaying increasing spine density between child and adolescent autistic patients (n=10) compared to controls (n=10). (G. Tang et al., 2014).

Tsc-deficient mouse models of ASD displayed spinepruning defects

Using Tsc2+/- mutated mice, since Tsc1 and Tsc2 mutations lead to mTOR-hyperactivation and ASD-like behaviors in mice (cite pg 1133 of paper), behavioral assays were performed to determine if mice displayed ASD-phenotypes. Tsc2+/- mice were found to display reduced social preference in a novel object recognition test compared to wild-type mice, later confirmed with a three-chamber social test, but ASD repetitive behaviors were not observed. Researchers then assessed

the cellular phenotypes of the mutated mice, finding a substantial amount of spine pruning between post-natal days (P)19-(P)20 and P29-P30 in wild-type mice, and a lack of this normal developmental pruning in Tsc2+/- mice. These increased dendritic spine density in later stages of life of Tsc2+/- mice are similar to patterns observed in human ASD subjects (Fig 2a,b).

Tsc2+/- mice were further administered rapamycin as an mTOR antagonist, with similar inhibitory activity as the functional Tsc2 gene, and found similar dendritic spine morphologies as Tsc2+/+ wild-type mice and no effects on these control mice as predicted (Fig 2b).

This result, in combination with earlier behavioral data suggests Tsc2+/- mice can be used as an ASD model organism as its behavioral and cellular phenotypes recapitulate those observed in human ASD subjects.

Rapamycin only decreased spine density in later stages of development; therefore its effects on spine pruning represented both early and late non-ASD mouse development (Fig 1b, 2b), suggesting the importance of mTOR inhibition in mammalian cortical development.

Figure 2. a) Confocal images of increased dendritic spine density in Tsc2+/- mice and reduce pruning between postnatal day 20-21 and 29-30. Reduced spine pruning was corrected to control levels with administration of rapamycin (Rapa). Scale bar, 2 um. b) Linear regression representing (a), n=7-10 mice per group, **compared with wildtype at P29-P30, p < 0.01 (two-way ANOVA). (G. Tang et al., 2014).

Impaired autophagy due to mTOR disinhibition underlies neuronal spine pruning defects in ASD-model mice with mutated Tsc2+/ Tang et al. could now begin to assess their prediction of a Tsc2/mTOR pathway controlling autophagy required for the pruning at synapses in their ASD-model mice. A western blot revealed highest mTOR activity in Tsc2+/- compared to 96

wild-type mice, and also found a strong reduction in synthesis of proteins involved in autophagy (Fig 3a), as predicted by the researchers. To further determine if autophagy is required for correct spine pruning in temporal lobe pyramidal neurons and if autophagy deficiency underlies ASD spine pruning pathology, autophagy knockout mice (Atg7CKO) coupled with mice over-expressing mTOR (Tsc2+/-) were compared to control mice (Tsc2+/+; Atg7flox/flox) (Fig 3b, c). Controls depicted typical early high spine density followed by a reduction in spine density later in development, and no differences were observed upon rapamycin administration for controls, and no ASDlike behaviors observed upon testing of sociability and social novelty in a three-chamber test (behavioral data figures not included). Tsc2+/-; Atg7flox/flox mice with functioning autophagy, were found to produce ASD-like spine densities that were corrected with rapamycin administration, similar to results in Fig 2b, and displayed ASD-behavioral phenotypes corrected to those of controls after rapamycin. Both Tsc2+/+ and Tsc2+/- mice with Atg7CKO experienced high spine density throughout early and late stages of development that were not reduced to control levels with rapamycin, and ASD-behavioral phenotypes were not corrected. This provides strong evidence supporting the predictions made by Tang et al., that mTOR hyperactivity caused by mutations in mTORinhibitory genes that effects downstream expression of autophagy proteins, inhibiting autophagy functioning, and that autophagy is the underlying mechanism behind neuronal synaptic pruning defects observed in ASD model mice.

Discussion and Conclusion Tang et al. analyzed spine density during development and found a greater dendritic spine density in ASD subjects at later developmental time points than non-ASD subjects. These atypical neurons were excitatory pyramidal cells found in layer V of the temporal lobe, suggesting that their increased dendritic spine density actually indicates strengthened local excitatory connectivity – a feature of ASD (Belmonte et al., 2004).

The approximately linear decrease in spine density continuing all the way into the 19th year of human development showed a greater reduction in synapses in normal controls than in ASD patients. This may explain why ASD patients often suffer from deficits in higher cognitive functions such as reasoning and judgment that develop during a person’s late teens (Sternberg & Berg, 1992), as well as provide clinicians with an idea for most efficacious times for therapeutic intervention.

Although it is likely that not all ASD subjects studied possessed TSC mutations, the researcher’s findings of pruning deficits along with mTOR signaling dysregulation suggests that mTOR is a common harmonizing pathway in ASD. The replication of these described dysfunctioning mTOR pathways along with increased spine numbers, decreased synaptic pruning, and ASD-behavioral phenotype in development designates Tsc2+/- mice as a strong mammalian model for ASD. This study also explored the amending role of rapamycin in ASD and its relation to autophagy, 97

Figure 3. a) Western blot revealing p-mTOR (mTOR activity) highest in Tsc2+/- mice and lead to decreased autophagy activity (LC3-II), which were corrected with rapomycin. b) Representative images of dendritic spines administered either DMSO vehicle or rapamycin. Scale bar, 2 um. c) Graphic depiction of (b) Mean +/- SD. (b-c) showed Tsc2+/- mice that also had no autophagy (Atg7CKO ) were treated with rapamycin and found no correction of pruning and dendrite density of the Tsc2+/-:Atg7CKO mice or just Ast7CKO mice, like the corrections observed with Tsc2+/- mice. (G. Tang et al., 2014).

and has already been implicated in clinical trials for treatment of ASD (Sahin, 2012). Tang et al. also confirmed that by blocking neuronal autophagy, ASDpathological declines in spine density and stereotypical ASD behaviors were observed and could be corrected by pharmacologically activating autophagy via mTOR kinase inhibition, indicating a link between mTOR and autophagy that is required for proper synaptic spine pruning. Therefore, Tang et al. demonstrated a novel foundational mechanism underlying ASD cellular and

behavioral phenotypes that entails mTOR hyperactivation inhibiting downstream neuronal autophagy that leads to structural and functional deficits in postnatal development of mice. This is not only the first mammalian evidence of synaptic remodeling through autophagy, but also emphasizes the importance of the role of autophagy in ASD that was previously overlooked. Greater understanding of this mTORdependent autophagy pathway may provide potential novel ASD therapeutic targets in previously unexplored autophagy pathways downstream mTOR signaling and acts to advance knowledge on the mechanisms underlying the debilitating ASD. Criticisms and Future Directions Although Tang et al. found their gene mutation studies to lead to mice with reduced social preference (characteristic of ASD) using a dyadic social interaction and reduced exploration in a novel-object recognition paradigm, researchers failed to demonstrate that their mice displayed self-grooming repetitive behaviors (also characteristic of ASD), decreasing the confidence in their ASD mammalian model. I purpose the researcher’s perform a marble-burying test to examine repetitive behavior further (Silverman, 2010).

Studies assessing similar developmental disorders such as idiopathic autism have found decreases in mTOR activity responsible for pathologies (Nicolini et al., 2015), where as Tang et al. are reporting hyperactive mTOR activity as a contributing mechanism responsible for decreased spine pruning in the temporal lobe. This discrepancy in ASD-related cellular functioning may be due to a confound created from the Tsc2 gene knockout in the mice used by Tang et al. Tsc2 inhibits mTOR activity, which is also a member of the Akt cell survival pathway – therefore Tsc2 knockout mice will result in widespread functional impairment of this critical cell survival pathway molecule in other brain and body areas, skewing the validity of experimental results. An improved means of gene mutational analysis lies in the use of a CRISPRCas9 knock-in mouse model recently described by the Zhang lab at MIT, a direct genomic editing method (Platt et al., 2014). With this model, researchers will be able to inject a virus targeting excitatory neuronal cells which will deliver single guide RNA’s that act to block multiple genes acting upstream to inhibit mTOR such as Tsc1/Tsc2, NF1, and Pten, instead of solely mTOR. This will allow for direct genome editing creating a loss of function for genes of interest and only in the areas that receive viral injection, such as the superior temporal gyrus and fusiform gyrus.

Researchers can also examine downstream effects of mTOR if multiple inhibitory genes were removed.

Further analysis can be done using the CRISPRCas9 mouse model to directly overexpress mTOR by creating a knock-in of a highly active promoter in front of the mTOR locus, instead of creating mTOR hyperactivity indirectly through mutating its regulatory genes. This will attenuate the effects of mTOR hyperactivity in specific brain regions to determine clearly how ASD biological and behavioral phenotypes can be seen.

Assessment of the mTOR pathway in association with valproic acid, a substance observed to activate dendritic spines, would be interesting as valproic acid induces autism in fetal mice (Nicolini et al., 2015), and increase the neuronal progenitor cell pool (Go et al., 2012).

In summary, there is much confusion and unknowns when it comes to the biological pathology of autism, but with advances in research like Tang’s group, more doors will open leading to greater findings in the future of autistic research.

ACKNOWLEDGEMENTS

Special thank you to Tang et al. 2014 for their work on ASD, as well as Dr. Bill Ju, Human Biology, at the University of Toronto Canada for the opportunity for this literature review, and the Neuroscience Specialist degree program in Human Biology, at the University of Toronto Canada for the opportunity to take HMB300H1S in 2015.

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Nature Medicine, 14(8), 843–848. doi:10.1038/nm1788 4. García-Peñas, J. J., & Carreras-Sááez, I. (2013).

[Autism, epilepsy and tuberous sclerosis complex: a functional model linked to mTOR pathway]. Revista de Neurologia, 56 Suppl 1, S153–61. Retrieved from http://www.ncbi.nlm.nih. gov/pubmed/23446718 5. Go, H. S., Kim, K. C., Choi, C. S., Jeon, S. J., Kwon, K. J., Han, S.-H., … Shin, C. Y. (2012). Prenatal exposure to valproic acid increases the neural progenitor cell pool and induces macrocephaly in rat brain via a mechanism involving the GSK-3β/β-catenin pathway. Neuropharmacology, 63(6), 1028–41. doi:10.1016/j.neuropharm.2012.07.028 6. Hutsler, J. J., & Zhang, H. (2010). Increased dendritic spine densities on cortical projection neurons in autism spectrum disorders. Brain Research, 1309, 83–94. doi:10.1016/j. brainres.2009.09.120 7. Kim, J., Kundu, M., Viollet, B., & Guan, K.-L. (2011).

AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nature Cell Biology, 13(2), 132–41. doi:10.1038/ncb2152 8. Nicolini, C., Ahn, Y., Michalski, B., Rho, J. M., & Fahnestock, M. (2015). Decreased mTOR signaling pathway in human idiopathic autism and in rats exposed to valproic acid.

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Identification of small exonic CNV from whole-exome sequence data and application to autism spectrum disorder. American Journal of Human Genetics, 93(4), 607–19. doi:10.1016/j. ajhg.2013.09.001 13. Purves, D., & Lichtman, J. (1980). Elimination of synapses in the developing nervous system. Science, 210(4466), 153–157. doi:10.1126/science.7414326 14. Rowland, A. M., Richmond, J. E., Olsen, J. G., Hall, D. H., & Bamber, B. A. (2006). Presynaptic terminals independently regulate synaptic clustering and autophagy of GABAA receptors in Caenorhabditis elegans. The Journal of Neuroscience : The Official Journal of the Society for Neuroscience, 26(6), 1711–20. doi:10.1523/JNEUROSCI.2279-05.2006 15. Sahin, M. (2012). Targeted treatment trials for tuberous sclerosis and autism: no longer a dream. Current Opinion in Neurobiology, 22(5), 895–901. doi:10.1016/j. conb.2012.04.008 16. Sakai, Y., Shaw, C. A., Dawson, B. C., Dugas, D. V., Al-Mohtaseb, Z., Hill, D. E., & Zoghbi, H. Y. (2011). Protein Interactome Reveals Converging Molecular Pathways Among Autism Disorders. Science Translational Medicine, 3(86), 86ra49–86ra49. doi:10.1126/scitranslmed.3002166 17. Shen, W., & Ganetzky, B. (2009). Autophagy promotes synapse development in Drosophila. The Journal of Cell Biology, 187(1), 71–9. doi:10.1083/jcb.200907109 18. Silverman, J. L., Yang, M., Lord, C., & Crawley, J. N. (2010). Behavioural phenotyping assays for mouse models of autism. Nature Reviews. Neuroscience, 11(7), 490–502. doi:10.1038/nrn2851 19. Sternberg, R. J., & Berg, C. A. (1992). Intellectual Development (Vol. 9). Cambridge University Press. Retrieved from https://books.google.com/books?hl=en&lr=&id=lEdlv 99Ql2gC&pgis=1 20. Tang, G., Gudsnuk, K., Kuo, S. H., Cotrina, M. L., Rosoklija, G., Sosunov, A., … Sulzer, D. (2014). Loss of mTOR-Dependent Macroautophagy Causes Autistic-like Synaptic Pruning Deficits. Neuron, 83(5), 1131–1143. doi:10.1016/j.neuron.2014.07.040 21. Tang, S. J., Reis, G., Kang, H., Gingras, A.-C., Sonenberg, N., & Schuman, E. M. (2002). A rapamycin-sensitive signaling pathway contributes to long-term synaptic plasticity in the hippocampus. Proceedings of the National Academy of 99

Sciences of the United States of America, 99(1), 467–72. doi:10.1073/pnas.012605299 22. Zhan, Y., Paolicelli, R. C., Sforazzini, F., Weinhard, L., Bolasco, G., Pagani, F., … Gross, C. T. (2014).

Deficient neuron-microglia signaling results in impaired functional brain connectivity and social behavior. Nature Neuroscience, 17(3), 400–6. doi:10.1038/nn.3641 Received Month, ##, 200#; revised ##, 200#; accepted Month, ##,

Month, 2013.

This work was supported by The Association for the Development of Undergraduate Neuroscience Education (SRA & RLN), The Endowment for Science Education (EA), and The Synaptic State Faculty Research Foundation (EA). The authors thank Mr. Spine L. Cord, Dr. Amy G. Dala, and the students in Neuroscience 101 for technical assistance, execution, and feedback on this lab exercise.

Address correspondence to: Dr. Rita L. Neurotrophin, Biology Department, 123 Growth Cone Avenue, Action Potential College, Hillock, IL 60101 Email: rln@apc.edu

The pivotal role of TNF-α in inducing cognitive dysfunction

Man Lai Ho

Inflammation, if prolonged, is known to cause adverse effects on the CNS and consequent cognitive dysfunction, via pro-inflammatory cytokines produced during the inflammatory process. The cytokine TNF-α is one of major mediators of the innate immune response, and is implicated in neurodegenerative diseases. Therefore, it is of interest to study the effects of TNF-α on the central nervous system and cognition. This review examines, in detail, Jing et al.’s investigation on the effects of intra-amygdala injection of TNF-α on fear conditioning in rats, provides insight on the study, and proposes future directions for further research.

Key words: tumor necrosis factor (TNF-α); glutamate toxicity; amygdala; NMDAR; fear learning Background Inflammation is an integral part of the innate immune system that aids the human body in its defense against harmful stimuli such as pathogens during infection, by attracting various immune cells to the site of infection to eradicate the invasive stimuli, removing damaged tissues and cells, and initiating tissue repair and recovery (Allan & Rothwell, 2003) However, if inflammation is left unchecked and prolonged, it may have detrimental instead of beneficial effects. Neurodegenerative diseases are usually accompanied by chronic neuroinflammation; as such, past studies have shown that pro-inflammatory cytokines produced during the inflammatory process, are consistently associated with several of these neurological disorders including Parkinson’s disease, Amyotrophic lateral sclerosis (ALS), and Alzheimer’s disease, and can cause intellectual deficiencies (Cunningham et al., 2009; Mogi et al., 1994; Paganelli et al., 2002; Poloni et.al, 2000; Reichenberg et al., 2001). The study by Jing, Hao, Bi, Zhang, & Yang (2015) examines, in particular, the pro-inflammatory cytokine tumor necrosis factor α (TNF-α) and its effects on cognition by injecting it directly into the brain of rats.

TNF-α is one of the major immune mediators generated during systemic infection, and it is known that TNF-α plays both pathophysiological and homeostatic roles in the central nervous system (CNS) (Montgomery & Bowers 2011). TNF-α has been shown to reduce long-term potentiation (LTP), an essential mechanism underlying learning and memory, in hippocampal slices (Tancredi et al., 1992). Furthermore, TNF-α overexpression appears to interfere with proper spatial learning and tasks in rats (Aloe et al, 1999). Jing et al. (2015) focuses specifically on the effects of TNF-α on fear learning, which is known to be facilitated by the amygdala (Maren, 2003) Past research has demonstrated that TNF-α induces excitotoxicity and that the glutamate cytotoxicity can be inhibited by the addition of NMDAR (ionotropic glutamate receptor) antagonists (Olmos & Lladó, 2014). Excitotoxicity refers to the neuronal damage or death that occurs due to excessive stimulation by excitatory neurotransmitters, with glutamate being the primary as it is the main excitatory neurotransmitter of the CNS. Thus, Jing et al.

(2015) further postulate that glutamatergic transmission mediates the cognitive effects induced by TNF-α injection.

Research Overview

Summary of Major Results

Indeed, Jing et al. (2015) have found that the intraamygdala injection of TNF-α, in which delivery was facilitated by the surgically implanted cannulae , can induce learning and memory deficits in rats.

Through the use of the classical auditory fear conditioning paradigm, learning and memory function of the rodents was assessed by scoring the freezing responses exhibited by the rats. The paradigm consists of a tone (CS) habituation session, a conditioning session where the tone was paired with a foot shock (US), and an extinction session. Rats that were administered arterial cerebrospinal fluid (ACSF), the control group, displayed a rapid increase and a gradual decrease in freezing behaviour, during the conditioning and extinction phases respectively. In contrast, the TNF-α-treated rats, the experimental group, demonstrated deficient acquisition and extinction of the fear response, as indicated by a much slower increase in freezing behaviour during the conditioning session and a delayed extinction of the freezing response. There were no differences in the foot shock sensitivities of both groups, therefore, increased foot shock sensitivity due to TNF-α as a possible confound, is eliminated.

In order to confirm determine the involvement of the glutamatergic pathway in TNF-α-induced cognitive impairment, Jing and his team subsequently 1) used high performance liquid chromatography (HPLC) to measure glutamate and GABA (control) levels in TNF-α-treated and vehicle-treated rats, and 2) performed a second auditory fear conditioning experiment in which the rodents were administered PBS+TNF-α (control), MK-801 (an NMDAR antagonist), or MK-801+TNF-α before undergoing the trials.

TNF-α-treated rodents were found to have higher levels of glutamate in the amygdala compared to those treated with the ACSF; however, there were no significant differences in GABA levels. In the latter experiment, rats injected with MK-801 showed appropriate acquisition and extinction of fear conditioning, while fear learning is impaired in PBS+TNF-α-treated rats, similar to the rodents infused with TNF-α solely. Furthermore, the administration of TNF-α with MK-801 to rats appears to be able to rescue proper fear learning.

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Conclusions and Discussion

Jing et al. (2015) have demonstrated that infusion of TNF-α into the amygdala impairs acquisition and extinction of fear conditioning, possibly through the overstimulation of the glutamatergic pathway since high glutamate levels were found in the amygdala and the application of an NMDAR antagonist is seen to be able to neutralize the detrimental effects of TNF-α on fear learning. This mechanism is supported by previous research showing that TNF-α elevates synaptic glutamate via 1) increasing glutamate release from astrocytes and 2) inhibiting glutamate reuptake by astrocytes. (Danbolt, 2001; Olmos & Lladó, 2014). Glutamate cytotoxicity is achieved via the activation of the NMDAR, initiating a cascade of downstream events that eventually leads to cellular death, either necrosis or apoptosis, and subsequent deterioration of cognitive function (Floden 2005).

As previously mentioned, there is an overabundance of circulating TNF-α in patients diagnosed with AD, a debilitating neurological disorder with symptoms of shortmemory loss, behavioral abnormalities, and learning deficits (Fillet et al., 1991). It is well-established that AD is characterized by amyloid-β plaques; in in vitro studies, TNF-α has been shown to increase the expression of amyloid-β precursor protein and Aβ, components of the extracellular aggregates, in glial and immune cells (Cunningham et al., 2009). Together with the fact that TNF-α induces glutamate-mediated neurotoxicity, it’s apparent that TNF-α plays a pivotal role in the development of AD pathology. Therefore, in regards to the treatment of AD, Jing et al. (2015)’s study is of significant importance as it provides further in vivo evidence that implicates TNF-α and the glutamatergic pathway as potential targets for therapeutical intervention.

Criticisms and Future Directions

Contrary to the results from Jing et al. (2015)’s study, there has been past evidence demonstrating TNF-α infusion into the CNS had beneficial rather than detrimental effects on cognition – administration of TNF-α centrally in the brain of rats resulted in improved performance on avoidance tasks, indicating that cognitive effects of TNF-α may vary depending on the site of infusion (Brennan & Tieder, 2006). Hence, for further research, an experiment testing hippocampaldependent spatial learning after intra-hippocampal TNF-α injection is proposed, and learning and memory function would be assessed using models such as the Barnes Maze or Morris Water Maze (MWM).

It is also known that, aside from NMDAR, AMPAR (glu is also capable of inducing glutamate cytotoxicity (Olmos & Lladó, 2014). It is of interest to see whether the pharmalogical blockade of AMPAR via antagonists yields the same recovery of fear learning like the application of MK-801 after intra-amygdala TNF-α injection.

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2. Aloe, L., Properzi, F., Probert, L., Akassoglou, K., Kassiotis, G., Micera, A., & Fiore, M. (1999). Learning abilities, NGF and BDNF brain levels in two lines of TNF-α transgenic mice, one characterized by neurological disorders, the other phenotypically normal. Brain Research, 840(1-2), 125-137. doi:10.1016/s0006-8993(99)01748-5 3. Brennan, F., & Tieder, J. (2006). Centrally administered tumor necrosis factor-α facilitates the avoidance performance of Sprague–Dawley rats. Brain Research, 1109(1), 142-145. doi:10.1016/j.brainres.2006.06.040 4. Cunningham, C., Campion, S., Lunnon, K., Murray, C., Woods, J., & Deacon, R. et al. (2009). Systemic Inflammation Induces Acute Behavioral and Cognitive Changes and Accelerates Neurodegenerative Disease. Biological Psychiatry, 65(4), 304-312. doi:10.1016/j.biopsych.2008.07.024 5. Cunningham, C., Campion, S., Lunnon, K., Murray, C., Woods, J., & Deacon, R. et al. (2009). Systemic Inflammation Induces Acute Behavioral and Cognitive Changes and Accelerates Neurodegenerative Disease. Biological Psychiatry, 65(4), 304-312. doi:10.1016/j.biopsych.2008.07.024 6. Danbolt, N. (2001). Glutamate uptake. Progress In Neurobiology, 65(1), 1-105. doi:10.1016/ s0301-0082(00)00067-8 7. Fillit, H., Ding, W., Buee, L., Kalman, J., Altstiel, L., Lawlor, B., & Wolf-Klein, G. (1991). Elevated circulating tumor necrosis factor levels in Alzheimer’s disease. Neuroscience Letters, 129(2), 318-320. doi:10.1016/0304-3940(91)90490-k 8. Floden, A. (2005). -Amyloid-Stimulated Microglia Induce Neuron Death via Synergistic Stimulation of Tumor Necrosis Factor and NMDA Receptors. Journal Of Neuroscience, 25(10), 2566-2575. doi:10.1523/jneurosci.4998-04.2005 9. Jing, H., Hao, Y., Bi, Q., Zhang, J., & Yang, P. (2015).

Intra-amygdala microinjection of TNF-α impairs the auditory fear conditioning of rats via glutamate toxicity. Neuroscience Research, 91, 34-40. doi:10.1016/j.neures.2014.10.015.

10. Maren, S. (2003). The Amygdala, Synaptic Plasticity, and Fear Memory. Annals Of The New York Academy Of Sciences, 985(1), 106-113. doi:10.1111/j.1749-6632.2003. tb07075.x 11. Mogi, M., Harada, M., Riederer, P., Narabayashi, H., Fujita, K., & Nagatsu, T. (1994). Tumor necrosis factor-α (TNF-α) increases both in the brain and in the cerebrospinal fluid from parkinsonian patients. Neuroscience Letters, 165(12), 208-210. doi:10.1016/0304-3940(94)90746-3 12. Montgomery, S., & Bowers, W. (2011). Tumor Necrosis Factor-alpha and the Roles it Plays in Homeostatic and Degenerative Processes Within the Central Nervous System. Journal Of Neuroimmune Pharmacology, 7(1), 42-59. doi:10.1007/ s11481-011-9287-2 13. Olmos, G., & Lladó, J. (2014). Tumor Necrosis Factor Alpha: A Link between Neuroinflammation and Excitotoxicity. Mediators Of Inflammation, 2014, 1-12. doi:10.1155/2014/861231 14. Paganelli, R., Di Iorio, A., Patricelli, L., Ripani, F., Sparvieri, E., & Faricelli, R. et al. (2002). Proinflammatory cytokines in sera of elderly patients with dementia: levels in vascular injury are higher than those of mild–moderate Alzheimer’s disease

patients. Experimental Gerontology, 37(2-3), 257-263. doi:10.1016/s0531-5565(01)00191-7 15. Poloni, M., Facchetti, D., Mai, R., Micheli, A., Agnoletti, L., & Francolini, G. et al. (2000). Circulating levels of tumour necrosis factor-α and its soluble receptors are increased in the blood of patients with amyotrophic lateral sclerosis.

Neuroscience Letters, 287(3), 211-214. doi:10.1016/ s0304-3940(00)01177-0 16. Reichenberg, A., Yirmiya, R., Schuld, A., Kraus, T., Haack, M., Morag, A., & Pollmächer, T. (2001). Cytokine-Associated Emotional and Cognitive Disturbances in Humans. Arch Gen Psychiatry, 58(5), 445. doi:10.1001/archpsyc.58.5.445 17. Tancredi, V., D’Arcangelo, G., Grassi, F., Tarroni, P., Palmieri, G., Santoni, A., & Eusebi, F. (1992). Tumor necrosis factor alters synaptic transmission in rat hippocampal slices.

Neuroscience Letters, 146(2), 176-178. doi:10.1016/03043940(92)90071-e 18. Yirmiya, R., & Goshen, I. (2011). Immune modulation of learning, memory, neural plasticity and neurogenesis. Brain, Behavior, And Immunity, 25(2), 181-213. doi:10.1016/j. bbi.2010.10.015

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AKAP150 Underlies Deficits Seen in Spatial Memory Following Short-Term Sleep Deprivation Patrick Hopper

Sleep is a process, which has been found to be vital for the consolidation of memories and synaptic plasticity, with even short-term sleep deprivation being sufficient to disrupt these processes. Hagewoud et al investigated whether or not short-term sleep deprivation would impact working spatial memory, when tested in a novel arm recognition task. They found that sleep deprived mice of 12 hours had significant declines in their working spatial memory, when compared to controls. Western Blot Analysis was conducted, to determine if there were any changes in cellular proteins and mechanisms, following the sleep deprivation. The researchers found that both the degree of phosphorylation on the serine 845 residue of the AMPA receptors and the level of AKAP150 protein both decreased significantly in the 12 hour sleep deprived mice. The authors concluded the decreased levels of phosphorylation were a result of decreased AKAP150, a scaffold protein which is responsible for tethering and moving PKA to the AMPA receptor for phosphorylation for serine 845. Furthermore, due to the importance of the AMPA receptor in LTP, the researchers believed these results were responsible for the memory deficits observed, Thus, the researchers provided evidence on how decreases in AKAP150 underlie deficits in hippocampal working spatial memory, produced by even short-term sleep loss.

Key words: A-Kinase Anchoring Proteins (AKAP150); Hippocampus; Short-term Sleep Deprivation; AMPA Receptor Background One of the most vital aspects for the health and proper function of neurons is detailed and precise control of signaling, both within time and across different locations1. These signaling events are able to trigger cascades that have dramatic effects within the cell, which if not controlled properly can lead to disruption of many critical pathways2. This crucial form of regulation is accomplished by using scaffold proteins, a subset of proteins which bind and transport signaling molecules to specific locations in the cell, in order to localize the signaling event and its actions1. One such family of proteins are called A-Kinase Anchoring Proteins (AKAPs) and are found in the post-synapses of many neurons in the brain. Three varieties of AKAPs exist: AKAP150 in rodents, AKAP79 in humans, and AKAP75 in bovine, with the majority of studies focusing on AKAP150 due to use of rats and mice as research subjects3. AKAP150 has been found to bind many intracellular signaling molecules, kinases, and receptor proteins, including PKA, PKC, calcium channels, and Calcineurin (CaN)4. Within the brain, AKAPs are expressed in almost every brain region, with highest expression occurring in the striatum, cerebral cortex, and the hippocampus, all of which are implicated in learning and memory3. Due to this finding and its ability to bind multiple signaling molecules, researchers focused on its possible involvement with synaptic plasticity and learning in the brain3.

Past research has found that AKAP150 is critical for delivering PKA to the GluR1 subunit of AMPA receptors for the phosphorylation of serine 845. This process is believed to cause incorporation of the AMPA receptor into the postsynaptic membrane, enhancing LTP and increasing synaptic plasticity5. Furthermore, another study found that AKAP150 can also function to deliver CaN to AMPA receptor for actions opposing those of PKA: dephosphorylation at the serine 845 residue, 103

resulting in channel closure and internalization6.

Further studies agree with these findings, such as Lu et al who injected a stop codon into the gene coding for AKAP150, eliminating the ability of the protein to bind and transport PKA, resulting in observable deficits in LTP and AMPA receptor phosphorylation5. Furthermore, Sanderson et al found the opposite when they knocked-out the CaN binding domain on AKAP150, decreasing the amount of LTD and increasing AMPA receptor phosphorylation6.

Sleep has been found to be a process critical for not only the elimination of waste products and neurotoxins, but also required for the formation and strengthening of neurons, during learning experiences7. Sleep deprivation (both long and short term), has been found to produce deficits in many types of hippocampal based memory and learning, including working spatial memory8. However, no studies have addressed whether AKAP150 could mediate these observed deficits, despite evidence of it mediating changes in LTP and LTD. Therefore, Hagewoud et al sought to determine the role (if any) AKAP150 played.

Research Overview

Summary of Major Results & Discussion

In their study, Hagewoud et al studied the impact of short-term sleep deprivation on working spatial memory. 10 week old mice were subjected to sleep deprivation, of either 6 or 12 hours, which was induced by mild stimulation of their cage. The mice were tested in a novel arm recognition task, using a Y shaped maze, in order to assess their level of working spatial memory following their period of sleep deprivation. The researchers found that even those short amounts of sleep deprivation were enough to induce deficits in the spatial memory of the mice8. While the

amount of exploration time and number of arm entries remained the same during both trials (removing motor deficits and lack of activity as a possible explanation), the amount of time spent exploring the novel arm was significantly higher for the control group when compared to the 12 hour sleep deprived mice (with the 6 hour sleep deprived mice showing deficits, which were at almost significant levels of impairment)8. This result agrees with the previous findings of Xie et al, who found that even small amounts of sleep loss were enough to produce significant deficits in hippocampal working memory7. Furthermore, Yoo et al found that sleep loss in general (whether short or long term) reduced hippocampal neuronal function, causing LTP impairment and consolidation deficits9.

Therefore, the results of this study agree with past work done in the field and provide evidence that even short amount of sleep loss are able to produce problems with synaptic plasticity and strength, leading to deficits in various forms of hippocampal memory. This finding is of particular relevance for students, as it provides evidence of how sleep loss for even short amounts of time can be detrimental to learning and thus how the practice of all-nighters (common among students during exams) can cause more harm than benefits. However, as detailed previously, many past studies have already investigated not only sleep loss (both short and long term) and its impact on hippocampal forms of memory. Therefore, this finding from Hagewoud et al, while interesting, offers no novel information on the effects of short-term sleep loss and thus is offers little impact in the field.

significantly less than controls, produced immediate and observable deficits in spatial memory10. As a result of their study, Hagewoud et al determined that changes in AMPA receptor level could not be responsible for the deficits in spatial working memory.

However, the two previous studies provide evidence for the decline level of AMPA receptor during sleep loss and declining AMPA receptors being responsible for spatial working memory deficits, standing against the results and hypothesis of the Hagewoud et al study, who concluded that since there was no decrease in AMPA receptor level, it cannot explain the decline working spatial memory. While this seems to be a logical conclusion, it disagrees with previous studies done in the field and thus further studies are need to explain this observed result. If the results are found to be in fact true, then this could be considered a novel and significant impact in the both memory and sleep research, overturning previously determined results and producing newer and more accurate lines of research, to better understand memory.

Additionally, the researchers found that the amount of phosphorylation of serine 845 on the GluR1 subunit of the AMPA receptor decreased significantly in the 12 hour sleep deprived mice, when compared to the controls (with 6 hour sleep deprived mice once again showing near significant levels of decline). The authors hypothesized this to underlie the deficits in spatial working memory during the novel arm recognition task. This hypothesis of their observed results agrees with previous findings, in which decreased levels of serine 845 phosphorylation has been noted in decreased learning and memory. Specifically, Lee et al created a mutant strain of mice, in which the phosphorylation sites on the GluR1 subunit where knocked out and found not only decreases in synaptic plasticity but also decreased levels of spatial memory, when tested in a morris water maze task13.

Figure 1) The exploration ratio (time spent in novel arm relative to the time spent in familiar arms) in control mice and 6 & 12 hour sleep deprived mice (Hagewoud et al. Exploration Ratio. (2010).)8

Using a Western Blot analysis, the researchers next measured the levels of AMPA receptors and AMPA receptor phosphorylation expressed in the hippocampus. Hagewoud et al found that the level of AMPA receptors did not change between the control and sleep deprived groups of mice. This result contradicts and stands against previous work done in sleep research. One study by Xie et al, found that sleep deprivation of only 4 hours was enough to cause a decrease in hippocampal AMPA receptor expression7. Furthermore, an additional study by Sanderson et al found that knocking out the expression of the AMPA receptor gene, so the receptor was expressed

Figure 2) The degree of phosphorylation of Serine AMPA receptors, in the hippocampus of control mice and 6 & 12 hour sleep deprived mice (Hagewoud et al. Exploration Ratio.

(2010).)8

Finally, the researchers used the Western Blot analysis to determine the levels of CaN, PKA, and AKAP150 present in the hippocampus, following the sleep deprivation of the mice. The results indicated that while the levels of CaN and PKA remained similar to levels present in control mice, there was a significant decline in the level of AKAP150 in the 104

12 hour sleep deprived mice (with a decline in the 6 hour sleep deprived mice, which was almost deemed to be significant). PKA is a protein kinase, responsible for phosphorylating the serine 845 residue on the AMPA receptor, while CaN is responsible for the dephosphorylation the same residue5,6. It is believed that the state of phosphorylation on serine 845, regulated by the opposing action of those two proteins, determines whether or not AMPA receptors are incorporated into the cell membrane, in addition to whether or not the receptor is opened. These changes have been observed to underlie whether LTP or LTD is induced and whether there are in impairments in memory11. However, the results of Hagewoud et al’s study show that levels of PKA and CaN expression remained the same, thus they could not be responsible for the working spatial memory deficits8. As previously described, AKAP150 is a scaffold protein, which is responsible for tethering and delivering both PKA and CaN to their proper location in the cell, thus acting as a regulator of their actions3. While no previous studies have investigated AKAP150 in relation to sleep induced memory impairments, there are numerous studies, which provide evidence for its role in various forms of memory impairment, due to its role as a scaffold protein. One study by Tunquist et al has found that mice who had the AKAP150 knocked out, experienced deficits in spatial memory, when tested in a Morris water maze. Using a combination of immunofluorescent and electrophysiological readings, it was that altered neuronal processes were underlying these changes in memory12. Therefore, when compared to previous research, AKAP150 mediating the changes in working spatial memory is a reasonable and logical conclusion. This result has little impact and significance, as AKP150 was previously known to be a regulator of LTP, LTD, and other types of memory5,6.

However, previous studies have not established for sleep deprivation is able to produce deficits in learning and memory, thus the results of this study are novel to sleep research and provide groundwork, in which future studies can take place.

Figure 3) The amount of AKAP150 scaffold protein in control mice and 6 & 12 hour sleep deprived mice (Hagewoud et al.

Exploration Ratio. (2010).)8

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Conclusions The authors concluded that like long amounts of sleep deprivation, even short amounts can be detrimental to learning and memory, with mice showing both behavioral and cellular deficits, as a result8. Furthermore, due to the importance of the AMPA receptor and GluR1 subunit in hippocampal forms of memory, Hagewoud et al. concluded that this decrease in spatial working memory was due a decreased degree of phosphorylation of the GluR1 subunit, on S8458. Finally, since the levels of the APAK150 protein (responsible for targeting the S845 kinase to the membrane) were lower, the authors concluded that this was responsible for the decreased phosphorylation levels8. These conclusions made by the authors agree with past work done in how sleep deprivation can affect learning and memory and the cellular machinery mediating those processes. However, the researchers found that the level of AMPA receptors did not decrease, contradicting past research. Therefore, further research is needed to solve this discrepancy. The conclusions determined by this study are significant for the field of neuroscience as a whole, because it offers greater insight into how learning and memory occur in the brain. Furthermore, it also illustrates how simple cellular processes in a specific region in the brain, can translate into larger behavioral and cognitive changes.

Criticisms and Future Directions

One criticism of Hagewoud et al’s study is that they did not measure the levels of AKAP150 in other brain areas, only measuring the amount in the hippocampus.

In their research on AKAP150, Ostroveanu et al found widespread amounts of the scaffold protein present in the entire brain, with the highest levels occurring in the striatum and cerebral cortex. These brains areas are not only involved with the storage and processing of memory, but also express higher amounts of AKAP150 than the hippocampus. In addition, AKAP150 is capable of binding many different proteins, including PKA, PSD-95, PKC, and CaN. Therefore, the short-term sleep deprivation could have possibly elicited changes in AKAP protein in other areas of the brain, which could be responsible for the decline in memory observed. Further experiments, using immunochemistry and western blot analysis could be conducted to view the levels of AKAP150 following sleep deprivation in those brain areas, in order to determine if the observed results in Hagewoud et al’s study were unique only to the hippocampus. This would allow for clarification on whether the observed results were truly due to changes in the hippocampus or widespread changes in the entire brain.

Another criticism of this paper, was that no in vivo experiments were conducted. This study and many similar ones, simply measured the levels of AKAP150 using Western Blot techniques, while neglecting in vivo techniques. Many other researchers have attempted to use in vivo models, such as Lu et al, who injected a stop codon sequence, causing AKAP150 to no longer bind and transport PKA to the membrane surface. They observed decreases in LTP and phosphorylation, as a result5. Additionally, Sanderson et al created a strain of mutant mice, in which the CaN binding domain was

knocked out and they found LTD decreased, while LTP and AMPAR phosphorylation levels were increased6.

However, further studies are need, in order to better understand how AKAP150 can produce the behavioral deficits in memory, observed in Hagewoud et al’s study. Therefore, a beneficial follow-up experiment would use a viral injection method, in order to create two new lines of mutated mice. One would lack both PKA and CaN domains, thus AKAP150 would be unable to bind both. One confusing aspect of Hagewoud et al’s experiment is that decreases in PKA and CaN delivery lead to a net decrease in phosphorylation, when one would expect them to have opposing actions and thus balance each other out. Thus, this in vivo model would help to clarify that abnormality. The second line would cause the total levels of AKAP150 to be overexpressed compared to controls. Havekes et al, recently found that after a period of sleep deprivation, by increasing the levels of cAMP, via injection, certain impairments in memory could be restored to levels similar to controls14. Furthermore, if decreases in AKAP150 was truly responsible for deficits in working spatial memory, then we would expect to see increases in working spatial working memory, when levels of AKAP150 are increased beyond endogenous levels. Therefore, in vivo studies as a whole would offer a better understanding of the relationship between AKAP150, sleep deprivation, and memory.

A final gap in the research presented by this paper is that it does describe or even attempt to find how sleep affects the expression levels of AKAP150 in the hippocampus. Vecsey et al used mice deprived of sleep for 5-6 hours and performed microarray, Western Blot, and PCR analyses, in order to examine the expression of various hippocampal genes following sleep deprivation and whether or not they were up or down regulated15. Vecsey et al found that 12 genes were upregulated and 9 genes down regulated, implicating the mTOR gene as a significant cause of cognitive impairment due to sleep deprivation15. An addition study would be to conduct a similar study, using solely microarray and PCR studies, to focus on the gene encoding for AKAP150 (akap5), as this was not reported or included in their study. Additionally, a functional analysis would be added, in order to see if it was related to and experienced changes similar to the genes reported in Vecsey et al’s study. These further experiments would provide in sight into whether lack of sleep caused changes in the translation of AKAP150 is responsible for the observed spatial memory decline.

4. Lu, Y. et al. A Kinase Anchor Protein 150 (AKAP150)associated Protein Kinase A Limits Dendritic Spine Density.

Journal of Biological Chemistry 286, 26496–26506 (2011).

5. Lu, Y. et al. Age-dependent requirement of AKAP150anchored PKA and GluR2-lacking AMPA receptors in LTP. The EMBO Journal 26,4879–4890 (2007).

6. Sanderson, J. et al. AKAP150-Anchored Calcineurin Regulates Synaptic Plasticity by Limiting Synaptic Incorporation of Ca2+-Permeable AMPA Receptors. Journal of Neuroscience 32, 15036–15052 (2012).

7. Xie, M. et al. Short-term sleep deprivation impairs spatial working memory and modulates expression levels of ionotropic glutamate receptor subunits in hippocampus. Behavioural Brain Research 286,64–70 (2015).

8. Hagewoud et al. Sleep deprivation impairs spatial working memory and reduces hippocampal AMPA receptor phosphorylation. Journal of Sleep Research 19, 280–288 (2009).

9. Sanderson, D. et al. Deletion of glutamate receptor-A (GluR-A) AMPA receptor subunits impairs one-trial spatial memory. Behavioral Neuroscience 121, 559–569 (2007).

10. Yoo, S., Hu, P., Gujar, N., Jolesz, F. & Walker, M. A deficit in the ability to form new human memories without sleep. Nature Neuroscience 10,385–392 (2007).

11. Snyder, E. et al. Role for A Kinase-anchoring Proteins (AKAPS) in Glutamate Receptor Trafficking and Long Term Synaptic Depression.Journal of Biological Chemistry 280, 16962–16968 (2005).

12. Tunquist, B. et al. Loss of AKAP150 perturbs distinct neuronal processes in mice. Proceedings of the National Academy of Sciences105, 12557–12562 (2008).

13. Lee, H et al. Phosphorylation of the AMPA Receptor GluR1 Subunit Is Required for Synaptic Plasticity and Retention of Spatial Memory. Cell 112, 631–643 (2003).

14. Havekes, R. et al. Transiently Increasing cAMP Levels Selectively in Hippocampal Excitatory Neurons during Sleep Deprivation Prevents Memory Deficits Caused by Sleep Loss.

Journal of Neuroscience 34,15715–15721 (2014).

15. Vecsey, C. et al. Genomic analysis of sleep deprivation reveals translational regulation in the hippocampus. Physiological Genomics 44,981–991 (2012).

References 1. Weisenhaus, M et al. Mutations in AKAP5 Disrupt Dendritic Signaling Complexes and Lead to Electrophysiological and Behavioral Phenotypes in Mice. PLoS ONE 5, (2010).

2. Jurado, S., Biou, V. & Malenka, R. A calcineurin/AKAP complex is required for NMDA receptor–dependent long-term depression.Nature Neuroscience 13, 1053–1055 (2010).

3. Ostroveanu, A., Dolga, A., Luiten, P., Eisel, U. & Nijholt, I. A-kinase anchoring protein 150 in the mouse brain is concentrated in areas involved in learning and memory. Brain Research 1145, 97–107 (2007).

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Maternal Behavior Hormone Receptor might be a crucial player in the development of social and mood disorders Patrick Hornlimann

The commonly with maternal behaviours associated hormone Oxytocin has more recently been associated with a variety of social and mood disorders. The cur-rent study intended to find effects of early life stress (ELS) on the susceptibility of certain genotypes to depression, anxiety and stress. The study involved 653 subjects that were genotyped, had to fill out an ELS questionnaire and were assessed on a depression, anxiety and stress scale (DASS). 4 tests were performed and one single nucleotide polymorphism (rs139832701) was found to be associated with higher DASS scores in the face of ELS. Also, brain tissue was gained to monitor downstream effects of specific SNPs, which revealed that rs3831817 alters OXTR levels. The current study supports the role of oxytocin in the development of mood and anxiety disorders.

Key words: Stress; Anxiety; Depression; DASS scale; Oxytocin; RNA expression; SNP Background Oxytocin is a mammalian hormone that is commonly associated with maternal behaviour, parturition and lactation. It is produced in the hypothalamus but also in other parts of the brain and is transported to the posterior pituitary before being releasing into blood circulation. Oxytocin targets Oxytocin receptors (OXTR) that are located in regions of the brain that are similar to the hormoneâ&#x20AC;&#x2122;s production sites. Various reports have suggested that Oxytocin is involved in social and brain disorders. Uvnas-Moberg1 found that social interactions are affected by Oxytocin, while Heinrichs et al.2 and Scantamburlo et al.3 suggested its involvement in anxiety and depression, respectively.

More recently, studies have revealed that Oxytocin concentrations differ as a result of adverse childhood experience4. Furthermore, its concentration seems to affect the severity of symptoms in patients with anxiety disorders5. On the basis of those findings, several studies intended to spot genetic variations that can be associated with mood and anxiety disorders in the face of adverse early life experiences. Besides the present study that examined the association between Oxytocin receptor (OXTR) variations and depression, anxiety and stress scale (DASS) score in the context of early life stress (ELS)6, there are a number of other studies that were conducted for similar purposes. rs2254298 and rs53576 were two of the identified SNPs that were reported. As a result of those being found, further research was conducted to identify the specific base changes that lead to higher susceptibility for certain disorders. Costa7 reported that OXTR rs2254298 GG carriers had higher levels of depression and anxiety compared to GA and AA groups. Likewise, Thompson et al.8 found that the same SNP in individuals who experienced early life adversity resulted in anxiety and depression occurring more often, however, this study reported the AG allele to be responsible for those effects. Similarly, for the rs53576 SNP, a correlation between GG allele carrier and reactivity to stress was found9. Those findings were confirmed and extended by McQuaid et al.10, who found that not only GG but also GA carriers displayed higher incidence of depres-sion in individuals that experienced early107

life adversity. In contrast, a higher susceptibility for depressive disorders was associated with AA and GA allele carriers by Saphire-Bernstein et al.11. Now, the present study does not attempt to clarify those issues but rather it intends to find new OXTR SNPs that might be causative for social and mood disorders in the context of ELS. Previous research of other SNPs could be used as a guideline for the conduction of future studies on newly detected gene variations.

Research Overview

Summary of Major Results

The current article suggests that OXTR variants indeed interact with ELS to determine an individualâ&#x20AC;&#x2122;s likelihood to develop anxiety, stress and depression symptoms6.

It is thus not only the genetics that deter-mine whether or not social and mood disorders mani-fest but much rather a combination of genetics and environmental influences. Furthermore, the present study revealed a new OXTR SNP that might be an important predictor for depression, which is the rs139832701 SNP. The findings suggest that rs139832701 is involved in all of the three measures of DASS score as displayed in table 16. They not only show the effects of the SNP on the symptoms but also the influence of ELS on the relationship between the genotype and the symptoms, which all depicted significant results. However, the findings suggest that this study has been conducted independently as this SNP appears not to be linked to previously found genetic variants6 (Figure 16). This either implies that there was a problem in the current study that led to those differing results or it is indeed the discovery of OXTR variants that is of importance in this context that previous studies have not been able to detect. To clarify this issue and thus the true relevance of these findings, further studies that examine rs139832701 must be conducted.

The authors came to their conclusion after having

Table 1: This table shows the correlations between genotypes and each of the DASS components without ELS interaction at the left side and with interaction at the right side. The RAW values display unadjusted p-values. SNP adjusted and Corrected values are adjusted. Bold values indicate p-values < 0.05.

Another SNP that did not display any distinct patterns in DASS outcomes, however, showed changing in OXTR expression levels in the brain cohorts was the rs3831817 SNP. The linkage disequilibrium displays a moderate value of 0.74 with the rs139832701 variant, suggesting that they are inherited more often together than not. Also, rs139832701 showed high linkage dis-equilibrium (r2â&#x2030;Ľ0.8) with previously found OXTR vari-ants (rs53576, rs237897)6. This indicates that rs53576 and rs237897 variants might be the cause for the changes in transcript levels.

Figure 1: This figure shows a linkage disequilibrium plot of the SNP found in the present study at the very right and other SNPs that were found in previous studies.

con-ducted four tests that were intended to detect new SNPs that resulted in higher DASS scores depending on ELS. The required data of the participants were attained by genotyping, ELS questionnaire and DASS score self-report. Moreover, brain tissue was required to determine downstream functional effects of certain SNPs. The tests were conducted according to Figure 2.

The SNP that was found as a result of those tests was then set up in a linkage disequilibrium plot to ex-amine its correlations with previously found SNPs in a similar context (Figure 16). The sample size was with 653 participants quite large, which increases the relevance of this study.

Figure 2: The four tests that were performed; Test 1: Relationship between ELS and DASS score; Test 2: Influence of covariates; Test 3: Models to determine the main effects of the SNP genotype on symptoms as well as the effect of ELS on that relationship; Test 4: Testing for downstream effects of SNPs

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Conclusions and Discussions The only OXTR variant that showed significant interaction with early life stress experience in order to trigger a higher severity in depressive and stress symptoms was rs139832701 (Figure 3). This led the authors draw their main conclusion that this specific genotype re-sults in increased incidence of anxiety and mood dis-orders in the face of ELS. As the current study repre-sents the first report of this SNP in the given context, it can be considered a novelty finding. This is further supported regarding the fact that rs139832701 is in linkage equilibrium with previously found SNPs, as it confirms the independence of the present study6. The large sample size is one of the major strengths of this study because it makes it more representative for the population as a whole. However, considering that the brain tissues were not gained from the same popu-lation as the actual participants, the generalizability of the study can be questioned due to the altering genetic variation between peoples. This can influence the outcome of the study and thus decreases its relevance. Despite the fact that the authors do a rather good in comparing their results to the ones from other studies, they poorly address how the current findings could be meaningfully integrated and used in the context. It does neither come out clearly what next step of research or follow-up study they suggest, nor in what respect this study might contribute to the progress of this field of research. As this study is the first one to find rs139832701 being involved in increasing the people’s likelihood to fall into depression when in combination with ELS, it is difficult to directly compare it to other literature, however, the authors do acknowledge similar findings and also try to relate them to the present study.

Figure 3: This figure shows the proposed model of this study

Criticism and Future Directions

Issues with the current study

The result section is very well structured. The different paragraphs that are arranged according to the tests make them easily understandable and interpretable.

However, the analysis seems to simplify some of the data slightly too much. The authors state in one of the paragraphs: “we found that none of our demographic variables (gender, age or years of education) were significant covariates”6 yet when having a close look at test 2 in Table 26, one can see that there are significant results displayed for age and education for 109

anxiety measures. Nevertheless, the other tests are analysed thoroughly and exactly. Test 1 and 3 showed that ELS correlates with DASS score and OXTR variants correlate with DASS score, respectively. For the latter association, it is clearly distinguished that only stress and depression scores are correlating but not anxiety parameters. As anxiety here again did not behave similar to depression and stress parameters, it might have been important to study the results of test 2 more exactly. Maybe anxiety cannot be put in context with OXTR variants in the way depression and stress can and should therefore be separately analysed. However, the rs139832701 SNP according to the re-sults seems to clearly have an association with DASS outcome when ELS was considered.

Table 2: This table displays the correlation between covariants and each of the DASS measures in p-values. The bold values are p<0.05.

Comparison to other studies

When compared to other studies that investigated similar models, several differences can be spotted.

First of all, as previously said, the sample size is with 5636 quite large when put in context with sample sizes of comparable studies that had 23612, 28810, 939 and 129 subjects8, respectively. All the subjects are Cau-casian decent and belong to the same ethnicity, which in other studies is often not the case and is of particu-lar relevance when it comes to genetic variations. The same applies to the brain tissues that were in this par-ticular study6 sampled from different population than the subjects themselves and can have implications for the results. Further, there are a lot of different measures for both ELS and depression, anxiety and stress parameters that will have an influence on the results of a study too. To give an example, McQuaid et al.10 used Beck Depression Inventory13 and Thompson et al.8 made use of Children’s Depression Inventory14, while in the present study DASS score was used as a measure. Moreover, the age of the subjects should be considered and in the present study displays a big spreading between 6 and 87 years. Norman et al.9 in comparison chose all subjects to be between 50 and 68.

Outlook

Further studies should try to achieve samples sizes similar to the present study and also to maintain the ethnic homology among the subjects. According to the already known SNPs such as rs53576, that is currently heavily investigated15,16,17, the way to go for this newly found SNP is already prepared. Not only is it important to now examine the downstream effects of rs139832701 but also should the findings be confirmed by further studies that might use brain

tissues from the same population as the subjects.

Moreover, the OXTR rs139832701 polymorphism should be further investigated in order to determine the base changes that are involved and thus which specific allele is responsible for the association.

As this study has also shown that rs3831817 has an influence on OXTR levels6 and silencing of OXTR were associated with autism18, it might also be interesting to further examine rs3831817 in that context.

References 1. Uvnas-Moberg, K.. Oxytocin may mediate the benefits of positive social interaction and emo-tions. Psychoneuroendocr inol;23:819e35. (1998) 2. Scantamburlo, G., Hansenne, M., Fuchs, S., Pit-chot, W., Marechal, P., Pequeux, C., et al. Plasma oxytocin levels and anxiety in patients with major depression. Psychoneuroendocrinol 2007;32:407e10. (2007) 3. Heinrichs, M., Baumgartner, T., Kirschbaum, C., Ehlert, U.. Social support and oxytocin interact to suppress cortisol and subjective responses to psychosocial stress. Biol Psychiatry;54:1389e98. (2003) 4. Heim, C., Young, L. J., Newport, D. J., Mletzko, T., Miller, A. H., Nemeroff, C. B.. Lower CSF oxy-tocin concentrations in women with a history of childhood abuse. Mol Psychiatry;14:954e8 (2009) 5. Hoge, E. A., Pollack, M. H., Kaufman, R. E., Zak, P. J., Simon, N. M.. Oxytocin levels in social anxiety disorder. CNS Neurosci Ther; 14:165e70.(2008) 6. Myers, A. J., Williams, L., Gatt, J. M., McAuley-Clark, E. Z., Dobson-Stone, C., Schofield, P. R., Nemeroff, C. B..

Variation in the oxytocin receptor gene is associated with increased risk for anxiety, stress and depression in individuals with a history of exposure to early life stress. J Psychiatr Res. 2014 Dec;59:93-100.

7. Costa, B., Pini, S., Gabelloni, P., Abelli, M., Lari, L., Cardini, A., Muti, M., Gesi, C., Landi, S., Galderisi, S., Mucci, A., Lucacchini, A., Cassano, G. B., Martini, C.. Oxytocin receptor polymor-phisms and adult attachment style in patients with depression.

Psychoneuroendocrinology;34(10):1506-14.

(2009) 8. Thompson, R. J., Parker, K. J., Hallmayer, J. F., Waugh, C. E., Gotlib, I. H.. Oxytocin Receptor Gene Polymorphism (rs2254298) Interacts with Familial Risk for Psychopathology to Predict Symptoms of Depression and Anxiety in Adoles-cent Girls. Psychoneuroendocrinology; 36(1): 144–147. (2011) 9. Norman, G. J., Hawkley, L., Luhmann, M., Ball, A. B., Cole, S. W., Berntson, G. G., Cacioppo, J. T.. Variation in the oxytocin receptor gene influ-ences neurocardiac reactivity to social stress and HPA function: a population based study.

Horm Behav.;61(1):134-9. (2012) 10. McQuaid, R. J., McInnis, O. A., Stead, J. D., Matheson, K., Anisman, H.. A paradoxical associ-ation of an oxytocin receptor gene polymorphism: early-life adversity and vulnerability to depression. Front Neurosci;7:128. (2013) 11. Saphire-Bernstein, S., Way, B. M., Kim, H. S., Sherman, D. K., Taylor, S. E.. Oxytocin receptor gene (OXTR) is

related to psychological re-sources. Proc Natl Acad Sci U S A; 108:15,118e15,122. (2011) 12. Malik, A. I., Zai, C. C., Abu, Z., Nowrouzi, B., Beitchman J. H.. The role of oxytocin and oxytocin receptor gene variants in childhood-onset aggression. Genes Brain Behav;11:545e51.

(2012) 13. Beck A. T., Ward C. H., Mendelson M., Mock J., Erbaugh J.. An inventory for measuring depres-sion. Arch. Gen. Psychiatry 4, 561–571. (1961) 14. Kovacs, M.. The Children’s Depression Inventory (CDI).

Psychopharmacol. Bull. 21, 995-1124. 1985) 15. Thompson S. M., Hammen C., Starr L. R., Najman J. M..

Oxytocin receptor gene polymorphism (rs53576) moderates the intergenerational transmission of depression. Psychoneuroendocri-nology;43:11-9. (2014) 16. Kim Y. R., Kim J. H., Kim C. H., Shin J. G., Treasure J. Association between the Oxytocin Receptor Gene Polymorphism (rs53576) and Bu-limia Nervosa. Eur Eat Disord Rev..

(2015) 17. Chang W. H., Lee I. H., Chen K. C., Chi M. H., Chiu N. T., Yao W. J., Lu R. B., Yang Y. K., Chen P. S.. Oxytocin receptor gene rs53576 polymor-phism modulates oxytocindopamine interaction and neuroticism traits--a SPECT study.

Psycho-neuroendocrinology;47:212-20. (2014) 18. Gregory S. G. , Connelly J. J., Towers A. J., Johnson J., Biscocho D., Markunas C. A., Lintas C., Abramson R. K., Wright H. H., Ellis P., Lang-ford C. F., Worley G., Delong G. R., Murphy S. K., Cuccaro M. L., Persico A., Pericak-Vance M. A.. Genomic and epigenetic evidence for oxytocin receptor deficiency in autism. BMC Med.;7:62. (2009)

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Consolidation of Memories Following Sleep is the Result of Synaptic Potentiation Justin Huang

Many studies have shown that sleep leads to consolidation of long-term memories. Yet what is unknown is how the mechanism by which sleep states affect memory. A popular theory over the past decade is the Synaptic Homeostasis Hypothesis coined by Tononi and Cirelli (2003, 2014). This theory postulates a role of sleep in the synaptic depression of unused synapses to rebalance the metabolic demands of neuronal activity. Although many studies do provide evidence for this theory, an equally impactful number of studies have suggested sleep is tightly linked to synaptic potentiation of various forms of memory. One of them, a study performed by Aton et al. (2014), has attempted to undeniably show this effect. By acutely affecting the sleeping behaviour of mice, Aton et al. (2014) were able to show a form of sleep-dependent synaptic plasticity. Subsequent experiments were performed to control for experimental conditions and test for undesired effects due to unexpected stresses.

However, the study has consistently shown a sleep-dependent synaptic potentiation in principal neurons and fast-spiking interneurons of the visual cortex. Given the novelty of this study, it is crucial to scrutinize the data in an attempt to understand what impact these findings will have on our current understanding of sleep, and how we should approach future studies linking sleep and memory consolidation.

Key words: in vivo recording, sleep, synaptic plasticity, thalamocortical oscillations, visual system, visual cortex, synaptic homeostasis hypothesis.

Background Sleep, although ubiquitous in nature, is puzzling in terms of its purpose and its mechanism of action.

In learning, the sleep state is viewed important in the consolidation of memories, the transformation of newly acquired memories during one’s wake into more long-term, robust forms of memory. Declarative and procedural memories are positively reinforced through sleep (Seehagen et al., 2015; Plihal and Born, 1997). Sleep also has a reinforcing effect on the formation of emotional memories (Wagner et al., 2001; Payne et al., 2008; Nishida et al. 2009).

These studies provide evidence for the importance of sleep in memory learning and consolidation. However, information regarding the possible mechanism by which sleep carries out its augmenting role in memory consolidation is less prevalent.

There has been an emerging theory known as the “Sleep Homeostasis Hypothesis” (SHY) (Tononi and Cirelli, 2003; 2014) which posits sleep as a mechanism for homeostatic renormalization, offsetting the net synaptic potentiation acquired during one’s wake. In this way, sleep would be consolidating memories and enhancing the ability to learn. The foundation of this hypothesis consists of three points. Firstly, increased synaptic strength is energetically unfavourable, and the visual awake in an awake rodent is dominated by synaptic inhibition (Haider et al., 2012). Second, the strengthening of synapses should occurring during an individual’s wake, given that this period is when he or she is interacting with the environment. Finally, given the first two components, the SHY suggests synaptic renormalization occurs during sleep through synaptic depression of underused synapses.

However, a series of studies have also emerged suggesting the opposite. One of them, by Aton et 111

al. (2014), challenges the SHY by suggesting sleep facilitates memory consolidation through potentiating synapses (as opposed to synaptic depression according to the SHY). Their previous study investigating ocular dominance plasticity through monocular deprivation demonstrated a sleep-dependent increase in neuron remodelling and synaptic potentiation (Aton et al., 2009), following a mechanism similar to longterm potentiation involving NMDAR and PKA activity (Frenkel et al., 2006). To further elucidate the role of sleep, Aton et al. (2014) examined orientation-specific response potentiation (OSRP) in mice – a naturally occurring form of synaptic plasticity whereby exposure to a visual stimulus of a specific manner results in a promoted response to stimuli of the same orientation.

The current study (Aton et al., 2014) is one of the first to explicitly show in vivo that cortical activity as a result of a specific visual stimulus can be reinforced through sleep. In this review, we will be examining the major findings and interpretations of this paper in an attempt to elucidate what future steps we should take towards understanding sleep. By refining our understanding of the sleep mechanisms, we may be able to better our treatments for common disorders involving sleep deficits, such as Alzheimer’s disease.

Research Overview

Sleep facilitates memory consolidation in mice by promoting OSRP

To evaluate how OSRP is affected by sleep behaviour, mice were either sleep-deprived (n=3) or allowed ad libitum sleep (n=4) for six hours following the stimulus (Figure 2A). OSRP was identified by measuring the % change in average orientation preference (Figure

2C) as well as the distribution of neurons experiencing increase and decreases during the experiment. OSRP was apparent in V1 principal neurons and fast-spiking interneurons, but only in mice in the ad libitum sleep group (Figure 2B, P<0.05). Mice who were sleepdeprived did not experience OSRP. These results also suggested a correlation between slow wave sleep (SWS) and REM sleep with OSRP in the visual cortex (Figure 2D, R = 0.78).

These findings provide in vivo evidence for the theory that sleep promotes synaptic potentiation, contrary to the SHY proposed by Tononi and Cirelli (2003; 2014). Originally, a prior study by Aton et al. (2009) had shown OSRP being driven by a mechanism similar to LTP. Despite the consistent findings of the 2009 study, Tononi and Cirelli (2014) had denied the implications for their hypothesis, citing an “unnatural experimental design” using monocular deprivation for ocular dominance plasticity studies in mice. In response, Aton et al. (2014) circumvented this issue by experimenting in a room devoid of light while still showing the naturally occurring OSRP, the expected response if sleep promoted synaptic potentiation and not synaptic depression.

Figure 1. The Synaptic Homeostasis Hypothesis as outlined by Tononi and Cirelli (2014).

The results were not impacted by any flaws in the experimental design

involved mice being equally sleep-deprived across either halves of the sleep phase (Figure 3A). Both interventions resulted in similar blocking of OSRP in principal neurons and fast-spiking interneurons (Figure 3C). In both experiments, the amount of SWS (R=0.58) and REM sleep (R=0.59) positively correlated with OSRP (Figure 2D).

These experiments were crucial to addressing issues brought by advocates of the SHY, namely the unnatural settings of the study. According to Tononi and Cirelli (2014), previous endeavours by Aton’s involving monocular deprivation were unrepresentative of the natural learning conditions of mice. Therefore they did not necessarily reflect the effects of sleep on memory learning and consolidation. These current findings serve to meet their criticisms by demonstrating the earlier results were in line with the original hypothesis, and that the resulting synaptic potentiation could not have been a result of the experiment.

Evoked visual responsiveness was increased during OSRP, proportional to slow wave sleep spindle oscillations

Recall orientation-specific response potentiation is a measurement of the responsiveness to specific visual elements for neurons in the visual cortex. As OSRP is mechanistically similar to LTP (Aton et al., 2009), Aton et al. (2014) were led to investigate the changes in neuronal activity in addition to their heightened orientation sensitivity. The evoked responsiveness index (ERI, maximum vs. spontaneous activity) was enhanced in accordance with the sleep-dependent OSRP (P<0.05, Figure 4A, 4B). Although no changes were observed in firing coherence to REM gamma or slow-wave sleep delta oscillations, there was a high spike-field coherence between slow wave sleep spindle oscillations compared to both OSRP and ERI (Figure 4C). These results suggest generation of slow wave sleep spindles by the V1 is important to OSRP, as other frequencies showed no correlation.

The influence of spindle oscillations was also implicated in Aton’s previous findings where sleepdependent plasticity following monocular deprivation was proportional to the synchronization of principal neuron firing to spindle oscillations (Aton et al., 2009). Recall of episodic-like memory was also associated with the amplitude of the spindle oscillations, while object recognition memory depending on the percentage of slow wave sleep during memory consolidation (Oyanera et al., 2014).

In order to evaluate the acute stress-related effects of the experimental design, as well as the time-ofday effects on OSRP, mice were provided the visual stimulus during the morning (AM, n=4) or evening (PM, n=4) with ad libitum sleep according to their natural behaviours. As seen in the prior experiment, both principal neurons and fast-spiking interneurons showed significant OSRP across a sleep phase (morning stimulus P<0.05, Figure 3C). An additional control for the acute effects of sleep deprivation 112

Figure 2.

Orientation-specific response potentiation is observed when post-stimulus sleep is permitted.

A: Timeline of the experiment. A 1-hour visual stimulus (visual gratings of a specific orientation) was presented to each mice, followed by a period of either ad libitum sleep or no sleep for a six hour period. Visual response measurements were recorded at the timepoints A (after baseline), B (following stimulus), C (following the intervention). B: Visual response data for sleeping (left) and sleep-deprived (right) mice. Changes in the firing responses at each were quantified as a measurement of OSRP. C: The percent change in orientation-specific response in V1 neurons (principal neurons and fast-spiking interneurons). OSRP was observed in both groups in sleeping mice. P<0.05, Holm-Sidak post hoc test. D: Correlation between sleep states (NREM, REM, Awake) versus the change in the orientationspecific response. OSRP was positively correlated to cumulative time spent in NREM and REM sleep, while negatively correlated to mice who stayed awake. From Aton et al. (2014).

Figure 3.

Orientation-specific response potentiation is present only following the natural sleep cycle in mice. A: Timeline of the experimental study. The visual response test was either performed in the morning (AM) or evening (PM) to experimentally test the effects of sleep according to the miceâ&#x20AC;&#x2122;s circadian time. An additional control experiment was conducted where mice were sleep-deprived for an equal amount early and late during their natural sleep. B: Visual response data for AM and PM conditions.

Changes in the firing responses at each were quantified as a measurement of OSRP. C: Percent change in orientation-specific response versus the treatment, which shows a detectable OSRP only following sleep. D: Sleep is positively correlated to OSRP, while sleep-deprivation is negatively correlated to OSRP. From Aton et al. (2014).

Figure 4. OSRP was proportional to spike-field coherence. A: % changes in evoked responsiveness index was evident following a sleep period. B: For all sleeping mice, the % change in ERI was positively correlated with the % change in orientation preference.

C: SWS spike-field coherences at baseline (solid) and 2h following the visual stimulus (dashed lines). Synchrony is observed poststimulus for spindle oscillations. D: Correlation between spindle spike-field coherence and the first two parameters (ERI, orientation preference). From Aton et al. (2014).

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Conclusions and Discussion

Discussion

Altogether, the findings in these experiments ultimately support the role of sleep in memory consolidation.

According to Aton et al. (2014), the data suggests sleep is necessary for OSRP to occur, as sleep deprivation impaired the response regardless of time and experimental effects (Figure 2). This is further supported by the positive correlation between OSRP and sleep (both SWS and REM sleep), and the negative correlation with an awake status. Two possible explanations arise from these results: either the state of sleep provides an appropriate, facilitative context for OSRP and therefore memory consolidation, or the awake phase serves detrimental to this process and the forms are permissive to its taking place. Therefore, although it can be concluded that sleep is necessary for OSRP, we are now prompted to elucidate how sleep promotes synaptic potentiation.

The experiments analyzing the state-specific activity patterns provide a small glint into the possibilities surrounding this newly defined focus. In addition to the increased neuronal firing of V1 principal neurons (Figure 4A, 4B), there was an observed synchronizing of their firing patterns to SWS spindle oscillations (Figure 4C).

Given the similarities between OSRP and other forms of synaptic plasticity (long-term potentiation, ocular dominance plasticity), Aton et al. (2014) hypothesizes a link between synaptic potentiation and the increased neuronal firing in accordance to thalamocortical oscillations.

The novelty within these studies lie in their contrast to the SHY which prescribes a synaptic depression event with sleep (Tononi and Cirelli, 2003). As a link in a chain of emerging studies, we are prompted to re-examine our current knowledge regarding the role of sleep in memory formation. Advances will provide new avenues to understanding and treating disorders comprised of cognitive and sleep deficits, such as Alzheimer’s disease.

Conclusion

In conclusion, the collection of these in vivo results by Aton et al. (2014) suggests, contrary to the synaptic homeostasis hypothesis, sleep promotes synaptic potentiation in the adult cortex. This response may be promoted by SWS oscillations. Further research is required to elucidate the mechanism by how sleep leads to synaptic potentiation.

Criticisms and Future Directions

The study by Aton et al. (2014) has demonstrated an experience-dependent synaptic potentiation in the primary visual cortex (V1) of rats that was promoted by sleep behaviour. Specifically, the tested perceptual learning was sleep-dependent, proportional to the time asleep. This OSRP from repeated sensory experience also requires an increased neuronal firing synchronized to SWS spindle oscillations. According to Cooke and Bear (2014), OSRP results in an increased V1 responsiveness in awake mice in the absence of reward or punishment (Cooke et al., 2015), perhaps from a mechanism similar to N-methyl-D aspartate receptor activity in long-term potentiation. The signifi-

cance in this study is in its implications: alongside other similar recent findings (Chauvette et al., 2012; Grosmark et al., 2012; Aton et al., 2009) this study effectively brings into question the reigning “synaptic homeostasis hypothesis” (SHY) which suggests sleep functions to reduce net synaptic potentiation during the waking period to baseline levels through a reduced noradrenergic system (Tononi and Cirelli, 2014). According to the SHY, synaptic potentiation is predominantly occurring during one’s wake, yet the previously described findings by Aton et al. (2014) clearly issue against this notion.

Regarding the methodology behind the study by Aton et al. (2014), drivable headstages (EIB-36, Neuralynx) were implanted into C57Bl/6j mice. Within the headstages, stereotrodes were implanted into various brain regions including the primary visual cortex to assess OSRP following different experimental sleep models.

Conceptually, Aton et al. (2014) provided sound experimental groupings by testing for the effects of sleep deprivation, its time of induction (early vs. late following stimulus), whilst controlling for acute stress and OSRP recovery by repeating the experiments while abiding to their natural sleep behaviour and allowing post-deprivation sleep respectively. As aforementioned, the weakness in the previous study

by Aton et al. was their choice in observing monocular deprivation, which was considered “unnatural” (Tononi and Cirelli, 2014). However, it should not be assumed that the synaptic homeostasis hypothesis has been overturned. Although synaptic potentiation would no doubt be important in the consolidation of long-term memories, a downscaling of unused synapses and short-term memories is also logically a plausible function of sleep. As more information emerges supporting either hypothesis, some have suggested we should instead be focusing on a combined hypothesis, where both are important to the role of sleep (Heller, 2014).

Nonetheless, given the meticulous experimental design by Aton et al. (2014), it would be ridiculous to discount synaptic potentiation to occur during sleep.

However, remaining is the question of how exactly sleep promotes synaptic potentiation. As the current study suggests a role of synaptic reinforcement during sleep, what is the mechanism behind the increase in OSRP under both SWS and REM sleep?

Previous studies have shown a lack of selectiveresponse potentiation correlating to decreases in expression of the Arc gene (McCurry et al., 2010), and that Arc null mutant mice retained no memories nor synaptic potentiation after 2 hours (Plath et al., 2006). In addition, Shepherd et al. (Shepherd et al., 2006) demonstrated an induction of Arc transcription in CA1 hippocampal neurons following 5 minutes of spatial exploration. These studies, in conjunction to the sleep-dependent nature of OSRP, implicate Arc expression in the promotion of synaptic potentiation and memory consolidation in sleep. In order to elucidate the potential role of Arc in the sleep-dependent OSRP, Arc(-/-) mice can be generated and subjected to the same experimental trials performed by Aton et al. (2014). The knockouts can be experimentally induced, such as through a Tet-Off system, to rule out the possibility of developmental compensatory effects.

In addition to the prospective results of the Arc(-/-) 114

mice, a western blot or qPCR experiment should be run to confirm the knock-out procedure. Lastly, since the competing SHY suggests sleep instead carries out long-term depression (LTD) (Tononi and Cirelli, 2014), an important control experiment is to test whether or not LTD occurs following sleep (with and without the induction of OSRP). This can be performed by monitoring the synaptic response under pharmacological intervention (Ca2+ chelators for example have been shown to block LTD [Brocher et al., 1992]).

In summary, modifications of the experiment by Aton et al. (2014) may provide a clearer investigation into the role of sleep in synaptic potentiation and memory consolidation.

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2. Aton SJ, Seibt J, Dumoulin M, Jha SK, Steinmetz N, Coleman T, Naidoo N, Frank MG. (2009). Mechanisms of sleep-dependent consolidation of cortical plasticity. Neuron.

61(3):454-66.

3. Brocher S, Artola A, Singer W. (1992). Intracellular injection of Ca2+ chelators block induction of long-term depression in rat visual cortex. Proc Natl Acad Sci USA. 89:123-127.

4. Chauvette S, Seigneur J, Timofeev I. (2012). Sleep oscillations in the thalamocortical system induce long-term neuronal plasticity. Neuron. 75(6):1105-13.

5. Cooke SF, Cooke SF, Bear MF. (2014). How the mechanisms of long-term synaptic potentiation and depression serve experience-dependent plasticity in primary visual cortex.

Philos Trans R Soc Lond B Biol Sci. 369(1633). [Pubmed: 20140021] 6. Cooke SF, Komorowski RW, Kaplan ES, Gavornik JP, Bear MF. (2015). Visual recognition memory, manifested as long-term habituation, requires synaptic plasticity in V1. Nat Neurosci. 18(2):262-71.

7. Frenkel MY, Sawtell NB, Diogo AC, Yoon B, Neve RL, Bear MF. (2006). Instructive effects of visual experience in mouse visual cortex. Neuron. 51(3):339-349.

8. Grosmark AD, Mizuseki K, Pastalkova E, Diba K, Buzsáki G. (2012). REM sleep reorganizes hippocampal excitability.

Neuron. 75(6):1001-7.

9. Haider B, Hausser M, Carandini M. (2013). Inhibition dominates sensory responses in the awake cortex. Nature.

493:97-100.

10. Heller C. (2014). Ups and Downs of Synapses During Sleep and Learning. Sleep. 37(7):1157-1158.

11. McCurry CL, Shepherd JD, Tropea D, Wang KH, Bear MF, Sur M. (2010). Loss of Arc renders the visual cortex impervious to the effects of sensory experience of deprivation.

Nat Neurosci. 13(4):450-7.

12. Nishida M, Pearsall J, Buckner RL, Walker MP. (2009).

REM sleep, prefrontal theta, and the consolidation of human emotional memory. Cereb Cortex. 19:1158–1166.

13. Oyanedel CN, Binder S, Kelemen E, Petersen K, Born J, 115

Inostroza M. (2014). Role of slow oscillatory activity and slow wave sleep in consolidation of episodic-like memory in rats.

Behav Brain Res. 275:126-130.

14. Payne JD, Stickgold R, Swanberg K, Kensinger EA. (2008). Sleep preferentially enhances memory for emotional components of scenes. Psychol Sci. 19:781–788.

15. Plath N, Ohana O, Dammermann B, Errington ML, Schmitz D, Gross C, Mao X, Engelsberg A, Mahlke C, Welzl H. (2006). Arc/Arg3.1 is essential for the consolidation of synaptic plasticity and memories. Neuron. 52(3):437-44.

16. Plihal W, Born J. (1997). Effects of early and late nocturnal sleep on declarative and procedural memory. J Cogn Neurosci. 9:534–547.

17. Seehagen S, Konrad C, Herbert JS, Schneider S. (2015).

Timely sleep facilitates declarative memory consolidation in infants. Proc Natl Acad Sci. 112(5):1625-1629.

18. Shepherd JD, et al. (2006). Arc/Arg3.1 mediates homeostatic synaptic scaling of AMPA receptors. Neuron.

52(3):475-84.

19. Tononi G, Cirelli C. (2014). Sleep and the price of plasticity: from synaptic to cellular homeostasis to memory consolidation and integration. Neuron. 81(1):12-34.

20. Tononi G, Cirelli C. (2003). Sleep and synaptic homeostasis: a hypothesis. Brain Res Bull. 62(2):143-50.

21. Wagner U, Gais S, Born J. (2001). Emotional memory formation is enhanced across sleep intervals with high amounts of rapid eye movement sleep. Learn Mem. 8:112–119.

Received April 06, 2015; revised ##, 200#; accepted Month, ##,

Month, 2013.

This work was supported by University of Toronto’s Human Biology Program. The authors thank Dr. Ju for technical assistance, execution, and feedback on this lab exercise.

Address correspondence to: Justin Huang, Human Biology Department, 300 Huron Street, Wetmore Hall, University of Toronto. Toronto, ON M5S 1C6. Email: justin.huang@mail. utoronto.ca

A novel pharmacogenetic approach: Transient neuronal activation through TRPV1 and capsaicin

Sonja Ing

The ability to precisely induce activity in neurons is essential in deciphering how a neuronal population might interact within complex circuits and in elucidating behavioural correlates. Various techniques have been developed to this effect, such as optogenetics and genetically engineered receptors. However, while these techniques have been incredibly successful in controlling neural activity in vivo, they are not without their limitations – the former is both labour-intensive and invasive, and the latter often has either low temporal resolution or lack of cellular specificity. In this paper, a novel noninvasive pharmacogenetic model developed by Güler et al is presented, where neural activity in genetically defined populations can be induced directly, rapidly and reversibly by selective expression of capsaicin receptor TRPV1. Demonstrating that capsaicin can successfully induce transient activity in two distinct neural populations indicates the translatability of Selective TRPV1 ExpressionMediated Activation (STEMA) across neural systems. Addition of STEMA to the repertoire of neural effectors may prove instrumental in elucidating complex neural circuits.

Key words: pharmacogenetics, neurogenetics, TRPV1, capsaicin, STEMA Background The ability to precisely and acutely control electrical activity in specific neuronal populations is essential in elucidating their function within complex circuits.

Precisely manipulating neural activity in vivo allows one to directly correlate behavioural responses. The earliest neuron perturbation techniques, conventional pharmacology and electrical stimulation, were unable to achieve both molecular specificity and spatial acuity, and so were inadequate to definitively assign functional circuit roles to neuronal populations1. In recent years, novel techniques have been developed that encompass both spatial and molecular specificity, primarily though taking advantage of advances in transgenic techniques and neuron-specific gene expression2. The current leading techniques include optogenetics and genetically engineered designer receptors. While each of these technologies has proven invaluable in studying causal relationships between neural activity and behavioural output, they are not without their limitations. For example, while optogenetics possesses high spatial resolution and extraordinarily precise control of neuron spike timing and firing patterns, it is highly invasive, labour-intensive, and not well-suited to controlling diffuse signalling networks1,2,3. On the other hand, while designer receptors have high spatial acuity and are non-invasive, they exhibit low to medium temporal resolution, depending on the phamacokinetic properties of the specific ligand2,3.

In their paper “Transient activation of specific neurons in mice by selective expression of the capsaicin receptor”, Güler et al present a novel pharmacogenetic technique that addresses some of the limitations posed by optogenetics and designer receptors. The authors demonstrate that Selective TRPV1 ExpressionMediated Activation (STEMA) is capable of transiently, rapidly and dose-dependently inducing neural activity in genetically defined neuronal populations through selective activation of the capsaicin receptor TRPV13.

TRPV1 is a non-selective cation channel, normally expressed in the peripheral nervous system, that

may be stimulated by capsaicin, noxious heat and pH4. When activated, TRPV1 depolarizes neurons and generates action potentials4. To avoid peripheral and painful TRPV1 activation, the authors engineered mice that expressed the receptor solely in genetically defined populations and not at the endogenous locus.

With STEMA, activation of TRPV1 by capsaicin, either by systemic injection or voluntary consumption, was sufficient to induce both neural activity and behaviour characteristics of the neurotransmitter system being activated3. In their paper, Güler et al successfully applied STEMA to the two distinct neuronal populations of dopamine (DA) and serotonin (5HT), respectively3.

STEMA is a valuable addition to the neuroscience toolbox of neuron perturbation techniques since it addresses some of the limitations presented by other methods. Similar to other orthogonal pharmacogenetic approaches, STEMA has high spatial acuity and is also non-invasive, as surgical procedures are required to neither express the TRPV1 receptor nor deliver its ligand capsaicin2,3. Further, while it does not act on the same kinetic timescale as optogenetics, STEMA’s temporal resolution is greatly improved compared to other designer receptor2,3.

Research Overview

Summary of Major Results

TRPV1 is exclusively expressed in DA neurons Güler et al genetically targeted TRPV1 to DA neurons through a triple transgenic approach. The first transgenic line consisted of Trpv1-knockout mice (B6.129X1-Trpv1tm1Jul/J), to eliminate peripheral expression of TRPV1. These mice were crossed to both Gt(ROSA)26Sor-stopflox-Trpv1-ires-ECFP and Slc6a3Cre mice. The resulting DAT-TRPV1 mice selectively expressed the Trpv1 gene in neurons expressing Cre-recombinase under the control of the dopamine transporter promoter Slc6a3 (or DAT) (Fig 1a)3.

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Selective expression of TRPV1 was confirmed in vitro in VTA slices through double immunohistochemistry of neural activity marker cFos and DA neuron marker tyrosine hydroxylase (TH) following capsaicin administration (Fig 1b)3. 96.2±0.7% of cells tested positive for both cFos and TH, compared to only 4.8±2.1% of vehicle-injected mice3. Furthermore, cFos levels were increased in most DA targets, such as the lateral habenula and piriform cortex, of DAT-TRPV1 mice3.

These results demonstrate that capsaicin is capable of eliciting activity-dependent gene expression specifically in DA neurons.

Capsaicin administration leads to specific activation of DA neurons and DA release

To confirm capsaicin-dependent activity of DA cells in DAT-TRPV1 mice, whole cell, voltage-clamp recordings were performed on midbrain slices in vitro.

Putative DA neurons were first identified by characteristic waveform dynamics and firing pattern criteria, and large inward currents were rapidly induced in these DA neurons following a large application of capsaicin, similar to endogenous capsaicin-induced currents in the periphery3,5.

In addition, comparable electrophysiological results were achieved in awake and freely moving mice. After using stereotactic coordinates to implant four-tetrode microdrives in the VTA, putative DA neurons were identified through characteristic baseline recordings3. The kinetics of the waveforms were not significantly different between DAT-TRPV1 and control mice, both before and after capsaicin administration, which indicates that STEMA does not interfere with normal physiological function3.

Both the peak firing rate and burst activity increased in DAT-TRPV1 mice following capsaicin administration in a dose-dependent manner, while no changes were observed in controls3. These results indicate that capsaicin is capable of inducing DA activity in vivo.

Moreover, in vivo fast-scan cyclic voltammetry confirmed that capsaicin is capable of enhancing DA release in DAT-TRPV1 mice3.

Behavioural responses characteristic of DA activity were induced by capsaicin

First, DA facilitates movement by modulating basal ganglia circuits, leading to a general increase in locomotion6. To monitor locomotor behaviour following capsaicin administration, mice were placed in an arena and consecutive beam breaks were measured3.

Compared to controls, DAT-TRPV1 mice demonstrated increased activity following capsaicin application, and activity levels subsequently returned to control levels within 15 minutes (Fig 1c)3. These results indicate that capsaicin can induce locomotion in a reversible manner, which is representative of DA’s normal role in motor behaviour6.

Second, DA facilitates goal-directed behaviour through the nigrostriatal pathway7. To investigate whether capsaicin-induced activity would modulate behavioural feeding responses, mice were trained to press a lever for food. Following high doses of capsaicin, lever-pressing was inhibited3; these results are in concordance with

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studies on DA agonists where the drugs were capable of inhibiting food motivation8. However, following low doses of capsaicin, lever-pressing increased3; augmented food incentive behaviours can similarly be seen in some hypodopaminergic mouse models9.

These results suggest that capsaicin-induced activity can either suppress or enhance incentive for food in a dose-dependent manner.

Third, DA plays a significant role in reward, also through the nigrostriatal pathway7. To examine whether capsaicin-induced activity influenced the reward associated with capsaicin in a two-bottle capsaicin preference test, mice were presented with two solutions with either almond or vanilla flavouring, where one flavour was paired with capsaicin and the other with vehicle3. Mice were first allowed access to only one of the solutions during an 8 day association phase, and then were given free access to both solutions3. As capsaicin is the active ingredient that makes chili peppers spicy, wild type mice will not normally consume capsaicin, whereas TRPV1-knockout mice are indifferent3,4. While control mice exhibited no preference for either flavour, DATTRPV1 mice greatly preferred the solution containing capsaicin, which indicates that capsaicin consumption was sufficient to induce DA activity that resulted in increased reward value3.

Capsaicin-dependent activation of 5HT neurons

To demonstrate the adaptability of STEMA across neural systems, triple transgenic ePet-TRPV1 mice were created by crossing R26-TRPV1 mice with ePetCre mice, where Trpv1 was selectively expressed in neurons expressing Cre-recombinase under the control of 5HT transporter ePet3,10. The selective expression in 5HT neurons was confirmed by capsaicin administration followed by double immunohistochemistry for cFos and tryptophan hydroxylase, where 71.2±3.4% of neurons were positive for both markers3.

Further, capsaicin-induced activity elicited behaviours characteristic of 5HT activation. For example, in an open-field anxiety test, ePet-TRPV1 mice spent less time in the center of the field as compared to controls, but returned to control levels within 10 minutes3. This result is comparable to the anxiogenic effects of 5HT agonists11, which indicates that capsaicin is capable of inducing 5HT-like effects in ePet-TRPV1 mice.

Discussion and Conclusions In both DAT-TRPV1 and ePet-TRPV1 lines, TRPV1 was selectively expressed in the appropriate neuronal population, where capsaicin-induced activity was representative of the appropriate neurotransmitter. The capsaicin-induced activity was both rapid and reversible, which indicates that STEMA possesses improved temporal resolution over other designer receptor systems1. Moreover, the transience of the activity highlights an advantage of this technique, as many of the agonists and drugs that can currently activate DA or 5HT systems exert effects on the timescale of hours2,3. Further, STEMA is noninvasive, as surgical procedures are required for neither insertion of TRPV1 nor administration of capsaicin.

Figure 1. (a) DAT-TRPV1 triple transgenic mice, created upon a TRPV1-knockout background, selectively express capsaicin receptor TRPV1 in neurons expressing Cre-recombinase under the control of DA transporter Slca3+/Cre. (b) Following capsaicin administration, double immunohistochemistry for cFos and tyrosine hydroxylase in VTA slices demonstrates selective expression of TRPV1 in DA neurons. (c) Capsaicin administration in DAT-TRPV1 mice is sufficient to induce DA-like locomotor activity, as indicated by beam breaks/15 s made by DAT-TRPV1 (red) or control (black, blue) mice following capsaicin (darker) or vehicle (lighter) injection.

Adapted from “Transient activation of specific neurons in mice by selective expression of the capsaicin receptor” by Güler et al, Nature commun, 3, 746 (2012).

These advantages, among others, help to distinguish STEMA from other techniques and allow it to be a valuable addition to the neuroscience toolkit.

In recent years, various studies have chosen STEMA as the most suitable neuron perturbation technique for their specific investigations. For example, Han et al created MrgprA3+-TRPV1 neurons to demonstrate that the MrgprA3+ nociceptors were specific to itch12, while Wang et al used STEMA to enhance the activity of medial prefrontal cortex neurons while examining their role in vulnerability and resistance to stress within a depression context13.

Critical Analysis

The usefulness of a pharmacogenetic technique to a neuroscience study relies on whether the technique’s specific performance characteristics are appropriately tailored to the unique and specific objectives of the experiment. Such characteristics can be evaluated at the receptor level, the ligand level or the receptor-ligand interaction level2. In many respects, STEMA possesses advantages, though it is not without its limitations.

Receptor: TRPV1 At the receptor level, TRPV1 can be expressed with high spatial resolution, since it can be specifically targeted to genetically defined neuronal populations via transgenic techniques1,2. While TRPV1 is normally endogenously present in the peripheral nervous system and is typically activated by noxious stimuli, the transgenic mice of STEMA were created on a Trpv1 knockout background4.

Thus, exogenous administration of capsaicin only exerts effects on TRPV1 receptors that have been engineered into the CNS, contributing to the high spatial acuity.

In addition, delivery of TRPV1 to specific cell types

is readily achieved, since the coding sequence for Trpv1 is approximately 2.5kb and this small size can be easily accommodated by either lentivirus or adenoassociated virus constructs2. Transgenic, rather than surgical, insertion of TRPV1 also indicates that STEMA is a noninvasive process3.

Furthermore, as a ligand-gated ionotropic channel (LGIC), TRPV1 activation directly affects membrane excitability, thereby bypassing some of the challenges inherent to designer GPCRs, which function through second messenger cascades. Such activation of designer GPCRs may result in sequestration of second messengers, which would indirectly affect the function of endogenous receptors14. G-proteinmediated cascades could also exert other unwanted side effects such as altering gene expression regulation, particularly if GPCR activation is sustained14.

Therefore, designer GPCRs, but not STEMA, may impose a constraint of compatibility with the endogenous system, since there is potential to interfere with normal physiological function2.

However, as a LGIC, the ionic selectivity of TRPV1 must be considered. While TRPV1 is a nonselective cation channel, it has a high permeability to Ca2+ ions1.

The resulting Ca2+ influx upon receptor activation may lead to confounding effects via Ca2+-mediated signaling pathways or even cell death2,3. While the former does require consideration, Ca2+-mediated cell death, however, is not a significant concern. Güler et al demonstrated that microglial activation, an early marker of cell damage, was not present 24 hours following capsaicin administration. Further, when capsaicin was repeatedly applied over a 30 day period, there was no significant reduction in DA neurons3. Nonetheless, it is possible that administration of higher capsaicin doses would result in TRPV1-dependent excitotoxicity.

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Ligand: capsaicin At the ligand level, capsaicin provides many advantages as an effector. First, capsaicin can be conveniently and noninvasively administered in a variety of ways, each of which results in high CNS penetration: orally, intraperitoneally and intravenously1,2. If delivered orally, capsaicin may be self-administered, which provides an opportunity to study reward and addiction models, particularly if TRPV1 is selectively expressed in dopamine neurons2,3.

In addition, capsaicin has high specificity and affinity for TRPV1, which contributes to the high spatial acuity of STEMA3. However, capsaicin is not the only ligand capable of acting on TRPV1, which limits some of the specificity of STEMA. For example, CNS endogenous ligands, such as endocannabinoid anandamide and N-arachidonoyl dopamine (NADA), have the potential to enhance neural activity independently of capsaicin, though they are not comparable to capsaicin in terms of potency at TRPV115,16. Although Güler et al did not observe changes in baseline firing rates of DA neurons in DAT-TRPV1 mice, the possibility of TRPV1 activation by endogenous ligands cannot be ruled out3.

Another advantage to using capsaicin is its rapid metabolism, which contributes to the improved temporal resolution of STEMA17. Following capsaicin administration, neural activity onset can be seen within 2-5 minutes and capsaicin can be cleared in less than 15 minutes2, which makes it an ideal effector if one is investigating transient effects.

Receptor-ligand: TRPV1-capsaicin The improved temporal resolution can be further explained at the interaction level, as the on and off kinetics of a system depends on both ligand and receptor properties. As described above, capsaicin has excellent pharmacokinetic properties due to its rapid metabolism and it also possesses high affinity for TRPV1,2,3.

Further, as an inducible effector system, STEMA provides advantages over traditional transgenic models of knockout/knockdown mice or over-expression mice18. These mice inevitably display abnormal behaviours, substantial neurotransmitter regulatory changes, and other side effects, which introduces significant confounds if one is trying to correlate neural signaling with behaviour18. Although TRPV1 transgenic mice extensively eliminate these problems and thus are hugely advantageous, the TRPV1-knockout mice are not completely identical to wild-type mice3. The most significant discrepancies include impaired sensitivity to noxious temperatures as well as decreased inflammation-induced thermal hyperalgesia4.

Future Directions While the kinetics of STEMA are superior to other designer receptor models, STEMA lacks the strong temporal resolution of optogenetics, where neural activity onset and termination occur on the order of milliseconds19. In the future, advancing this technique could involve improving the kinetic acuity by incorporating some elements of optogenetics. One possibility 119

involves activating TRPV1 with photo-releasable ligands such as caged vanilloids, which are biologically inert precursors that yield active ligands when photolyzed20. A second prospective activation strategy uses the intrinsic thermosensitivity of TRPV1, whereby a short pulse of infrared light may be sufficient to stimulate the receptor21. Future experiments would investigate if either of these activation strategies in DAT-TRPV1 mice is capable of sufficiently improving temporal resolution, through stimulating the receptor while making electrophysiological recordings. Further, these experiments would explore whether the activation strategies could elicit characteristic dopamine activity and behaviour, through tests such as electrophysiology in freely moving mice, locomotor or food consumption assays.

Additional future experiments could involve addressing some of the limitations of STEMA and trying to clarify any possible confounds, such as potential effects of endogenous ligands on TRPV1, excessive activation of Ca2+-mediated signaling pathways, or behavioural side effects resulting from TRPV1-knockout mice15,16,18.

In the case of the former, future experiments could be conducted on endocannabinoid anandamide-knockout or NADA-knockout lines to see if there are any significant differences in baseline firing rates compared to control wild type lines. Such an experiment could eliminate the potential confound of endogenous ligands, which is essential when trying to establish causal relationships.

References 1. Sternson, S.M. & Roth, B.L. Chemogenetic tools to interrogate brain functions. Annu Rev Neurosci, 37, 387-407 (2014).

2. Shapiro, M.G. et al. Unparalleled control of neural activity using orthogonal pharmacogenetics. ACS Chem Neurosci.

3(8), 619-629 (2012).

3. Güler, A.D. et al. Transient activation of specific neurons in mice by selective expression of the capsaicin receptor. Nat commun. 3, 746 (2012).

4. Caterina M.J. et al. Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science. 288, 306–313 (2000).

5. Vellani V., Mapplebeck S., Moriondo A., Davis J.B. & McNaughton P.A. Protein kinase C activation potentiates gating of the vanilloid receptor VR1 by capsaicin, protons, heat and anandamide. J Physiol. 534, 813–825 (2001).

6. Kravitz A.V. et al. Regulation of parkinsonian motor behaviours by optogenetic control of basalganglia circuitry.

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7. Wise R.A. Dopamine, learning and motivation. Nat Rev Neurosci. 5, 483–494 (2004).

8. van der Hoek G.A. & Cooper S.J. The selective dopamine uptake inhibitor GBR 12909: its effects on the microstructure of feeding in rats. Pharmacol Biochem Behav. 48, 135–140 (1994).

9. Beeler J.A., Frazier C.R. & Zhuang X. Dopaminergic enhancement of local food-seeking is under global homeostatic control. Eur J Neurosci 35(1), 146-59 (2012).

10. Scott M.M. et al. A genetic approach to access serotonin neurons for in vivo and in vitro studies. Proc Natl Acad Sci USA. 102(45), 16472â&#x20AC;&#x201C;16477 (2005).

11. Stiedl O, et al. Activation of the brain 5-HT2C receptors causes hypolocomotion without anxiogenic-like cardiovascular adjustments in mice. Neuropharmacol. 52, 949â&#x20AC;&#x201C;957 (2007).

12. Han, L. et al. A subpopulation of nociceptors specifically linked to itch. Nat Neurosci, 16(2), 174-182 (2013).

13. Wang, M., Perova, Z., Arenkiel, B.R. & Li, B. Synaptic modifications in the medial prefrontal cortex in susceptibility and resilience to stress. J Neurosci. 34(22), 7485-7492 (2014).

14. Nichols, C.D. & Roth, B.L. Engineered G-protein coupled receptors are powerful tools to investigate biological processes and behaviors. Front Mol Neurosci. 2 (2009).

15. Smart, D. et al. The endogenous lipid anandamide is a full agonist at the human vanilloid receptor (hVR1). Br J Pharmacol. 129(2), 227-30 (2000).

16. Huang, S.M. et al. An endogenous capsaicin-like substance with high potency at recombinant and native vanilloid VR1 receptors. Proc Natl Acad Sci USA. 99(12), 8400-5 (2002).

17. Chanda, S., Bashir, M., Babbar, S., Koganti, A. & Bley, K. In vitro hepatic and skin metabolism of capsaicin. Drug Metab Dispos. 36, 670-675 (2008).

18. Haenisch, B. & Bonisch, H. Depression and antidepressants: insights from knockout of dopamine, serotonin or noradrenaline re-uptake transporters. Pharmacol Ther. 129, 352-368 (2011).

19. Yizhar, O. et al. Optogenetics in neural systems. Neuron.

71, 9-34 (2011).

20. Zhao, J. et al. Caged vanilloid ligands for activation of TRPV1 receptors by 1-and 2-photon excitation. Biochem.

45(15), 4915-4926 (2006).

21. Tzabazis, A.Z. et al. Selective nociceptor activation in volunteers by infrared diode laser. Mol Pain. 7, 18 (2011).

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Differential Brain Activation in Sommeliers: Effects of expertise on flavour integration Sylvia Jennings

The purpose of this study was to study the effects of expertise on perception of flavour. The study uses wine experts to understand the integration of a complex stimulus across modalities. Using fMRI and subtracting from control wine novices, the authors were able to isolate key differences in brain activation when experts and novices tasted wine. Experts had more immediate, stronger, and widespread activation, and activated more areas involved in memory. Experts also activated perceptual areas not involved in this study, which suggests that the brains of wine experts are wired to detect stimuli with many modalities. Further studies should investigate this integration using more modalities to truly demonstrate wine experts’ adaptations. This study was an important step in our knowledge pertaining to the structural and functional changes that occur in the brain as a result of expertise. It highlights that expertise is affecting both perception and cognition, making an expert more adept, and also more efficient.

Key words: fMRI, flavour, expertise, olfaction pathways, wine, taste Background The purpose of this study is to examine the effects of expertise on taste perception. The authors investigated this using neuroimaging during wine consumption. The investigating these effects involves three major areas of research: taste perception, integration of modalities, and the neurological and psychological effects of expertise.

There are many ways in which taste perception can be modified, and each of these has an effect on eating behaviour. Eating behaviour is a complicated but urgent issue, as it impacts obesity, nutrition, and the environment (Pazart et al., 2014). Flavour integration has a direct impact on eating behaviour (Okamato and Dan, 2013). Understanding the mechanisms behind flavour integration will further our insight into eating behaviour. Studying expertise’s effects is a convenient way to explore this integration and how it can differ depending on prior experience.

Taste alone has been found to activate areas of the brain involved in both perception and affect (O’Doherty et al., 2001). Tasting activates areas of the thalamus, orbitofrontal cortex, amygdala, and insular taste cortex.

Location and intensity of activation in the amygdala and orbitofrontal cortex will vary depending if the stimulus is negative or positive (O’Doherty et al., 2001). Some fMRI studies have shown that there is a significant top-down effect on taste perception (Kobayashi et al., 2004). In particular, taste imagery will affect perception.

The other most important influence on flavour perception is smell. Although many factors such as sight, hearing and semantic cues can influence perception, the integration of taste and olfaction is the main mode of taste perception (Auvray and Spence, 2008). PET studies have shown that when olfaction and gustatory senses are stimulated simultaneously, their primary cortices are less active than when stimulated alone (Small et al., 1997). These studies also showed that novel taste and smell experiences activate the basal forebrain and amygdala. FMRI studies show that the insula, caudal orbitofrontal cortex, anterior cingulate cortex, and frontal operculum are involved in the 121

integration, especially when confronted with a novel stimulus (Small et al., 2004). The fact that novel stimuli affect activation implies that familiarity is an important factor in taste perception.

The differences between experts and novices has long been studied in psychology, and more recently in neuroimaging. Most heavily studied area of expertise is visual recognition. Neuroimaging studies have shown that experts have much more activation in response to their domain-specific stimulus than novices, but some studies have shown that this effect is modified by level of engagement (Harel et al., 2010). In odor experts, it’s been shown that activation is modified compared to novices such that their brains are structurally and functionally different (Thomas-Danguin et al., 2014).

Odor training results in increased sensitivity and identification ability. Experts have also shown to develop odor mental imagery, not found in novices. This implies that the structural changes are creating a very different experience towards odorants, which would heavily impact flavour perception/eating experience.

In this study the authors examined the differential brain activation between wine experts and novices while consuming wine. The consumption was tightly controlled, such that the participants could only taste and retronasally smell the wine. FMRI monitored activation during a taste phase and after-taste phase.

Research Overview

Summary of Major Results

The results of this study highlight key differences between experts and novices. All participants had activation in the insula, frontal lobe, pallidum, parahippocampal gyrus and the thalamus. Activation persisted in the insula and frontal lobe during the after-taste phase. To analyze the differences between experts and the control novices, the authors subtracted one group’s activation from the other. This showed that during the taste phase, experts had twice as many

brain regions activated. During the after-taste phase, the opposite effect was shown: novices had many more brain regions activated compared to controls.

See figure 3 in appendix for full results of activation.

There was also a difference in type of brain activation. Novices showed widespread parietal activation during the taste phase, whereas experts activated many areas of the temporal lobe, brain stem, and the occipital associative cortex. Not until the after-taste phase do the novices show greater activation in the temporal lobe. They also show greater activation in the frontal and parietal lobe. Experts show greater activation in the hippocampus during this phase.

This difference in activation is significant because it shows that experts are processing stimuli faster, and with a more complex perception. Because of the differential brainstem activation, it’s clear that experts are processing stimuli differently at the most basic levels.

During the after-taste phase, experts had completed the processes related to flavour integration and only memory processes remain. A clear difference between experts and controls is that only experts activated the hippocampus or parahippocampal region. Controls were still activating integration processes during this phase.

Conclusions and Discussion

Wine is a particularly good test of expertise. It’s been shown in previous studies that wine experts have a superior sense of smell, and have developed scent imagery – a skill not ordinarily possessed (Royet et al., 2013). Experts in this area have developed a semantic component to the integration of scent and taste that others have not. This ability shows that experts are different from novices in a perceptual and intellectual way. Wine experts are intensely practiced such that they can integrate information from sight, taste, smell and semantic cues to quickly assess many qualities.

Wine experts had an immediately different reaction to wines from the instant of perception. Their brainstem was more activated, implying they had a stronger perception of flavour. Their temporal lobe was very strongly activated in many areas, implying that processing of the stimuli was happening immediately. The orbitofrontal, operculum, and insula, all essential for the integration process (Small and Prescott, 2005), were activated during the earlier phase in experts, but not as clearly activated in controls until the after-taste phase. Experts had a much earlier reaction and ability to integrate flavours. Their activation was also much more concise. Though they had much more activation in the first phase, it was located mostly in the temporal lobe, with specific projections into the frontal lobe, presumably to enable complex processing. The controls showed little temporal lobe activation, but widespread parietal lobe activation, which the experts demonstrated almost none of. This suggests that this entire portion of the cortex is unnecessary for extensive wine analysis.

By the after-taste phase there are very clear differences between the experts and controls. Novices were still integrating senses via the operculum, orbitofrontal cortex and insula. Novices had begun to activate the temporal lobe in a more widespread manor, but the experts appear to have only memory-related processes left over. Activation in the occipital association cortex,

prefrontal lobe, and hippocampus demonstrate the experts’ extensive memory for wine, as they’re all associated with memory processing , and not directly with perception (Parr et al., 2004). Thus, experts are automatically recalling and comparing the wines.

Figure 1. Graph shows experts (red) vs. controls (blue).

Highlights the conciseness of the expert’s activation in the key areas for integrating taste and scent (Pazart et al., 2014).

Figure 2. Graph shows experts (green) vs. controls (blue).

Highlights the extensive activation in key areas for memory found in experts compared to controls (Pazart et al., 2014).

Conclusions The main conclusions to be drawn from the study are that expertise promotes faster and more efficient flavour processing. Further, the brain areas involved appear to be memory-related, implying a deeper understanding of the flavour (associated origins, familiar characteristics). Expertise implies faster processing at the appropriate levels of the brain to integrate flavour from various modalities.

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Criticisms and Future Directions

A large problem with this study was the absence of stimuli. Wine tasting traditionally has three phases: swirling and visually examining, sniffing, and then tasting. The difference between wine experts and novices would be most apparent when other sources of information about the wine (such as orthonasal scent and sight) are present. Wine experts have developed a particular skill of integrating these senses, and the more senses that are integrated, the faster and more refined the experts’ activation would be compared to novices. Assessing wine experts activation in an unnatural way could severely impact their ability to succinctly perceive and retrieve information.

Sight is a major component to wine tasting particularly pertaining to colour. Previous studies have investigated the general effects of colour on flavour (Spence et al., 2010), and these effects would certainly be present in wine tasting. As the results of this study showed, the visual association cortex was activated even without any visual stimulus, demonstrating that the automatic reaction of experts includes visualization as a key component.

Very importantly, wine experts will sniff the wine before tasting. Omitting this component is a large flaw in understanding the effects of expertise. Studies have shown that taste and smell are not simply additive (Seubert et al., 2014). The combined effects activate a different pathway. Including an orthonasal component in this study could make an even faster, more efficient, and profound effect between experts and novices.

Another issue with this study could be the perceived pleasantness between wine experts and novices.

It’s been shown that wine experts have a greater preference for dry wines, such as used in this study (Blackman et al., 2010). The novice participants might have had an aversive reaction to the wines, and the experts a positive reaction. This difference in preference would have a large effect on the contrasting activation. In particular, the temporal lobe is highly associated with responding to positive or negative stimuli, and this might be responsible for some of the widespread temporal activation in experts. To control for this, a future study could pre-screen the wines to be sure there is no difference in preference between a typical novice compared to experts.

Future studies need to integrate as many aspects of the traditional wine tasting process as possible.

Studies should also consider differences in wine preference, as inappropriate stimuli for novices could be a confounding variable.

References 1. Auvray M, Spence C (2008) The multisensory perception of flavor. Conscious Cogn 17:1016–1031.

2. Blackman J, Saliba A, Schmidtke L (2010) Sweetness acceptance of novices, experienced consumers and winemakers in Hunter Valley Semillon wines. Food Qual Prefer 21: 679–683.

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3. Harel A, Gilaie-Dotan S, Malach R, Bentin S (2010) Topdown engagement modulates the neural expressions of visual expertise. Cereb Cortex 20:2304–2318.

4. Kobayashi M, Takeda M, Hattori N, Fukunaga M, Sasabe T, Inoue N, et al (2004) Functional imaging of gustatory perception and imagery: “top-down” processing of gustatory signals. Neuroimage 23:1271–1282.

5. O’Doherty J, Rolls E T, Francis S, Bowtell R, McGlone F (2001) Representation of pleasant and aversive taste in the human brain. J Neurophysiol 85:1315–1321.

6. Okamoto M, Dan I (2013) Extrinsic information influences taste and flavor perception: a review from psychological and neuroimaging perspectives. Semin Cell Dev Biol 24:247–255.

7. Parr WV, White KG, Heatherbell DA (2004) Exploring the nature of wine expertise: what underlies wine experts’ olfactory recognition memory advantage? Food Qual Prefer 15: 411–420.

8. Pazart L, Comte A, Magnin E, Millot J-L and Moulin T (2014) An fMRI study on the influence of sommeliers’ expertise on the integration of flavor. Front Behav Neurosci 8:358.

9. Royet JP, Plailly J, Saive AL, Veyrac A, Delon-Martin C (2013) The impact of expertise in olfaction. Front Psychol 4:928.

10. Seubert J, Ohla K, Yokomukai Y, Kellermann T, Lundström JN (2014) Superadditive opercular activation to food flavor is mediated by enhanced temporal and limbic coupling.

Hum. Brain Mapp. doi: 10.1002/hbm.22728 11. Small DM, Jones-Gotman M, Zatorre RJ, Petrides M, Evans AC (1997) Flavor processing: more than the sum of its parts. Neuroreport 8:3913–3917.

12. Small DM, Voss J, Mak YE, Simmons KB, Parrish T, Gitelman D (2004) Experience-dependent neural integration of taste and smell in the human brain. J Neurophysiol 92: 1892–1903.

13. Small DM, Prescott J (2005) Odor/taste integration and the perception of flavor. Exp Brain Res 166: 345–357.

14. Spence C, Levitan CA, Shankar MU, Zampini M (2010) Does Food Color Influence Taste and Flavor Perception in Humans? Chemosensory Perception 3(1): 68-84.

15. Thomas-Danguin T, et al. (2014) The perception of odor objects in everyday life: a review on the processing of odor mixtures. Cogn Sci 5: 504.

Modeling and Treatment of Familial Parkinson’s Disease Using iPSCs

Nimara Dias

This review will discuss the way in which induced pluripotent stem cell (iPSC)-derived neurons, generated using skin fibroblasts from individuals with Parkinson’s disease (PD), are used as a means of observing the neurodegenerative phenotype associated with familial PD. How these derived neurons interact with specific cellular stressors, particularly with mutations for example, found in the PTEN-induced putative kinase 1 (PINK1) gene as well as the leucine-rich repeat kinase 2 (LRRK2) gene that are commonly involved in the familial, or inherited, form of Parkinson’s disease will also be analysed. By using iPSC-derived neurons from PD patients, it becomes possible to create a closely representative model of PD and from there, see what factors cause or drive the progression of the disease as well as attempt to rescue the ensuing phenotype. Pharmacological treatment for individual patients who may respond differently depending on their specific genotype is just one method of treatment among other possible future directions.

Key words: Parkinson’s Disease (PD); induced pluripotent stem cells (iPSCs); fibroblasts; dopamine (DA); PTEN-induced putative kinase 1 (PINK1); leucine-rich repeat kinase 2 (LRRK2); sporadic PD; familial PD; mitochondrial reactive oxygen species (mROS); cellular stressors Background Parkinson’s disease (PD) is characterized by an ongoing degeneration of dopamine-generating neurons and synapses of the nigrostriatal pathway1.

This neurodegeneration leads to a slow onset of clinical symptoms such as resting tremor, bradykinesia and a shuffling gait2, as well as nonmotor, cognitive symptoms as the disease progresses like depression, hallucination, and dementia among others3. The majority of PD cases are sporadic (idiopathic or lateonset PD), meaning that they may be brought forth by both genetic and environmental factors4. However, there are rare cases of familial, or inherited/earlyonset PD, and through studies of this form, causative mutations have been identified and knowledge of Parkinson’s disease has significantly expanded1.

The most prominent mutations that have been identified are found in the leucine-rich repeat kinase 2 (LRRK2) gene, the PTEN-induced putative kinase 1 (PINK1) gene, the parkin gene (PARK2) and the α-synuclein gene (SNCA)4. The dominant mutation of LRRK2, that is involved in both familial (predominantly) and sporadic PD, causes the impairment of the function of mitochondria and is involved with loss of dopamine neurons5. Like LRRK2, PINK1 also encodes a kinase, this one particularly found in the membrane of the mitochondria4. It is thought to play a part in neuroprotection, and recessive mutations in this gene causes disruption in its kinase-signalling pathway6 leading to early-onset, familial PD1. Parkin and α-synuclein are highly associated in that they are commonly found localized together in Lewy bodies (LB), a hallmark of PD pathology7. Parkin may actually be involved in LB formation and aggregates of α-synuclein are characteristic of Lewy bodies7.

The most problematic factor of understanding the true pathology of PD was that an appropriate model that closely resembled Parkinson’s disease in humans was not possible to find. Cultures of neurons from

animal models were difficult to sustain and differences in genes provided an unreliable model. With the use of iPSCs derived from the fibroblasts of the patients themselves which were reprogrammed back into a pluripotent state, and with the correct factors, differentiated into dopaminergic neurons, the mutations identified in the familial form of PD could be understood much more clearly8. Mechanisms by which the phenotype of PD arose, caused by the aforementioned mutations, could now be defined4.

Typical experiments involving these iPSCs include the addition of cellular stressors to the iPSCs of patients that contain mutations in PD-associated genes, to observe what generates the pathology of PD4. Mitochondrial reactive oxygen species (mROS) levels are then analysed as a measure of vulnerability of the derived cells, mROS being a chemically inducible source of cellular stress1. From here, ways of rescuing the phenotype are then studied, for example, through gene correction5 or pharmacological treatment1.

Research Overview

Summary of Major Results

Cooper et al. conducted experiments looking at specific mutations in genes that impact the activity of kinases involved with mitochondrial function: LRRK2 and PINK1, that seem to increase the risk of developing familial Parkinson’s disease. By using iPSCs derived from fibroblasts of familial PD patients as well as healthy patients without the disease and patients with the mutations who have not yet shown signs of PD, to form dopaminergic (DA) neural cells, researchers from several labs were able to combine assay results to compare the cell phenotypes from each1.

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Chemical Stressors and Vulnerability

The mitochondria and proteins of the derived DA neurons from all subjects were first tested for vulnerabilities (dysfunction and degradation respectively) using ten different chemical stressors, where the vulnerability was measured in terms of the amount of lactate dehydrogenase (LDH) released from the cells1.

The neural cells containing the recessive homozygous Q456X mutation in PINK1 showed higher vulnerability to valinomycin, MPP+, and concanamycin A among others compared to healthy individuals1. This was shared with both heterozygous and homozygous LRRK2 mutations, demonstrating that both asymptomatic individuals carrying the mutation and PD patients show vulnerabilities to similar chemical stressors1. In addition, after immunocytochemistry and cell count assays, it was observed that these same neural cells also contained less DA neurons after application of chemical stressors1. In keeping with a separate study observing the association of MPP+ with DA neuronal cell death, a significant loss of DA neurons after exposure to a low dosage of MPP+ was also shown2.

mROS

Neural cells from patients with PD carrying the PINK1 Q456X mutation showed increased levels of mROS when a low concentration of valinomycin was applied, however this increase did not occur with the addition of any other chemical stressors1. Typically the antioxidant GSH acts as a protective measure against harm that arises from increased levels of mROS1. These neural cells were observed to have GSH present, and at lower levels in comparison to healthy individuals, after addition of valinomycin, MPP+ and concanamycin A, meaning that cellular oxidative stress also causes high vulnerability for those carrying the PINK1 mutation1 (Figure 1).

A study that looked at GSH distribution in the substantia nigra (SN) confirmed that there is a depletion of GSH within the SN of PD patients through mercury orange histofluorescence on samples of brain tissue9 (Figure 2).

Graziotto, J. et al. Pharmacological Rescue of Mitochondrial Deficits in iPSC-Derived Neural Cells from Patients with Familial Parkinsonâ&#x20AC;&#x2122;s Disease.

Sci Transl Med. 4, 141ra90-141ra90 (2012).

Figure 1 - Measuring levels of GSH in response to increasing concentrations of cellular stressors added. D,E,F, and G show a reduction in GSH levels in PINK1 Q456X mutant neural cells at varying concentrations of particular cellular stressors in comparison to healthy subjects, whereas H, and I show no change between healthy and PD patients.

Mitochondrial Respiration

The oxygen consumption rates of derived neural cells were determined by the use of compounds targeting ATP synthase and components of the electron transport chain (ETC) in mitochondria1. The basal rate of oxygen consumption in those carrying the PINK1 mutation was increased in comparison to healthy individuals and was not changed with the addition of oligomycin (targets ATP synthase). However those carrying either the heterozygous or homozygous mutation of LRRK2 showed decreased basal oxygen consumption rates and similar reactions to oligomycin, FCCP and rotenone (the latter two targeting components of the ETC)1.

Mobility of Mitochondria

Using live cell imaging, it was found that PD patients only carrying the LRRK2 mutations showed an increase in mobility of mitochondria in a more bidirectional manner as well as a decrease in length, where the mitochondria in axons of neural cells containing this mutation were 125

Pearce, R.K.B., Owen, A., Daniel, S., Jenner, P., Marsden, C.D. Alterations in the distribution of glutathione in the substantia nigra in Parkinsonâ&#x20AC;&#x2122;s disease.

J Neural Transm. 104, 661-677 (1997).

Figure 2 - (A,C) transmitted light, (B,D) fluorescence. A and B show PD substantia nigras stained for GSH with less expression of GSH in comparison to the control (C and D) substantia nigras.

20% shorter, in comparison to both healthy individuals and those carrying the PINK1 mutation1. Interestingly, the axons themselves of the iPSC-derived PD neural cells, or those carrying the mutations, were not of a different length in comparison to healthy subjects, however this does not seem to match the literature in which a decrease in length of neurites is often seen in LRRK2 mutant cell forms of PD patients10.

Pharmacological Rescue

To see if vulnerability of neural cells that carried the PINK1 and LRRK2 mutations could be rescued pharmacologically, coenzyme Q10 or rapamycin (antioxidants), or an LRRK2 inhibitor was used1.

The results showed that coenzyme Q10 reduced the release of LDH in neural cells carrying any mutation (if exposed to low concentrations of valinomycin but not high concentrations), rapamycin reduced the vulnerability of neural cells with the LRRK2 mutation only to valinomycin, but no other mutant neural cell, and the LRRK2 inhibitor reduced LDH release of any neural cell mutant from exposure to valinomycin1. In addition rapamycin and the LRRK2 inhibitor reduced mROS levels from neural cell PINK1 mutants exposed to valinomycin1. Several studies have also discussed possible herbal remedies that can be used to reduce oxidative damage, such as green tea component epigallocatechin 3-gallate (EGCG) that was shown to prevent loss of DA neurons from exposure to MPTP11, however these experiments use mice models and so this may not be representative of human PD. Discussion From the initial experiments that assessed vulnerability levels in iPSC-derived neural cells from patients with familial PD and pre-symptomatic patients who carry the mutations as well, it was shown that these cells displayed higher vulnerability when exposed to low concentrations of chemical stressors such as valinomycin1. Valinomycin is a chemical stressor which causes the depolarization of mitochondria with an influx of K+ ions1. However they do not show vulnerability to FCCP, a chemical stressor that does the same function except using protons. This may show that the mutations in the derived neural cells cause the inability to respond to mitochondria that has been damaged by K+ ions1.

Oxidative stress is one of the major contributors to the characteristic loss of DA neurons causing Parkinson’s Disease.12 Nitric oxide (NO) and H2O2 are substances known as reactive oxygen species, and these are formed as metabolism by-products12. An increase in these substances will cause DNA, lipid and protein damage (oxidative damage)12. Antioxidants such as reduced GSH counteract the effects of oxidative damage12, however experiments show that PD patients, or those carrying the familial PD mutations, have depleted levels of GSH in response to chemical stressors1. This is to be expected and confirms that a proper PD model is being used as autoxidation of dopamine is what both generates H2O2 and reduces GSH allowing free radicals to accumulate, oxidative damage to continue and loss of DA neurons to progress12.

It was shown that the basal rate of oxygen consumption in those carrying the PINK1 mutation was increased in comparison to healthy individuals but remained unchanged with the addition of oligomycin that inhibits ATP synthase1. This is showing that ATP-independent respiration was taking place and that protons were increasingly moving passively from the membrane of the mitochondria1. However in LRRK2 mutant neural cells there was a decreased basal oxygen consumption rate and similar reactions to oligomycin, FCCP and rotenone1. So therefore in opposition to those carrying the PINK1 mutation, proton leakage was not enhanced1.

In terms of the mobility of mitochondria, only the neural cell lines that carried the LRRK2 mutations showed any differences from the normal axonal transport process1. Studies using Drosophila as a model of PD clearly showed that PINK1 had strong implications as a regulator of mitochondrial transport and that knockdowns of it would cause dysfunctional mitochondria and impaired mitochondrial distribution13.

This could possibly be considered an example of discrepancies that occur between models, where the iPSC line derived specifically from the fibroblasts of the patient themselves is more accurate in describing the mechanisms behind human PD, as opposed to animal or insect models.

Using iPSCs offers a major benefit in understanding what products could be used to rescue a diseased phenotype. Pharmacological rescue of vulnerability was displayed by antioxidants like coenzyme Q10 and rapamycin, as well as an LRRK2 inhibitor1, but there are so many more compounds that can be used to target different aspects of cellular stress that may be influencing the neurodegenerative phenotype of PD. For example, glial-derived neurotrophic factor (GDNF) was shown in a separate study to reverse the effects of MPP+ (an inhibitor of mitochondrial complex I also involved in loss of DA neurons1) where the presence of GDNF allowed for 90% cell survival (as opposed to the 50% cell survival in the presence of MPP+ alone)2.

Conclusion It is through the use of iPSCs that the most accurate information can be gathered about human Parkinson’s disease. Throughout the literature discrepancies between this model representing the patients themselves, and animal models could be identified. The continuous degeneration of dopamine-generating neurons in the nigrostriatal pathway is the key feature of Parkinson’s disease and understanding how this disease arises is necessary for understanding how to rescue the phenotype. The iPSCs generated in this study were efficient in responding to low concentrations of cellular stressors that can be thought to mimic the gradual amassing of dysfunctional mitochondria (as you would see in aging PD patients) and so they provide an opportunity to delve into how these significant mutations affect the degenerative aspects of this disease1. Individual treatment is possible by using iPSCs, and can be done by using cellular reprogramming technology as seen from the data collected of 126

which mutations respond to which antioxidants for pharmacological rescuing. In short, these neural cells derived from iPSCs allow for major advances in understanding the foundation of PD and possible treatments.

Criticisms and Future Directions

The literature surrounding PD iPSC-derived neural cells as a proper model of neurodegenerative diseases and as a therapeutic model is slowly becoming more significant. This is just the beginning of recognizing the true potential of understanding the mechanisms by which Parkinson’s disease arises and can be stopped.

There are limits to what animal models, cellular models and in vitro models can offer4 and in an era of individualized medicine, the use of iPSCs has no end.

From Cooper et al.’s paper what can be seen already is a more defined mechanism by which loss of DA neurons occurs, a difference in what phenotypes arise from each familial PD mutation and differences in the pharmacological substances that will rescue each phenotype the best. Critically speaking though, even though this paper was about pharmacological rescue of mitochondrial deficits, a lot of the focus was put on rescuing vulnerability to two particular chemical stressors1. Focusing on specific aspects of the overall degeneration that comes with PD was repeated often in the literature and represents the huge gap in knowledge of all the known aspects put together.

Little research has contemplated how many unknown factors actually come in to play.

The most important future direction that the majority of the literature surrounding the rare familial cases of PD point to, is how to use this information to understand and potentially diminish the much more common cases of sporadic PD. Since both familial and sporadic PD forms have similar implications in impaired mitochondrial function, axonal transport, improper protein aggregation, etc. all eventually leading to loss of neurons, and both are related to age14, the use of iPSCs has also given an incredible advantage in the ability to extrapolate what can be learned from genetic cases, and apply it to idiopathic ones. Also, although pharmacological treatment for individual cases is often researched, another plausible method of treatment is gene correction. Sanders et al. found that the damage done to mtDNA by the LRRK2 G2019S mutation was rescued by ZFN-mediated correction of the mutation in the derived iPSC lines15. This opens up a brand new pathway for treatment and one in which a mixture of pharmacological products would not be required to rescue the disease phenotype.

With the transition from animal models to iPSCs, research has surged forward in an unprecedented manner. An accurate model has provided much more potential to identify the cause of PD and finally establish safe and effective treatment methods.

References 1. Graziotto, J. et al. Pharmacological Rescue of Mitochondrial Deficits in iPSC-Derived Neural Cells from Patients with Familial Parkinson’s Disease. Sci Transl Med. 4, 141ra90-141ra90 (2012).

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2. Peng, J., Liu, Q., Rao, M.S., Zeng, X. Using Human Pluripotent Stem Cell– Derived Dopaminergic Neurons to Evaluate Candidate Parkinson’s Disease Therapeutic Agents in MPP+ and Rotenone Models. J Biomol Screen. 18, 522-523 (2013).

3. Chaudhuri, K.R., Healy, D.G., Schapira, A.H.V. Nonmotor symptoms of Parkinson’s disease: diagnosis and management. Lancet Neurol. 5, 235–245 (2006).

4. Beevers, J.E., Caffrey, T.M., Wade-Martins, R. Induced pluripotent stem cell (iPSC)-derived dopamingergic models of Parkinson’s disease. Biochem.Soc Trans. 41, 1503-1508 (2013).

5. Xu, Q., Shenoy, S., Li, C. Mouse models for LRRK2 Parkinson’s disease. Parkinsonism Relat D. 18S1, S186-S189 (2012).

6. Gandhi, S, et al. PINK1 protein in normal human brain and Parkinson’s disease. Brain. 129, 1720-1731 (2006).

7. Schlossmacher, G.M. et al. Parkin Localizes to the Lewy Bodies of Parkinson Disease and Dementia with Lewy Bodies.

Am J Pathol. 160, 1655-1667 (2002).

8. Soldner, F. et al. Generation of Isogenic Pluripotent Stem Cells Differing Exclusively at Two Early Onset Parkinson Point Mutations. Cell. 146, 318-331 (2011).

9. Pearce, R.K.B., Owen, A., Daniel, S., Jenner, P., Marsden, C.D. Alterations in the distribution of glutathione in the substantia nigra in Parkinson’s disease. J Neural Transm.

104, 661-677 (1997).

10. Burke, R.E., O’Malley, Karen. Axon degeneration in Parkinson’s disease. Exp Neurol. 246, 72-83 (2013).

11. Choi, J. et al. Prevention of Nitric Oxide-Mediated 1-Methyl-4-Phenyl-1,2,3,4-Tetrahydropyridine-Induced Parkinson’s Disease in Mice by Tea Phenolic Epigallocatechin 3-Gallate. NeuroToxicology. 23, 367-374 (2002).

12. Surendran S., Rajasankar S. Parkinson’s disease: oxidative stress and therapeutic approaches. Neurol Sci. 31, 531-540 (2010).

13. Liu, S. et al. Parkinson’s Disease-Associated Kinase PINK1 Regulates Miro Protein Level and Axonal Transport of Mitochondria. PLoS Genet. 8, e1002537 (2012).

14. Sanchez, G. et al. Unaltered Striatal Dopamine Release Levels in Young Parkin Knockout, Pink1 Knockout, DJ-1 Knockout and LRRK2 R1441G Transgenic Mice. PLoS ONE. 9, e94826 (2014).

15. Sanders, L.H. et al. LRRK2 mutations cause mitochondrial DNA damage in iPSC-derived neural cells from Parkinson’s disease patients: Reversal by gene correction. Neurobiol Dis. 62, 381-386 (2014).

Received April 6, 2015; revised April 6, 2015; accepted April 6, 2015.

This work was supported by The Association for the Development of Undergraduate Neuroscience Education (SRA & RLN), The Endowment for Science Education (EA), and The Synaptic State Faculty Research Foundation (EA).

The author thanks Dr. Ju, and the students in HMB300 for technical assistance, execution, and feedback on this assignment.

Address correspondence to: Department, 40 Willcocks Toronto, ON M5S 1C5

Nimara Dias, Human Biology St, University of Toronto, Email: dias.nimara@gmail.com

Copyright © 2015 Dr. Bill JU, Neurosciences, Human Biology Program

To accomplish more or loss less: the story of sleep deprivation and Alzheimer’s disease

Xin Yue Kou

It is a difficult question to define what is sleep, and would take a whole field of research to try to explain that question, which we still don’t quite have the answer to yet. What we do know is that sleep is vital, many processes occurs during sleep, restoring our body to optimal condition. Based on experiences, we know that when we temporarily loose a large amount of sleep we lack the ability to physically and cognitively function properly, however when we spread the amount of sleep lost over a longer period of time, it might be harder to feel the same effects, but chronic sleep deprivation is in fact more detrimental than how we physically feel about it.

Previous studies have looked at the link between sleep deprivation and neurodegenerative diseases such as Alzheimer’s disease (AD), where sleep abnormalities can increase the risk of an individual to develop AD, although there is also evidence vice versa suggesting changes in sleep-wake cycles can cause significant increase in AD progression through increased amyloid-beta plaques. However it is uncertain whether SD is a causal risk factor or simply a disease biomarker, due to that lack of intensive knowledge for effects on other important AD phenotypes. Hence this review will set to determine the role of sleep deprivation in AD. Key words: Sleep deprivation, chronic stress, Alzheimer’s disease, amyloid precursor protein, tau metabolism, astrocytes.

Background Neurological dementias such as Parkinson’s disease, dementia with Lewy bodies, Alzheimer’s disease and many more are becoming increasingly common in our society. As more and more death are caused by these diseases, it has became the pressing concern to find an effective cure. Alzheimer’s disease being the most common of the neurodegenerative diseases have definitely been intensively studied, and we have learned much more about the disease since it was first diagnosed, however we have still yet to find the defining evidence that will lead us to the cure.

Defining features of AD include the amyloid-beta plaques caused by the cleaved form of the amyloid precursor protein (APP), amyloid-beta 40 and 42, and the neurofibrillary tangles caused by aggregating hyper-phosphorylated tau proteins [1,2]. However recent studied show that neuropathological events such as the loss of neurons occur far before the disease is clinically recognized [3,4]. Hence it is critical to find the appropriate biomarker that can detect the earliest onset of AD. One way to look for early associative biomarkers is to find potential risk factors that may lead to AD, which would be useful in terms of narrowing down subjects who may be more likely to develop AD, and therefore track the development of AD. Finding a risk factor is also important in terms of prevention, and developing treatment as well.

Studies have shown evidence of chronic stress associated with cognitive decline similar or related to AD [5,6], especially the link between sleep deprivation and AD have caught the attentions of many scientists. Many evidences show that chronic sleep deprivation or changes in sleep patterns such as sleep fragmentation is associated with phenotypes of AD [7], for example amyloid-beta plaques were shown

to correlate positively with sleep deficits [8]. Even though many evidence have illustrated that there is a relationship between sleep deficits and AD, there still lacks very clear evidence showing that chronic sleep deficits do in fact cause an increased risk of AD rather than just an associative biomarker as the disease progresses.

One of the first study conducted to look at the various AD phenotypes in relation to sleep deprivation is by Di Meco et al, where they used the 3xTg mouse model and introduced the mice to chronic sleep deprivation over 2 month trial. The mice were then later sacrificed for brain examination by biochemistry assays as well as immunohistochemistry studies, and results demonstrated that chronic sleep deprivation do in fact likely contribute to the development of AD [9].

This review will critically analyze the results of the aforementioned article as well as expanding on related evidences provided by other studies.

Research Overview

Major Results and Discussion

Sleep deprivation decrease cognitive functions The 2 different groups of 3xTg mice were tested using the Y-maze, fear conditioning which showed insignificant result, and only in the Morris water maze where sleep deprived mice showed significantly decreased spatial memory [9].

Most studies confirms that cognitive function such as learning and memory is greatly affected by sleep deprivation [10], where not only spatial memory is affected, but episodic memory as well, as sleep plays an important role in memory consolidation and improves synaptic plasticity [11].

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Sleep deprivation and Amyloid-beta Surprisingly the assay results did not show significant difference of the plaque forming amyloid-beta 1-40 and 1-42, although the results suggested to be not very reliable due to the young age of the mice [9].

This definitely did not support other studies, previous evidence shown in APP-PS1 mice suggested that amyloid-beta plaque deposition is significantly associated with sleep deprivation [8]. This association has been tested in human subjects as well based on recording of decreased REM sleep and increased SWS fragmentation. The study by Sanchez-Espinosa et al looked at the relationship between sleep deficits and plasma amyloid-beta level in amnestic mild cognitive impairment (aMCI) subjects, often considered the prestage of developing AD, as well as normal healthy old (HO) adults. Results show that higher level of amyloidbeta deposition is significantly positively correlated with increased SWS arousals only in aMCI subjects, not HO (figure1) [12]. Therefore it is likely that sleep deficits facilitate plaque deposits in AD individuals, however is it still unsure whether sleep deficits can cause plaque deposition under non-AD condition.

conformational change, which correlates to the results of increased MC-1 antibody immunoreactivity, which is used to recognize forms of pathogenic tau, in sleep deprivation mice[9].

However other studies do suggest that tau metabolism is not significantly affected by sleep deficits [13].

Sleep deprivation and neuroinflammation

Glial fibrillary acidic protein (GFAP) as an astrocytosis marker were found at higher expressions in the brains of the sleep deprived animals.

Astrocytes may also be a progressive marker for AD, some study actually consider that astrocytes deteriorate prior to the actual neurons [14].

Conclusion Everyone knows that sleep is important, we need a good night sleep to function properly and to be physically awake during the day. However as our society evolved, we tend to prioritize other things before adequate sleep, for example, workers have overtime, students cram for exams, and sometimes enjoying the night life with friends is just more exciting to us than going to bed before midnight. But not many people is aware of the long term consequences that comes with chronic sleep deprivation Based on current evidences, it is still difficult to clarify that whether sleep deprivation leads to the various AD phenotypes or vice versa, especially the underling mechanisms are yet to be unveiled.

Even though there is no definite proof that chronic sleep deprivation leads to AD, but it is still a risk factor that do in fact facilitate progression of AD. So whether to accomplish “more” or lose “less”, the choice is yours.

Criticisms and Future Directions

Figure 1. a)HO have significantly more REM sleep and less SWS fragmentation. b)HO have significantly less amyloid-beta 40 and 42 plaque forming peptides. c) Positive correlation between SWS arousal and amyloid-beta 42 in aMCI subjects.

Sleep deprivation and tau metabolism

Brain assay of the sleep deprivation group showed decreased tau phosphorylation at several AD-relevant epitopes. After investigation, a significant decrease of cdk-5 kinase activity in sleep deprived mice seems to be responsible[9].

Overall sleep deprived animals had significantly higher amount of total insoluble tau, possibly due to 129

The main article was interesting that it covered almost complete aspects of the AD phenotype and how it is affected by chronic sleep deprivation, however potentially due to that the study covered such a wide spectrum, each separate individual experiments was not performed very detailed or thoroughly explained, and did not clearly test for confounding factors.

Another factor to note is that this article is using an AD mouse model, meaning that the subject itself is predetermined to develop or have developed AD. Hence even if the results can be supported, it would only mean that sleep deprivation can increase the progression of AD, and we still would not be able to say that chronic sleep deprivation can cause onset of AD in a previously healthy individual. In other words, it has not been confirmed that sleep deprivation can causes development of AD, but it does in fact increase the risk and progression of developing AD. Other studies also suggest that not only sleep deprivation contribute to AD, in fact chronic stress in general such as depression and anxiety also pose as risk factors of AD [10]. Therefore a more direct way of looking at chronic stress factors is to directly monitor changes in cortisol level [15].

Even though genetic causes of AD is only about 5%, prevention for all forms of AD may have a genetic solution, with the increasing advancements in genetic technologies, developing a genetic solution for the prevention as well as treatment of the disease seems to be an appropriate next step [16].

References 1. Wurtman, R. Biomarkers in the diagnosis and management of Alzheimer’s disease. Metabolism Clinical And Experimental. 64, 547-550 (2015).

2. Agostiho, P., Pliassova, A., Oliveira, C.R., Cunha, R.A. Localization and trafficking of amyloid-β protein precursor and secretases: Impact on Alzheimer’s disease. Journal of Alzheimer’s Disease. 45, 329-347 (2015). a7 3. Bernard, C. et al. Time course of brain volume changes in the preclinical phase of Alzheimer’s disease. Alzheimers Dement. 10,143–51 (2014).

4. Sperling, R.A. et al. Toward defining the preclinical stages of Alzheimer’s disease: recommendations from the National Institute on Aging-Alzheimer’s Association workgroups on diagnostic guidelines for Alzheimer’s disease. Alzheimers Dement. 7, 280–292 (2011).

5. Wilson, R.S., Begeny, C.T., Boyle, P.A., Schneider, J.A., Bennett D.A. Vulnerability to stress, anxiety, and development of dementia in old age. Am J Geriatr Psychiatry. 19, 327-334 (2011).

6. Ravona-Springer, R., Beeri, M.S., Goldbourt, U. Younger age at crisis following parental death in male children and adolescents is associated with higher risk for dementia at old age. Alzheimer Dis Assoc Disord. 26, 68-73 (2012).

7. Pan, W., Kastin A.J. Can sleep apnea cause Alzheimer’s disease? Neuroscience and Biobehavioral Reviews. 47, 656-669 (2014).

8. Roh, J.H. et al. Sleep-wake cycle and diurnal fluctuation of amyloid-β as biomarkers of brain amyloid pathology. Sci Transl Med. 4(150), 150ra122 (2012).

9. Di Meco, A., & Joshi, Y. B., & Pratico, Domenico. Sleep deprivation impairs memory, tau metabolism, and synaptic integrity of a mouse model of Alzheimer’s disease with plaques and tangles. Neurobiologi of Aging. 35, 1813-1820 (2014).

10. Kumar, A., Chanana, P. Sleep reduction: A link to other neurobiological diseases. Sleep and Biological Rhythms. 12.

150-161 (2014).

11. Tononi, G., Cirelli, C. Sleeo and the price of plasticity: from synaptic and cellular homeostasis to memory consolidation and integration. Neuron. 81, 12-34 (2014).

12. Sanchez-Espinosa, M.P., Atienza, M., Cantero, J.L. Sleep deficits in mild cognitive impairment are related to increased levels of plasma amyloid-β and cortical thinning. NeuroImage.

98, 395-404 (2014).

13. Rothman, S.M., Herdener, N., Frankola, K.A., Mughal, M.R., Mattson, M.P. Chronic mild sleep restriction accentuates contextual memory impairments, and accumulations of cortical Aβ and pTau in a mouse model of Alzheimer’s disease. Brain Res. 1529, 200–8 (2013).

14. Sica, R.E. Could astrocytes be the primary target of an offending agent causing the primary degenerative diseases of the human central nervous system? A hypothesis. Medical Hypotheses. 84. 481-489 (2015).

15. Duncan, M. J. et al. Effects of aging and genotype on circadian rhythms, sleep, and clock gene expression in APPxPS1 knock-in mice, a model for Alzheimer’s disease.

Experimental Neurology. 236, 249-258 (2012).

16. Alonso Vilatela, M.E., Lopez-Lopez, M., Yescas-Gomez, P. Genetics of Alzheimer’s disease. Arch Med Res. 43(8), 622–31 (2012).

Copyright © 2015 Dr. Bill JU, Neurosciences, Human Biology Program

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Reconsolidation and Extinction Are Dissociable and Mutually Exclusive Processes: Behavioral and Molecular Evidence The Importance of Specificity in Neuroscience

Alexandra Kubica

Understanding how much the processes of extinction and reconsolidation differ is important for the increase in knowledge of the memory system as well as improvement of anxiety therapies. Merlo, Milton, Goozée, Theobald, & Everitt, (2014) were able to show that extinction and reconsolidation are mutually exclusive processes by observing how calcineurin levels change based on amount of exposure to conditioned stimulus. However, more research needs to be completed as lack of specificity of calcineurin and confounding factors such as increased anxiety were not taken into account in the execution of this experiment.

Key words: reconsolidation; extinction; calcineurin; NMDA receptors; fear conditioned learning; Lister Hooded rat; memory; neuroscience; Background Originally it was thought that memory once encoded would remain stable even if retrieved. However, with extensive research, memories have been found to change and can be reconsolidated once retrieved. Reconsolidation is the process of a memory being reactivated and thus being liable and susceptible to change (Nader & Hardt, 2009). This updated version of the memory is then stored again, affecting the original memory. Extinction, however also uses the same mechanism of retrieving memories in order to change them. In the process of extinction the organism is exposed to the stimulus it was trained to react to in a certain way, over and over again until it is habituated and does not respond in the same way it did when it was originally trained (Lin et al., 2003). This new memory of not reacting to the stimulus then inhibits the old memory of reacting to it and the behaviour is extinct.

In order for a memory to be consolidated a kinase must be activated so that protein transcription can start (Glanzman, et al., 1989). Only once this occurs can a memory be stable and eventually be stored in the cortex, and if a protein is not formed the memory cannot be consolidated, as in the case of short term memory (Nader, Schafe, & LeDoux, 2000). Calcineurin is a protein that has been found to help with the extinction of fear based memories by inhibiting phosphorylation and not allowing protein synthesis to occur (Lin et al., 2003). NMDA receptors in the amygdala release calcineurin and were found to be responsible for facilitating fear memory consolidation (Falls, Miserendino, & Davis, 1992).

Researchers then became interested in discovering if these two processes could occur at the same time.

Merlo, Milton, Goozée, Theobald, & Everitt, (2014), were interested in observing if these two processes, reconsolidation and extinction, were mutually exclusive or if they could occur at the same time.

Research Overview

Summary of Major Results

Merlo et al., (2014) trained rats to anticipate a shock

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through the floor of the box they were placed in, when a certain auditory stimulus was presented. 24hours later the rats were injected with either saline solution, a NMDA receptor agonist DCS (D-cycloserine) or a NMDA receptor antagonist MK-801. The researchers believed that when the NMDA receptors were inhibited using MK-801 it would release less calcineurin and extinction should not occur. However, when NMDAR activity was increased with DCS it meant that there was more calcineurin and thus extinction should occur.

After being injected, the rats either received the shock stimulus 1, 4 ,7 or 10 times. Rats were then either tested 24 hours after reactivation of the memory or were biopsied an hour after to observe calcineurin levels.

Results found that in the 1CS (control stimulus) paired with DCS condition, the rats showed the same amount of freezing as with saline (Figure 2).

1CS paired with MK-801 showed significantly less freezing responses. In the 10CS with MK-801 a significant amount freezing is observed compared to saline and DCS. In 10CS and DCS condition there is a decrease in freezing compared to control, but a very large decrease compared to MK-801. In 4CS time spent freezing appeared to be about the same over all injection conditions. In 7CS condition similar to 10CS MK-801 had significantly more freezing with a decrease in saline condition and even more of a decrease in DCS condition. In western blots performed after the second day more calcineurin was found in the 10CS condition rats compared to 1CS (Figure 1). About the same amount of calcineurin was found across all CS conditions with MK-801. Brain activation was observed to be the same for both processes in the basolateral amygdala.

Theses results are in line with a previous study done

by Pérez-Cuesta and Héctor Maldonado in 2009, where they tested reconsolidation and extinction in crabs and found extinction cannot happen with only one exposure to the stimulus, only reconsolidation will occur. However, they also found reconsolidation and extinction can occur simultaneously when there is more than one exposure to the conditioned stimulus, going against what Merlo et al., found in their 10 and 7 condition stimulus conditions.

Figure 1. Interpretation of western blots done one hour after re-exposure to conditioned stimulus on the second day for varying amount of exposure to the stimulus .

Conclusions and Discussion With these results the researchers concluded that extinction and reconsolidation cannot happen at the same time, they are mutually exclusive due to the effects of calcineurin on fear memory. Calcineurin does not affect reconsolidation as increasing calcineurin with DCS in the one conditioned stimulus exposure condition, did not affect the freezing behaviour compared to control. In the 10CS with MK-801 there was and large increase in freezing because the lack of calcineurin does not allow extinction to occur and thus the fear-based memory from previous trails is remembered and so fear responses are not extinguished. With 10CS and DCS there is less freezing than even the control as increasing calcineurin production allows for extinction to take place and the rats no longer associate the stimulus with fear and show less fear responses when presented. In the 4CS freezing responses are very similar when injected with MK-801 because calcineurin is not needed for reconsolidation, but is for extinction, no matter how many exposures to the stimulus happens the calcineurin levels will be similar to reconsolidation levels as activation of any more calcineurin is being inhibited.

In western blots more calcineurin was found in extinction or 10 conditioned stimulus rats as calcineurin is needed for extinction and not reconsolidation so the levels in the 1CS were lower. Calcineurin levels may be specific, but the brain activation seems to be the same for both processes.

Though this does help to explain some things on how memories transition from a state of reconsolidation to extinction based on amount of exposures to stimulus.

It is still not understood how the distinction is made in the body in situations where there is not enough exposures for extinction, but too many for reconsolidation and which process should take place.

How exactly reconsolidation or extinction happen molecularly is not entirely known. The processes neurologically appear to be similar, however, which has made researchers try to understand if they these processes can happen in conjunction or cannot happen while the other is occurring. Being able to make this distinction will allow for improvements in therapy when considering therapies associated with both processes.

Systematic desensitization is used for anxiety disorders where people who have learned adverse reactions to harmless stimuli and learn to not be scared of it anymore by being exposed to it over and over again in

stages and realize they are not in danger and they no longer feel anxiety in those situations (Wolpe, 1958).

Reconsolidation therapies have used by researcherâ&#x20AC;&#x2122;s interested in those suffering from post traumatic stress disorder (Brunet, 2008). These individuals are unable to continue living normal lives with intrusive thoughts constantly bombarding them throughout the day. Interrupting this reconsolidation in humans by administering propranolol, a beta-blocker that inhibits fear responses and thus interferes with fear-based consolidation; after reactivation that were trained with aversive stimulus has been found to inhibit the response behaviour to the stimulus (Kindt et al., 2009) Interrupting reconsolidating and allowing these traumatizing memories of past events to become less distressing would permit these individuals to live normal lives.

Criticisms and Future Directions Though mutual exclusion was proven, the experiment failed to show if calcineurin was specifically affecting extinction or if it was just responsible for LTP (long term potentiation) in general. Is it actually creating this new memory that inhibits the old one and allows for this new knowledge to be stored and thus the freezing responses are less or is it that calcineurin allows for LTP to take place and when it is blocked then the memory is not stored as well. This may also be the case with reconsolidation as with just one exposure may not allow for more strengthening of the synapses. If calcineurin helps with LTP and strengthening of the synapse then blocking it will lead to more freezing, learning is not occuring in general. An example of this was seen with PKMzeta which was thought to be the protein made in LTP and this protein needed to be reactivated in order to use the memory again. Researchers thought that ZIP inhibited PKMzetaâ&#x20AC;&#x2122;s activation thus inhibiting the memory (Pastalkova et al., 2006) It was found much later that ZIP actually had a lack of specify for PKMzeta and decreased LTP in other proteins as well even if the mice did not possess the PKMzeta protein (Volk et al., 2013). To test this further an experiment needs be done to see if doing the reconsolidation test or 1CS condition over multiple days leads to more calcineurin as well showing that it may just be responsible for LTP. Three days may not be enough time for the memory to be consolidated as well as reconsolidated in the cortex. Most of the experiments done for the last session were only 24 hours later with one being done 96hours later, but there have been experiments where rats are tested 25 days later with associated memories still intact (Shelma, Sacktor & Dudai, 2007). Seeing if these high calcineurin levels are found many days later or if it is just seen as the learning occurs would be another future experiment.

Researchers have recently discovered that people with anxiety disorders do not inhibit or reconsolidate fear memories as well as people without these disorders (Soeter, Kindt, & Perales, 2013). These researchers showed participants an aversive image and then had an eye-blink stimulus presented after it and these memories were reactivated on the second day in conjunction with being given propranolol. On 132

Figure 2. Rats were trained with fear-conditioning to perform freezing behaviour in response to a conditioned stimulus. Rats were trained on the first day and were re-exposed to the stimulus on the second day as well as injected with either saline, MK-801 or DCS. Rats were exposed to the conditioned stimulus on the second day either, once (B), four (C), seven (D), or ten times (E). Merlo et al., (2014)

the third day of testing those given propranolol that had high trait anxiety had very little reduction in blink response, previous experiments done like this showed a lot more reduction in those without anxiety. An experiment needs to be done on rats that are genetically modified to be more anxious and observe if the same affects of calcineurin are seen.

The discovery of proteins in correlation with memory has a very complex history. Though proteins may have been discovered in conjunction with memory the extent to which they affect it is still up for debate.

References 1. Brunet, A., Orr, S., Tremblay, J., Robertson, K., Nader, K., & Pitman, R. (2008). Effect Of Post-retrieval Propranolol On Psychophysiologic Responding During Subsequent Scriptdriven Traumatic Imagery In Post-traumatic Stress Disorder.

Journal of Psychiatric Research, 42, 503-506.

2. Falls, W. A., Miserendino, J. D., Davis, M. (1992) Extinction of fear-potentiatedstartle: blockade by infusion of an NMDA antagonist into the amygdala. J Neurosci, 12, 854–863.

3. Glanzman, D., Mackey, S., Hawkins, R., Dyke, A., Lloyd, P., & Kandel, E. (1989). Depletion of serotonin in the nervous system of Aplysia reduces the behavioral enhancement of gill withdrawal as well as the heterosynaptic facilitation produced by tail shock. The Journal of Neuroscience, 9(12), 4200-4213.

4. Kindt, M., Soeter, M., & Vervliet, B. (2009). Beyond Extinction: Erasing Human Fear Responses And Preventing The Return Of Fear. Nature Neuroscience, 12(3), 256-258.

5. Lin, C., Yeh, S., Leu, T., Chang, W., Wang, S., & Gean, P. (2003). Identification of Calcineurin as a Key Signal in 133

the Extinction of Fear Memory. The Journal of Neuroscience, 23(5), 1574 –1579-1574 –1579.

6. Merlo, E., Milton, A., Goozée, Z., Theobald, D., & Everitt, B. (2014). Reconsolidation and Extinction Are Dissociable and Mutually Exclusive Processes: Behavioral and Molecular Evidence. The Journal of Neuroscience, 34(7), 2422–2431.

7. Nader, K., & Hardt, O. (2009). A single standard for memory; the case for reconsolidation.Nature Reviews, 10, 224-234.

8. Nader, K., Schafe, G. E., LeDoux, J. E. (2000) Fear memories require protein synthesis in the amygdala for reconsolidation after retrieval. Nature, 406, 722–726.

9. Pastalkova, E., Serrano, P., Pinkhasova, D., Wallace, E., Fenton, A., & Sacktor, T. (2006). Storage of Spatial Information by the Maintenance Mechanism of LTP. Science, 313(5790), 1141-1144.

10. Perez-Cuesta, L., & Maldonado, H. (2009). Memory reconsolidation and extinction in the crab: Mutual exclusion or coexistence? Learning & Memory, 16, 714-721.

11. Shema, R., Sacktor, T., & Dudai, Y. (2007). Rapid erasure of long-term memory associations in the cortex by an inhibitor of PKM zeta. Science, 317(5840), 951-953.

12. Soeter, M., Kindt, M., & Perales, J. (2013). High Trait Anxiety: A Challenge for Disrupting Fear Memory Reconsolidation. PLoS ONE, 8(11), E75239-E75239. Retrieved from http://journals. plos.org/plosone/article?id=10.1371/journal.pone.0075239 13. Wolpe, J. (1958). Psychotherapy by Reciprocal Inhibition. Stanford, CA.: Stanford University Press.

14. Volk, L., Bachman, J., Johnson, R., Yu, Y., & Huganir, R. (2013). PKM-ζ is not required for hippocampal synaptic plasticity, learning and memory. Nature, 493(7432), 420-423.

Copyright © 2015 Dr. Bill JU, Neurosciences, Human Biology Program

The Effects Of Kynurenic Acid On The Brain And Its Implications In Schizophrenia

Shikha Kuthiala

Schizophrenia is a neurological disorder that has been shown to appear in late adolescence and is characterized by positive, negative and cognitive symptoms. Recently, kynurenic acid, a metabolite made from the breakdown of tryptophan, has been implicated in the development of schizophrenia. Kynurenic acid is an antagonist of both NMDA receptors and alpha-7 nicotinic acetylcholine receptors and as such may interfere with signaling in the brain. It is also thought to play a role in the development of cognitive symptoms in schizophrenia by acting upon the hippocampus. The article by DeAngeli et al. looked at the changes in the hippocampus upon administration of elevated levels of L-KYN, a kynurenic acid precursor and its implications in schizophrenia. They demonstrated that there are deficiencies in learning and memory as well as changes in reward-seeking behaviors in rats treated with L-KYN during early adolescence. They also highlighted that the effects of kynurenic acid on the brain were seen only when the elevated levels were applied in adolescence and not when applied in adulthood.

Overall their results showed that kynurenic acid exposure caused neural changes, which may play a causal role in schizophrenia. It is postulated that treatment centered on reducing KYNA can be beneficial to the afflicted individuals.

Key words: kynurenic acid (KYNA); hippocampus; schizophrenia; reward-seeking; long term potentiation (LTP); neuroscience; physiology Background Schizophrenia is a syndrome that affects the brain leading to positive symptoms such as hallucinations and delusions. Additionally, there are negative symptoms such as social withdrawal and cognitive symptoms like increased addictive behavior (Jones et al., 2011). Medications have been developed to help deal with positive symptoms but there is a lack of treatment for cognitive symptoms, which are often debilitating to the individual (Holder & Wayhs, 2014).

Kynurenic acid (KYNA) has been shown to play a role in the development of cognitive symptoms. It is made by astrocytes during the breakdown of tryptophan and in normal human brains it is found in concentrations of 10-150nM (Rozsa, 2008). It acts on both NMDA and alpha7 nicotinic receptors in the brain. When exposed to high levels of KYNA as an adolescent, rats will display schizophrenic like symptoms after they mature. As adults, these rats presented with cognitive deficits. They also exhibited addictive behavior, which is thought to be due to alterations in the neural reward pathway, and they showed diminished ability to learn and to undergo long-term potentiation (LTP) (DeAngeli et al., 2015). Therefore, understanding the mechanism and downstream effects of KYNA in neural functions is important to elucidate its role in schizophrenia and to create effective treatments. DeAngeli et al. showed that cognition was altered by administering L-KYN, a KYNA precursor to young rats and observing changes in their abilities as adults. They first tested for addictive behavior by testing sign-tracking behaviors in the rats, and found that those exposed to L-KYN displayed greater reward-seeking actions. They then tested the ability to undergo LTP by stimulating the Schaffer Collateral neurons in vitro and measuring the change in the CA1 neurons. It was found that treated rats had a decreased ability to potentate, and therefore

had diminished learning and memory capabilities. It was also shown that KYNA has temporal specificity, if exposure to the metabolite happened in adolescence the rat became schizophrenic. Conversely, if the exposure happened in adulthood there were no observable detrimental effects. Therefore, DeAngeli et al. provided strong evidence that KYNA plays a role in schizophrenia development. These findings are impactful because this study was the first to show how KYNA had a negative effect during development and it provided the initial research towards helping cognitive symptoms. Other experimenters have also investigated the role of KYNA in the development of schizophrenia. Additional studies have provided insight into the mechanism of action used by KYNA during development (Rosza et al., 2008). The study

by Rosza et al. showed how KYNA affects the brain and how its modulation can be used as a potential treatment for schizophrenia. Pocivavsek et al. used behavioral testing to see how learning and memory are changed in response to KYNA, which expands the conclusion put forward by the original study. Kozak et al. and Potter et al. used different methods to show that inhibiting enzymes in the KYNA formation pathway can have positive effects, therefore further implicating the role of KYNA in schizophrenia. It has also been shown in some individuals that stress increases KYNA in-vivo (Chiappelli et al., 2014). This result shows a potential endogenous way in which KYNA levels can become deleterious and provides insight on how KYNA can be modulated in the body. The spatial effects of the metabolite through the brain have been highlighted by Sathyasaikumar et al., who studied the prefrontal cortex, to show that it was affected differently than the hippocampus. The primary article has shown that KYNA can cause cognition to change and supplementary studies have helped to further understand the implications of KYNA in both development and 134

schizophrenia. There is still a great deal of work to do, but the understanding of this metabolite in relation to the diseased state has come a great way.

Research Overview

Summary of Majore Results

Kynurenic Acid and Cognition The primary article by DeAngeli et al. showed that increased levels of KYNA during adolescence lead to cognitive impairments in rats. The experimental rats showed a lack of ability to undergo LTP when using electrical stimulation on hippocampal slices. These changes showed that excess KYNA, which antagonizes NMRA and alpha-7-nAChR, can cause behavioral and cognitive deficits. The decline in cognition was demonstrated by the inability of experimental rats to learn and perform other hippocampal behaviors, such as social behaviors, when compared to controls. This study also showed the temporal specificity of KYNA; if the increased exposure occurred during adolescence cognitive changes were seen, whereas exposure as an adult did not cause these problems (DeAngeli et al., 2015). This result was supported by another study by Trecartin and Bucci. This experiment was conducted on 16 adolescent male rats, which were treated with L-KYN. These rats showed that social behavior decreased after KYNA exposure. When the same experiment was repeated on 16 adult rats there were no apparent deficits. Social behavior was measured by noting the number of times the treated rat approached the area near an unfamiliar rat when both were placed in a tub (Trecartin & Bucci, 2011).

This result is important because it provides a potential cause of cognitive symptoms, which are currently neglected in treatment.

Kyneuric Acid and Changes To the Brain The original paper states that the changes in the treated rats are due to the effects of KYNA on the hippocampus. It is postulated that KYNA binds to and antagonizes the NMDA and alpha-7-NAChR, thus changing the functionality of the hippocampus (DeAngeli et al., 2015). This conclusion is furthered

by Pocivavek et al., who showed that when KYNA synthesis was inhibited, more glutamate was released in the hippocampus. This was shown by administering ESBA, which inhibits the formation of KYNA, and then measuring the amount of glutamate in the extracellular environment. The levels of glutamate were compared in treated and control animals. This result indicated that KYNA does impair the ability of the hippocampus and altered behavior by blocking normal glutamate functioning (Pocivavek, 2011). Changes to the hippocampus were also shown more directly by Pershing et al., who conducted a morphological study of the hippocampus. Rats were treated with KYNA and the structure was analyzed by Golgi staining hippocampal slices and comparing the slices from control and treated animals. The study found decreased dendritic density in rats treated with KYNA (Pershing et al., 2015).

This result showed how the hippocampus changed, furthering the conclusion presented by DeAngeli et al.

135

Overall, it was shown that KYNA affects the structure, function and output of the hippocampus and early treatment can help preserve hippocampus-mediated behaviors.

Kynurenic Acid and Dopamine Sensitivity In the study, DeAngeli et al. state that addiction and schizophrenia are co-morbid due to changes in the reward system of the brain. The article posited that the dopamine receptors increase in sensitivity in response to adolescent exposure to KYNA. This hypothesis was tested by training the rats to press a lever to receive a food reward. The experiment revealed that the treated rats display more sign-tracking and more addictive behaviors. This finding is impactful because it shows a particular region of the brain that is altered by the metabolite and could therefore be targeted for therapeutics. Erhardt et al. also showed a similar conclusion by increasing the KYNA concentration three to nine times the normal amount in the brain. They found that dopamine neurons in the ventral tegmental nucleus (VTA), part of the reward pathway, increased their firing rate, and remained depolarized for greater periods of time in response to the KYNA stimulus. However, they also showed that dopamine neurons fired less in the prefrontal cortex (Erhardt et al., 2007). Therefore, this study showed how hyperactivity of dopamine can cause the positive symptoms of schizophrenia in one region and hypoactivity of dopamine can cause negative symptoms in another. These results show that treatment for KYNA may benefit the full spectrum of symptoms seen in schizophrenia.

Figure 1. This figure shows the diminished ability to undergo LTP in rats exposed in adolescences to L-KYN, in comparison to control rats treated only with vehicle (DeAngeli et al, 2015).

Discussion and Conclusion DeAngeli et al. studied the changes in adolescent rats exposed to high levels of kynurenic acid using both electrophysiology and behavioral tasks. They concluded in their experiments that KYNA played a causal role in the development of schizophrenia and its associated cognitive symptoms. Their results are significant because the study showed a metabolite that could be the target of new therapeutics. These treatments have the potential to surpass current medications like antipsychotic drugs, which only help positive symptoms of schizophrenia like hallucinations

Table 1. KYNA treated rats showed more sign tracking behaviors than the control rats when the conditioned stimulus was present (DeAngeli et al, 2015).

and delusions (Holder &Wayhs, 2014). Treatments to antagonize KYNA have the potential to help cognitive symptoms and also negative symptoms, another currently untreated aspect of schizophrenia (Erhardt et al., 2007). This study was also the first to analyze how changes in metabolite concentration during development can be implicated in schizophrenia. This finding provided insight into a potential early marker for schizophrenia. In addition, measuring higher levels of KYNA in adolescence may be indicative of the individualâ&#x20AC;&#x2122;s susceptibility to developing a schizophrenic phenotype as an adult. By targeting KYNA, treatments for schizophrenia can be preventative instead of symptomatic treatments after the syndrome has already developed (DeAngeli et al., 2015).

The authors presented 3 major conclusions in the study. First, was that KYNA exposure will decrease learning and memory abilities by down-regulating LTP in the hippocampus. This conclusion showed the potential basis for cognitive symptoms and has been expanded on by Pershing et al., who showed that dendritic density changes when exposed to KYNA. This conclusion is important because it presents a location and a mechanism that treatments can target to up-regulate LTP and help cognitive symptoms.

The result also showed the specific changes in the hippocampus, which affect behavior. Therefore, this conclusion can be extrapolated to other neurological disorders that stem from hippocampal changes (Sublette et al., 2011). Overall, the evidence from these studies can help develop protective therapies that can benefit individuals with schizophrenia as well as individuals suffering from other syndromes.

The second conclusion of the study was that the KYNA exposure would lead to a change in the reward circuitry of the brain, including the VTA and nucleus accumbens, increasing reward-seeking behaviors.

This finding was shown using behavioral tests and indicated that KYNA can alter the firing of dopamine neurons in the VTA. A study by Erhardt et al. proved this by measuring the changes in dopamine neuron firing in the VTA with and without KYNA exposure.

They demonstrated that the application of KYNA lead to hypoactivity of the dopamine neurons and that administration of a stimulating substance, which increased dopamine, can reverse the effect. This finding may indicate why rats in the original study had more reward seeking behavior; since the rats are continually in a state of low dopamine they are more likely to become addicted to any behavior that will increase their dopamine levels (Erhardt et al., 2007).

This finding allows for the development of a dopa-

mine agonist treatment that will treat the root cause of addiction in schizophrenic individuals.

The final conclusion presented in the paper was that KYNA has temporally specific effects. The study showed that treatment in adolescence lead to cognitive and behavioral changes, but exposure did not have the same effect in adults. This conclusion highlights the importance of detecting potential risk factors early so that treatments can be put into place before the disorder develops in late adolescence (Uhlhaas and Singer, 2011). This suggests that there is vulnerability at a younger age that is no longer applicable in adults.

This conclusion was supported by Pocivavsek et al., who showed that pre and postnatal exposures to high levels of KYNA lead to cognitive deficits when older.

The conclusion was also corroborated by showing that adult injections of KYNA would not change social behaviors but adolescent exposure lead to increased avoidance and decreased interactions (Trecartin & Bucci, 2011). These findings show that inhibitory treatments towards KYNA must be done early. This provides not only a target, but also a critical window in which treatment must be administered so that symptoms of schizophrenia can be minimized and potentially eliminated. The significance of this study is that it progresses the current treatment plan for schizophrenia into a more holistic treatment. By targeting KYNA we can treat cognitive symptoms, which have thus far been a debilitating but untreatable component of schizophrenia. KYNA treatments have also been postulated to work on negative symptoms, but further research is required confirm this conclusion (Erhardt et al., 2007). Additionally, treatment that antagonizes KYNA can help addictive behaviors, which allows this treatment to not only help in schizophrenia, but possibly also other disorders with addictive components. These studies provide evidence to advance current schizophrenia treatments as well as information to help in the development of preventative therapies.

Criticisms and Future Directions While the work done by DeAngeli et al. has shown a great deal about KYNA and its functions there are points at which the study can be furthered. These additional experiments would allow for a greater understanding of the changes caused by KYNA and the eventual treatments that could be developed. The first experiment, which could lead to a more complete understanding, is to conduct behavioral testing for learning and memory. While the researchers used electrophysiological experiments to show that LTP 136

ability had decreased, by adding a behavioral test such as a Morris Water Maze it could show the behavioral implications of KYNA (Ma et al, 2014). In the second experiment, conducting an in-vitro experiment could have furthered the conclusion. While it was shown that addictive behaviors become more prominent, measuring the change in the firing rate of VTA neurons would elucidate if the VTA was directly being effected and how so. This could be done by exposing the VTA neurons to a typical stimulant and measuring the changes in firing patterns of the dopamine neurons in treated and control rats (Erhardt et al., 2007).

Outside of the experimental design, some of the conclusions presented in the paper could also be further investigated. The authors concluded that the hippocampus and its associated behaviors were affected by KYNA as shown by the change in learning and memory.

Expanding on this conclusion by testing other areas often associated with schizophrenia, like the pre-frontal cortex (Jones et al., 2011), would show how other brain regions are affected by KYNA exposure. Additionally, analyzing hippocampal changes at the cellular level in response to KYNA would elucidate the basic unit of change that could be targeted with preventative therapeutics.

The researchers also concluded that reward-seeking behaviors had changed. This conclusion could be expanded to see if it is simply dopamine neurons in the VTA that are being effected or dopamine neurons throughout the brain. This study would eliminate other possibilities for observed behaviors and would strengthen the presented conclusion. The study could be conducted by comparing the dopamine released from regions like the substantia nigra in KYNA treated animals and controls.

This would show if the changes in dopamine neurons were limited to the reward pathway or if it was a diffuse effect. The final conclusion presented is that KYNA has an age specific effect; while this conclusion has been supported it could be advanced by administering KYNA at different time points throughout adolescence instead of during the whole time frame (Trecardin & Bucci, 2011). This would develop a more precise time scale as to when KYNA has an effect and when it does not. The collected data would allow for a better understanding of the critical time period in which changes occur, and could provide a treatment window. In addition, by administering different doses, the deleterious level of KYNA could be uncovered. Ultimately, these studies could be expanded in the future to develop preventative measures that are both temporally specific and given at the optimal dosage.

KYNA levels can be targeted multiple ways; its effect can be blocked with an antagonist or its formation can be prevented by inhibiting enzymes in the tryptophan metabolism pathway. Once the best method to inhibit KYNA is found, it could be combined with the current anti-psychotic treatments for positive symptoms leading to a treatment that is able to act on positive, negative and cognitive symptoms (Jones et al., 2011). This can increase the quality of life for those with schizophrenia, as they are able to regain lost functions and eliminate unwanted positive symptoms.

Another future goal for KYNA and schizophrenia is to develop a test, analogous to an amniocentesis, which would be able to see if KYNA levels are increased in fetuses (Nicolaides et al., 2012). This information can 137

be used to develop a treatment to administer while in utero or a treatment to administer once the child is born to decrease KYNA levels. If there is no increase in KYNA in fetuses, another future direction would be to develop methods to continually check the levels of KYNA in children. This could potentially done by sampling CSF using a lumbar puncture (Sharief, 2006). This would allow for early detection of elevated levels, which can then be treated. These future plans would not only treat schizophrenic symptoms but also prevent the cortical changes seen in the disorder. Overall, there is a great deal of work to be done on schizophrenia, and expanding the work done in the original article can lead to very positive, life long effects.

References 1. DeAngeli, NE et al. Exposure to kynurenic acid during adolescence increases sign-tracking and impairs long-term potentiation in adulthood. Front Behav Neurosci 8:451-60 (2015).

2. Jones, C.A., Watson, D.J.C., Fone, K.C.F. Animal Models of Schizophrenia. Br J Pharmocol 164:1162-94 (2011).

3. Potter, M.C. et al. Reduction of endogenous kynurenic acid formation enhances extracellular glutamate hippocampal plasticity and cognitive behavior. Neuropsychopharacology 35:1734-42 (2010).

4. Holder, S.D., Wayhs, A. Schizophrenia. American Family Physician 90: 775-82 (2014).

5. Rozsa, E., Robotka, H., Vecsei, L., Toldi, J. The Janusface kynurenic acid. J Neural Transm 115:1087-91 (2008).

6. Pocivavsek, A. et al. Fluctuations in Endogenous Kynurenic Acid Control Hippocampal Glutamate and Memory.

Neuropsychopharamology 36:2357-67 (2011).

7. Kozak, R. et al. Reduction of Brain Kynurenic Acid Improves Cognitive Function. J Neurosci 34:10592-602 (2014).

8. Chiappelli, J. et al. Stress-Induced Increase in Kyneuric Acid as a Potential Biomarker for Patients With Schizophrenia and Distress Intolerance. JAMA Psychiatry 71:761-8 (2014).

9. Sathyasaikumar, A. et al. Impaired Kynurenine Pathway Metabolism in the Prefrontal Cortex of Individual With Schizophrenia. Schizophrenia Bull 37:1147-56 (2011).

10. Trecartin, K., Bucci, D. Administration of Kynurenine during Adolescence, but not during Adulthood, Impairs Social Behavior in Rats. Schizophr Res 133:156-58 (2011).

11. Pershing, M.L. et al. Elevated levels of kynurenic acid during gestation produce neurochemical, morphological, and cognitive deficits in adulthood: implications for schizophrenia.

Neuropharmacology 90:33-41 (2015).

12. Erhardt, S., Schwieler, L., Nilsson, L., Linderholm, K., Engberd, G. The kynurenic acid hypothesis of schizophrenia.

Stockholm, Sweden: Elsevier (2007).

13. Sublette, M. et al. Plasma Kynurenine Levels are Elevated in Suicide Attempters with Major Depressive Disorder. Brain Behav Immun 25:1272-78 (2011).

14. Pocivavsek, A., Wi, H.Q., Elmer, G.I., Bruno, J.P., Schwarcz, R. Pre- and Postnatal Exposure to Kynurenine causes Cognitive Deficits in Adulthood. Eur J Neurosci 35:1605-12 (2012).

15. Uhlhaas, P.J., Singer, W. The development of neural synchrony and Large Scale Cortical Networks During Adolescence: Relevance for the Pathophysiology of Schizophrenia and Neurodevelopmental Hypothesis. Schizophrenia Bull 37: 514-23 (2011).

16. Ma, Q.L. et al. Loss of MAP Function leads to Hippocampal Synapses Loss and Deficits in the Morris Water Maze with Aging. J Neurosci 34:7124-36 (2014).

17. Nicolaides, K.H. et al. Noninvasive prenatal testing for fetal trisomies in a routinely screened first-trimester population. Am J Obstet Gynecol 207:374e1-e6 (2012).

18. Sharief, M. Lumbar puncture and CSF examination.

Medicine 32: 44-46 (2006).

Received March, 05 20 2015 accepted

2015; April,

revised 02,

March, 2015.

This work was supported by The Association for the Development of Undergraduate Neuroscience Education (SRA & RLN), The Endowment for Science Education (EA), and The Synaptic State Faculty Research Foundation (EA). The authors thank Mr. Spine L. Cord, Dr. Amy G. Dala, and the students in Neuroscience 101 for technical assistance, execution, and feedback on this lab exercise.

Address correspondence to: Dr. Rita L. Neurotrophin, Biology Department, 123 Growth Cone Avenue, Action Potential College, Hillock, IL 60101 Email: rln@apc.edu Copyright Š 2015 Dr. Bill JU, Neurosciences, Human Biology Program

138

Selectively Activating Endogenous A3 Receptors is The New Therapeutic Solution to Chronic Pain Soonji Kwon

Most effective treatments of chronic pain typically involve endogenous opioids, adrenergic and calcium channels.

These clinical treatment methods however, have detrimental side effects and addictive qualities over time. The study conducted by Little et al. highlights the potentials of using a highly specific agonist of a G protein-coupled adenosine receptor (AR) subtype A3 MRS5698 in reversing chronic neuropathic pain in a state- and dose-dependent manner.

Although there have been developments in the analgesic uses of other AR subtype agonists, specifically A1AR and A2AAR agonists, there were unwanted cardiovascular side effects. Conversely, agonists of A3AR produce only positive results. They are highly expressed in cells important for carrying out neuroprotective effects, such as inflammatory cells and peripheral sensory nerves and have anti-tumor capacity. Additionally, there are already clinical trials using A3AR agonist IB-MECA [N6-(3-iodobenzyl)-adenosine-50-N-methyluronamide] as a means to decrease pain caused by chemotherapeutic agents and constriction of sciatic nerve without serious side effects. Furthermore, activation of A3AR following administration of MRS5698 at spinal and supraspinal (rostral ventromedial medulla (RVM)) sites show promising signs of becoming the most effective treatment of chronic pain without side effects.

Key words: chronic pain; neuropathic; adenosine; A3AR agonist; IB-MECA; MRS5698; anti-tumor; neuroprotective Background Early theories of pain and pain perception date back to the 1800’s1. With the advancement in technology including many imaging techniques, descending pathways involved in pain are now generally understood as the anti-nociceptive or analgesic system1. Chronic pain is distinct from normal pain since it can alter activity in the CNS, lead to sensitization and even rearrangements within networks of neurons1. This modulation of pain stimuli typically begins in the dorsal horn of the spinal cord where nociceptive-specific neurons and wide dynamic range (WDR) neurons are found2. In chronic pain states, WDR neurons receive persistent C-fiber (unmyelinated) stimulation and they remain turned “on”, continually relaying pain information to the brain2. Within the brain, the major regions involved in noxious stimuli processing include the primary and secondary somatosensory cortices, anterior cingulate cortex (ACC), insula, prefrontal cortex (PFC), thalamus and cerebellum3.

Additionally, projections from the frontal lobe and the amygdala are received by the periaqueductal gray (PAG), which govern nociceptive neurons of the spinal cord through the rostral ventromedial medulla (RVM) and the dorsolateral pontine tegmentum (DLPT)2.

Chronic neuropathic pain is a prevalent health concern but the plastic nature of the pain pathway adds complexity to the development of successful treatment strategies. Pain management studies show that injection of cholesystokinin (CKK) antagonists into supraspinal RVM reverses allodynia resulting from spinal nerve ligation2. Injection into the spinal cord itself is also another important site of injection and it contains important modulatory systems and contains many opioid receptors2. Historically, opioid, adrenergic and calcium channel pathways were common targets for treatment of chronic pain4. However, these drugs are often highly costly with incomplete pain relief and accompanied by adverse side effects that lead to its discontinued use4.

In contrast, mechanisms involving adenosine may be the best therapeutic solution to chronic pain.

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There are four subtypes of the G-protein-coupled adenosine receptors (AR), A1, A2A, A2B, and A35.

Under normal conditions, it has been shown that there are tonic low activation levels of A3 receptors6.

When the extracellular concentrations of adenosine increase, it acts as a signal to neighbouring cells to induce protective responses5. Previous studies of A3AR agonists IB-MECA [N6-(3-iodobenzyl)adenosine-5’-N-methyluronamide] and its chlorinated counterpart Cl-IB-MECA [2-chloro-N6-(3-iodobenzyl)-adenosine-5’-N-methyluronamide] shows it blocks the development of neuropathic pain induced by chronic constriction injury (CCI)7. An extension of this study has been conducted by Little et al. using multiple models of pain and has shown that these results are reproducible. Additionally, the translational capacity of activating A3 receptors as a means to prevent chronic pain is very high since A3R agonists have also been shown to have anti-cancer effects4.

Research Overview

Summary of Major Results

The first major finding of the study conducted by Little et al. shows an increase in extracellular concentration of adenosine following administration of ABT-702, a selective adenosine kinase inhibitor4. This increase in adenosine, which was previously shown to act as a protective signal5, reversed mechano-allodynia in a rodent model of CCI and chemotherapy-induced peripheral neurpathy (CIPN)4.

Behaviourally, the ABT-702 treatment group had a higher paw withdrawal threshold compared to vehicle and A3AR antagonist MRS1523 treated groups4.

Most notably, the administration of MRS5698, a highly specific A3AR agonist, was shown to be effective across many well-characterized models of pain4. It is bioavailable orally, its effects have a fast onset of less than 30 minutes and Rotarod tests do not show any debilitating motor effects4. There were three models of pain used to

A Figure 1 (A) Dose-dependent decrease in behavioural indication of spontaneous pain Day 10 post-cancer-induced-bone pain. (B) A comparison of A3AR agonist MRS5698 with morphine in daily injections from Days 8-15. Adapted from “Endogenous adenosine A3 receptor activation selectively alleviates persistent pain states,” by Little JW, et al. 2014. Brain. doi:10.1093/brain/awu330.

B

Figure 2 Western blot showing lower lumbar (L5-6) spinal cord and RVM expression levels of mRNA. Hprt1 and β-actin used as endogenous control gene.

Adapted from “Endogenous adenosine A3 receptor activation selectively alleviates persistent pain states,”

by Little JW, et al. 2014. Brain. doi:10.1093/brain/awu330.

support its efficacy: spared nerve injury, spinal nerve ligation and cancer-induced bone pain. MRS5698 reversed mechano-allodynia and decreased behavioural indication of pain (such as guarding and flinching as shown in Figure 1A) in a dose-dependent manner4. While repeated injections of morphine on Days 8-15 post-CCI resulted in drug tolerance, MRS5698 retained its anti-nociceptive effects (Figure 1B)4. Additionally, the therapeutic effects of MRS5698 treatment did not compromise the ability to respond to normal physiological pain. Thus, there was no effect on flick tail latency and hot-plate test.

One of the most important findings in this paper is that Little et al. determined two main optimal injection sites of injection to induce anti-nociceptive effects: the spinal cord and the supraspinal RVM4. A3AR mRNA transcript and protein levels were measured and compared by Western blot and determined that both sites were important for A3AR activation, as seen in figure 2. Then, they showed that subcutaneous injection of MRS5698 was also able to reduce the activity of WDR neurons involved in chronic pain, as described in the introduction. There was a significant reduction in neuronal excitability compared to the baseline levels of activity following spinal nerve ligation4. Lastly, to test whether this adenosine pathway involves endogenous opioid and cannabinoid pathways, antagonists of opioid and cannabinoid receptor antagonists, Naloxone, rimonabant and SR144528, were used4. Pretreatment of the antagonists did not affect the MRS5698 A3AR activation4. In figure 3, a flow chart summarizes and depicts the interaction between adenosine & spinal and RVM A3AR activation.

Systemic injection of MRS1523 if given with ABT-702 will diminish the anti-nociceptive effects of ABT-702.

Conversely, systemic injection of MRS5698, which are A3AR agonists, will increase the anti-nociceptic effects by decreasing the neuronal excitability to normal levels.

Figure 3 Summary flow chart depicting the effects of extracellular adenosine concentration at spinal and supraspinal RVM sites.

MRS1523 is the selective inhibitor of A3AR and MRS5698 is the selective agonist of A3AR. Decreased neuronal excitability results in anti-nociceptive effects. Adapted from “Endogenous adenosine A3 receptor activation selectively alleviates persistent pain states,” by Little JW, et al. 2014. Brain. doi:10.1093/brain/awu330.

Discussion and Conclusion The anti-nociceptive function of adenosine was initially discovered and examined in receptor agonists of subtypes A18,9 and A2A10. However, further in vivo studies using animal models of pain demonstrated adverse cardiovascular side effects. Therefore, combining the results stated above, the most important implication of the highly specific A3AR agonist, MRS5698, is the translational capacity of the therapeutic drug for the management of cancer-induced pain as well as having anti-tumour effects. Although chemotherapy may be effectively removing the tumour, in some instances, unbearable pain will result in termination of the treatment. Paclitaxel, or its trade name Taxol, is currently a chemotherapeutic drug used to treat breast, ovarian, non-small cell lung carcinomas, and Kaposi sarcoma11.

However, paclitaxel causes neuropathic pain when given in optimal dosages11. A recent study has demonstrated that administration of IB-MECA, an A3AR agonist, prevents paclitaxel-induced neuropathic pain by inhibiting the activity of spinal NADPH oxidase and downstream redox systems11. Notably, the anti-tumor function of paclitaxel was not compromised. Similar effects of IB-MECA administration was found in other chemotherapeutic drugs such as oxaliplatin, used for metastatic colon cancer, and bortezoimib, used for multiple myelomas11.

The use of A3AR agonists to reverse chemotherapy140

induced peripheral neuropathy (CIPN) is a new and upcoming field of research interest. Another study conducted by Yao et al. examined the anti-cancerous effects of IB-MECA and Cl-IB-MECA against HL-60 leukemia and U-937 lymphoma cells6. Flow cytometry and immunofluorescent staining showed that these incubating A3AR agonists with HL-60 and U-937 cells resulted in an increase in apoptotic cells6. Thus, the antitumor effects of administrating A3AR agonists in patients experiencing CIPN would benefit from its dual actions.

Pain management studies have finally paved way for a new potential drug to combat chronic/persistent pain without detrimental side effects. This study by Little et al. demonstrated the effectiveness of a non-narcotic agonist of A3AR, MRS5698, in many different pain models4.

Although previous research that focused on targeting A1AR and A2AR activation has resulted in cardiovascular side effects in preclinical and clinical trials, no serious side effects have been reported in clinical trials of A3AR agonists, IB-MECA and Cl-IBMECA. Thus, clinical implications of MRS5698 is significant since patients will not become dependent or sensitized to the chronic pain treatment as they would using opioid and cannabinoid agents, such as morphine. With A3AR activation, normal pain thresholds were not compromised and inherent reward is not activated in healthy rats, which suggests highly selective alleviation of persistent neuropathic pain4. It is state- and dose-dependent and is able to reverse mechano-allodynia in CCI-induced neuropathic pain and even against CIPN4.

Clinical trials of using A3AR agonists for cancer treatments are already underway, meaning this drug has potential duality of eliminating both pain and cancerous cells.

Criticisms and Future Directions The paper mentions that injection of MRS5698 reversed mechano-allodynia in spared nerve injury and spinal nerve ligation, similar to the CCI pain model4. However, almost all of the figures only show results of post-CCI. In order to effectively convince the readers of the widespread effects of this new potential therapeutic drug, figures showing efficacy in multiple pain models should be included.

The next steps that should be taken are to examine whether the results are reproducible across all (available) models of chronic pain and chemotherapeutic agents. Additional CIPN animal models that are available for further tests include vincristine-, toxol-, and cisplatin-induced peripheral neuropathy models (VIPN, TIPN, CIPN respectively)12,13. Given that A3AR should always be activated in chronic pain states, the prospect of using A3AR agonists as a universal treatment for all chemotherapeutic-induced nociception is remarkable.

Currently, further advancements are being made in the molecular design of better, more specific A3AR agonists.

Tosh et al. have made modifications to MRS5698 and have synthesized multiple new drugs that surpass all the benefits of the existing A3AR agonists14. Some of the modifications include decreasing the molecular weight of the compound in order to overcome the blood brain barrier and maximize delivery throughout the body. Thus, further molecular advancements will result in the creation of a therapeutic drug that has translational capacity of treating chronic neuropathic pain and ultimately increase the quality of life in affected individuals.

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2. Jay GW. Chronic Pain. London, GBR: CRC Press.

(2007). Retrieved from: http://www.ebrary.com 3. Bushnell M, Čeko M, & Low L. Cognitive and emotional control of pain and its disruption in chronic pain. Nature Reviews Neuroscience 14, 502-511 (2013).

4. Little JW, Ford A, Symons-Liguori AM, Chen Z, Janes K, Doyle T, Xie J, Luongo L, Tosh DK, Maione S, Bannister K, Dickenson AH, Vanderah TW, Porreca F, Jacobson KA, & Salvemini D. Endogenous adenosine A3 receptor activation selectively alleviates persistent pain states. Brain (2014) doi: 10.1093/brain/awu330.

5. Fredholm BB, IJzerman AP, Jacobson KA, Linden J, & Muller CE. International Union of Basic and Clinical Pharmacology. LXXXI. Nomenclature and classification of adenosine receptors–an update. Pharmacol Rev 63, 1–34 (2011).

6. Yao Y, Sei Y, Abbracchio MP, Jiang J-L, Kim Y-C, & Jacobson KA. Adenosine A3 Receptor Agonists Protect HL-60 and U-937 Cells from Apoptosis Induced by A3 Antagonists. Biochemical and Biophysical Research Communications 232(2), 317-322 (1997).

7. Chen Z, Janes K, Chen C, Doyle T, Bryant L, Tosh D, Jacobson KA, Salvemini D. Controlling murine and rat chronic pain through A3 adenosine receptor activation. The FASEB Journal 26, 1855-1865 (2012).

8. Kiesman WF, Elzein E, and Zablocki J. A1 adenosine receptor antagonists, agonists, and allosteric enhancers. Handb.

Exp. Pharmacol. 193, 25–58 (2009).

9. Zylka MJ. (2011) Pain-relieving prospects for adenosine recep- tors and ectonucleotidases. Trends Mol. Med. 17, 188–196 10. Loram LC, Harrison JA, Sloane EM, Hutchinson MR, Sholar P, Taylor FR, Berkelhammer D, Coats BD, Poole S, Milligan ED, Maier SF, Rieger J, & Watkins LR. Enduring reversal of neuropathic pain by a single intrathecal injection of adenosine 2A receptor agonists: a novel therapy for neuropathic pain. J. Neurosci. 29, 14015–14025 (2009).

11. Janes K, Esposito E, Doyle T, Cuzzocrea S, Tosh DK, Jacobson KA, Salvemini D. A3 adenosine receptor agonist prevents the development of paclitaxel-induced neuropathic pain by modulating spinal glial-restricted redox-dependent signaling pathways. PAIN 155(12), 2560-2567 (2014).

12. Wang L & Wang Z. Animal and cellular models of chronic pain.

Advanced Drug Delivery Reviews 55(8), 949-965 (2003).

13. Mogil J. Animal models of pain: Progress and challenges.

Nature Reviews Neuroscience 10, 283-294 (2009).

14. Tosh DK, Finley A, Paoletta S, Moss SM, Gao Z-G, Gizewski ET, Auchampach JA, Salvemini D, & Jacobson KA. In Vivo Phenotypic Screening for Treating Chronic Neuropathic Pain: Modification of C2-Arylethynyl Group of Conformationally Constrained A3 Adenosine Receptor Agonists. Journal of Medicinal Chemistry 57(23), 9901-9914 (2014).

This work was supported by The Association for the Development of Undergraduate Neuroscience Education (SRA & RLN), The Endowment for Science Education (EA), and The Synaptic State Faculty Research Foundation (EA). The authors thank Mr. Spine L. Cord, Dr. Amy G. Dala, and the students in Neuroscience 101 for technical assistance, execution, and feedback on this lab exercise.

Address Biology Potential

correspondence to: Dr. Rita L. Neurotrophin, Department, 123 Growth Cone Avenue, Action College, Hillock, IL 60101 Email: rln@apc.edu

Copyright © 2015 Dr. Bill JU, Neurosciences, Human Biology Program

Working memory training is most effective in healthy young adults to improve cognitive skills

Shonali Lakhani

Previous studies suggest that training working memory activates the medial temporal lobe, which can contribute to better episodic memory by stimulating interacting brain areas. Most studies do not examine the small effects of training on episodic memory processes, such as familiarity in the perirhinal cortex and recollection in the hippocampus. A recent finding indicated that a high demand on executive function to process complex spatial information, increased prefrontal cortex activity.

Cognitive training of working memory typically involves a variety of tasks that may be too complicated or take up too much time. A new spatial memory task was developed to improve the episodic memory abilities for familiarity and recollection as well as fluid intelligence. Development of a new spatial task targeting both episodic memory processes necessitates participants to adjust their viewpoint accordingly. Thus, multiple training tasks are not required to improve cognition, as their spatial working memory task led to stronger long-term memory and fluid intelligence for abstract reasoning.

Key words: cognitive neuroscience; working memory; fluid intelligence; performance training Background The use of cognitive training was investigated to improve the speed and accuracy of cognitive functions (Thorndike and Woodworth, 1901). Several functions were part of the focus, namely training working memory influenced attention, observation and discrimination.

This led to many more studies by other researchers into the interaction between working memory and how it is the foundation for many other cognitive functions, such as spatial representation.

More recently, Lee and Rudebeck (2010) used functional neuroimaging to show the effects of increasing the demand on working memory during complex spatial processing. The effects included activation of posterior medial temporal lobe structures, which was greater when there was a high demand on spatial processing, irrespective of the demand on working memory. Thus, this finding is significant because it implies that making the most of working memory training requires the incorporation of spatial tasks. In addition, Huntley and colleagues (2010) continued this investigation by administering a range of spatial and verbal tasks to Alzheimer’s patients in sequences that were not structured randomly. In this way, strategic methods of encoding data, such as chunking information, reduced the demand on working memory and improved recollection. They also found that it is most beneficial to target patients in the earliest stages of the disease for cognitive training gains.

Another factor may affect cognition in those carrying certain versions of the LMX1A gene may have an advantage during training, as their working memory is enhanced more than the individuals carrying a different version of the gene. This research is yet to be confirmed by comparing the results obtained from a future study study repeated on a larger population in order to generalize the results. Nonetheless, this is promising for those who carry the advantageous allele into old age (Bellandera et al., 2011).

On the one hand, cognitive training that focused on improving major deficits in recollection, for those with

mild to moderate forms of Alzheimer’s disease, has shown general benefits to working memory. The use of verbal and visual modalities, along with spatial and temporal memory, allowed Boller and colleagues (2012) to take a multi-domain approach. On the other hand, a domain-general paradigm was used to conduct working memory training on healthy groups of young and old adults (Brehmer, Westerberg & Bäckman, 2012). They found that both groups had better performance, but the younger adults gained more than the older adults overall because they initially made greater improvement.

However, when Lee et al. (2012) trained young adults on a video game that required working memory processes, they did not see much benefit to other cognitive processes when those processes did not bear any similarity to the trained tasks. By comparison, Cheng et al. (2012) found that healthy elders benefit more from multi-domain training that targets diverse capacities than from a single-domain approach. This research supports Rudebeck et al.’s (2012) development of a single spatial working memory task that targeted overlapping pathways, but they only trained healthy young adults. In contrast to these findings, a study published a year earlier by Bergman Nutley and colleagues (2011) revealed that working memory in young children improved with training, but did not affect problem-solving skills. Therefore, the use of visual and spatial tasks in this study has demonstrated that such training is not as effective in all age groups, as the study being reviewed only included adults.

Research Overview

Summary of Major Results

Both the high gain and low gain training groups achieved greater scores for episodic memory processes with time, but there are significant differences between them, as well as the high gain compared to the control, and the low gain versus the control. The training group 142

performed significantly better on a reasoning task, in comparison with the control group. However, low pre-training scores lead to higher reasoning scores, irrespective of training task gain.

In addition, the gain scores correlate positively with the recognition tasks for both episodic memory processes, so there is a significant difference between the high gain group compared with the control, or compared with the low gain group. Even if the object and scene tasks are considered separately, there is still no significant difference between the scores for the low gain and control groups.

Therefore, training score improvement is linked to increased recognition, which is demonstrated by the scores for the episodic memory process of familiarity, whereas poor recollection pre-training predicted a marked improvement only in recollection scores, regardless of training scores (Rudebeck et al., 2012).

Conclusions and Discussion

For optimal encoding and retrieval of episodic memory, the ability to maintain information in working memory is crucial. Also, neural mechanisms overlap as medial temporal lobe lesions affect working memory when the stimuli are difficult to verbalize or relational processing is complicated. Moreover, improved reasoning is due to the task-induced activation of a common network from the lateral prefrontal cortex to the parietal cortex, leading to better episodic memory (Rudebeck et al., 2012). Likewise, Cheng et al. (2012) showed improved reasoning in healthy older adults with multi-domain training, which may be due to connections between different brain areas. Hence, the more working memory is improved, the larger transfer effect on episodic memory, which is why the prefrontal cortex and medial temporal lobe structures are involved in non-specific gains.

As a result, depending on the cognitive skills required, appropriate training tasks must be created based on existing ability and training task gains pursued (Rudebeck et al., 2012). This may be why Boller et al. (2012) carefully designed their training task to address Alzheimer’s patients’ specific cognitive impairments and measure the extent of transfer to episodic memory processes, such as recall and recognition. This is important, as it allowed participants to improve recollection at mild to moderate stages of the disease. Yet, Lee et al.’s (2012) results suggest that older adults may benefit from video game training, only if improved working memory is desired.

Furthermore, the hippocampus is important for both recollection and familiarity as well as object and scene recognition (Rudebeck et al., 2012), implying that further research would allow generalization to all forms of episodic memory in various populations by using different kinds of stimuli and targeting certain neural processes. When Brehmer, Westerberg & Bäckman (2012) studied the difference between younger and older adults, adaptive training groups had higher gains in near transfer tasks for both groups. Whereas younger adults had more gains in comparison with the active control group, who were not challenged progressively by an adaptive training procedure. Also, young adults in adaptive training performed the best in 143

Figure 1. Lee et al. (2012)’s results show that the hybrid-variablepriority training group that focused on learning each rule of the game one by one outperformed the group that was instructed to give full emphasis to all aspects of the training task. This suggests that the specific skills to do well on the game are retained when participants focus on grasping one skill at a time.

the far transfer tasks, such as reasoning.

Bergman Nutley et al.’s (2011) results in children may be explained by the gender imbalance, as more boys were a part of the study and tended to be less motivated to engage with the task. Still, their results do indicate that training on non-verbal reasoning tasks improved fluid intelligence in young children, so it seems that the domains involved in training must be specific.

Figure 2. Brehmer, Westerberg & Bäckman (2012) measured working memory performance over a period of four weeks, in which they found that younger adults made more improvement than the older adults between the first and second week.

Conclusions The research conducted by Rudebeck et al. is significant because it can be applied to help the aging population maintain good cognitive function for independence. This has a great impact for patients who suffer from memory impairments or dementia. Thus, this research may have led Kanaan S et al. (2014) to portray that it is feasible to transfer improved performance on practiced training tasks.

This achieves better cognition in Alzheimer’s patients, which carries over to general tasks, even after two to four months of training. Knowledge of different neural pathways was integrated to find an overlap between the ones for working memory and episodic memory (Rudebeck et

al.). This led to the development of a new task that targets the neural correlates for both types of memory. Thus, the type of advance made in the field of cognitive science is incremental.

Criticisms and Future Directions

Rudebeck et al. could take their study further by carrying out experiments to measure the amount of oxygen and energy delivered to highly active brain areas. For instance, Buschkuehl, Garcia, Jaeggi, Bernard, & Jonides J (2014) report increased blood flow in areas of the brain during training, which is maintained to an extent afterwards. However, Rudebeck et al did not test for a transfer from the n-back training task to other sensory modalities, such as audition to fully predict the participant’s real potential.

Thompson et al. (2013) disagree that training working memory transfers to a higher speed of processing, but their participants had a gender imbalance, a high IQ and were from top universities. Unlike Rudebeck et al’s study, training was not done daily to be consistent over three weeks, which is the optimal period to improve cognition. Also, testing was carried out in a stressful university environment. Rudebeck et al’s study recruited participants from the community and allowed them to train from home at their own convenience, which makes it more representative of the general population in terms of age and mental health.

Stamenova et al. (2014) found that older adults with better cognitive function were more likely to benefit from recollection training when verbal memory was used to encode information. On the contrary, there is no substantial evidence that the gains in recognition memory transfer to any other types of stimuli that may be used for the rest of the cognitive domains. Thus, the task must be very precise in order to improve cognitive abilities in lower functioning older adults, which also requires that the task is manageable enough for them to make any progress. In contrast, Rudebeck et al.’s study used spatial working memory training on purpose, in order to avoid verbal encoding of data because it would not transfer well to other untrained tasks.

Thus, Rudebeck et al. could consider why Bergman Nutley et al.’s study found that young children did not improve their cognitive abilities across different tasks the case and if it is possible to design a task that elicits the same results across all age groups. This is important because older adults tend to improve more with training when their existing cognitive capacities are good.

To take future directions, Rudebeck et al. can add more dimensions to their procedure by measuring the effects of lifestyle factors that may contribute to the biological correlates for improved cognition. For example, a study by Scullin et al. (2012) found that when Parkinson’s disease patients took dopaminergic medication and got enough sleep at night, the effects of training working memory were enhanced. This is a confounding variable that can affect the results of the study, so it should be taken into account.

In addition, Hyer, Scott, Lyles, Dhabliwala, & McKenzie (2014) used a comprehensive approach to help older adults with mild to moderate memory impairments retain and improve working memory. The participants applied

mnemonic strategies to their daily life, while engaging in various activities that affect memory processes. This involved leading a healthier lifestyle by exercising four times a week, eating a Mediterranean diet, reducing stress to affect beta amyloid plaques, socializing more, meditating, and identifying their purpose in life. Therefore, it is evident that targeted cognitive training is most effective for adults with unimpaired cognition from the findings, which show that those who have strong cognition in old age and are at the lowest risk of dementia are the ones who benefit the most from training. This finding can inform the measures taken in the present to provide training for the population, so that when they have aged, they will not be at a disadvantage.

A recent study by Jiang et al. (2015) illustrated how spatial training induced memory improvement in a rat model of Alzheimer’s disease has an underlying process that is dependent on CaMKII, leading to dendrite growth and spine generation. Hence, future studies could investigate the effects on the levels of this molecule during different types of memory training in different populations to objectively measure the effects on cognitive processes.

References 1. Bellandera M, Brehmera Y, Westerberga H, Karlssona S, Fürtha D, Bergmanb O, Erikssonb E, Bäckman L (2011) Preliminary evidence that allelic variation in the LMX1A gene influences training-related working memory improvement. Neuropsychologia 49:1938–42.

2. Bergman Nutley S, Söderqvist S, Bryde S, Thorell LB, Humphreys K, Klingberg T (2011) Gains in fluid intelligence after training non-verbal reasoning in 4-year-old children: a controlled, randomized study. Developmental Sci 14:591–601.

3. Boller B, Jennings JM, Dieudonné B, Verny M, Ergis A-M (2012) Recollection training and transfer effects in Alzheimer’s disease: Effectiveness of the repetition-lag procedure. Brain Cognition 78:169-177.

4. Brehmer Y, Westerberg H, Bäckman L (2012) Workingmemory training in younger and older adults: training gains, transfer, and maintenance. Front Hum Neurosci 6:63. doi: 10.3389/ fnhum.2012.00063.

5. Buschkuehl M, Garcia L, Jaeggi S, Bernard J, Jonides J (2014) Neural Effects of Short-Term Training on Working Memory. Cogn Affect Behav Neurosci 14:147-60 6. Cheng Y, Wu W, Feng W, Wang J, Chen Y, Shen Y, Li Q, Zhang X, Li C (2012) The effects of multi-domain versus single-domain cognitive training in non-demented older people: a randomized controlled trial. BMC Med 10:30. doi:10.1186/1741-7015-10-30.

7. Huntley J, Bor D, Hampshire A, Owen A, Howard R (2011) Working memory task performance and chunking in early Alzheimer’s disease. Br J Psychiatry 198:398–403.

8. Hyer L, Scott C, Lyles J, Dhabliwala J, McKenzie L (2014) Memory intervention: the value of a clinical holistic program for older adults with memory impairments. Aging Ment Health 18:169-78 9. Jiang X, Chai G-S, Wang Z-H, Hu Y, Li X-G, Ma Z-W, Wang Q, Wang J-Z, Liu G-P (2015) CaMKII-dependent dendrite ramification and spine generation promote spatial training-induced memory improvement in a rat model of sporadic Alzheimer’s disease. Neurobiol Aging 36:867-76 10. Kanaan S, McDowd JM, Colgrove Y, Burns JM, Gajewski B, Pohl PS (2014) Feasibility and Efficacy of Intensive Cognitive Training in Early-Stage Alzheimer’s Disease. Am J Alzheimers Dis Other Demen 29:150-8.

11. Lee AC, Rudebeck SR (2010) Investigating the interaction between spatial perception and working memory in the human medial temporal lobe. J Cogn Neurosci 22:2823-35.

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12. Lee HK, Boot WR, Basak C, Voss MW, Prakash RS, Neider M, Erickson KI, Simons DJ, Fabiani M, Gratton G, Low KA, Kramer AF (2012) Performance gains from directed training do not transfer to untrained tasks. Acta Psychol 139:146-58.

13. Rudebeck S, Bor D, Ormond A, Oâ&#x20AC;&#x2122;Reilly J, Lee A, Chao L (2012) A Potential Spatial Working Memory Training Task to Improve Both Episodic Memory and Fluid Intelligence. PLoS ONE 7:e50431.

14. Scullin MK, Trotti LM, Wilson AG, Greer SA, Bliwise DL (2012) Nocturnal sleep enhances working memory training in Parkinsonâ&#x20AC;&#x2122;s disease but not Lewy body dementia. Brain 135:2789-97 15. Stamenova V, Jennings JM, Cook SP, Walker LAS, Smith AM, Davidson PSR (2014) Training recollection in healthy older adults: clear improvements on the training task, but little evidence of transfer.

Front Hum Neurosci 8:898. doi: 10.3389/fnhum.2014.00898 16. Thompson TW, Waskom ML, Garel K-LA, Cardenas-Iniguez C, Reynolds GO, Winter R, Chang P, Pollard K, Lala N, Alvarez GA, Gabrieli JDE (2013) Failure of Working Memory Training to Enhance Cognition or Intelligence. PLoS ONE 8:e63614.

17. Thorndike EL, Woodworth RS (1901) The influence of improvement in one mental function upon the efficiency of other functions: III. Functions involving attention, observation and discrimination. Psychol Rev 8:553-564.

Address correspondence to: Shonali Lakhani, Human Biology Department, University of Toronto, Toronto, ON 60101 Email: Shonali.lakhani@mail.utoronto.ca

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Neural Correlates of Artistic Imagination through the Visual Modality

Dong-Eun Lee

Honing imaginative skills allows us to adapt/form novel paradigms for perceiving incoming stimuli. But investigating potential co-occurring alterations in extant neural mechanisms to effect such perceptual changes has been a difficult feat, due to multiple confounds that render imagination an individually unique experience. To simplify matters, then, Schlegel and colleagues approached this investigation by focusing on a simplified art form: visual representational art, where an artist must create an expressive image faithful to the actual object or scene.1 As such, three types of cognition associated with the visual art process were considered: intrinsic imagination, perception of visual stimuli, and skilled action, for outward expression of such perception. To investigate, undergraduates enrolled in a drawing/painting class were compared to undergraduates studying other subjects similar in intensity, with fMRI and DTI recordings throughout the four-month class. As courses progressed, changes in the frontal lobe activation were observed with co-occurring changes in cerebellar activity in art learners, establishing that neural circuits rewire as representational art skills are learned. However, for these findings to imply a potential mechanism in which imaginative abilities are cultivated, visual representational art must have construct validity as a model for artistic creativity. In addition, the control group’s organic chemistry course, considered as acquiring problemsolving skills in closed systems, must control for only the confounding effects, and must itself discernibly be a closed system in comparison. This review evaluates Schlegel and colleagues’ experimental design and robustness in their consequent results for offering transferable insights about central mechanisms effecting artistic creativity.

Key words: representational art; functional magnetic resonance imaging (fMRI); diffusion tensor imaging (DTI); frontal lobes; cerebellum Background Creativity is ubiquitous and necessary in a human life. It allows humanity to adapt new ideas that enrich quality of life and/or provide new tools for a particular niche, like Marie-Guillemine Benoist’s Portrait d’une négresse2 (Figure 1a), and Hama and colleagues’ optical method, ScaleA23 (Figure 1b). Although both are creative products of the imaginers’ novel perceptions about the world, as sclerotic vessel4 is to fibrotic interstitium-endothelium5, both are also born from different mechanisms (Figure 1). Then, are there universal neural activation motifs in a creative process or is the mechanism giving rise to Benoist’s work discernibly different from that producing Hama’s group’s ScaleA2?

Schlegel and colleagues decide to examine brain activations in individuals learning visual representational art to instigate a response. Choosing one art form, and this one in particular, affords advantages for the experimenters. Firstly, studying a single style in a single modality eliminates obscurity in definitions and consequent subjective gradations between different styles of artistic expressions.6 Furthermore, also known as figurative art, a representational artist expresses concepts or processes in depictions of his/ her observations of the world.7 Thus, confounding contextual factors, such as culture, environment of upbringing, and affect, which otherwise would be focal points for other styles, such as pop art,6,8 are remote and peripheral as a representational artist must be faithful to the observed scene/subject (Figure 1a).

Extant studies examine corresponding neural circuits to representational art appreciation. In a TMS & EEG study, disrupting the left dorsolateral prefrontal cortex and right posterior parietal cortex significantly reduced intrinsic windbags are great esthetic appreciation of

representational art.10 In another study, frontotemporal dementia patients, with disrupted semantic knowledge and sense of self, exhibited preserved preferences in esthetic appreciation of art.11 However, neural circuits associated with learning and producing representational art is a novel investigation.

Schlegel and colleague focus on three aspects of cognition considered in visual art production: creative cognition, perception via visual modality, and translation into precise motor ability.

The first aspect, as implied, is the imaginative process in which original ideas and patterns are derived. This is the mechanism least understood and principally significant. Debates about mechanistic categorizations range from cogent to divergent,12,13 but meta-analysis of studies about creativity indicated diffuse findings of lower frontal lobe white matter fractional anisotropy (FA).14 Moving on, visual perceptual abilities, with respect to representational art, requires the artist to create precise, unassumed two-dimensional images of the world. To achieve this, Bayesian inferences acting to infer from subjective experiences must be tightly regulated. There is no agreement on whether artists have rewired mechanisms to regulate inferential processes,15,16 leaving Schlegel’s group to approach with an open mind. Lastly, the perceptuomotor abilities of an artist to translate her/his perception to an artwork is important. Visual system, as with other modalities, contains the dorsal or “where” stream, about spatial arrangement and movement, and the ventral or “what” stream, about object identity. For the purpose of artwork creation, the dorsal, translation to action stream was thought to be more relevant.

Therefore, these factors and previous findings were considered for constructing the authors’ experimental design.

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Figure 1. (a) Marie-Guillemine Benoist’s Portrait d’une négresse, 1800, housed in Musée du Louvre.9 (b) Hama and colleagues’ ScaleA2 technique: c. mouse brain incubated in ScaleA2 reagent for >2 weeks; d. preserved fluorescent labeling of >2 week ScaleA2 reagent-incubated mouse brain; e. mouse embryos incubated in phosphate buffered saline (PBS, left) and ScaleA2 reagent (right) for >2 weeks.3

Research Overview

Summary of Major Results

35 undergraduate students within 19-22 years of age, 17 enrolled in one of two introductory observational drawing or painting class – the treatment group – and 18 enrolled in introductory organic chemistry class – the control group – participated as subjects.

To behaviorally evaluate creative cognition, an artistic creative thinking analysis measure, called the Torrence Tests of Creative Thinking Figural Form A (TTCT), were administered to subjects twice, before the course began and after the course ended to show significant change in the treatment group (Figure 2a).

By dividing the TTCT scale into 5 factors, and of them, the treatment group increased in factors 1, 2, and 3, which translate to: divergent thinking, or the ability to produce multiple original constructs; effective systematic illustrative modeling; and forming rich, complex imagery, respectively (Figure 2b).

To track progressive neurological changes from before the course to the last month of the course, diffuser tensor imaging data were obtained for both treatment and control groups. The results indicated that, white matter FA in the voxel areas of the prefrontal cortex significantly declined in the treatment group students while control group students showed no change throughout in those voxels (Figure 3).

To track progress in visual perception skills, optical illusion tasks were administered to subjects longitudinally throughout course progression: the Craik-O’Brien-Cornsweet illusion with differences in luminance, and the Mueller-Lyer illusion. In conjunction, functional magnetic resonance images (fMRI) were also acquired. No significant changes were observed, in behavioral and fMRI data.

Lastly, to longitudinally determine trends in perceptuomotor skills throughout course progression, gesture drawing tasks were administered to subjects while DTI were also acquired in tandem. From the gesture drawing tsk results, treatment group subjects exhibited a steady 147

Figure 2. Results of artistic creative thinking from figure synthesis to colorfulness of imagery. (a) TTCT results at the end of course compared to results of TTCT taken at before start of course. (b) Factor analyses of TTCT submeasures. The asterisks indicate significant differences between exp (treatment group) and con (control). F1 = divergent thinking; F2 = effective systematic modeling; F3 = complex imagery; F4 = verbal creativity; F5 = originality and synthesis of lines

increase, while control did not exhibit significant changes (Figure 4a). And by statistically comparing trends, the treatment group exhibited significant improvement in the gesture drawing scores at the second month of classes onwards (Figure 4b). In the subsequent DTI data that followed, the treatment group subjects exhibited significant increase in white matter activation in the right anterior cerebellum throughout course progression (Figure 4c).

Discussion & Conclusions In keeping with previous findings, Schlegel and colleagues found lower white matter FA in the frontal lobes, while behaviorally observing improved creative cognition within the visual art paradigm (Figure 2).

However, decreased white matter activation in a particular cluster of voxels may indicate multiple reasons. Secondly, authors report no significant change in neural activations or in behavior relevant to visual perception, indicating that changes with regards to visual perception are not required for cultivating visual representational art skills (Figure 3). Lastly, authors observed significant increase in gesture drawing skills and rewiring changes for the art learners (figure 4), making perceptuomotor skills within the dorsal visual stream an important cognition in honing visual representational art. The implicated brain area of which rewiring was observed was the right anterior cerebellum, as higher white matter FA was observed (Figure 4). This area, from previous works, is correlated to proprioceptive feedback and is found to project to hand and arm areas of the left

Figure 3. DTI results, longitudinally tracked throughout course progression. (a) Significant voxel areas that exhibited progressively decreased activation in the treatment group (exp). (b) Time course of FA normalized to DTI results obtained before the beginning of courses in voxels exhibiting significant changes within the treatment group data. Exp = treatment group; con = control group.

primary motor cortex, and enhance hand and eye movements.17,18 This work expresses that rewiring changes in the prefrontal cortex causes artistic creativity in cultivation of artistic skills, and an artist need not adapt novel ways to perceive exteroceptive cues but perfect fine tune motor abilities to externally display her/his point of view more accurately.

Criticisms and Future Directions From here on, Schlegel and colleagues’ work is evaluated in their experimental design and analyses of results. Also, suggestions for potential post hoc studies are discussed.

Experimental Design

Schlegel and colleagues effectively approach a novel question by longitudinally mapping different cognitive changes with neural activation changes.

In comparison to previous works, this experimental design offers more robust evidence, from functional (fMRI), connectivity (DTI), and behavioral/cognitive data. The findings are made more salient by the authors’ choice to study subjects who are acquiring or improving on pre-existing artistic creativity, since baseline recordings would also exist in the treatment group subjects as well for comparison.

However, issues lie in the authors’ definition of visual representational art with respect to the choice of control. Authors contend that this art form is problem-solving in an open system more so than organic chemistry, which is claimed to foster problemsolving in a closed system. Representational artists must produce two-dimensional images recognizable as objects or scenes in reality; they must evoke their point of view within the constraints of the realized world. Similarly, organic chemists must formulate a string of reactions to target a product molecule within constraints of electron sinks and sources. The visual representational artist, unlike surrealist or abstract art,6,8 is limited in her/his expressive freedom by

extant objects and scenes in the world, not unlike the limitations placed by electrostatic and other chemical interactive properties of compounds and atoms for synthetic chemists. Two groups of undergraduates taking either groups of classes would undoubtedly require creativity to produce new ideas and solutions within these disciplines. They would only differ about the context with which creativity is accessed, artistic vs. scientific. Instead, choosing undergraduate students in an intense program not involved in artistic endeavours may have been better. In doing so, by including variable disciplines, no biased results would arise from control conditions.

In addition to the issues with defining control conditions’ program/class of enrollment, including a third condition not undertaking intensive programs or not actively learning in class, perhaps young professionals, could also solidify appropriateness in their choice of control group conditions to be undergraduate students in an intensive program. Also adding a cohort of professional representative artists to compare potential differences in maturity level of skills and creativity may have afforded more insight about their question.

Furthermore, including follow-up behavioral, fMRI, and DTI data a month or more after courses ended may have also outlined longevity of the learned artistic creativity.

Analyses of Results

Since huge degree of cognitive problem-solving would require the prefrontal cortex, one could argue that, in learning the specific skills for transforming a 3-D image into a 2-D one, a more skilled person may not need to calculate or guess as much, hence having less activation in the prefrontal cortex, and perhaps more activation in the cerebellum and neurons imprinted with automatic procedural memory (perhaps parietal lobes). In this regard, the rewiring observed may predominately be due to maturation of certain skills, and less to do with increased artistic creativity, as authors have defined.

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Figure 4. (a) Time course results of the gesture drawing tasks administered to both treatment and control subjects throughout class progression. (b) Statistical analysis based on data patterns compared between gesture drawing task results of treatment and control groups, where the neon green color indicates significant increase of treatment group results compared to control. (c) Significant voxel areas selected per monthly DTI acquisition, green showing significantly higher FA in treatment group subjects and orange showing significantly lower FA, compared to control. Exp = treatment group; con = control group.

Potential Post hoc Studies

Since connectivity and structural studies were done, a stimulation study should be implemented on both cohorts using repetitive transcranial magnetic stimulation (rTMS). As 1Hz frequency stimulation (low freq) inhibits targeted areas and 10Hz-20Hz stimulation (high freq) activates targeted areas,19 the cohorts should be given stimulations reciprocal to findings before performing the behavioral tasks to discern whether the implicated areas are causally responsible. Therefore, the art learners should be subjected with low freq stimulation of the right anterior cerebellum (as accurately as possible) and high freq stimulation of the frontal lobes, while the control group should be given the opposite. If the art learners perform poorly or exhibit reduced performance, then the rewiring changes are indeed responsible.

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To further investigate implications of the lower white matter FA in prefrontal cortex, lesion studies should be conducted. By examining patients experiencing schizophrenia, known to have cortical thinning and overall volume decrease,20,21 or those with dementia,22 the ambiguities associated with the aforesaid result can be explored.

In addition, post-mortem cellular studies should also be performed to discern whether extra-axonal network is responsible for the lower white matter FA in the frontal lobes. By immunostaining a cryosection of the implicated voxel area for glial fibrillary acidic protein (GFAP) of a professional visual representational artist and of a layman would also offer insight about the observed result.23 If the artist cryosection indicates significantly higher glial cell support system compared to a laymanâ&#x20AC;&#x2122;s, then the decreased prefrontal white

matter activation is due to a more developed glial cell population.

Lastly, different affective and cognitive skills have also been implicated with creativity, such as mood disorders, personality disorders,21,24 and meditation.25,26 Since there is a wealth of studies dedicated to these, a meta-analysis study scanning for works investigating creativity and these conditions or topics might also offer insight about potential rewiring changes or neural circuits responsible for creativity.

References 1. Schlegel, A. et al. The artist emerges: Visual art learning alters neural structure and function. Neuroimage 105, 440–451 (2015).

2. Farrington, L. E. Reinventing Herself: The Black Female Nude.

Woman’s Art J. 24, 15–23 (2004).

3. Hama, H. et al. Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Nat. Neurosci. 14, 1481–1488 (2011).

4. Lahoute, C., Herbin, O., Mallat, Z. & Tedgui, A. Adaptive immunity in atherosclerosis: mechanisms and future therapeutic targets.

Nat. Rev. Cardiol. 8, 348–358 (2011).

5. Zeisberg, E. M. et al. Endothelial-to-mesenchymal transition contributes to cardiac fibrosis. Nat. Med. 13, 952–961 (2007).

6. Gartus, A., Klemer, N. & Leder, H. The effects of visual context and individual differences on perception and evaluation of modern art and graffiti art. Acta Psychol. (Amst). 156, 64–76 (2015).

7. Figurative art. A Dictionary of Media and Communication (2011).

8. Costello, D. Kant and the problem of strong non-perceptual art.

Br. J. Aesthet. 53, 277–298 (2013).

9. Benoist, M.-G. Portrait d’une negresse. (1800). at <http:// en.wikipedia.org/wiki/Marie-Guillemine_Benoist#/media/ File:Marie-Guillemine_Benoist_-_portrait_d%27une_negresse.jpg> 10. Cattaneo, Z. et al. The role of prefrontal and parietal cortices in esthetic appreciation of representational and abstract art: A TMS study. Neuroimage 99, 443–450 (2014).

11. Halpern, a R. & O’Connor, M. G. Stability of Art Preference in Frontotemporal Dementia. Psychol. Aesthetics, Creat. Arts 7, 95–99 (2013).

12. Sternberg, R. J. Creativity or creativities? Int. J. Hum. Comput.

Stud. 63, 370–382 (2005).

13. Dietrich, A. & Kanso, R. A review of EEG, ERP, and neuroimaging studies of creativity and insight. Psychol. Bull. 136, 822–848 (2010).

14. Jung, R. E., Grazioplene, R., Caprihan, A., Chavez, R. S. & Haier, R. J. White matter integrity, creativity, and psychopathology: Disentangling constructs with diffusion tensor imaging. PLoS One 5, (2010).

15. Perdreau, F. & Cavanagh, P. Is artists’ perception more veridical? Front. Neurosci. 7, 1–11 (2013).

16. Graham, D. & Meng, M. Lightness constancy in visual artists.

J. Vis. 11, 371 (2011).

17. Miall, R. C. & Reckess, G. Z. The cerebellum and the timing of coordinated eye and hand tracking. Brain Cogn. 48, 212–226 (2002).

18. Chamberlain, R. et al. Drawing on the right side of the brain: A voxel-based morphometry analysis of observational drawing. Neuroimage 96, 167–173 (2014).

19. Cudeiro, J. et al. Effects on EEG of Low (1Hz) and High (15Hz) Frequency Repetitive Transcranial Magnetic Stimulation of the Visual Cortex: A Study in the Anesthetized Cat. Open Neurosci. J. 1, 25–30 (2007).

20. Buchsbaum, M. S. The frontal lobes, basal ganglia, and temporal lobes as sites for schizophrenia. Schizophr. Bull. 16, 379–389 (1990).

21. Carson, S. H. Creativity and psychopathology: A shared vulnerability model. Can. J. Psychiatry 56, 144–153 (2011).

22. Crutch, S. J. & Rossor, M. N. Artistic Changes in Alzheimer’s Disease. Int. Rev. Neurobiol. 74, 147–161 (2006).

23. Andreiuolo, F. et al. GFAPδ immunostaining improves visualization of normal and pathologic astrocytic heterogeneity.

Neuropathology 29, 31–39 (2009).

24. Motzkin, J. C., Newman, J. P., Kiehl, K. a. & Koenigs, M. Reduced Prefrontal Connectivity in Psychopathy. J. Neurosci. 31, 17348–17357 (2011).

25. Horan, R. The Neuropsychological Connection Between Creativity and Meditation. Creat. Res. J. 21, 199–222 (2009).

26. Deshmukh, V. D. Cognitive pause-and-unload hypothesis of meditation and creativity. 5, 217–231 (2013).

Received March, 12,

February, 2015;

10, accepted

2015; April, 06,

revised 2015.

This work was supported by The Neuroscience Association of Undergraduate Students (NAUS), The University of Toronto Human Biology Faculty (HMB), and The Neurobiology of Behavior 1 Series (HMB300H1). The author thanks Dr. Bill Ju, Dr. Alexander Schlegel, Dr. Prescott Alexander, and colleagues of Dartmouth College for execution, experiments, and results explored in this review.

Address correspondence to: Miss Dong-Eun Lee, Neuroscience and Human Physiology Undergraduate Program, University of Toronto, Toronto, ON M5S 3J6 Email: donge.lee@utoronto.ca.

Copyright © 2015 Dr. Bill JU, Miss Dong-Eun LEE, Neurosciences, Human Biology Program

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Overcoming social difficulties with the help of medications

Victor Lee

Social deficits have been shown to occur in a variety of psychiatric disorders such as schizophrenia and autism spectrum disorder. IRSp53 is an excitatory synaptic signaling protein and mice that lack the protein demonstrated impaired social interaction and communication as well as hyperactive NMDA receptors. Treatment of the IRSp53 knockout mice with memantine, which is a NMDA receptor antagonist, or MPEP, which is a metabotropic glutamate receptor 5 antagonist, rescued social interaction. NMDA receptor function, hippocampal plasticity, and neuronal firing in the medial prefrontal cortex was also normalized along with social interaction.

Reduced NMDA receptor function has been implicated in social impairments as well, which suggeset that deviation of NMDA receptor activity from a regular level can lead to social deficits and that correction of the receptor activity can rescue social interaction.

Key words: Social deficit, IRSp53, NMDA receptor, mGluR5 Background Social deficits are characteristic of many neuropsychiatric disorders such as attention deficit hyperactive disorder, autism spectrum disorder, and schizophrenia.1,2,3 IRSp53 is an excitatory synaptic signaling protein that plays a role in the control of the cytoskeletal actin filaments.4,5 IRSp53 knockout mice have been shown to have lower AMPA/NMDA ratio.6 The knockout mice also demonstrate increased NMDA receptor function, increased long term potentiation dependent on NMDA receptors, and decreased hippocampus-dependent learning and memory. The drugs used in this paper are the NMDA receptor antagonist memantine7 and the metabotropic glutamate receptor 5 antagonist MPEP.8 Memantine has been shown to be effective in treating Alzheimerâ&#x20AC;&#x2122;s Disease in humans, which is a neurodegenerative disorder,9 and MPEP has been shown to have antidepressant like effects on mice.10 In this paper, wild type mice and IRSp53 knockout mice were used and drugs were delivered to the mice by injection. Various behavioural experiments were used to assess the social aptitude of both mice. The three-chambered test is a prominent social interaction assay which measures the amount of time a subject mouse spends in either the chamber with a novel stranger mouse or the chamber with a novel object.11 Long term depression (LTD) was measured to test for plasticity. Brain slices have long been used to study long term potentiation and LTD as indicators of synaptic plasticity.12 Experiments in this paper that tested for LTD utilized sagital and coronal brain slices.

Drugs were delivered to the slices by infusion into the artificial cerebrospinal fluid bathing the slices. Whole cell patch clamp recordings and field potential recordings were used to measure excitatory post synaptic currents. Neurons were stained with antibodies and phalloidin, which is commonly used to stain for actin,13 to measure F-actin stability.

The F-actin stability may contribute to the hyperactivation of NMDA receptors by reducing LTD of NMDA receptors. Currently, it is known that F-actin is severed by cofilin, which is activated through phosphorylation.14 The stabilized F-actin becomes resistant to

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activated cofilin but the mechanism of how this occurs is unclear. There are many proteins that are upstream of cofilin such as p21- activated kinase, LIM kinase, Rho-associated kinase, Rac/cdc42, and Rho.15 Though this paper has observed the effects of drug treatment on social behaviour in knockout mice, drug treatment on cofilin resistant actin in knockout mice remains unexamined. Unraveling the mechanism of the cofilin resistance is a potential next step in determining new treatments for synaptic dysfunction (by revealing novel drug targets) and ultimately treatment of social deficits.

Research Overview

Summary of Major Results

Social deficits and rescue of social interaction Measurements of social interaction demonstrated that IRSp53 knockout mice had social deficits compared to wild type mice. The three chambered test revealed that knockout mice spent less time with the stranger mouse than the wild type mice. Knockout mice spent less time sniffing cages and other mice despite demonstrating normal olfactory function. Knockout mice moved greater distances than wild type mice within a 48 hour period. Knockout mice also emitted less ultrasonic vocalizations. All of these results confirm deficits in many different aspects of social interaction in the knockout mice. Heterozygotic mice were also tested for social function and were confirmed to have social behaviour similar to that of wild type mice.

All mice were given intraperitoneal injections of memantine (10 mg/kg) or MPEP (30 mg/kg) and it was found that memantine and MPEP rescued certain aspects of social deficits in the knockout mice, such as performance in the three chambered test (Figure 1). However, some forms of social function such as hyperactivity and ultrasonic vocalizations were not normalized. Furthermore, a dose of MPEP at 10 mg/ kg was not sufficient to rescue social deficit.

Figure 1. Quantification of time the wild type mice (WT) and IRSp53 knockout mice (KO) spent in a chamber with a novel object (O) and a chamber with a stranger mouse (S).

LTD of NMDA receptors in hippocampus Excitatory postsynaptic potentials were measured in the SC-CA1 synapses of the hippocampus and it was found that LTD was impaired for NMDA receptors in the synapses for IRSp53 knockout mice but there was normal LTD of AMPA receptors.

It has been shown that retention of NMDA receptors in the synapse requires stable F-actin16 and that depolymerization of actin is needed for long LTD of NMDA receptors.17 In this study, it was found that reduced LTD was also associated with stable F-actin.

It was found that F-actin depolymerization in synapses was less in knockout mice as compared to wild type mice, and even less in Shank positive (excitatory) synapses (Figure 2a-d). It was also found that cofilin, a negative regulator of F-actin, was found to be expressed at similar levels in both wild type and knockout mice (Figure 2e). Furthermore, it was found that the IRSp53 knockout mice had higher levels of cofilin phosphorylation (Figure 2f), a measure of cofilin inactivation, but higher levels of NMDA-induced cofilin dephosphorylation (Figure 2g,h).

Treatment of brain slices with memantine was found to have restored LTD of NMDA receptors and normalized NMDA receptor activity levels in IRSp53 knockout mice. These drugs did not show such an effect in wild type mice. MPEP was found to normalize AMPA/ NMDA ratios in knockout mice as well. Memantine was not used to test for normalization of AMPA/ NMDA ratio due to its voltage dependent blockade of NMDA receptors.

Reduced dendritic complexity and firing rate of mPFC The medial prefrontal cortex (mPFC) is an area that is known to be involved in social function.18 The layer II and III neurons was found to have reduced dendritic complexity in the apical dendrites (but not the basal dendrites). The firing rate and amplitude of the miniature excitatory postsynaptic currents was also found to

be lower in the IRSp53 knockout mice layer II and III neurons when compared to the wild type mice layer II and III neurons.

When single unit recordings were used on live anesthesized mice, it was found that IRSp53 deficient mice had reduced firing only in excitatory synapses and inhibitory synapse firing rates were left unchanged.

Memantine was found to increase the firing rate of neuronal firing in both wild type and IRSp53 knockout mice but the effect was found to be larger in the knockout mice. These results suggest that the removal of IRSp53 reduces firing rate in the mPFC but can be rescued with memantine.

Furthermore, it was found that memantine does not incraese the firing rate of hippocampal CA1 neurons in both wild type and IRSp53 knockout mice. However, firing rate of memantine treated IRSp53 knockout mice was significantly higher than the firing rate of memantine treated wild type mice. This suggests that memantine induces a difference in firing rate between the wild type mice and IRSp53 knockout mice.

Conclusions and Discussion All the data provided seems to suggest that social deficits in IRSp53 knockout mice are caused by hyperactivity of NMDA receptors in the hippocampus.

Memantine rescued social interaction and LTD of NMDA receptors in the hippocampus. MPEP also rescued social interaction but was shown to increase AMPA/NMDA ratios as well. However, LTD of metabotropic glutamate receptor 5 in the hippocampal synapses appear to be normal so this suggests that the mechanism of how MPEP rescues social deficits does not involve modulating LTD of metabotropic glutamate receptor 5 but rather, works indirectly to modulate NMDA receptor activity.

The drugs used in this study, memantine and MPEP, could rescue some aspects of social function, such as time spent with stranger mouse in a three chambered test, but could not rescue some other aspects of social function, such as ultrasonic vocalizations or hyperactivity. Drug induced social rescue in other mouse lines have rescued symptoms such as hyperactivity.

For example, Cntnap2 knockout mice have had their hyperactivity reduced with the antipsychotic drug risperidone.19 The selectivity of the rescues by these drugs may ease further study of the mechanisms.

The hippocampus of the IRSp53 knockout mice showed less LTD of the NMDA receptors and this may be due to stabilized F-actin in those mice which prevent NMDA from leaving the membrane. However, F-actin may also be stimulating the activity of the NMDA receptors on the membrane through interaction mediated by alpha actinin.20 The results that show that there is significant decrease in dendritic complexity of mPFC neurons are the first to show in vivo that IRSp53 upregulates dendritic spines. Interestingly enough, the hippocampal neurons display normal dendritic complexity in the knockout mice despite the fact that there is hyperactivity of NMDA receptors in the synapses of the hippocampus. Furthermore, memantine restoring 152

Figure 2. Data reflecting the stabilized F-actin and lower basal cofilin activity in IRSp53 knockout mice hippocampal synapses. (a,b) Select images of hippocampal synapses of both wild type and knockout mice. MAP2 and Shank were used to stain for dendrites and excitatory synapses respectively and phalloidin was used to stain for F-actin. (c,d) Quantified results of a and b. (e,f) Cofilin levels were found to be similar in wild type and knockout mice but basal levels of phosphorylated cofilin appeared to be higher in knockout mice. (g,h) Greater levels of dephosphorylation were found in knockout mice than wild type mice when stimulated with NMDA.

the low firing rates of the mPFC neurons implies that the restoring neuronal firing may play a role in restoring social interaction in the knockout mice.

These results suggest that NMDA receptor hyperfunction is implicated in social deficit. However, other results have shown that NMDA receptor hypofunction is also implicated in social deficit.21 As is the case with many biological models, balance is key in maintaining proper function for the organism.

IRSp53 has been implicated with Autism spectrum disorder (ASD).22 However, it has not yet been established as to whether NMDA receptor hyperactivity is associated with ASD. Memantine has been shown to help with social deficits in ASD,23 which suggests that NMDA receptor hyperactivity may indeed contribute to social deficits in ASD.

Conclusions

In conclusion, the data shows that NMDA receptor hyperactivity in IRSp53 knockout mice causes social deficits. However, there is potential for therapeutic treatment through NMDA receptor inhibition. This study has unraveled part of the mechanism that causes social dysfunction but there is still more work to be done in the field. Further understanding of molecular 153

mechanisms involved in social deficits can help reveal more drug targets that can potential treat conditions such as ASD. Criticisms and Future Directions While this paper has examined many perspectives of the problem, many aspects of it remain unexamined.

The study acknowledged that memantine and MPEP could not rescue some social dysfunction symptoms such as hyperactivity but did not attempt to rescue them with drugs that are known to rescue such symptoms such as risperidone. Regardless of outcome, conducting such experiments would provide insight of mechanisms of social deficit and reveal potential drug targets.

The data revealed that IRSp53 knockout mice had reduced long term depression of NMDA receptors in the hippocampus. This may have been caused by the stabilization of F-actin in the dendrites. The experiments demonstrated that the stabilization of F-actin is in part due to the F-actin becoming resistant to cofilin.

However, the mechanism was unexplored. The paper neglected to check expression of genes upstream of cofilin such as LIM kinase, p21- activated kinase, Rho-associated kinase, Rac/cdc42, and Rho.

Overall,. it would be a good next step to uncover what may be causing the F-actin stability and resistance to cofilin. Upregulating and downregulating these genes is another approach that may be done. Discovering other proteins that are involved in this pathway would offer insight onto what proteins to create drug targets for to treat social deficits and can potentially be used as treatments for conditions such as ASD. References 1. Rich, E. C., Loo, S. K., Yang, M., Dang, J. & Smalley, S. L. Social functioning difficulties in ADHD: Association with PDD risk.

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4. Soltau, M. et al. Insulin receptor substrate of 53 kDa links postsynaptic shank to PSD-95. J. Neurochem. 90, 659–665 (2004).

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6. Kim, M.-H. et al. Enhanced NMDA receptor-mediated synaptic transmission, enhanced long-term potentiation, and impaired learning and memory in mice lacking IRSp53. J. Neurosci. Off. J. Soc. Neurosci. 29, 1586–1595 (2009).

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Received November, 12, 2014; accepted December, 16, 2014.

This study was supported by the National Research Foundation of Korea (to D.K., 2012-0008795; to Y.C.B., 2012-0009328) and the Institute for Basic Science (IBS-R002-D1 to E.K. and IBS-R002-G1 to M.W.J.).

Address correspondence to: Dr. Eunjoon Kim, Department of Biological Sciences, KAIST, Daejeon, Korea. Email: kime@kaist.ac.kr Copyright © 2015 Nature America, Inc. All rights reserved.

154

Role of mu-opioid receptors in stress affecting vulnerability to substance abuse

Ella Lew

The mu-opioid receptor (MOR) plays a significant role in executive functioning in the orbitofrontal lobe as well as the reward system in the mesolimbic area. Stress increases production of endogenous opiates that bind to MOR implicating both regions of executive functioning and rewards. This notion was shown recently in a study on male mice where social defeat stress inhibited behavioral flexibility from a decrease in MOR binding. This review aims to evaluate the literature on MOR binding and the association with mental illness and drug addictions. This review will focus on the role of MOR, the SNP MOR variant A118G, effects of social defeat, and the implications MOR has on an individual’s vulnerability to substance abuse. This review will also look at the efficacy of treating alcoholism with the popular MOR antagonist, Naltrexone. An often overlooked area of sex differences in stress-induced MOR binding will be evaluated which calls for a paradigm shift in future research and treatment to accommodate for sex-dependent MOR binding and stress effects.

Key words: drug abuse, stress, sex differences, social defeat, mu-opioid receptors (MOR), Barnes maze Background Behaviors can normally be driven autonomously but is difficult in reward-seeking addictions. The mu-opioid receptor (MOR), part of the G-protein coupled opioid receptor family, has a role in both reward (Thorsell, 2013; Wang et al., 2012) and behavioral flexibility in changing environments (Legardo et al., 2015). MOR is expressed widely in the brain from the locus coeruleus, orbitofrontal cortex, hippocampus, and VTA making it a challenge to completely elucidate MOR’s role.

MOR bind to endogenous ß-endorphins and act on neurons through G-proteins to open presynaptic inward rectifying K+ channels and inhibit pre-synaptic Ca2+ channels reducing membrane potential and neurotransmitter release (Thorsell, 2013). In GABAergic neurons, reduced GABA transmission resulting in disinhibition and increased dopamine release in the ventral striatum (Thorsell, 2013). MOR is also the binding site for common opiates in drug abuse such as morphine and heroin (Bond et al., 1998; Thorsell, 2013). This makes MOR a probable mediator in opiate addictions and alcoholism with research showing MOR inactivation can help to ameliorate reward reinforcements from drugs and alcohol (Bond et al., 1998; Johnston, Herschel, Lasek, Hammer, & Nikulina, 2015; Komatsu et al., 2011; Wand et al., 2002). Many groups have looked at possible MOR-antagonists and knock-downs to elucidate the stress and addictions pathway and help explain the variability in treatment efficacy among people.

Normally, stress is a signal for aversive events and it is adaptive to remember for future encounters. However, MOR binding and learning is implicated by stress, in particular social defeat stress. Studies evaluating MOR knockout mice under chronic defeat stress find male mice have decreased BDNF levels in the hippocampus and are unable to learn to avoid aversive situations (Komatsu et al., 2011; Wang et al., 2012). Interestingly, while distressed male mice exhibited decreased MOR binding and were unable to adapt to a reversal phase of the Barnes Maze, this impairment was not seen in females (Legardo et al., 2015). The inability to properly adapt in MOR modulated stress mirrors the struggle of drug addictions.

MOR SNPs have been identified in humans and 155

animals which prompted investigation in the functional differences among the MOR SNPs (Xu et al., 2014).

Most extensively studied is the A118G SNP in the first exon of the OPRM1 MOR gene where Asn from A118 is replaced by Asp in the G118 allele affecting the N-glycosylation site (Bond et al., 1998; Thorsell, 2013). While the types of ligand bound to the receptor were unaffected, some studies find that G118 increases binding affinity to ß-endorphin and potency on G-protein signaling by 3 times (Bond et al., 1998; Komatsu et al., 2011). Difference in opioid sensitivity may explain the variation in stress response and severity of substance abuse among individuals. This is supported by substance dependent individuals with the 118G allele being more responsive to MOR-antagonist treatments, is more prevalent in normal no-drug abuse populations, and has greater HPA axis suppression than individuals with the A118 allele (Wand et al., 2002).

However, Befort et al. (2001), Beyer et al. (2004), Ramchandani et al. (2011) were unable to replicate findings of increased binding affinities from the A118G SNP (as reviewed in Thorsell, 2013). This suggests that the G allele itself may not be enough to cause functional change but that there are other variables to consider for future research such as stressor type, other opioid receptors, serotonin levels to consider in stress and addiction management.

Research Overview

Results & Discussion

MOR on GABAergic neurons regulates dopamine release and is important in reinforcing behaviors through the mesocorticolimbic reward pathway.

When MOR activation is limited, there is less positive reinforcement and dopamine release. Tanda and Di Chiara found that inactivating MOR with a MORantagonist, naloxonazine injections in the VTA, can reduce and abolish the rewarding dopamine release from morphine and nicotine in the NAcc of male rats (1998). MOR’s role in the reward system is also seen in mice models with alcohol dependence

where lacking MOR prevents experiencing the positive effects of alcohol seen in wild-type mice and the KO mice discontinue self-administered alcohol (Ghozland, Chu, Kieffer, & Roberts, 2005). Furthermore, through chronic morphine treatment, a MOR agonist, an enhanced basal MOR signaling was developed that may lead to forming an addiction (Wang et al., 2004). These findings suggest that MOR is involved in positive reinforcements for substances in the reward pathway where drug addictions can develop.

MOR’s involvement in the reward system strongly supports the MOR-antagonist therapeutic drugs to interfere with the reward system that has shown to have powerful effects in reducing drug/alcohol-seeking behavior. Naltrexone is an opioid receptor antagonist drug approved by the US FDA since 1994 that has effectively reduced chances of alcohol relapse (O’Malley, Jaffe, & Chang, 1992), alcohol craving, the number of heavy drinking days, and pleasure from alcohol in dependent animal models (Swift, Whelihan, & Kuznetsov, 1994).

Dopamine released from alcohol consumption was suppressed in a dose-dependent manner by naltrexone (Thorsell, 2013). However, naltrexone is not a perfect drug with unwanted symptoms and low compliance (Thorsell, 2013). Differences in drug efficacy are likely due to the variation in MOR SNPs. MOR SNPs were first investigated by Bond et al. in 1998 by DNA sequencing 113 former heroin addicts and 39 controls with no prior drug abuse. The MOR A118G SNP was the most prevalent in the total study population. The same group reported that the G118 variant did not change the type of ligands the receptor can bound to but had a 3 times higher affinity and potency in G-protein signaling to B-endorphins. Notably, the G allele was found significantly higher in the control group than the heroin group. The authors believe that through increased B-endorphin affinity the G allele confers a form of protection from addiction. Some other groups after have been unable to replicate these findings and suggest that the G-allele alone may not be definitive in whether an addiction will or will not be established.

However, the G-allele has shown to have functional differences in stress and addiction. Wand et al. investigated the difference in individuals with A versus G allele with a MOR-antagonist Naloxone (2002). In the absence of MOR activity, G118 individuals had an increase in cortisol response compared to A118. This indicates that G118 acts as a greater HPA axis inhibitor and lowers stress response. This study was conducted without a stressor and measured basal levels however, it would be significant to perform the same study with different forms of stressors and evaluate the impact stress has on the G variant compared to A. This study was only conducted on male mice. However, females are known to have different hormone levels that influence stress, increased stress sensitivity, and cope with different stress responses, it would be worthwhile to investigate the different effects G-allele has between the sexes. Similar findings were found in rhesus monkeys with a C77G SNP in the MOR similar to the human A118G SNP exhibited increased B-endorphin affinity by 3.5 times and lowered cortisol levels (Miller, Bendor, Tiefenbacher, Yang, Novak, & Madras, 2004)/ Chronic HPA activation can lead to disease states and also heightened catecholamine effects and can make drug abuse more susceptible

(Wand et al., 2002). Furthermore, naltrexone treatment is more effective in the G allele compared to negligible effects in the A allele. This explains the drug’s report in low compliance since the G-allele is less frequent in the addict populations and only the few who carry the G-allele will see significant treatment effects. In a meta-analysis, individuals with the 118G variant reported less relapse in heavy drinking (Chamorro, Marcos, & Miron-Canelo, 2012). These findings support Bond et al.’s proposal that the G variant can better protect from drug addictions than the A variant.

Studies have shown that stress can negatively impact learning leading to maintenance of a drug addiction.

Komatsu et al. investigated male C57BL/6J control mice and MOR knock-outs (KO) under social defeat stress (2011). After defeat stress control mice exhibited social aversion and spent less time in the social interaction zone with an unfamiliar peer in the open field test. This may also explain the hesitancy for depressed or stressed individuals seeking help from a therapist in times of stress. In the MOR KO, mice showed reduced social aversion and also a reduction in BDNF mRNA in the hippocampi compared to controls. It can be speculated that MOR KO mice were unable to accommodate learning from the initial social defeat condition. This supports the finding that MOR agonists in VTA increase Fos expression in dopaminergic projections necessary for learning (Nikulina et al., 2008). Likewise, socially defeated male mice showed decreased MOR binding in an autoradiography and impaired behavioral flexibility in a reversed Barnes Maze trial (Legardo et al., 2015).

These findings suggest that MOR activation is necessary to facilitate learning from stressful events. These findings are highly relevant in stress-induced mental illnesses like PTSD, GAD, and depression where MOR binding is reduced from chronic stress and learning and seeking help from others can be challenged in these conditions. These findings suggest that the popular cognitive-behavior therapy that utilizes behavioral changes may be less effective in socially defeated individuals with downregulated MOR binding.

Conclusions

Understanding the role MOR plays in addictions and stress can reduce vulnerability in developing drug addictions and mental illness as well as creating more targeted and efficacious therapeutics. Research in MOR has been very progressive in understanding its function, the A118G SNP, and translating findings into efficacious treatments. MOR influences the reward system and dopamine release relevant in drug addictions.

MOR activation also has a role in learning by increased Fos expression in dopaminergic projections and BDNF mRNA in the hippocampi. Thus, abnormal MOR functioning can decrease the ability to learn and can make one more susceptible to drug addictions. Social defeat stress that reduces MOR binding and its downstream effects exhibits decreased learning and behavioral flexibility. MOR proves to be responsive to pharmacological agents and this understanding is significant in finding therapeutic treatments for drug addiction and mental illness. Research in MOR-antagonists has proven to be powerful treatments but is limited to select G118 individuals. However, further investigations of the differ156

ences between the A and G118 SNP can help devise more targeted treatments between the phenotypes.

Criticisms and Future Directions There is a large paucity in the sex differences that exist in MOR modulated stress and drug addiction research. The majority of studies are done on male mice subjects and individuals and thus the results cannot be confidently translated to female subjects.

Especially, with the general understanding that women respond to stress differently from men and that levels of estrogen hormones can influence stress. Milner et al. performed a labelled immunoelectron microscopy with silver-intensified gold particles to evaluate the MOR density and trafficking in the hippocampus of male and female rats (2013). They found that the sexes responded differently in both acute and chronic stress.

While females showed increased density and trafficking of MOR in PARV-labeled dendrites after chronic stress, males saw no change in MOR density. This implicates that learning occurs in females but not males under chronic stress. In another study, MOR binding was downregulated and behavioral flexibility was impaired in socially defeated male mice but not female mice (Legardo et al., 2015). Future research should look at how stress and MOR binding affects the sexes differently and devise more appropriately sex-targeted treatments to accommodate for the different stress mechanisms.

While many studies employ different stress conditions, future studies should clearly identify the types of stressors (social, physical, emotional) to determine whether different stressor types have a significant impact on MOR binding and learning.

Additionally, while the MOR-antagonist Naltrexone has limited responses to the extracellular modification in A118G SNPs, future therapeutics can look at terminating receptor signaling intracellularly through phosphorylation by a cytoplasmic kinase or through endocytosis or desensitization of MOR. Furthermore, knowing that the G allele is suggested to confer protective properties against addiction, CRISPR can be utilized to modify the 118 SNP in individuals with the A allele to express the G allele. Individuals with the G variant are more responsive to treatment and fare better at stress management. Other variables that would influence reward systems or interact with MOR binding should also be studied such as serotonin receptors and other types of opioid receptors.

References 1. Bond, C., LaForge, K.S., Tian, M., Melia, D., Zhang, S., Borg, L., …

Yu, L. (1998). Single-nucleotide polymorphism in the human mu opioid receptor gene alters B-endorphin binding and activity: Possible implications for opiate addiction. Proc Natl Acad Sci USA, 95, 9608-9613.

2. Chamorro, A.J., Marcos, M., & Miron-Canelo, J.A. (2012).

Association of micro-opioid receptor (OPRM1) gene polymorphism with response to naltrexone in alcohol dependence: a systematic review and meta-analysis. Addict Biol, 17, 505-512.

3. Ghozland, S., Chu, K., Kieffer, B.L., & Roberts, A.J. (2005).

Lack of stimulant and anxiolytic-life effects of ethanol and accelerated development of ethanol dependence in mu-opioid receptor knockout mice. Neuropharmacology, 49(4), 493-501.

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4. Greenfield, S.F., Pettinati, H.M., O’Malley, S., Randall P.K., & Randall, C.L. (2010). Gender differences in alcohol treatment: an analysis of outcome from the COMBINE study. Alcohol Clin Exp Res, 34(10), 1803-12.

5. Iniquez, S.D., Riggs, L.M., Nieto S.J., Dayrit, G., Zamora, N.N., Shawhan, K.L., Cruz, B., & Warren, B.L. (2014). Social defeat stress induces a depressionlike phenotype in adolescent male c57BL/6 mice. Stress, 17(3), 247-55.

6. Johnston, C.E., Herschel, D.J., Lasek, A.W., Hammer, R.P., & Nikulina, E.M. (2015). Knockdown of ventral tegmental area mu-opioid receptors in rats prevents effects of social defeat stress: implications for amphetamine cross-sensitization, social avoidance, weight regulation and expression of brain-derived neurotrophic factor. Neuropharmacology, 89, 325-34.

7. Komatsu, H., Ohara, A., Sasaki, K., Abe, H., Hattori, H., Hall, F.S., Uhl, G.R., & Sora, I. (2011). Decreased response to social defeat stress in µ-opioid-receptor knockout mice. Pharmacol Biochem Behav, 99(4), 676-82.

8. Laredo, S.A., Steinman, M.Q., Robles, C.F., Ferrer, E., Ragen, B.J., & Trainor, B.C. (2015). Effects of defeat stress on behavioural flexibility in males and females: modulation by the mu-opioid receptor. European Journal of Neuroscience, 1-8.

9. Miller, G.M., Bendor, J., Tiefenbacher, S., Yang, H., Novak, M.A., & Madras, B.K. (2004). A mu-opioid receptor single nucleotide polymorphism in rhesus monkey: association with stress response and aggression. Molecular Psychiatry, 9, 99-108.

10. Milner, T.A., Burstein, S.R., Marrone, G.F., Khalid, S., Gonzalez, A.D., Williams, T.J., … Waters, E.M. (2013). Stress Differentially Alter Mu Opioid Receptor Density and Trafficking in Paravalbumin-Containing Interneurons in the Female and Male Rat Hippocampus. Synapse, 67, 757-772.

11. Nikulina, E.M., Arrillaga-Romany, I., Miczek, K.A., & Hammer R.P. (2008). Long-lasting alteration in mesocorticolimbic structures after repeated social defeat stress in rats: time course of mu-opioid receptor mRNA and FosB/DeltaFosB immunoreactivity. Eur J Neurosci, 27(9), 2272-84.

12. Nikulina, E.M., Hammer, R.P., Miczek, K.A., & Kream, R.M. (1999). Social defeat stress increases expression of mu-opioid receptor mRNA in rat ventral tegmental area. Neuroreport, 10(14), 3015-9.

13. O’Malley, S.S., Jaffe, A.J., & Chang, G. (1992). Naltrexone and coping skills therapy for alcohol dependence. A controlled study.

Arch Gen Psychiatry, 49, 881-7.

14. Swift, R.M., Whelihan W., & Kuznetsov, O. (1994). Naltrexone-induced alterations in human ethanol intoxication. Am J Psychiatry, 151, 1463-1467.

15. Tanda, G., & Di Chiara, G. (1998). A dopamine-mu1 opioid link in the rat ventral tegmentum shared by palatable food (Fonzies) and non-psychostimulant drugs of abuse. Eur J Neurosci, 10(3), 1179-87.

16. Thorsell, A. (2013). The µ-Opioid Receptor and Treatment Response to Naltrexone. Alcohol and Alcoholism, 48(4), 402-408.

17. Verzillo, V., Madia, P.A., Liu, N.J., Chakrabarti, S., & Gintzler, A.R. (2014). Mu-opioid receptor splice variants: sex-dependent regulation by chronic morphine. J Neurochem, 130(6), 790-6.

18. Wand, G.S., McCaul, M., Yang, X., Reynolds, J., Gotjen, D., Lee, S., & Ali, A. (2002). The Mu-Opioid Receptor Gene Polymorphism (A118G) Alters HPA Axis Activation Induced by Opioid Receptor Blockade. Neuropsychopharmacology, 26, 106-114.

19. Wang, D., Raehal, K.M., Lin, E.T., Lowery, J.J., Kieffer, B.L., Bilsky, E.J., & Sadee, W. (2004). Basal signaling activity of mu opioid receptor in mouse brain: role in narcotic dependence. J Pharmacol Exp Ther, 305, 512-520.

20. Xu, J., Lu, Z., Xu, M., Pan, L., Deng, Y., Xie, X., … Pan, Y.X. (2014). A heroin addiction severity-associated intronic single nucleotide polymorphism modulates alternative pre-mRNA splicing of the µ opioid receptor gene OPRM1 via hnRNPH interactions. J Neurosci, 34(33), 11048-66.

Received April 2015.

Address correspondence to: Ella Lew, Human Biology Department, 30 Wilcox Street, University of Toronto, ON, Canada Email: ella.lew@mail.utoronto.ca Copyright © 2015 Dr. Bill JU, Neurosciences, Human Biology Program

Seeking Autism-Linked Performance Within the Synaptic World: Effect of Neurexins and Related Proteins

Vivian Liu

Autism spectrum disorder (ASD), a neurodevelopmental disorder tagged with high genetic concordance, is a concern among many parents. Given the genetic correspondence, researchers have been exploring many candidate genes and testing for behavioral changes upon mutations. That being said, autism has a wide range of disorders which vary form patient to patient, and is most likely the result of a multifactorial cause. It is important to note that many co-occuring conditions are also associated with autism. Therefore, there is currently no â&#x20AC;&#x153;trueâ&#x20AC;? animal model of autism, but rather multiple mouse models of autism. Previous studies have implicated autism as a result of synaptic transmission defects, especially mutations (in most cases, knockouts) involving synaptic proteins. Many mouse models involving the knockout of certain synaptic proteins serve to recapitulate some, but not all of the autistic-associated phenotypes. These potential synaptic proteins at hand include as neuroligins, synapsins, SHANK and neurexins, all which contribute to proper facilitation and organization of neuronal synapses. Behavioral measurements such as the open field test, novel object recognition task, ultrasonic vocalizations and three-The discovery of causal genes can then allow researchers and clinicians to apply the innovative diagnostic methods to young patients or pregnant women, encouraging early detection and therapeutic approaches. The focus of the present literature review aims to analyze past research on synapticrelated proteins and their relations to autism, with a strong emphasis on neurexins, a recently explored gene.

This review will also suggest next steps to enhance the current knowledge of autism, and the issues at hand with gene elucidation using mouse models.

Key words: autism; neuroligins; synapsins, SHANK; neurexins; ultrasonic vocalizations; synaptic; multifactorial Background The Autism Spectrum Disorder (ASD) has one of the highest genetic rates within neurodevelopmental disorders, constituting about 0.85-0.92 concordance among monozygotic twins1. ASD is well established prior to when the child turns three years of age, producing an early onset of core symptoms. The crucial three symptoms include insufficiency in social interactions including gestures and communication, repetitive actions or behaviors and limited or self-deprivation of interests. Comorbid conditions that are seen occurring with autism, but are not the main diagnostic markers include anxiety disorders, depression and fragile X syndrome. Recent genomic analysis have narrowed down hundreds of genetic variants, some common and some rare, which are all to some extent, associated with ASD. That being said, previous research has explored a variety of candidate genes related to synaptic functioning that may be in association with autism. Four of the major ones include: Neuroligins, synapsins, SHANK3 and neurexins. Each of these proteins have demonstrated relevance to autistic behavior in one aspect or another.

To begin, neuroligins function as cell-attachment proteins that reside on the post-synaptic membrane, interacting with their pre-synaptic partner, the neurexins. Prior research done on neuroligin-3 (NL3) mutations within mice displayed an increase in repeated motor actions of adult mice, a prominent autistic-like behavioural phenotype2. It was depicted that elimination of NL3 in the dorsal stratium has little effect on rotarod execution, but the same deletion in the NAc resulted in increased performance on the rotarod, suggesting that the NAc-NL3 interactions could facilitate repetitive behaviors2.

In addition, mutated synapsins also serve to cause autistic-like phenotypes in mice with the co-presence of epilepsy, which has a quarter chance of occurring. Synapsins are neuronal phosphoproteins, which contact lipid counterparts of presynaptic vesicles and actin3. Researchers compared Syn1 and Syn3 KO mice prior and subsequently to epilepsy occurrence, and concluded that the deficits in social preferences and temporary social recognition existed independent of epilepsy4. Excessive self-grooming activity and repetitive locomotion behaviours were also apparent in Syn2 KO mice4. Earlier research claimed synpasins to be one of the synaptic proteins enhanced by neurexins3.

To extend, prior research have also investigated the Shank proteins (1-3), a group of scaffold that consist of sites for protein-protein contact, especially membrane proteins. These Shank proteins reside on the postsynaptic area of excitatory synapses and is said to be involved in transduction processes, especially in metabotropic contexts5. Anxiety characteristics and exploratory activity were noted in Shank1 KO mice in comparison to Shank 1 heterozygote and Shank 1 WT mice6. This synaptic dysfunction resulted in the absence of social affiliation and repetition in peeking behavior7.

It was also previously found that ProSAP2/ Shank 3 could affect synaptic proteins through neurexinneurolgin co-operation.

Given that neurexins seem to have a connection with many other synaptic proteins such as the three aforementioned, it was thought to have a crucial role in contributing to autistic behaviours. It is well known that neurexins are a category of synaptic linkage proteins that reside at the pre-synaptic terminal.

The family of neurexins consist of NRXN1-3 genes, yielding ether promoters a or b. Graytonâ&#x20AC;&#x2122;s team 158

focused their research with the knockout of Nrxn1a in mice, depicting that male Nrxn1a KO mice present a more violent phenotype towards other rodents, female Nrxn1a KO mice partake in reduced social care (nest-building) and both genders had decrease in locomotion8. Measurement tests included openfield test, elevated plus maze, three-chamber social approach task and the social investigation task. Further research in regards to neurexins were then pursued, one of them being a major article reviewed here.

In the primary article of interest, researcher J Dachtler and his team tested eight weeks old Nrxn 2a KO mice for autistic performance. The assumed hypothesis was that elimination of the fist exon of the Nrxn2 gene would display some autistic consistent behavior. It is important to explore not only one specific gene of the Nrxn family of proteins, but the other available genes as well. Nrxn1a KO may provide some of the spectrum of autistic-like traits, while Nrxn2a KO may provide the other missing characteristics that were not observed. Assaying as much candidate genes as possible allows scientists to have a scale of the most dangerous mutations to the least, some causing more severe symptoms than others.

Research Overview

Summary of Major Results

Limited Social Interests The current study being reviewed had a dual approach in confirming their hypothesis: Behavioural measurements and quantification assays. The experimental mice at hand all exceeded eight weeks of age. To begin, the common three-chambered assay test was employed to measure the range of social interactions among mice. The target mice were placed in the middle sector, where the chambers to the side contained a novel conspecific and a vacant wire cage.

Openings in between the chambers allow for social exploration, but results evidently displayed that Nxn2a KO mice failed to show interest in their new neighbor9.

Moreover Nrxn2a KO mice were depleted in terms of curiosity for novel object awareness in comparison to WT mice9. Past studies of Syn1 KO mice showed that they roamed less in novel bedding and covered buried lower numbers of marbles4. Both studies seem to converge on one point: Synaptic defects seem to contribute to environmental ignorance of mutated mice, which essentially can include social contexts, in which contact and interests are limited.

Elevated Anxiety Levels To extend, researchers also tested for state anxiety levels. State anxiety is a temporary response to a danger stimulus or fear. The open-field and EPM test were employed to measure state anxiety levels. The open field test depicted thatNrxn2a KO mice would much rather commit to thigmotaxis (wall-hugging tendency) than enter the centre zone, where it is both exposed and bright9. The same results were recapitulated in NL2 KO mice, where they approached the centre zone much less than the WT mice even 159

Figure 1.Nrxn2aKO and WT mice behaviour during the ThreeChambered Assay. Nrxn2a KO mice showed a lack of interest for the stranger conspecific, in comparison to the WT mice.

For the vacant cage, both types of mice had insignificant differences in exploration time, suggesting the Nrxn2a KO most likely stayed put in their initial cage for the rest of the elapsed time.

though the distance roamed were almost equivalent10.

The Nrxn2a seems to be more bothered presence of a stressful stimulus, making them more vulnerable to rises in anxiety levels.

Figure 2. Nrxn2a KO spent drastically less time in the centre zone, where they are exposed to unrestricted, luminous space

In terms of the Elevated Plus Maze (EPM), this task consisted of enclosures and open arms, allowing mice to peek and explore the area above them (open, luminous space). As predicted, Nrxn2a KO mice preferably spent a large amount of time within enclosed arms, whereas WT mice showed much more exploration towards the novel area9. The latency to emerge to a new surrounding was undoubtedly much longer in Nrxn2a KO mice than in WT mice.

Hippocampal Protein Munc18-1 Loss Real-time PCR was used to assay the mRNA levels of various synaptic genes that either interact with

Figure 3. NL2KO mice clearly display elevated latency times to cross in the centre zone in comparison to WT littermates. The NL2KO mice also lingered longer in the dark zones of the open field test

neurexins at the presynaptic terminal or neuroligins at the postsynaptic level. An evident decrease was seen with the Stxbp1 gene within the hippocampus9.

Western blotting was then applied to quanitify the Munc18-1 protein presence, which is encoded by the Stxbp1 gene, and low levels were found9. However, special deficits were not discovered when the fearmotivated task was applied. Both Nrxn2a KO and WT mice could still subsequently recall that the dark chamber was associated with a shock one day later9.

This was an alarming result, as this major loss of Munc18-1 in the hippocampus did not seem to affect hippocampal-dependent activities.

Figure 4. Homogenates of the frontal cortex and the hippocampus were isolated and compared against for Munc18-1 levels. Western blotting was used for quantification. Blotting showed that the difference in Munc18-1 proteins had almost undetectable changes in the frontal cortex, but apparent changes in the hippocampus.

Discussions and Conclusions To this day, the etiological means of autism are still not transparent. However accumulation of research results have shed some insight on aligning and contradictory results.

One major finding that Dachtler and his team seem to duplicate in their Nrxn2a KO research is the lack of

social interaction and contact. NL2 KO mice2, Syn1 KO mice3 and Shank3B7 KO mice were all shown to pay less attention towards social novelty than their WT littermates. Nrxn2a KO mice did not offer curiosity towards their fellow conspecific either, emphasizing the alignment of results9. In the case that the Nrxn2a KO mice may have had olfactory deficits, the researchers employed the food burial task. Both groups of mice had the ability to search out the food in a reasonable amount of time, proving that an inability to sense social odors did not contribute to the Nrxn2a KO miceâ&#x20AC;&#x2122;s reluctance to interact with a novel conspecific9. For future references, it is likely that the causal gene(s) of autism would have to accomplish deficits in social behavior and engagement in order to be of significance.

Anxiety-associated phenotypes were observed in the open field test and elevated plus maze. Within the open field test, Nrxn2a KO showed increased thigmotaxis to the periphery of the arena in comparison to WT mice9. On the contrary, WT mice wandered into the centre zone much more frequently. In other words, Nrxn2a KO mice elicited more anxiety in response to uncomfortable situations. ASD can be characterized by sensitivity to selective contexts, which in return produce rising anxiety levels. Similar results were observed in the elevated plus maze, where the Nrxn2a KO mice took substantially longer to emerge out of the closed arms than the WT mice9.

This suggests that Nrxn2a may have more fear to new contexts, therefore having higher state anxiety levels, resulting in a delay in emergence. One way of interpreting this may be that WT are less prone stir up anxiety due to changes in surroundings, and are more adaptive than Nrxn2a KO mice. These results are in alliance with Shank1-/- mice6 and Syn2 -/mice3, where exploratory activity was reduced in open field testing, proposing that the measure of anxiety could be a reliable indicator of autism if more mouse models continue to show this trend.

Although Western Blot revealed that Munc18-1 had a dramatic decrease in the hippocampus, researchers found no interference with spatial recall a day after training for passive evasion9. On the contrary, previous research has shown that NL1 KO mice resulted in an 160

approximate 20% decrease in Munc18-1 protein within the hippocampus, and presented special deficits in the Morris Water Maze10. A day after training, WT mice spent elongated periods in the target area while Nlgn1 KO mice did not, showing reduced spatial memory.

So far, the knowledge about Munc18-1 is limited- it guides and solidifies SNARE complex fusion, meaning that is an important protein for both presynaptic and postsynaptic processes. This suggests that possibility that Munc18-1’s effects could be context-dependent, and that its’ synaptic role differs when interacting with ether neuroligins or neurexins. Consequently, this could inconclusively explain why special processing interference was seen in one study but not the other, but further research is this area would be necessary.

Aforementioned in the introduction, three of the four candidate genes of autism had consistent self-grooming actions upon targeted mutations. Ironically, the Nrxn2a KO study did not reproduce such results; both types of mice were indifferent in terms of the amount of self-grooming performed. Prior research has strong linkage to genetic defects and repetition: NL3-KO and NLR451R1C mutant mice achieved higher performance on the rotarod, a repetitive task of staying on a spinning rod, than WT companions2. Syn2 KO mice self-combed much more than WT mice when left in a cage and Shank3 KO mice engaged in more head pokes to exposed space. The light-dark emergence and zero-maze testing also showed greater number of head peeks for Shank3 KO mice7. It is important to note that the mice did not actually enter the “light” or “exposed” zones, suggesting that they were NOT drawn to novelty but rather performing habitual movements. If the Shank3 KO were truly curious about their surroundings, a high number of entries into the light zones or open arms would have occurred. The discussion of why the Nrxn2a got the null result for self-grooming will be discussed in the critical analysis section.

Although no clear establishment exists, the Nrxn2a KO study has succeeded in showing that neurexin dysfunction does serve some relevance to recapitulate anxiety-like behaviors, such as interference with social interactions and prominent anxiety levels. Just like all the other previous studies, however, the correspondence is still premature and requires much more credible evidence to build upon these surface level findings. It would make sense to extend the research to Nrxn3 and its relevance to autism, because only then can scientists classify which autistic-like traits are shared within defects of the neurexin family and which are not.

Critical Analysis

Like all scientific studies, there are notable aspects, as well as room for improvement. To begin, Dachtler and his team did a thorough job in testing and analyzing autistic-associated factors and symptoms for Nrxn2a KO mice. Similar to many other behavioral studies, a variety of tests were utilized to test the different aspects of autism. This included the major behavioral measures such as the open field test and elevated plus maze to analyze state anxiety, the novel object recognition task for trait anxiety, the three –chambered assay to rate social interactions, and even the quantification of 161

Munc18-1 levels in the hippocampus. That being said, it seemed as if the experiment had concentrated more on the comorbid associations rather than the fundamental symptoms of autism. These disorders may be present along with autism, but are not early determinants of autism itself. The hallmark characteristics of autism such as repetition (in this grooming) failed to be measured properly and social callings (communication deficits) were not considered. Within this study, selfgrooming was measured alongside with the open field test when the mice were allowed to roam. However, it is logical that self-grooming usually occurs under optimal conditions when the mice feels comfortable with the surrounding. Measuring self-grooming during the open field test makes the data flawed because external factors are being introduced, such as the lighting being adjusted to a high level 200 lux and the mice being exposed to an open area. Hence, there may be a possibility that the mice is much more concentrated with natural avoidance rather than performing self-grooming. A preceding Nrxn1a removal study showed that under optimal conditions, twice the grooming can be observed in comparison to control mice – in that case, it was red light set at 40 lux in a vacant cage with clean bedding11. In addition, a direct testing on mice social callings would have given researchers some insight into genetics mutations that can cause verbal deficits in humans, a hallmark for autism. In prior research done with SHANK -/- isolated mice pups, the amount of “calling” for the mother was certainly less in comparison to the WT conspecifics.

For future experiments, if the Nrxn3 gene were to be explored, ultrasound vocalizations should be recorded and examined.

What was also unclear within this experiment was what kind of role did Munc 18-1 play in contributing to the autism-like happenings, if any. Deletion of Nrxn2a KO mice did manifest into autistic-like performance, but it is uncertain whether this effect is direct or indirect. Such abnormal behaviors could have risen from the depletion of the Nrxn2 gene itself, or from the indirect reduction of Munc18-1.

Future Directions

To properly measure repetition in behavior, any sort of separate testing will be fine, as long as it is not combined with another behavioral measure.

An example would be placing mice would in a vacant cage with clean bedding and soft lighting for half an hour. Inside the cage will be a large cotton fibre ball.

The amount of cotton remaining intact depicts the level of repetitive, compulsive behavior in the mice.

For future experiments, if the Nrxn3 gene were to be explored, ultrasound vocalizations should be recorded and examined. A proposed approach would be to remove target mice from mother at day eight, as researchers should aim to recapitulate the early onset of social impairments seen with toddlers or young children. Detached young mice will be placed in a vacant cage with clean bedding for about a half hour, with a recording microphone placed above the cage to track noises made. It is expected that the mutated mice will have reduced calling due to insensitivity of social expression.

To solve this hippocampal Munc18-1 dilemma, researchers can do a similar study as Dachtler and his team, but instead truncate the Stbxp1 gene without deleting the Nrxn2a gene. Deletion of the Stbxp1 gene should not occur because that will result in a “silent” mouse with zero neurotransmitter release and termination to exocytosis14. If the same autisticassociated behaviors can be observed, then that signifies that Munc18-1 is the crucial protein at hand and not Nrxn2a. Nrxn2a would just then serve as one of the ways to affect Munc18-1 levels, but it is really Munc18-1 that encourages the abnormal behavior.

One recent review discussed the efficacy for mice models to improve autism research. 90 million years have passed since rodents and humans have had a mutual ancestor, and so the idea that of having a true mouse model of autism seems impossible15. Steven Hyman, the author, brought two key issues to light.

The translation from genotypes to phenotypes due to crucial mutations may not be accurate using mouse lines because behavioral testing between the animal and humans differ greatly15. This raises the second concern: How do researchers know for a fact that the mouse molecular targets serve the same purposes as they do in humans; are there cases of evolutionary maintenance? It is important to consider the relevancy, because drugs that target molecular pathways of the mice may not be as effective in humans due to evolutionary changes. Ongoing research on autism have proposed many possible genes related to autism and with the new technical advancements, our future does look promising as long as every step is a careful and evaluative one.

9. Dachtler J, Glasper J, Cohen RN, Iovrra J L, Swiffen D J, Jackson A J et al. Deletion of a-neurexin 2 results in autism-related behaviours in mice. Translational Psychiatry. 2014; 4: 484; doi:10.1038/tp.2014.123 10. Blundell, J., Tabuchi, K., Bollinger, M., Brose, N., Liu, X., Sudhof C. and Powell C. Increased Anxiety-like Behaviour in Mice Lacking the Inhibitory Synapse Cell Adhesion Molcule Neuroligin 2. Genes Brains Behaviour. 2009; 8(1): 114-126; doi: 10.1111/j.1601183X.2008.00455.x 11. Etherton, M., Blaiss, C., Powell, C. and Sudof, T. Mouse Neurexin1a Deletion Causes Correlated Electrophsyiological and Behavioural Changes Consisten with Cognitive Impairment. 2009 Proc. Natl. Acad.

Sci. USA 106, 17998–18003; doi: 10.1073/pnas .0910297106.

12. Wohr M, Roullet F, Hung A, Sheng M and Crawley J. Communication Impairments in Mice Lacking Shank1: Reduced Levels of Ultrasonic Vocalizations and Scent Marking Behaviour. PLoS One. 2011;6(6): doi: 10.1371/journal.pone.0020631 13. Hamdan F, Gautheir J, Dobrzeniecka S, Lortie A, Mottron L, Vanasse M et al. Intellectual disability without epilepsy associated with STXBP1 disruption. European Journal of Human Genetics. 2011; 19: 607-609: doi:10.1038/ejhg.2010.183; 14. Toonen, R. Role of Munc18-1 in Synaptic Vesicle and Large DenseCore Vesicle Secretion. Biochem. Soc. Trans. 2003; 31: 848-850 15. Hyman, S. How Far Can Mice Carry Autism Research? Journal Cell Science. 2014; 158: 13-14 doi:10.1016/j.cell.2014.06.032

References 1. Miles, J.H. Autism spectrum disorder-A genetics review. Genetics in Medicine. 2011; 13: 278-294 2. Rothwell, P., Fuccillo, M., Mexeiner, S., Hayton, S., Gockce, O., Lim, B.K., Fowler, S., Malenka, R. and Sudhof, T. Autism-Associated Neuroligin 3 Mutations Commonly Impair Striatal Circuits to Boost Repetitive Behaviours. Journal of Cell Science. 2014; 158: 198-212; doi:10.1016/j.cell.2014.04.045 3. Chen, J., Yu, S. Fu, Y., and Li X. Synaptic Proteins and Receptor Defects in Autism Spectrum Disorders. Front Cell Neurosci. 2014; 8:276; doi: 10.3389/fncel.2014.00276 4. Greco B., Manago, F., Tucci, V., Kao, H.T., Valtorta, F. and Benfenati.

Behaviour Brain Resources. Autism Related Behaviour Abnormalities in Synapsin Knockout Mice. 2013; 251(100): 65-74; doi: 10.1016/j. bbr.2012.12.015 5. Sheng, M. and Kim, E. The Shank Family of Scaffold Proteins.

Journal of Cell Science. 2000; 113: 1851-1856 6. Silverman, J., Turner, S.M., Barkan, C.L., Tolu, S.S., Saxena, R., Hung, A., Sheng, M. and Crawley, J. Sociability and Motor Functions in Shank1 Mutant Mice. Behaviour Brain Resources. 2011. 1380; 120-137. doi: 10.1016/j.brainres.2010.09.026 7. Wang, X., McCoy, P., Rodriguez M., Shawn J., Roberts, A., Colvin, J., Rousquet-Moore, D., Weinberg, R., Philpot, B.D., Beaudet, A.L. Wetsel, W.C. and Jiang, Y.H. Synaptic Dysfunction and Abnormal Behaviours in Mice Lacking Major Isoforms of Shank 3. Human Molecular Genetics.

2011; 20(15): 3093-3108; doi: 10.1093/hmg/ddr212 8. Grayton, H.M., Missler, M., Collier, D.A. and Fernandes, C. Altered Social Behaviours in Neurexin1a Knockout Mice Resemble core Symptoms in Neurodevelopmental Disorders. PloSOne. 2013; 28(8): 86-92; doi: 10.1371/journal.pone.0067114.

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Effects of systems consolidation, optogenetic inhibition, and adult neurogenesis in hippocampal memory traces

Yi Xuan Li

Distinct memories are thought to be encoded in sparse neural networks called memory traces. A transgenic mouse line is created to distinctively label neural activity during encoding and retrieval of contextual fear conditioning (CFC) memories in the dentate gyrus (DG) and CA3. Greater freezing and signal overlap was observed in mice re-exposed to the fear inducing context as opposed to a novel context. Signal overlap decreased over time while extent of freezing remained the same in re-exposure mice. This provided physical evidence for systems consolidation. Optogenetic inhibition of neurons recruited during CFC reduced extent of freezing during re-exposure. Based on this result the authors believe these neurons form the memory trace of the fear memory. An unaddressed alternative explanation is that the neurons do not encode the memory, but merely serve as a pointer to the true memory trace downstream.

Optogenetic activation of downstream neocortical neurons in an unrelated context is recommended for future experiments. Ablation of adult neurogenesis in DG reduced extent of freezing and signal overlap in CA3 but surprisingly not DG. The result may relate to the hypothesis that adult born neurons (ABN) are modulators of CA3 activity. The role of ABN in CFC is still poorly understood and a gain of function test on neurogenesis is recommended for future studies.

Key words: memory trace; dentate gyrus; transgenic mice; CA3; contextual fear conditioning; optogenetic inhibition; adult neurogenesis; social defeat; neuroscience Background Memories are believed to be physically stored in defined, sparse networks of brain neurons called memory traces(1). Due to the sparse distribution these neurons have been difficult to locate and study(2).

Recently studies on lateral amygdala (LA) neurons have observed defined neuron ensembles that activate during both encoding and retrieval of distinct fear conditioning memories(3). Furthermore, memory expression is eliminated when the neurons are selectively ablated or inhibited, suggesting that these neurons form an essential part of the memory trace. The hippocampus (HPC) has since been identified as another structure ideal for identifying and studying memory traces(4).

HPC is crucially involved in the formation and consolidation of declarative memories(5). Aside from LA, HPC also plays a critical role in fear conditioning by facilitating the association of the fear to the related context(6).

The two HPC subfields analyzed for this study, DG and CA3, are involved in pattern separation, which is the disambiguation of sensory inputs about similar contexts into different representations downstream(7)(8).

Immediate early genes (IEG) Arc and c-fos were used as markers for neuron activation due to their rapid and transient expression following input-specific activation(9). A transgenic mouse line was designed to induce a permanent labeling with enhanced yellow fluorescent protein (EYFP) in neurons that expressed IEG in the presence of tamoxifen (TAM). Mice were subjected to CFC training shortly after TAM injection to tag neurons activated during encoding with EYFP. This allows the encoding signal to be distinguished from and merged with immunohistochemical signals of Arc or c-fos that represent the retrieval signal(10). Retrieval of declarative memories gradually becomes HPC independent over time, so in order to observe changes in hippocampal memory trace over the long term, retrieval test is delayed for a subset of mice(11).

To to selectively inhibit neurons activated during

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encoding, the same transgenic model was used but the position of EYFP was replaced by the optogenetic inhibitor Archaerhodopsin-3 (Arch). This allowed convenient alternation between inhibited/uninhibited states by switching on/off a photostimulator and enabling real-time observations of changes in memory expression(10).

The subgranular zone (SGZ) of DG is among the few brain regions where neurogenesis continues into adulthood. ABN are incorporated both anatomically and functionally into the the hippocampal circuit (2a).

Previous studies assessing the role of ABN in CFC have demonstrated that mice with arrested adult neurogenesis have decreased response and colabeled DG signals in one-shock CFC, but not three-shock CFC(12). Although not the site of neurogenesis, signals in CA3 appear to be affected in the same way as DG(13). This study examines both DG and CA3 in the same experiment to compare the role of ABN in both regions.

Research Overview

Effect of Context and Time on Activation Patterns and Memory Expression

The transgenic mouse line ArcCreERT2 x R26RSTOP-floxed-enhanced yellow fluorescent protein (EYFP) was created to enable separate signaling of activation during CFC encoding and retrieval. Arc was co-expressed with a Cre recombinase â&#x20AC;&#x201C; estrogen receptor fusion protein (Cre-ER). Upon binding to TAM, an ER agonist, Cre-ER would localize to the nucleus and interact with loxP to delete the STOP codon upstream of EYFP (Figure 1A). In CA3, c-fos was used instead of Arc as the neuron activation marker, due to Arc labeling in CA3 being mostly dendritic(10).

Mice were injected with TAM and subjected to CFC in Context A 5 hours later. After 5 (recent) or 30 days (remote), mice were either re-exposed to Context A or introduced to a novel Context B. Extent of freezing was

Figure 1. (A), Schematic representation of the two transgenes.

The upward kink flanked by loxP is a STOP codon. (B), Schematic view of a dorsal HPC coronal slice under a confocal microscope, showing encoding signals (EYFP+) in green, retrieval signals (Arc+/c-fos+) in red, and co-labeled signals in yellow

recorded, and coronal slices of dorsal HPC were obtained from mice shortly after and put under a confocal microscope (Figure 1B). The amount of EYFP+ and Arc+/c-fox+ signals was counted and signal overlap calculated.

Therefore, ArcCreERT2 x R26R-CAG-STOP-floxedAch-3–GFP line was created. The same transgenic system as outlined in Figure 1A was used but the position of EYFP was replaced by Arch. Mice were surgically implanted with fibre optics above either DG or CA3 at 8-12 weeks of age.

Mice underwent CFC in Context A 5 hours after TAM injection to induce Arch-GFP expression in activated cells. Mice were then re-exposed to Context A two weeks later, where photostimulation at 593.5 nm was turned on for the first 3 minutes to elicit strong hyperpolarization in Arch-GFP+ neurons, and turned off during the remaining 3 minutes. Two days later mice were introduced to Context B with the same light epochs. Control mice that lacked the ArchCreER transgene underwent the same paradigm.

To address the alternative explanation that inhibition of any group of neurons may disrupt function of the region as a whole and impair memory expression, the experiment was repeated on another group of mice with the exception of undergoing CFC training in a distinct Context C as opposed to Context A. In mice that underwent conditioning in Context A, optogenetic inhibition of DG and CA3 both reduced the extent of freezing as compared to control when re-exposed to context A (p = 0.02). When lights

Figure 2. Number of EYFP+ signals is shown in (C), (F), (I), (L), Arc+/c-fos+ in (D), (G), (J), (M), and signal overlap as % co-labeled neurons in (E), (H), (K), (N). White bar represents re-exposure to Context A, black represents novel Context B.

Among the recent retrieval cohort, significantly more freezing were observed in those placed in Context A (P < 0.001). In the DG and CA3, while EYFP+ and Arc+/c-fos+ signal was similar, the signal overlap was much higher in Context A (Figure 2E, H). These results demonstrated high input specificity of DG and CA3 neurons, supporting their role in pattern separation(7).

In contrast to the recent cohort, similar levels of freezing and signal overlap were observed in the remote cohort (Figure 2K, N). This indicates a generalization of similar inputs over time(11). Among mice re-exposed to Context A, signal overlap was significantly lower in the remote cohort for both CA3 and DG in mice from Context A (p<1), while freezing level and by implication, memory retrieval remained strong. This provides the physical evidence for systems consolidation, the process which memories become HPC-independent and encoded in more permanent areas in the cortex(11).

Effect of Optogenetic Inhibition on Memory Expression

To truly determine if the tagged neurons are part of the memory trace, selective inhibition is needed to see if these neurons are necessary for memory retrieval.

Figure 3. Extent of freezing is shown with control mice in Context A set as 100%. Mice that underwent CFC training in Context A is shown in (F – H) and Context C in (K – N). The lighter green bar represents control mice.

were turned off at minute four, freezing immediately resumed in ArcCreER+ mice to the same level as control (Figure 3F, H). In contrast, in mice that underwent conditioning in Context C, optogenetic inhibition failed to reduce extent of freezing (Figure 3K, M). The results from Context B were not significant (10. Since memory expression remained intact when neurons recruited for unrelated memories were blocked, the alternative explanation could be rejected. Based on these results, the the authors believe these neurons form the memory trace of the fear memory(10).

Effect of Adult Neurogenesis Ablation on Activation Patterns

ArcCreERT2 x R26R-STOP-floxed-EYFP mice were irradiated with x-ray to ablate ABN six weeks prior to CFC. X-ray irradiated mice and sham mice then underwent the same CFC paradigm as the recent cohort 164

described earlier, with the exception of three shocks administered during encoding phase of 3-Shock CFC.

Figure 4. Number of EYFP+ signals is shown in (E), (H), (K), (N), Arc+/c-fos+ in (F), (I), (L), (O), and signal overlap in (G), (J), (M), (P). The lighter red bar represents sham mice.

Significant reduction in freezing (p < 0.05) was observed in x-ray irradiated mice from 1-Shock CFC and was in accordance with previous studies. The unexpected result was the significantly lower signal overlap compared to sham x-irradiated mice in CA3 (p < 0.01) but not DG (Figure 4G, J). While the result was surprising, it might relate to the recent hypothesis that ABN function as a modulator of CA3 neurons(14).

If ABN did not modulate DG, then the lack of signal overlap difference was unsurprising since ABN only made up 5 – 10% of total granule cell populations(10) No significant difference in freezing and CA3 signal overlap was observed in 3-Shock CFC. This indicates that stronger encoding can rescue fear conditioning, in accordance with previous studies (12).

Future Directions The role of ABN in CFC remains poorly understood after this study mice. Instead of ablation, a gain of function experiment can be conducted to see if enhanced ability to distinguish similar contexts results. The same transgenic system is used but with loxP flanking the pro-apoptotic gene Bax(15). Increase in adult neurogenesis can be monitored with immunostaining for Dcx, a marker for ABN. While memory expression deficits from optogenetic inhibition have demonstrated the essentiality of recruited neurons in memory retrieval, a further step can be taken to test if activation of these neurons is sufficient to elicit the memory in the absence of environmental cues. Optogenetic activators such as ChEF can be expressed in place of Arch using the same transgenic machinery. Subject mice to CFC in a particular context, then directly activate the tagged neuron populations when mice are in an unrelated context to test if memory response can be elicited(4).

An alternative explanation for the optogenetic results is that the neurons do not encode the memory itself.

Instead the neurons merely serve as a gateway for the true memory trace downstream. This can be tested via optogenetic activation of neocortical areas directly downstream of HPC. If direct activation elicits the full memory response, it indicates that the hippocampal neurons are not part of the memory trace(16).

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Critical Analysis The experimenters designed a very flexible transgenic system consisting of just two transgenes. Expression of different genes could occupy the same position in the system and be expressed in the same manner.

TAM as a regulator was flexible temporally.

The experiment cleverly addressed in an alternative explanation for optogenetic inhibition results that silencing any group of hippocampal neurons would inhibit the specific memory. However, it did not address another alternative explanation that hippocampal neurons might be pointers to memory trace instead of being part of the trace.

References 1. Josselyn SA (2010). Continuing the search for the engram: examining the mechanism of fear memories. J Psychiatry Neurosci 35(4): 221–228.

2. Han JH, Kushner SA, Yiu AP, Hsiang HL, Buch T, Waisman A, Bontempi B, Neve RL, Frankland PW, Josselyn SA (2009). Selective Erasure of a Fear Memory. Science 323 (5920): 1492-1496.

3. Reijmers LG, Perkins BL, Matsuo N, Mayford M (2007). Localization of a Stable Neural Correlate of Associative Memory. Science 317 (5842): 1230-1233.

4. Liu X, Ramirez S, Pang PT, Puryear CB, Govindarajan A, Deisseroth K, Tonegawa S. (2012). Optogenetic stimulation of a hippocampal engram activates fear memory recall. Nature 484(7394):381-5. doi: 10.1038 5. Eichenbaum H. (2000). A cortical–hippocampal system for declarative memory. Nat Rev Neurosci 1(1):41-50.

6. Phillips RG, LeDoux JE. (1992). Differential Contribution of Amygdala and Hippocampus to Cued and Contextual Fear Conditioning. Behav Neurosci 106(2):274-85.

7. Bakker A, Kirwan CB, Miller M, Stark CE (2008). Pattern separation in the human hippocampal CA3 and dentate gyrus. Science.

319(5870):1640-2. doi: 10.1126 8. McHugh TJ, Jones MW, Quinn JJ, Balthasar N, Coppari R, Elmquist JK, Lowell BB, Fanselow MS, Wilson MA, Tonegawa S. (2007).

Dentate Gyrus NMDA Receptors Mediate Rapid Pattern Separation in the Hippocampal Network. Science 317 (5834): 94-99.

9. Kubik S, Miyashita T, Guzowski JF. (2007). Using immediate-early genes to map hippocampal subregional functions. Learn Mem 14(11): 758-70 10. Denny CA, Kheirbek MA, Alba EL, Tanaka KF, Brachman RA, Laughman KB, Tomm NK, Turi GF, Losonczy A, Hen R. (2014).

Hippocampal memory traces are differentially modulated by experience, time, and adult neurogenesis. Neuron 83(1): 189-201.

11. Goshen I, Brodsky M, Prakash R, Wallace J, Gradinaru V, Ramakrishnan C, Deisseroth K. (2011). Dynamics of retrieval strategies for remote memories. Cell 147(3): 678-89.

12. Drew MR, Denny CA, Hen R. (2010). Arrest of adult hippocampal neurogenesis in mice impairs single- but not multiple-trial contextual fear conditioning. Behav Neurosci. 124(4): 446-54.

13. Niibori Y, Yu TS, Epp JR, Akers KG, Josselyn SA, Frankland PW. (2012). Suppression of adult neurogenesis impairs population coding of similar contexts in hippocampal CA3 region. Nat Commun 3:1253 14. Sahay A, Wilson DA, Hen R. (2011). Pattern separation: a common function for new neurons in hippocampus and olfactory bulb. Neuron 26;70(4):582-8. doi: 10.1016 15. Hill AS, Sahay A, Hen R. (2015). Increasing Adult Hippocampal Neurogenesis is Sufficient to Reduce Anxiety and Depression-Like Behaviors. Neuropsychopharmacology. doi: 10.1038 16. Cowansage KK, Shuman T, Dillingham BC, Chang A, Golshani P, Mayford M. (2014). Direct reactivation of a coherent neocortical memory of context. Neuron 84(2):432-41.

Adult hippocampal neurogenesis and its role in Alzheimer’s disease in transgenic mice models

Ziteng Li

The hippocampus is a critical brain structure involved in learning and memory and is particularly susceptible to damage during the early stages of Alzheimer’s disease (AD). There is a growing body of evidence that suggests impaired adult hippocampal neurogenesis in diseased AD patients is a major underlying factor contributing to the cognitive decline and memory impairments in affected individuals. Conversely, studies have also shown that enhanced neurogenesis contributes to memory rescue. Several key AD associated molecules such as APP, AopE, and PS1 and variants have been identified in the positive and negative regulation of these new neuronal cells while many social and lifestyle factors have been implied in increased endogenous neurogenesis. Here, in this review, we will summarize the current research on the neurogenic roles of molecules that cause adult hippocampal neurogenesis decline in AD, conditions that stimulate endogenous neurogenesis as well as the potential application of these new neurons in the treatment and diagnosis of AD. Key words: Adult neurogenesis, Alzheimer’s Disease, mouse models, memory decline, hippocampus, APP, AopE, PS1 Introduction First described by German physician Dr Alois Alzhimer in 1906, Alzhimer’s disease (AD) is an age related debilitating neurodegenerative disease that is characterized by progressive dementia throughout the affected persons life (Goedert M, Spillantini MG, 2006). At present, the mechanisms and the neuro-circuits underlying the disease is largely unknown and there is no one definitive reason for its onset. However, it has is become increasingly clear that it develops due to a complex series of events and is likely due to a combination of environmental, genetic and lifestyle factors life (Goedert M, Spillantini MG, 2006). Therefore reason of onset and the risk of developing AD will vary from person to person. Despite this, the majority of patients who do develop Alzheimer’s will do so later in life in which the apolipoprotein E (apoE) genotype is the greatest risk factor (Nichol K et al., 2009). The rare early-onset familial form of AD (FAD) represents less then 5% of AD patients and is cause by mutations in genes encoding presenilin-1 (PS1), presenilin-2 (PS2), and amyloid precursor protein (APP) (Wen PH et al.,2004) Individuals with the disorder usually suffer from severe cognitive decline, memory loss, attention deficits and changes in mood and personality (Snyder JS et al., 2001). The gradual accumulation of extracelluar β-amyloid (Aβ) plaques and intracellular neurofibrillary tangles (NFTs) along with massive neuronal death are the neuropathological hallmarks of AD (Gadadhar A et al., 2011). As the disease progresses, neurofibrillary tangles and amyloid plaques spread throughout the brain and in the late stages of AD, all patients have significantly shrunken brains due to the gross atrophy of neurons. These pathologies are evident in specific areas of the brain including the cerebral cortex, ventricles and the hippocampus (Caille I et al. 2004).

In particular, the hippocampus is one of the first brain structures to be affected by AD pathology (Goedert M, Spillantini MG, 2006). The hippocampus is located in the medial temporal lobe of the brain and is known to have trisynaptic circuitry (Fig 1) (Stone SS et al.,

2011). Briefly, the information from the entorhinal cortex (EC) is received by the dente gyrus (DG) a structure found within the hippocampus. The information flow then proceeds from DG to CA3 to CA1 and finally to the subicululm, which sends the information, back into the deep layers of the EC (Stone SS et al., 2011). It has been suggested that the hippocampus is involved in learning and memory as well as long term potentiation (LTP) (Wen PH et al.,2004). The discovery of neurons being produce de novo in the DG of the adult hippocampus has suggested a new from of neural plasticity that can be involved in memory processes (Van Praag H et al., 2002). Currently there is a growing body of evidence that suggests that adult hippocampal neurogenesis promotes improved spatial and episodic memory while a decline in adult hippocampal neurogenesis maybe underlying the cognitive impairments associated with neurodegenerative diseases such as AD (Van Praag H et al., 2002) In this review we will summarize the current research on the neurogenic roles of molecules that cause adult hippocampal neurogenesis decline in AD, conditions that stimulate endogenous neurogenesis as well as the potential application of these new neurons in the treatment and diagnosis of AD. Results Molecules affecting Neurogenesis in the Hippocampus Adult neurogenesis has been established to occur consistently in two regions of the adult brain; the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus (DG) in the hippocampus (Hsiao et al. 2014). In the SGZ, new cells are differentiated into glial cells and neurons (Hsiao et al.

2014). The de novo neurons are is what is incorporated in to the granule cell layer of the dentate gyrus (Hsiao et al. 2014) Fig 2. Various studies have shown that these newborn neurons contribute to hippocampal dependent memories and learning demonstrated in tasks such as trace eyeblink conditioning and spatial tests (Shors et al., 166

Fig 1. Neural circuitry and network in a mouse hippocampus (Deng W et al 2010). a. Illustration of the trisynaptic circuitry in the hippocampus b. network and information of the trisynaptic pathway from the entorhinal cortex (EC) to the dente gyrus to CA3 then to CA1 Schaffer collaterals which ultimately project information back into the EC

2001). Ablation of neurogenesis have shown to impair contextual fear conditioning, but conflicting reports have also been made citing that ablation has no effect on the Morris water maze nor contextual fear conditioning (Shors et al., 2002; Snyder et al., 2005).

In more recent years, many molecules that are central to AD have been found to have regulatory roles in adult neurogenesis. In P117L familial AD (FAD) mice model, PS1 mutants impaired de novo neuron production in the adult hippocampus by decreasing neural progenitor survival (Wen PH et al.,2004). In the PS1M146V knockin mice, PS1 mutations exhibited impaired hippocampus dependent learning measured by contextual fear conditioning—which correlated with decreased adult neurogenesis in the hippocampus (Ghosal K et al., 2010). PDAPP mutants with APP mutations also demonstrated decrease in neurogenesis in the SGZ and the soluble form of APP (sAPP) has a positive regulatory role in the proliferation of progenitor cells in the adult SGZ (Donovan M et al., 2006; Caille I et al. 2004). Lastly, a knockout mutant of ApoE and a knockin mutant of ApoE4 also demonstrated reduced levels of neurogenesis (Li G et al., 2009).

These studies using various transgenic mice models of AD have generated a surmounting body of evidence that supports the idea that adult hippocampal neurogenesis plays a role in the cognitive dysfunction of AD. We have also identified key AD-associated molecules that play a regulatory role in the synthesis of these new neurons including APP, AopE, and PS1 and their variants.

Social Interaction, Exercise and Environmental Enrichment Although at present, there is no definitive evidence to support any one treatment in delaying the progres167

sion of AD. Maintaining strong social contacts, healthy lifestyle and being mentally active as one ages has been strongly implicated to decrease risk of cognitive decline and onset of the disease. Recent studies have found that social interaction, environment enrichment (eg. improved standard of living), as well as exercise can rescue deficits of AD by promoting neurogenesis.

Hsiao et al. (2014) found in the APP/PS1 animal mouse model, social interactions between mutant mice and wild type mice rescued memory deficits and decreased the progression of AD in mutant mice. The researchers found increased BDNF-mRNA and protein production after cohousing, which lead to neurogenesis and ultimately rescue of memory in mutant mice. They also found that overexpression of BDNF mimicked memory-improving effect while genetic knockdown and chemically blocking cell proliferation blocked this memory improvement. These findings provide evidence that social interactions improve memory and cognition in the mouse model of AD via BNDF expression and associated neurogenesis in the hippocampus. Alternatively, Hsiao et al. (2011) also looked into what would happen in the absence of social interaction or rather what would occur if the APP/PS1 mice were placed in social isolation. They found elevated levels of hippocampal Aβ detected by increasing β-and γ-secretase activities which contributes directly to the pathogenesis of AD including neurodegeneration. The researchers also found that isolated mice had lower levels of LTP in hippocampal CA1 neurons, which exacerbated the already present memory deficits. Barrientos et al. (2003) also found that social isolation following memory and cognitive impairment significantly decreased BDNF-mRNA in the dentate gyrus and the CA3 region of the hippocampus. These findings suggest that social isolation may accelerate the progression of AD in diseased individuals while social interactions may rescue the impairments via neurogenesis.

Physical exercise such as wheel running in mice models have been found to improve cognition and hippocampal plasticity (Nichol K et al., 2007). In one study, ApoE mutant mice that previously exhibited deficits in cognition on the radial arm water maze test (RAWM) (a hippocampal dependent spatial memory task) showed significant improvement in the tasks as well has increased BDNF levels after 6 weeks of wheel running (Nichol K et al., 2009). Similarity, the aged Tg2576 AD mice model also demonstrated that exercise can improve cognitive performance and promote neurogenesis even after the development of AD pathology (Nichol K et al., 2007).

Enriched environments (EE) have also been found to be positive regulators for adult neuronal hippocampal neurogenesis and have been shown to increase improve cognitive performance, decrease Aβ levels an also increase hippocampal LTP in APPswe/PS1DE9 mice (Hu Y et al., 2010). Exercise along with EE has also been shown to improve water maze performance and also increase newborn granule cells in the DG of APP23 mice (Mirochnic S et al. 2009). However there are contradicting studies that have shown that EE does not enhance neurogenesis in PS1 knock out mice or FAD-linked PS1 variants (Feng R et al.

2001). In a more recent study, EE has been even shown to suppress neurogenesis in ApoE4 mice (Levi O, Michaelson DM, 2007). These results imply that EE has various effects on different mice models of AD.

Fig 2. Adult hippocampal neurogenesis in the SGZ (Mu Y, Gage F 2011). Type 1 and Type 2 neural stem cells generate astrocytes and neuroblasts. The neuroblasts will migrate to the granule cell layer of the dentate gyrus where it will become dentate granuale cells.

These new cells will then form extensive dendritic trees that receive information of the entorhinal cortex and project to CA3 neurons.

Conclusion and Discussion In summary, we have concluded by using various mutant mouse model of AD, that known AD-associated molecules such APP, AopE, and PS1 also play a important role in the regulation of hippocampal neurogenesis in the adult mice brain. These finding further support the ongoing hypothesis that a decline in neurogenesis in SZG of adults play a crucial role in the onset and progression of early and late-onset Alzheimer’s disease. Notably, alteration of these new neurons occurs in the very early stages of AD even prior to neuronal atrophy, amyloid deposition, and tau tangles (Goedert M, Spillantini MG, 2006). This suggests that neurogenesis is one of the key elements in the disease pathology of AD. Currently, although there is no conclusive drug or therapeutic treatment of AD, there have been strides in research to prevent its early onset. In particular, it has been found that there are extrinsic conditions that can facilitate endogenous neurogenesis in the hippocampus including; maintaining strong social interactions, physical exercise as well as being in an enriched environment as we age. Although the number newly synthesized neurons in the SZG are pale in comparison to the number degenerating neurons, we have found promising results in the rescuing of memory impairments from these de novo cells (Hsiao et al. 2014; Shors et al., 2001). While it is very unlikely that neurogenesis will provide global repair to the deficits caused by AD, it is very plausible that it can aid in slowing down the progression of the disease. Lastly due to its very early onset, declining neurogenesis may in the future act as a neuro-biologal marker for the diagnosis as well as facilitate understanding the underlying neuromechanisms of the disease.

Critical Analysis and Future Directions Although there have been many comprehensive studies in this field of research that support hypothesis that neurogenesis is occurring and is an underlying factor in AD. There are also many gaps and inconsistencies in the literature that needs to be addressed.

Firstly, although the majority of studies have found an increased number of new neuronal cells synthesized, there are many opposing articles that have reported the opposite. This disjuncture maybe explained by the different AD mutant mice models used in the respective studies. Since different mutations and promoters of various transgenic mice lines express different neuronal cell populations of the transgenes, they are likely to express distinct levels AD-related proteins.

Therefore, it is very hard to compare the results from two different transgenic lines. As a result, a standard or systematic comparison of transgenic mice with consistent gender, age, genetic background, progression in AD and neurogenic analysis should be conducted so conclusive results can be drawn.

Secondly, in the current mice models of AD, most transgenic lines only exhibit one or partial pathology of AD. This is unrealistic portrayal of the disease, since combined pathologies may interact and yield widely different results from a single or partial diseased state.

Therefore the discovery of a new strain of transgenic mice that accurately reflect all AD pathologies can be a future initiative in this field of research. Lastly it is hard to generalize mice models to humans due to the intrinsic physiological and genetic differences and much of the research reviewed in this paper have never been tested in human trials. Therefore, to accurately assess the applications of this research to the human AD, human trials and post-mortem examinations should be conducted on willing diseased individuals.

Some possible future directions in this field of research include a drug therapy that specifically targets and promote adult neurogenesis. Recently, allopreganolone has been cited as a neurosteroid that can aid neurogenesis in the brain (Brinton RD et al., 2006). However promising, this compound has not been pursued for clinical use due to its short half-life (Brinton RD et al., 2006). Current active drug therapies mask the symptoms of AD rather then treat the underlying disease by stopping its progression. Therefore an effective therapy for AD is still unavailable for diseased patients. However the current research and existing animal models that were reviewed in this paper may provide a basis and better insight into the prevention, treatment and management of cognitive decline such as Alzheimer’s disease associated dementia.

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Improved cognitive function through the elucidation of alcoholically induced changes in the brain

Bernie Longange

Alcohol has the ability to affect both the body and the brain in a variety of ways. In recent years studies have found that moderate levels of alcohol may have positive effects on cognition. Susceptibility to these effects may be related to the period of mental development, gender, drinking history, as well as many other unforeseen factors. At the moment the proper levels of alcohol consumption for cognitive improvement are being determined, and eventually the pathways that cause changes in mental functioning may be elucidated.

Key words: alcohol, consumption, moderate, pathways, nondrinkers and cognitive function Background Cognitive function serves not only as a reflection into the way the mind works and how intelligence is measured or attained, but also a glimpse into the future of how the brain will develop1. Studies have been conducted in attempts to improve cognitive functionality, but few definitive answers have been obtained2. Even previously rigid observations are being called into question with new discoveries in the field. For years, alcohol consumption was believed to be the cause of reduced mental ability due to both short term cognitive impairments and damage caused to the brain in the long run3.Studies had shown that increased alcohol consumption had an inverse relationship with increases in cognitive function.

The more alcohol you would drink the less intelligent you would become4. Drinking alcohol increased the risk of cognitive impairment5 and dementia6. Further evidence shows that drinking increases the risk of cardiovascular diseases, and cardiovascular diseases are in turn related with “cognitive aging” or decreases in mental ability7.

Although being considered fact for many years, this observation has been under scrutiny as of late, as more recent studies have been showing contrary results.

People who drink moderately appear to exhibit increased cognition8. This does not extend to those who are considered problem drinkers, as too much alcohol destroys the brain9. This shift has changed the focus of studies relating alcohol consumption to cognition. Research has been considering what levels of alcohol consumption resulted in the highest increases in mental ability10. Studies have continued to attempt to explain why and how alcohol improves cognitive function at certain levels. This relates to the many experiments being conducted in understanding brain function in relation to moderate doses of different drugs, as similar research is also at the forefront of studies in methamphetamine usage and dosage11. The changes the brain undergoes after consumption are the main targets in studies of this nature today.

The paper by Pia Horvat et al. “Alcohol consumption, drinking patterns, and cognitive function in older Eastern European adults” looked at the association between quantity, frequency and changes in alcohol consumption and changes in mental ability. This is an in depth look at the correlation between cognitive function and alcohol intake than has been taken in the past and is the one of the first steps in discovering the pathways in which cognitive improvements can be made.

Research Overview

Summary of Major Results

Moderate consumption of alcohol accompanied increased cognitive function in the studied individuals.

Approximately 28,947 men and women between the ages of 45 – 69 were randomly selected from regions of Eastern Europeans where alcohol is one of the leading causes of illnesses and premature death.

Individuals were first categorized by levels of selfreported alcohol consumption. There were 4 levels of intake that ranged from nondrinkers (0 g/d) to heavy drinkers (≥20/40 g/d), and a separate category for binge drinking, which was defined as consumption of ≥60/100 g of alcohol in one session, at least once a month. Four cognitive tests were conducted by trained nurses. These tests included word recall tests (immediate and delayed recall), a verbal fluency test, and a letter cancellation test. These examinations looked at verbal memory, learning, verbal fluency, attention, mental speed and concentration. After 1-6 years had passed participants were reexamined for cognitive function, and had to report any changes in their alcohol intake. Here individuals were placed into 6 categories; stable nondrinkers, ex-drinkers, reduced drinkers, increased drinkers, and people who abstained during the first test but had started drinking.

Cognitive function in men Nondrinkers were found to have lower cognitive scores that moderate drinkers, but after adjusting for socioeconomic and lifestyle confounds, these results were not significant. An exception was found when the cognitive scores of nondrinkers were cross-sectionally compared to the reference group (the lowest level of drinking) which showed significant results.

In the follow-up, participants who had stopped drinking had “significantly lower cognitive scores than stable drinkers. This effect was observed especially in men.

Verbal performance was shown to be significantly lower for those who had stably abstained from drinking.

Cognitive function in women Light drinkers scored consistently higher than nondrinkers. Moderate drinking correlated with better performance in comparison to lower levels of alcohol consumption. Drinking a few times a month seemed 170

1

2

3

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Figure 1. Male cognitive test scores for immediate word recall. This tested for verbal memory and learning.

Figure 2. Male cognitive test scores for delayed word recall. This tested for verbal memory and learning.

Figure 3. Male cognitive test scores for verbal fluency recall. This tested for verbal fluency.

Figure 4. Male cognitive test scores for letter fluency. This tested for attention, metal speed nd concentration.

to have a positive effect on the cognitive test scores.

In the follow-up, participants who had stopped drinking had “significantly lower cognitive scores than stable drinkers. Women who had started drinking as well as consistent nondrinkers had lower cognitive scores than they had during the first examination.

Discussion

The effects of drugs on humans are never as straight forward as they appear. There always seems to be a negative side effect to drugs that are deemed to be “good”, but as of late there is increasing evidence of “bad” drugs having positive side effects. Alcohol research in particular has taken a large turn in the last decade. The focus has shifted from all the negative effects alcohol has on the body, to the potential positive effects it has. The Pia Horvat et al. study continues along the lines of the paper “Alcohol Consumption and Cognitive Function in the Whitehall II Study” by Annie Britton and colleagues. The paper looks to the change in cognition caused by different levels in alcohol consumption. The more recent study is one of the first to look at quantity and frequency in the association between alcohol and the functioning of the brain.

Overall the authors are showing that moderate levels of 171

alcohol serve for better mental functioning. Although in males some of the results were not significant, the same patterns are still present. Increases in cognitive ability are still observed in those who drink alcohol in moderation, in comparison to those who drink obscene amounts of alcohol, as well as those who do not drink at all. With these results, the authors are eluding to the idea that moderate levels of alcohol are involved in a pathway that increases cognitive function, lesser amounts of alcohol does not have a strong enough effect, and too much alcohol is detrimental to the brain’s function. An exact amount that produces the best results was not discovered, but an approximate range was. With the data from the follow-up it was observed that continued moderate drinkers had the best results. Those who didn’t drink at all, or that had only started drinking had worse scores in comparison to their original scores. Looking at those who started drinking after the original assessment it is possible that after a certain amount of time improvements are no longer possible with alcohol and it actually decreases mental functioning. This could mean that at certain levels of mental development, such as adolescence, the consumption of alcohol actually aids brain development.

The beneficial effects of alcohol on cognitive function are also stronger in women than in men. It is possible that the male and female brains have minute differ-

ences in development that are facilitated differently through alcohol. On the contrary, this may also mean that the differences in the male and female anatomy affect how the development of their brains is altered (ie. Levels of fat in the body change the overall effect of alcohol in the body).

Conclusions A study in the frequency and quantity of alcohol consumed in relation to changes in cognitive function is the avenue this area of study was expected to take.

Knowing that alcohol increases mental functioning, is strong but dangerous information, as it may promote alcoholism. This topic needs to be properly researched so that facts can be elucidated from it. Once optimal levels of alcohol consumption have been discovered the next step in the study will be at the forefront of research, and an answer that has evaded civilization for centuries will finally be answered. What substance can undoubtedly make humans smarter?

Criticisms and Future Directions

A problem with this study is that people were asked to self-report their levels of alcohol consumption. This could greatly affect the results, as some people may be ashamed of how much they drink, and would report lower levels of intake, or would boast about how much they consumed and inflate their numbers. Although confounds for lifestyle and socioeconomic status were taken into consideration, this study remains open to a lot of potential errors. This study should be replicated, with a different animal, with more controls to reduce confounds.

Mice have been commonly used to observe the effects of alcohol and other drugs on the brain. Using mice, the effects of moderate levels of alcohol consumption on the brain could be observed. These results would then be compared to the brains from past studies of alcoholic mouse brains. Considering the differences in the two brain types (such as changes in receptors, production of proteins etc.), the cause of cognitive improvement could be assumed.

Once confounds in the results are eliminated the actual pathways that cause increases and decreases in mental function can be considered. Certain properties of ingesting ethanol can be tested in different areas of the brain, to see what actually causes the differences in mental functioning. Learning what changes in the brain would open a field of research, focused on breaking down boundaries on the human brain. This could be the first step in making the cognitive abilities of humans limitless.

on Adolescents and College Students. Prev Med 40: 23-32.

4. Sabia S (2014) Alcohol Consumption and Cognitive Decline in Early Old Age. Neurology 82: 332-39.

5. Virtaa JJ et al. (2010) Midlife alcohol consumption and later risk of cognitive impairment: a twin follow-up study. J Alzheimers Dis. 22:939–948.

6. Järvenpää T, Rinne JO, Koskenvuo M, Räihä I, Kaprio J (2005) Binge Drinking in Midlife and Dementia Risk. Epidemiology 16: 766-71.

7. Rehm J, Sempos CT, Trevisan M (2003) Alcohol and cardiovascular disease--more than one paradox to consider. Average volume of alcohol consumption, patterns of drinking and risk of coronary heart disease--a review. J Cardiovasc Risk 10:15-20 8. Bond GE et al. (1999) Alcohol, Aging, and Cognitive Performance in a Cohort of Japanese Americans Aged 65 and Older: The Kame Project Int Psychogeriatr 13: 207-23.

9. Harper C (2009) The Neuropathology of Alcohol-Related Brain Damage. Alcohol Alcoholism 44: 136-40.

10. Britton A et al. (2004) Alcohol Consumption and Cognitive Function in the Whitehall II Study.” Am J Epidemiol 160: 240-47.

11. Ricaurte GA, Schuster CR, Seiden LS (1980) Long-term Effects of Repeated Methylamphetamine Administration on Dopamine and Serotonin Neurons in the Rat Brain: A Regional Study.

Brain Res 193: 153-63.

12. Horvat P et al.(2014) Alcohol consumption, drinking patterns, and cognitive function in older Eastern European adults. Neurology 84: 287-295

References 1. Amieva H (2005) The 9 Year Cognitive Decline before Dementia of the Alzheimer Type: A Prospective Population-based Study. Brain 128:1093-101.

2. Hillman CH, Erickson KI, and Kramer AF (2008) Be Smart, Exercise Your Heart: Exercise Effects on Brain and Cognition.” Nat Rev Neurosci 9: 58-65 3. Zeigler DW, et al (2005) The Neurocognitive Effects of Alcohol 172

Down-Regulation of Amyloid-Beta Peptide Binding P75 in Basal Forebrain Cholinergic Neurons Rescued Neurodegeneration and Behavioral Deficits in AD Mouse Models Tong Mai

The neurotrophin receptor p75 binds to different ligands to induce various functions including cell survival, cell death, and differentiation. It’s interaction with the amyloid-beta peptide is believed to contribute to the neurodegeneration of cholinergic neurons of basal forebrain in Alzheimer’s disease. Studies on mouse models showed that down-regulation of p75 level can rescue the neuronal functions and cognitive deficits. Its limited expression in the brain served as a good therapeutic target for AD. Key words: Alzheimer’s Disease, p75 neurotrophin receptor, amyloid-beta, mouse models, neurodegeneration, LM11A-331, Tg2576, cognitive deficits, Cholinergic neurons Background Alzheimer’s disease is an age-related neurodegenerative disorder that is affecting many of the elders worldwide. It is characterized by the accumulation of extracellular amyloid plaques and formation of intracellular tangles. The neuronal degeneration often leads to cognitive impairment and functional deficit (Xia et al, 2014). The major regions of the brain damaged in AD include the entorhinal cortex and the basal forebrain (Fombonne et al, 2009). The cholinergic neurons of basal forebrain are enriched with the low-affinity neurotrophin receptor p75, which is sparsely distributed in other areas of the brain. Previous researches have showed that p75 have multifaceted roles to induce either cell survival or cell death depending on the expression of co-receptors and the ligands (Ovsepian et al, 2013). It is a transmembrane receptor with a death domain that could activate cell apoptosis upon binding of ligand (Xia et al, 2014). The amyloid cascade hypothesis of Alzheimer’s disease suggests that the amyloid plaque formation resulting from the overproduction or the failure to break down Aβ is the major cause for the dementia (Ovsepian et al, 2013).

The binding of soluble oligomeric form of amyloid-beta to p75 induces toxicity results in neuronal dysfunction and cell apoptosis in AD (Xia et al, 2014; Fombonne et al, 2009). Elevated level of amyloid-beta is associated with aggravated memory impairment, LTP inhibition, and cell death in AD patients (Xia et al, 2014). The enhanced expression of amyloid-beta in AD also showed an upregulate effect on the number of p75 in basal forebrain (Chakravarthy et al, 2010). Previous studies on the relationship of p75 and AD using mouse models have shown many conflicting results. Careful manipulation on the genetic background of the mice is important (Greferath et al, 2000). Various transgenic and knock out mouse models researchers used to study for p75. Upregulation of p75 enhanced cell death and increase vulnerability to AD and amyloid-beta toxicity whereas p75 knockout mice have increased neuronal size, slowed neurodegeneration, and increased cholinergic innervation (Yeo et al, 1997; Barrett et al, 2010).

Yet most of the studies have focused on the brain pathologies upon p75 manipulation, few have demonstrated the effect of changing p75 level on behavioral and cognitive functions.

173

Research Overview

Summary of Major Results

Murphy et al. (2014) conducted a study on the Tg2576 transgenic mice containing human APP gene with different levels of p75 expressions. Several memory experiments were performed: fear conditioning testing for fear memory; Y maze testing for short term memory; and Barnes maze testing for spatial memory.

The mice were between 4 to 8 months old (early phase of AD) in the tests except for the Barnes maze test which used 2 age groups (4-8 months and 12-14 months). They were able to show that reducing the level of p75 receptors in the Tg2576 mice showed significant improvement in performance in all of the cognitive tests. The Tg2576 mice exhibited less shock response in the contextual fear conditioning test compare to the other three groups with p75 reduction. The Tg2576/ p75+/- mice preferred to use more spatial search strategy in the Barnes Maze and have longer memory retention about the familiar arms in the Y maze.

The synaptic transmission of the hippocampal CA1 neurons was enhanced after the reduction of p75. The LTP of the Tg2576/p75+/- and wild type exhibited similar magnitudes whereas the Tg2576 had a reduction in LTP. There was an increase in human Ab expression in the Tg2576 mice with reduction of p75.

Conclusions and Discussion Reduction of p75 on AD mouse models rescued the neurodegeneration as well as the behavioral/cognitive deficits. The level of p75 negatively regulates the cholinergic system, which inhibit hippocampal function and impair spatial memory (Barrett et al, 2010). The p75 knockout mice consistently performed greater in Barnes maze task compare to the control. The p75 enables amyloid-beta peptide to induce cell apoptosis. Downregulation of p75 was showed to enhance the neuronal size and function but have very little effect on the neuronal numbers (Greferath et al, 2010; Boskovic et al, 2014). The change in p75 expression only affected the basal forebrain cholinergic neurons but have no effect on neurons of other regions of the brain (Yeo et al, 1997).

Because of its limited and specific expression, it is a very good therapeutic target for treating early- and mid- stage of AD. Mouse models expressing various levels of p75 exhibit gene dosage dependent effect on their functional impairments. The heterozygous mice with only one functional p75 had intermediate performance on cognitive tasks, whereas the p75-deficient mice performed the best and the worst for the control (Barrett et al, 2010; Murphy et al, 2014). The direct effect of p75 on the cholinergic neuronal function is undoubtable, whereas the effect is dependent on the co-receptor and ligand. The NGF binds to p75 to promote cell survival or cell death depending on the presence of the co-receptor TrkA (Simmons et al, 2014). While reduction on p75 has no effect on levels of amyloid-beta peptide, number of amyloid-beta is positively correlated with p75 (Chakravarthy et al, 2010).

A small non-peptide ligand LM11A-31 was found to bind p75 and reversed the neurodegeneration in mouse models at the mid- and late AD stage (Simmons et al, 2014). After oral administration, mice had increased performance in Y maze tasks and decreased in synaptic loss (Knowles et al, 2013). The binding of LM11A-31 to p75 activates the survival signalling pathway and inactivates the cell death cascade.

Conclusions

Amyloid-beta peptides bind to p75 at the cholinergic neurons of the basal forebrain causing synaptic dysfunction and impair cognitive performance. Downregulation of p75 in various AD mouse models with different genetic backgrounds has shown that p75 exhibits a gene-dose dependent effect. Lower expression of p75 in previous studies has shown the reversed brain pathologies. Murphy et al. (2014) further showed that reduction of p75 in Tg2576 not only attenuated the neurodegeneration; the behavioral deficits were also rescued and the performances of the Tg2576/ p75+/- mice were comparable to the wild type.

Criticisms and Future Directions

Previous studies provide good evidence that reduction of p75 expression can rescue neurodegeneration and cognitive deficits in AD mice models. However, most of mice in the studies were at the early or midprogression of AD, more research is needed to be done on the late AD phase. Furthermore, the cognitive tests conducted in the studies were mostly tasks associated with spatial memory. It is known that p75 caused degeneration of cholinergic neurons which interfere with hippocampal function leading to deficit on spatial memory (Barrett et al, 2010). Yet in human patients with Alzheimer’s disease, the cognitive declines are more serious and multi-dimensions. While previous studies could only showed that reduction of p75 could enhanced performance of mice on tasks related to spatial memory, it is hard to conclude that downregulation of p75 will be able to rescue other cognitive deficits (i.e. episodic memory, language). Although the small ligand LM11A-31 has shown to prevent the cell loss in AD mouse models, researchers need to further test on the specificity and side-effect of the ligand and whether the change in neuropathology have any impact on the functional impairment.

References 1. Barrett, G. L., Reid, C. A., Tsafoulis, C., Zhu, W., Williams, D. A., Paolini, A. G., Trieu, J., Murphy, M. (2010). Enhanced Spatial Memory and Hippocampal Long-Term Potentiation in p75 Neurotrophin Receptor Knockout Mice. Hippocampus, 20, 145-152.

2. Boskovic, Z., Alfonsi, F., Rumballe, B. A., Fonseka, S., Windels, F., Coulson, E. J. (2014). The Role of p75NTR in Cholinergic basal forebrain structure and function. J Neurosci., 34(39), 1303313038.

3. Chakravarthy, B., Gaudet, C., Menard, M., Atkinson, T., Brown, L., LaFerla, F. M., Armato, U., Whitfield, J. (2010). Amyloid-β peptides stimulate the expression of the p75NTR neurotrophin receptor in SH-SY5Y human neuroblastoma cells and AD transgenic mice. J Alzheimers Dis, 19, 915-925.

4. Fombonne, J., Rabizadeh, S., Banwait, S., Mehlen, P., Bredesen, D. E. (2009), Selective vulnerability in Alzheimer’s Disease: Amyloid Precursor Protein and p75NTR interaction. Ann. Neurol. 65, 295-303.

5. Greferath, U., Bennie, A., Kourakis, A., Bartlett, P. L., Murphy, M., Barrett, G. L. (2000). Enlarged cholinergic forebrain neurons and improved spatial learning in p75 knockout mice. Eur. J. Neurosci., 12, 885-893.

6. Knowles, J. K., Simmons, D. A., Nguyen, T. V., Griend, L. V., Xie, Y., Zhang, H., Yang, T., Pollak, J., Chang, T., Arancio, O., Buckwalter, M. S., Wyss-Coray, T., Massa, S. M., Longo, F. M. (2013). A small molecule p75NTR ligand prevents cognitive deficits and neurite degeneration in an Alzheimer’s mouse model. Neurobiol Aging, 34, 2052-2063.

7. Murphy, M., Wilson, Y. M., Vargas, E., Munro, K. M., Smith, B., Huang, A., Li, Q., Xiao, J., Master, C. L., Reid, C. A., Barrett, G. L. (2014). Reduction of p75 neurotrophin receptor ameliorates the cognitive deficits in a model of Alzheimer’s disease. Neurobiol Aging, 1-13.

8. Ovsepian, S. V., Antyborzec, I., O’Leary, V. B., Zaborszky, L., Herms, J., Dolly, J. O. (2013). Neurophin receptor p75 mediates the uptake of the amyloid beta (Aβ) peptide, guiding it to lysosomes for degradation in basal forebrain cholinergic neurons. Brain Struct Funct, 219, 1527-1541.

9. Simmons, D. A., Knowles, J. K., Belichenko, N. P., Banerjee, G., Finkle, C., Massa, S. M., Longo, F. M. (2014). A small molecule p75NTR ligand, LM11A-31, Reverses cholinergic neurite dystrophy in Alzheimer’s Disease mouse models with mid- to late-stage disease progression. Plus one, 9(8).

10. Xia, M., Cheng, X., Yi, R., Gao, D., Xiong, J. (2014). The Binding Receptors of Aβ: an Alternative Therapeutic Target for Alzheimer’s Disease. Mol. Neurobiol. Doi: 10.1007/s12035-0148994-0.

11. Yeo, T. T., Chua-Couzens, J., Butcher, L. L., Bredesen, D. E., Cooper, J. D., Valletta, J. S., Mobley, W. C., Longo, F. M. (1997). Absence of p75NTR Causes Increased Basal Forebrain Cholinergic Neuron Size, Choline Acetyltransferase Activity, and Target Innervation. J. Neurosci., 17(20), 7594-7605.

Received Month, ##, 200#;

##, accepted

This work was supported by Undergraduate Neuroscience ment for Science Education Research Foundation (EA).

Dr. Amy G. Dala, and the nical assistance, execution,

200#; Month,

revised ##,

Month, 2013.

The Association for the Development of Education (SRA & RLN), The Endow(EA), and The Synaptic State Faculty The authors thank Mr. Spine L. Cord, students in Neuroscience 101 for techand feedback on this lab exercise.

Address correspondence to: Dr. Rita L. Neurotrophin, Biology Department, 123 Growth Cone Avenue, Action Potential College, Hillock, IL 60101 Email: rln@apc.edu

174

Discovering Biomarkers to Detect Early Onset of Stroke

Fazila Malek

One of the highest death rate is associated with stroke world wide and especially in third world countries where certain diets consist of unhealthy saturated fats and oils, and lack of MRI machines or CT scans. Detecting biomarkers for stroke in the blood serum can be an easy tool to determine whether or not a person has suffered from stroke, upon which rapid treatment would be available preceding quick diagnosis. Biomarkers like NSE have been found to correlate strongly with stroke onset, and can be a determinant of stroke occurrence. NSE is an enzyme released in the CSF upon cell death and crosses the blood brain barrier (BBB) into the blood stream. A control group with people of no stroke symptoms and a study group of people with some signs and symptoms of acute stroke were chosen. Taking blood samples from these groups and measuring serum levels of NSE, showed that all controls had NSE levels below 25 ng/ml and stroke patients had most levels arising in the greater than 25 ng/ml range. NSE is a promising biomarker in serum allowing to detect stroke occurrence in countries where medical imaging procedures or not available or are too costly for people to afford.

Key words: neuron specific enolase (NSE); acute and ischemic stroke; degree of disability; biomarker Background As stroke is one the leading causes of death and long term rehab hospitalization of patients, it would be affective to diagnose and treat stroke early to increase survival rates and prevent severity in stroke patients to rise. Many countries in the world do not have access to expensive technology such as CT scans or MRI machines. Some areas of the world may not be able to afford expensive machinery, and the first world countries which are able to provide these services could greatly cut down the costs and make use of the finances elsewhere in the healthcare field. A study done in the United Kingdom found that £8.9 billion were used just in stroke treatment and diagnosis alone1. Thus, being able to use biomarkers within the blood would be an ideal and easy solution to diagnosing and treating stroke.

The paper by Bharosay et al. looks at biomarkers in the blood to discover early onset of stroke in patients within 72 hours. Taking blood samples from a control group with no clinical signs and symptoms of stroke, and then repeating the same with patients with stroke occurrence within 72 hours. Using an antibody against the γ, γ-Neuron specific enolase, they were able perform an enzyme immunoassay. They also used National Institute of Health Stroke Scale (NIHSS), which is a set of questions, and the score obtained would determine severity of stroke.1 Many studies have been done to look at specific biomarkers to use to diagnose stroke, which will be discussed in this review for future directions. Some biomarkers discovered and studied were microRNAs3, ubiquitin fusion degradation protein 1 (UFD1)4, and glutathione S-transferase-π (GST- π)5.This paper will review some of the advantages and disadvantages to each type of biomarker for early detection of stroke.

had signs and symptoms of stroke within 72 hours, the following results were found. The control group consisted of 101 people, and 82% of them were in the 46-65 years age range. In the study group of 156 stroke patients, consisted of 59% of people in the 46-65 age range. Also, the study group consisted of 63% males. Other risk factors like hypertension, atrial fibrillation, and diabetes mellitus were generally found more with in the study group compared to the control.

Neuron Specific Enolase (NSE) levels in Control Group vs. Study Group In the study group, 47% of the population had NSE levels greater than 25 ng/ml and ~9% of the people had NSE levels greater than 35 ng/ml. In contrast, everyone in the control group had lower than 25 ng/ ml of NSE in serum. See Figure 1.

NSE levels and Severity of Stroke Elevated levels of NSE were 92% positively correlated with the severity of stroke, suggesting NSE level being important for cerebrovascular stroke.

NSE and Degree of Disability Degree of disability was determined using the

Research Overview

Summary of Major Results

Control vs. Cerebrovascular Stroke Patients By having a control group of people who had no signs and symptoms of stroke and a group of people who 175

Figure 1. Control group consisted of less than 25 ng/ml NSE level (red). Study group had highest NSE level in 25.1 – 35 ng/ ml range, and some in greater than 35 ng/ml.

Source: 1) Bharosay et al.

National Institute of Health Stroke Scale (NIHSS), and categorized into mild, moderate, and severe degree of disability. There was a high correlation between NSE levels and the degree of disability.

NSE levels and Neurological Worsening Neurological worsening was determined by comparing the NIHSS score at the time of admission and after 7 days of admission. If the score at 7 days was more than 2 points greater, the patient was categorized as having neurological worsening. There was a high correlation found between NSE levels and neurological worsening also.

Conclusions and Discussion In this study, NSE levels were found to be increased greater levels in stroke patients than in normal individuals who had no sings or symptoms of stroke. Firstly, there are a number of studies, which use NSE as a biomarker, such as concussion studies, seizures, and last but not least stroke studies. NSE indicates to be a useful biomarker because it has a half-life of about 48 hours, and is released upon cell death â&#x20AC;&#x201C;apoptosis. Its ability to cross the blood brain barrier is important as it can now be detected in the blood and measured for diagnosis of acute stroke1. In the study done by Gelderblom et al., in vivo stroke animal models were used to measure NSE levels, and found that there were significant amounts of increased NSE in the stroke patients both in acute and ischemic6. Another study done by Hatfield and McKernan focused on NSE being a biomarker for ischemic stroke, and also found that post 3 days of stroke occurance, there were significantly elevated levels of NSE in the serum7. See figure 2. Therefore, there is significance to NSE levels after stroke and seems like the same hypothesis has been proven with multiple studies.

Figure 2. Source: Hatfield and McKernan.

Increase levels of CSF NSE in ischemic stroke rats post 3 days of stroke.

NSE levels were also an indicator for the degree of disability, where a strong positive correlation was found between the NSE levels and degree of disability in Bharosay et al. Another study, by Pandey et al. looked at levels of NSE and also at the degree of disability. Pandey et al. using NSE and C-recactive protein (CRP) as biomarkers. CRP was used since it is normally not present in the blood, but is a targeted

production by the liver after stroke onset and released in the bloodstream, therefore this could potentially be a good marker for stroke in addition to NSE. This study is similar to Bharosay et al., since Pandey et al. also used the NIHSS scoring at the admission time and 7 days after stroke. It was found that both NSE and CRP levels were elevated in the bloodstream after stroke, but CRP levels were particularly elevated with the degree of disability. The more severe disability from stroke onset, the greater amounts of CRP in the blood8.

Conclusions

Neuron specific enolase (NSE) shows to be a very effective and important biomarker of stroke and other brain injury conditions. Numerous studies such as Bharosay et al., and Gelderblom et al.

The importance of finding a good biomarker in serum is due to the fact that many countries do not have access to CT scan machines or MRI machines. Using a biomarker to detect stroke can be as simple as drawing blood from the patient and testing for NSE levels.

Conclusions and Discussion This study by Bharosay et al., used a biomarker, specifically, neuron specific enolase (NSE) available in the cerebrovascular system, as it is found in the cerebrospinal fluid. Being easily accessible in the blood serum, an immunoassay was done. This is quicker than the typical CT or MRI scans that may take more time or simply the wait time to have these tests done is lengthy2,3. One of the strengths of this study is that the method of diagnosing stroke is very rapid. It saves time and costly machines are not needed. This would increase rates of quick treatment for patients, and decrease death rates from stroke due to not treating patient on time. A limitation of this study was that diagnosis of stroke using biomarkers occurs after the onset.

A gap to fill for future directions would be to try and detect stroke early on before it actually occurs.5 NSE equally present in low amounts and moderatehigh amounts in stroke patients The results from Bharosay et al. are quite convincing that NSE is a good biomarker for detecting stroke, however a drawback for the paper was Figure 1, which is presented above. 67 cases of the study group have NSE levels below 25 ng/ml and 70 cases of the study group have NSE levels between 25-35 ng/ ml, and only 13 cases in the greater than 35 ng/ ml range. This can weaken the conclusion that NSE is indeed a good biomarker for stroke occurrence, since there were similar number of cases for NSE below 25 ng/ml and for 35-45 ng/ml. There is not a large statistical difference between the case numbers, suggesting that NSE can be both in low amounts and in moderate-high amounts in stroke patients2. miRNA as biomarker In addition, for future studies, the authors could target miRNAs in stroke patients that could differ from normal people, who have not had stroke. This can be done by collecting plasma from stroke patients and controls,

176

and isolating RNA using TRIzol reagent, as done in the study by Wang et al. Next, a microarray and qRT-PCR can be used to determine significant differences between the stroke patients and controls. The miRNAs that had 2-fold difference were chosen to be tested for biomarkers. By using SYBRGreen dye, out of all the miRNAs, it was found by Wang et al. that has-miR-1065b-5P and has-miR-4306 had significant increase in acute stroke patients as compared to controls. Looking at miRNA as biomarkers would be very good because certain miRNA can be specific to certain injuries and tissues/cells, or brain damage. In addition, miRNAs are also quite stable structures, making it easy to work with without having to worry about degradation or making cohort with other molecules. 3 NSE, on the other hand is an enzyme and authors can run the risk of NSE being degraded easily.

Ubiquitin Fusion Degradation Protein 1 (UFD1) Another biomarker that could be used to detect stroke is ubiquitin fusion degradation protein 1 (UFD1) in blood samples and compare to it to cohort studies. A study by Allard et al. looked at a Swiss cohot, Spanish cohort, and North American cohort to compare. UFD1 was found to be elevated in all three cohorts and is also associated with brain injury, by being present in cerebrospinal fluid.4 UFD1 can be found in the blood stream, once there is damage to the blood brain barrier, allowing UFD1 to spill into the blood stream.

Note, this could be a drawback to use as a potential biomarker, as brain injury does not necessarily guarantee damage of blood brain barrier.9 Glutathione S-Transferase-π (GST-π) Further, the researchers could look at trying to detect stroke by looking at a pattern or window of stroke occurrence. A study done by Turck et al. suggested looking at glutathione s-transferase-π, which was an elevated enzyme within 3 hours of occurrence of stroke in patients. It was an immediate increase in stroke patients as compared to controls. This could allow early treatment of thrombolysis, which would cause breakdown of clots.5 References 1. Saka, O et al. Cost of Stroke in the United Kingdom. Age and Ageing. (2009). 38: 27-32.

2. Bharosay, A et al. Correlation of Brain Biomarker Neuron Specific Neolase (NSE) with Degree of Disability and Neurological Worsening in Cerebrovascular Stroke. Ind J Clin Biochem (Apr-June 2012) 27(2): 186-190.

3. Wang, W et al. Circulating MicroRNAs as Novel Potential Biomarkers for Early Diagnosis of Acute Stroke in Humans.

Journal of Stroke and Cerebrovascular Diseases. 23(10).

(Nov-Dec 2014). 2607-2613.

4. Allard, L et al. Ubiquitin Fusion Degradation Protein 1 as a Blood Marker for The Early Diagnosis of Ischemic Stroke.

Biomarker Insights (2007). 2: 155-164.

5. Turck, N et al. Blood Glutathione S-Transferase- π as a Time Indicator of Stroke Onset. Plos One. (Sept 2012).

7(9):1-9.

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6. Gelderblom et al. Plasma levels of neuron specific enolase quantify the extent of neuronal injury in murine models of ischemic stroke and multiple sclerosis. Neurobiology of Diease.

(2013) 59: 177-182.

7. Hatfield, R and McKernan, R. CSF neuron-specific enolase as a quantitative marker of neuronal damage in a rat stroke model. Brain Research. (1992) 577: 249-252.

8. Pandey, A et al. Neuron Specific Enolase and C-Reactive protein Levels in Stroke and its subtypes: Correlation with Degree of Disability. Neurochemical Research. (2014) 39: 1426-1432.

9. Laborde, C et al. Potential Biomarkers for Stroke.

(2012) Expert Review of Proteomics. 9.4: 437-439.

Received April, 6, 2015; revised Month, ##, 200#; accepted Month, ##, 2013.

This work was supported by Undergraduate Neuroscience ment for Science Education Research Foundation (EA).

Dr. Amy G. Dala, and the nical assistance, execution,

The Association for the Development of Education (SRA & RLN), The Endow(EA), and The Synaptic State Faculty The authors thank Mr. Spine L. Cord, students in Neuroscience 101 for techand feedback on this lab exercise.

Address correspondence to: Dr. Rita L. Neurotrophin, Biology Department, 123 Growth Cone Avenue, Action Potential College, Hillock, IL 60101 Email: rln@apc.edu

Divya Mamootil

Erasing Fear Memories– Is it possible?

Using Pavlonian conditioning, rats can readily learn to fear a neutral stimulus such as a tone when paired with an aversive stimulus like foot shock. This learned fear of the tone (CS) can be reduced by extinction, a process where fear responses decline due to the repeated presentations of the CS alone11. Sometimes, extinguished fear responses can be renewed with a change of context, and the hippocampus regulates this fear renewal following extinction3. The paper we will review by Stephen Maren (2014)8 explores the intermediary role of the hippocampus in novelty-induced fear. Rats were injected with the GABAA inhibitor, muscimol, or saline to see if they would remember extinguished fear with a novel stimulus or context. This study provides potential for eliminating fear memories in clinical applications, such as PTSD. Although it may not be possible to completely erase a painful memory, reducing its aversive effects on an individual can be of critical significance in psychological and medical fields. Here we review the main results of Maren’s research and examine related studies that have explored neural substrates involved in the renewal of extinguished fear memories.

Key words: hippocampus; fear conditioning; PTSD; extinction; amygdala; muscimol; renewal; GABA; prefrontal cortex Background Fears that are maladaptive or inappropriate can be reduced through extinction training. Extinction is at the heart of exposure-based therapies, which are a main treatment choice for anxiety disorders2. However, studies have found that extinction cannot completely erase memories of the original tone-shock pair; instead, it temporarily masks the expression of the original fear memory and reduces fear responses2.

Extinction is highly context-sensitive; for example, exposure to a CS outside of the extinction context often causes the renewal of fear. Lesion and inactivation studies have shown that the contextualization of extinction depends on the hippocampus15. Therefore, understanding the neural mechanisms of fear renewal may provide insight into the long term efficacy of exposure therapy.

Along with the hippocampus, the amygdala and medial prefrontal cortex are also important brain regions that mediate fear learning, extinction, and renewal10. The amygdala is an important part of the brain for regulating one’s emotions and motivational behaviour. In relation to fear, Walker & Davis (2002)13 described the importance of NMDA and AMPA glutamatergic receptors in mediating fear learning.

Their studies found that injecting the AMPA receptor antagonist, NBQX, into the basolateral amygdala led to less freezing behaviour in rats, whereas the NMDA receptor antagonist did not have this effect of inhibiting fear renewal. Therefore, AMPA receptors seem to be the most critical for expressing fear13. The medial prefrontal cortex interacts closely with the amygdala and is also involved in fear regulation. Lebron et al (2004)7 experimented with lesions in this brain region and found that lesions of the ventromedial prefrontal cortex resulted in poor extinction memory in rats. This led them to believe that the vmPFC has an important role in housing extinction neurons, which inhibit the expression of fear responses, such as freezing. Thus, many brain regions are involved in fear learning and the loss of fear; however, specific receptors and pathways are still being studied.

Previous research by Cole et al (2013)2 studied the

kappa opioid receptors (KOR) in the hippocampus, which are associated with fear learning and anxiety.

They found that the KOR antagonist norbinaltorphamine hydrochloride (norBNI) reduces fear renewal in rats when injected into the ventral hippocampus (but not the dorsal), compared to saline controls. Another study by Baker-Andresen et al (2013)1 looked at DNA methylation of BDNF exon IV in the medial prefrontal cortex of female mice (whom are resistant to fear extinction). This methylation reduced BDNF signalling that is essential to fear learning and memory. However, they found that the trkB agonist 7,8-dihydroxyflavone increases BDNF signalling and thus, blocks the return of fear in female mice after extinction training. These studies represent a novel approach to treating fearrelated anxiety disorders such as PTSD1, however, they are very invasive methods that are difficult to use in human clinical treatments.

Other research has explored more non-invasive strategies such as the use of pharmacological drugs.

Zelikowsky et al (2013)15 studied the effects of the drug Scopolamine, a cholinergic antagonist, in inhibiting the contextualization of fear extinction. They administered the drug systemically into the hippocampus in low doses, and it proved to significantly reduce fear renewal in both the original training context as well as a novel context.

This drug also slowed the rate of long-term extinction memory formation. Therefore, low doses of Scopolamine may be combined with exposure therapy to make extinction more relapse-resistant. Similarly, Haaker et al (2013)5 administered a single dose of the dopamine precursor, L-dopa, and found that it makes extinction memories context-independent, thereby preventing fear renewal in both rats and humans. They predicted that extinction memory may be dopamine-dependent, so increasing dopamine levels may be promising for future anxiety therapies5.

Thus, a plethora of previous studies have shown multiple factors that seem to be implicated in the retrieval of original fear memories and subsequent relapse of fear. Specific receptors and neurotransmitters involved in these processes need to be researched further. We will begin this review with a discussion of Stephen Maren’s experiments on hippocampus-mediated fear renewal.

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Research Overview

Summary of Major Results

Maren used non-invasive experimental methods in this study through the injection of the drug muscimol, instead of employing mass lesions of the brain. Rats were first fear conditioned with a tone-shock pair, then learned to extinguish the fear a day later. Then they were injected with either the GABAA agonist muscimol or saline (control). Following the injection, rats were given a retrieval test in either the same or different context from extinction training. The researchers also presented a novel or familiar tone to the rats in a subsequent experiment. It was hypothesized that blocking the hippocampal pathway that detects associative novelty would inhibit fear renewal in rats.

In experiment one (where the context was manipulated), saline-injected rats showed higher levels of freezing in the novel context compared to the context where the extinction occurred during the retrieval test. In contrast, the muscimol-injected rats had consistently low freezing behaviour in both contexts.

In experiment two (where the conditioned stimulus was manipulated), saline-injected rats showed higher levels of freezing when the new conditioned stimulus was presented compared to the familiar one. The muscimol-injected rats showed similar behaviour but to a much lesser degree. In both cases, musicmol inactivated the hippocampal pathway that mediates fear renewal following extinction.

Figure 1. The visuals above indicate the extent to which rats retrieve previously extinguished fears in either the same or novel conditions. Since the muscimol injected rats can no longer detect novelty, they should have impaired memory for previously learned fears in the novel conditions, as shown in Figures A) and B).

Conclusions and Discussion The results of Marenâ&#x20AC;&#x2122;s experiment fall in line with those of the previously discussed studies, since they all involve blocking receptors and signaling pathways in the hippocampus to prevent the renewal of fear.

Rats will respond strongly to novelty, but the GABAA agonist, muscimol, inhibits the normally heightened response to novel stimuli. Therefore, there seems to be a strong correlation between the hippocampusâ&#x20AC;&#x2122; detection of novelty and fear renewal, because when the hippocampus is inactivated the fear response declines or disappears entirely. Since the hippocampus is very important for spatial memory, it is no surprise that it has a preference for detecting novelty of context compared to stimuli. Both the dorsal and ventral hippocampus seem to be involved in fear renewal, as previous studies have found relevant receptors and pathways in both areas2,8. As mentioned in the background, fear extinction is most likely an inhibition or down regulation of processes rather than the erasure of fear memories. Therefore, research should continue to focus on long-lasting suppression of fear renewal pathways rather than find ways to eliminate fear memories10.

Studies on inhibition of fear renewal are extremely important to understand if we want to help patients with anxiety cope with their unwanted feelings of worry.

One of the most salient pieces of information we intake when we encounter a traumatic experience is the place or context where it took place. This is why people with PTSD and other anxiety disorders feel overwhelmed when they come across situations similar to the context they originally encountered the aversive experience in.

Essentially, they experience a fear renewal, and this response is very difficult to eliminate once the individual has gone through a panic attack. In fact, the failure to inhibit fear is a triggering factor in the development of PTSD1. Although much progress has been made in this field of research, more studies need to be done to investigate the details of the fear renewal pathways. Determining the underlying mechanisms of fear renewal can help us develop treatments that weaken or completely erase learned fears and memories of traumatic experiences in the future.

Table 1. The two graphs above show the freezing behaviour of saline vs. muscimol injected rats with a familiar (white) or novel (grey) stimulus/context. The first graph shows results from experiment 1, while the second graph depicts results from experiment 2. The differences in behaviour for the familiar conditions were not statistically significant.

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Criticisms and Future Directions Further research should prompt experimenters to look at how the time of muscimol administration affects the renewal of fear memories. Wang et al (2014)14 did a study where rats were given high doses of corticosterone, and this resulted in an inhibition of the renewal of fear by reducing anxiety levels. They examined the temporal context of injecting corticosterone by using different time delays, such as 1 or 24 hours after the fear conditioning, and in both cases there was reduced renewal of fear. Also, one of the limitations of Maren’s paper was that the researchers only studied short term effects of muscimol on renewal of fear. On the other hand, Wang et al. looked at long term effects of inhibition by assessing anxiety levels in rats a week later through an elevated arm maze task, which continued to result in inhibition of fear renewal.

In future studies, the researchers should test how long the hippocampal inactivation lasts by doing more trials over a week or two, so that even if the fear is inhibited in the initial trial, they will know how long it will resist renewal despite various changes in stimulus/context.

Although Maren’s paper studied dorsal hippocampal inactivation in suppressing the return of fear, they could study other areas of the brain that are involved in fear renewal. For example, Sharpe and Killcross (2015)12 studied how inactivation of the prelimbic cortex can also inhibit the renewal of fear in novel contexts. They used muscimol and saline injections as well, and varied the time of injection (during or after extinction). They found that rats with lesions in the prelimbic cortex exhibited less freezing in the novel context, similar to the dorsal hippocampal inactivation in the original paper by Maren. Parallel studies

by Orsini et al (2011)9 looked at the how projections of the ventral hippocampus and prelimbic cortex to the basolateral amygdala mediate context-dependent fear renewal. They used a retrograde tracer for c-Fos expression in this pathway and found high expression of c-Fos when fear is renewed in a new context.

However, inhibiting projections to the basolateral amygdala eliminated this fear renewal.

Finally, many neurobiological methods such as stereotactic lesion and pharmacological drug injections have been discussed as non-invasive ways of exploring the renewal of extinguished fear memories; however there are other approaches that can be used to study this issue as well. For example, Drexel et al (2014)4 used a psychological viewpoint to study inhibition of fear renewal through exposure therapy, by exposing the aversive stimuli to the patient on a regular basis so that they get used to it and no longer perceive it as a threat. This kind of method can be used in future studies by exposing the rats to the previously conditioned fear stimulus and different variations of that stimulus or the context it was in after the extinction phase. In this way, even if the context changes or there’s a slight change in the stimulus itself, the rats will longer be affected by it, and there won’t be a renewal in extinguished fear response.

Figure 2 This figure from the paper by Jingji & Maren (2015)6 shows the injection of various dyes and markers in the brain where BA-projecting neurons are red, PL-projecting neurons are green, and Fos-positive neurons are blue. You can see from the merge that Fos is being expressed in the PL to BA projection pathway, and the same results are seen in the VH to BA pathway.

References 1. Baker-Andresen, D. et al. Learn Mem 20, 237-240 (2013).

2. Cole, S. et al. PLos ONE 8: e58701. doi:10.1371/journal. pone.0058701 (2013).

3. Delamater, A.R. et al. J Exp Psychol Anim Behav Process 35, 224-237 (2009).

4. Drexler, S. M. et al. Behav Neurosci 128, 474-481 (2014).

5. Haaker, J. et al. Proc Natl Acad Sci 110: e2428-2436. doi:10.1073/pnas.1303061110 (2013).

6. Jingji, J. & Maren, S. Scientific Reports 5: 8388. doi:10.1038/srep08388 (2015).

7. Lebron, K. et al. Learn Mem 11, 544-548 (2004).

8. Maren, S. Neurobiol Learn Mem 108, 88-95 (2014).

9. Orsini, C.A. et al. J Neurosci 31, 17269-17277 (2011).

10. Palomares-Castillo, E. et al. Brain Res 1476, 211-234 (2012).

11. Sah, P. & Westbrook, R.F. Nature 454, 589-590 (2008).

12. Sharpe, M. & Killcross, S. Neurobiol Learn Mem 118, 20-29 (2015).

13. Walker, D.L. & Davis, M. Pharmacol Biochem Be 71, 379-392 (2002).

14. Wang, H. et al. Pharmacol Biochem Be 124, 188-195 (2014).

15. Zelikowsky, M. et al. Biol Psychiat 73, 345-352 (2013).

This work was supported by The Association for the Development of Undergraduate Neuroscience Education (SRA & RLN), The Endowment for Science Education (EA), and The Synaptic State Faculty Research Foundation (EA). The authors thank Divya Mamootil, and the students in Neuroscience 101 for technical assistance, execution, and feedback on this lab exercise.

Address correspondence to: Divya Mamootil, Human Biology Department, University of Toronto, Toronto, CA Email: divya. mamootil@mail.utoronto.ca Copyright © 2015 Dr. Bill JU, Neurosciences, Human Biology Program

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Bridging the Gap in Traumatic Brain Injury: The promise of the Collagen Matrix

Catherine B. Matolcsy

Traumatic brain injury (TBI) is a multifaceted pathophysiology that kills or leaves millions of individuals with mental deficits worldwide each year. TBI affects more men than women, which is typically attributed to its association with impulsive decision-making and behaviour. TBI can be classified as a dual-insult pathology, whereby the primary injury occurs due to mechanical force being applied to the head as a whole or a localized region, and a temporally delayed and spatially more diffuse inflammatory and metabolic insult to cells in surrounding and functionally connected areas. Current treatment options for traumatic brain injury focus on preventing further deterioration, aiming to inhibit the action of the secondary metabolic insult, such as ensuring adequate blood flow, oxygenation, and scanning for stray skull fragments or foreign objects. Very little can be done currently to reverse or rehabilitate deficits resulting from primary impact. Current animal model studies focus on anti-inflammatory based minimization of tissue loss, as well as neurotrophic and stem cell therapies to promote migration and reintroduce new cells into the lesioned area to conceivably improve behavioural and cognitive abilities post-trauma. Exogenous collagen matrix grafting has proven to be a promising mechanism to promote recovery of endogenous cells in many somatic systems, including bone, teeth, liver and bladder. Most recently, collagen grafts have been employed following brain injury in rodents and have resulted in encouraging outcomes including cognitive improvement.

Key words: collagen matrix graft, traumatic brain injury (TBI), controlled cortical impact (CCI), spatial memory, Morris water maze (MWM), spatial learning, motor function, neurotrophic factors, glial scar, induced pluripotent stem cells-(iPSCs) Background TBI is a top cause of death in individuals under 35 years of age in developed countries, yet very few treatment options are available. TBI, which encompasses penetrating and non-penetrating occurrences of severe mechanical force applied to the head, is often characterized by neuronal loss, inflammation, and neurological deficits both acutely and long-term.1 Researchers have focused on providing a steppingstone for neuronal tissue to repair itself and to allow the tissue to potentially reform connections that were lost due to injury. Some have focused on purely structural models2, whereas others have integrated known neurotrophic factors and or stem cells into their constructs.2,3,4,5 Alternative approaches include noninvasive anti-inflammatories, beta-adrenoreceptor antagonists, and even cognitive-rehabilitation.6,7,8,9 Shin et al. elucidate the benefits of an exogenous bovine collagen scaffold in a rodent model following controlled cortical impact (CCI), which is used as a model of TBI.1 The results suggest a decrease in injury size due to increased neuronal survival, and ameliorated spatial learning in treated vs. non-treated TBI mice.1 Collagen has intrinsic abilities to aid in neuronal survival, and cellular migration, yet whether this is the means of action of collagen in ameliorating outcome is yet to be confirmed.2 No exogenous growth factors are used to promote regeneration in this study. These findings demonstrate a progressive advance in the field, they give hope that matrix grafts are beneficial in TBI, but leave many questions unanswered.

Shin et al. have managed to answer the ‘what’ of the effects of collagen matrices in TBI but have not yet tackled the ‘how’. The field must work to elucidate the mechanisms underlying these benefits and seek further ways to ameliorate the efficacy of the grafts, potentially incorporating other therapeutics or technologies.

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Research Overview

Summary of Major Results and Discussion

Experiments were carried out in male rats. Four cohorts were used; TBI rats with collagen matrix graft treatment (IC), sham surgery rats with collagen matrix graft treatment (SC), TBI rats with no treatment (IN) and sham rats with no treatment (SN). TBI was induced under anesthesia using craniectomy CCI procedure; CCI exposes brain tissue by disrupting the meninges; thus facilitating collagen matrix graft implantation.1 II.i MOTOR FUNCTION In beam balance and beam walking tests for motor function, IN and IC performed equally poorer than both SC and SN rats.1 However, no significant difference between IN and IC, or SC and SN, confirming that collagen implantation does not intrinsically alter motor function in days 1-5 post injury. Beam time was normalized to a pre-treatment trial time on an individual basis.1 Further precautions to ensure equivalent motor capabilities between groups included measurement of swim speeds, which were similar in all cohorts.

Other treatment approaches have accomplished amelioration not limited to cognitive function but also including motor function. Introduction of an enriching environment versus a standard environment during recovery promotes cognitive & motor rehabilitation. Furthermore, introducing iPSCs into lesion site one week post trauma have proven more effective than collagen grafts in promoting motor function recovery aswell.8 II.ii SPATIAL MEMORY & RETENTION Amelioration in spatial memory and retention was observed with collagen implantation, as was assessed by a hidden platform Morris water maze. IC rats localized a submerged platform in reduced time versus IN

rats (Figure 1).1 A probe trial, whereby animals were placed in the MWM from which the platform had been removed, was used as a measurement of memory retention, where longer time spent in the correct quadrant equates to better retention. IN and IC spent equally less time in the correct quadrant versus both SC and SN rats.1 Spatial memory retention is distinct from spatial memory not only temporally but also by the structural and anatomical cortical connectivities being employed. Retention relies on a more expansive circuitry within the hippocampus, meaning on a crude level, it requires more functionally intact hippocampi than spatial memory formation alone.

Specific areas of the hippocampus, especially the CA3 and longitudinal axons of these cells connecting them to one another in CA3-CA3 networks, as well as in connecting CA3-CA1 neurons are essential for spatial memory retention.10 These details indicate that potentially not only cell number should be considered but also the functionality of their connections, as the number of cells present does not fully encompass the function of the hippocampus.

Swim speed tests were revisited to confirm differences in MWM performance were not due to motor capability disparities.1 No significant variations in speed were noted between groups, eliminating ability to swim as an explanation of differences in performance.

both reduced versus SN and SC, as expected (Figure 3).1 The improvements in cell numbers are significant yet remain substantially reduced versus sham-surgery mice, being that IC remained approximately 35-40% below sham levels in hippocampal neuron count. To ensure lesion volume reduction did not result due to increased glial scarring, GFAP staining of astrocytes at injury site was performed. No differences in IN versus IC astrocyte number or morphology were observed in the pericontusional regions indicating that no extensively abnormal glial scaring was visible due to collagen graft impantation.1 This however, does not dismiss the potential involvement of astrocytes or other glial cells in the recovery and increased rehabilitation in collagen graft treated rats. Ensuring that glial scaring, which is typically known to contain injuries within boarders in the central nervous system, did not increase following collagen graft implantation does not eliminate their possible involvement, as it only measures their level and morphology at a distinct time point well into the recovery process (19d). It can be postulated that potentially collagen provides a means of attracting and promoting scar formation more quickly and efficiently, resulting in less penumbral damage and less spread of injury.11,12 Therefore, not necessarily resulting in any heterogeneity of the glial scar at 19 days post-trauma yet, variations may be visualizable at an earlier time point in recovery. Further studies with earlier sacrifice would allow the definitive rejection of astrocytes as an involved party in the differences in recovery between IN and IC groups.

Finally, trichrome staining of lesion site slices was used to visualize remnants of exogenous collagen.

No collagen matrix remained bordering the CCI site.

Many other studies have provided similar reductions in lesion volume and increased neuronal survival employing both collagen and other ECM molecules in various cortical injury models including stroke and surgical brain trauma .7,9 Conclusions and Discussion

Conclusions Figure 1. Spatial memory assay using MWM task. Collagen matrix treatment rats locate hidden platform in less time than non-treated rats, demonstrating better spatial memory in IC versus IN cohorts..1

II.iii LESION SITE PROPERTIES Post-mortem dissections were fixed, sliced and coverslipped for lesion volume studies using light microscope.

Lesion volume was significantly reduced in IC versus IN rats (Figure 2). Although lesion volume reduction seems triumphant at first appraisal, it is possible that scar tissue, or non-functional cell types have infested the penumbral region of the injury, resulting it what appears to be a less extensive lesion. However, this maintenance of tissue correlated to enhanced neuronal survival/migration as CA1 and CA3 hippocampal neuron numbers were significantly higher in IC versus IN, yet

Improvements in spatial memory, hippocampal cell number and reduced lesion volume have all been attained using collagen matrix grafting in a rat model following TBI.1 Stem cells and environmental stimulation have proven advantageous in motor function recovery.8 The field of TBI treatment is divided by multiple approaches which seek therapeutics stemming from various disciplines including, cellular, molecular, behavioural and pharmacological strategies. Historically, medicine has demonstrated that a unification of methodologies typically proves superior to any single approach alone, this should be considered in future studies.

Criticisms and Future Directions

Much potential exists in the field of TBI treatment for question and explanation that must be addressed by future research. Firstly as mentioned throughout the literature, head injury leading to TBI is not typically predicted, and is typically not treated immediately after 182

Figure 2. Tissue lesion after sacrifice,19 days post-injury. Collagen graft treatment (B) resulted in reduced lesion volume versus non-treatment group (A). Quantified lesion volume differences are significant (C).1

Figure 3. Hippocampal CA1 and CA3 neuronal cell numbers. Both CA1 (A) and CA3 (B) neuronal counts were significantly less diminished in IC versus IN cohorts, however cell number remained significantly decreased in IC verus SN.1

the injury occurs. Shin et al. used an experimental design whereby the grafts were implanted immediately following controlled cortical impact (CCI), which would be highly unlikely to replicate in clinical application of the technique.1,2 This time delay in implantation would foreseeably affect the efficacy of the treatment.

Certain studies have allowed between 24 hours and 7 days time between injury and surgical implantation.2,5 183

Future experiments should allow a reasonable time delay, congruent with what would be observed in human clinical application, before treatment application.

Secondly, as data regarding cell numbers and growth was only measured at one time point, which was considered the end of the study, the progression of the cellsâ&#x20AC;&#x2122; migration and integration into the

grafted area was not fully tracked. The Shin et al. study investigated only the absolute number of neurons present after the allotted time post-injury had passed, others have used multiple staining and visualization techniques to localize exact cell types at and around the lesion and graft site.2 Factors such as von Willebrand factor(vWf), NeuN, DCX and Tau-1 have been utilized by other members of the field to visualize endothelial cells, neurons, neural progenitors and axons respectively.2 Capturing only the final result of implantation of this graft fails to explain the progression of how cells came to be more numerous in the case of a collagen graft implantation.

Additionally the study failed to explain why collagen was so efficacious as a matrix for minimizing injury in TBI. The authors hypothesized that this could be attributed to collagenâ&#x20AC;&#x2122;s intrinsic tendency to stimulate neurogenesis through its degradation by other cell types, which other applications of collagen have demonstrated in the past.1,4,7 Using techniques that allow the elucidation of interactions between collagen and the surface receptors on the cells interacting with collagen would allow for a more conclusive argument regarding its function in this model. The effect of collagen in this study however, due to itâ&#x20AC;&#x2122;s immediate introduction post trauma, could possible be due to an inherent anti-inflammatory property that would not prove clinically relevant, as immediate implantation is not realistic. Comparison of the effects of collagen scaffold graft to the effects observed by a known substance that prevents inflammation, and immune system mediated propagation of damage, such as statins, progesterone, PPAR agonists, or minocycline would aid in explicating the effects of collagen.11 Finally, the world of regenerative medicine is becoming increasingly more focused on stem cell therapy and an inter-disciplinary approach. Past research has demonstrated that the benefits of incorporating more pro-growth factors in the injury site along with a graft.2,4, as well as stem cell treatment in combination with collagen scaffolding results in lesser inflammation and cytokine response.5 Future investigations regarding graft implantation with neural and perivascular stem cells, and potential neurotrophic factors would aid to bridge the gap between cellular and molecular therapeutics.3,5,11

5. De Freitas, H.T. et al. (2015) Effect of the treatment of focal brain ablation in rat with bone marrow mesenchymal stromal cells on sensorimotor recovery and cytokine production. J Neurol Sci. 348(1-2):166-73. doi: 10.1016/j. jns.2014.11.032 6. Park, H.Y., Maitra, K., Martinez, K.M. (2015) The Effect of Occupation-based Cognitive Rehabilitation for Traumatic Brain Injury: A Meta-analysis of Randomized Controlled Trials. Occup Ther Int. [Epub ahead of print] doi: 10.1002/oti.1389.

7. Huang, K.F., Hsu, W.C., Chiu, W.T., Wang, J.Y. (2012).

Functional improvement and neurogenesis after collagen-GAG matrix implantation into surgical brain trauma. Biomaterials.

33(7): 2067-75. doi: 10.1016/j.biomaterials.2011.11.040.

8. Dunkerson, J. et al. (2014) Combining enriched environment and induced pluripotent stem cell therapy results in improved cognitive and motor function following traumatic brain injury. Restor Neurol Neurosci. 32(5):675-87. doi: 10.3233/RNN-140408.

9. Ning, R. et al. (2014) Neamine induces neuroprotection after acute ischemic stroke in type one diabetic rats. Neurosci.

257:76-85. doi: 10.1016/j.neuroscience.2013.10.071.

10. Steffenach, H.A., Sloviter, R.S., Moser, E.I., Moser, M.B. (2002) Impaired retention of spatial memory after transection of longitudinally oriented axons of hippocampal CA3 pyramidal cells. Proc Natl Acad Sci. 99(5):3194-8.

11. Kumar, A., Loane, D.J. (2012) Neuroinflammation after traumatic brain injury: opportunities for therapeutic intervention. Brain, Behaviour, and Immunity. 26(8):1191-1201. doi: 10.1016/j.bbi.2012.06.008 12. Burda, J.E., Bernstein, A.M., Sofroniew, M.V. (2015) Astrocyte roles in traumatic brain injury. Exp Neurol. [Epub ahead of print] doi: 10.1016/j.expneurol.2015.03.020.

This work was supported by The Association for the Development of Undergraduate Neuroscience Education (SRA & RLN), The Endowment for Science Education (EA), and The Synaptic State Faculty Research Foundation (EA). The authors thank Mr. Spine L. Cord, Dr. Amy G. Dala, and the students in Neuroscience 101 for technical assistance, execution, and feedback on this lab exercise.

References 1. Shin, S.C., et al. (2015) Neuroprotective effects of collagen matrix in rats after traumatic brain injury. Restorative Neurology and Neuroscience. [Epub ahead of print] doi: 10.3233/RNN-140430 2. Elias, P.Z., Spector, M. (2012) Treatment of penetrating brain injury in a rat model using collagen scaffolds incorporating soluble Nogo receptor. Journal of Tissue Engineering and Regenerative Medicine. 9:137-150. doi: 10.1002/term.1621 3. Crapo, P.M., Tottey, S., Silvka, P.F., Stephen, F. (2014) Effects of biological scaffolds on human stem cells and implications for CNS tissue engineering. Tissue Engineering. 20(1-2):312-23.

4. Han, S. et al. (2015) The linear-ordered collagen scaffoldBDNF complex significantly promotes functional recovery after completely transected spinal cord injury in canine. Biomaterials. 41:89-96. doi: 10.1016/j.biomaterials.2014. 11.031.

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The potential for epigenetic treatment of neuropsychological disorders.

Lucy McPhee

Epigenetics are a rising area of research for neuropsychological disorders such as depression, Alzheimerâ&#x20AC;&#x2122;s Disease and Huntingtonâ&#x20AC;&#x2122;s Disease. Hypermethylation of genes can lead to reduced expression by the formation of heterochromatin. By determining which genes of interest are differentially methylated from the average, it is possible to determine which genes are being underexpressed leading to the disordered phenotype. This leads to the potential for epigenetic treatments for neurological disorders. The most studied and most promising of these is histone deacteylase inhibitors, which are currently most commonly studied in cancer research. Other treatments which warrant more research include DNMT inhibitors and non coding RNAs. Key words: neuropsychological disorders; epigenetics; methylation; Histone deacetylases (HDAC); HDAC inhibitors Background Neuropsychological disorders are often discussed in psychology and neuroscience through changes in structure and activation, or neurotransmitter levels seen in the brain of disordered individuals, but it is not clear why or when these changes occur (Schwartz and Monk, 2014). With an average age of onset of 12 years, anxiety disorders are likely rooted in childhood, but much more research still needs to be done to determine the real cause. A promising epigenetics study of the amygdalas of young, anxious temperament disorder rhesus monkeys opens up the possibility that methylation of genes in development may be a factor in ATD and later anxiety disorders (Alisch et al., 2014).

Epigenetics are not a part of the actual genetic code, but can be maintained through mitotic division, and can therefore affect an entire cell type or area (Mensaert, et al. 2014). DNA methylation is the most commonly studied variety of epigenetic modification, and it also the area examined in the aforementioned paper. In general, hypermethylation leads to reduced gene expression which is more often the cause of disorder than hypomethylation. This is a fairly new area of study when it comes to mental disorders, but several other studies have also implicated that epigenetics are involved. It is difficult because brain tissue is required to run epigenetic tests, so most work is done in animal models. However there have been human twin studies that implicate epigenetics in major depressive disorder (Davies et al., 2014), and other human studies have suggested that changes in genetic methylation are related to schizophrenia diagnosis (Chase et al., 2013).

The current gold standard of determining epigenetic modifcations is whole genome bisulphite sequencing (WGBS), which detects hydroxymethylation of cytosines in the genome with good resolution. Analysis involved comparison of CpG regions to the average to determine whether they are hyper- or hypo-methylated (Mensaert, et al. 2014). The drawbacks of this technique are that it is very expensive and not very efficient. However it does provide a chance to identify epigenetic areas for risk for mental illness, which could lead to pharmacological treatments. The main treatment focus currently is on histone deacetylase (HDAC) inhibitors, which can reverse hypermethylation. These molecules reduce DNA methylation and 185

can open up chromatin structure leading to enhanced gene expression (Narayan & Dragunow, 2009).

Research Overview

Summary of Major Results

Anxious temperament (AT) rhesus monkeys were analyzed for differential methylation of the amygdala using reduced representation bisuphate sequening by Alisch et al. AT phenotype was associated with hypermethylation and reduced gene expression of BCL11A and JAG1 genes. BCL11A is a downstream glutamate receptor effector that plays a role in neurite branching.

JAG1 is a NOTCH receptor which is involved in plasticity and the formation of spatial memories. Sites on these genes with the most statistically different methylation patters look as though they will impact binding sites, or noncoding RNA regulatory sites (Alisch et al, 2014).

Using immunoprecipitation and ultra-deep sequencing, Davies et al discovered that the gene ZBTB20 is hypermethylated in individuals with major depressive disorder. It normally plays a role in the development of the hippocampus, long term potentiation and NMDA receptor functioning. This method is not as reliable as the gold standard of bisulphate sequencing, but it uses periphery blood samples instead of brain tissue and can therefore be used in living patients (Davies, 2014).

Schizophrenia is the mental illness which is most commonly associated with gene transcription errors.

Using post mortem immunoblotting, Chase et al found increased methylation of the gene H3K9 in individuals with schizophrenia compared to controls. H3K9me2 can lead to formation of even more heterochromatin, further silencing genes in the parietal cortex, which could be an explanation for the disordered thought and disrupted sensory perception observed in many individuals with schizophrenia (Chase et al, 2013).

Low transcription levels of reelin is one of the most commonly found signs in post mortem analysis of schizophrenia patients, which is important in cortical development and hippocampal functioning via interaction with NMDA receptors. Hypermethylation of the reelin promoter has been observed in schizophrenia patients, which is thought to be the cause of the low

expression of reelin, suggesting it may be a potential cause of schizophrenia itself (Grayson, et al., 2006).

As reduced gene expression due to hypermethylation seems to be a possible cause for neuropsychological disorder, new treatment theories are emerging which attempt to reverse harmful methylation and return gene expression to normal. One of the most popular mechanisms for this is HDAC inhibitors, such as valproic acid (VPA), which reduce the removal of methyl groups from histones (Figure 1), allowing increased access to transcription factors (Fuchikami et al, 2015). HDAC expression increases with age, and inhibitors have demonstrated the ability to help protect aging axons from damage and ischemic injury in the optic nerves of mice (Baltan, 2012). They have also been used as a treatment for transgenic Huntington Disease mice.

This study found that the HDAC inhibitor treatment led to beneficial changes in cognition and motor function compared to controls (Figure 2). The main systems affected seem to the related to ubiquitin, which is important in homeostasis, division, and neuronal functioning (Jia et al, 2012). In mouse models, HDAC inhibitors have been shows to have antidepressant effects, even more so when combined with SSRI treatment (Schroeder, et al., 2007).

Conclusions and Discussion

Conclusions

These preliminary studies suggest that hypermethylation of genes related to neuronal growth and differentiation may be responsible for part of the pathology of neuropsychological disorders. Methylation levels in general or for specific genes are correlated with affect in all of the discussed disorders. Following from this theory, demethylation of genes is a possible novel treatment for these disorders. Histone deacetylase inhibitors have been suggested as a possible method through which this could be achieved, but

Figure 1: DNA methylation and histone acetylation (Narayan & Dragunow, 2010)

there is still much to be learned. However this field holds the potential to understanding the genetic and molecular causes of neuropsychological disorders, which are currently very much in the dark. The most popular area of epigenomic research right now is in cancer treatment, where the goal of the treatment is to kill off the cancer cells to treat the patient (Minucci & Pelicci, 2006). This method does not translate well to neuropsychological disorder treatment, because neuronal death is much more likely to be the cause of the disorder than any helpful treatment. Therefore new methods must be created in order to use epigenetics to treat neurological disorders.

Criticisms and Future Directions

While there is increasing research into the theory that hypermethylation may be a cause of these disorders, there is a dearth of research related to possible treatments if this is the case. Most studies indicating promising

Figure 2: Treatment with HDAC inhibitor improved open field test activity in HD mice (Jia, et al., 2012).

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effects use animal models, but a clinical study treating Alzheimer’s Disease patients with valproic acid showed a worsening of aggressive symptoms compared to a control. However AD is a very complex disease with many molecules involved, this one trial does not mean that HDAC inhibitors will never work for any neurological disorder (Narayan & Dragunow, 2010). Further trials are needed, both animal and human, for all neuropsychological disorders before this can be considered as a useful therapy possibility.

A recurring problem with HDAC inhibitor treatments is that they often have trouble bypassing the blood brain barrier (BBB). One recent study addresses this problem by developing BBB permeable HDAC inhibitors, which would reduce the doses needed for effective treatment if this does become a neuropsychological therapy in future (Seo et al., 2014).

DNA methyltransferase (DNMT) inhibitors are also an area of interest in epigenetic treatments. They have been shown to stop apoptosis in cultured mouse motor neurons, which is promising, but there is very little research on their potential for neurological disorder treatment (Chestnut, et al., 2011). DNMTs have been implicated in the GABA hypothesis of major depression, but no studies have been done investigating antidepressant effects of DNMT inhibitors (Luscher, et al., 2011). Non coding RNAs are also being developed, which can help stop pathological mRNA expression, but this is an even newer field which needs further exporation as well (Qureshi & Mehler, 2013).

References 1. Alisch RS., Chopra, P., Fox AS., Chen K., White ATJ., Rosebloom PH., Keles S., Kelin NH. (2014). Differentially Methylated Plasticity Genes in the Amygdala of Young Primates are Linked to Anxious Temperament, an at Risk Phenotype for Anxiety and Depressive Disorders. The Journal of Neuroscience, 34(47), 15548-15556.

2. Baltan S. (2012) Histone deacetylase inhibitors preserve function in aging axons. J Neurochem. 123(S2):108-115.

3. Chase KA., Gavin DP., Guidotti A., Sharma RP. (2013) Histone methylation at H3K9: evidence for restrictive epigenome in schizophrenia. Schizophren Res. 149(1-3):15-20.

4. Chestnut BA., Chang Q., Price A., Lesuisse C., Wong M., Martin LJ. (2011) Epigenetic regulation of motor neuron cell death through DNA methylation. J Neurosci. 31(46):1661916636.

5. Davies MN., Krause L., Bell JT., Gao F., Ward KJ., Wu H et al. (2014) Hypermethylation in the ZBTB20 gene is associated with major depressive disorder. Genome Biol.

15(4):R56 6. Fuchikami M., Yamamoto S., Morinobu S., Okada S., Yamawaki Y., Yamawaki S. (2015) The potential use of histone deacetylase inhibitors in treatment of depression. Prog Neuro-Psychopharmacol Biol. Psychiatry. In press S02785846(15) 7. Grayson DR., Chen Y., Costa E., Dong E., Guidotti A., Kundakovic M., et al. (2006) The human reelin gene: Transcription factors (+), repressors (-), and the methylation 187

switch (+/-) in schizophrenia. Pharmacol Ther. 111(1):272286.

8. Jia H., Kast RJ., Steffan JS., Thomas EA. (2012) Selective histone deacetylase (HDAC) inhibition imparts beneficial effects in Huntington’s disease mice: implications for the ubiquitin – proteasomal and autophagy systems. Human Molecular Genetics. 21(24):5280-5293 9. Luscher B., Shen Q., Sahir N. (2011) The GABAergic deficit hypothesis of major depressive disorder. Mol Psychiatry.

16(4):383-406.

10. Melucci S., Pelicci PG. (2006) Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer. Nat Rev Cancer. 6(1):38-51.

11. Mensaert, K., Denil S., Trooskens G.,Van Criekinge W., Thas O., De Meyer T. (2014) Next-Generation Technologies and Data Analytical Approaches for Epigenomics. Environmental and Molecular Mutagenesis. 55:155-170.

12. Narayan P., Dragunow N. (2009) Pharmacology of epigenetics in brain disorders. British Journal of Pharmacology. 159:285-303.

13. Qureshi IA., Mehler MF. (2013) Developing epigenetic diagnostics and therapeutics for brain disorders. Trends Mol Med. 19(12):online.

14. Schroeder FA., Lin CL., Crusio WE., Akbarian S. (2007) Antidepressant-like effects of the histone deacetylase inhibitor, sodium butyrate, in the mouse. Biol Psychiatry. 62(1):55-64.

Distinguishing the neurobiological features of resilient cognition in Alzheimer’s Disease

Amaara Mohammed

The presence of neuropathological plaques and tangles in the aging population has long been associated with Alzheimer’s Disease (AD). Yet, this pathology does not always result in impaired cognition. This article reviews the neurobiological differences found in the resilient cognition of those with AD pathology. Several synaptic, cellular and biochemical features were found to be distinct in those with resilient cognition and those with AD Dementia.

Key words: Alzheimer’s Disease; Synaptophysin; Synaptopodin; Cognition Background The neurodegenerative disorder Alzheimer’s Disease (AD) is currently the most common cause of dementia and affects millions of people around the world.¹ There are multiple risk factors associated with AD, including genetic factors, hypertension, diet and most significantly, age. Individuals over the age of 65 are most vulnerable to the disease and at this point, the risk increases every 5 years.¹ Alzheimer’s Disease was first described over 100 years ago by Alois Alzheimer in Germany, characterising the first case with memory impairments and the presence of neuropathological plaques and tangles, which today, are major indications of the disease.² Progressive memory loss is the clinical trademark of AD but eventually, cognitive function also deteriorates.³ The neuropathological trademarks of AD involve the accumulation of β amyloid (Aβ) proteins expressed as plaques and the phosphorylation of tau proteins expressed as neurofibrillary tangles.³ The formation of these plaques and tangles are estimated to begin 20 years before clinical symptoms arise.² MRI studies have shown the association of AD with hippocampal atrophy, however, it remains difficult to distinguish from other forms of dementia.⁴ However, this pathology is also known to be present without the impairment of cognitive function.⁵ Recently, there has been a number of studies investigating this incongruity between pathology and cognition, all of which reported similar dissonance, remarkably in older individuals.⁶⁻⁸ It was found that one third of older individuals have plaques and tangles that meet the National Institute on Aging criteria for the likelihood of developing AD, despite them having normal cognition.⁹⁻¹⁰ There is evidently a spectrum or range of brain pathology that occurs amidst cognitive function: there are those with healthy cognition with no signs of neurodegeneration, those with intact cognition despite evidence of AD pathology and those with impaired cognition with AD pathology.¹¹ While this inconsistency between pathology and cognition has been recognised for many years, there has been little postmortem examination into the the neurobiological factors responsible. Arnold et al. (2013) describe and identify the cellular, biochemical and synaptic composition that distinguishes resilient cognition in the presence of AD pathology.

Research Overview

Summary of Major Results

After characterising cognition levels and clinical diagnoses of participants annually, brain autopsies and neuropathological diagnoses of AD were made.

Participants were grouped according to pathology and cognition including those with high cognition but highest quartile for pathology were categorised as “AD Resilient”, those with low cognition and high pathology as “AD Dementia” and those with high cognition scores and low pathology as “Normal Comparison” (NC).

After measuring the densities of Aβ plaques and PHF-tau tangles in the mid frontal gyrus cortex, it was found that these densities were considerably larger in the AD-Resilient and AD-Dementia groups than in the NC. While the differences were not very significant, overall brain weight of AD-Dementia group were lower than the others, and NeuN neuron density of NC group was lower than that of the other two groups.

The densities of GFAP astrocyte in AD-Resilient was greater than those of AD-Dementia and NC groups.

The AD-Dementia group showed low measures of postsynaptic spine count densities and presynaptic vesicle synaptophysin immunoreactivity while they were retained in the AD-Resilient and NC groups (Fig. 1).

In an attempt to investigate the relation between the pathologic lesions with neurons, astrocytes and synapses, Aβ plaques were also positively correlated with GFAP astrocytes but negatively correlated with NeuN neurons, synaptopodin spines and synaptophysin immunoreactivity. PHF-tau tangles were negatively correlated, most significantly with synaptopodin spines and positively correlated with GFAP astrocytes.

The antibody microarray analysis showed a clear distinction between the groups. There was a tight congregation of proteins in the NC group while it was more dispersed in the other groups, suggesting a clear distinction in the protein expression profile of healthy brain tissue from those with AD pathology. 16 proteins were identified, distinguishing the AD-Resilient and AD-Dementia groups.

These results indicate a clear distinction of the cellular, synaptic and biochemical features of resilient cognition in AD pathology from dementia and controls.

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Fig 1. Photomicrographs illustrating the immunostained appearance of typical case from each group. Graphs representing the means along with standard of error bars. A: Aβ plaques, B: tau neurofibrillary tangles, C: NeuN neurons, D: glial fibrillary acidic protein astrocytes, E: Synaptopodin spines, and F: Synaptophysin spines in AD-Resilient (AD-R), AD-Dementia (AD-D) and Normal Comparison (NC).

Conclusions and Discussion Understanding the neurobiological foundations of expression in Alzheimer’s disease may be what is essential in developing new advances in maintaining cognitive function.

Cellular and synaptic features Astrocytes play an important role in the repair and maintenance of a healthy brain by providing biochemical, structural, detoxifying and nutritional support to endothelial cells and neurons.¹¹ The observed increase of GFAP astrocytes in the AD-Resilient group suggests that cognition is rectified in AD pathology through these functioning’s.

The severity of dementia in AD is often correlated to synaptic loss and has been associated with the collection of Aβ plaques and PHF- tau tangles.¹² Synaptophysin is an extensively studied synaptic vesicle membrane protein present in the presynaptic terminals whose normal expression demonstrates intact connectivity. This is consistent with the findings of normal synaptophysin expression in the AD-Resilient group, showing conserved connectivity while the AD-Dementia group showed a reduction of synaptophysin expression, indicating severed connectivity.

Dendritic spines are postsynaptic membrane with specialised roles and are important to the connectivity of neurons in the brain. Using synaptopodin to label dendritic spines, it was found that spine densities were reduced in the AD-Dementia group indicating reduced connectivity while the AD-Resilient group showed normal densities. This preservation of both postsynaptic synaptopodin and presynaptic synaptophysin in AD-Resilient supports the notion of conserved synaptic connectivity. Synaptic connectivity plays an important role in neurotransmission, signal transduction, longterm potentiation, learning, and memory¹, all of which are severely comprised in dementia.¹³ 189

Biochemical- protein expression The tight congregation of protein expression in the NC suggests a coherent profile while the dissipated protein expression in AD-Resilient and AD-Dementia indicates pathology. Proteins distinguishing AD-Resilient from AD-Dementia were identified, including the Aβ precursor binding protein, which is shields against memory loss and interleukin-3 which inhibits neuron death brought about by Aβ, were increased in the AD-Resilient group. This explains the preservation of cognition in this group. However, proteins involved in phosphorylation of tau (Serine/threonine protein phosphatase) and apoptosis (tumour necrosis factor receptor) were expressed more in the AD-Dementia group, resulting in impairment of cognition.

Criticisms and Conclusions

Like every experiment, it is essential to take into account the strength and weaknesses. The sample classifications functioned to enhance the contrast of pathology and cognition between the AD-Resilient and AD-Dementia groups. The participants were part of the Religious Orders Study (ROS), making them quintessential due to their consistency in nutrition, education, health care and other lifestyle factors. These factors may cause generalisation among other populations but for this study, a more divergent lifestyle factors may pose to confound the experiment.

While postmortem neuropathology continues to be the leading method in studying brain diseases and its neurobiology, it only provides information about pathology near the time of death and postmortem changes can alter the brain chemically and cellularly.

Finally, the use of two-dimensional profile counting, optical density and an automated computer-assisted analyses of fractional area determination resulted in a minimisation of operator bias and finding betweengroup differences. However, it may have also formed

systematic inaccuracies that could have been avoided using a reference volume-based approach instead.

Regardless, the findings of this study clearly shows that the resilient brain can be distinguished on a cellular and biochemical level that can form the foundation in the development of prevention and treatment procedures for dementia.

Future Directions

The identification of the candidate proteins, (interleukin-3, β-amyloid precursor binding protein, and serine/threonine protein phosphotase) found in AD-Resilient and AD-Dementia require further analysis, but these findings have provided a framework in which diagnostic, prevention, and treatment methods for dementia can be developed.

These proteins can serve as potential biomarkers in the diagnoses in early stages of AD. There has been evidence which showed a significant increase in the concentrations of interleukin-3, and interleukin-11 in the serum of AD patients when compared to controls and decreased interleukin-3 in the the CSF of AD patients.¹⁴ This suggests that interleukin-3 can be a strong candidate for a biomarker in Alzheimer’s Disease.

Evidence also shows that the treatment of metal ionophores (PBT2, Prana Biotechnology) on transgenic mice resulted in a decrease of Aβ plaques and PHF tau tangles, which significantly improved learning and memory performance on the Morris water maze.¹⁵ A follow up study analysed the proteins associated in the synaptic conditions involved with the PBT2 treatment.¹⁶ LTP, which involves the strengthening of dendritic spines are dependent on NMDA receptors and CaMKII. Both these proteins were increased consistently with the increase of spine density after PBT2 treatment. In addition, there was an increase in the elements involved in the BDNF pathway which is responsible for the health of dendritic spines, including pro-BDNF and TrkB after treatment.

The discovery of the proteins that distinguishes resilient cognition in AD pathology is only a stepping stone as to what can be accomplished in the future. As it currently stands, by the age of diagnosis, the brain is already severely damaged, therefore improvement and development of early diagnostic methods are essential.

Recognising and establishing the neurobiological differences between AD-Dementia and AD-Resilient can lead to boundless opportunities in the evolution of new and early prevention and treatment options for AD. References 1. Oboudiyat, C., Glazer, H., Seifan, A., Greer, C. & Isaacson, R. Alzheimer’s Disease. Seminars in Neurology 33, 313-329 (2013).

2. Blennow, K., J de Leon, M. & Zetterberg, H. Alzheimer’s Disease. Lancet 368, 387-403 (2006).

3. Bennett, D. et al. Epigenomics of Alzheimer’s disease.

Translational Research 165, 200-220 (2015).

4. Frisoni, G. et al. Hippocampal and entorhinal cortex atrophy in frontotemporal dementia and Alzheimer’s disease.

Neurology 52, 91-91 (1999).

5. Hyman, B. et al. National Institute on Aging–Alzheimer’s Association guidelines for the neuropathologic assessment of Alzheimer’s disease. Alzheimer’s & Dementia 8, 1-13 (2012).

6. O’Brien, R.J., Resnick, S.M., Zonderman, A.B., Ferrucci, L., Crain, B.J., Pletnikova, O., Rudow, G., Iacono, D., Riudavets, M.A., Driscoll, I., Price, D.L., Martin, L.J., Troncoso, J.C. Neuropathologic studies of the Baltimore Longitudinal Study of Aging (BLSA). J. Alzheimers Dis. 18, 665–675 (2009).

7. White, L. Brain lesions at autopsy in older JapaneseAmerican men as related to cognitive impairment and dementia in the final years of life: a summary report from the HonoluluAsia Aging Study. J. Alzheimers Dis. 18, 713–725 (2009).

8. Haroutunian, V., Schnaider-Beeri, M., Schmeidler, J., Wysocki, M., Purohit, D.P., Perl, D.P., Libow, L.S., Lesser, G.T., Maroukian, M., Grossman, H.T. Role of the neuropathology of Alzheimer disease in dementia in the oldest-old.

Arch. Neurol. 65, 1211–1217 (2008).

9. Bennett, D.A., Schneider, J.A., Arvanitakis, Z., Kelly, J.F., Aggarwal, N.T., Shah, R.C., Wilson, R.S. Neuropathology of older persons without cognitive impairment from two community-based studies. Neurology 66, 1837–1844 (2006).

10. Schneider, J.A., Arvanitakis, Z., Bang, W., Bennett, D.A. Mixed brain pathologies account for most dementia cases in community dwelling older persons. Neurology 69, 2197–2204 (2007).

11. Arnold, S. et al. Cellular, synaptic, and biochemical features of resilient cognition in Alzheimer’s disease. Neurobiology of Aging 34, 157-168 (2013).

12. Arendt, T. Synaptic degeneration in Alzheimer’s disease.

Acta Neuropathol. 118, 167–179 (2009).

13. Alvarez, V.A., Sabatini, B.L. Anatomical and physiological plasticity of dendritic spines. Annu. Rev. Neurosci. 30, 79–97 (2007).

14. Bahl JMC, Simonsen AH, Larsen SO, Skogstrand K, Waldemar G, et al. Putative Biomarkers For Alzheimer’s Disease; Interleukin- 3, Interleukin-11 and Macrophage Inflammatory Protein-1 Delta in Serum and Cerebrospinal Fluid. Supplement 4(6): 34 (2010).

15. Adlard PA, Cherny RA, Finkelstein DI, Gautier E, Robb E, et al. Rapid restoration of cognition in Alzheimer’s transgenic mice with 8-hydroxy quinoline analogs is associated with decreased interstitial Abeta. Neuron 59: 43–55 (2008).

16. Adlard PA, Bica L, White AR, Nurjono M, Filiz G, et al. Metal Ionophore Treatment Restores Dendritic Spine Density and Synaptic Protein Levels in a Mouse Model of Alzheimer’s Disease. PLoS ONE 6(3): e17669 (2011).

Copyright © 2015 Dr. Bill JU, Neurosciences, Human Biology Program

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Musical experience enhances cognitive performance among the aging population Arinda Muntean

Activities such as playing an instrument can have some serious implications on enhancing the brain as we age.

It can improve our memory, speech perception and cognitive performance. It is important to begin developing these skills at an earlier age as it is easier to take up new interests and learn quickly and practice should be continued even until adulthood in order to attain the aforementioned result. This will only become more crucial as the population shifts towards higher life expectancy rates so that declines in cognition function due to agerelated causes can be mitigated. The purpose of this study was to investigate if adults from ages 45-65 that have had previous musical experience show an enhanced overall performance in three areas related to cognition: speech in noise perception, auditory and visual working memory, and auditory acuity. Various tests have been conducted for each of the previous assessments and results have shown that musically experienced older adults do exhibit improved results in some of the tasks. Further research must be conducted in this growing area of study in order to understand the underlying mechanisms that drive cognitive functioning.

Key words: Auditory memory, Auditory acuity, Age, Hearing in noise, Musical experience, Speech in noise perception.

Background As we age, it becomes increasingly difficult to understand what is being said as background noise makes it harder to pay attention. Some of these conditions that we are aware of vary from the simple increase in volume in background noise, to mechanisms within the brain affiliated with the reduction of memory and attention. This study focused on how the understanding of speech in noise (SIN) differed between aging musicians and non-musicians, specifically if there were significant brain changes that have eventually come to adapt to noise, thus making it easier to hear in this type of environment (Parbery-Clark, et al., 2011). This topic becomes especially relevant to how effective communication can be as people age and as the average lifespan is extended due to technological advances, suggesting that musical experience can mitigate the effects of age-related cognitive decline.

A previous study by Jentschz et al., has indicated that among a younger cohort, musical experience provides an advantage in improving behavioral responses in the frontal cortex as cognitive conflict appears. Not only are various brain structures affected during ageing (Huang et al., 2010), life experiences such as physical activity and any other activities that engage the brain, have an impact on cognitive abilities, which should be taken into effect for future studies (Anderson et al., 2013). Bugos et al. specified that musical practice for an extensive amount of time (i.e. six months) even for amateurs have led to an increased and effective performance in working memory, though the results were not sustained after several months of delay, suggesting that practice is key when cognitive responses are in focus. It is already known that the strength of auditory processing can be enhanced through time and that the amount of musical experience can actually increase cognitive functioning; however, Parbery-Clark et al. aimed to test these findings on an older group of adults between the ages of 45 and 65, which is not specifically unique among this type of study (Bugos et al., 2007). Various studies have aimed at a wide range of ages in order 191

to compare their results pertaining to age, as well as maintaining a consistent method (Strait et al., 2010).

A study focused on a specific age range is significant because much of previous research has only focused on a younger cohort of musicians, providing evidence that there is in fact an enhancement in cognitive functioning due to musical experience; however, research on older adults can answer questions in regards to being able to extend this cognitive performance as musical experience continues throughout oneâ&#x20AC;&#x2122;s lifetime. It is important to understand what is happening in older adults, considering that as the average length of life increases, one will inevitably experience decline in cognitive function for a longer amount of time. Thus, this is one of many fields of studies that can aid in improving the aging populationâ&#x20AC;&#x2122;s lifestyle.

Research Overview

Summary of Major Results

Approximately half of the participants that were considered musicians played an instrument for most of their lives and were asked to continue practicing at least thrice a week, while the rest of the participants had either never played an instrument or had less than 3 years of musical experience. There were 3 parameters of the brain in which participants were asked to engage in various tasks: speech perception in noise, working memory, and auditory temporal acuity.

3 different tests were used to determine the perception of speech in noise. The first test was called the Hearing in Noise Test (HINT) which played varying intensity level sentences in conjunction with a preset intensity level noise, and repeated the task. Similarly, the next test, QuickSIN, also appointed the task of repeating sentences through varying levels of noise. Sentences were played at 70 dB and had a signal to noise ratio of 25dB which then decreased as the task progressed.

The last test was called the Words in Noise Test (WIN) was similar to QuickSIN but started at a different signal to noise ratio. For all three tests, a better performance

was portrayed by obtaining a low score based on the signal to noise ratio. Results showed that musicians did achieve a lower score than non-musicians (Figure 1).

SIN results correlated with auditory memory and the backward masking task. The three SIN tests were also analyzed among each other where QuickSIN and HINT showed no correlation while no significant relationship was observed between HINT and WIN. Parbery-Clark et al. suggested that this may be due to the difference in the mechanism these tests target.

Auditory memory was assessed through the WoodcockJohnson III test of Cognitive Abilities which consisted of spoken words and numbers that were then recalled in the same order or reversed order. Visual working memory was assessed through the Visual Working Memory subtest (VWM). There were 8 boxes that changed colour and participants were asked to click on the boxes in the order that they changed colour, either in the forward or reverse order. Higher scores were indicative of better performances on both tests. The correlation found between the SIN results and the auditory memory tests were analyzed between musicians and non-musicians in Table 1. Figure 2 presents findings that show that while auditory working memory in musicians produced higher results, visual working memory did not have an impact on musicians or non-musicians.

In order to test for auditory acuity, a backward masking task from the IHR Multi-center Battery for Auditory Processing was used to determine to what degree the participants’ hearing can pinpoint certain changes in noise perception. Musicians demonstrated a better performance for this task, in which a lower dB target sound and therefore, a lower threshold signified the enhanced ability to perceive sounds amid various noises.

Correlations were also analyzed between musicians and age of when they began practicing; however, since this study particularly looked at a specific age range (3-8 years), there was not significant correlation observed.

Additionally, the researchers analyzed whether years of musical experience among musicians had an impact on cognitive function and found that there was no significant relationship as well. While comparing musicians and non-musicians exclusively, results indicated that musicians did have better cognitive performance, which is a result that is in line with previous studies on this subject.

Figure 1. Musicians demonstrated improved performance (i.e. a lower score) among the SIN tests (QuickSIN, HINT, and WIN) as well as the backwards masking task that assessed auditory temporal acuity. Source: doi:10.1371/journal.pone.0018082.g001

Figure 2. Results indicate that participants with musical experience had an increase in efficacy of auditory working memory, though no change was observed among the two groups when assessed for visual working memory. Source: doi:10.1371/ journal.pone.0018082.g002

Discussion The authors concluded that among an older cohort, participants with extensive amounts of musical experience had improved SIN results and auditory working memory and acuity compared to non-musicians (Parbery-Clark et al., 2011). SIN perception has not only been tested on older adults, but also young adults in order to compare results and see if there is in fact a decline due to age. This type of perception test is a good assessor of auditory cognition function as the participant must be able to focus on a sentence being read while distracted by various background noise thresholds (Parbery-Clark et al., 2011). The effects of the SIN perception test are almost equivocal to situations involving communication with others in one’s daily life. Other studies such as, Zendel et al., have shown enhanced auditory performance not only in SIN perception but also gap detection among musicians, suggesting that musical experience may have other positive advantages over those who haven’t practiced music. Parbery-Clark et al. indicate in their study that SIN perception can be slightly dependent on auditory working memory. For example, example, musicians had better SIN scores due to an increased auditory working memory capacity targeted by musical training. It was also discussed that SIN perception was modulated by an extensive period of musical training (Parbery-Clark et al., 2011).

A strength found within this study was that the researchers aimed to focus their question towards an older cohort, considering that musical experience could have a potential long-term effect rather than studying the effects only on young adults. This information can provide essential knowledge for mitigating the hearing impairments that come with a decline in age. Additionally, this study provided further evidence that cognitive function can be strengthened through the practice of musical instruments , which was the subject of another study done by Wong et al. Their results demonstrated that musicians had enhanced cortical responses within the brainstem and of auditory information processing (Wong et al., 2007).

The results of this study can be an indication that having musical experience can be beneficial and could compensate for the decline in hearing due to age. By taking a simple aspect in everyone’s daily life (communication amid background noise) this study was able to demonstrate that there was a positive relationship between musical experience and better performance on the various auditory tasks.

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Table 1. Performance at auditory and visual tasks for musicians and non-musicians. HINT, QuickSIN, and WIN were the SIN perception tests. The backward masking test (BM) assessed auditory acuity. A lower score in the previous four tests indicates better performance. If scores were higher for auditory working memory (AWM) and visual working memory (VWM), this indicated better performance as well. The most significant results regarding improved cognitive function in musicians was observed in BM and AWM tasks. Source: doi:10.1371/journal.pone.0018082.t003

Conclusions

Although this study found a positive correlation, the results demonstrate that this was incremental knowledge gained because many previous studies have already determined that having some sort of musical experience enhances cognitive performance, despite the age range. In addition the correlation between musical experience and enhanced performance did not seem strong enough to determine that musical experience can aid in adapting to the aging process; however, it is important to note that this could be due to age-related impairments that could already setback cognitive performance.

Even through there is still more research to be done in this field, the new knowledge coming from this investigation and multiple others can aid in ways to treat and mitigate the effects of decline in hearing and can be further extended to how strong the impact could be compared with learning music at different ages.

Criticisms and Future Directions

A limitation to this study was that the correlation between the age the musician began practicing an instrument and measuring cognitive skills was a weak relationship because all musiciansâ&#x20AC;&#x2122; starting age ranged from 3-8, which presented a limited set of values to analyze. Thus, future studies could investigate whether picking up an instrument at various ages, such as during early and late adolescence, or early and late adulthood, could impact cognitive and auditory performance. One study by Bugos et al. focused on the older adult cohort, ranging from ages 60 to 85, to determine whether training in piano practice could improve working memory for those that havenâ&#x20AC;&#x2122;t had much musical experience throughout their lifetimes.

The results provided evidence that those participants that di d engage in Individualized Piano Instruction or the IPI program showed an improved attention and concentration for cognitive tasks over a sustained amount of time (Bugos et al., 2007). What could be extrapolated from this study is the IPI program, which not only provides instruction in musical theory and literacy, it also included dexterity exercises as soon as the participant was ready to play an instrument. Therefore, an extension to Parbery-Clark et al. study is to apply this method to other age groups and investigate its effect on cognitive skills.

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In the Parbery-Clark et al. study, attention was not a primary parameter measured, however, it is important to note that the simple task of paying attention to a speaker aids in the overall understanding of speech during communication in daily life. Strait et al. investigated the effect that experience in music had on auditory and visual attention. An experimental method that could be incorporated for future studies include tasks that were computer simulated based and compared reactions times of various cues (Strait et al., 2010). For example, the task that measured visual attention required the participants to observe a character and to respond by pressing a button if the character moved (Strait et al., 2010). The task eventually became difficult when participants were asked not to respond to certain cues that occurred before characterâ&#x20AC;&#x2122;s movement. The auditory attention task was the same as the visual task, though the cues were sounds instead.

Finally, in the current study, how there was no correlation between musical experience and an improvement in visual working memory. With no valid reason to explain this, there leaves no room but to further investigate the cause. Perhaps there is no true correlation found in older adults, but a study by Huang et al. demonstrated that when young adult, musically experienced participants were asked to perform a verbal memory task, there was activation in the visual cortex compared to no activation in non-musicians (Huang et al., 2010). Parbery-Clark et al. measured visual memory through computer-based visual exercises; however, there could be a link to inducing the visual cortex by another area of the brain. As a result, an experimental method that could be used consists of various tasks in which participants were asked to listen to 20 simple words (that were related to fruits, animals, tools, and landscapes) and remember as many words as possible as well as categorize the words as natural or artificial objects (this would induce stronger encoding) (Strait et al., 2010). Then, they were asked to retrieve as many words as they can silently while pressing a button each time a word was remembered. The recalling process was done in an MRI scanner in order to visualize what was occurring in the brain. This method could be employed on older adults as well, whether they were musicians or not, for activations in the visual cortex.

References 1. Anderson, S., White-Schwoch, T., Parbery-Clark, A., & Kraus, N. (2013). A dynamic auditory-cognitive system supports speech-in-noise perception in older adults. Hearing Research. 300:18-32.

2. Bugos, J. A., Perlstein, W.M, McCrae, C.S., Brophy, T.S., & Bedenbaugh, P.H. (2007) Individualized Piano Instruction Enhances Executive Functioning and Working Memory in Older Adults. Aging & Mental Health. 11:464-71.

3. Huang, Z., Zhang, J.X., Yang, Z., Dong, G., Wu, J., Chan, A.S., & Weng, X. (2010) Verbal Memory Retrieval Engages Visual Cortex in Musicians. Neuroscience 168: 179-89.

4. Jentzsch, I., Mkrtchian, A., & Kansal, N. (2014).

Improved effectiveness of performance monitoring in amateur instrumental musicians. Neuropsychologia, 52:117-124.

5. Parbery-Clark, A., Strait, D.L., Anderson, S., Hittner, E.,& Kraus, N. (2011) Musical Experience and the Aging Auditory System: Implications for Cognitive Abilities and Hearing Speech in Noise. PLoS One 6.

6. Strait, Dana L., Kraus, N., Parbery-Clark, A., & Ashley, R. (2010) Musical Experience Shapes Top-Down Auditory Mechanisms: Evidence from Masking and Auditory Attention Performance. Hearing research. 261: 22-29.

7. Wong, P., Skoe, E., Russo, N.M., Dees, T., & Kraus, N. (2007). Musical Experience Shapes Human Brainstem Encoding of Linguistic Pitch Patterns. Nature neuroscience.

10(4): 420-422.

8. Zendel, B.R., & Alain, C. (2012). Musicians Experience Less Age-related Decline In Central Auditory Processing. Psychology and Aging, 27(2), 410-417.

This work was supported by The Association for the Development of Undergraduate Neuroscience Education (SRA & RLN), The Endowment for Science Education (EA), and The Synaptic State Faculty Research Foundation (EA). The authors thank Mr. Spine L. Cord, Dr. Amy G. Dala, and the students in Neuroscience 101 for technical assistance, execution, and feedback on this lab exercise.

Address correspondence to: Dr. Rita L. Neurotrophin, Biology Department, 123 Growth Cone Avenue, Action Potential College, Hillock, IL 60101 Email: rln@apc.edu Copyright Š 2015 Dr. Bill JU, Neurosciences, Human Biology Program

194

Visualizing anxiety through mGlu7 receptor immunocytochemistry Jena, L Niceforo

Anxiety is a disorder which is thought to decrease the afflictedâ&#x20AC;&#x2122;s quality of life through compromised mental functioning. It can associate with comorbidities like diabetes and hypertension, and become a significant economical burden. Estimated to cause around 97.4 billion dollars in losses globally due to individualâ&#x20AC;&#x2122;s inability to function correctly. Not only are anxiety disorders very difficult to diagnose, but it is thought that about two thirds of cases are misdiagnosed due to either overlapping symptoms with depression or individuals not seeking help when they need it. In addition, it is known that if left untreated, anxiety can lead to suicidal thoughts and behaviors1. Some physical manifestations and symptoms of anxiety include problems sleeping, cold or sweaty hands and feet, shortness of breath, heart palpitations, an inability to be still or calm, dry mouth, numbness or tingling in the hands or feet, nausea, muscle tension, and dizziness. While its cause is unknown, A general study by Meszaros et al. associated negative life events with increased anxiety symptoms these studies used questionnaires to find that the symptoms of anxiety manifested themselves when many negative life events occurred2. These symptoms are believed to come from molecular changes in mGlu7 receptors in the hippocampus. An understanding of anxiety at the molecular level could lead to pharmacological treatments which will dramatically increase the quality of life for those suffering from anxiety. Through some of the molecular techniques mentioned in this review, we will be able to visualize anxiety through immunocytochemistry. We will then extrapolate the results to mouse models and perform tests for anxious behaviors once knockouts have been performed.

Key words are : Anxiety, immunocytochemistry, mice models, mGlu7 receptors, adenyl-cyclase Background Currently scientists have developed drug targets for mGlu2 and mGlu5 receptor subunits, however mGlu7 receptor subunits have not been examined pharmacologically. It is thought that these specific subunits play a large role in causing anxious behaviors in individuals that suffer from the condition. When mGlu7 receptors are activated, they trigger downstream effects in the hippocampus that cause anxious behaviors in people.

Finding the mechanisms of this pathway has significant implications for treatment of CNS disorders, not just anxiety and depression, but the sensation of pain, and even types of brain tumors, can be treated by finding a specific blocker for mGlu7 receptors3. The pathway in which glutamate acts to bind to ionotropic NMDA and AMPA receptors is the same in which it effects metabotropic glutamate receptors (mGlur). Available today are antidepressants or antianxiety medication that act as NMDAR agonists, which have significant adverse side effects like memory dysfunction, ataxia, neurodegeneration and drug dependence. This receptor is ineffective for attempting to alleviate symptoms of depression and anxious behaviors, attention has been shifted from NMDARs to mGlurs because of their specific role in causing anxious behaviors.

When attempting to target mGlurs pharmacologically you can create safer neuropharmacological drugs free of negative side effects4. mGlu7 receptors are coupled to G1/G0 proteins and when they are activated they are known to play a role in anxiety and depression. It is known that presynaptic mGlu7 autoreceptors control glutamate release in the CNS, while heteroreceptors control GABA release in the hippocampus. What scientists have inferred from immunohistochemistry performed on mGlu7 receptors in hippocampal slices is that mouse GABAergic nerve terminals have presynaptic mGlu7 heteroreceptors, and when they are activated it inhibits GABA exocy195

tosis. It was found that most nerve endings that had mGlu7 receptors were negatively coupled to adenyl cyclase activity5. When mGlu7 inhibits adenyl cyclase it effects glutamate release, it was also found that when mGlu7 receptors were exposed to agonists they facilitate glutamate release. In addition it is known that a genetic deletion of mGlu7 receptors reduces anxiety and depression by causing an increase in BDNF levels in the hippocampus6. This has significant implications for individuals who suffer from anxiety, studies in the past have showed that when mGlu7 knockouts in mice were performed it alleviated anxious behaviors. If a specific enzyme can be targeted to treat anxiety and depression than a pharmacological solution to anxiety can be presented without negative side effects, you can effectively prevent an individual from continuing to suffer from anxiety, depression panic attacks etc Research Overview

Summary of Major Results

Lacovelli et al. discovered that through the use of monoclonal anti-HA antibodies to bind to HEK293 cells that mGlu7 proteins are coupled to Gi and activate multiple signaling pathways, in addition it was found that adenyl cyclase activity was inhibited along with MAPK stimulation. When mGlu7 receptor expressing cells were given an agonist (L-AP4), the agonist stimulated cAMP formation, but when the blocker PTX was given to the cells it was found that inhibition of adenyl cyclase was prevented. Confirming that mGlu7 receptors were coupled to Gi proteins Suggesting that the Gi coupled protein is independent when it activates mGlu7 receptors. The researchers transfected cells with mGlu7 cDNA, it was found that cells that had inhibition of adenyl cyclase when the blocker L-AP4 was applied, its effect was neutralized

by cells expressing GRK4. In mGlu7 expressing cells L-AP4 stimulated ERK1/2 phosphorylation was fully desensitized. These couplings are shown on immunoblots. In addition it was discovered that β-arrestins could amplify ERK1/2 phosphorylation, and that the naturally occurring β-arrestin is required for the coupling of mGlu7 receptors to ERK1/2 activation6.

These results are consistent with the fact that there is a presence of mGlu7 receptors in the hippocampus, mice were tested using immunohistochemistry where mGlu7 receptors were thought to appear in the brain by using a specific antibody for them in rat brains. It was detected that the most abundant mGlu7 receptors were found in parts of the brain that were correlated with GABAergic synapses6.

Figure 1. Shows that when JNK is applied to mGlu7 coupled to β-arrestin knockouts that mGlu7 receptors are not as effectively activated6.

glutamate to be released instead7. In their 2010 study Weironska et al. found that when mGlu7 receptors were knocked out in mice GABAergic neurons could not function as well, which lead to some significant conclusions about mGlu7 receptors. That as the level of GAD proteins associated with mGlu7 receptors decreased in the hippocampus that the level of GABA enzymes also decreased8. This study filled in the gaps from the past, it monitored presynaptic GABAergic nerve terminals to visualize mGlu7 receptors mediating GABA release. mGlu7 receptors were found to inhibit GABA release and modulate NMDA receptors as well. This has a significant impact for the rest of the brain because of 5-HT receptors and NMDA agonists effect on controlling GABA outflow to the nerves6. It is significant if mGlu7 receptors play a role in modulating these GABAergic nerve terminals as well, not only would we have another target for treating dysfunction in the brain but we could specifically target mGlu7 receptors, in this pathway to attempt to develop a drug that has minimal amount of side effects while still targeting the specific over reactive receptor in your brain that causes you to experience anxiety. Not only are mGlu7 receptors affected by GABA release in the brain but other mGlu receptors found in that family too.

Each specific mGlu is responsible for a different type of behavior, what is specific about the mGlu7 receptor is that it is abundant in the brain and is responsible for the antidepressant quality of drugs in neurons that express GABA. When mGlu7 gets activated since it is coupled to Gi proteins that two cascade pathways are initiated by the same molecule, such that when knockouts are performed in mGlu7 pathway you have to be very specific which molecule you target. By specifically targeting a downstream effect of something initiated by the mGlu7 receptor (like β-arrestin) vs the Gi coupled protein. An individual would be able to theoretically eliminate what causes the symptoms of anxiety. The next step would be to extrapolate these results from cell cultures into mouse models and perform knockouts of each downstream effect from the pathway, and determine the phenotype of each mouse.

Perform tests for anxious behaviors and if the mouse cannot experience anxiety based on which molecule is knocked out, finally design a specific inhibitor for that molecule, which would be defined as a drug to cure anxiety or depression.

Criticisms and Future Directions Figure 2. Shows that when GRKs were added in mGlu7 receptors, ERK decreases, when L-AP4 is added, it strengthens mGlu7 receptors, the ERK response is stronger.

Research Overview

Conclusions and Discussion

Studies before this one have inferred that mGlu7 receptors exist presynaptically on GABAergic neurons, and because of that it was thought that mGlu7 receptors played a role in inhibiting GABA release, causing

The study showed that mGlu7 receptors are coupled to Gi proteins, yet a main criticism is that there was no inhibition of Gi protein at all, only mGlu7 receptors were inhibited in the study. Leaving a fairly wide gap, since Gi are coupled to mGlu7 receptors, once the receptors become activated that also turns on the pathway which the Gi proteins are coupled to. The researchers could have attempted to knock out the Gi coupled proteins using miRNA or a specific inhibitor for them. To see which effects of the pathway would still be viable. You could also attempt to visualize, once the Gi pathway has been neutralized using immunocytochemistry if only the mGlu7 receptors still produced downstream effects and where the effects would be.

If the entire pathway can be silenced from using an 196

inhibitor for Gi proteins, you need to target something farther downstream of mGlu7 receptors, you could act on β-arrestin. The next step after immunocytochemistry is performed is that you would want to extrapolate the results to mice models. You could silence any of the genes associating with mGlu7 like ERK1/2 and JNK using miRNA, you would have to sequence the gene in mice, then create a miRNA complementary to it, inject the mouse and then perform tests for anxious behaviors9. Some commonly used tests for anxious behaviors in mice would be the elevated plus maze or the open field test. If the mouse is feeling anxious it will tend to run along the walls of the maze, but if it is feeling relaxed it will run in the center. Ideally should the pathway be specific for anxiety, if you perform a knockout of the pathway in a mouse model you would have the mouse running throughout the maze regardless of what stimulus you present it with. The mouse will not be able to feel anxiety anymore once you silence the mGlu7 pathway. You could also delete a gene in a mouse that is specific for β-arrestin and see how that will effect the phenotype of the mouse, if the mouse will still be able to feel anxiety. Ideally if the science is sound behind these models, you could one day design a specific inhibitor for this mGlu7 pathway, in a pill form and give it to a person, to completely erase all anxious behaviors without negative side effects. Which would have huge implications for any individual that suffers from anxiety to one day be free from their disease permanently and to be able to lead a normal life, is what any person would want.

References 1. Olariu E, Forero CG, Castro-Rodriguez JI, Rodrigo-Calvo MT, Alvarez P,Martin-Lopez LM, Sanchez-Toto A, Adroher ND, Blasco-Cubedo MJ, Vilagut G, Fullana MA, Alonso J(2015) Detection of anxiety disorders in primary care: A Meta-analysis of assisted and unassisted diagnoses. Depress Anxiety. [Epub ahead of print] 2. Meszaros V, Ajtay G, Fodor K, Komlosi S, Boross V, Barna C, Udvardy-Meszaros A, Perczel Forintos D. From life events to symptoms of anxiety and depression: the role of dysfunctional attitudes and coping. Neurological Review. 67:397-408 3. Nicoletti F, Bruno V, Ngomba RT, Gradini R, Battaglia G, Metabotropic glutamate receptors as drug targets: What’s new. Current Opinion in Pharmacology. 20:89-94 4. Weironska JM, Lequtko B, Dudys D, Pilc A. Olfactory Bulbectomy and amitriptyline treatment influences mGlu receptors expression in the mouse brain hippocampus. Pharmacological Reports. 6:844-55 5. Summa M, Di Prisco S, Grillir M, Usai C, Marchi M, Pittaluga A. Presynaptic mGlu7 receptors control GABA release in mouse hippocampus. Neuropharmacology.66:215-24 6. Lacovelli L, Felicioni M, Nistico R, Nicoletti F, De Blasi A. Selective Regulation of recombinantly expressed mGlu7 receptors metabotropic glutamate receptors by G-protein coupled receptor kinases and arrestins. Neuropharmacology. 77:303-312 7. Schoepp D. Unveiling the functions of presynaptic metabotropic glutamate receptors in the central nervous system.

Journal of Pharmacological Experimental Theory. 299:12-20 197

8. Weironska M, Branski P, Siwek A, Dybala M, Nowak G, Pilc A. GABAergic dysfunction in mGlu7 receptor-deficient mice as reflected by decreased levels of glutamic acid decarboxylase 65 and 67kDa and increased reelin proteins in the hippocampus. Brain Research. 1334:12-14 9. Y, Zhang. Y, Wang. L, Wang. M, Bai. X, Zhang. X, Zhu. Dopamine receptor D2 and associated microRNAs are involved in stress susceptibility and resistance to escitalopram treatment.

2015. Int. J. Neuropsychopharmacology.[EPub Ahead of Print].

This work was supported by The Association for the Development of Undergraduate Neuroscience Education (SRA & RLN), The Endowment for Science Education (EA), and The Synaptic State Faculty Research Foundation (EA). The authors thank Mr. Spine L. Cord, Dr. Amy G. Dala, and the students in Neuroscience 101 for technical assistance, execution, and feedback on this lab exercise.

Address correspondence to: Dr. Rita L. Neurotrophin, Biology Department, 123 Growth Cone Avenue, Action Potential College, Hillock, IL 60101 Email: rln@apc.edu Copyright © 2015 Dr. Bill JU, Neurosciences, Human Biology Program

The Next Step in Antidepressant Therapy: BDNF Oscillation Patterns as a Potential Early Predictor for Therapy Response

Yuki Nishimura

Numerous studies have shown evidence for the critical role that brain-derived neurotrophic factors (BDNF) play in the development of stress-related mood ailments like major depressive disorders. Due to its association with potential antidepressant qualities, BDNF has been targeted in treatments for depression. One such method of antidepressant therapies includes partial sleep deprivation (PSD), which has been shown to cause a rapid increase in BDNF levels, and a short lived positive effect on patients suffering from depression.

Giese et al. (2014) treated 28 patients suffering from major depressive disorders using PSD, and evaluated blood serum levels of each patient before, during, and after antidepressant treatment. They found that after a 2-week follow up of these patients, all patientsâ&#x20AC;&#x2122; BDNF levels increased immediately after treatment, though the overall therapy response was short-lived. More importantly, they found that patients who had higher levels of diurnal BDNF prior to treatment had more significant increases in serum BDNF levels post-treatment, and that these patients reported better treatment responses than those patients who had lower, more un-responsive BDNF levels.

These results not only show that PSD treatments result in a boost in the BDNF levels, but also that pretreatment levels of BDNF in each patient could potentially predict therapy response. However, further studies are needed in order to examine whether stress hormone deregulation and pre-treatment cortisol levels could also be playing a role in antidepressant treatment responses.

Key words: BDNF; Major Depressive Disorder (MDD); Partial Sleep Deprivation; Antidepressant Therapy Response; Early Predictor.

Background Fluctuating levels of neurotrophic factors, such as BDNF levels in the hippocampal region of the brain, have been correlated with many stress-induced mood disorders (Duman and Monteggia, 2006). For example, when placed in a stressful environment where rats were subject to randomized foot shocks, BDNF levels decreased rapidly, suggesting a link between high stress levels and declined BDNF levels (Rasmussen et al., 2002). Furthermore, prenatal maternal deprivation of BDNF led to the birth of rats that had more trouble adjusting to stressful environments like the swim stress test (Roceri et al., 2002). This suggests that depriving BDNF during critical periods of fetal brain development could lead to drastic functional deficiencies and increased susceptibility to stress-related mood disorders like generalized anxiety (Cutuli et a., 2015).

Yet many of these deficiencies are changeable, as shown by the studies done in which boosting BDNF levels led to varying degrees of reversal or recovery of decreased brain function and stress management. One major application of this comes in the form of antidepressant treatments for major depressive disorder (MDD) patients. Many patients with depression show inherently lowered levels of BDNF (Shimizu et al., 2003), and antidepressant therapies have started to shift their focus towards increasing these BDNF levels as a potential method of treatment. For example, classical antidepressant therapies like monoamine oxidase inhibitors (MAOIs) and selective serotonin reuptake inhibitors (SSRIs) have both been shown to result in a slow increase of BDNF levels in patients (Nibuya et al, 2003; Coppell et al 2003). Although MDD does not necessarily result as a response to stressful environments, it has been well recognized that stress exacerbates MDD incidents (Gold

and Chrousos, 2002), and further studies have shown that MDD patients suffer not only a decreased BDNF level, but also a subsequent decrease in hippocampal size (Phillips et al., 2015).

Because of the growing consensus that BDNF has a critical role in many stress-related mood disorders like MDD, BDNF has been proposed as a potential biomarker for both diagnosing depression, and gauging antidepressant therapy efficacy (Karege et al., 2002). Although most studies have shown that post-treatment levels of BDNF rise in patients, some studies have also reported a group of non-responder patients, who show little to no evidence of increased BDNF levels (Molendijk et al., 2011). These nonresponder patients were also shown to have less success in antidepressant therapies. These studies suggested that the presence of an immediate surge in BDNF levels after antidepressant treatment was strongly correlated to treatment success. Although this allows clinicians to predict antidepressant therapy success, it can only be done after such treatment has been performed, which ultimately does little to help the already burdensome healthcare system. Instead, being able to predict whether antidepressant therapy will be effective prior to actual treatment will allow clinicians to make more economical and less timeconsuming treatment choices.

Research Overview

Summary of Major Results

In order to find an earlier predictor for antidepressant therapy treatment, Giese et al. (2014) examined patient serum BDNF levels prior to treatment admin198

istration to see whether predisposal to higher or lower BDNF levels resulted in improved treatment success.

In order to have rapid BDNF increase results, Giese et al. (2014) used partial sleep deprivation (PSD) treatments on 28 patients suffering from MDD, and all participants were evaluated using the Hamilton Depression Rating Scale (HDRS) prior to treatment.

The researchers collected blood serum from these patients for seven time periods, because BDNF is known to follow a daily circadian rhythm oscillation pattern in which BDNF levels are the highest in the morning and lowest during midnight (Begliuomini et al., 2008). During Day 0 (before PSD treatment was performed), they collected blood at 8am, 2pm, and 8pm to assess the baseline BDNF oscillation patterns for each patient. Then, during PSD (Day 1), they collected blood serum at 1:30am (right after patients were awoken for PSD), 8am, 2pm, and 8pm.

Two weeks after PSD treatment, all 28 patients were evaluated again using the HDRS scale in order to see how successful the treatment response was.

Results showed that all of the participants’ BDNF oscillation patterns on Day 0 prior to PSD treatment showed the highest levels in the morning (8am) and lowest at midnight (Day 1--1:30am), which was expected because previous studies found that BDNF levels oscillate in accordance with the circadian rhythm. After PSD treatment (Day 1), mean BDNF levels increased 10.4% (8am), 16.2% (2pm), and 20.7% (8pm) when compared to pre-treatment levels, showing that PSD resulted in rapid BDNF level increases. This result is in keeping with other similar studies done in which PSD treatments led to transient increases in BDNF levels (Beck et al., 2010).

However, two weeks after PSD, all participants took the HDRS questionnaire and those who scored lower on it, and therefore had better antidepressant results from the PSD, were classified as “long-term responders” (n=10), whereas those who did not show lasting treatment effects and scored higher HDRS scores were classified as “non-responders” (n=18).

Long-term responders not only had higher post-PSD

Figure 1. Diurnal serum BDNF levels. (A) Mean BDNF levels for all participants showed highest levels in the morning that steadily declined and reached its minimum levels around midnight, as was expected. (B) Post PSD treatment (Day 1) mean BDNF levels were higher than pre-PSD treatment (Day 0), showing that PSD treatment results in rapid BDNF level upsurges. (Giese et al., 2014)

(Day 1) BDNF levels than their counterparts, but also had higher serum BDNF levels pre-PSD treatment (Day 0), and had a general predisposition to higher levels of BDNF prior to any antidepressant treatment.

Conclusions and Discussion Partial sleep deprivation (PSD) is effective in inducing rapid increase in BDNF levels and garnering some relief from depressive episodes, yet both of these outcomes are short-lived, as was also seen in other studies done using PSD as the choice of antidepressant therapy (Giedke and Schwarzler, 2002).

Furthermore, similarly to other studies conducted by Lee and Kim (2008), Giese et al. found that antidepressant therapy resulted in some patients who had less obvious increases in BDNF levels (“nonresponder” patients), and that these patients had less success in antidepressant therapy effectiveness, as evidenced in the lack of significant difference in HDRS ratings before and after two weeks of treatment.

More importantly, however, Giese et al. showed that patient predisposition to stable BDNF circadian rhythms

Figure 2. Post-PSD Treatment serum BDNF levels. (A) Day 1 (after treatment) BDNF levels show little diurnal patterns. (B) Responder patients who had shown significant HDRS rating scale improvement after two weeks of treatment show that their BDNF levels oscillate in a more noticeable fashion than their counterpart, non-responder patients. (C) Relationship between HDRS improvement and posttreatment BDNF levels. The higher the BDNF level was after treatment, the more likely the PSD treatment effects would last.

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in which BDNF levels show clear signs of levels being highest in the morning and lowest at midnight, as well as a generally higher BDNF level prior to therapy are good predictors of antidepressant treatment efficacy and patient response to therapy. In short, clear diurnal BDNF oscillation patterns, coupled with higher levels of BDNF prior to treatment are good predictors of certain antidepressant therapy success. Therefore, BDNF’s role in neurogenesis and long-term potentiation are critical to mood disorders like major depressive disorder, and targeted studies into BDNF activity can prove worthwhile in Neuroscience and Psychiatry.

Criticisms and Future Directions

Although Giese et al. (2014) were not the first to posit that BDNF levels play a central role in antidepressant therapy efficacy, their study further supports that BDNF circadian rhythms could be an early predictor for how effective partial sleep deprivation treatment (and potentially other antidepressant therapies) will be. This study not only enhances the growing literature on the relationship between antidepressant therapy and BDNF levels, but also adds a new perspective by suggesting the potential of BDNF as a biomarker for early treatment efficacy predictor.

Unlike the SSRI study conducted by Wolkowitz et al.

(2011) that found similar BDNF potential as an early predictor of SSRI success, Giese et al.’s study was effective because it measured serum BDNF levels more frequently and allowed a thorough investigation into the patients’ diurnal patterns of BDNF prior to PSD treatment. However, the number of patients in this study was much lower in comparison to most other studies done on antidepressant therapies, and Giese et al. themselves saw the need for the same study to be conducted on a much wider patient scale. Prior studies like Tardic et al. (2011) have already suggested that post-treatment lack of increase in BDNF levels predict treatment ineffectiveness, so Giese et al. should focus their study on earlier, pre-treatment predictors of treatment efficacy, by collecting more data and blood samples prior to the beginning of treatment. Furthermore, because sleep deprivation has been linked to stress hormone deregulation by the hypothalamic-pituitary-adrenal (HPA) axis (Schule et al., 2001), further studies could be performed to see whether the patterns of cortisol levels relate to daily BDNF levels, and see whether pre-treatment cortisol levels and/or BDNF levels better predict the effectiveness of antidepressant treatments. By studying the HPA stress hormone regulation patterns, Giese et al. will better be able to explain the essential mechanisms underlying how to make antidepressant therapies more effective for patients.

References 1. Beck J et al. (2010) Modafinil reduces microsleep during partial sleep deprivation in depressed patients. J Psychiatr Res 44:853-64.

2. Begliuomini S et al. (2008) Plasma brain-derived neurotrophic factor daily variations in men: correlation with cortisol circadian rhythm. J Endorinol 197: 429-35.

3. Coppell A et al. (2003) Bi-phasic change in BDNF gene expression following antidepressant drug treatment. Neuropharmaology 44: 903-10.

4. Cutuli A et al. (2015) Pre-reproductive maternal enrichment influences rat maternal care and offspring developmental trajectories: behavioral performances and neuroplasticity correlates. Front Behav Neurosci 12:66-72.

5. Duman RS, Monteggia LM (2006) A neurotrophic model for stress-related mood disorders. Biol Psychiatry 12: 1116-27.

6. Giedke H, Schwarzler F (2002) Therapeutic use of sleep deprivation in depression. Sleep Med Rev 6: 361-77.

7. Giese M. et al. (2014) Fast BDNF serum level increase and diurnal BDNF oscillations are associated with therapeutic response after partial sleep deprivation. J Psychiatr Res 59: 1-7.

8. Gold PW, Chrousos GP. Organization of the stress system and its dysregulation in melancholic and atypical depression: high vs low CRH/NE states. Mol Psychiatry 7:254-75.

9. Karege F et al. (2002) Postnatal developmental profile of brain-derived neurotrophic factor in rat brain and platelets.

Neurosci Lett 328: 261-4.

10. Lee HY, Kim YK (2008) Plasma BDNF as a peripheral marker for the action mechanism of antidepressants. Neuropsychobiology 57: 194-9.

11. Molendijk et al. (2011) Serum levels of BDNF in MDD: State-trait issues, clinical features and pharmacological treatment. Mol Psychiatry 16: 1088-95.

12. Nibuya et al. (1995) Regulation of BDNF and trkB mRNA in rat brain by chronic electroconvulsive seizure and antidepressant drug treatments. J Neurosci 15: 7539-47.

13. Phillips JL et al. (2015) A prospective, longitudinal study of the effect of remission on cortical thickness and hippocampal volume in patients with treatment-resistant depression. Int J Neuropsychopharmacol.

14. Rasmussen A et al. (2002) Down-regulation of BDNF mRNA in the hippocampal dentate gyrus after re-axposure to cues previously associated with footshock. Neuropsycho 27: 133-42.

15. Roceri et al. (2002) Early maternal deprivation reduces the expression of BDNF and NMDA receptor subunits in rat hippocampus. Mol Psychiatry, 2: 609-16.

16. Schule C et al. (2001) Attenuation of HPA axis hyperactivity and simultaneous clinical deterioration in a depressed patient treated with mirtazapine. World J Biol Psychiatry 2: 103-5.

17. Shimizu et al. (2003) Alterations of serum levels of brainderived neurotrophic factor (BDNF) in depressed patients with or without antidepressants. Biol Psychiatry 54: 70-75.

18. Tardic A et al. (2011) The early non-increase of serum BDNF predicts failure of antidepressant treatment in patients with major depression: a pilot study. Prog Neuropsychopharmacol Biol Psychiatry 35: 415-20.

19. Wolkowitz OM et al. (2011) Serum BDNF levels before treatment predict SSRI response in depression.

Prof Neuropsychopharmacol Biol Psychiatry 35: 1623-30.

Copyright © 2015 Dr. Bill JU, Neurosciences, Human Biology Program 200

Further Insight on Using Mean Diffusivity as a Potential Biomarker to Identify Mild Cognitive Impairment Converters to Alzheimer’s Disease Original Article: Changes in White Matter Integrity Before Conversion From Mild Cognitive Impairment to Alzheimer’s Disease Miranda Nong

Alzheimer’s disease is a progressive neurodegenerative disease that causes memory and cognitive degeneration which eventually leads to dementia. Differences of gray matter and white matter has been studied in individuals with mild cognitive impairment that eventually develops Alzheimer’s Disease and those who do not.

Analysis techniques such as voxel-based morphometry allows for brain volumes to be examined and compared between the different groups. The apparent diffusion technique allows for mead diffusivity values to be obtained and once again compared with the different groups since it is a good indication of neuronal loss in brain structures. It can potentially be a biomarker for diagnosing individuals with Alzheimer’s disease years before clinical symptoms occur and can provide an early treatment for those individuals.

Key words: Alzheimer’s Disease (AD); white matter (WM); gray matter (GM); mean diffusivity (MD); mild cognitive impairment (MCI); voxel-based morphometry (VBM); apparent diffusion technique (ADC) Background Alzheimer’s disease (AD) is a progressive neurodegenerative disease that is a very common cause of dementia1. AD symptoms are not limited to memory loss, but also plays a role in the decline of many cognitive dependent components such as language dysfunction, loss of sight, decrease of attention, visuospatial difficulty, and personality changes usually associated with depression2. The neuropathology of AD is biochemically characterized to be due to the β amyloid and tau protein accumulation in the brain2, however there are still many other processes that underlies the pathology if AD. Through neuroimaging techniques, it has been found that AD is associated with the loss and atrophy of gray matter (GM) in many cortical and subcortical structures as well as the loss of white matter (WM) in the brain3. These processes and molecular changes within the brain could potentially act as a biological marker to identify those that have an onset of AD. Since AD is a progressive disease, there are different classifications of stages until it fully develops. Mild cognitive impairment (MCI) is a clinically identified early determinant stage of which an individual may or may not continue to develop AD4. MCI individuals has some cognitive impairment, but it is not enough to be identified as dementia and typically has memory deficit that is greater than their average age group5.

Individuals who exhibit MCI and continue on to developing AD are called converters, and those who do not, are called non-converters4. Studies distinguished that MCI converters show greater GM atrophy in the parietal and left temporal lobe6 and more WM lesions in periventricular brain regions7. If individuals with AD onset can be identified at the MCI stage, then an earlier treatment can be provided since it is estimated that AD pathology occurs 10 to 20 years before clinical symptoms show2.

Clark et al. used florbetapir-PET imaging was used to look at the density of β amyloid protein within the brain. This techniques allows the protein aggrega201

tion plaques to be identified in individuals that are approaching the end of their life. The purpose of this technique was to see the consistency of β amyloid plaques that are found in autopsies of AD individuals.

The identification of β amyloid aggregation in the brain could potentially be a biomarker to find individuals with AD onset. However this study was limited to a small sample size and the use of individuals that are capable of providing the data instead of MCI individuals.

As previously mentioned, GM and WM lesions differences has been observed in previous research, thus in the paper by Defrancesco et. al4, they focused on identifying the differences of MCI converters, MCI non-converters, and healthy individuals through neuroimaging techniques. The data obtained from different analyses based from neuroimaging could provide further insight on identifying individuals with early onset of AD. Research Overview Data from fifty five German speaking patients aged 62 or older were collected. They included 13 MCI converters, 14 MCI non-converters, and 28 healthy individuals. All patients were categorized into groups based on an interview and multiple psychological assessments which include the MMSE and CERAD battery tests to determine cognitive function levels.

The main method to attain the neuroimaging was through diffusion weighted imaging and T1-weighted MRI scans for each patient. A comparison between the scans would then be done through a voxel-based morphometry (VBM) analysis, which is a technique to compare brain volume4. Apparent diffusion Coefficient (ADC) maps were also constructed to reflect the mean diffusivity (MD) values. MD values are a scalar measurement3 of the total amount of diffusion within a VBM analysis. High MD values suggests neuronal loss and increased brain water content3.

Summary of Major Results and Discussion

Multiple Regression Analysis A multiple regression analysis is a comparison of variables. Figure 1 is a comparison between the neuropsychological tests taken by the participants and GM atrophy within the brain in a T1-weighted image of all participants. A positive correlation was found between worse test results for MMSE and verbal memory with higher GM atrophy of the left putamen/sublobar and left inferior frontal gyrus, respectively. MMSE8 is a psychological test that grades the cognitive state, a low test score suggests cognitive impairment which is in accordance with lesions of the brain structures.

Figure 1. The yellow is an indication of a positive correlation between low psychological test scores (MMSE and verbal memory) and high GM atrophy4.

VBM Analysis VBM analysis is a method to look at the brain volume and do a comparison between the three different groups: MCI converters, MCI non-converters, and healthy individuals. Figure 2 indicates that there is more GM atrophy in MCI converters in the left parietal lobe, the left putamen, the left insula, the right parahippocampal gyrus, the frontal lobe, and the left temporal lobe. Based on a comparison between MCI non-converters and normal healthy individuals, no difference between the brain volumes were seen.

Analysys of ADC Maps and Mean Diffusivity A comparison analysis is done with the MD values reflected from ADC maps from diffusion weighted images of the three group of patients. MD values are measured using gradient labelling techniques of water protons. An increase in MD value indicates atrophy of brain areas.

A comparison between MCI converters and nonconverters (Figure 3, left) showed a significant increase in MD values of GM in the left limbic lobe, right middle temporal lobe, basal ganglia, and increased MD values of WM in the parietal, frontal, and temporal lobe. The lesions of both GM and WM of MCI converters are very wide spread.

A comparison between MCI converters and healthy individuals (Figure 3, right) presented similar data, although the increase of MD values was slightly higher, it was not significant. Which can conclude that the difference between MCI non-converters and healthy individuals are not significant.

An overlap between the VBM analysis where brain volume is measured and ADC maps where MD values are calculated, there is overlap seen. Increase MD values and GM volume loss occur in most of the same structures. This suggests that volume loss is associated with neurodegeneration of the structure.

Conclusions Defrancesco et al. showed the differences between MCI converters, MCI non-converters, and healthy individuals using a neuroimaging technique that can potentially be used to diagnose the early onset of AD in the population. From this study it showed that changes in MD values reflected neuronal loss, thus can be used as an indication of neurodegeneration in different areas of the brain. From the difference of GM and WM lesions within the brain seen between MCI converters versus non-converters, it can be used as a biomarker to accurately identify the early onset of AD to allow for earlier treatment of AD. Criticisms and Future Directions

Figure 2. This is a T1 MRI scan that is superimposed to show the different areas (in yellow) where GM is lost in MCI converters versus MCI non-converters. GM loss is seen in the bilateral frontal lobe, the left parietal lobe, left temporal lobe, the left putamen, the left insula, and the right parahippocampal gyrus4.

Defrancesco et al provided solid results on the difference between MCI converters and non-converters based on neuroimaging analysis techniques. However, the paper still does not provide the rate at which MCI converters will develop a full onset of AD later in life.

With this, a long term analysis of current participants should be followed to show the growth of increasing GM and WM lesions as the disease progresses.

This paper provides substantial evidence on the widespread lesions of WM seen in the parietal, frontal, and temporal lobes by measure MD values, but these lesions could also be attributed to other neurodegenerative diseases that is not necessarily AD. Based on research by Nowrangi et al.10, they examined the fornix, which is a region of WM that connects the medial temporal lobes to the hypothalamus. The fornix also plays a role in semantic and episodic memory and is shown to have an altered structure from normal 202

Figure 3. The left side shows an increase in MD values between MCI converters and MCI non-converters. The right side shows an increase in MC values between MCI converters and healthy individuals.

in MCI and AD patients11. Diffusion tensor imaging is a preferred type of neuroimaging for examining WM and can be used to do a comparison between MCI converters, non-converters, and healthy individuals to look at specific regions of WM differences.

References 1. Barker, W. et al. Relative Frequencies of Alzheimer Disease, Lewy Body, Vascular and Frontotemporal Dementia, and Hippocampal Sclerosisin the State of Florida Brain Bank.

Alzheimer Dis Assoc Disord 16, 203-212 (2002).

2. Holtzman, D., Morris, J. & Goate, A. Alzheimer’s Disease: The Challenge of the Second Century. Sci Transl Med 77, 1-17 (2011).

3. Gold, B.T., Johnson, N.F., Powell, D.K. & Smith, C.D. White matter integrity and vulnerability to Alzheimer’s disease: preliminary findings and future directions.Biochimica et Biophysica 1822, 416-422 (2012).

4. Defrancesco, M. et al. Changes in White Matter Integrity before Conversion from Mild Cognitive Impairment to Alzheimer’s Disease. PLoS ONE 9, e106062. (2014).

5. Petersen, R. C. Mild cognitive impairment as a diagnostic entity. Journal of internal medicine 256, 183–194 (2004).

6. Karas, G, Sluimer, J & Goekoop, R. Amnestic mild cognitive impairment: structural MR imaging findings predictive of conversion to Alzheimer disease.American Journal 10, 944-949 (2008).

7. Defrancesco, M & Marksteiner, J. Impact of white matter lesions and cognitive deficits on conversion from mild cognitive impairment to Alzheimer’s disease. Journal of Alzheimer’s Disease 34, 665-672 (2013).

8. Folstein, M. F., Folstein, S. E. & McHugh, P. R. Mini-mental state: a practical method for grading the cognitive state of patients for the clinician. Journal of psychiatric research12, 189–198 (1975).

9. Nowrangi M.A. & Rosenberg P.B. The fornix in mild cognitive impairment and alzheimer’s disease. Front Aging Neurosci 7. 1-7 (2015) 203

10. Kehoe E.G. et al. Fornix white matter is correlated with resting-state functional connectivity of the thalamus and hippocampus in healthy aging but not in mild cognitive impairment – a preliminary study. Front Aging Neurosci 7. 1-10 (2015)

This work was supported by The Association for the Development

of Undergraduate Neuroscience Education (SRA & RLN), The Endowment for Science Education (EA), and The Synaptic State Faculty Research Foundation (EA). The authors thank Mr. Spine L. Cord, Dr. Amy G. Dala, and the students in Neuroscience 101 for technical assistance, execution, and feedback on this lab exercise.

Address correspondence to: Dr. Rita L. Neurotrophin, Biology Department, 123 Growth Cone Avenue, Action Potential College, Hillock, IL 60101 Email: rln@apc.edu Copyright © 2015 Dr. Bill JU, Neurosciences, Human Biology Program

Don’t Stress About it: 5HTT Genotype and Epigenetics Daria Pacurariu

Kumsta et al (2010) examined the relationship between 5HTT genotype, post-natal stress, and emotion regulation abilities in 127 participants. Their results show that s allele carriers are more susceptible to stress and have more emotion regulation difficulties. Early life stressors caused by institutional deprivation result in neurobiological alterations which impede proper affective processing, leading to emotional problems and possibly increasing the carrier’s vulnerability to developing psychopathologies. These neurobiological alterations may include epigenetic mechanisms such as methylation of the promoter region of the 5HTT gene. The field of epigenetics has recently been gaining importance due to its ability to provide a link between early life adversity and later depression-associated behavior (Dalton et al. 2014).

Key words: early life adversity, institutional deprivation, emotional regulation, 5HTT polymorphism, epigenetics Background The serotonin transporter protein (5HTT) has two polymorphisms in the 5’ flanking region known as 5-HTTLPR. The protein’s transcriptional activity is a direct result of which version of the allele an individual possesses. The short (s) allele has low transcriptional activity compared to the long (l) allele (Kumsta et al., 2010). This gene has been associated with emotional regulation and stress response. As previously observed, if the transporter is blocked with fluoxetine, a serotonin selective reuptake inhibitor, early after birth, it can lead to depressive behavior later in life (Ansorge et al. 2004). This indicates that a transient decrease in serotonin uptake during the postnatal period can have lasting impacts on the developing brain.

There have been both genetic and environmental factors associated with the expression of the 5HTT gene.

Early adverse experiences may have many negative long term effects, including maladaptive coping style, emotional regulation difficulties, and increased reactivity to stress (Sanchez et al., 2001). These effects are induced through epigenetics: alterations in the activity of genes due to processes like methylation and acetylation. A cytosine-phosphate-guanosine (CpG) island overlaps with the transcription start site of the 5HTT gene (Kinnally et al.,2010), so it is considered one of the key candidates for the epigenetic mechanisms brought on by stress. Methylation of this area will drastically reduce gene transcription and expression, and increase the possibility of developing depression.

An example of early life stress is disorganized parental attachment and maternal deprivation. If the offspring-parent bond is disorganized, as would be the case with a child reared in an institution, this can lead to increased incidence of psychopathology (Sroufe, 2005) such as personality disorders (Carlson, 1998). Early life stress has been studied in rodents and has been shown to increase glucocorticoid levels, elevate stress response, and worsen cognitive functioning (Kundakovic & Champagne, 2015). Moreover, maternal deprivation has also been demonstrated to have a weighty effect on rhesus macaques, as those raised without a mother displayed similar maladap-

tive phenotypes as the rodents (Harlow et al., 1965).

Although the evidence using animal models is plentiful, not many researchers have looked at the effects of early life stress on the psychological well-being of humans. The experiment by Kumsta et al. (2010) had two purposes: to discover a relationship between the different 5HTT polymorphisms and emotional regulation abilities of 11 year old individuals that have been exposed to early life deprivation, and to see whether this relationship relies on the occurrence of stressors during adolescence (age 11-15).

Research Overview

Summary of Major Results

The longitudinal experiment by Kumsta et al (2010) included 127 participants. The experimental group consisted of 51 participants born in Romania during the communist regime and placed in orphanages for varying time periods (6-42 months), after which they were adopted by wealthy families in the UK. The control group (n=74) consisted of Romanian babies that were institutionally deprived for less than 6 months and of within-UK adoptees. DNA was collected from buccal swabs and the polymorphism was sequenced using a PCR reaction.

Emotional regulation abilities were measured at ages 11 and 15 using the Rutter scale, and depression symptoms were measured using the Child and Adolescent Psychiatric Assessment Interview. As illustrated in Figure 1, the participants with genotypes s/l or s/s in the severe institutional deprivation group had the most emotional problems, but there was no significant difference between them. l/l genotypes had the least emotional problems. There were no significant differences in the comparison group, where genotype didn’t matter because individuals were not subjected to early life stressors. These results were replicated by Peterson et al (2012): they observed that adolescents with low transcriptional efficiency of the 5HTT gene (s/s genotype) whose mothers reported more stressful life events had a higher number of depressed/anxious behaviors (illustrated in Figure 2).

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Furthermore, the authors observed that cognitive and emotional impairments were relatively unwavering in the institutionally deprived group (Kumsta et al, 2010). This result is consistent with research

by Kinnally (2014), whose experiments with rhesus macaques demonstrate that early life adversity causes long lasting changes and can have pervasive effects on individuals.

Figure 1: Number of emotional problems (as measured by the Rutter Scale) associated with the 5HTT polymorphism in the institutionally deprived and control groups.

Kumsta R, Stevens S, Brookes K, Scholts W, Castle J, Beckett C, Kreppner J, Rutter M, Sonuga-Marke E (2010) 5HTT genotype moderates the influence of early institutional deprivation on emotional problems in adolescence: evidence from the English and Romanian Adoptee (ERA) study. Journal of Child Psychology and Psychiatry 51(7): 755–762

Conclusions and Discussions Kumsta et al (2010) demonstrated a relationship between severe institutional deprivation and emotional problems later in life, which is moderated by the individual’s 5HTT genotype. Their results reveal that stress has more adverse effects on individuals with a low transcriptional efficiency of the 5HTT gene.

This finding, while intriguing, is not novel. Evidence from Caspi et al. (2003) also shows that early life stress may play a critical role in the development of depressive behaviour, an effect which is mediated by the 5HTT genotype. According to their research, teenagers with the s allele are more sensitive to adverse life events and more prone to committing suicide.

In stating that cognitive and emotional impairments were relatively resolute in the institutionally deprived group, Kumsta et al (2010) are suggesting that early adverse experiences lead to stable neurobiological alterations which exert their effects later in life, perhaps through sensitization to stressors. Primate studies carried out by Kinnally (2014) examined epigenetic modifications of the 5HTT gene in response to varying levels of maternal care in rhesus macaques. Those with poorer maternal care experienced more postnatal stress and therefore more 5HTT methylation.

This epigenetic change continued manifesting itself 8 years later, and the macaques had lower overall health (defined as lower body weight and increased prevalence of diarrhea). Their results suggest that epigenetic modifications are long lasting and can have pervasive effects on individuals. By advancing our knowledge in this field, we could not only discover new markers for detecting susceptibility to depression, but also improve diagnosis and treatment of this psychopathology.

It has long been known that institutionally-raised offspring can develop disorganized attachment relationships which can drastically affect their adult lives (Kundakovic & Champagne, 2015). Through their research, Kumsta et al. (2010) shine light on the importance of creating positive caretaker-offspring relationships in order to augment attachment and elevate long term outcomes of institutionalized children.

Criticisms and Future Directions

Figure 2: Interaction between transcription efficiency of the 5HTT gene and the number of stressful life events moderates the amount of anxious depressed symptoms exhibited by adolescents Petersen, I. T., Bates, J. E., Goodnight, J. E., Dodge, K. E., Lansford, J. E., Pettit, G., Latendresse, S. J., Dick, D. M. (2012). Interaction Between Serotonin Transporter Polymorphism (5-HTTLPR) and Stressful Life Events in Adolescents’ Trajectories of Anxious/Depressed Symptoms. Developmental Psychology. 48 (5): 1463–1475 205

Critical Analysis The Kumsta et al (2010) paper had three weaknesses. First, the authors are not basing their reports on clinical depression diagnoses, simply on a wide range of emotional problems determined via measures like the Rutter Scale. These self report measures are completed by parents and children, and there is a possibility for bias and deception. Second, DNA information was only available for 127 participants, so the study had limited power. The final criticism is that while the authors alluded to a “neurobiological alteration”, they did not study this process in detail.

Future directions The main future direction suggested for Kumsta et al. would be to try to examine the altered genes of these Romanian orphans. While we are unable to inspect epigenetic changes in the brains of living individuals, it can be done in peripheral tissues (Kundakovic &

Champagne, 2015). A possible experiment that they should carry out involves comparing the amount of DNA methylation between the institutionally deprived and control groups. This can be done by extracting genomic DNA from blood leukocytes and quantifying it using bisulfide pyrosequencing (Zhao et al. 2013).

This process could elucidate part of the mechanism by which early life adversity affects later behavior.

Naumova et al (2012) obtained blood samples from adolescents that were raised by institutions or by their biological parents and analyzed whole genome methylation sequences. They discovered that children raised in institutions had a greater amount of methylation, particularly in genes involved in immune responsiveness and cellular signalling. Kinnally et al (2010) found similar patterns in rhesus macaques.

It is known that institutionally-raised children have a maladaptive stress response (Kundakovic & Champagne, 2015; Kumsta et al., 2010). Bernard and Dozier (2010) carried out an experiment examining salivary cortisol changes in children using hierarchical linear modeling. Their results demonstrated that infants with a disorganized attachment style (raised in an institution) showed a heightened cortisol response in comparison to the control group (those raised by their biological parents). It would be interesting to see whether there is a difference in salivary cortisol between participants with s/s, s/l, and l/l 5HTT genotypes. A possible future direction would be to measure the change in the level of cortisol before and after exposure to a stressor in a similar manner to Bernard and Dozier (2010), and to correlate that with the participant’s 5HTT genotype.

Lastly, the authors could explore the relationship between 5HTT genotype and amygdale activity in response to stress. Niklova et al. (2014) used blood oxygen level-dependent fMRI and found a positive correlation between 5HTT promoter methylation and amygdale activity. Although their results demonstrate that institutionally raised children who were exposed to post-natal stress have increased threat related amygdale activation, Kumsta et al should use this method to examine amygdale activation and its relationship with the 5HTT polymorphism.

References 1. Ansorge MS, Zhou M, Lira A, Hen R, Gingrich JA (2004) Early-life blockade of the 5-HT transporter alters emotional behavior in adult mice. Science 306: 879–881.

2. Carlson EA (1998) A prospective longitudinal study of attachment disorganization/ disorientation. Child Dev 69: 1107–1128.

3. Caspi A, Sugden K, Moffitt T E, Taylor A, Craig I W, Harrington H, Poulton R (2003) Influence of life stress on depression: Moderation by a polymorphism in the 5-HTT gene. Science 301: 386– 389.

4. Dalton V S, Kolshus E, Loughlin, D M (2014) Epigenetics and depression: return of the repressed. Journal of Affective Disorders. 155: 1–12 5. Harlow HF, Dodsworth RO, Harlow MK (1965) Total social isolation in monkeys. Proc Natl Acad Sci USA 54: 90–97.

6. Kinnally EL (2014) Epigenetic Plasticity Following Early Stress Predicts Long-Term Health Outcomes in Rhesus Macaques. AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 155:192–199 7. Kinnally EL, Capitanio JP, Leibel R, Deng L, LeDuc C, Haghighi F, Mann JJ (2010) Epigenetic regulation of serotonin transporter expression and behavior in infant rhesus macaques. Genes, Brain and Behavior 9: 575–582 8. Kumsta R, Stevens S, Brookes K, Scholts W, Castle J, Beckett C, Kreppner J, Rutter M, Sonuga-Marke E (2010) 5HTT genotype moderates the influence of early institutional deprivation on emotional problems in adolescence: evidence from the English and Romanian Adoptee (ERA) study. Journal of Child Psychology and Psychiatry 51(7): 755–762 9. Kundakovic M, Champagne FA (2015) Early-Life Experience, Epigenetics, and the Developing Brain. Neuropsychopharmacology REVIEWS 40: 141–153 10. Naumova OY, Lee M, Koposov R, Szyf M, Dozier M, Grigorenko EL (2012) Differential patterns of whole-genome DNA methylation in institutionalized children and children raised by their biological parents. Dev Psychopathol 24: 143–155.

11. Nikolova YS, Koenen KC, Galea S, Wang C, Seney ML, Sibille E, Williamson D E, Hariri A R (2014) Beyond genotype: serotonin transporter epigenetic modification predicts human brain function. Nature Neuroscience 17(9): 1153-1158 12. Petersen IT, Bates J E, Goodnight JE, Dodge KE, Lansford J E, Pettit G, Latendresse SJ, Dick DM (2012) Interaction Between Serotonin Transporter Polymorphism (5-HTTLPR) and Stressful Life Events in Adolescents’ Trajectories of Anxious/Depressed Symptoms. Developmental Psychology 48 (5): 1463–1475 13. Sanchez MM, Ladd CO, Plotsky PM (2001) Early adverse experience as a developmental risk factor for later psychopathology: evidence from rodent and primate models.

Dev. Psychopathol 13: 419–449.

14. Sroufe LA (2005) Attachment and development: a prospective, longitudinal study from birth to adulthood. Attach Hum Dev 7: 349–367.

15. Zhao J, Goldberg J, Bremner JD, Vaccarino V (2013) Association Between Promoter Methylation of Serotonin Transporter Gene and Depressive Symptoms: A Monozygotic Twin Study. Psychosomatic Medicine 75, 523-529.

This work was supported by The Association for the Development of Undergraduate Neuroscience Education (SRA & RLN), The Endowment for Science Education (EA), and The Synaptic State Faculty Research Foundation (EA). The authors thank Mr. Spine L. Cord, Dr. Amy G. Dala, and the students in Neuroscience 101 for technical assistance, execution, and feedback on this lab exercise.

Copyright © 2015 Dr. Bill JU, Neurosciences, Human Biology Program

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Chronic Sleep Deprivation is Enough Induce Neuronal Degeneration Hyun Park

The rapidly developing technology industry has enabled modern society to shorten sleep times, yet the consequences of these conveniences on the brain are largely unknown. The locus ceruleus neurons (LCn) are metabolically wake-active neurons that fire during wakefulness. Impairment in this area from a sleep deprivation stressor was hypothesized to result in metabolic response failures. Dinucleotide-dependent deacetylase sirtuin type 3 (SirT3) is a pathway responsible for regulating metabolic processes such as redox homeostasis and energy production. In SirT3wt mice, SirT3 was up-regulated during brief wakefulness (Sh Wake) whereas mice lacking SirT3 function encountered oxidative injury from the inability to respond with antioxidants. In extended wakefulness (Ext Wake), both SirT3wt and SirT3-/- mice showed an accumulation of oxidative stress, in the form of superoxide, and acetylation of mitochondrial proteins. Apoptosis was also activated and LCns were lost.

This implies the importance of maintaining mitochondrial metabolic homeostasis in LCns. Failure to maintain the SirT3 pathway can result in permanent neuronal degeneration.

Key words: sleep deprivation, mitochondria, oxidative stress, SirT3, locus ceruleus (LC), metabolic homeostasis Background Modern society is very vulnerable to sleep deprivation from increasing prevalence of irregular work schedules, electronic media exposure, and artificial lighting (Shochat, 2012). They all in turn affect sleeping schedules, usually resulting in sleep loss. The significance of sleep loss is unclear but evident that it has its consequences. The disturbances to regular sleeping habits from sleep deprivation has shown to negatively influence cognitive performance (Alhola P and Polo-Kantola, 2007) and could ultimately lead to dementia (Spira et al, 2014). Such impairments are derived from homeostatic influences, (Blutstein and Haydon, 2013) such as the role of adenosine in the cortex to encourage wakefulness. An imbalance of homeostasis alters the drive to sleep, and elicits physiological consequences.

During wakefulness a set of wake active neurons fire at high frequencies, and these neurons in particular are believed to modulate activities during wakefulness, such as meeting metabolic demands. Wake active neurons in the locus ceruleus are essential for cognitive function and show sensitivity to metabolic stressors. Their firing rates are optimal during wakefulness and reach quiescence during their transition from NREM to REM sleep (Aston-Jones and Bloom, 1981). This implies that these neurons are susceptible to stress from prolonged wakefulness to meet metabolic demands. A recent study (Stern and Naidoo, 2015) has suggested that in terms of neurodegeneration, wake active neurons are the most vulnerable in number and function.

The mitochondrion is implicated in many metabolic pathways, including apoptosis (Filosto et al., 2011).

Neurodegenerative diseases has been implicated with mitochondrial abnormalities, and proposes that impairments in the mitochondria to play a role in the pathology of these disorders. Therefore this emphasizes the importance of the mitochondria, especially in wake active neurons.

Sirtuin type 3 (SirT3) is a protein located on the inner mitochondrial membrane. It is responsible for coordinating redox homeostasis and energy produc207

tion. When ATP levels deplete, SirT3 attempts to restore ATP production through deacetylation of ETC complex I proteins, hence metabolic stressors acetylate proteins (Ahn et al., 2008). It also up-regulates antioxidant defenses when faced with oxidative stress.

However, when faced with severe oxidative stress, SirT3 becomes impaired and is unable to properly maintain metabolic homeostasis. In normal physiological circumstances, SirT3 couples with NAD+ to coordinate ATP production and antioxidant production.

However when NAD+ levels are low and oxidative stress is elevated (high superoxide concentrations), SirT3 can be impaired. The hypothesis proposed was that Sh Wake would promote SirT3-dependent antioxidant response to maintain redox homeostasis, and in Ext Wake, SirT3 activity would decline and metabolic homeostasis fails to maintain itself. In addition, failure to maintain homeostasis elicits neuronal degeneration in the locus ceruleus.

Research Overview

Summary of Major Results

To determine the importance of the SirT3 pathway for antioxidant responses, mRNA marked with superoxide dismutase (SOD) were measured (Figure 1.). In wild type mice, Sh Wake showed a higher demand for SirT3, from elevated superoxide levels. In SirT3-/mice, Rest and Sh Wake did not show any significant differences. This suggests that the SirT3 pathway is an essential pathway to maintain redox homeostasis in Sh Wake and knockouts of it may lead to failure to maintain homeostasis.

Mitochondrial acetylation was measured to determine whether a reduction in SirT3 protein correlated with reduced SirT3 activity in Ext Wake. Mitochondrial acetylation showed a statistically significant increase in acetylation at the mitochondria than the cytosol (Figure 2.).

In light of SirT3, its activity in Ext Wake was examined. CC-3 was used to determine if apoptosis was induced in Ext Wake using a confocal microscope.

Statistically significant increase in CC-3 was observed

in SirT3 wild type only (Figure 3.). This resulted in >25% neuronal loss from Ext Wake compared to Rest. In contrast, CC-3 activity in SirT3-/- was not increased in Ext Wake relative to Rest.

To assess the influence Ext Wake has on dendritic morphology, LCns on the ventral lateral side were observed (Figure 4.). SirT3wt showed greater dendritic segments than SirT3-/- mice at Rest. Ext Wake showed a reduction in dendritic segments in SirT3wt mice, unlike the SirT3-/- mice. In SirT3-/mice, dendritic morphology in Ext Wake was varied from Rest. Beading and vacuolization was evident.

There was no significant difference between rest, Sh Wake wakefulness, and Ext Wake in terms of corticosterone levels (Figure 5.). This suggested that Rest, Sh Wake, and Ext Wake were not associated with elevated plasma coricosterone.

Figure 3. Confocal images of LCns (TH in red) and caspase activity (CC-3 in green) of SirT3-/- and SirT3wt in Rest and Ext Wake.

Figure 1. Mean mRNA copies in response to Sh Wake (black) and Rest (gray) for SirT3wt and SirT3-/- mice (n=8-10/group).

Figure 4. Ventral lateral images to show the effects of sleep loss on LCn dendrites in SirT3wt and SirT3-/-

Figure 2. Integrated densities of acetylated proteins (n=20/group) (left) Mitochondrial acetylation in Rest, Sh Wake, and Ext Wake (right) cytosolic acetylation in Rest, Sh Wake, and Ext Wake.

Discussion The study was designed to test the hypotheses: (1) the wake active neurons in the LCns respond to wakefulness as a metabolic stressor; (2) SirT3 activation and short term sleep loss protects LCns and metabolic homeostasis; (3) extended wakefulness results in failure to protect homeostatic responses and results in LCn injury.

Neuronal loss in the LCns have been associated with neurodegenerative diseases such as Parkinsonâ&#x20AC;&#x2122;s and Alzheimerâ&#x20AC;&#x2122;s Disease (Zarow et al., 2013). The cumulative loss of LCns has shown to influence cognition and accelerate neurodegenerative processes.

Figure 5. Plasma corticosterone level measured from Rest, Sh Wake, and Ext Wake. No significant differences detected with ANOVA with 94% statistical power to detect changes of 100ng/ml of corticosterone levels. (n=8-14/group) 208

To address a metabolic stress, sleep deprivation was induced. Although it is an effective approach to gain insight on the biology of sleep, it may also be a source of stress (Redwine et al., 2013). Studies have shown elevated corticosterone levels in model organisms from sleep deprivation, thus for this study, there was an attempt to minimize stress as a confounding variable by providing mice with enriched environments and allowing for spontaneous wakefulness. Plasma corticosterone levels were not significantly different within the three paradigms: Sh Wake, Ext Wake and Rest.

It was expected that SirT3-/- to yield in a greater neuronal loss in Ext Wake than wild type however, SirT3wt mice had a reduction of >25% and a smaller dendritic field than SirT3-/-. This outcome may have been due to the relatively lower metabolic demand in SirT3-/- than wild type. Also, LCns may be been lost prior to Ext Wake.

Although neuronal loss was lower in SirT3-/- in Ext Wake, it presented neurite beading and vacuolization.

Sh Wake and Ext Wake showed two reciprocal results: Sh Wake upregulated SirT3 activity whereas Ext Wake reduced it. The differences in the two groups involved mitochondrial acetylation, ATP production, and regulation of other proteins, of which were all under the influence of the SirT3 protein. This proposes that the duration of wakefulness can alter how the brain manipulates SirT3 activity.

Although it may be surprising to see that antioxidant response stimulated from only 3 hours of wakefulness, it is important to note that metabolic demands during wakefulness is high (Oswald, 1980). Hence their name, wake active neurons, they are active throughout wakefulness and their activity drops to quiescence during REM sleep. It is believed that during sleep, there is restoration of metabolic homeostasis.

This suggests that sleep is a vital function for LCns and possibly other neuronal groups.

Conclusions The undefined consequences of sleep deprivation can lead to detrimental damages in the brain. This study has suggested that neuronal damage may be irreversible, and may allow individuals to become susceptible to neurodegenerative diseases. It also addressed the importance of SirT3 activity for maintaining redox homeostasis in response to oxidative stress and its vulnerability to degeneration in the LCn from extended wakefulness. In brief wakefulness, SirT3 activity can be upregulated by increasing antioxidant enzymes thus maintaining homeostasis.

In extended wakefulness, SirT3 activity declines as mitochondrial acetylation and superoxide concentrations are elevated. The redox homeostasis to maintain a metabolic balance requires proper SirT3 activity. If SirT3 can be manipulated to protect cells, it could lead to promising therapeutic methods for sleep-deprived individuals. It is important to note that it is not just SirT3 activity that has an influence on brain health, but many more to be revealed. Although more research is needed to settle the underlying principles involved in sleep deprivation and its link to brain health, a general consensus can be established: sleep is more important better than we thought.

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Criticisms and Future Directions The study has made implications on neurodegenerative diseases. It is widely known that a characteristic of a neurological disorder is the loss of neurons (Spira et al., 2014). If neuronal loss is induced by poor SirT3 function, overexpression of SirT3 can be put into the test to determine the impact it has on sleep deprivation. In such study, possible theoretical outcomes can be: expressing a relatively high immune response to sleep deprivation due to the efficient ability to combat oxidative stress, or even habituation of elevated SirT3.

Due to excessive increases of SirT3 at rest, and little oxidative stress to combat, whether it will have negative implications on health.

Moreover, it would be interesting to investigate if in the case SirT3 is knocked out from the embryo, if other proteins or mechanisms are able to cope or replace SirT3â&#x20AC;&#x2122;s original function post-natally.

In addition, the study observed the effects of wakefulness on wake active neurons. Previous studies (Stern and Naidoo, 2015) have shown that wake active neurons are heavily involved in sleep disturbances and promoting neurodegenerative disorders. If the use of wake active neurons in short term wakefulness induces up-regulation of antioxidant production, this would imply that sleep active neurons during sleep has an important role in maintaining redox homeostasis as well. Another approach to study the importance of sleep may be examining whether a reduction in sleep active neurons relay a similar outcome. The importance of sleep, especially for restorative properties has been established (Everson et al., 2005). This may be able to clarify whether neuronal loss is initiated from overuse of wake active neurons or underuse of sleep active neurons.

The authorsâ&#x20AC;&#x2122; approach to investigate SirT3, a mitochondrial protein and its function to maintain metabolic homeostasis was plausible but there was no direct evidence in whether the inability to maintain redox homeostasis resulted in cognitive deficiencies. It would have been better to assess behavioural tests before and after inducing sleep deprivation in both genotypes: SirT3wt and SirT3-/-.

Also, observing LCn following sleep recovery, long term, may confirm whether or not neuronal loss is permanent. If sufficient neurotrophins and neurogenesis is initiated, neuronal loss may be reversible.

References 1. Ahn BH, Kim HS, Song S, Lee IH, Liu J, Vassilopoulous A, Deng CX, Finkel T. (2008). A role for the mitochondrial deacetylase Sirt3 in regulating energy homeostasis. Proc Natl Acad Sci USA 105(38): 14447-52.

2. Alhola P. and Polo-Kantola P. (2007). Sleep deprivation: Impact on cognitive performance. Neuropsychiatr Dis Treat.

3(5):553-67.

3. Aston-Jones G, Bloom FE. (1981). Activity of norepinephrine-containing locus coeruleus neurons in behaving rats anticipate fluctuations in the sleep-waking cycle. The Journal of Neuroscience 1(8):876-86.

4. Blutstein T, Haydon PG. (2013). The Importance of astrocyte-derived purines in the modulation of sleep. Glia 61(2):129-39.

5. Everson CA, Laatsch CS, Hogg N. (2005). Antioxidant defense reponses to sleep loss and sleep recovery. Am J Physiol Regul Integr Comp Physiol 288(3):R374-83.

6. Filosto M, Scarpelli M, Cotelli MS, Vieimi V, Todeschini A, Gregorelli V, Tonin P, Tomelleri G, Padovani A. (2011). The role of mitochondria in neurodegenitive diseases. Journal of Neuroscience 258(10):1763-74.

7. Oswald I. (1980). Sleep as a Restorative Process: Human Clues. Progress in Brain Research 53:279-88.

8. Redwine L, Hauger RL, Gillin JC, Irwin M. (2013). Effects of Sleep and Sleep Deprivation on Interleukin-6, Growth Hormone, Cortisol, and Melatonin Levels in Humans. Journal of Clinical Endocrinology and Metabolism 85(10):3597-603.

9. Stern AL, Naidoo N. (2015) Wake-active neurons across aging and neurodegeneration: a potential role for sleep disturbances in promoting disease. Springplus. doi: 10.1186/ s40064-014-0777-6.

10. Shochat T. (2012). Impact of lifestyle and technology developments on sleep. Nature and Science of Sleep, 2012(4):19-31.

11. Spira A.P., Chen-Edinboro L.P., Wu M.N., Yaffe K. (2014). Impact of sleep on the risk of cognitive decline and dementia. Curr Opin Psychiatry. 27(6):478-83.

12. Zarow C, Lyness SA, Mortimer JA, Chui HC. (2003).

Neuronal Loss is Greater in the Locus Coeruleus Than Nucleus Basalis and Substantia Nigra in Alzheimer and Parkinson Diseases. Archives of Neurology 60(3):337-341.

13. Zhang J., Zhu Y., Zhan G., Fenik P., Panossian L., Wang M.M., …Veasey S. (2014). Extended Wakefulness: Compromised Metabolics in and Degeneration of Locus Ceruleus Neurons. Journal of Neuroscience, 34(12), 4418-31

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Can Neurogenesis Using Stem Cells Be the Key to Post-Stroke Functional Recovery? A Review of Neurogenesis and Stroke Recovery in Animal Models Hemish Patel

Neural cells that are damaged or destroyed do not partake in neuron self-regeneration process. Once these cells are damaged by any means, can elicit responses downstream that may be irreversible. These alterations in the cellular mechanism can lead to disabilities in motor function and other impairments, and ultimately lead to changes in behavior. One of the main reasons behind the death of these neurons is stroke. Neurogenesis via stem cells has been a promising approach to find solution to reestablish the normal cellular functions of damaged neural cells. The essence of the paper, Mine et al. (2013) in Neurophysiology of Disease, demonstrates an experiment using neural stem/progenitor cells (NSPCs). The authors administer NSPCs in T-cell rat models, allowing for neurogenesis to occur in the rat brain. The NPSCs, followed by one hour of middle cerebral artery occlusion (MCAO) are administered in the sub ventricular zone (SVZ) of the rat brain. The rat brain is endures two main changes: reduction in inflammation and an increase in neuroblast and mature neuron proliferation. Functional recovery of neural cells in the brain is observed via behavioral tests performed following the administration of grafted NSPCs. Neural stem cell research states to aim replacing dead cells and fixing neural circuitry. Stem cell researchers administer the neural cells to the right area in the brain such that it elicits progressive response downstream causing cell proliferation, neuroprotection and immunomodulation. The crux of the paper Mine et al. aims to stress, using several experimental approaches, that neurogenesis and stem cells can be used as a potential candidate for neural cell recovery post-stroke within animal models.

Key words: stroke; neurogenesis; sub ventricular zone; stem cells; middle cerebral artery occlusion (MCAO) Background Stroke is a debilitating, yet a common disease with its rate of incidence steadily rising with advancing age. It is ranked amongst the highest causes of death and disabilities in Europe, United States and Canada (Wagner et al., 2014). Its rate of occurrence tends to be higher in males compared to females of up to the age 75, at which point the rate of incidence amongst both genders tends to equal out. However, it has been shown that women over the age of 85 are likely to be more prone to the disease, compared to males of age 85 and up (Wagner et al., 2014). Thus, stroke can influence both genders equally depending on the age. It leads to several changes in the brain, such as death of neural cells as well as alterations in other areas of the brain making them venerable to subsequent strokes (Merson & Bourne 2014). Stroke is also linked to inflammation in the brain (Tobin et al., 2014). Stroke can lead to variety of life style changes in many individuals. Despite the severity of the disease, there are currently no treatments for stroke in regards to damaged neurons.

Neural cells and brain circuitry protection are crucial, as they do not have the ability to regenerate in the adult brains. However, in past decade, new theories have emerged which allow scientists to replace the damaged neuronal cells. This process of so-called neurogenesis has opened up a field of research that could possibly lead to recovery post-stroke. Neurogenesis is believed to occur in the sub ventricular zone (SVZ) of the brain in the hippocampus (Frankland et al., 2013 & Tobin et al., 2014). Thus, it can then be hypothesized that administration of neural progenitor cells in the brain could possibly reverse the adverse effects of stroke.

Mine et al. (2013) devised a study that would allow 211

them to observe the effects of neural stem/progenitor cells (NSPCs) derived from the fecal striatum, when administered in the SVZ in rats following MCAO. The authors were successful in explaining how NPSCs gave rise to many different changes in the brain, such as reduction of inflammation and proliferation of neural cells through a variety of different experimental approaches.

Numerous studies have performed to show the use stem cells in neurogenesis post-stroke. NSPSCs administered endogenously generated new neuroblasts and migrated to the injury site in the striatum (Arvidsson et al., 2002). Human fecal NSPCs transplantation has also shown to give rise to neurons that migrate towards ischemic lesions in rats (Kelly et al., 2004).

Daadi et al. (2009) showed that human embryonic derived stem cells when administered into the ischemic boundary in rats following stroke, also migrated towards the lesion and showed improvements in forelimb functioning. Finally, a study conducted by Doeppner et al.

(2014), depicted that there was a reduction in functional impairment with administration of stem cells in rats following ischemic stroke. The heart of this paper, Mine et al. demonstrates that administration of NSPCs not only gives rise to new neuronal cells but also leads to a decrease in inflammation, an increase in axonal projections and functional recovery post-stroke.

The crux of this review paper will focus on the results of the study performed by Mine et al. (2013), as it tries to determine a relationship of post-stroke induced neurogenesis for functional recovery and proliferation of new neuronal cells within the adult rat brain. The paper will also gives a brief overview of the relation between inflammation and macrophage activity within the SVZ zone.

Research Overview Animal Models The study used T cell deficient rats. The rats were kept in 12-hour cycles of light and dark. They were provided with food and water. All of the experimental procedures followed Malmo-Lund Ethical Committee guidelines. There were 2 experimental groups. In the first experimental group, 10 rats were injected with 5-bromo-2â&#x20AC;&#x2122;-deoxyuridine (BrdU) as a marker for proliferation after MCAO twice daily for 14 days. NSPCs were administrated in the contralateral striatum. The rats were sacrificed after 4 weeks. In the second experimental group, following MCAO, 33 rats were introduced to NSPCs 48-hours to the ipsilateral striatum. This group was split into 2 others groups. The first group received BrdU injections for 14 days and sacrificed after 6 weeks.

The second group received BrdU injections for 14 days after 9 weeks and sacrificed after 14 weeks. One group was given grafted NSPCs while the other group was given implanted (vehicular) NPSCs. Neural Stem Cells/Progenitor Cells Culturing The stem cells were acquired from striatal tissue from 7.5-8 week aborted human fetuses from Lund and Malmo University. The culture of stem cells was passaged several times for over 2 years before transplantation.

Behavioral Tests A. Stepping Test The stepping test was used to observe the function of the forelimb. The rat was held with its hind limbs joined with one hand, one forelimb with the other. The other forepaw was touching the table surface. The number of adjustments was counted as the rat moved sideways along the table surface. The test was performed twice daily for 5 days before MCAO and repeated every 3 weeks after MCAO. The pre-treatment score was the mean average of the last 3 days preceding MCAO. B. Cylinder Test This test was used to observe the effects of the asymmetrical forelimb usage. The rats were placed in a cylinder. They were allowed to roam freely while being observed. Two perpendicular mirrors were placed behind the cylinder. A recording would be made every time the rat used its paw to touch the cylinder wall.

An average recording was taken, either every 15 min intervals or once rat made 20 touches.

Summary of Major Results

Mine et al. (2013) observed numerous consequences of NSPCs through their experimental design. The results are concisely summarized in the following sections that are important for discussion. To avoid rejection by the rat body, all of the experiments were performed on T cell deficient rats. MCAO performed on the rat caused damage in both the striatum and cerebral cortex.

Neurogenesis and Inflammation in the striatum In the first experimental group, after 7 weeks there was a steady increase in neuroblasts and an increase in the number of cells that expressed mature neuron markers.

In the second experimental group, there was a perceived increase in cell proliferation. It was observed that NSPCs promoted the recruitment of new striatal neuroblasts after MCAO in the grafted group (Fig. 1). There was a stable decrease in inflammation of the brain, however the number of macrophages and microglia remained constant in the area of injury; they did not increase or decrease in their numbers.

Mine et al. (2013) were also able to demonstrate that some of the neurons had the functional ability to make axonal projections to several areas of the brain.

Behavioral Recovery A. Stepping Test The results of this test showed functional recovery of both back and forelimb function following 6 weeks after the administration of NSPCs (Fig. 2). Before giving the rats stem cells, it was seen that MCAO left the rats impaired in their left forelimb function compared to the right side.

B. Cylinder Test The cylinder test results showed an increase in percentage of left paw touches after 12 weeks of MCAO in the group with grafted NSPCs (Fig. 2).

Mine et al. (2013) show that there is a partial motor recovery after transplantation of NPSCs in rats with induced stroke.

Figure 1. An increase in the number of neuroblasts is seen in the ipsilateral striatum following NSPCs transplanting compared to the vehicle subjects. Immunofluorescence was used to mark the cells. (Mine et al. 2013)

Conclusions and Discussion Mine et al. (2013) described that NSPCs could lead to partial functional recovery after 1 hour of MCAO. They also exemplified that the transplanting of stem cells leads to two major changes in the brain: they allow for a decrease in brain inflammation and an increase in neurogenesis. NSPCs have also been seen to proliferate into astrocytes and oligodendrocytes. However, they do not become neurons (Kallur et al., 2006). The stepping test and cylinder test illustrated that the rats could reestablish motor function in their forelimbs after administration of neural stem cells. The authors also observed grafted neural stem cells express markers for mature neurons. They also 212

Figure 2. A and B depict the stepping test results. Vehicle rats are those that do not have administered NSPCs. Left forelimb shows partial recovery following MCAO in rats with NSPCs. There is some recovery in the right forelimb but it is not as evident as the left forelimb motor movement. C shows the cylinder test which shows an increase in the percentage of left forelimb touches in rats with transplanted NSPCs. (Mine et al. 2013)

observed these neurons make axonal projections to the globus pallidus (Mine et al., 2013). This suggests that stem cells work in conjunction to fix neural circuitry damaged following stroke. Inflammation is a critical mechanism that must be looked at for brain protection.

By observing the macrophage and microglia in the injured area of the rat brain, the authors proposed that inflammation is controlled and has long-lasting effects.

Human grafted stem cells have a diminishing effect on the inflammatory response in the damaged striatum.

Horie et al. (2011) also observed and recorded this decrease in inflammatory response when human fetal NSPCs were delivered intra-cortically giving rise to a short decrease in inflammation following distal MCAO.

Significance/Relevance to the Field

The conclusions made by Mine et al. (2013) describe that stem cells can be used to gain fractional behavioral recovery. The authors also illustrate the practicality of stem cells to decrease inflammation and increase proliferation of neural cells that can have beneficial outcomes in the brain.

213

Moreover, they show that stem cells can induce plentiful benefits to the brain, such as replacement of dead cells and repair of neural circuitry. NSPC transplanting can allow for repairing of neural circuitry, which involves the increase in dendritic spines and axonal growth (Andres et al. 2011). Stem cells also lead to neurons expressing of trophic factors and provide neuroprotection. The results from this study give an insight on the power of NSPC transplantation in the brain and the advantageous effects on the brain following stroke.

Criticisms and Future Directions The study by Mine et al. (2013) provides the scientific community with a thought-provoking hypothesis and chance to effortlessly replicate the experiments conducted to obtain the results. A main confound of the study was the usage of T cell deficient animals. They were used to avoid the NSPCs from being rejected by the immune system of the animal. A similar study

was performed by Chau et al. (2013), where they demonstrated that after inducing stroke in neonatal rats, induction of pluripotent stem cells increases regeneration and recovery. Many of the studies look at human NSPC administration into animal models, however it is quite difficult to reproduce these experiments in human models. The effect of stem cells in animals might not be the same in a human system.

A challenging prospect in human subjects is the location of transplantation, as stem cells may not migrate to the desired location and cause beneficial effects.

An ethical debate is also important factor to consider when it comes to humans.

Previous studies, however, have shown functional recover in humans following stroke, which has been associated with a functional reorganization of the damaged area (Zhang et al., 2014). This recovery is also due to recruitment of neighboring circuits to the damaged region. Thus, the solution lies in fixing the circuitry that was damaged (Hermann & Chop, 2012).

The damaged neural circuitry post-stroke should be a primary region for further examination.

A promising method would rely on the identification of a favorable area where stem cells can be transplanted for maximal affect to the CNS and brain circuitry.

However, after careful administration of stem cells, it would need to be made sure that the stem cells have not been rejected by the immune system. Stem cells could not only be used in stroke injury but they could also be used in other brain diseases and deficits associated with neurons. Stem cells are not only limited to the brain but they could also differentiate into a wide variety of cells and the possibilities become limitless once the initial obstacles are overcome.

7. Horie N, Pereira MP, Niizume K, Sun G, Keren-Hill H, Encarnacion A, Shamloo M (2011). Transplanted stem cellsecreted vascular endothelial growth factor effects poststroke recovery, inflammation, and vascular repair. Stem Cells, 29: 274-285.

8. Kallur T, Darsalia V, Lindvall O, Kokaia Z (2006).

Human fetal cortical and striatal neural stem cells generate region-specific neurons in vitro and differentiate extensively to neurons after instrastriatal transplantation in neonatal rats. J. Neurosci. Res. 84:1630-1644.

9. Merson, TD, Bourne JA (2014) Endogenous neurogenesis following ischaemic brain injury: Insights for therapeutic strategies. The International Journal of Biochemistry & Cell Biology, 56: 4-19.

10. Mine, Y, Tatarishvili, J, Oki, K, Monni, E, Kokaia, Z, Lindvall, O. (2013). Grafted human neural stem cells enhance several steps of endogenous neurogenesis and improve behavioral recovery after middle cerebral artery occlusion in rats. Neurobiology of Disease, 52: 191-203.

11. Tobin, MK, Bonds JA, Minshall RD, Pelligrino, DA, Testai FD, Lazarov O (2014). Neurogenesis and inflammation after ischemic stroke: what is known and where we go from here.

Journal of Cerebral Blood Flow & Metabolism, 34: 15731584.

12. Wagner AP, Buga A, Doeppner TR, Hermann DM (2014).

Stem cell therapies in preclinical models of stroke associated with aging. Frontiers in Cellular Neuroscience, 8: 347.

References 1. Andres RH, Horie N, Slikker W, Keren-Gill H, Zhan K, Sun G, Manley NC, Pereira MP, Sheikh LA, McMillan EL, Schaar BT, Svendsen CN, Bliss TM, Steinberg GK (2011). Human neural stem cells enhance structural plasticity and axonal transport in the ischaemic brain. Brain, 134: 1777-1789.

2. Arvidsson A, Collin T, Kirik D, Kokaia Z, Lindvall O (2002). Neuronal replacement from endogenous precursors in the adult brain after stroke. Nat. Med. 8: 963-970.

3. Chau M, Deveau TC, Song M, Gu X, Chen D, Wei L (2013). IPS Cell Transplantation Increases Regeneration and Functional Recovery after Ischemic Stroke in Neonatal Rats.

Stem Cell, 10: 1002.

4. Daadi MM, Li Z, Arac A, Grueter BA, Sofilos M, Malenka RC, Wu JC, Steinberg GK (2009). Molecular and magnetic resonance imaging of human embryonic stem cell-derived neural stem cell grafts in ischemic rat brain. Mol. Ther. 17: 1282-1291.

5. Doeppner TR, Kaltwasser B, Bahr M, Hermann DM (2014). Effects of neural progenitor cells on post-stroke neurological impairment â&#x20AC;&#x201C; a detailed and comprehensive analysis of behavioral tests. Frontiers in Cellular Neuroscience, 8: 338.

6. Frankland P.W., Kholer S., & Josselyn S.A. (2013).

Hippocampal neurogenesis and forgetting. Trends in Neuroscience. 36(9):497-503.

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Lateral entorhinal cortex encodes associations of past experience and location Maryna Pilkiw

The lateral entorhinal cortex (LEC) is a structure that provides major input into the hippocampus and, in turn, receives hippocampal outputs. The LEC has been seen to predominantly process object and stimulus-based information, however, new evidence suggests that the LEC may process higher order information. Tsao et al.

(2013) examined single cell activity in the LEC of rats in the open field with or without a familiar object present.

The authors characterized two distinct neuronal populations: cells that increase their activity in the vicinity of an object (object cells) and cells that increase their firing rate in the location of the familiar object but only during the trials when the object is removed (trace cells), thus coding for an association of a past experience and a specific location. Tsao et al. demonstrated that trace cell activity is not unique to physical objects but can be elicited by such experiences as rewarding microstimulation administered at a specific location. Trace cell activity points to the critical role of the LEC in the hippocampal-entorhinal memory network.

Key words: hippocampus, lateral entorhinal cortex, learning and memory, trace cells, single unit activity Background One of the most famous and important clinical cases in neuroscience is the case of patient H.M. The surgical removal of H.M.’s temporal lobes gave scientists an initial understanding of the importance of the hippocampus in memory formation and retrieval (Scoville & Milner, 1957). However, whereas most of the attention of the researchers was focused on the hippocampus as a central memory structure, the recent report of the post-mortem histology of H.M.’s brain revealed that while his hippocampi were more intact than had been previously assumed, the entorhinal cortices were completely removed during his surgery, prompting the authors to suggest that the main reason for H.M.’s amnesia could be traced to the disconnection between entorhinal inputs into the hippocampus, and not the hippocampi removal per se (Annese et al., 2014). Clinical cases in which patients’ entorhinal cortices (EC) get damaged due to trauma or neurodegeneration (e.g., Alzheimer’s disease), provide strong evidence for the importance of the intact EC for the formation of memories of daily experiences (Khan et al., 2014; Bayley et al., 2006; Kapur & Brooks, 1999). The EC is a cortical structure that is a part of the larger hippocampal formation, surrounding the hippocampus proper (HPC) and reciprocally sending inputs to and receiving outputs from the hippocampus (Schultz & Engelhardt, 2014).

Because the EC receives and processes inputs from neocortical regions before sending information to the HPC, and then, in turn, passes the information received from the HPC back to the neocortex (Suzuki & Amaral, 2004; Agster & Burwell, 2013), the EC can be seen as a hub of cortical information processing and a structure crucial for the functioning of the hippocampus-dependant memory network (e.g., see Takehara-Nishiuchi, 2014). Recently, substantial evidence has accumulated suggesting that the EC has two distinct regions based on their cytoarchitecture, connectivity, and the type of information that is being predominantly processed. The medial region of 215

the EC (MEC) has been shown to mainly process spatial information (e.g., grid cells) and lateral regions have been suggested to mediate sensory, objectbased and contextual information (e.g., Cauter et al., 2013; Yoganarasimha et al., 2011; Deshmukh & Knierim, 2011). Specifically, the LEC has been shown to be necessary for retention of sensory information in trace-eyeblink conditioning (Morrissey, 2012), object-context recognition task (Wilson et al, 2013a), associative recognition memory (Wilson et al, 2013b), and cross-modal association learning (Boisselier et al., 2014). Whereas initial models have been dividing the roles of the MEC and the LEC into “what” and “where” modules that provide corresponding inputs into the HPC, it appears that such division is an oversimplification, and the LEC, while predominantly modulating object/experience-based information, is also important for spatial processing (Knierim, 2014). In order to test how neurons in the LEC respond to physical features of an object within a spatial location and whether the LEC encodes a higher order representation of an object, such as previous experience with it in conjunction with a specific location, Tsao et al. (2013) recorded in vivo single unit activity from behaving rats that encountered either an object or a rewarding stimulation within a determined location of a testing arena. The animals were given three consecutive 10 min trials: “object-free” trial (to determine baseline activity of the cells), “object” trial with an object placed at a specific location, followed by the “object-free trial” again. The initial task was then modified such that the object trials included novel objects in different locations, a familiar object in the novel environment (the same testing box but with the walls of different colour and different spatial cues), a familiar object in a familiar environment but different locations, and, finally, a familiar object was substituted with the rewarding microstimulation of the medial forebrain bundle (MFB) when a rat entered a specific location.

Research Overview

Summary of Major Results

Activity of the single units was quantified as the mean frequency of cell firing. The firing rate of a neuron when a rat was in the location of the object was compared to the firing rate outside of the object area.

The researchers identified one group of cells that increased the firing rate when the animal entered the location of the object during the “object” trials and a second group of neurons that responded to the location of the object on the following “object-free” trials, i.e., the cells were representing a trace of the familiar object. When tested with the novel object, the activity of the new “object” and the “object-trace” cells could be identified. When the authors moved the familiar object to different locations within the box, the activity of the trace cell increased at every location where the familiar object was encountered during the previous trial, thus following the location of the object. However, when a familiar object was placed in the novel environment, the trace cell activity did not emerge, suggesting that the trace cells code for experience in a unique environment. Finally, when the rats were tested with the MFB microstimulation delivered at a specific location instead of an object encounter, one set of the LEC neurons responded to the experience of stimulation itself and a distinct set of trace cells increased the firing rate when the animal entered the location where during the previous trial, but not the current trial, the stimulation was delivered. Tsao et al. also demonstrated that the differential neuronal activity was not due to the animal’s movement behavior.

Conclusions and Discussion With the series of experiments Tsao et al. identified a distinct group of LEC neurons that increase their activity in response to an animal entering a location of a previous encounter with an object. It has been previously demonstrated that the LEC contains cells that preferentially respond to objects in the environment

Figure 2. The top panel indicates a location within a recording box where MFB stimulation was delivered to a rat. Bottom figures show activity of different cells that increase their activity either during stimulation (middle) or post-stimulation (right). (Source: Tsao et al., 2013)

Figure 1. The top panel shows representation of the recording box during the three trials, a trial without an object, with the object, and with the object removed. The panels below show activity of two cells: object cell that increases its firing rate around the object during the “object” trial and the trace cell that is not active during the object trial but increases its activity in the post-object trial in the location where the object was previously present. (Source: Tsao et al., 2013)

(Deshmukh & Knierim, 2011). However, newly identified cells represent a unique, non-overlapping population of neurons that become active only when the previously encountered object or experience is not present. Tsao et al. argue that activity of the identified trace cells is also distinct from previously described paired-association cells that show specific object response, “mismatch” cells that transiently respond to misplaced objects, and the hippocampal place cells that show remapping when an object is moved. The authors point out that the activity of the trace cells most likely corresponds to the retrieval of object-location association in the absence of the object. Additionally, the authors showed that the trace cell activity is not unique to physical objects but rather to specific experiences as they demonstrated with the rewarding stimulation in a given location which would generate trace cell activity to the same extent as the encounter with physical objects. Tsao et al. also demonstrated that the LEC cell activity represents both “what” and “where” information which gives support to newly proposed models that suggest that a division between the MEC and the LEC should be seen not as a “where” and “what” dichotomy but rather as a difference in context (MEC) and content (LEC) that are generated from different reference frames: global, internally generated cues (MEC) vs. local, generated by external sensory input cues (LEC) (Knierim, 2014).

216

Future Directions The first identification of place cells in the CA1 by O’Keefe and Dostrovsky (1971) set a steady trend for the discovery of functionally different cell types, or neurons that code for a representation of a single spatial, temporal or object-related feature, in the hippocampus. Since the discovery of place cells, which are groups of neurons representing a specific location that an animal occupies, scientists have identified grid cells which act as an animal’s coordinate system, border cells that represent edges of the animal’s space (Moser et al. 2015), head-direction cells which fire when the animal turns its head in a specific direction (Finkelstein, 2015), time cells, the firing of which corresponds to specific temporal intervals (Eichenbaum, 2014), etc.

All of these cell types seem to code for a specific feature which later is integrated into a multi-level multimodal representation that can be encoded as a memory. Can trace cells be seen as yet another type of neuron in the array of functional hippocampal cell types? An important difference between trace cells and the other types mentioned above is that the trace cells represent already integrated information which includes both place and object/experience. One important question is about the role that the LEC plays: does the LEC contribute to feature integration, i.e., combination of location information and object/experience or does it represent information processed by the hippocampus and is a place for hippocampal memory storage or retrieval?

Projections from the superficial layers of the LEC reach the dentate gyrus and the CA3, whereas neurons from the subiculum synapse in the deep layers of the LEC (Igarashi et al., 2014a). Based on anatomical connectivity and evidence that the CA1 integrates spatial and object-based information (Lu et al., 2013) we can ask to what degree information gets integrated in the LEC before it reaches the hippocampus? In the study by Sargolini et al. (2006) who recorded single cell activity from the medial entorhinal cortex (MEC), researchers found different degrees of spatial feature integration based on the layer of the MEC in which the recording was done, with deep (receiving the HPC outputs) but not superficial layers (sending input to the HPC) showing a conjunction of features. If the same pattern is true in the LEC, we would expect to see integration of object/experience and location in the deep but not superficial layers which would mean that the LEC receives information already processed by the hippocampus. However, because Tsao et al. observed trace cell activity in both deep and superficial layers it is not clear what specific role the LEC plays, and a further differentiation of trace cell activity in different layers of the LEC should be done. Specifically, oscillatory activity between superficial and deep layers and the CA1 could be examined. A synchronized ensemble activity between the LEC and the CA1 has been shown to correlate with successful retrieval of associative memories (Igarashi et al., 2014). Differentiation of the deep and superficial layers of the LEC based on the coupling of oscillatory activity with the HPC can provide a better understanding of the information processing and direction of information flow.

If the LEC, however, does not play a major role 217

in feature integration, can it be seen as a place of memory encoding and/or retrieval? There is evidence that the coordination of the entorhinal-hippocampal circuit is important for memory network (e.g., Suh et al., 2011). Can activity of trace cells within LEChippocampal network be seen as a representation of episodic memory? Episodic memory is characterized as mental time travel that includes dimensions of “what”, “where”, and “when”. Not surprisingly, the study of episodic memory is challenging in non-human animals because of the mental travel component (Hampton & Schwartz, 2004). There are many studies that manipulated what-where-when features in animal experience to allow the extrapolation that animals can indeed mentally “time travel” in order to solve given tasks (e.g., Erquorul & Eichenbaum, 2004). Demonstrated by Tsao et al. activity of trace cells shows integration of the where and what components that correspond to past experience. To further this idea, the experiment could be performed to include a temporal component to Tsao et al.’s design, such that the experience of the animal is contingent not only on a specific location but also a specific timing. If trace cells can incorporate all of the temporal, spatial and object/experience features, their activity could be considered as a proxy for episodic memory in animals.

References 1. Agster, K. L., & Burwell, R. D. (2013). Hippocampal and subicular efferents and afferents of the perirhinal, postrhinal, and entorhinal cortices of the rat.Behavioural brain research, 254, 50-64.

2. Annese, J., Schenker-Ahmed, N. M., Bartsch, H., Maechler, P., Sheh, C., Thomas, N., ... & Corkin, S. (2014).

Postmortem examination of patient HM’s brain based on histological sectioning and digital 3D reconstruction. Nature communications, 5.

3. Bayley, P.J., Hopkins, R.O., Squire, L.R., 2006. The fate of old memories after medial temporal lobe damage. J. Neurosci. 26, 13311–13317.

4. Boisselier, L., Ferry, B., & Gervais, R. (2014). Involvement of the lateral entorhinal cortex for the formation of cross‐modal olfactory‐tactile associations in the rat. Hippocampus, 24(7), 877-891.

5. Van Cauter, T., Camon, J., Alvernhe, A., Elduayen, C., Sargolini, F., & Save, E. (2013). Distinct roles of medial and lateral entorhinal cortex in spatial cognition. Cerebral Cortex, 23(2), 451-459.

6. Eichenbaum, H. (2014). Time cells in the hippocampus: a new dimens1ion for mapping memories. Nature Reviews Neuroscience, 15(11), 732-744.

7. Ergorul, C., & Eichenbaum, H. (2004). The hippocampus and memory for “what,” “where,” and “when”. Learning & Memory, 11(4), 397-405.

8. Finkelstein, A., Derdikman, D., Rubin, A., Foerster, J. N., Las, L., & Ulanovsky, N. (2014). Three-dimensional headdirection coding in the bat brain. Nature. 517, 159-164.

9. Hampton, R. R., & Schwartz, B. L. (2004). Episodic memory in nonhumans: what, and where, is when?. Current Opinion in Neurobiology, 14(2), 192-197.

10. Igarashi, K. M., Ito, H. T., Moser, E. I., & Moser, M. B. (2014a). Functional diversity along the transverse axis of hippocampal area CA1. FEBS letters 588(15), 2470-2476.

11. Igarashi, K. M., Lu, L., Colgin, L. L., Moser, M. B., & Moser, E. I. (2014b). Coordination of entorhinal-hippocampal ensemble activity during associative learning. Nature, 510(7503), 143-147.

12. Kapur, N., Brooks, D.J., 1999. Temporally-specific retrograde amnesia in two cases of discrete bilateral hippocampal pathology. Hippocampus 9, 247–254.

13. Khan, U. A., Liu, L., Provenzano, F. A., Berman, D. E., Profaci, C. P., Sloan, R., ... & Small, S. A. (2014). Molecular drivers and cortical spread of lateral entorhinal cortex dysfunction in preclinical Alzheimer’s disease. Nature neuroscience, 17(2), 304-311.

14. Knierim, J. J., Neunuebel, J. P., & Deshmukh, S. S. (2014). Functional correlates of the lateral and medial entorhinal cortex: objects, path integration and local–global reference frames. Philosophical Transactions of the Royal Society B: Biological Sciences, 369(1635), 20130369.

15. Lu, L., Leutgeb, J. K., Tsao, A., Henriksen, E. J., Leutgeb, S., Barnes, C. A., ... & Moser, E. I. (2013). Impaired hippocampal rate coding after lesions of the lateral entorhinal cortex.

Nature neuroscience, 16(8), 1085-1093.

16. Morrissey, M. D., Maal-Bared, G., Brady, S., & Takehara-Nishiuchi, K. (2012). Functional dissociation within the entorhinal cortex for memory retrieval of an association between temporally discontiguous stimuli. The Journal of Neuroscience, 32(16), 5356-5361.

17. Moser, M. B., Rowland, D. C., & Moser, E. I. (2015).

Place Cells, Grid Cells, and Memory. Cold Spring Harbor perspectives in biology, 7(2), a021808.

18. O’Keefe, J., & Dostrovsky, J. (1971). The hippocampus as a spatial map. Preliminary evidence from unit activity in the freely-moving rat. Brain research,34(1), 171-175.

19. Sargolini, F., Fyhn, M., Hafting, T., McNaughton, B. L., Witter, M. P., Moser, M. B., & Moser, E. I. (2006). Conjunctive representation of position, direction, and velocity in entorhinal cortex. Science, 312(5774), 758-762.

20. Schultz, C., & Engelhardt, M. (2014). Anatomy of the Hippocampal Formation.The Hippocampus in Clinical Neuroscience, 34, 6-17.

21. Suh, J., Rivest, A. J., Nakashiba, T., Tominaga, T., & Tonegawa, S. (2011). Entorhinal cortex layer III input to the hippocampus is crucial for temporal association memory.

Science, 334(6061), 1415-1420.

22. Suzuki, W.A., Amaral, D.G., 2004. Functional neuroanatomy of the medial temporal lobe memory system. Cortex 40, 220–222.

23. Takehara-Nishiuchi, K. (2014). Entorhinal cortex and consolidated memory.Neuroscience research, 84, 27-33.

24. Tsao, A., Moser, M. B., & Moser, E. I. (2013). Traces of experience in the lateral entorhinal cortex. Current Biology, 23(5), 399-405.

25. Wilson, D. I., Langston, R. F., Schlesiger, M. I., Wagner, M., Watanabe, S., & Ainge, J. A. (2013). Lateral entorhinal cortex is critical for novel object‐context recognition. Hippo-

campus, 23(5), 352-366.

26. Wilson, D. I., Watanabe, S., Milner, H., & Ainge, J. A. (2013). Lateral entorhinal cortex is necessary for associative but not nonassociative recognition memory.Hippocampus, 23(12), 1280-1290.

27. Yoganarasimha, D., Rao, G., & Knierim, J. J. (2011).

Lateral entorhinal neurons are not spatially selective in cue‐rich environments. Hippocampus, 21(12), 1363-1374.

The author thanks Dr. Ju for his enormous support and invaluable teaching.

Correspondenceto: MarynaPilkiw,Email:maryna.pilkiw@mail.utoronto.ca Copyright © 2015 Dr. Bill JU, Neurosciences, Human Biology Program

218

Reviewing glutamate mediated excitotoxicity in miR-1000 Drosophila mutants

Padmesh Ramanujam

Despite several advances in the field of medicine, the ability to cure and slow the degeneration process in various neurodegenerative conditions is yet to be identified. In recent years, non-coding RNAs (ncRNAs) have emerged as a key regulator of many cellular and molecular events in the central nervous system (CNS). Off these, microRNAs (miRNAs) have received considerable attention and great deal of research has been done in inferring their role in the CNS. The miRNAs have been shown to regulate synaptic signaling and excitation of neurons. Several studies have investigated the postsynaptic and retrograde role of miRNAs in the CNS. However, studies investigating the presynaptic role of miRNAs in regulating cell excitation have been scarce. Verma and colleagues recently have showed the presynaptic neuroprotective role of miRNA-1000 in Drosophila through its regulation of vesicular glutamate transporter (VGlut) gene expression.

Verma and coworkers concluded that miR-1000 plays a role in regulating glutamate release and regulating cell excitation in the postsynaptic cell. Using miR-1000 Drosophila mutants and knockouts, the authors were able to show increased VGlut gene expression, evidence of neurodegeneration, increased amplitude and frequency of miniature excitatory junction potential (mEJPs) and impaired movement. From these findings, the authors drew the potential link of using miRNA functionality to treat various neurodegenerative conditions like Parkinson’s and amyotrophic later sclerosis.

Key words: microRNA (miRNA), glutamate, vesicular glutamate transporter VGlut, excitotoxicity, neurodegeneration Background Neurodegenerative diseases and stroke are incapacitating neurological disorders and are one of the leading causes of long-term disability in the population1,2.

The inability to cure and delay the pathogenesis of neurodegenerative diseases and stroke have imposed financial burden on the health care system, driving the quest to identify the mechanisms of these neurological disorders1. Research on the pathogenesis of these diseases in various animal models has prominently shown the role of non-coding RNAs (ncRNAs) and ncRNA mediated regulation of gene expression1.

NcRNAs represent varied group of RNA molecules (miRNAs, rRNAs, tRNAs and lncRNAs (long noncoding RNAs)) that are involved in numerous cellular functions in addition to regulating gene expression, these interfere with protein translation and in some cases also mediate toxic responses1,3. Off these, microRNAs (miRNAs) have received substantial attention and have been best studied3,4.

MicroRNAs are short ncRNAs that post-transcriptionally repress gene expression1. The mRNAs are usually ~ 21-23 nucleotides long and bind to the 3’ UTR of the target mRNA for its degradation, destabilization or translational silencing1,3. The formation of mature miRNA involves series of nuclear and cytoplasmic processes that modify a primary miRNA transcript to a mature miRNA through the association of various proteins1,4.

In fact, dysfunction in the key proteins involved in the miRNA biogenesis pathway has been implicated in neurodegenerative diseases like amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD)1. MicroRNAs are vastly distributed in the nervous system and are vital for driving brain development3. Furthermore, miRNAs are shown to mediate synaptic plasticity and maturation of dendritic spines 219

and synapses. Indeed, several studies have documented formation of mature miRNA from pre-miRNA transcripts within the dendritic spines and processing of miRNA within those sites with local stimulation4.

In addition to neurodegenerative diseases, the miRNAs also play a key role in addiction, psychiatric disorders and excitotoxicity cell death. Several studies have noted an increase in the expression of miR-181a within the nucleus accumbens medium spiny neurons in facilitating addiction5. MicroRNA-181a represses the expression of GluA2- a subunit of AMPA-type glutamate receptor5. The non-GluA2 AMPA receptor at the synaptic membrane and its trafficking has been noted in literature to mediate addiction behavior5. Furthermore, substantial evidence has shown dysregulation in the expression of miRNAs in psychiatric disorders such as schizophrenia, bipolar disorder, depression and anxiety disorders4. Numerous studies have implicated the role of miRNAs in glutamate mediated excitotoxicity6.

Experiments using hypoxia/ reoxygenation (H/R) injury animal models for stroke have noted an increase in the expression of miR-107 2. MiR-107 represses glutamate transporter-1 (GT-1) gene expression and thus, its overexpression leads to excitotoxicity2. In fact, up-regulation of miR-181and miR-29 that repress glutamate transports have been noted in astrocytes upon cerebral ischemia7. However, thus far, the role of miRNA in presynaptic release of glutamate in glutamate mediated excitotoxicity has not been noted8.

In a paper published on Nature’s website in February 2015, Verma and coworkers show the neuroprotective role of miR-1000 in Drosophila against excitotoxicity through its role in regulating the expression of vesicular glutamate transporter (VGlut) 8. Using miR-1000 Drosophila mutant models, Verma and colleagues showed the dysregulation of the vesicular

glutamate transporter, excess glutamate release, and glutamate mediated excitotoxicity and cell death8. With the implication of miRNAs in excitotoxicity and with the evidence of excitotoxicity in Parkinsonâ&#x20AC;&#x2122;s and ALS, a miR-1000 similar gene in mammalian brain can be used a possible target8.

Research Overview

Summary of Major Results

Dysregulation of the vesicular glutamate transporter Verma and coworkers noted the misregulation in the expression of VGlut in miR-1000 Drosophila mutants8.

The scientists studied VGlut gene expression by generating two different Drosophila mutants- KO1 and KO2. In the KO1 mutant, the authors created a knockout of miR-10008. In the KO2 mutants, the authors first created a targeted homologous recombination of mini-white cassette for the miR-1000 gene, and then they replaced it either with GFP and GAL4 reporters or miR-1000 rescue allele by recombinasemediated cassette exchange (RMCE) using a vector8.

The miR-1000 rescue allele restored half the function of miR-1000 and was used to study the recovery from neurodegeneration in Drosophila mutants8.

In the miR-1000 mutants, the expression levels of VGlut were found to be increased by four fold from the baseline8. However, in the rescued mutant this effect was found to be one-halved (Fig. 1)8. Also, using a Luciferase reporter assay to the 3â&#x20AC;&#x2122;UTR of VGlut mRNA, the authors noticed a decrease in the Luciferase activity confirming the specificity of miR-1000 to its target8. Since Luciferase reporter assay has been used in numerous studies to validate the specificity gene targets, the use of this technique in this paper corroborates the dysregulation in VGlut expression5.

Figure 1 A qualitative PCR showing VGlut transcript levels from the heads Drosophila of the indicated genotypes. Source: Verma, P., Augustine, G.J., Ammar, M.R., Tashiro, A. & Cohen, S.M. A neuroprotective role for microRNA miR-1000 mediated by limiting glutamate exitotoxicity. Nat Neurosci 18, 379-385 (2015).

Excess presynaptic glutamate release The authors investigated the Drosophila neuromuscular junction (NMJ) activity to examine the changes in presynaptic glutamate release. Using miR-1000 KO2 mutant expressing GAL-4 reporter gene, the authors noted an increase in the size and the number of glutamate vesicles (Fig. 2)8. Moreover, recordings of NMJ spontaneous miniature excitatory junction potentials (mEJPs)

in miR-1000 mutants showed an increase in the mEJP median amplitude and frequency, but these effects were significantly rescued with the use of miR-1000 rescue allele (Fig. 3)8. However, when the VGlut mRNA was silenced using VGlut-RNAi, the Drosophila mutants showed significantly reduced mEJP median amplitude and frequency compared to the miR-1000 mutants8.

Several studies that have investigated miRNA involved glutamate mediated excitotoxicity have shown similar changes in miniature excitatory postsynaptic potential currents (mEPSCs) validating the role of miR-1000 in the current study in excess glutamate release5,6.

Figure 2 The confocal micrographs illustrating the changes in vesicle size and number at the Drosophila NMJ. The top shows the NMJ of Drosophila heterozygous for miR-1000 and the bottom shows NMJ of Drosophila homozygous for miR-1000.

The muscle was labelled for mysosin heavy chain (MHC) in blue, nuclei were labled with DAPI, VGlut was labelled in red with antiVGlut and green was used to visualize the NMJ that incorporated within the recombinded KO2 gene. Source: Verma, P., Augustine, G.J., Ammar, M.R., Tashiro, A. & Cohen, S.M. A neuroprotective role for microRNA miR-1000 mediated by limiting glutamate exitotoxicity. Nat Neurosci 18, 379-385 (2015).

Glutamate facilitated excitotoxicity and cell death The role of glutamate in excitotoxicity and cell death was investigated with modifications of glutamate receptors at the NMJs in the Drosophila mutants. The miR-1000 Drosophila mutants that carried a mutation in the NMDA type glutamate receptor showed improved survival, climbing performance and reduced cell death compared to non-NMDA mutated Drosophila mutants (Fig. 4)8.

The climbing performance was used in the current study to investigate neurodegeneration in Drosophila by measuring the percentage of miR-1000 mutant flies that were able to climb >5cm in a 30-cm column 8. However, with the use of Drosophila inhibitor of apoptosis protein (DIAP1) in metabotrophic glutamate receptor (mGluR) expressing cells significantly improved climbing performance in miR-1000 mutants8. These results collectively prove glutamate mediated excitotoxicity in miR-1000 mutants facilitated by glutamate receptors.

Conclusions and Discussion Verma and colleagues collectively demonstrated the role of miR-1000 in regulating VGlut expression by verifying both excess glutamate release and glutamate mediated excitotoxicity.

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Figure 3 The meadian mEJPs at the Drosophila NMJ of the indicated genotype. Source: Verma, P., Augustine, G.J., Ammar, M.R., Tashiro, A. & Cohen, S.M. A neuroprotective role for microRNA miR-1000 mediated by limiting glutamate exitotoxicity. Nat Neurosci 18, 379-385 (2015).

Excess glutamate release The authors associated excess glutamate release with increased median mEJP amplitude and frequency8.

These effects were significantly minimized by silencing VGlut mRNA with VGlut-RNAi or rescuing the effects of miR-1000 KO with miR-1000 rescue allele8. The authors have gone further to comment that increased mEJP amplitude and frequency were probably due to increased size and number of vesicles respectively8.

Previous studies in mice have suggested that VGult2 expression regulates the quantal size of glutamate vesicles9. Furthermore, electrophysiological studies in mice using VGlut2 heterozygous and homozygous mutants have shown reduced EPSPs to a single glutamate vesicle fusion9. Despite the lack of EPSP measurement in the current study of a single glutamate vesicle, findings from these studies are compelling to admit that increased mEJP amplitudes are due to increased vesicle size. In fact, confocal micrographs of the Drosophila NMJ showed previously further warrantees the increase in vesicle size after VGlut overexpression.

However, the different roles of VGlut isoforms question the reasoning behind the increase in vesicle size. Several studies have found VGut1 and VGlut2 isoforms to be involved in synaptic plasticity and synaptic release, with VGlut3 shown to be involved in intracellular glutamate storage10. Though the findings are predominately from mammalian central nervous system (CNS), it still questions whether the increase in vesicle size and mEJP measurements observed are mediated by VGlut1-related isoforms or VGlut-3 related isoform.

Similarly, several studies have found differences in the properties of VGlut isoforms in their release probability. In the rodent CNS, the VGlut1 isoform has been associated with increased docked and reserved vesicles in addition to greater release probability10,11.

This is consistent with observation of enhanced vesicle release in the current study. However, the lack of differentiation between VGlut isoforms and the initial definition of release probability in the current study, questions whether the increase in frequency of vesicle release is due to overexpression of VGlut or due to other mechanisms that are unknown.

In addition to differences in VGlut isoforms, the examination of Drosophila NMJ in the current study doubts 221

Figure 4 The lifespan of adult Drosophila miR-1000 mutants with and without carrying a mutation at the NMDA type glutamate receptor gene. Source: Verma, P., Augustine, G.J., Ammar, M.R., Tashiro, A. & Cohen, S.M. A neuroprotective role for microRNA miR-1000 mediated by limiting glutamate exitotoxicity. Nat Neurosci 18, 379-385 (2015)

the validity of the results. It is known that Drosophila muscle fibres are polyinnervated, with each muscle fibre being innervated by more than one neuron12,13.

Hence, it is plausible that the observed increase in the mEJP amplitude and frequency could have been due to spontaneous release of two or more quantum from more than one neuron innervating the muscle. But several studies that have used isopotential muscle fibres from Drosophila like in the current study were able to record accurate EPSP values from the muscle fibre13. These findings further validate the results of the current study.

Glutamate mediated excitotoxicity The authors associated movement disorders and vacuole formation in the mutant Drosophila with signs of neurodegeneration. Numerous studies that have used Drosophila as a model neurodegenerative organism have demonstrated failure in movement coordination and irregularity in movements8,14. This has been shown to cause by disruption in axonal transport of motor neurons14. In similar studies that have investigated brain degeneration in Swiss cheese (sws) mutant flies have found associations between vacuole formation with severity of neurodegeneration and number of apoptotic cell death1,15. These findings are suggested to be indicative of the results from the current study.

The improvements in climbing performance and cell survival in miR-1000 mutants in glutamate receptor knockouts in the current study, demonstrated that neurodegeneration is specific to glutamate release. In fact, several studies that investigated excitotoxicity in stroke models and the role of glutamate transporters in excitotoxicity consistently showed similar results6,8.

Also, experiments that reduced NMDA receptor activity using NMDA receptor antagonists, have shown reduced nociceptive-elevated pain perception in VGlut2 knockouts of neuropathic rodent pain models9.

Even though inconsistent results were observed between knockouts of mGluR and ionotropic glutamate receptors in miR-1000 mutants in the current study, several experiments have noted variable roles for mGluR and ionotropic receptors in Drosophila15.

Thus, altogether these findings demonstrate that excitotoxicity is specific to glutamate release.

Significance of the work

The current study investigated the presynaptic role of miR-1000 in Drosophila in regulating expression of vesicular glutamate transporter (VGlut) and its neuroprotective effects in the CNS. Several studies have investigated the postsynaptic roles of miRNA in regulating glutamate receptors, synaptic plasticity and its retrograde release at NMJs6,8. As such, only few studies have investigated the presynaptic role of miRNAs and work by Verma and colleagues demonstrate one of its kind.

Only in the recent years, evidence is starting to emerge to reveal the role of miRNAs in various neurodegenerative diseases and psychiatric disorders1,4.

Extensive research especially with stroke and cerebral ischemia models have revealed numerous miRNA genes involved in excitotoxicity and neurodegeneration1,2,3. In fact, with excitotoxicity been noted in various neurodegenerative diseases like PD, Alzheimerâ&#x20AC;&#x2122;s, MS and ALS, there has been great search in the field of neurodegenerative sciences to cure or at least delay the degenerative process1. As such, the emergence of miRNAs has showed a new plausible target to treat such neurodegenerative conditions.

In addition to neurodegenerative conditions, psychiatric disorders such as anxiety disorders, depression and schizophrenia have shown changes in the expression of certain miRNAs4. In fact, dysregulation in miR-181 family of miRNAs have shown to be associated with schizophrenia and addiction behaviors4,5.

As such, studies by Verma et al. and others have become significant in attempts to treat various neurological conditions. However, these findings demand further research as several studies have demonstrated the roles of miRNAs to be diverse and in some cases to be shown to mediate toxic responses1.

Criticisms and Future Directions Overall, Verma and coworkers showed evidence of excess glutamate release and glutamate specific excitotoxicity and neurodegeneration in miR-1000 mutants. However, there are caveats in the current study that questions the validity of the results and requires further research.

The authors associated the increase in mEJP amplitude and frequency with increase in vesicle size and quanta respectively8. However, findings from other studies on the properties of VGlut isoforms doubt the results10,11. Several studies have found VGlut1 and VGlut2 isoforms to predominately play a role in synaptic plasticity and synaptic release and VGlut3 to be involved in presynaptic intracellular storage of neurotransmitters10,11. As the current study did not identify the exact type of VGlut isoform studied, the reason to increased vesicle size/ mEJP amplitude is unknown. That is, it is unclear whether increased vesicle size is due to overexpression of VGlut3 or VGlut1/VGlut2 related isoforms.

Furthermore, studies have also noted differences in the properties of VGlut1 and VGlut2 in rodent models10,11. The VGlut1 isoform has been associated with increased release probability compared to VGlut2.

However, without the identity of the studied isoform of VGlut in the current experiment, it is hard to distinguish whether the observed increase in frequency of mEJPs were due to increased release of vesicles that were bound by VGlut2 or reflects the normal functionality of VGlut1. As such, to clearly infer the results, future experiments involving the study of single VGlut isoform with the knockouts of other two VGlut isoforms must be studied.

In addition, reduction in muscle-specific ionotropic glutamate receptor activity was not effective in Drosophila mutants in improving mEJP amplitude and climbing performance. Both ionotropic (AMPA and kainate) and metabotropic glutamate receptors (mGluRs) are present at the Drosophila NMJ15.

Previous studies have shown metabotropic and ionotropic receptors to mediate the effectiveness of short-term plasticity and fast synaptic transmission at the NMJ respectively15. Though overall reduction in NMDA, AMPA and mGluR receptors were able to improve mEJP amplitude and climbing performance in miR-1000 mutants; however, only the reduction of mGluR specifically at the NMJ produced such improvements in miR-1000 mutants.

Numerous studies have shown various homeostatic compensatory mechanisms in Drosophila NMJs that carry mutated glutamate receptors15. Previous studies have shown that motoneurons were able to compensate for disrupted postsynaptic glutamate receptors by increasing their vesicle release and quantal content upon an action potential15. Specifically, these compensatory effects were shown to be mediated in a retrograde fashion through the action of ionotropic receptor with the retrograde signal assuming to be Ca2+. Thus, it can be hypothesized that retrograde compensatory actions are largely mediated from mutated ionotropic receptors and muscle excitation are mediated by intact mGluRs. In order to observe the opposing effects of ionotropic and metabotropic glutamate receptors at the Drosophila NMJs, the compensatory mechanism in mutated glutamate receptors must be examined16. An experiment can be done by mutating the ionotropic glutamate receptor in a miR-1000 mutant at the NMJ to note the compensatory effect in the motoneuron15.

A similar experiment can be done by mutating the metabotropic receptor.

References 1. 1. Szafranski, K., Abraham, K.J. & Mekhall, K. Noncoding RNA in neural function, disease, and aging. Front Genet, 1-16 (2015).

2. Yang, Z.B. et al. Up-regulation of brain-enriched miR-107 promotes excitatory nuerotoxicity through down-regulation of glutamate transporter-1 expression following ischaemic stroke.

Clin Sci (Lond) 127, 679-689 (2014).

3. Saugstad, J.A. Non-coding RNAs in stroke and neuroprotection. Front Neurol, 1-11 (2015).

4. Kocerha, J., Dwivedi, Y. & Brennand, K.J. Noncoding RNAs and neurobehavioral mechanisms in psychiatric disease.

Mol Psychiatry, 1-8 (2015).

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5. Saba, R. et al. Dopamine-regulated microRNA miR-181a controls GluA2 surface expression in hippocampal neurons.

Mol Cell Biol 32, 619-632 (2012).

6. Harraz, M. M. et al. MicroRNA-223 is neuroprotective by targeting glutamate receptors. Proc Natl Acad Sci U S A. 109, 18962-18967 (2012).

7. Ouyang, Y. B. et al. Role of astrocytes in delayed neuronal death: GLT-1 and its novel regulation by microRNAs. Adv Neurobiol 11, 171-188 (2014).

8. Verma, P., Augustine, G.J., Ammar, M.R., Tashiro, A. & Cohen, S.M. A neuroprotective role for microRNA miR-1000 mediated by limiting glutamate exitotoxicity. Nat Neurosci 18, 379-385 (2015).

9. Moechars, D. et al. Vesicular glutamate transporter VGlut2 expression levels control quantal size and neuropathic pain. J Neurosci 26, 12055-12066 (2006).

10. Fremeau, R.T., Voglmaier, S., Seal, R. P. & Edwards, R.H. VGluts define subsets of excitatory neurons and suggest novel roles for glutamate. Trends Neurosci 27, 98-103 (2004).

11. Santos, M.S., Foss, S.M., Park, C.K. & Voglmaier, S.M. Protein interactions of the vesicular glutamate transporter VGLUT1. PLoS One 9, 1-13 (2014).

12. Keshishian, H., Broadie, K., Chiba, A. & Bate., M. The Drosophila neuromuscular junction: A model system for studying synaptic development and function. Annu Rev Neurosci 19, 545-575 (1996).

13. Jan, L.Y. & Jan, Y.N. L-Glutamate as an excitatory transmitter at the Drosophila larval neuromuscular junction. J. Physiol. 262, 215-238 (1976).

14. Bilen, J. & Bonini, N.M. Drosophila as a model for human neurodegenerative disease. Annu Rev Genet 39, 153-171 (2005).

15. DiAntonio A. Glutamate receptors at the Drosophila neuromuscular junction. Int Rev Neurobiol 75, 165-179 (2006).

16. Adler, E.M., Augustine, G.J., Duffy, S.N. & Charlton, M.P. Alien intracellular calcium chelators attenuate neurotransmitter release at the squid giant axon. J Neurosci. 11, 1496-1507 (1991).

Copyright Š 2015 Dr. Bill JU, Neurosciences, Human Biology Program

223

The impact of Social Defeat Stress on behavior and the Dopaminergic system

Joravir Riar

This review focuses on the effects of social defeat stress on behavior and the dopaminergic system in animals. It discusses experiments that induced social defeat stress on experimental mice and observed behavioral changes as compared to control mice. Certain experiments also tested to see how social defeat stress changes dopamine receptor levels and affinity in certain mouse brain region while also keeping track of changes DARPP levels.

The results of most experiment agreed on the fact that social defeat stress leads to symptoms of depression and anxiety in behavior while also cause a decrease in dopamine receptor and affinity levels. One experiment additionally found an increase in DARPP levels in socially defeated mice as compared to control mice.

Key words: social defeat; dopamine; dopaminergic pathway; D1/D2 receptor; cyclic adenosine 3′,5′-monophosphate-regulated phosphoprotein-32 (DARPP-32) Background Social defeat is the phenomenon where an animal loses a confrontation to another of its own kind. Figure 1, given below, shows the process of inducing social defeat stress on experimental mice. Inducing social

Figure 1. (Berton et al., 2006). Social Defeat Procedure Model.

Diagram showing the social defeat procedure that is followed in order to inflict social defeat stress on experimental mice.

defeat stress, as shown in figure 1, involves repeated social defeat for a prolonged period of time. Previous experiments have shown that implementing the social defeat procedure on an animal induces several symptoms related to depression and anxiety. Such symptoms include increased sucrose dislike and a tendency to spend more time in the dark in dark/light preference test, respectively (Yu et al., 2011; Kinsey et al., 2007). Results from experiments have also concluded that when socially defeated mice are put through behavioral tests they tend to show symptoms of depression and anxiety (Jin et al, 2015).

Substantial evidence denoting social defeat as a cause of depression and anxiety like symptoms has led to further research investigating its impact on the dopaminergic pathway. Dopamine is a neurotransmitter that regulates rewards and motivation (de Jong et al., 2015), making it an obvious choice for research on the impact of social defeat on depression and anxiety like behaviors as they both involve decreases in motivation and rewards. Previous research has been focused on the effect of social defeat stress

on dopamine receptor levels. It has also shown that social defeat stress causes changes in the sensitivity of dopamine neurons while also up regulating their expression and binding in certain parts of the brain (Avgustinovich and Alekseyenko, 2010; Kudryavtseva et al., 2008). But there are other components of the dopaminergic pathway that could potentially be tested in order to find the impact social defeat stress has on the dopaminergic pathway. One such component is a cytosolic protein called cyclic adenosine 3′,5′-monophosphate-regulated phosphoprotein-32 (DARPP-32). DARPP-32 is known to play a vital role in normal functioning of dopaminoceptive neurons by acting as a mediator between dopamine and several other neurotransmitters and the dopaminoceptive neurons (Jin et al, 2015). Hence any significant changes in DARPP-32 levels would indicate a noteworthy change in the dopaminergic pathway. Currently not a lot of research has been done using DARPP-32 as an indicator of changes in the dopaminergic pathway making it a possible candidate for future research concerning the topic in discussion.

In an article by Jin et al. (2015), the authors conducted research testing changes in mouse behavior, dopamine receptor expression and DARPP levels in curtains parts of the brain. The findings of their experiment made clear connection between the changes in behavior caused by social defeat stress with changes in dopaminergic pathways by using DARPP-32 levels as an indicator. Whereas the research conducted in articles by Avgustinovich and Alekseyenko, (2010) and Kudryavtseva et al., (2008) both recorded the changes in dopamine receptor sensitivity as a method of determining the effect of social defeat stress on the dopaminergic pathway. Both these experiments were successful in showing that social defeat stress resulted in a decrease in dopamine receptor sensitivity. Hence providing clear evidence that social defeat stress impacts the dopaminergic pathway.

Considering humans are also animals that frequently get involved in social dispute, these finding could potentially provide useful information for research on the effects of social defeat stress on humans.

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Research Overview

Summary of Major Results

In the article by Jin et al. (2015), researchers conducted an experiment that compared socially defeated mice to control mice to test for changes in behavior, dopamine receptor and DARPP levels.

A locomotion test, light/dark preference test, novel object recognition test, social integration test, Morris water maze test and the forced swimming test were conducted on both the control and the mice subject to the social defeat procedure as part of the experiment. Finally, western blotting procedure was used on mice brains after conducting the above experiment to test dopamine levels in the prefrontal cortex (PFC), amygdala (AMY) and hippocampus (HIP) of both the defeated and control mice.

The experiment conducted by Jin et al. (2015) lead to observations showing significant reduction in the total movement of defeated mice as compared to the control mice when they were put through the above mentioned behavioral test. They also found that defeated mice preferred spending much longer time in dark and congested areas as compared to the control mice.

Additionally, Defeated mice were found to perform worse on novel recognition tests as compared to control mice. Similarly, in the social interaction test defeated mice spent a decreased amount of time participating in social sniffing as compared to the control mice.

As for the difference in dopamine 1 and 2 receptor, no significant differences were seen between the control and defeated mice. The DARPP-32 levels showed a significant difference between the defeated and control mice, as the defeated mice were observed to have much higher levels of DARPP in their prefrontal cortex (PFC) and amygdala (AMY) as compared to the control. These results are shown graphically in Figure 2 below.

Figure 2. (Jin et al., 2015). DARPP-32 levels in control vs socially defeated mice. Graph showing DARPP-32 levels in the prefrontal cortex, hippocampus and amygdala of socially defeated and control mice.

The experiment conducted in the article by Jin et al., (2015) showed that a defeated animal displays symptoms that are similar to a depressed or anxious animal with signs of cognitive impairment. However based on the results of the conducted experiments these symptoms cannot be attributed to the changes in dopamine receptor levels in the prefrontal cortex (PFC), amygdala (AMY) and hippocampus (HIP) 225

of defeated mice as compared to control mice. But considering the observed increase in DARPP levels it can be concluded that the dopaminergic pathway is involved in the behavioral symptoms that were observed in socially defeated mice. Other researchers found that dopamine receptor activation played a fundamental role in acquiring social defeat behavior (Gray et al., 2015). Another study found that increase in dopamine receptor 1 (DR1) coincides with resilient behavior whereas decrease in DR1 leads to depression like behavior. While also showing that repeated activation of Dopamine receptor 2 cause social avoidance behavior (Francis et al., 2015). Another study found a direct relation between normal dopamine and the recovery of socially defeated mice as blocking dopamine receptor lead to socially defeated mice not gaining back aggression (Rillich and Stevenson 2014). The results of all these experiments differ from the experiment in discussion above as they show a direct link between changes in dopamine receptors expression and social defeat behavior. At the same time these results reinforce the results of the experiment conducted by Jin et al. (2015) by confirming the involvement of the dopaminergic pathways in causing behavioral changes in socially defeated mice.

Conclusions and Discussion In conclusion, the research conducted using the social defeat procedure mentioned earlier, has been a valuable source for gaining a better understanding of the impacts social defeat stress has on animals.

Majority of research on this topic has used several behavioral tests to determine changes in behavior of socially defeated mice. The results of these experiments have been complementary to each other as almost all of them confirm that social defeat stress can lead to animals showing depression and anxiety like symptoms. Several researchers have also looked at the biochemical changes caused by social defeat stress. The results of this research have convincingly showed changes in dopamine receptor expression and DARPP levels to correspond with social defeat stress. Some experiments have also shown changes in dopamine receptor and DARPP levels to accompany behavioral changes in socially defeated mice.

Since social defeat stress has been proven to cause changes in dopamine receptor and DARPP levels we can conclude that a change in both these factors indicates a fluctuation in the dopaminergic system, which regulate motivation and rewards. This fluctuation in motivation and rewards coincides with the results of many experiment discussed above that show increased depression and anxiety like behavior in socially defeated mice. Considering social defeat is a part of the lives of all animals including humans, this research gains more significance as it relates to all animals. The use of DARPP-32 as an indicator for changes in the dopaminergic pathway induced by social defeat stress is a new and innovative technique that has been used for the first time by Jin et al.

(2015) in the study they conducted. Looking at the results of their experiment and it is safe to say that DARPP-32 protein is a potential new tool to study the impacts social defeat has on the dopaminergic system. Furthermore their

experiment involved the use of several different types of behavioral test, as listed above, giving more credibility to their results concerning behavioral changes.

Criticisms and Future Directions

Stress is a derivative of human interactions and has been proven capable of causing changes in behavior pertaining to depression and anxiety. The experiment under consideration tested the effects of chronic social stress on dopamine levels in mouse models, furthering the existing knowledge of the impacts of stress on rewards, motivation and behavior. This information can be theoretically extended to be relevant in explaining the impacts of stress on daily human life.

The researchers induced stress associated with social defeat by subjecting mice to an aggressor for 10 days.

They followed with a series of behavioral tests in order to detect resulting depression and anxiety-like symptoms. In order to determine dopamine levels post stress procedures, the mice were then sacrificed and levels of dopamine receptor proteins were assessed using western blotting techniques. Results showed a significant change in depressive and anxious behavior of defeated mice as compared to the control group, with minor differences being observed in the dopamine receptor levels.

Overall, the paper showed that chronic social stress is capable of disrupting normal behavior and motivation in mice, suggesting for an incremental advancement in its contribution to the prevailing literatures.

Critical Analysis of Results However, there are limitations to the contributions of the paper. The experiment conducted lacked a mechanism to measure the affinity and density of dopamine receptors which if tested may resolve the discrepancy between measured dopamine receptor protein levels and observed behavioral changes. Techniques such as molecular dynamic simulations on homology models and the molecular docking method (Kanagarajadurai, 2009) are possible methods to detect dopamine receptor affinity and could serve to enhance the findings of the experiment in discussion. Alternatively, radio ligand binding assay, as used in an experiment by Ahring et al. (2015), can be used to achieve similar results, testing for receptor affinity and functional effectiveness.

Another flaw of the experiment is the lack of consideration for possible confounds. As reported by other researchers, changes in compounds such as KLF11 (Duncan et al., 2015) and reduced glucorticoid receptor resistance (Jung et al., 2015) have been shown to emulate the depression and anxiety like behavior observed in socially stressed mice of the experiment in discussion. This is an indication of a flaw in the study, as it does not prove that the depression and anxiety like features observed in socially stressed mice are solely dependent on changes in dopamine receptors. Researchers should take step towards confirming the source of change in behavior by controlling for compounds that may play a role in inducing depression and anxiety like behavior in mice. Furthermore brain-imaging techniques such as functional magnetic resonance imaging and positron emission tomography could also prove as useful tools in localizing the effects of social defeat stress on areas of the brain associated with these behavioral changes.

References 1. Ahring, P. K., Olsen, J. A., Nielsen, E. Ø., Peters, D., Pedersen, M. H., Rohde, L. A., ... & Balle, T. (2015). Engineered α4β2 Nicotinic Acetylcholine Receptors as Models for Measuring Agonist Binding and Effect at the Orthosteric Low-affinity α4-α4 Interface. Neuropharmacology.

2. Avgustinovich, D. F., Alekseyenko, O. V., & Tenditnik, M. V. (2001). Fighting among C57BL/6J mice and its implications for [3 H] 8-hydroxy-N, N-dipropyl-2-aminotetralin binding in various brain regions. Neuroscience letters, 305(3), 189-192.

3. -Berton, O., McClung, C. A., DiLeone, R. J., Krishnan, V., Renthal, W., Russo, S. J., ... & Nestler, E. J. (2006).

Essential role of BDNF in the mesolimbic dopamine pathway in social defeat stress. Science, 311(5762), 864-868.

4. -de Jong, J. W., Roelofs, T. J., Mol, F. M., Hillen, A. E., Meijboom, K. E., Luijendijk, M. C., ... & Adan, R. A. (2015). Reducing Ventral Tegmental Dopamine D2 Receptor Expression Selectively Boosts Incentive Motivation.Neuropsychopharmacology.

5. -Duncan, J., Wang, N., Zhang, X., Johnson, S., Harris, S., Zheng, B., ... & Wang, J. M. (2015). Chronic Social Stress and Ethanol Increase Expression of KLF11, a Cell Death Mediator, in Rat Brain. Neurotoxicity Research, 1-14.

6. -Francis, T. C., Chandra, R., Friend, D. M., Finkel, E., Dayrit, G., Miranda, J., ... & Lobo, M. K. (2015). Nucleus accumbens medium spiny neuron subtypes mediate depression-related outcomes to social defeat stress. Biological psychiatry, 77(3), 212-222.

7. -Gray, C. L., Norvelle, A., Larkin, T., & Huhman, K. L. (2015). Dopamine in the nucleus accumbens modulates the memory of social defeat in Syrian hamsters (Mesocricetus auratus). Behavioural brain research, 286, 22-28.

8. -Jung, S. H., Wang, Y., Kim, T., Tarr, A., Reader, B., Powell, N., & Sheridan, J. F. (2015). Molecular mechanisms of repeated social defeat-induced glucocorticoid resistance: Role of microRNA. Brain, behavior, and immunity, 44, 195-206 9. -Kanagarajadurai, K., Malini, M., Bhattacharya, A., Panicker, M. M., & Sowdhamini, R. (2009). Molecular modeling and docking studies of human 5-hydroxytryptamine 2A (5-HT2A) receptor for the identification of hotspots for ligand binding. Molecular BioSystems, 5(12), 1877-1888 10. -Kinsey, S. G., Bailey, M. T., Sheridan, J. F., Padgett, D. A., & Avitsur, R. (2007). Repeated social defeat causes increased anxiety-like behavior and alters splenocyte function in C57BL/6 and CD-1 mice. Brain, behavior, and immunity,21(4), 458-466.

11. -Kudryavtseva, N. N., Avgustinovich, D. F., Bondar, N. P., Tenditnik, M. V., & Kovalenko, I. L. (2008). An experimental approach for the study of psychotropic drug effects under simulated clinical conditions. Current drug metabolism, 9(4), 352-360.

12. -Rillich, J., & Stevenson, P. A. (2014). A fighter’s comeback: Dopamine is necessary for recovery of aggression after social defeat in crickets. Hormones and behavior, 66(4), 696-704.

13. -Yu, T., Guo, M., Garza, J., Rendon, S., Sun, X. L., Zhang, W., & Lu, X. Y. (2011). Cognitive and neural correlates of depression-like behaviour in socially defeated mice: an animal model of depression with cognitive dysfunction.International Journal of Neuropsychopharmacology, 14(3), 303-317.

226

An investigation of the facilitative effects of exercise on learning and memory

Ashkan Salehi

While exercise’s enhancement of learning and memory has been greatly investigated in the past, the question of how long these benefits endure after exercise has yet to be determined. The research article in question has sought to demonstrate the aforementioned endurance of the effects of exercise on cognition by investigating BDNF protein concentrations, analyzing performance in a Radial Arm Water Maze (RWM) with exposures to different delay periods, and analyzing overall improvements in memory acquisition, retention and strength. Firstly, authors divided 43 mice into two groups: sedentary and exercising. Authors provided the latter with running wheels, and analyzed how both groups learned the RWM at different times after exercise (immediately after, or with 1 or 2 week delays). Secondly, investigators determined the levels of BDNF protein in hippocampus. Investigators noted a correlation between exercise and lower errors, lower latency, more retention of memory, and more BDNF release.

Delay periods led to better acquisition but reducing delays led to better retention and strength. In regards to investigating how long the effects of exercise endure, average BDNF protein levels across all exercising mice were observed to increase for 3-4 weeks after exercise, before declining to baseline. The research article undertaken

by Berchtold et. al thereby illustrated the endurance of exercise’s effects on cognition by tracking BDNF protein levels, studied the effect of delay periods, and noted overall improvements in memory function due to exercise.

These findings thereby suggest a critical link between exercise and improvements in mental health.

Key words: Brain-Derived Neurotrophic Factor (BDNF); exercise; temporal endurance; Radial-Arm Water Maze (RWM); memory; acquisition; retention; delay period.

Background The effect of exercise on the human mind has been the subject of numerous past studies. Much research has been done to investigate the role of exercise on cognitive functions, often seeing improvements in test performance (Weuve et al., 2004), reduction of dementia (Larson et al., 2006), reduction of brain atrophy (Colcombe et al., 2003), and increases in cortical brain volume (Erickson and Kramer, 2008). Therefore, past studies of the effect of exercise on human cognition have demonstrated many beneficial effects.

Studies have also investigated the effects of exercise on memory, often using rodent models to establish precursors for human studies. These past studies have shown improved memory acquisition and retention using different cognitive tasks like the Morris water maze (van Praag et al., 2005), radial arm maze (Schweitzer et al., 2006), active avoidance test (Green- wood et al., 2007), object recognition test (O’Callaghan et al., 2007), passive avoidance test (Radak et al., 2006), and also the radial-arm water maze (RWM) (Khabour et al., 2008).

During these trials, it has been demonstrated that BDNF is a molecular mechanism that is up-regulated in response to exercise (Cotman and Berchtold, 2002).

Furthermore, blocking BDNF’s binding to TrkB by antibodies has been shown to attenuate the acquisition of spatial learning tasks (Vaynman et al., 2004).

In another past study, the genetic mechanisms behind exercise’s effects on learning and memory were described as the up-regulation in the gene expression of BDNF and synaptic trafficking proteins (synapsin I, synaptotagmin and syntaxin) (Molteni et al., 2002).

In addition, many intracellular signaling pathways in the hippocampus are also up-regulated in response to 227

exercise; Studies by Tong et al. and Molteni et al. have demonstrated increases in the induction of calciumcalmodulin kinase II (CaMK II), mitogen-activated protein kinases (MAPKI and MAPKII), transcription factors like cAMP response element-binding protein (CREB), as well as increased functioning of both glutamatergic and GABAergic systems.

However, regardless of the insight from past studies, researchers have yet to determine exactly how long the positive effects of exercise on learning (being improved memory acquisition, retention, strengthening etc.) can actually endure. In addition, one has yet to investigate the influence of time gaps between exercise, and subsequent performance in cognitive tasks like the RWM. This paper hereby investigates the influence of exercise on memory and acquisition and retention in the RWM, the effects of introducing a delay period after exercise on performance, and the persistence of exercise-induced BDNF protein levels, which is used as an indicator of the endurance of exercise’s positive effects on cognition.

Research Overview

Summary of Major Results

Berchtold et. al have found that the fastest memory acquisition in the RWM cognitive task was by EX1 mice, followed closely by EX2 mice, both of which have had a delay period between exercise and testing.

This is in contrast to EX and Sedentary mice, which demonstrated slower acquisition of the task, the latter having the worst performance. All exercising mice had fewer errors in the RWM and less total failures of task than sedentary mice. All exercising mice also showed better retention of memory during the probe

Table 1. In (A), a figure demonstrating a correlation between exercise and decreased latency in maze. In (B), decreased errors in maze are correlated with exercise. In (C) failures are demonstrated to occur less in exercising groups. In (D), a figure demonstrating that exercising mice, with special regard to the EX group, spend less time in maze than sedentary groups.

trial. Interestingly, EX mice showed better retention than EX-delay mice in the probe trial, perhaps due to the recency of training as this group experienced no waiting period. Also important to note, EX mice spent less time on average in arms of the maze than EX-delay mice, suggesting stronger memory.

In accordance to measuring the endurance of exercise’s effects on cognition, average BDNF protein levels across all exercising mice were observed to increase in response to exercise, and then decrease back down to baseline after 3-4 weeks from the end of exercise. BDNF protein levels were highest in the EX group, followed

by EX-delay groups and Sedentary groups respectively.

Performance scores showed a steep increase in response to small BDNF increments but reached a plateau with more BDNF levels, suggesting a saturation limit to the effect of BDNF on cognitive performance.

The findings of Berchtold et. al fall in line with the discoveries of past papers in that the general hypothesis of exercise having a facilitative effect on memory and learning has been validated. For example, authors have noted that exercise causes an increase in the rodent memory acquisition and retention, which is similar to the findings of Vaynman et al., van Praag et al. and Schweitzer et al. In addition, authors have demonstrated this enhancement of acquisition and retention, using the RWM cognitive task just like Nichol et al. and Khabour et al. have done previously.

Berchtold et. al have also successfully demonstrated that BDNF is up-regulated in response to exercise just like Cotman and Berchtold have noted previously. In summary, the major results of the study undertaken by Berchtold et. al fall in line with other studies and therefore are validated by previously-pioneered findings.

protein levels as a measuring tool for exercise’s positive effects: BDNF is involved with synaptogenesis, and in this study, has led to better performance after exercise. Therefore, the persistence and eventual decrease of BDNF does reflect the persistence of an obvious effect of exercise. Exercise therefore does have a rather beneficial and long-lasting effect on memory that persists for as long as a month. One now has a critical piece of knowledge regarding the persistence of learning improvements, which has not been previously investigated by other authors who have studied exercise’s effects on memory. Also contributing to knowledge regarding endurance of these effects was the finding that performance showed a steep increase in response to small BDNF increments at first, but that performance plateaued eventually, suggesting a saturation limit to the effect of BDNF on performance. This has been helpful in elucidating more about the role of BDNF and helps clarify the understanding that BDNF is not a magic drug of any sorts that could be exploited to infinitely enhance learning and memory.

Berchtold et. al were also the first to investigate the effect that delay periods between exercise and testing have on cognitive performance. It was found that BDNF protein levels were highest in the EX group, followed by EX-delay groups and Sedentary groups respectively. This can be interpreted as meaning that exercise’s most powerful molecular drive occurs if learning of memory-tasks is done immediately, as it is only then that exercise is able to objectively release

Conclusions and Discussion The most important inquiry undertaken by Berchtold et. al was investigating how long the positive effects of exercise on learning and memory persist; no other past study had systematically analyzed this beneficial effect of exercise on cognition. This main question was thereby addressed when authors found that average BDNF protein levels across all exercising mice remained elevated for 3-4 weeks after exercise and only then showed a decrease back down to baseline.

It is important to note why authors chose to use BDNF

Figure 1. A chart demonstrating a statistically significant correlation between hippocampal BDNF protein levels and exercise.

Exercising mice demonstrate higher BDNF protein levels whilst the sedentary group has the lowest amounts. Non-delay exercise mice had more BDNF than those with delays.

228

the most amount of BDNF: the molecular mechanism behind synaptic growth and expansion. This has helped elucidate more about the role of BDNF with specific regard to how it is altered by delay periods.

Therefore like the novel investigation of the endurance of BDNF, this finding is a new investigation in the field of exercise and learning and paves the way for future studies that can even provide more knowledge about the aforementioned field.

In accordance to the findings of previous papers on the matter of exercise and cognition, Berchtold et al. were able to demonstrate that all exercising mice had fewer errors and also less failures of the RWM task than sedentary mice. One can interpret this to mean that exercise clearly allows better performance in learning tasks that involve memory, simply because mice that were not exposed to exercise wheels did not perform as well.

Berchtold et. al found that the fastest acquisition was

by EX1 mice, followed closely by EX2 mice. This is in contrast to EX and sedentary mice, the latter having the worst performance. It can be interpreted that a small delay period between exercise and training will lead to fastest improvements in performance since mice with a one-week delay demonstrated the fastest acquisition. However, it is important to note that this does not necessarily mean that delays allow for stronger memory. Another major result of this study was that EX mice spent less time on average in arms of the maze than EX-delay mice. This is interpreted as EX mice having stronger memory, therefore meaning that delay periods can take away from the total strength of memory. This is also reflected in yet another result, in which EX mice performed better in another aspect: although all exercising mice showed better retention of memory during the probe trials, EX mice performed better than EX-delay mice due to the recency of training.

Interpreting these 2 findings allows one to speculate that although delay periods allow faster acquisition, immediate testing after exercise will have other positive aspects like more memory strength and retention.

The findings of Berchtold et al. in regards to acquisition, retention and overall performance in a RMW are able to once again fall in line with the findings of previous studies. Exercise leads to improved acquisition and retention, as shown previously by van Praag et al., Schweitzer et al., Green- wood et al., O’Callaghan et al. and Radak et al. Furthermore, performance in RMW was seen to improve as a result of exercise, falling in line with the investigation of Khabour et al.

In summary, exercise is once again, like the many preceding studies in this area, interpreted to lead to an enhancement in learning and memory.

The aforementioned results of Berchtold et. al contribute significant knowledge to the field of memory and learning in that it has been the first to investigate exactly how long the enhanced performance on cognitive tasks endures after exercise. The findings has also been significant in contributing more knowledge about the neurotrophic role of BDNF on top of previous findings, and have been the first to investigate how different delay periods influence the effects of exercise on learning. There are therefore many significant implications in terms of discovering more about the role of exercise, and future studies can benefit from replicating 229

the investigation of the time-course of BDNF release and the influences of delay periods.

Conclusions

Authors Berchtold et al. have provided further evidence about the facilitative effect of exercise on memory acquisition, retention and strengthening. This relates greatly to the field of neuroscience as it serves to expand our currently limited understanding of the important effects of exercise on cognitive functions. In addition, this article provides new findings by investigating the effect of different delay periods between exercise and testing, on cognitive performance: enabling future research inquiries in this area by neuroscientists. The authors of this paper also demonstrate another new finding: the time-course of the endurance effect of exercise on molecular mechanisms of the synapse like BDNF, which is relevant to neuroscience by providing another potential area to investigate in the future. In summary, the findings of Berchtold et. al introduce a new frontier for potential cognitive enhancement in the future by demonstrating the positive effects of exercise on learning and memory.

Criticisms and Future Directions

A potential problem regarding the methodology employed by Berchtold et. al is that the radial-arm water maze may have a confounding stress factor due to the fact that mice are forced to swim and thus performance may be negatively affected because of the anxiety placed on mice. In addition, there may be another confound as swimming during performance tests may be a form of exercise. This means that sedentary mice will accidentally be exposed to exercise and their BDNF and performance levels will not be representative, probably being higher levels than that of a true sedentary mouse.

Other potential issues involve the conclusions that Berchtold et. al derived from their findings. BDNF was taken to be the indicator of exercise-induced synaptogenesis, and the persistence of its protein levels were taken as indicative of the endurance of exercise’s effects. But what if BDNF is not the only mechanism involved in synaptogenesis and only measuring its endurance can take away from the actual persistence of exercise’s effects? The persistence of exercise’s effect could include actual measurement of synapse numbers, up-regulation of genes for synaptic trafficking of BDNF (Synapsin etc.), and the number Trk B receptors. All of the aforementioned should have been measured to see if they attenuate 3-4 weeks after exercise like BDNF had done.

Considering that BDNF release correlated with improved performance, Berchtold et. al can conduct future studies that use BDNF blockers in exercising mice, and investigate whether subsequent performance matches that of sedentary mice. Previous studies have demonstrated that BDNF-blockers have an attenuating effect on memory formation (Vaynman et al., 2004). This will be an important future step as it serves to further describe the role of BDNF in memory facilitation by showing how the lack thereof causes deficits in learning.

Berchtold et. al can also try to identify potential genes involved in exercise’s effects on memory. A past study by Molteni et. al has demonstrated that exercise up-regulates gene expression for BDNF and synaptic trafficking proteins (Molteni et al., 2002). Identifying genetic causation is important as it helps one to understand the causal mechanism behind learning and can provide future targets for manipulation and treatment.

Authors can also perform future investigations on whether Trk B receptors are up-regulated in response to exercise. Previous studies have shown the importance of TrkB by showing the attenuating effect of anti-Trk B antibodies on memory (Vaynman et al., 2006). In the future, Berchtold et. al could use GFP tags on Trk B receptors to determine if there is an increase in the numbers of this receptors, or whether the BDNF ligand is the only factor increased. This is important as Berchtold et. al have demonstrated the response of increased BDNF release, but lack information on the receptor that BDNF binds in the first place.

References 1. Berchtold NC, Castelo C, Cotman CW (2010) Exercise and time-dependent benefits to learning and memory. J Neurosci 167:588–597.

2. Colcombe SJ, Erickson KI, Raz N, Webb AG, Cohen NJ, McAuley E, Kramer AF (2003) Aerobic fitness reduces brain tissue loss in aging humans. J Gerontol A Biol Sci Med Sci 58:176–180.

3. Cotman CW, Berchtold NC (2002) Exercise: a behavioral intervention to enhance brain health and plasticity. Trends Neurosci 25:292–298.

4. Erickson K, Kramer AF (2008) Exercise effects on cognitive and neural plasticity in older adults. Br J Sports Med 43:22–24.

5. Greenwood BN, Strong PV, Dorey AA, Fleshner M (2007) Therapeutic effects of exercise: wheel running reverses stress-induced interference with shuttle box escape.

Behav Neurosci 121:992–1000.

6. Khabour OF, Alzoubi KH, Alomari MA, Alzubi MA (2008) Changes in spatial memory and BDNF expression to concurrent dietary restriction and voluntary exercise. Hippocampus 20(5):637-45.

7. Larson EB, Wang L, Bowen JD, McCormick WC, Teri L, Crane P, Kukull W (2006) Exercise is associated with reduced risk for incident dementia among persons 65 years of age and older. Ann Intern Med 144:73–81.

8. Molteni R, Ying Z, Gomez-Pinilla F (2002) Differential effects of acute and chronic exercise on plasticity-related genes in the rat hippocampus revealed by microarray. Eur J Neurosci 16:1107–1116.

9. O’Callaghan RM, Ohle R, Kelly AM (2007) The effects of forced exercise on hippocampal plasticity in the rat: a comparison of LTP, spatial and non-spatial learning. Behav Brain Res 176:362–366.

10. Radak Z, Toldy A, Szabo Z, Siamilis S, Nyakas C, Silye G, Jakus J, Goto S (2006) The effects of training and detraining on memory, neurotrophins and oxidative stress markers in rat brain. Neuro- chem Int 49:387–392.

11. Schweitzer NB, Alessio HM, Berry SD, Roeske K, Hagerman AE (2006) Exercise-induced changes in cardiac gene expression and its relation to spatial maze performance.

Neurochem Int 48:9–16.

12. Van Praag H, Shubert T, Zhao C, Gage FH (2005) Exercise enhances learning and hippocampal neurogenesis in aged mice. J Neurosci 25:8680 – 8685.

13. Vaynman S, Ying Z, Gomez-Pinilla F (2004) Hippocampal BDNF mediates the efficacy of exercise on synaptic plasticity and cognition. Eur J Neurosci 20:2580 –2590.

14. Vaynman S, Ying Z, Yin D, Gomez-Pinilla F (2006) Exercise differentially regulates synaptic proteins associated to the function of BDNF. Brain Res 1070:124–130.

15. Weuve J, Kang JH, Manson JE, Breteler MM, Ware JH, Grodstein F (2004) Physical activity, including walking, and cognitive function in older women. JAMA 292:1454–1461.

Received

April

2nd

2015;

accepted

April

29th

2015.

This work was supported by The Canadian Neurological Society (CNS), as well as the Progressive Neurological Society (PNS), the latter providing the Awesome Nerds System (ANS) and Somatic (S) programs. The authors thank Dr. M. Otto.

Rneuron, Dr. Hip O’Campus, and the students of HMB300 for technical assistance and feedback on this lab exercise.

Address correspondence to: Dr. Beady N. Eff, Human Biology Department, 666 Dendrite Spine Avenue, Institute of Axonomy, Hillock, IL 60101, Email: HPC@CNS.edu Copyright © 2015 Mr. Ashkan SALEHI, Neurosciences, Human Biology Program

230

Brain Inflammation, A Link Between Obesity and Cognitive Deficits

Husain Shakil

Obesity has been linked to chronic inflammation and irregular cytokine production. Clinical research has shown that obese patients, on average, have deficits in learning and memory when compared to non-obese individuals. However, the mechanism by which obesity affects the brain is unclear. Studies have indicated that brain inflammation is detrimental to cognitive function. Pro-inflammatory cytokines have been found to impair memory. A study conducted by Pistell et al. examined the relationship between obesity and impaired cognitive ability, and proposed a role for inflammation as the link between the two. In the study, different mice were administered the Western diet (WD), the High Fat Lard diet (HFL), or respective low fat control diets. The cognitive ability of mice was assessed with a stone T-maze, and markers of brain inflammation such as IL-6, TNF-α, MCP-1, and BDNF were measured with ELISAs. Mice given the WD diet were not found to have either abnormal brain inflammation or cognitive impairment. By increasing the fat content to an HFL diet, mice were then found to have both increased brain inflammation and reduced cognitive ability. When taken together these results suggest that cognitive decline associated with obesity requires the presence of higher amounts of inflammation in the brain. This supports the model in which inflammation is the mechanism through which obesity induces cognitive deficits.

Key words: Obesity; Inflammation; Stone T-maze; Spatial memory test; Interleukin 6 (IL-6); Tumor necrosis factor alpha (TNF-α); Monocyte Chemotactic Protein 1 (MCP-1); Brain-derived neurotrophic factor (BDNF) Background “Corpulence is not only a disease itself, but the harbinger of others”, a quote from Hippocrates of ancient Greece, often considered to be the father of western medicine1. This quote signifies that obesity has been recognized as a debilitating condition since antiquity. It is associated with a myriad of comorbidities including diabetes, and cardiovascular disease, which are among the leading causes of mortality in North America. The Globe and Mail has estimated that obesity costs the Canadian economy up to $7 billion annually2. This figure may increase further as the incidence of the disease is rising1. For these reasons, research into understanding the pathology of this metabolic condition is vital.

Among the many complications associated with obesity is altered brain physiology3. Statistical studies have found that increased body mass index (BMI) is correlated with reduced cerebral volume4. As well, brain abnormalities, such as reductions in neural grey matter volume, have been found in individuals suffering from obesity5. Some studies have investigated the effect of obesity on cognitive ability. Elias et al. compared obese and non-obese individuals with regard to their performance on a variety of memory and logic tests6. The results from this study indicated that obese males had lower cognitive performance compared to non-obese males. Similarly, Sorsen et al. compared the cognitive ability of obese individuals to non-obese individuals through intelligence tests, and found that people with the condition had significantly lower scores7.

Scientists have also investigated the relationship between obesity and the immune system. Obesity has been characterized as a state of chronic inflammation, involving abnormal cytokine production2. This irregularity is thought to be due to the inflammation of adipose tissue. This inflammation is caused by the infiltration of pro-inflammatory immune cells including Th1 cells, CD8+ cytotoxic T cells, M1 macrophages, Mast cells, and Neutrophils into the tissue8. When activated these cells release pro-inflammatory cytokines such as Interleukin 6 (IL-6), Tumor necrosis factor alpha (TNF-α), 231

and Monocyte Chemotactic Protein 1 (MCP-19.

There is a wide body of evidence that illustrates the adverse effects of inflammation on the brain10,11. A study conducted by Sandi et al. has shown that in a state of high stress, where inflammatory cytokines such as IL-1 and IL-6 circulate in the brain, spatial memory is impaired12. These same cytokines are also thought to be responsible for age associated cognitive decline13.

A study done on patients with psychosis has shown that increased levels of IL-6 and TNF-α causes reductions in brain derived neurotrophic factor (BDNF)14. BDNF is necessary for the survival of neurons in the central nervous system, and in its absence neurons undergo apoptosis. A reduction in levels of BDNF is thought to be the cause of memory loss associated with old age15.

Currently, the mechanism through which obesity causes cognitive deficits is poorly understood. In their article, Pistell et al. have investigated whether inflammation is the cause of altered brain function found in the obese state. They use a mouse model to compare the obese and non-obese state. In their study they measure cerebral cortical levels of IL-6, TNF-α, MCP-1, and BDNF, as these proteins are known to affect learning and memory. Pistell et al. use a behavioral test to assess the cognitive performance of both obese and non-obese mice. This approach is well founded; as previous studies have shown that peripheral immune activation in mice impedes spatial learning in the Morris water maze, a behavioral task16. This review will investigate the findings of Pistell et al. in their paper “Cognitive impairment following high fat diet consumption is associated with brain inflammation”, and discuss their proposed model of inflammation as the cause of cognitive decline due to obesity.

Major Results and Implications The Western Diet (WD) Does Not Alter Cognitive Function or Inflammation: One cohort of 12-month-old C57Bl/6 mice (n = 22)

was placed on a WD for a total of 21 weeks. 41% of the total calories from the diet were derived from fat. An equal number of mice of the same age were placed on a corresponding low-fat control diet (cWD).

An unpaired t-test revealed that WD mice gained significantly more weight than cWD mice (p<0.0001) after each diet was administered. WD mice displayed the obese phenotype while cWD mice did not.

The cognitive ability of each cohort of mice was tested with the stone T-maze spatial memory task. The task required mice to correctly navigate 13 consecutive left and right turns within the maze. Every mouse was pretrained in the maze and then given 15 consecutive trial runs. The number of errors committed by each mouse during each trial was used as an unbiased measure of performance. A two-way analysis of variance (ANOVA) was used to test whether certain factors significantly affected trial performance. Trial number was found to significantly affect performance for both WD and cWD mice (p<0.0001), indicating that mice were able to learn the task. Diet group was not found to have a significant effect on performance in the behavioral task (Figure 1). This result suggests that there were no differences between WD obese mice and cWD control mice with respect to spatial memory. This finding highly contrasts previous studies that indicate obesity is associated with lower cognitive ability.

Figure 12. The effect of WD on performance in the stone T-maze. Data represents mean ± SEM of the number of mistakes committed by mice from each cohort over all trials in the stone T-maze. Trials are split into 5 blocks of 3 trials each.

After spatial memory testing, mice were euthanized to retrieve brain tissue from the cerebral cortex. Tissue samples were tested with ELISAs to determine IL-6, TNF-α, MCP-1, and BDNF levels to evaluate the inflammatory state of each mouse brain. An unpaired t-test did not reveal any significant difference between WD and cWD mouse brain cytokine levels (Figure 2). These results suggest that obesity induced by the western diet does not drastically alter the inflammatory state of the mouse brain, which opposes the hypothesis set forth by Pistell et al.

High Fat Lard Diet (HFL) Alters Cognitive Function and Inflammation: Another cohort of 12-month-old C57Bl/6 mice (n = 23) was placed on a HFL for a total of 16 weeks. 60% of the total calories from the diet were derived from fat.

An equal number of mice of the same age were placed on a corresponding low-fat control diet (cHFL). HFL mice displayed the obese phenotype, and an unpaired t-test revealed that these mice were significantly heavier

Figure 22. The effect of WD on brain cytokine and BDNF levels. Data represents mean ± SEM of each protein in each mouse diet group.

than non-obese cHFL mice (p<0.0001).

The cognitive ability of mice from each diet group was tested and compared in the same manner as WD mice. Trial number was found to have a significant impact on performance for both HFL and cHFL mice (p<0.0001), indicating that mice were able to learn the task. Diet group was also found to have a significant effect on performance in the behavioral task (p<0.001), such that obese mice were found to perform more poorly (Figure 3). Therefore a difference in cognitive ability was found between the HFL obese mice and the cHFL control mice. This supports previous evidence that obesity causes deficits in cognitive function.

Figure 32. The effect of HFL on performance in the stone T-maze. Data represents mean ± SEM of the number of mistakes committed by mice from each cohort over all trials in the stone T-maze. Trials are split into 5 blocks of 3 trials each.

Cerebral cortical tissue samples were also tested as they were for WD mice. An unpaired t-test revealed significant differences between HFL and cHFL brain IL-6 (p< 0.01), TNF-α (p<0.01), MCP-1 (p<0.001), and BDNF levels (p<0.05), (Figure 4a). Collectively, these results suggest that obesity induced by the High Fat Lard diet alters the inflammatory state and cognitive ability of the mouse brain. This result supports the hypotheses set forth by Pistell et al.

Discussion In their study Pistell et al. investigated the effects of obesity caused by high fat diets on cognitive function, brain cytokines, and neuronal growth factors. The WD obese mice did not significantly differ from controls in their cognitive abilities. In addition these mice did not 232

Figure 42. The effect of HFL on brain cytokine and BDNF levels. Data represents mean Âą SEM of each protein in each mouse diet group

differ in their cytokine or growth factor levels. Together these results suggest that inflammation is necessary for the cognitive decline associated with obesity. This result supports the model hypothesized by Pistell et al., which proposes that inflammation is the key factor mediating the adverse effect of obesity on brain function.

The fact that the WD diet was unable to promote brain inflammation was a peculiar result, since previous studies have shown that ingestion of a high fat diet increases brain inflammation in mice17. The western diet itself has been found to alter brain physiology in animal models of Alzheimerâ&#x20AC;&#x2122;s disease18. The age of the mice could have caused this irregularity. Many studies have shown that aging increases the levels of pro-inflammatory cytokines in the brain, including IL-6. Mice used in this study were middle aged, which may have caused control mice to have increased levels of cytokines, and decreased levels of BDNF in the brain. This may have rendered any increase in the markers of brain inflammation produced by the western diet negligible.

HFL mice were found to perform significantly worse than cHFL mice in the stone T-maze. HFL mice were also found to have increased levels of markers for inflammation. These results give strong support to previous findings that obesity modulates the inflammatory state of the body, and that it has adverse affects on learning and memory.

As well, these finding support the model that brain inflammation is the cause of cognitive decline due to obesity.

The HFL and WD diets produced contrasting results, despite both being high in fat content. HFL mice were found to gain more weight than WD mice. This may be the reason why HFL obese mice were significantly different from control mice, while WD mice were not.

The level of adiposity present in the obese state may be important in determining the outcome of the condition. This suggests that obesity exists on a spectrum, and is a dynamic rather than static condition.

Previous studies have found that the cognitive decline associated with aging is due to a shift in neuronal cell metabolism. This shift involves the increase lipid and ketone body oxidation in neuronal cells19. Fat content in the HFL diet was much higher than in the WD diet. This may have contributed to the different results seen. The higher levels of fat in the HFL diet may have caused more of a shift in metabolism toward lipid oxidation than the WD diet. This would explain why the HFL diet caused poorer performance in the behavioral task, while WD did not.

The higher fat content in the HFL diet could also have increased serum concentrations of triglycerides.

Previous research indicates that high levels of serum triglycerides are able to reduce cognitive function20. The 233

higher fat content in the HFL diet may have been able to increase serum triglyceride to a level that could impair cognitive function, whereas fat content of the WD diet may not have been sufficient to do so. This suggests an alternate model for the pathology of obesity in which the increased level of serum triglycerides as opposed to brain inflammation mediates the reduced cognitive ability seen. However, it may just be the case that an increase in serum triglycerides causes an increase in brain inflammation, but this remains unclear.

The results of this study are highly relevant, as obesity is still prevalent throughout the world. The findings have great impact because they provide support to a model that explains the mechanism through which obesity negatively affects cognitive ability. This model can help clinicians devise potential treatments for the adverse effects of obesity on the brain. According to this model, provision of anti-inflammatory medication to patients with this condition may reverse their cognitive deficits.

Conclusions The rising incidence of obesity across North America is cause for concern, due to the array of diseases associated with the condition. Pistell et al. have aimed to uncover the mechanism through which obesity affects the brain. They were able to show that mice given a high fat lard diet performed significantly worse than control mice on a spatial learning task. Further they showed that poor performance was correlated with multiple markers of brain inflammation. Ultimately, their paper provides evidence for the model that obesity induces brain inflammation, which in turn causes deficits in cognition. Pistell et al. have made an important contribution to the understanding of the pathological mechanisms involved in obesity. The paper provides evidence for a link between obesity and neurodegeneration, using inflammation as the connection.

Criticisms and Future Directions

Pistell et al. were able to provide strong evidence for the notion that brain inflammation is the cause of cognitive decline due to obesity. This was seen when HFL mice were found to perform significantly worse on the Stone-T maze when compared to control groups, along with having significantly higher levels of IL-6, TNF-Îą, MCP-1, and lower levels of BDNF. However a point of contention could be that the brain inflammation seen was due to stress rather than the weight gain in the mice. The stone T-maze requires mice to wade through water, which can be perceived as stressful conditions.

Stress is known to increase markers of brain inflammation. As well, under conditions of high stress spatial learning ability declines12. Therefore stress is able to account for all the results seen for HFL mice. A possible strategy to eliminate this confound would be to re-conduct the experiment using the radial arm maze as the behavioral task instead. The radial arm maze uses reward as the motivation for learning rather than stress. Therefore any brain inflammation found in mice after such a test is unlikely to be due to stress.

An obvious criticism of the study conducted by Pistell

et al. is that their proposed model failed to hold for WD mice. These mice displayed the obese phenotype, but did not display abnormal brain inflammation or cognitive deficits when compared to the respective control group.

This result was explained by considering the age of the mice. It was proposed that inflammation caused by the age of the mice masked any adverse effects produced by obesity. Repeating the experiment with younger mice can test this hypothesis. If no difference is found between younger obese mice and control mice, then age cannot be used to explain the peculiar result. It would then be necessary to further investigate why WD obese mice do not display the same inflammatory and cognitive differences seen in HFL mice.

A possible future experiment that could be performed would be to perform lipectomies and fat transplants on HFL mice. Erion et al. have previously performed both lipectomies and fat transplants to adjust adiposity in db/db obese mice21. In their study they found that peripheral inflammation in these obese mice was increased by fat transplants, and reduced by lipectomies. Investigating the effect of these procedures on performance in the stone T-maze would be an important experiment for evaluating their proposed model. The Pistell et al. model would be supported if increasing adiposity and inflammation through a fat transplant decreased the performance of an HFL mouse in a stone T-maze. Similarly, their model would also be supported if decreasing adiposity and inflammation through a lipectomy, increased performance in the stone T-maze. Any other result would weaken their model, and necessitate adjustments to it.

Another useful experiment that could be performed would be to compare markers of brain inflammation in human obese individuals and controls. Pistell et al. were able to suggest a mechanism for how obesity adversely affects the brain using an animal model. It would be very useful to try and replicate the results of their experiments in a human context. The ultimate goal of this research is to understand the human obese condition. Therefore, it is important to investigate whether the model of immune mediated cognitive decline holds true for the human case.

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14. Mondelli, V. et al. Stress and inflammation reduce brain-derived neurotrophic factor expression in first-episode psychosis: A pathway to smaller hippocampal volume. J. Clin.

Psychiatry 72, 1677–1684 (2011).

15. Shimada, H. et al. A large, cross-sectional observational study of serum BDNF, cognitive function, and mild cognitive impairment in the elderly. Front. Aging Neurosci. 6, 1–9 (2014).

16. Chen, J. et al. Neuroinflammation and disruption in working memory in aged mice after acute stimulation of the peripheral innate immune system. Brain. Behav. Immun. 22, 301–311 (2008).

17. White, C. L. et al. Effects of high fat diet on Morris maze performance, oxidative stress, and inflammation in rats: Contributions of maternal diet. Neurobiol. Dis. 35, 3–13 (2009).

18. Hooijmans, C. R. et al. DHA and cholesterol containing diets influence Alzheimer-like pathology, cognition and cerebral vasculature in APPswe/PS1dE9 mice. Neurobiol. Dis. 33, 482–498 (2009).

19. Brinton, R. D. Estrogen regulation of glucose metabolism and mitochondrial function: Therapeutic implications for prevention of Alzheimer’s disease. Adv. Drug Deliv. Rev. 60, 1504–1511 (2008).

20. Farr, S. a. et al. Obesity and hypertriglyceridemia produce cognitive impairment. Endocrinology 149, 2628–2636 (2008).

21. Erion, J. R. et al. Obesity elicits interleukin 1-mediated deficits in hippocampal synaptic plasticity. J. Neurosci. 34, 2618–31 (2014).

Received March, 12,

February, 2015;

10, accepted

Address correspondence to: Bay Street, Email:

2015; April, 06,

revised 2015.

Mr. Husain Shakil, 832 Husain.Shakil@mail.utoronto.ca

Copyright © 2015 Dr. Bill JU, Neurosciences, Human Biology Program 234

The role of cAMP in mediating hippocampal-dependent spatial memory loss following periods of acute sleep deprivation

Arman Shekari

Very little is known about sleep in general. It is common practice in science to eliminate what you don’t understand and observe the results; such is the logic behind sleep deprivation research. This review assesses the infancy of this field of research up until today, and into the future. As of today, researchers have come to the consensus that sleep regulates gene expression though the modulation of the cAMP-PKA-CREB pathway. This review focuses on a seminal paper done by Havekes et al. (2014) that manipulated cAMP levels during periods of sleep deprivation. Increasing cAMP levels essentially eliminated the negative effects of sleep deprivation.

Considerations to areas of future study based on these results are given.

Key words: Hippocampus (HPC); learning; memory; spatial; sleep; octopamine; cAMP; Background

The Molecular Basis of Sleep Deprivation: Past and Present

The effects of sleep deprivation are an unfortunate reality for about 50 million people in the US alone (Hublin et al., 2001). While the gross physiological symptoms of sleep deprivation are familiar to many, little is known about the molecular basis of sleep deprivation (SD). Giuditta, Rutigliano, & Vitale-Neugebauer (1980) were first to break ground in this area of research by demonstrating that sleep accelerates the process of DNA transcription by comparing levels of radio-labeled RNA in sleep-deprived vs. control animals. Their results were confirmed 24 years later by Cirelli, Gutierrez, & Tononi (2004) who observed a 10% increase in RNA expression during sleep through the analysis of RNA microarrays taken from either sleeping or awake animals. Research done in the 1990s suggested that sleep might play a role in regulating translation as well, as Ramm & Smith (1990) observed that sleep is highly correlated with relative rates of protein expression in the cortex. Recent research has confirmed this notion as well. Vecsey et al.

(2012) demonstrated that 5 hours of SD reduced the levels of a phosphorylated form of the protein mTOR, a key regulator of protein synthesis. Subsequent microarray analysis by this group revealed that more than 500 genes were altered in their expression following SD. From this, researchers have come to the general consensus that sleep regulates gene expression. With this in mind, research today is focused on elucidating exactly how sleep achieves this on a molecular level.

Work done by Wang et al. (2010) demonstrated that a large majority of the genes affected by SD contain the cAMP response element (CRE). Further work done by Vecsey et al. (2009) demonstrated that cAMP levels in the hippocampus decrease significantly following periods of acute SD. This group went on to discover that this decrease in cAMP during SD was mediated by the increased levels of the enzyme PDE4(A5), an enzyme responsible for the breakdown of cAMP. From their findings, Vecsey et al. (2009) hypothesized that SD causes an increase in hippocampal PDE4(A5) levels, reducing cAMP levels and subsequently catalytic PKA levels, resulting in a deficit of phosphorylated CREB binding to CRE-containing DNA (See Fig. 2). A

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groundbreaking study by Havekes et al. (2014) aimed to confirm this hypothesis by directly manipulating cAMP levels in the hippocampus of mice during periods of SD. To do this, they created a viral vector containing a gene coding for the octopamine receptor (DmOctβ) found natively in Drosophila. Once bound to octopamine, this GPCR activates adenylyl cyclase, which goes on to produce cAMP from ATP. They restricted the expression of this receptor solely to excitatory hippocampal neurons by using a CAMKIIa promoter. Octopamine could then enter the brain via intra-peritoneal (IP) injection and increase cAMP levels solely in the hippocampus. By doing this, they could directly assess the impact of their experimental manipulation by having mice perform the hippocampal-dependent spatial novel object recognition task following periods of SD.

Testing the Efficacy of Hypothesized Treatments

As indicated above, once researchers hypothesize a candidate pathway mediated by SD, they create a treatment based on their hypothesis and subsequently test it using a behavioral task. The articles reviewed here focus mainly on hippocampal dependent tasks.

Tests of hippocampal-dependent spatial memory are used to assess the impact of SD on animals (usually mice). One such test is the spatial novel object recognition test. In this test, mice are placed in a chamber with 3 objects and are given time to explore.

The mice are removed from the test chamber and the location of one of the objects is changed. In SD research, animals are sleep-deprived after their initial round of exploration and are then placed back into the chamber where their “% preference for the displaced object” is assessed (Havekes et al., 2014).

Research Overview

Summary of Major Results

Through the immunohistochemical analysis of the brain tissue of harvested mice, Havekes et al. (2014) were able to confirm the presence of the octopamine receptor on excitatory neurons in the CA1, CA2, CA3, and dentate gyrus regions of the hippocampus.

Separate analyses done on brain tissue from the pre-

frontal cortex revealed an absence of the receptor.

The absence of the octopamine receptor in unrelated brain areas such as the pre-frontal cortex served to confirm the efficacy of the CAMKIIa promoter in restricting the expression of the octopamine receptor to the area of interest. Hippocampal tissue harvested from receptor-expressing mice revealed a significant increase in cAMP levels from baseline upon octopamine ligand injection. As expected, control mice showed no response to octopamine in terms of hippocampal cAMP levels. These results served as a proof-of-concept and confirmed that cAMP levels could be reliably controlled through simple IP injections of octopamine. Octopamine receptor-expressing mice that received octopamine injections during a 5-hour period of sleep deprivation showed no significant difference in their performance on a spatial novel object recognition task compared to control mice that were not sleep deprived. As expected, control mice who were subject to 5 hours of sleep deprivation performed significantly worse on the novel objection recognition task compared to the other experimental groups (See Fig. 1). These results are in line with the hypothesis put forth by Vecsey et al. (2009). If SD reduces cAMP levels, actively increasing cAMP in an area of the brain should essentially eliminate any difference between a control animal and one who ahs been sleep deprived. This is exactly what was observed by Havekes et al. (2014).

Conclusions and Discussion

Validity of the Authors’ Claims

Although discussed in passing by Havekes et al. (2014), their use of the CAMKIIa promoter paired with the spatial novel object recognition task added great validity to their experimental design. As previously mentioned, the CAMKIIa promoter ensures that the octopamine receptor is only expressed in excitatory hippocampal neurons.

The activity of these neurons has been shown to be key in the acquisition of spatial memories (McHail & Dumas, 2015). By restricting the effect of their treatment to this population of neurons, and by using a test of spatial memory to determine the efficacy of their treatment, the conclusion made by the authors that boosting cAMP levels in the hippocampus effectively reverses the spatial memory deficits caused by sleep deprivation is valid.

Figure 1. Sleep-deprived mice injected with octopamine (DmOctβ SD) performed significantly better on a novel object recognition task compared to control sleep deprived mice (eGFP SD). Non sleep-deprived control mice did not differ significantly in their performance compared to sleep-deprived octopamine-injected mice. (Figure from: Havekes et. al 2014)

Critical Analysis of the Results

One thing that was not included in the analysis done by Havekes et al. (2014) was a direct assay of hippocampal cAMP levels during the periods of SD. Being able to illustrate in real time that SD causes decreases in hippocampal cAMP and mice treated with the receptor+octopamine perform just as well as control mice would have beautifully illustrated the success of their model. Past work done

by Vecsey et al. (2009) illustrating that cAMP levels do indeed fall during SD in the hippocampus along with the original authors’ aforementioned immunohistochemical analyses done in vitro may have been why the authors felt that a direct assay of cAMP levels in real time was unnecessary. Another aspect absent from their study was an assay of downstream genetic targets. As previously stated, the cAMP-PKA signaling pathway serves to phosphorylate CREB in order to trigger gene transcription.

Havekes et al. (2014) have essentially started from the top of the signaling cascade. As shown by Vecsey et al.

(2012), sleep mediates the regulation of more than 500 genes, most of which contain the CREB-binding CRE element. Concluding that cAMP levels mediate memory deficits gives no insight into the genes and associated proteins that are actually mediating this loss of memory.

FUTURE DIRECTIONS As evident from the above section, one of the biggest

Figure 2. Image showing the hypothesized impact of SD on the cAMP pathway. During periods of SD, PDE4(A5) activity increases, causing a decrease in the amount of cAMP and subsequently active PKA. As a result, less phosphorylated CREB is present and the expression of cAMP responsive genes is reduced. Reduced expression of the protein mTOR serves to further imbalance gene expression as mTOR itself plays a role in modulating protein expression. (Image from Vecesey et al. 2009)

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problems with the study done by Havekes et al. (2014) is their lack of elucidation of downstream targets to cAMP. Previous research seems to implicate the role of GABA receptors in mediating memory impairments following SD. A study done by Tretter et al. (2009) observed that mice engineered to overexpress the GABAa receptor have impairments in spatial memory consolidation. Furthermore, a study done by Tdavarty et al. (2011) observed that periods of acute SD caused levels of the GABAb receptor to increase. A future study that manipulates cAMP levels along with keeping track of GABAa and GABAb receptor protein expression through immunochemical analysis would be helpful in determining if the observed increases in GABA activity are the result of the deregulation of the cAMP-PKA-CREB pathway caused by SD. One potential problem with the actual experimental design of Havekes et al. (2014) study has to do with how the test animals were sleep-deprived. As evident from this review, SD research has been going on for a long time, and within that time several standardized methods have been developed. The most common method (and the one employed by Havekes et al. (2014)) is the gentle stimulation method, which involves gently disturbing the surroundings of a mouse to ensure that it does not fall asleep (Havekes et al., 2014). Another method is the novel environment method, where new materials are continually placed in the animal’s cage to keep it awake (Kopp et al., 2006). The first and most obvious problem with both of these methods is the stress that the animals experience during periods SD confounding results. Work done by Tiba et al. (2008) demonstrated that animals that lack adrenal glands still exhibit memory deficits following periods of acute SD. Although stress may not directly confound results, previous research has not ruled out the role of external confounds associated with SD methods.

For example, both Kopp et al. (2006) and Vecsey et al. (2009) conducted a studies observing the effect of 5 hours of SD on NMDA receptor composition. While both studies sleep-deprived animals for a total of 5 hours, only Kopp et al. (2006) observed significant changes in NMDA receptor composition. The only significant difference between was their use of different SD techniques.

With advances in optogenetic technology, it is now possible to sleep-deprive animals solely through the optogenetic stimulation of orexin/hypocretin neurons in the hypothalamus. Rolls et al. (2011) was able to observe memory consolidation impairments in SD mice using this method. In order to avoid any unnecessary confounds, Havekes et al. (2014) should redo their experiment with the use of the gentle stimulation technique swapped for newer optogenetic methods that do not involve physically disturbing the mice repeatedly in order to achieve SD. Finally, it is important to note that increasing hippocampal cAMP levels is not the only known method of protecting spatial memories. A study done by Alesia et al. (2011) demonstrated that acute nicotine treatment during periods of SD was effective in protecting spatial memories. It may be possible that cross-talk between the cholinergic system and the cAMP-PKA-CREB pathway would be occurring.

A follow-up study assessing changes in both cAMP levels along with hippocampal cholinergic activation would help identify novel downstream effectors mediating this potential crosstalk.

This review has analyzed research that implicates sleep’s vital role in regulating gene expression through 237

Final Words the cAMP-PKA pathway. It is important to realize that research in this area has been largely restricted to the hippocampus. This is mainly because the hippocampus is a fairly straightforward structure in terms of its internal circuitry and it is extremely easy to test for hippocampal deficits. Anyone who has had a bad night of sleep knows that the effects of SD are not restricted to memory. SD also has emotional implications, with people feeling more irritable if they have not slept well. Hopefully future research will branch out and observe SD-related changes in other areas such as the limbic system in order to determine if similar cAMP-mediated gene regulation changes are occurring. Validating these hypotheses will be a great challenge as testing for emotional changes in animals is far more complicated than testing for spatial memory. Still, research in this area is vital to understanding the molecular basis of sleep.

References 1. Aleisa, A., Helal, G., Alhaider, I., Alzoubi, K., Srivareerat, M., Tran, T., ...& Alkadhi, K. (2011). Acute nicotine treatment prevents REM sleep deprivation-induced learning and memory impairment in rat. Hippocampus 2, 899-909 2. Cirelli, C., Gutierrez, C., & Tononi, G. (2004). Extensive and Divergent Effects of Sleep and Wakefulness on Brain Gene Expression. Neuron, 41(1), 35-43.

3. Giuditta, A., Rutigliano, B., & Vitale-Neugebauer, A. (1980). Influence of Synchronized Sleep on the Biosynthesis of RNA in Neuronal and Mixed Fractions Isolated from Rabbit Cerebral Cortex. Journal of Neurochemistry, 35(6), 12671272.

4. Havekes, R., Bruinenberg, V., Tudor, J., Ferri, S., Baumann, A., Meerlo, P., & Abel, T. (2014). Transiently Increasing cAMP Levels Selectively in Hippocampal Excitatory Neurons during Sleep Deprivation Prevents Memory Deficits Caused by Sleep Loss. The Journal of Neuroscience, 34(47), 15715-15721.

5. Hublin, C., Kaprio, J., Partinen, M., & Koskenvuo, M. Insufficient sleep –a population-based study in adults. (2001).

Sleep. 24(4), 392-400 6. Kopp, C., Longordo, F., Nicholson, J., & Luthi, A. (2006). Insufficient Sleep Reversibly Alters Bidirectional Synaptic Plasticity and NMDA Receptor Function. Journal of Neuroscience 26, 12456-12465 7. Mchail, D., & Dumas, T. (2015). Multiple forms of metaplasticity at a single hippocampal synapse during late postnatal development. Developmental Cognitive Neuroscience, 23(3), 145-154.

8. Ramm, P., & Smith, C. (1990). Rates of cerebral protein synthesis are linked to slow wave sleep in the rat. Physiology & Behavior, 48(5), 749-753.

9. Rolls, A., Colas, D., Adamantidis, A., Carter, M., LanreAmos, T., Heller, H., & Lecea, L. (2011). Optogenetic disruption of sleep continuity impairs memory consolidation.

Proceedings of the National Academy of Sciences 108, 13305-13310

10. Tadavarty, R., Rajput, PS., Wong, JM., Kumar, U., & Sastry BR. (2011). Sleep-deprivation induces changes in GABA(B) and mGlu receptor expression and has consequences for synaptic long-term depression. PloS One. 6, e24933 11. Tiba, PA., Oliveira, MG., Rossi, VC., Tufik, S., Suchecki, D. 2008. Glucocorticoids are not responsible for paradoxical sleep deprivation-induced memory impairments. Sleep. 31 12. Tretter, V., Revilla-Sanchez, R., Houston, C., Terunuma, M., Havekes, R., Florian, C., ... Moss, S. (2009). Deficits in spatial memory correlate with modified -aminobutyric acid type A receptor tyrosine phosphorylation in the hippocampus. Proceedings of the National Academy of Sciences. 106, 20039-20044 13. Vecsey, C., Baillie, G., Jaganath, D., Havekes, R., Daniels, A., Wimmer, M., ... &Abel, T. (2009). Sleep deprivation impairs cAMP signalling in the hippocampus. Nature. 461, 1122-1125 14. Vecsey, C., Peixoto, L., Choi, J., Wimmer, M., Jaganath, D., Hernandez, P., . . . Abel, T. (2012). Genomic analysis of sleep deprivation reveals translational regulation in the hippocampus. Physiological Genomics, 44(20), 981-991.

15. Wang, H., Liu, Y., Briesemann, M., & Yan, J. (2010).

Computational analysis of gene regulation in animal sleep deprivation. Physiological Genomics, 42(3), 427-436

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Treating Alzheimer’s Disease with Magnetic Resonance ImagingGuided Focused Ultrasound

Jaclin Simonetta

Repeated magnetic resonance imaging-guided focused ultrasound has risen in popularity as a non-invasive treatment for Alzheimer’s disease. Several studies have shown how this technique has been successful for delivering drugs across the blood brain barrier, a challenge that has been of great concern for treating neurological diseases.

A study done by Burgess et al. used focused ultrasound with the delivery of microbubble contrast agent intravenously, to show how this technique could reduce behavioural impairments and pathological abnormalities in AD, specifically the amyloid-β peptide deposits, Tg CRND8 mice were treated with focused ultrasound every week for three weeks, targeting the ultrasound at the hippocampus. The transgenic mice, as well as the nontransgenic mice were assessed in a Y-maze test post-treatment, assessing spatial memory in the mice. After treatment with focused ultrasound in the bilateral hippocampus, the mice spent more time in the novel arm of the maze, indicative of improved memory. Immunohistochemical analysis and plaque analysis were also completed. There was shown to be an increase in neurons and decreased plaque load in the region exposed to sonication.

It is recommended that to provide further evidence that focused ultrasound is the ideal non-invasive therapy for AD, it is essential that this technique be applied in clinical trials. Studies done by McDannold and others on the macaque brain has shown that the temporary disruption of the blood brain barrier by sonication has relatively low risk for neurological impairment post-treatment.

Key words: MR imaging-guided focused ultrasound, Alzheimer’s disease, Y-maze test, amyloid plaques Background Alzheimer’s disease (AD) is a neurodegenerative disorder that affects millions of people worldwide.

Despite the many years of research, there is currently no disease-modifying therapies available. AD is most notably associated with decreased spatial memory and cognition, as well as its pathological abnormalities that have been shown to be the disease-causing agent.

Specifically, amyloid-β (Aβ) peptide deposits in the extracellular AD brain and forms amyloid plaques due to impaired removal of the peptide10. There are also intraneuronal neurofibrillary tangles consisting of hyperphosphorylated tau protein, an important feature in the neuropathological diagnosis of AD1,13.

Research on magnetic resonance (MR) imagingguided focused ultrasound (FUS) began as a search for a non-invasive method to deliver drugs passed the blood brain barrier (BBB). Anti-Aβ drugs were looked at as a potential therapeutic for AD, but only a small portion of the drug was able to arrive in the brain due to the limited permeability of the BBB8.

In addition, the only option for drug delivery into the brain involved highly invasive procedures, such as injecting patients with a needle into the brain, which would require the opening of the skull5. These procedures exposed patients to the risks of permanent neurological damage, bleeding and infection. In 2001, Hynynen and others were able to show that MR imaging-guided ultrasound increases the permeability of the BBB, allowing the delivery of therapeutics into the brain without damaging surrounding brain tissue5.

MR imaging-guided focused ultrasound requires the administration of microbubble contrast agents, composed of either a lipid or polymer shell, outside the brain3. The microbubbles oscillate outside the BBB due to lowfrequency sonication. During low-frequency sonication, microbubble ultrasonography (US) contrast agents are 239

acoustically activated, oscillate outside the BBB, and open the tight junctions of the BBB. This transient opening of the tight junctions allows the microbubbles to enter the brain3. The focused ultrasound is directed at the brain area of interest. For instance, when studying the effects of this approach in the AD brain, the ultrasound is typically targeted at the bilateral hippocampus, specifically the entorhinal cortex and dentate gyrus2,7 (Figure 1).

It was noticed that even when the microbubbles containing therapeutic drugs were replaced with microbubbles without additional drug delivery, there was also shown to be decreased plaque load in the AD brain9. It was also shown that this non-pharmacological technique could increase plasticity in the hippocampal neurons.

However, this was only observed in healthy mice, making it difficult to validate MR imaging-guided focused ultrasound as a therapy to alleviate AD memory-related impairments. Thus, further investigation was initiated by Burgess and others to see if MR imaging-guided FUS alone was sufficient to cause an improvement in physiological and behavioural symptoms of AD in a transgenic mouse model. Specifically, they looked at whether focused ultrasound could increase hippocampal plasticity in the AD brain and improve memory impairments2.

Although this paper gives great insight into the potential for MR imaging-guided focused ultrasound as a therapeutic for AD, more studies need to done to confirm it as being responsible for the behavioural and pathological improvements in AD transgenic mice.

It would be ideal to test the efficacy of MR imagingguided focused ultrasound in AD patients, in regards to behavioural changes. For instance, at least one other experimenting testing spatial memory should have been performed to confirm the behavioural changes seen in the Y-maze post-focused ultrasound treatment. One such test to assess memory and cognition is the Morris Water Maze test.

Research Overview

Summary of Major Results

Y-Maze Test The TgCRND8 transgenic mouse model for AD were used in the Burgess study. These mice were treated with MR imaging-guided focused ultrasound to open the blood-brain barrier once per week for three weeks, and were put in the Y-maze. Without treatment, transgenic (Tg) mice spent 61% less time than the nontransgenic (non-Tg) mice in the new environment, or novel arm of the Y-maze. After MR imaging-guided focused ultrasound, Tg mice spent about 99% more time in the novel arm of the Y-maze (Figure 2a). In other words, the TgCRND8 mice spent 48 seconds in the novel arm after treatment, while without treatment, they spent only about 24 seconds exploring the novel arm.

The Y-maze test is a great indicator of spatial memory improvements or deficits, however, as mentioned in the background, it would have been to the authors’ benefit to perform another spatial memory-dependent task to confirm their results.

Physiological Abnormalities The mice behaviour in the Y-maze was reflected by the significant reduction in the number and size of amyloid plaques in the focused ultrasound-treated mice.

Immunohistochemical analysis showed that there was a significant plaque size reduction in the treated TgCRND8 mice by about 20%, and a 19% decrease in plaque load in the hippocampus (Figure 2b, c). When this method was applied in one hemisphere of the TgCRND8 mouse model of Alzheimer’s disease, plaque load significantly declined even without the delivery of therapeutic agents.

Hippocampal Plasticity There was an increase in the number of newborn neurons in the hippocampus in the treated mice group, and a 227% increase in dendrite length in both treated non-Tg and TgCRND8 mice. The increase in dendritic length is indicative of plasticity in the hippocampus, specifically in the dentate gyrus. Like all neurodegenerative disease, AD is associated with the rapid loss of neurons, specifically the cholinergic neurons15. The increase in the number of neurons in the hippocampus may be the cause of improved behaviour demonstrated in the performance in the Y-maze test.

Safety of MR imaging-guided focused ultrasound Not only is the efficacy of the technique important, but also the safety, since the goal is for focused ultrasound to be used clinically in patients with AD. Focused ultrasound used at low power showed no neuronal damage, and was far safer than most clinical ultrasounds imagers5,16. In addition, in a larger mammalian model, McDannold and others showed that the temporary disruption of the blood brain barrier by ultrasound does not have adverse effects12. After focused ultrasound treatment, the animals did not show any significant behavioural deficits or visual impairments.

Overall, the findings in the study by Burgess and others fits in line with other publications in the field, and have helped to carry through the search for a therapeutic for AD by using focused ultrasound. A more recent study by Leinenga and Gotz provided further support to the effectiveness of focused ultrasound to remove amyloid-β from

the AD mice brain, and increase memory after treatment10.

Specifically, just like in the Burgess and others study, they also performed the Y-maze test, in addition to two other memory tasks. The mice treated with this non-pharmacological treatment displayed increased performance in these tasks, indicative of improved spatial memory.

Figure 1. The positioning of the MR imaging-guided focused ultrasound during treatment (Burgess et al. 2014).

Conclusions and Discussion The results from this experiment suggested that the mice treated with MR imaging-guided focused ultrasound had an increase in spatial memory and cognition, which are diminished in almost all cases of AD. This incremental advance was driven by previous studies using MR imaging-guided focused ultrasound as a method to deliver drugs by previous studies using MR imagingguided focused ultrasound as a method to deliver drugs, such as anti–Aβ amyloid (anti-Aβ) antibodies, from the blood to the brain in an AD mouse model2.

Increase in dendrite length post-treatment with fo1cused ultrasound suggests that this technique could have a role in increased neuronal plasticity in the rapidly degenerating brain in AD. This can have great implications in terms of improved behaviour, for instance, increased spatial memory and cognition, if focused onto the hippocampus, an area of the brain well-documented as the area for the consolidation of memories into long-term memories6.

Despite previous work on this technique, the Burgess et al. study has had a large impact in terms of confirming that MR imaging-guided focused ultrasound reverses some of the pathological abnormalities in the transgenic mice, as well as improves behaviour in terms of memory and cognition.

This work has led to further investigation on MR imagingguided focused ultrasound, and its use in a vast number of neurodegenerative diseases and tumors therapies11.

Criticisms and Future Directions

The vast amount of research done and relevant findings published on focused ultrasound has made it very encouraging to proceed with performing clinical trials with AD patients to validate this technique. Since it is a non-invasive treatment, there is a relatively low risk for testing its effects on AD patients. Furthermore, in a study done by McDannold and others on the macaque brain, the temporary disruption of the blood brain barrier by ultrasound was shown to not have any significant adverse effects12. Although there are more studies that need to be done to ensure the safety of the treatment in clinical trials, it is essential that they act on this in the 240

Figure 2. Figure: Relevant findings post-treatment with focused ultrasound. (a) Comparing time spent in novel arm before and after treatment. (b, c) Highlighting the reduction in plaque size and plaque number after treatment. (Burgess et al. 2014).

near future. In addition, in a response to the Burgess et al study, Jolesz proposes that these results need to be confirmed in clinical trials, to see whether focused ultrasound could be a treatment implemented in clinics to effectively reduce plaque load and improve behavioural deficits in these patients7. This provides further evidence for the pressing need for developing diseasemodifying therapies for AD, and MR imaging-guided focused ultrasound appears to be a likely candidate to have significant effects in the AD brain.

One of the limitations to their study was that only one experimental test was used to assess the behavioural changes on the treated animals. Another experiment they could perform to assess spatial, hippocampal-dependent learning and memory is the Morris Water Maze (MWM) test. The MWM test is a spatial memory task, requiring the animal to remember its way to the platform in the water after multiple trials. Performance is impaired in all the different strains of AD transgenic mice, and thus would be appropriate to use to provide further support to the results of the Y-maze test4. It is expected that treated TgCRND8 mice will require less time to locate the platform in the pool after multiple trials compared to non-treated TgCRND8 mice, exemplifying an improvement in spatial memory. It would also be beneficial to assess other cognitive changes characteristic of AD, such as anxiety. This could be done by evaluating the performance of ultrasound treated TgCRND8 mice in the open field test. In the open field test, animals that are more anxious will stay closer to the edges of the box, while animals that are feeling curious will cross the open field14.

One factor that is not addressed in the Burgess et al. article is the effect focused ultrasound has on the neurofibrillary tangles (NFTs). NFTs are comprised of hyperphosphorylated tau protein, and is another pathological feature of AD. It is crucial that further investigation be done in regards to MR imaging-guided focused ultrasound and its effect, if any, on tau protein accumulation.

Amyloid-beta and tau have been recently discovered to have a synergistic role in the effects of AD, and possibly plays a role in amyloid-beta toxicity6. Therefore, targeting the hippocampus with focused ultrasound could possibly alleviate both pathological aspects of the disease.

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2. Burgess, Alison, Sonam Dubey, Sharon Yeung, Olivia 241

Hough, Naomi Eterman, Isabelle Aubert, and Kullervo Hynynen.

“Alzheimer Disease in a Mouse Model: MR Imaging-Guided Focused Ultrasound Targeted to the Hippocampus Opens the Blood-Brain Barrier and Improves Pathologic Abnormalities and Behavior.” Radiology 273, no. 3 (December 2014): 736–45. doi:10.1148/radiol.14140245.

3. Choi, James J., Kirsten Selert, Fotios Vlachos, Anna Wong, and Elisa E. Konofagou. “Noninvasive and Localized Neuronal Delivery Using Short Ultrasonic Pulses and Microbubbles.” Proceedings of the National Academy of Sciences 108, no. 40 (October 4, 2011): 16539–44. doi:10.1073/ pnas.1105116108.

4. Galeano, Pablo, Pamela V. Martino Adami, Sonia Do Carmo, Eduardo Blanco, Cecilia Rotondaro, Francisco Capani, Eduardo M. Castaño, A. Claudio Cuello, and Laura Morelli. “Longitudinal Analysis of the Behavioral Phenotype in a Novel Transgenic Rat Model of Early Stages of Alzheimer’s Disease.” Frontiers in Behavioral Neuroscience 8 (September 16, 2014). doi:10.3389/fnbeh.2014.00321.

5. Hynynen, Kullervo, Nathan McDannold, Natalia Vykhodtseva, and Ferenc A. Jolesz. “Noninvasive MR Imaging–guided Focal Opening of the Blood-Brain Barrier in Rabbits.” Radiology 220, no. 3 (September 1, 2001): 640–46. doi:10.1148/ radiol.2202001804.

6. Ittner, Lars M., and Jürgen Götz. “Amyloid-Β and Tau — A Toxic Pas de Deux in Alzheimer’s Disease.” Nature Reviews Neuroscience 12, no. 2 (February 2011): 67–72. doi:http:// dx.doi.org.myaccess.library.utoronto.ca/10.1038/nrn2967.

7. Jolesz, Ferenc A. “Science to Practice: Opening the BloodBrain Barrier with Focused Ultrasound—A Potential Treatment for Alzheimer Disease?” Radiology 273, no. 3 (November 24, 2014): 631–33. doi:10.1148/radiol.14142033.

8. Jordão, Jessica F., Carlos A. Ayala-Grosso, Kelly Markham, Yuexi Huang, Rajiv Chopra, JoAnne McLaurin, Kullervo Hynynen, and Isabelle Aubert. “Antibodies Targeted to the Brain with Image-Guided Focused Ultrasound Reduces Amyloid-Β Plaque Load in the TgCRND8 Mouse Model of Alzheimer’s Disease.” PLoS ONE 5, no. 5 (May 11, 2010): e10549. doi:10.1371/journal.pone.0010549.

9. Jordão, Jessica F., Emmanuel Thévenot, Kelly MarkhamCoultes, Tiffany Scarcelli, Ying-Qi Weng, Kristiana Xhima, Meaghan O’Reilly, et al. “Amyloid-Β Plaque Reduction, Endogenous Antibody Delivery and Glial Activation by BrainTargeted, Transcranial Focused Ultrasound.” Experimental Neurology 248 (October 2013): 16–29. doi:10.1016/j. expneurol.2013.05.008.

10. Leinenga, Gerhard, and Jürgen Götz. “Scanning Ultrasound Removes Amyloid-Β and Restores Memory in an Alzheimer’s Disease Mouse Model.” Science Translational Medicine 7, no. 278 (March 11, 2015): 278ra33–278ra33. doi:10.1126/scitranslmed.aaa2512.

11. Lin, Chung-Yin, Tzu-Ming Liu, Chao-Yu Chen, Yen-Lin Huang, Wei-Kai Huang, Chi-Kuang Sun, Fu-Hsiung Chang, and Win-Li Lin. “Quantitative and Qualitative Investigation into the Impact of Focused Ultrasound with Microbubbles on the Triggered Release of Nanoparticles from Vasculature in Mouse Tumors.” Journal of Controlled Release: Official Journal of the Controlled Release Society 146, no. 3 (September 15, 2010): 291–98. doi:10.1016/j.jconrel.2010.05.033.

12. McDannold, Nathan, Costas D. Arvanitis, Natalia Vykhodtseva, and Margaret S. Livingstone. “Temporary Disruption of the Blood-Brain Barrier by Use of Ultrasound and Microbubbles: Safety and Efficacy Evaluation in Rhesus Macaques.”

Cancer Research 72, no. 14 (July 15, 2012): 3652–63. doi:10.1158/0008-5472.CAN-12-0128.

13. Raymond, Scott B., Lisa H. Treat, Jonathan D. Dewey, Nathan J. McDannold, Kullervo Hynynen, and Brian J. Bacskai.

“Ultrasound Enhanced Delivery of Molecular Imaging and Therapeutic Agents in Alzheimer’s Disease Mouse Models.”

PLoS ONE 3, no. 5 (May 14, 2008). doi:10.1371/journal. pone.0002175.

14. Romano, Adele, Lorenzo Pace, Bianca Tempesta, Angelo Michele Lavecchia, Teresa Macheda, Gaurav Bedse, Antonio Petrella, et al. “Depressive-like Behavior Is Paired to Monoaminergic Alteration in a Murine Model of Alzheimer’s Disease.”

The International Journal of Neuropsychopharmacology / Official Scientific Journal of the Collegium Internationale Neuropsychopharmacologicum (CINP) 18, no. 4 (2014). doi:10.1093/ijnp/pyu020.

15. Winner, Beate, and Jürgen Winkler. “Adult Neurogenesis in Neurodegenerative Diseases.” Cold Spring Harbor Perspectives in Biology 7, no. 4 (April 1, 2015): a021287. doi:10.1101/cshperspect.a021287.

16. Yoo, Seung-Schik, Alexander Bystritsky, Jong-Hwan Lee, Yongzhi Zhang, Krisztina Fischer, Byoung-Kyong Min, Nathan J. McDannold, Alvaro Pascual-Leone, and Ferenc A. Jolesz. “Focused Ultrasound Modulates RegionSpecific Brain Activity.” NeuroImage 56, no. 3 (June 1, 2011): 1267–75. doi:10.1016/j.neuroimage.2011.02.058.

This work was supported by The Association for the Development of Undergraduate Neuroscience Education (SRA & RLN), The Endowment for Science Education (EA), and The Synaptic State Faculty Research Foundation (EA). The authors thank Mr. Spine L. Cord, Dr. Amy G. Dala, and the students in Neuroscience 101 for technical assistance, execution, and feedback on this lab exercise.

Address correspondence to: Jaclin Simonetta. Human Biology Department, 123 Microbubble Road, Blood Brain Barrier College, Toronto, ON 60101 Email: jaclin,simonetta@mail.utoronto.ca Copyright © 2015 Jaclin Simonetta, Human Biology Program

242

Novel Metabotropic Function of NMDARs in Alzheimer’s Disease

Olivia Singh

AB oligomers have been shown to cause a synaptotoxic effects including blocking LTP- a process needed for memory and learning. LTP is largely regulated by AMPA and NMDA receptors at synapses. In connection with this, one study has investigated the relationship between AB oligomers and NMDARs. This study found that AB oligomers are able to cause AMPA synaptic depression by increasing the ratio of GluN2A:GluN2B subunit containing NMDARs at the synapse in an ion flux independent manner. This provides evidence for a novel function of NMDARs and the results of this study are supported by additional studies on the metabotrophic function of NMDARs, subunit switching during development, and endocytosis of GluN2B subunit containing NMDARs. While the authors did not consider that the size of the AB oligomers could alter the effects on NMDARs, this research may be furthered by investigation of tyrosine dephosphorylation of the GluN2B subunit containing NMDARs. The results of future studies may lead to the identification of new drug targets for patients experiencing Alzheimer’s disease.

Key words: N-methyl d-aspartate (NMDA); AMPA;long term potentiation (LTP); amyloid –beta (Aβ) oligomers; GluN2B (R2B); memantine; endocytosis; tyrosine dephosphorylation Background Alzheimer’s disease (AD) is characterized by a progressive loss of cognitive ability, memory loss, aphasia, agnosia and a disruption to daily living (1).

These declines are attributed to extracellular amyloid plaques and intracellular neurofibrillary tau tangles; along will the loss of neurons and synaptic plasticity in the hippocampus and cerebral cortex.

Unfortunately, the mechanism underlying the changes seen in the brains of AD patients is still unknown.

Since its proposal over 20 years ago, the most widely accepted theory for the pathological changes seen in AD patients is the amyloid cascade hypothesis (2).

This hypothesis involves the abnormal splicing of the amyloid precursor protein (APP) by β and γ secretases into the 40-42 amino acid amyloid β-peptide (Aβ).

It is thought that the accumulation of the Aβ peptide into extracellular plaques is the main causative agent for the rest of the pathological changes seen in AD brains. However there is increasing evidence that the Aβ oligomers may be more neurotoxic than the fibrils found in plaques. Some in vitro, in vivo and in silico studies have pointed to oligomers as being correlated with AD pathology better than plaques (3). These studies warrant greater investigation on Aβ oligomers and their role in producing neurological changes.

One area that has received considerable attention is the synaptotoxic effects caused by Aβ oligomers and their effects on N-methyl D-aspartate receptors (NMDARs).

There is experimental evidence that blocking NMDARs is able to alleviate the effects of Aβ on synapses (4).

NMDARs are glutamate dependent receptors that contribute to hippocampal dependent learning and long term potentiation (LTP) (5). They have an ionotropic function mediated by a voltage-dependent Mg2+ block (6). When activated simultaneously by binding of presynaptic glutamate and a co-agonist, and post synaptic depolarization, they allow the influx of Ca2+ which acts on downstream targets for learning and memory.

NMDARs are heterotetrameric complexes composed of two glycine/serine binding GluN1 subunits and two glutamate binding GluN2 subunits (7). While the GluN1 243

subunit is ubiquitously expressed, the GluN2 subunit is coded by four genes (GluN2A-D) which are temporally and spatially regulated throughout development (8). It has been observed that while GluN2B subunits are found in the prenatal brain, their numbers are quickly compromised by increased expression of GluN2A subunits with age (9). The GluN2A and GluN2B subunits are predominantly found in the adult hippocampus.

Not including cholinesterases, memantine is the only other drug approved by the FDA for the treatment of Alzheimer’s (10). Memantine is a low-affinity noncompetitive anatagonist of the NMDAR that is used to treat moderate to severe AD (11). However it has been shown that Aβ peptides preferentially act on NMDARs containing the GluN2B subunit to cause decreases in LTP and there may be benefits for using subtype selective NMDAR antagonists instead (12). Unfortunately, the mechanism of the interaction between Aβ and GluN2B-NMDARs is still unknown.

The current study by Kessels et al. (13) proposes that oligomeric Aβ causes a conformational change in NMDARs in an ion flux independent manner which induces a switch from the GluN2B subunit to the GluN2A subunit resulting in synaptic depression.

Research Overview

Summary of Major Results

AB oligomers cause synaptic depression First the authors showed that Aβ oligomers are able to induce synaptic depression in neurons. Hippocampal CA1 slices were sparsely infected with APP-CT100, the product of β-secretase cleavage and the precursor of Aβ. The AMPAR-mediated excitatory postsynaptic current (EPSC) and the NMDAR-mediated currents were measured in the infected CA1 pyramidal neurons and the neighbouring control neurons. Synaptic depression was observed in the APP-CT100 infected neurons but not when scyllo-inositol (blocks effects of Aβ oligomers) was applied (Figure 1).

AB oligomers act on the GluN2B subunit of NMDAR Slices were then incubated with a variety of drugs during APP-CT100 expression to elucidate the connection between Aβ and different subunits of the NMDAR. Synaptic depression was reduced when slices were incubated with d-APV or R-CPP (acts at glutamate binding site on GluN2 subunit) during APP-CT100 expression. The same effect was observed when an antagonist for the GluN2B subunit (Ro or ifenprodil) was applied. However, when an antagonist for the GluN2A subunit (PEAQX) was used, synaptic depression still occurred. These results show Aβ is acting on GluN2B rather than the GluN2A subunit.

AB oligomers induce a conformational change of the NMDAR independent of ion flux Next, the authors showed that the synaptic depression is not related to the ionotropic function of the NMDAR. When slices were incubated with MK-801 or ketamine (ion-channel blockers) during APP-CT100 expression, there was no change in synaptic depression compared to control. This suggests that a conformational change in the NMDAR rather than ionflux through the channel is needed to for Aβ mediated synaptic depression. This conformational change is dependent on switching of the GluN2B subunit to the GluN2A subunit as observed by an increase in the GluN2A:GluN2B ratio with the application of the drug Ro (Figure 2).

Figure 1. (A) Model for whole-cell recording of affected and unaffected neurons in the CA1 (F) Student t test of log transformed data to reveal differences in EPSC with the application of different drugs.

Conclusions and Discussion Together, the results of the experiments suggest that Aβ mediated synaptic depression relies on the activation of the GluN2B subunit but not ion flow through the NMDAR. This implies a novel metabotropic function of the NMDAR compared to its traditional ionotropic function. Although research in this field is new, there is some evidence supporting this claim.

One study examined the difference in signaling ability of GluN2A and GluN2B subunits to induce LTP (14).

The authors shortened the C terminal domain of the GluN2A subunit and introduced this truncated version in a mouse line. Then using GluN2B antagonists, it was shown that even though the NMDAR could form the channel, localize to the synapse and allow Ca2+ influx, there are subunit specific downstream signaling pathways that are needed to contribute to LTP. It was also proposed that Aβ induces a conformational switch from the GluN2B subunit to the GluN2A subunit.

From developmental studies, this switch has been shown to occur naturally with age (15) and has been implicated to be the reason for decreased plasticity and the refinement of neuronal circuits in mature animals (16).

However, there are other factors apart from Aβ that can also increase the ratio of GluN2A:GluN2B . One study has investigated the mechanisms behind subunit specific trafficking to synapses (17). Increasing levels of expression of the GluN2A containing receptors and synaptic activity is able to promote GluN2A insertion into synapses and replacement of synaptic GluN2B containing receptors.

Similar to the effects of AB observed in the study in question, the process of switching seems to be regulated by binding to GluN2 subunits but ion flux through the NMDAR is not required. This supports the finding that AB is able to cause an increase in the GluN2A:GluN2B ratio in an ion channel independent manner.

The authors of the study suggest switching of the GluN2A subunit for the GluN2B subunit of NMDARs as an explanation for the increase of the GluN2A:GluN2B subunit ratio. However this increase in ratio may be due to a variety of reasons such as selective targeting of GluN2B subunits for degradation, increased production of GluN2A subunits or switching of the subunits at the synapse. The most likely explanation is endocytosis of the GluN2B subunit containing NMDAR. A study by Snyder et al. (18) found that applying AB decreased the surface expression of the GluN2B and GluN1 subunits of the NMDAR but there was no change in the overall amounts of these subunits. They also verified that AB causes endocytosis of the NMDARs using a cleavable biotin but did not cause endocytosis of another ionotropic GABA receptor indicating that AB is selective for NMDARs. The effect of endocytosis of GluN2B and GluN1 subunit containing NMDARs was reversed with a y-secretase inhibitor which reduces levels of AB. These results corroborate the findings of Kessels et al. and propose Aβ mediated endocytosis of GluN2B subunit containing NMDARs as an explanation for the increased ratio of GluN2A:GluN2B subunits.

While the authors attribute AB-mediated synaptic depression to maturation of the synapse by switching of the GluN2B subunit for the GluN2A subunit, other studies suggest that AB can influence NMDARs by dysregulation of glutamate. The distinction is between these two mechanisms seems to be the location of the NMDAR (19). When AB acts on synaptic NMDARs, it causes endocytosis and switching of the subunits as described in the reviewed paper. However AB can also act on extrasynaptic NMDARs to induce prolonged excitation by impairing glutamate uptake and increasing glutamate release. The excess glutamate at the synapse will spill over to the extrasynaptic NDMARs and can produce excitotoxicity resulting from chronic mild activation of NMDARs and leads to loss of synaptic function, synap244

Figure 2. (A) GluN2A and GluN2B currents are affected differently in APP CT-100 affected and unaffected neurons. (B) Paired affected and unaffected neurons to reveal a selective loss of GluN2B currents. (C) GluN2A to GluN2B current ratio in pairs of affected and unaffected neurons.

totoxicity and cell death (20). One study has shown that low concentrations, soluble AB oligomers are able to inhibit LTP by enhancing the currents of GluN2B subunit containing NMDARs (21). This is proposed to function similarly to a glutamate reuptake inhibitor (TBOA). While it is assumed the study by Kessels et al. focused on synaptic NMDARs, this point should be made explicit since AB can have varying consequences depending on the location of the NMDAR. Conclusions The authors were able to show that Aβ includes AMPAR synaptic depression by activating the GluN2B subunit of the NMDAR and not through ion flux of the NMDAR. They also showed that the Aβ oligomers were able to increase the GluN2A:GluN2B ratio and interpreted this as subunit switching by endocytosis of GluN2B NMDARs from synapses. The conclusions of this study have been validated through studies showing that NMDARs can have a metabotropic function and it is needed for LTP to occur, subunit switching of the GluN2B subunit containing NMDARs for the GluN2A subunit containing NMDARs, and endocytosis of GluN2B NMDARs. A point the authors should consider is the effects of Aβ on extrasynaptic NMDARs since it has been shown that Aβ can have different effects on NMDARs depending on its location.

In conclusion, this study provides a preliminary mechanism for Aβ function on synapses. Memantine is currently the only FDA approved drug for Alzheimer’s disease that targets NMDARs however the results of this review indicates it may be more beneficial to use a more subunit selective drug. If components of this pathway are identified upon further investigation, they may serve as potential drug targets in AD patients to reduce synaptic depression and thereby learning and memory problems in this population.

Criticisms and Future Directions

While the authors did include many controls to confirm their data, one limitation pertains to the fact that the authors assumed their viral infection of the Aβ precursor led to Aβ oligomers. They did not test whether the Aβ accumulated into extracellular aggregates over the 30 hour incubation time. Determining whether the intracellular Aβ oligomers or the extracellular Aβ plaques conferred AMPA synaptic 245

depression would greatly affect any future studies on the mechanism by which this occurs. In addition, another study has shown that even the size of the oligomers could change the effects of Aβ on a neuron, with a height of 1-2 significantly more toxic to neurons than oligomers with a height above 4-5 nm (22).

One study has shown that NMDAR endocytosis can be regulated by tyrosine dephosphorylation in an ion flux independent manner (23). By using site-directed mutagenesis, the authors were able to identify tyrosine residues in the C terminal domain of the GluN2A subunit responsible for the observed endocytosis of the NMDAR. Based on this finding, a separate study has identified that dephosphorylation of Tyr1472 in the GluN2B subunit of the NMDAR is correlated with receptor endocytosis (18). Therefore, future studies should focus on potential tyrosine phosphatases that interact with AB as well as GluN2B containing NMDARs. One such tyrosine phosphatase that has received some attention is STEP (18, 23, 24).

An alternate approach to study tyrosine dephosphorylation is to inhibit the kinase responsible for phosphorylating GluN2B subunits. EphB2 is a receptor tyrosine kinase that phosphorylates GluN2B subunits to promote synaptic localization and the formation of LTP. One study has shown that AB oligomers are able to bind to EphB2 at its fibronectin III repeats domain which then tags it for degradation by the proteasome (25). Future studies should investigate the potential for inhibitors of EphB2 to block AB-mediated synaptic depression. It would also be interesting to study whether the interaction between EphB2 and AB causes AMPAR depression at synapses.

References 1. Brayne, C., Mayeux, R., & Reitz, C. Epidemiology of Alzheimer disease. Nature Reviews Neurology 7, 137-152 (2011).

2. Hardy, J.A. & Higgins, G.A. Alzheimer’s disease: The amyloid cascade hypothesis. Science 256, 184-185 (1992).

3. Hayden, E.Y. & Teplow, D.B. Amyloid β-protein oligomers and Alzheimer’s disease. Alzheimers Res Ther 5, 60 (2013).

4. Kamenetz, F. et al. APP Processing and Synaptic Function. Neuron 37, 925-937 (2003).

5. Dingledine, R., Borges, K., Bowie, D., & Traynelis, S.F. The glutamate receptor ion channels. Pharmacol Rev 51, 7-61 (1999).

6. Rauner, C. & Kohr, G. Triheteromeric NR1/NR2A/ NR2B Receptors Constitute the Major N-Methyl-D-aspartate Receptor Population in Adult Hippocampal Synapses. J Biological Chem 286, 7558-7566 (2011).

7. Rambhadra, A., Gonzalez, J., & Jayaraman, V. Subunit Arrangement in N-Methyl-D-aspartate (NMDA) Receptors. J Biological Chem 285, 15296-15301 (2010).

8. Monyer, H., Burnashev, N., Laurie, D. J., Sakmann, B., & Seeburg, P. H. Developmental and regional expression in the rat brain and functional properties of four NMDA receptors.

Neuron 12, 529-540 (1994).

9. Sheng, M., Cummings, J., Roldan, L. A., Jan, Y. N., & Jan, L. Y. Changing subunit composition of heteromeric NMDA receptors during development of rat cortex. Nature 368,144-147 (1994).

10. Kumar, A., Singh, A., & Ekavali. A review on Alzheimer’s disease pathophysiology and its management: an update.

Pharmacol Rep 67, 195-203 (2015).

11. Alligaier, M. & Alligaier, C. An update on drug treatment options of Alzheimer’s disease. Front Biosci 19, 1345-1354 (2014).

12. Hu, N.W., Klyubin, I., Anwyl, R., & Rowan, M.J. GluN2B subunit-containing NMDA receptor antagonists prevent Abetamediated synaptic plasticity disruption in vivo. Proc Natl Acad Sci U.S.A. 106, 20504-20509 (2009).

13. Kessels, H.W., Nabavi, S. & Malinow R. Metabotropic NMDA receptor function is required for β-amyloid-induced synaptic depression. PNAS 110, 4033-4038 (2013).

14. Kohr, G. et al. Intracellular Domains of NMDA Receptor Subtypes Are Determinants for Long-Term Potentiation Induction. J Neurosci 23, 10791-10799 (2003).

15. Tovar, K. R., & Westbrook, G.L. The incorporation of NMDA receptors with a distinct subunit composition at nascent hippocampal synapses in vitro. J Neurosci 19, 4180–4188 (1999).

16. Sanz-Clemente, A., Nicoll, R.A., & Roche, K.W. Diversity in NMDA receptor composition: many regulators, many consequences. Neuroscientist 19, 62-75 (2013).

17. Wang, Z., Zhao, J., & Li, S. Dysregulation of synaptic and extrasynaptic N-methyl-D-aspartate receptors induced by amyloid-β. Neuroscience Bulletin 29, 752-760 (2013).

18. Vissel, B., Krupp, J.J., Heinemann, S.F., & Westbrook, G.L. A use-dependent tyrosine dephosphorylation of NMDA receptors is independent of ion flux. Nat Neurosci 4, 587-596 (2001).

19. Wenk. G.L., Parsons, C.G., & Danysz, W. Potential role of N-methyl-D-aspartate receptors as executors of neurodegeneration resulting from diverse insults: focus on memantine.

Behav Pharmacol 17, 411–424 (2006).

20. Li, S. et al. Soluble Aβ oligomers inhibit long-term potentiation through a mechanism involving excessive activation of extrasynaptic NR2B-containing NMDA receptors. J Neurosci 31, 6627-6638 (2011).

21. Snyder, E.M. et al. Regulation of NMDA receptor trafficking by amyloid-β. Nature Neuroscience 8, 1051-1058 (2005).

22. Cizas, P. et al. Size-dependent neurotoxicity of β-amyloid oligomers. Archives of Biochemistry and Biophysics 496, 84-92 (2010).

23. Venkitaramani, D.V., Moura, P.J., Picciotto, M.R., & Lombroso, P.J. Striatal-enriched protein tyrosine phosphatase (STEP) knockout mice have enhanced hippocampal memory.

Eur J Neurosci 33, 2288–2298 (2011).

24. Zhang, Y. et al. Genetic reduction of striatal-enriched

tyrosine phosphatase (STEP) reverses cognitive and cellular deficits in an Alzheimer’s disease mouse model. Proc Natl Acad Sci U.S.A. 107, 19014–19019 (2010).

25. Cisse, M. et al. Reversing EphB2 depletion rescues cognitive functions in Alzheimer model. Nature 469, 47-52 (2011).

This work was supported by The Association for the Development of Undergraduate Neuroscience Education (SRA & RLN), The Endowment for Science Education (EA), and The Synaptic State Faculty Research Foundation (EA). The authors thank Mr. Spine L. Cord, Dr. Amy G. Dala, and the students in Neuroscience 101 for technical assistance, execution, and feedback on this lab exercise.

Address correspondence to: Dr. Rita L. Neurotrophin, Biology Department, 123 Growth Cone Avenue, Action Potential College, Hillock, IL 60101 Email: rln@apc.edu Copyright © 2015 Dr. Bill JU, Neurosciences, Human Biology Program

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Dopamine D1/D5 Receptor mediated tLTP Pathway in the Dentate Gyrus and Implications in Spatially-Dependent Learning and Memory

Pranay Siriya

D1 and D5 receptors have been considered to underlie timing-dependent LTP (tLTP) in the dentate gyrus and hippocampus, as these receptors have been found using real-time PCR. The medial perforant pathway brings spatial information about the environment into the dentate gyrus, thus it has been hypothesized there is a role for D1/D5 receptors in facilitating tLTP. Yang and Dani showed using D1 or D5 knockout mice and with pharmacological interventions that these receptors prolong the time delay between pre and postsynaptic neuronal firing facilitating tLTP. They decrease the threshold for tLTP and have a significant role in spatial learning and memory. Moreover, they have proposed a mechanism by which tLTP may be facilitated, specifically closing of IA current channels via downstream effectors. This suggests there are physiological implications, as the hippocampus is crucial for consolidating long-term memories.

Studies support this showing D1 and D5 receptors to be imperative in spatial context drug-addiction, in behavioral tasks requiring spatial learning, in memory tasks and in cognitive performance. However, Yang and Dani’s paper has some shortcomings which need to be further investigated, and implications which need to be given more consideration in order to understand the greater significance of D1 and D5 receptors in various aspects of spatial dependent learning and memory. Mechanisms of D1/D5 receptors are still not completely understood and are only correlational.

Key words: tLTP; hippocampus; DA1; DA5; spatial learning Background Previous studies have used real-time PCR and showed that D1 and D5 receptors in the perforant pathway1 and their role have been crucial in explicating spatial learning and memory2. However, it has been unclear how D1 and D5 receptors are involved in mediating spatial learning and memory, for it lacks a mechanism by which they lead to spatial memory. It known that the learning process occurs through LTP but the time delay between pre and postsynaptic neurons was unclear. Moreover, D1 and D5 receptors have become important systemically in the cortex, and not just the hippocampus.

In the auditory cortex, D1 and D5 receptors have been implicated in the learning for their activation in the auditory cortex, hippocampus, and striatum facilitate learning of frequency modulated tones; thus, expanding the role of D1 and D5 receptors beyond spatial learning and memory3 in the hippocampus.

Instead, learning discrepancies in frequencies has real-life application, such as the Doppler effect, thus this facet of receptor activation can be related back to spatial learning. Since the medial perforant pathway begins with input from the entorhinal cortex, it is not surprising that spatial learning paradigms are dependent on LTP in the dentate gyrus, but it is confirmed now that tLTP is dependent on D1/D5 receptors.

Consequently, it is important to understand the role of dopamine and how it is related to memory consolidation in the hippocampus. It is known that stimulus salience is dependent on intrinsic dopamine release when an unexpected stimulus is pleasant4. This also explains why dopamine levels are increased in novel environments and memory tasks that are spatially dependent will be facilitated with dopamine release. However, other studies have showed that D1 but not D5 receptors are also important in contextual fear learning conditioning5.

It is known that there is “dopamine depression”4 when there are negative prediction errors or stimuli are aver247

sive4. Thus, this may be important in future studies that look to distinguish the role of D1 and D5 since they are quite similar biochemically.

Primarily D1 and D5 receptor research has been limited to the hippocampus and memory. Studies are investigating the role of these receptors in working memory, short term memory, long term memory as well cognitive performance. There are neuropharmacology studies that look at D1 and D5 receptors in cognitive disorders such as Schizophrenia6,7. The impairments associated with Schizophrenia are often correlated with activity of the prefrontal cortex, and the hippocampus is hypothesized to be important in consolidating memories and relaying memories to the cortex.

Thus, D1 receptors are being investigated to understand working memory disorders, and consequently, long term memory and Schizophrenia6,7. Given the diffuse role of dopamine in the human brain, it is critical to understand how D1 and D5 receptor expression is transient in the brain, and how they are involved in various aspects of learning and memory. Studies showed that these receptors are capable of producing drug-associated spatial memories in a single pairing cocaine conditioned place preference paradigm8. Thus, it may also have implications in Parkinson’s disease.

Most importantly, the downstream pathway of D1 and D5 receptors must be identified, as currently, there is only partial confirmation of previous hypotheses. The hypothesis set forth by Yang and Dani suggests a possible role for D1/D5 receptor mRNAs but how they are transported or involved in LTP is still unclear9.

Research Overview

Summary of Major Results

D1/D5 receptors important for tLTP in the dentate gyrus

Yang and Dani found that using a STDP protocol, D1 receptors increased the time delay between firing for which timing-dependent LTP (tLTP) could occur.

Specifically, using a D1 receptor agonist, at Δt = ±30ms tLTP was strongest. When postsynaptic firing preceded presynaptic firing by 100ms, tLTP was weakest, and closest to baseline. Likewise, when the postsynaptic neuron fired 60ms after the presynaptic, LTP was almost at baseline9.

Figure 1. Summary of results involving D1 receptor agonist (red) and antagonist (blue) demonstrate variability of tLTP in a time-dependent manner9. Blocking the receptors leads to tLTD.

D5 receptors also participate in tLTP, evident through knockout rats. Moreover, they showed greater deficits in tLTP than D1 receptor knockouts. Together, these findings support what had been hypothesized earlier10 in studies

by Hamilton et. al and Kesner et. al. They proposed that novelty and salience is mediated by D1/D5 receptors and that such information is gated by the dentate gyrus10.

Furthermore, it supports experiments conducted by da Silva et. al which looked at behavioral tests in rats with D1/D5 knockout in spatial dependent memory tasks2.

Mechanism for tLTP mediated by tLTP

Using Kv4.2 knockout rats, those lacking a gene to generate IA currents, a specific potassium ion-based current, tLTP was induced. Similarly, using a pharmacological antagonist to Kv4.2 channels, tLTP was comparable to that of D1 antagonist with Δt = +10ms.

This result is a consistent as it has been shown before IA current generating channels are abundant in the rat dentate gyrus9,1. The same result was not observed when non-IA currents were blocked using pharmacological techniques in rat hippocampal perforated-clamp slices. Therefore, it is hypothesized that downstream effector molecules of D1

activation cause closing K+ ion channels generating IA currents. Lastly, studies have showed MAPK and ERKs are implicated in LTP and it is known that MAPK activates channels generating IA currents.

Using another pharmacological inhibitor specific to MEK, tLTP in postsynaptic neurons was inhibited. There had been no previous hypotheses concerning D1/ D5 receptors and MAPK-ERK pathway therefore, this was a significant observation by the authors. Previous behavioural tests showed blocking ERK, a downstream molecule in the MAPK pathway led to impaired contextual fear learning in behavioural tasks11 but they did not specify a role for dopamine. Yang and Dani hypothesized that D1 receptor should be causing a downstream effector to block MAPK in the dentate gyrus to inhibit IA currents to observe tLTP. They investigated the role of NMDA and hypothesized that changes in IA currents may be due to NMDA activation upstream.

Conclusions and Discussion

D1 Receptors and D5 Receptors in tLTP

The authors have shown that inactivation of either D1 or D5 dopamine receptors impairs tLTP and more importantly, the duration for which tLTP can occur is significantly longer when D1 receptors are activated.

However, there is a caveat. If postsynaptic neurons fire 100 milliseconds or more before the presynaptic neuron, tLTD is observed instead of tLTP. This can serve as a mechanism to ensure synaptic efficacy is not increased for two neurons firing randomly. That is, random neuronal activity could cause two neurons to be firing within a short time duration and this could cause tLTP that is not warranted. Thus, it is important that D1 receptors do not increase the time delay for tLTP between two neurons indefinitely. Conversely, 100ms is long enough such that salient stimuli from the environment can cause a neuron to fire and activation of D1 receptors can increase the time for the second neuron to fire and increase the synaptic strength between the two neurons, providing a basis for memory. Although this finding is not entirely new, as it was observed through behavioral tests2, it is the first to demonstrate at a molecular level, the significance of D1 receptor activation in tLTP and specifically the effect of D1/D5 receptor activation in potential downstream mechanisms.

One significant impact of this is conclusion is its relation to impaired cognitive activity. In a study done by Tsang

Figure 2. Panels A and D show results from a paper by Kelleher et. al.11 Using MEK knockout mice, compared to controls, the knockout mice took longer to learn to swim out of a water maze task to a platform that was spatially dependent. Panel D shows the knockout mice spent less time freezing 24 hours later in a contextual fear dependent task. Both these paradigms are hippocampal dependent forms of spatial memory and thus are dependent on D1/D5 receptors found at the gateway of the hippocampus as proposed by Yang and Dani.

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et. al12, individuals with gene mutations coding for D1 receptors have showed poorer neurocognitive performance. D1 receptor mRNA is known to decline with age, therefore, some the models used in this study, namely perforated-clamp and STDP protocol can be used to test specific agonists to recover D1-related impairments with age and in individuals with Schizophrenia12.

Another implication of the study is the importance of D1 activation in spatial dependent drug-tolerance13. If a single dose of cocaine can confer a conditioned place preference8, D1 activation can have a significant role in drug-tolerance that is spatially dependent. Again, the same experimental setup in this study can be used to test antagonists for D1 receptors to treat addictions, specifically, spatially primed withdrawal urges.

The authors suggest endogenous dopamine release is variable and D1 and D5 receptors lower the threshold for tLTP by the presence of mRNA. This model is not properly elaborated on and is a shortcoming of the paper. How mRNA gets transported into the dendrites and its functional importance are not discussed nor a method to test it. Nonetheless, the idea the authors discussed is novel and can be used as a test for abnormalities that may arise from deficiencies in D1/ D5 mRNA in individuals with learning deficits.

Mechanism for tLTP in the Dentate Gyrus

The authors provide correlational evidence of downstream effectors and how they may be involved in mediating tLTP. Specifically, the model proposed is NMDA-dependent and acts as a positive feedback loop with IA currents. That is, closed IA currents cause depolarization of the cell, which in turn help overcome the magnesium block experienced by NMDA channels. The NMDARs in turn cause more IA current-generating channels to close and the process repeats. Yang and Dani are the first to provide an actual mechanism that suggest how D1/D5 receptors actually cause tLTP threshold to be lowered. This makes their paper unique as previous attempts have only hypothesized which G-protein pathway D1 and D5 receptors might work with10.

Specifically, Hansen and Manahan-Vaughan had done a review on D1/D5 receptors and they had proposed D1 receptors work through adenylyl cyclase and protein kinase A while D5 receptors activates phospholipase C. This hypothesis was partially supported by Yang and Dani with their pharmacological interventions on MAPK, a proposed downstream target of the D1 receptor pathway. MAPK can be influenced through PK-A activation, but there is no evidence from Yang and Dani that it is PK-A is actually activated when D1 receptors are bound to dopamine. The only conclusion they can be certain of is D1 somehow inactivates IA currents to induce tLTP, and that D1 activation is dependent on dopamine. The former is a novel conclusion and supports results of behavioural studies using Kv4.2 knockout mice in spatial learning tasks and proposes an explanation of why the behaviours were observed.

Physiological Implications

In addition to drug tolerance, cognitive impairments, the authors predict D1/D5 receptors would be important in forming associations between external stimuli, 249

the environment, and resources such as food and drugs, which cause endogenous release of dopamine.

Moreover, this model would suggest that neurodegenerative conditions which lower oneâ&#x20AC;&#x2122;s endogenous dopamine levels would be associated with learning and memory difficulties, such as in Parkinsonâ&#x20AC;&#x2122;s. This is a novel way of understanding learning and memory disorders, whereas traditionally, the focus has been on NMDARs and glutamate. Consequently, there are implications of dopamine in learning and memory that have not been considered before. Moreover, there is a potential way to test drug therapies using a similar model as in the experiment by Yang and Dani because it is able to preserve intracellular environments and downstream effectors, which previous studies have been unable to do9.

Criticisms and Future Directions There are some significant hypothesizes in the original study that need to be further explored. Firstly, the authors were unable to distinguish the different roles of D1 and D5 receptors biochemically, yet the two receptors have been shown to have different degree of effect in rat models. Even in this study, D5 receptors seem to be more important than D1 receptors in inducing tLTP, for D5 knockouts had greater tLTD than D1 knockouts. This may be associated with the different molecular pathway10,14 associated with D5 receptors as hypothesized by Hansen and Manahan-Vaughan, or differ in memories they consolidate14. This can be tested this, using a similar model Yang and Dani used in their experiment. By using pharmacological antagonists against PLC and CAMKII in D1 knockout mice, which are proposed downstream molecules of D5 receptors10, one can observe the effects on tLTP and strength of tLTP due to D1 alone. Likewise, in D1 knockouts, using adenylyl cyclase antagonists will show the effect of activating D5 receptors and elucidate mechanisms of D5 alone.

Additionally, in discussing downstream effectors, Yang and Dani can only correlate their data to what they expected to happen. There is no direct evidence that it is actually D1 activation that leads to IA currents to close. They simply compared tLTP of Kv4.2 knockouts to D1 knockout mice to draw conclusions by comparison.

Hannnan et. al have demonstrated one way to determine protein-protein interactions by using GST-pulldown with co-immunoprecipitaion. Molecules that bind to D1 or D5 receptors will be attached because the receptors are coated with an antibody and resin15. By spinning and washing the solution, only proteins attached to the antibody will remain, and once eluted, the antibody dissociates leaving the downstream effector15. Using co-immunoprecipiation allows for the collection of proteins binding to the receptor, and not just the first downstream effector.

Another aspect of the paper that requires clarification and further investigation is how D1/D5 receptor mRNA is relevant in tLTP. They suggest the mRNA may have a functional role in facilitating tLTP. To test this, one can use histochemistry16. By labelling dopamine receptor genes oligonucleotides, they can be used as probes to detect expression of mRNA in the dentate gyrus neurons and input from entorhinal cortex. Furthermore, a plasmid with a promoter, D1/D5 receptor gene and GFP, provides

a method of visualizing movement of the receptor to determine under which conditions it is transcribed and translated16. This can allow the authors to see how dopamine causes a change in the number of receptors and where expression of D1/D5 receptors is localized.

Lastly, behavioural tests are easy to conduct using the knockout mice by doing spatial water maze tasks—spatial cues providing the location of a surface in a pool of opaque water2. With a bilateral lesion of CA3 and CA1 neurons2, the authors can analyze just the effect of D1 or D5 receptors in the mPP on spatial learning and whether they are relevant in learning and memory tasks. Likewise, potential drug treatments for Schizophrenia, or how to increase cortical activity using D1/D5 agonists can be tested using the model in the experiment with a behavioural model suggested.

A similar approach can be used for treating drugaddictions that are spatially dependent and potentially curing spatially primed withdrawal symptoms. Treatments drugs that inhibit D1/D5 receptors will make it harder to form learned associations between environment and drug; but theoretically, the individual will still have the experience for taking the drug. In doing so, drug-resistance and physiological compensatory mechanisms due to spatial drug-tolerance are not as easily activated preventing death from extreme doses.

References 1. Mu, Y., Zhao, C. & Gage, F. H. Dopaminergic Modulation of Cortical Inputs during Maturation of Adult-Born Dentate Granule Cells. J Neurosci. 31(11), 4113-4123 (2011).

2. da Silva, C. N. W., Köhler, C. C., Radiske, A. & Cammarota, M. D1/D5 dopamine receptors modulate spatial memory formation. Neurobiol Learn Mem. 97(2), 271-275 (2012).

3. Reichenbach, M., Herrmann, U., Kähne, T., Schicknick, H., Pielot, R., Naumann. M., et al. Differential effects of dopamine signalling on long-term memory formation and consolidation in rodent brain. Protemoe Sci. 13(13), (2015); doi:10.1186/s12953-015-0069-2.

4. Gebauer, L. & Kringleback, M. L. Ever-Changing Cycles of Musical Pleasure: The Role of Dopamine and Anticipation.

Psychomusicology: Music, Mind, and Brain. 22(2), 152-167 (2012).

5. Heath, F., Jurkus, R., Bast, T., Pezze, M. A., Lee, J. L. C., Voigt, J. P., et al. Dopamine D1-like receptor signalling in the hippocampus and amygdala modulates the acquisition of contextual fear conditioning. Psychopharmacology. (2015); doi. 10.1007/s00213-015-3897-y.

6. Rosell, D. R., Zaluda, L. C., McClure, M. M., PerezRodriguez, M. M., Strike, K. S., Barch, D. M., et al. Effects of the D1 Dopamine Receptor Agonist Dihydrexidine (DAR0100A) on Working Memory in Schizotypal Personality Disorder. Neuropsychopharmacol. 40, 446–453 (2014).

7. Goldman-Rakic, P. S., Castner, S. A., Svensson, T. H., Siever, L. J. & Williams, G. V. Targeting the dopamine D1 receptor in schizophrenia: insights for cognitive dysfunction.

Psychopharmacology. 174, 3-16 (2004).

8. Krammer, C. P., Barbano, M. F. & Medina, J. H. Dopamine D1/D5 receptors in the dorsal hippocampus are required for the acquisition and expression of a single trial cocaine-

associated memory. Neurobiol. Learn. Mem. 116, 172-180 (2014).

9. Yang, K., & Dani, J.A. Dopamine D1 and D5 Receptors Modulate Spike Timing-Dependent Plasticity at Medial Perforant Path to Dentate Granule Cell Synapses. J Neurosci.

34(48), 15888-15897 (2014).

10. Hansen, N. & Manahan-Vaughan, D. Dopamine D1/ D5 Receptors Mediate Informational Saliency that Promotes Persistent Hippocampal Long-Term Plasticity. Cereb. Cortex.

24(4), 845-858 (2014).

11. Kelleher, R. J., Govindarajan, A., Jung, H.Y., Kang, H. & Tonegawa, S. Translational control by MAPK signaling in long-term synaptic plasticity and memory. Cell. 116, 467–479 (2004).

12. Tsang, J., Fullard, J. F., Giakoumaki, S. G., Katsel, P., Karagiorga, V. E., Greenwood, T. A. et al. The relationship between dopamine receptor D1 and cognitive performance.

Npj. Schizophrenia. (2015); doi:10.1038/npjschz.2014.2.

13. Martin-Iverson, M. T. & Burger, L. Y. Behavioral sensitization and tolerance to cocaine and the occupation of dopamine receptors by dopamine. Mol. Neurobiol. 11, 31-46 (1995).

14. Furini, C. R., Myskiw, J. C., Schmidt, B. E., Marcondes, L. A. & Izquierdo, I. D1 and D5 dopamine receptors participate on the consolidation of two different memories. Behav. Brain.

Res. 271, 212-217 (2014).

15. Hannan, A. M., Kabani, N., Paspalas, D. C. & Levenson, R. Interaction with Dopamine D2 Receptor Enhances Expression of Transient Receptor Potential Channel 1 at the Cell Surface. Biochim Biophys Acta. 1778(4), 974-982 (2008).

16. Mengod, G., Martinez-Mir, I. M., Vilaró, T. M. & Palicios M. J. Localization of the mRNA for the dopamine D2 receptor in the rat brain by in situ hybridization histochemistry. Proc. Natl. Acad. Sci. 86, 8560-8564 (1989).

This work was supported by The Association for the Development of Undergraduate Neuroscience Education (SRA & RLN), The Endowment for Science Education (EA), and The Synaptic State Faculty Research Foundation (EA). The authors thank Mr. Spine L. Cord, Dr. Amy G. Dala, and the students in Neuroscience 101 for technical assistance, execution, and feedback on this lab exercise.

Address correspondence to: Dr. Rita L. Neurotrophin, Biology Department, 123 Growth Cone Avenue, Action Potential College, Hillock, IL 60101 Email: rln@apc.edu Copyright © 2015 Dr. Bill JU, Neurosciences, Human Biology Program

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Suppression of α-syn in Mice Model of Human Lewy Body Disorders Reverses Detrimental Effects of α-syn Accumulation

Stephanie Strug

Background High levels of α-synuclein (α-syn) – an unfolded protein, found in the central nervous system, believed to be apart of synaptic plasticity (Games, 2012) – are an indicator of Lewy body (LB) disorders, including dementia with Lewy bodies (DLB) and Parkinson’s disease with dementia (PDD) which has an increased risk of development based on triplication of the α-syn gene (Halliday, 2014). A common symptom of α-syn accumulation is cognitive impairment. High levels of α-syn can cause problems with synaptic function, such as issues with neurotransmitter release, synaptic vesicle recycling, endocytosis/exocytosis, and protein levels within the synapse (Lim, 2011). In the research discussed in Lim’s (2011) paper it was tested if suppression of α-syn would stop and/or reverse the problems associated with its accumulation. The subjects tested were mutant (A53T) and wild-type (WT) α-syn transgenic mice. A53T mutant α-syn transgenic mice are models of human α-syn accumulation diseases (Bencsik, 2014). To ensure that the mice would represent DLB similar distribution of α-syn at high levels must be found in multiple regions within the forebrain, specifically the limbic region, the correct levels were found in the A53T mice.

Four phenotypically different types of mice were put on a 200mg/kg of doxycycline diet from insemination to postnatal day 21 as to ensure that there would be no developmental defects due to early expression of α-syn. The mice were then behavioral tested (contextual and cued fear) at different stages of development (4 months, 8 months, 12 months, and 12 months with doxycycline diet from 9-12 months – to stop the expression of α-syn) to monitor their memory. The amount of α-syn accumulation was tested through brain tissue collection, which then underwent immunohistochemistry, and finally pathology grading where the amount of α-syn accumulation in 13 different areas were graded on an interval from 0 (none present) to 3 (intense accumulation) in increments of 0.5 to determine the correlation between α-syn accumulation and memory (Lim, 2011).

Some variables that accompany α-syn accumulation were not taken into account at the time of Lim’s 2011 research. Such as Beta amyloid (Aα) interactions with α-syn were not taken into consideration, which they should as these interactions can lead to degeneration of specific neurons found in the limbic system (Overk, 2014), the same system which was found to have partial recovery once α-syn suppression had occurred.

And the type of α-syn accumulation was not taken into account; α-syn can form monomers, oligomers, and fibrils. Different types of α-syn are prone to express different forms, which may accumulate in specific areas of the brain (Rockenstein, 2014). The neurotoxicity of α-syn may depend on the species (Games, 2012).

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Research Overview

Summary of Major Results

The tTA/α-syn mice (which produce an accumulation of A53Tα-syn) begin showing accumulations of α-syn at the age of 4 months in their cingulate cortex, hippocampus, dentate gyrus and mammillary body; all these sections are part of the limbic areas. At the age of 8 months the accumulation of α-syn has not spread, but the areas are now more concentrated figure 1. This distribution of α-syn found in the strain of mice is very much like the pattern of α-syn found in humans with DLB. Showing that the mutant mice tTa/α-syn are indeed a good model for the humans with Lewy body disorders (Lim, 2011).

A53Tα-syn seems to be accepted as a good model as it is continuously used in experiments there after, such as Martin 2013, Bencsik 2014, Rockenstein, 2014, Chen 2015, and Liu 2015 as a representative of the human model for Lewy body disorders.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3144489/figure/F1/ Figure 1. The cingulate cortex, mammillary body, dentate gyrus molecular layer, and hippocampus at CA2/3 region from the controlled mice at 8 months, the tTa/α-syn mice at 4 months, and the tTa/α-syn mice at 8 months are stained to show the accumulation of α-syn. The accumulation in the tTa/α-syn mice at months has a similar spread as the accumulation at 4 months, but is more concentrated (Lim, 2011)

The tTA/α-syn mice did significantly poorer with the contextual fear memory test than compared to the other mice genotypes. Contextual fear memory relies on the hippocampus, therefore the lack of contextual fear memory is due to impairment in the hippocampus caused by the accumulation of α-syn figure 2 (Lim, 2011). This falls in line with the expected theory that α-syn accumulation in the limbic system leads to memory deficits (Games, 2012).

Mice were put on 200mg/kg of doxycycline from month 9-12 to supress α-syn transgene, and compared to the same phenotype where α-syn was expressed. The mice on the doxycycline diet that did have α-syn accumulation at 8 months no longer had α-syn accumulation in their hippocampal region when sacrificed at 12 months figure 3. Showing that suppression of the expression of the transgene α-syn not only stops the accumulation of α-syn, but also removes α-syn from the hippocampus (Lim, 2011).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3144489/figure/F6/

Figure 3. CA1, CA3/mossy fibres, and dentate gyryus molecular layer (DG mol) are shown from the control (nTg) and the mice genotype (tTA/A53Tα-syn) [that is a model for dementia with Lewy Bodies (DLB) found in humans] at 8 months, 12 months, and 12 months on a doxycycline diet from month 9-12 [12m(dox9 -12m)]. The accumulation of α-syn is being compared. The α-syn accumulations from sections found in the nTg and tTA/ A53Tα-syn 12m(dox9-12m) are very similar as the diet causes α-syn accumulation to be supressed and removes the α-syn accumulation that had already taken place (Lim, 2011).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3144489/figure/F5/

Figure 2. (A) When A53Tα-syn was fully expressed in mice with phenotype tTA/α-syn they froze for less time at 4 months, and significantly less time at 8 months compared to the other phenotypes. (C) % freezing time (contextual fear) versus pathology in the hippocampus is graphed indicating a negative

When mice of the same genotype on different diets are compared, those who are suppressing the transgene expression of α-syn perform better on contextual fear memory tests figure 4. This shows that not only does the diet remove the accumulation of α-syn, but it also reverses the poor effects as memory function is improved (Lim, 2011).

Conclusion and Discussion It is always difficult to find animal models of human neurological diseases and it looks as though the mouse with genotype tTA/α-syn expressing A53T α-syn used in this experiment is a good model (Lim, 2011). Now that this genotype is known, this model can be used for further testing regarding understanding of Lewy body diseases and hopefully a cure or medication can be found for the human population. In order for developmental abnormalities not to occur in utero such as mitochondrial defects

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3144489/figure/F8/

Figure 4. All genotypes tested are being compared between 12 months and 12m(dox9-12m). In contextual fear there is a significant increase in % freezing in the tTA/α-syn genotype. This is because the removal of α-syn accumulation in the hippocampal region aided in the reversion of the hippocampal memory defect. In cued fear there is no significant increase in % freezing from any of the genotypes as cued fear memory is based in the amygdala, which suppression of α-syn accumulation has no effect on (Lim, 2011).

that were found to take place in transgenic mice with an overexpression of A53T α-syn, which is the predecessor of dopamine neuron deterioration (Chen, 2015) Lim and his team used a diet of 200mg/kg doxycycline from insemination to postnatal day 21, and were able to

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avoid developmental defects (2011). This technique is important in future research as the mice need to be as close to the human model as possible, and humans do not develop Lewy body disorders til late in life, therefore α-syn accumulation in the mice brain can only take place once maturity has been reached.

Lim’s research showed that suppressing the expression of transgene α-syn in mice, not only stops the progression of DLB, but also removes previous accumulation of α-syn and restores some of the lost function both of the physical synapse and memory deficit that had incurred (2011). Proteases kallikrein-6 and calpain-1 naturally get rid of α-syn, so that accumulation doesn’t take place, however in Lewy body disorders the activity of these proteases are considerably diminished hence the accumulation of α-syn (Miners, 2014). If these proteases could restart their activity level or if more proteases could be placed into the brain, then α-syn accumulation would be diminished. The diminished α-syn may have the same response as in the mice in which case this technique could lead to amazing treatment options for patients to relieve the symptoms of Lewy body disorders.

Criticisms and Future Directions

In Lim’s research it was clearly stated that the long-term suppression of α-syn expression in the transgenic mice had not been tested (2011). Therefore it is unknown whether the suppression of α-syn or removal of its accumulation may have detrimental effects on the subject.

This is definitely something that needs to be tested before any more research in α-syn suppression is done.

For if there are negative effects to long-term suppression of α-syn than this route may no longer be a viable treatment, or at the very lest more research will need to be done on how to relieve any deleterious effects.

While the mice model is a very close representation of humans with Lewy body disorders it is only that way because of the doxycycline diet it was put on to stop the expression of α-syn before maturity. That diet was used once again to suppress the expression of α-syn from months 9-12, which is when accumulation of α-syn diminished and memory function was recovered (Lim, 2011). While that gives great hope to the treatment, further research must be done to determine how to suppress α-syn in humans. It is unknown if a αdoxycycline diet would be effective on humans, or if it may even be harmful. Proteases kallikrein-6 and calpain-1 are natural reducers of α-syn accumulation, but in subjects with Lewy body disorders their activity is so impaired that α-syn builds up (Miners, 2014). Research efforts should put in place to determine a way to increase the activity of the proteases when Lewy bodies are present so that the accumulation of α-syn will not be as great. This could lead to repairs in synaptic structure and reduce the memory impairments that α-syn accumulation caused.

References 1. Bencsik, A., Muselli, L., Leboidre, M., Lakhdar, L., Baron, T. (2014).

Early and persistent expression of phosphorylated α-synuclein in the enteric nervous system of A53T mutant human α-synuclein transgenic mice. Journal of Neuropathology and Experimental Neurology, 73(12), 1144-51. doi: 10.1097/NEN.0000000000000137 253

2. Chen, L., Xie, Z., Turkson, S., Zhuang, X. (2015). A53T human α-synuclein overexpression in transgenic mice induces pervasive mitochondria macroautophagy defects preceding dopamine neuron degeneration. Journal of Neuroscience, 35(3), 890-905. doi: 10.1523/JNEUROSCI.0089-14.2015 3. Gallea, J. I., Celej, M. S. (2014). Structural insights into amyloid oligomers of the Parkinson disease-related protein α-synuclein. Journal of Biological Chemistry, 289(39), 2673342. doi:10.1074/jbc.M114.566695 4. Games, D., Seubert, P., Rockenstein, E., Patrick, C., Trejo, M., Ubhi, K., … Masliah, E. (2013). Axonopathy in an α-Synuclein Transgenic Model of Lewy Body Disease Is Associated with Extensive Accumulation of C-Terminal–Truncated α-Synuclein. The American Journal of Pathology, 182(3), 940–953. doi:10.1016/j.ajpath.2012.11.018 5. Halliday, G. M., Leverenz, J. B., Schneider, J. S. and Adler, C. H. (2014), The neurobiological basis of cognitive impairment in Parkinson’s disease. Mov. Disord., 29, 634–650. doi: 10.1002/mds.25857 6. Lim, Y., Kehm, V. M., Lee, E. B., Soper, J. H., Li, C., Trojanowski, J. Q., Lee, V. M.-Y. (2011). α-Syn Suppression Reverses Synaptic and Memory Defects in a Mouse Model of Dementia with Lewy Bodies. The Journal of Neuroscience, 31(27), 10076-10087. doi: 10.1523/JNEUROSCI.0618-11.2011 7. Liu, F. T., Chen, Y., Yang, Y. J., Yang, L, Yu, M., Zhao, J., … Wang, J. (2015). Involvement of mortalin/GRP75/mthsp70 in the mitochondrial impairments induced by A53T mutant α-synuclein. Brain Research, 16;1604, 52-61. doi: 10.1016/j.brainres.2015.01.050 8. Martin, L. J., Semenkow, S., Hanaford, A., Wong, M. (2013).

Mitochondrial permeability transition pore regulates Parkinson’s disease development in mutant α-synuclein transgenic mice. Neurobiology of Aging, 35(5), 1132-52 doi: 10.1016/j.neurobiolaging.2013.11.008 9. Melnikova, T., Fromholt, S., Kim, H., Lee, D., Xu, G., Price, A., …

Borchelt, D. R. (2013). Reversible pathologic and cognitive phenotypes in an inducible model of Alzheimer-amyloidosis. The Journal of Neuroscience : The Official Journal of the Society for Neuroscience, 33(9), 3765–3779. doi:10.1523/JNEUROSCI.4251-12.2013 10. Miners, J. S., Renfrew, R., Swirski, M., & Love, S. (2014).

11. Accumulation of α-synuclein in dementia with Lewy bodies is associated with decline in the α-synuclein-degrading enzymes kallikrein-6 and calpain-1. Acta Neuropathologica Communications 2, 164. doi:10.1186/s40478-014-0164-0 12. Overk, C. R., Cartier, A., Shaked, G., Rockenstein, E., Ubhi, K., Spencer, B., … Masliah, E. (2014). Hippocampal neuronal cells that accumulate α-synuclein fragments are more vulnerable to Aα oligomer toxicity via mGluR5 – implications for dementia with Lewy bodies. Mol. Neurodegener., 9, 18. doi: 10.1186/1750-1326-9-18 13. Regensburger, M., Prots, I., & Winner, B. (2014). Adult Hippocampal Neurogenesis in Parkinson’s Disease: Impact on Neuronal Survival and Plasticity. Neural Plasticity, 2014, 454696. doi:10.1155/2014/454696 14. Rockenstein, E., Nuber, S., Overk, C. R., Ubhi, K., Mante, M., Patrick, C., … Masliah, E. (2014). Accumulation of oligomer-prone α-synuclein exacerbates synaptic and neuronal degeneration in vivo. Brain, 137(5), 1496-1513. doi: http://dx.doi.org/10.1093/brain/awu057 15. Swirski, M., Miners, J. S., de Silva, R., Lashley, T., Ling, H., Holton, J., … Love, S. (2014). Evaluating the relationship between amyloid-αand α-synuclein phosphorylated at Ser129 in dementia with Lewy bodies and Parkinson’s disease. Alzheimer’s Research & Therapy, 6(9-9), 77. doi:10.1186/s13195-014-0077-y 16. Wang, Y., Yu, Z., Ren, H., Wang, J., Wu, J., Chen, Y., Ding, Z. (2014). The synergistic effect between α-amyloid(1-42) and α-synuclein on the synapses dysfunction in hippocampal neurons. Journal of Chemical Neuroanatomy, 63, 1-5. doi: 10.1016/j.jchemneu.2014.11.001

A reserve pool of glutamate receptors is required for LTP

Ola Taji

The cellular mechanism of forming new memories is by having strengthening synapse between two neuron which occurs through long-term potentiation (LTP). It is believed that LTP requires the cytoplasmic tail of the AMPAR subunit GluA1. This paper used single-cell replacement assay, outside-out patches, and electrophysiology to see the minimal requirement of the GluA1 C-tail for LTP in the CA1 pyramidal neurons. In this research, it was found that GluA1 C-tail is not required for LTP, but when replaced with GluA2 LTP was expressed normally. Also when another family of glutamate receptor was expressed called kainite receptor (KAR) it also expressed LTP normally. However the only time LTP was impaired was during synaptic expression. These results demonstrate that in order for LTP to be formed it just needs a reserved pool of glutamate receptors, also from this we can see how flexible the synapse can be.

Keywords: long-term potentiation (LTP); AMPAR; GluA1 C-tail; Background The important region of our brain, the hippocampus, increasing synaptic strength between two neurons for a long time which is called long-term potentiation (LTP).

Thus, allowing us to store newly forming memory and information. However, LTP can only be triggered in the presence of AMPA receptors (AMPAR) since it is largely dependent on them (Opazo.et.al. 2012).

AMPA receptors are a type of ionotropic glutamate receptors that has a fast synaptic transmission, and it is the primary reason for postsynaptic depolarization (Lu.et.al. 2012). AMPAR is a tetramer that consist of 4 subunit isoforms GluA1-4 which have a distinct C-terminal tail (Bredt.et.al. 2003). Both GluA1 and A4 have long C-tail compared to GluA2 and A3 which have shorter C-tails (Malinow.et.al. 2002). In the CA1 neurons of the hippocampus, they mostly consist of GluA1/2 heteromer receptors and a minor fraction of GluA2/3 heteromer receptors (Lu.et.al. 2012).

These heteromer receptors get trafficked to synapse with the aid of the C-terminal tail interacting with proteins (Widagdo.et.al. 2015). AMPAR trafficking is known to be a two-step process: 1) delivery of receptors to the surface, 2) targeting of the receptors to the synapse (Lu.et.al. 2010). One of the main AMPAR subunits that have been seen to affect LTP and trafficking dramatically is the GluA1 subunit. Many researchers examined how GluA1 containing receptors such as GluA1/2 got trafficked to the synaptic membrane. What they found was that different proteins interacted with the C-terminal tail in order to help with GluA1 trafficking (Opazo.et.al. 2012). Within the C-terminal tail, there is two important phosphorylation site and they are called S831 and S845 (Lu.et.al.

2012). Each of these phosphorylation site get phosphorylated by different kinase proteins for example S831 is phosphorylated by CaMKII and protein kinase C (PKC), while S845 only gets phosphorylated by protein kinase A (PKA) (Kessels.et.al. 2009). These proteins were seen to be important for the expression of LTP (Selcher.et.al. 2011).Previous research have showed that when they fully knocked down GluA1 subunit, the result that they gained was that LTP was reduced (Selcher.et.al. 2011). In this paper, they go on to see the minimum requirement of the GluA1 C-tail for LTP that was done by mutating the C-tail and observing

the effect on the three stages of trafficking. Also in this paper they replace GluA1/2 receptors with homomeric GluA2 or with kainite-type glutamate receptor (KAR) to see how that will affect LTP. Research Overview

Summary of Major Results

The requirement of the GluA1 C-tail in trafficking stages AMPAR trafficking is a two stage process: surface expression, synaptic targeting. Within this research, they produced two different GluA1 truncation, where when GluA1’s C-tail was fully truncated (GluA1∆C) and the other we have a truncation up to the amino acid 824 (GluA1∆824). Granger and colleagues they looked at the role of GluA1 C-tail on surface expression. Using a mouse line that had the genes for GluA1, A2, and A3 lined by loxP (Gria1-3fl/fl) sites, when Cre is induced it eliminates AMPAR from the CA1 neurons of these mice. That was proven by using outside-out patches recording which confirmed the complete absence of AMPAR by seeing the elimination of the glutamate current. They then inserted GluA1 which rescued the current, however, when GluA1∆C was inserted there was rescue but not to the same extent as GluA1 because there is a decrease in surface expression which is due to impaired trafficking in GluA1∆C (Figure 1a).

Subsequently Granger and colleagues went on to see if GluA1 C-tail is required for synaptic transmission.

Using Gria1-3fl/fl CA1 neuron they inserted GluA1, and then recorded the excitatory postsynaptic current (EPSC) after 17 days. What they found was that the EPSC was rescued to ~68% of the control cell, also when they did this process again, instead using GluA1∆C the same results were obtained (Figure 1b). This illustrates that dispute the sever truncation synaptic transmission was still able to be rescued.

GluA1 C-tail is not required for LTP After seeing the result from synaptic expression and transmission, Granger and her colleagues were wondering if GluA1 C-tail is important for LTP. Using electroporation they were able to transfect at first both Cre (complete absence of AMPAR) and full-length 254

GluA1, then measure LTP. The results that they obtained exhibited normal LTP, they did the procedure again twice, but this time instead of transfecting GluA1∆C they transfected both GluA1∆841 and GluA1∆MPR (together will represent GluA1∆C). However, LTP was still seen to be normal as the control (Figure 1c). From this experiment, we can conclude that C-tail is not required for LTP. Other AMPAR is sufficient for LTP Since Granger et.al. found that GluA1 C-tail is not necessary for LTP, they went on to see if there is other AMPAR subunits that might affect LTP. One of the subunits that they looked at is GluA2 because it is found both in GluA1/2 and GluA2/3 heteromer receptors which are contained in CA1 neurons of the hippocampus.GluA2 also has a C-tail, but it is short.

By going through the same experiments as GluA1, GluA2∆C showed impaired synaptic expression, but it was able to be trafficked to the synapse. However, LTP in GluA2∆C was impaired l (Figure 2a).This meant that the C-tail of GluA2 is not important for LTP. The experiments conducted so far revealed that when GluA1 is knocked out LTP is impaired, but when GluA2 or 3 is deleted that had no effect. When we inserted either GluA1∆C or GluA2∆C in Gria1-3fl/fl LTP was seen to be substantially impaired. These findings suggest that in order for LTP to be generated all it needs is a reserve pool of AMPAR subunit.

Granger et.al. they then turned to another family of glutamate receptors called Kainate receptors (KAR).

Using the Gria1-3fl/fl CA1 neurons with Cre that removed all types of AMPAR, which allowed them to insert only KAR (GluK1). When GluK1 was inserted, they realized that LTP was similar to control neurons (Figure 2b). To ensure that the current is only from GluK1, ACET was applied and it caused EPSC to be eliminated in neurons that only contained GluK1 and it did not affect control neurons (Figure 2c). This experiment demonstrated that even without AMPAR itself, neurons can still undergo LTP as long as there is an alternative glutamate receptor.

Discussion Using the single-cell molecular approach by utilizing Gria1-3fl/fl neurons that helped in giving full control over what to express in the CA1 neurons. In previous research they showed that when amino acid S831 was deleted that cause CAMKII not to be able to

Figure 1. a) Measuring the amount of expression of GluA1 by measuring glutamate current. When Cre is added in Gria1-3fl/fl cells it eliminates current, but when full-length GluA1 is expressed the current is almost like the control while GluA1-∆C the current is much less. b) Measuring the EPSP to see GluA1 containing receptor are transferred to the synapse. When have GluA1-∆C EPSP amplitude was seen to increase. c) Even if there is GluA1-∆C, LTP was still generated but not the same extent as control.

phosphorylate at all, which lead to decrease in the trafficking of GluA1 and also caused LTP to be diminished (Opazo.et.al. 2012). However, Granger.et.al. found that when full-length GluA1 was inserted that rescued LTP and trafficking was seen to be as control.

On the other hand, when GluA1∆C was inserted synaptic expression was impaired but the synaptic transmission was not impacted, and when they used outside-out patches to record EPSC they found that LTP was slightly impaired. Many research proved that when GluA1 is not present that cause LTP to vanish.

In this research, they observed that when GluA2 is present with or without the short C-tail, LTP was still able to be generated but compared to control it was fractionally impaired. This allowed Granger.et.al. to take it a step further by seeing if LTP can be generated by other glutamate receptor, so they looked at Kainate receptor (KAR) by using GluK1 subunit. What was observed is, when GluK1 was inserted into Gria1-3fl/fl

Figure 2: a)when GluA2∆C was inserted in Gria1-3fl/fl LTP was formed but it is reduced compared to control b) when using a different family type of glutamate receptor called KAR (GluK1), LTP was still able to be formed by that neuron, and when ACET was added EPSC decreased 255

LTP was generated and it was indistinguishable to the control neuron’s LTP. From the results we observed that synaptic transmission was not affected despite GluA1∆C was inserted, this could indicate that there is probably some other region in the GluA1 that is helping in the trafficking.

Conclusions LTP is important because when LTP is present it increases synaptic strength between two neurons and through that learning and memory can occur. Therefore, from this paper several things can be concluded, firstly is that C-tail of the GluA1 is not important for synaptic transmission nor is it significant for LTP, which was proven when GluA1∆C got inserted into Gria1-3fl/fl CA1 neuron. Secondly is that LTP can still be generated even if GluA2 is only present, but the LTP would be slightly impaired compared to control.

Third of all is that LTP can be generated as long as there is some type of glutamate receptor such as KAR. Criticisms and Future Directions In this research, it was concluded that the GluA1 C-tail has no role in LTP generation. However, one question remains a mystery, and that is whether there are other specific interactions, which can cluster AMPAR at the synapse. Solving this question will help us understand synaptic modification during learning and memory. In order to investigate whether there are specific interactions that cause clusters of AMPAR in the synapse, we can probably observe kinases, because when LTP is induced it activates different kinases, which then phosphorylate the AMPAR at different locations, therefore, which leads AMPAR to be trafficked to the synapse and increasing LTP. So the kinase that we can concentrate on is cGMP-dependent kinase (cGK), this kinase phosphorylates GluA1 at serine 845 position (Kim.et.al. 2015). Therefore, to observe if cGK has an effect on AMPAR trafficking we can first knockout cGK (Kim.et.al. 2015), then using single-molecule targeting technique such as using Gria1-3fl/fl CA1 neurons, we can observe what happens to AMPAR’s trafficking (Constals.et.al. 2015). At the same time use Electrophysiology, and Outside-out patches to record both the voltage and the current, and from these recordings we can see whether LTP decreases or increases. Another experimental approach is truncate AMPAR at the S845 position, at the same time we can immunostain AMPAR to see if there is an increase of AMPAR on the surface of the synapse, or instead we can use single-molecules targeting, so we can observe whether AMPAR trafficking occurs. Also within that experiment we can measure the voltage and current using Electrophysiology and Outside-out patches to see if there is an increase in LTP. In the research, they showed that synaptic transmission was not affected when GluA1∆C was inserted in Gria1-3fl/fl CA1 neurons, so there is probably another region in GluA1 subunit that may also help in GluA1’s trafficking. Many recent research have come across a region of the GluA1 that helps in its trafficking to the

synapse. That region is called the cytoplasmic domain loop1 (Lu.et.al. 2010). Within this GluA1 loop1, research, found that there is a phosphorylation site called S567, which could only be phosphorylated by CAMKII (Lu.et.al. 2010). When S567 is phosphorylated by CAMKII it only causes synaptic trafficking and not synaptic transmission, which was proven when they deleted S567 GluA1 what not trafficked to the synapse (Lu.et.al. 2010). Another recent research found that there is another kinase that can also phosphorylate S567 in Loop1 of GluA1 That kinase is called casein kinase 2 (CK2), and this kinase only regulate surface expression (Lussier.et.al. 2014). When S567 was mutated into alanine they observed a decrease in GluA1 surface expression (Lussier.et.al. 2014).

Previous research showed that when GluA1 is knocked down LTP disappears. However, a research came out showing that there is a gene in the neurons that can affect the expression of GluA1, this gene is called LRP1 (Gan.et.al. 2014).This research that was done by using western blot analysis they showed that when they deleted LRP1 gene there was a significant decrease in the level of GluA1. Gan.et.al. concluded that when LRP1 is deleted GluA1 is not expressed then there is no phosphorylation on S845 and S831 sites.

References 1. Opazo.P, Sainlos.M, Choquet.D. Regulation of AMPA receptor surface diffusion by PSD-95 slots. Elsevier.

2012;22:453-460 2. Lu.W, Roche.K.W. Posttranslational regulation of AMPA receptor trafficking and function. Elsevier. 2012;22:470-479 3. Bredt.D.S., Nicoll.R.A. AMPA Receptor Trafficking Review at Excitatory Synapses. Neuron. 2003;40:361-379 4. Malinow.R., Malenka.R.C. AMPAR Receptor Trafficking and Synaptic Plasticity. Annu.Rev.Neuroscience.

2002;25:103-126 5. Widagdo.J., Chai.Y.J., Huganir.R.L., Anggono.V. ActivityDependent Ubiquitination of GluA1 and GluA2 Regulates AMPA Receptor Intracellular Sorting and Degradation. Cell Reports.

2015;10: 783-795 6. Lu.W., Isozaki.K., Roche.K.W., Nicoll.R.A. Synaptic targeting of AMPA receptors is regulated by a CaMKII site in the first intracellular loop of GluA1. PNAS. 2010;107: 22266-22271 7. Kessels.H.W., Malinow.R. Synaptic AMPA Receptor Plasticity and Behavior. Neuron. 2009;61: 340-350 8. Selcher.J.C., Xu.W., Hanson.J.E., Malenka.R.C., Madison.D.V. Glutamate receptor subunit GluA1 is necessary for long-term potentiation and synapse unsilencing, but not long-term depression in mouse hippocampus. Elsevier. 2011; 1435: 8-14 9. Lussier.M.P., Gu.X., Lu.W., Roche.K.W. Casein kinase 2 phosphorylates GluA1 and regulates its surface expression.

European Journal of Neuroscience. 2012; 39:1148-1158 10. Gan.M., Jiang.P., McLean.P., Kanekiyo.T., Bu.G. LowDensity Lipoprotein Receptor-Related Protein 1 (LRP1) Regulates the Stability and Function of GluA1 a-Amino-3256

Hydroxy-5-Methyl-4-Isoxazole Propionic Acid (AMPA) Receptor in Neurons. PLoS ONE. 2014; 9: 1-18 11. Granger,A.J. Shi,Y. Lu,W. Cerpas,M. Nicoll,R.A. LTP requires a reserve pool of glutamate receptors independent of subunit type. Nature. 2013; 493:495-500 12. Kim,S. Titcombe,R.F. Zhang,H. Khatri,L. Girma,H.K. Hofmann,F. Arancio, O. Ziff, E.B. Network compensation of cyclic GMP-dependent protein kinase II knockout in the hippocampus by Ca2+-permeable AMPA receptors. PNAS. 2015; 112:3122-3127 13. Constals,A. Penn, A.C. Hosy,E. Choquet,D. GlutamateInduced AMPA Receptor Desensitization Increases Their Mobility and Modulates Short-Term Plasticity through Unbinding from Stargazin. Neuron. 2015; 85:787-803 14. Woolfrey.K.M., Sanderson.J.L., Dellâ&#x20AC;&#x2122;Acqua.M.L. The Palmitoyl Acyltransferase DHHC2 Regulates Recycling Endosome Exocytosis and Synaptic Potentiation through Palmitoylation of AKAP79/150. Jour, Neuroscience. 2015; 25: 442-456 15. Borgdorff.A.J., Choquet.D. Regulation of AMPA receptor lateral movements. Nature. 2002; 417: 649-653 This work was supported by The Association for the Development of Undergraduate Neuroscience Education (SRA & RLN), The Endowment for Science Education (EA), and The Synaptic State Faculty Research Foundation (EA). The authors thank Mr. Spine L. Cord, Dr. Amy G. Dala, and the students in Neuroscience 101 for technical assistance, execution, and feedback on this lab exercise.

Address correspondence to: Dr. Rita L. Neurotrophin, Biology Department, 123 Growth Cone Avenue, Action Potential College, Hillock, IL 60101 Email: rln@apc.edu Copyright Š 2015 Dr. Bill JU, Neurosciences, Human Biology Program

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Daniel Takla

Demyelination: Prevention and Restoration

Remyelination and other treatments of neurodegenerative diseases are a relatively new area of research. Very little is known about the specific role of glia in disease specific cases. In order to further advance in finding clinically relevant treatments, much more knowledge is needed about the mechanism and interplay of environmental cues and oligodendrocytes. This review discusses the current paradigm in the field focusing on the preventative approach to demyelination. It then goes on to focus on a proof of concept paper done by Windrem et al. (2014) and remyelination. Windrem et al. were able to show a complete remyelination of a demyelinated mouse brain using xenografted hGPCs. Potential future studies in the area of research based on the results are given.

Key words: Xenograft; Cell transplantation; Remyelination; Shiverer Mice; Neurodegenerative Diseases; Human Growth Progenitor Cells (hGPC); Background Myelin and their integrity are crucial for the functionality of the CNS. The oligodendrites and schwann cells wrap around axons to enhance communication in the nervous system1. Another paper by Gibson et al. showed that not only do myelin affect the electrical activity of neurons but also that neurons themselves modulate myelination2. Neurodegenerative diseases as well as following a serious injury to the spinal cord (SCI), demyelination occurs, which leads to initially believed irreversible loss of function3. Previous studies shows that structural damage to the nodes of Ranvier causes blocked or delayed conduction and loss of function prior to visible myelin degradation4. This was then followed by apparent myelin degeneration and the inability for spontaneous regeneration. Researchers previously believed this was due to the establishment of a non-permissive environment for myelin regeneration3.

Remyelination is an critical feature of regeneration, this is the generation of new myelin by using resident precursor/ stem cells as well as foreign precursors to replace the lost oligodendrocytes (OL)5,6. A paper by Duncan et al. showed that remyelination limited axonal vulnerability to deleterious environment tissue cues as well as restoring proper function of nodal and internodal regions in the remyelinated axon7. SCI damages have been relatively minimized using current knowledge. Yune et al. showed that by inhibiting the p38 mitogen activated protein kinase (p38MAPK)-dependant pro-NGF production in the microglia, you are able to protect the OL’s from apoptosis8.

Yune et al. also showed that the administration of fluoxetine (an SSRI) following SCI also inhibited the p38MAPK dependent pro-NGF and protected OL’s9. Fluoxetine also inhibited caspase-3 activation which also reduced cell death of OL’s9. Polyethylene Glycol administration is also a recognized membrane sealant used to silence oxidative stress and decrease membrane permeability as well as decrease neuroinflammation10. Immundomodulation is also an approach shown by Davalos et al. to aid in mitigating the damage done by the SCI11.

However, following SCI and a failure to limit the deleterious effects on the myelin sheath, as well as in many neurodegenerative diseases, therapeutic techniques need to be looked at to remyelinate the demyelinated nervous system. In a paper by Windrem et al, “A

competitive advantage by neonatally engrafted human glial progenitor yields mice whose brains are chimeric for human glia.”, Windrem looked at xenografting human glial progenitor cells (hGPCs) and placing them into the brains of hypomyelinated immunodeficient (rag2-/-) mice in an attempt to repair and restore myelin and ultimately better neuronal electric activity6. This study, if successful would allow us to assess the role of glial cells in demyelination pathology as well as possible therapeutic uses. The paper briefly referenced huntington’s disease (HD) and the use of mutant hGPCs in elucidating the role of glia in pathology.

Research Overview

Summary of Major Results

Windrem et al. took immunodeficient hypomyelinated and immunodeficient wild type (wt) mice and xenografted hGPCs as well as allografted mGPCs. After transplantation, phenotypic analysis was conducted on the corpus callosum using an optic fractionator.

The development was compared at 3, 4.5, 6, 8 and 12 months. Four major results were found from the experiment conducted. hGPCs progessively expanded in the mouse forebrain The hypomyelinated mice were able to be myelinated by both the allografted mGPCs and the xenografted hGPCs however, only the hGPCs showed greater expansion into the mouse neocortical gray matter as well the subcortical region. hGPCs actively removed the resident glial progenitors There was a progressive expansion of the hGPC pool relative to the host. In one year time, the mouse progenitors had been entirely replaced by the transplanted hGPCs. Windrem found that not only did the hGPCs almost entirely differentiate into OLs, restoring the myelin but they also had dominance over the mGPCs as well as behaviorally performing better on cognitive tasks than the native allografted mice.

Context-dependant nature of differentiation The hGPCs were transplanted into mice that contain a myelin sheath as well as into those that lacked one 258

(shiverer mice). This allowed the researchers to test the environmental effect on differentiation of progenitor cells. NG2+ progenitor cells are precursor cells to oligodendrocytes which will ultimately form the myelin sheath. Results found that when comparing NG2+ levels between the two types of mice (shiverer & wt), NG2+ levels were lower in wt mice. This meant that fewer GPCs became oligodendrocytes due to the lack of environmental necessity through cues. The number of donor derived oligodendrocytes were substantially greater in the shiverer mice than in the wt. These results showed that the hypomyelinated environment affected the GPCs in determining their differential pathway. In myelinated mouse brains, there was less of a need for oligodendrocytes so the transplanted GPCs remained progenitor cells. The mechanism by which these results occurred were not explained.

Figure 3. At 4 and 8 months, mGPCs exhibit substantially lower mitotic indexes than the xenografted hGPCs. (Image from Windrem et al. 2014).

Greater mitotic activity between hGPCs and mGPCs After analysis of the mitotic indices of hGPCs and mGPCs, results showed that hGPCs remained mitotically active long after the mitotic expansion of allografted mGPCs had ended. This mitotic advantage may in part explain the competitive advantage that hGPCs have over mGPCs. These results set precedent for the therapeutic potential of remyelination. The ability to create human glial chimeras particular to each patient and disease allows for further insight into regenerative effect of hGPCs. It also allows us to generate GPCs, oligodendrocytes, and astrocytes from pluripotent cells in order to gain further knowledge into the role of glia in neurodegenerative pathology.

Conclusions and Discussion

Figure 1. Shows the systematic expansion and of hGPCs into the hypomyelinated immunodeficient mice from the callosum to the cortical mantel. (Image from Windrem et al. 2014).

The analysis of the results showed that the hGPCs dominated and had such a competitive advantage over the mGPCs that, by 9 months almost all the mouse progenitors in the mouse brain were replaced by hGPCs. This is in part due to the evolution of the myelin sheath that allowed for larger body sizes and faster electrical conduction12. Evolution has lead to the expansion in the functional roles of glia. Astrocytes maintain synaptic density as well as being needed for synaptogenesis (information processing in the CNS)13.

More than discussing the advantage hGPCs possess over mGPCs, this experiment acts of a proof of concept.

It showed that it is possible to completely remyelinate the CNS assuming that certain conditions are met in order to make it a possibility. The context-dependent nature of differentiation also allows for more follow up studies to be conducted. These results are relevant to the area of demyelination pathology and therapeutics in terms of simply gaining greater understanding as well as potential first steps into clinical trials.

Criticism and Future Directions

Critical Analysis of Results

Figure 2. At 1 year, the total number of NG2+ hGPCs are almost double in the shiverer mice than in the myelinated wt mice. (Image from Windrem et al. 2014).

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One concept referenced in the discussion section is the need for certain conditions in order to illustrate this proof of concept. Windrem et al. required the mice to be immunodeficient, this allowed for no immune response to occur when xenografting hGPCs into the mouse brain. This permanent immune system suppression was definitely a shortcoming of the experiment conducted

because it is clinically unrealistic14.The experiment also obtained hGPCs which were taken from aborted fetuses from the second trimester. This lack of availability of embryotic human tissue is also a serious draw back to the extrapolation of these results for clinical purposes.

There is also the ethical implications that arise when using aborted fetuses as the source of clinical therapy.

Another major criticism of the paper is the lack of next steps. Granted that the paper is a proof of concept, demonstrating a complete remyelination of the mouse brain, it offered very little in terms of where to go from there. The potential use of hGPCs and xenografting to understand more about disease pathology was suggested but nothing in terms of the shorting comings of their paper or how these results could be specifically used further. The discussion of this paper was little more than a reiteration of the major results. The use of iPPCs or olfactory ensheathing cells to remyelinate the demyelinated CNS have been of interest and shown in numerous studies in the past such as that by Franklin et al. in 199615. Although Windrem et al. went further in showing a complete remyelination and an improvement in cognition, these results are not entirely revolutionary.

Future Directions

As made evident in the previous section, the greatest problem of the experiment by Windrem et al. is the lack of next steps and clinical significance. Experiments by Lavdas et al. showed the transplantation of myelinating cells or their precursors can lead toward a myelinating phentoype and supplement recovery16. This shows other similar experiments that are clinically relevant. Further understanding the role of glia to disease pathology as well as potential clinical trials are the next steps. Current research is ongoing relating to the use of autologous cell transplantation to override graft rejection. Several clinical trials are underway but results indicate that further research is needed17.

Using Huntington’s Disease (HD) model mice, which involve using hGPCs from pluripotent cells that carry the polyglutamine repeat that is characteristic of HD, and reproducing this experiment should allow us to understand the role of glia in HD. It will elucidate the differentiation pathway and the role the environment of HD plays on the hGPCs6.

A study by Constantinescu et al. used animal models for multiple sclerosis (MS)18, using experimental autoimmune encephalomyelitis (EAE) in conjunction with xenografting may provide greater insight into the clinically relevant role of glia in MS pathology.

Last Remarks This review has researched the current therapies that aim to mitigate some of the demyelination caused through CNS injury. This review also look at current knowledge in the field of remyelination and treatment aimed at neurodegenerative diseases. It is important to note however, that this review was primarily an analysis of an experiment that acted as a proof of concept.

This means that many more follow up experiments or extrapolations of the results to more clinically relevant treatments are required. Nonetheless, research in this area is crucial because it allows us to gain insight into neurodegenerative disease pathology.

References 1. Edgar, J.M, et al. Oligodendroglial modulation of fast axonal transport in a mouse model of hereditary s p a s t i c paraplegia. J Cell Biol. 166:121–131 (2004) 2. Gibson, E.M. et al. Neuronal activity promotes oligodendrogenesis and adaptive myelination in the mammalian brain.

Science. 344:1252304 (2014).

3. Fitch, M.T, Silver, J. CNS injury, glial scars, and inflammation: Inhibitory extracellular matrices and regeneration failure. Exp Neurol 209:294–301 (2008).

4. Hartline DK, What is myelin? Neuron Glia Biol. 4:153–163 (2008).

5. Franklin, R.J, French-Constant, C. Remyelination in the CNS: From biology to therapy. Nat Rev Neurosci. 9:839–855 (2008).

6. Windrem, M.S. et al, Neonatal chimerization with human glial progenitor cells can both remyelinate and rescue the otherwise lethally hypomyelinated shiverer mouse. Cell Stem Cell. 2:553-565 (2008).

7. Duncan, I.D. et al. Extensive remyelination of the CNS leads to functional recovery. Proc Natl Acad Sci USA. 106:6832–6836 (2009).

8. Yune, T.Y. et al. Minocycline alleviates death of oligodendrocytes by inhibiting pro-nerve growth factor production in microglia after spinal cord injury. J Neurosci.

27:7751–7761 (2007).

9. Yune, T.Y, Lee, J.Y, Kang, S. Fluoxetine prevents oligodendrocyte cell death by inhibiting microglia activation after spinal cord injury. J Neurotrauma. doi: 10:1089/ neu.2014.3527 (2014).

10. Baptiste, D.C. et al. Systemic polyethylene glycol promotes neurological recovery and tissue sparing in rats after cervical spinal cord injury. J Neuropathol Exp Neurol. 68:661–676 (2009) 11. Davalos, D. et al. ATP mediates rapid microglial response to local brain injury in vivo. Nat Neurosci.8:752–758 (2005) 12. Zalc, B, Goujet, D, Colman, D. The origin of the m y e l i n a tion program in vertebrates. Curr Biol. 18:R511–R512 (2008).

13. Kang, et al. Astrocyte-mediated potentiation of inhibitory synaptic transmission. Nat Neurosci. 1:683–692 (1998).

14. Mathieux, E. et al. IgG response to intracerebral Xenotransplantation: specificity and role in the rejection of porcine neurons. Am. J. Transplant. 14:1109-1119 (2014).

15. Franklin, R.J, Gilson, J.M, Franceschini, I.A, Barnett, S.C. Schwann cell-like myelination following transplantation of an olfactory bulb-ensheathing cell line into areas of demyelination in the adult CNS. Glia. 17:217–224 (1996).

16. Lavdas, A.A, Papastefanaki, F, Thomaidou, D, Matsas, R. Schwann cell transplantation for CNS repair. Curr Med Chem.

15:151–160 (2008).

17. Harrop, J.S. et al. Evaluation of clinical experience using cellbased therapies in patients with spinal cord injury: A systematic review. J Neurosurg Spine 17 (1 Suppl):230–246 (2012).

18. Constantinescu, C.S. et al. Experimental autoimmune encephalomyelitis (EAE) as a model for multiple sclerosis (MS). Br J Pharmacol. 164:1079–1106 (2011) 260

Study Shows How Transcranial Magnetic Stimulation Changes Depressed Brains

Eugene C. Tang

Major depression is a common mental disorder that is poorly understood. As a result, our understanding regarding the treatments for this disorder, including antidepressant medication and brain stimulation, is also poor. A recent study investigated the effect and mechanism of repetitive transcranial magnetic stimulation treatment in patients with treatment-resistant major depression. In this article, results and implications of findings of the study are presented, and potential issues and future directions are discussed.

Key words: major depression; transcranial magnetic stimulation (TMS); resting state network; default mode network (DMN); central executive network (CEN); dorsolateral prefrontal cortex (DLPFC) Background Major depression has been one of the most common psychiatric conditions, affecting millions in Canada alone (Pearson, Janz, & Ali, 2013). However, many of the current antidepressant medication have been shown to be limited in terms of treatment efficacy compared to placebo (Kirsche et al., 2008). One of the reasons for the lack of effective treatment is our little understanding of the pathophysiology of the disorder. The extend of implication of major depression ranges from neurotransmitters to neural networks.

Neurotransmitters were traditionally believed to be primarily responsible for major depression; however, the increasing evidence that reveals the complex nature of the disorder does not support this belief.

Instead, neuroimaging studies using magnetic resonance imaging (MRI) identify resting state networks in the brain, and find abnormal functioning of some of these resting state networks in depressed individuals.

For example, the default mode network (DMN), which is mainly involved in self-referential processing during wakeful rest, is found to be dysregulated in depressed individuals (Di Simplicio, Norbury, & Harmer, 2012; Guo et al., 2014; Sheline et al., 2009). The knowledge of resting state neural networks causes a shift in focus away from the traditional â&#x20AC;&#x153;neurotransmitter hypothesisâ&#x20AC;? of major depression. Whereas antidepressant medication acts on various neurotransmitters and their receptors, some brain stimulation techniques deliver a treatment effect through a different mechanism that is not yet understood. One example of such technique is the transcranial magnetic stimulation.

Transcranial magnetic stimulation (TMS) appears to be a safe and tolerable treatment alternative that is effective in treating depressed individuals, including those resistant or averse to antidepressant medication (Slotema, Blom, Hoek, & Sommer, 2010). Despite its apparent treatment efficacy, its mechanism of change is largely unknown. A recent study by Liston et al. (2014) investigated the effects of repetitive TMS (rTMS) as a treatment of depression and the mechanisms in which it modulates brain activity in depressed individuals. TMS applies magnetic current to a small brain region via electrical coils placed onto the head. In the study, rTMS treatment was applied to the left dorsolateral prefrontal cortex (DLPFC) for 25 sessions over 5 weeks at 10-Hz.

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Research Overview

Summary of Major Results

The study by Liston et al. (2014) compared 17 treatment-resistant depressed individuals with 35 healthy controls using resting state functional MRI (rs-fMRI) scans. Results show that pre-treatment depressed and healthy individuals differ in connectivity of neural networks. Specifically, pre-treatment depressed individuals show hypoconnectivity between DLPFC and the Central Executive Network (CEN), and hyperconnectivity between the subgenual anterior cingulate cortex (sgACC) and DMN. After 25 sessions of 10-Hz excitatory rTMS treatment, a number of results were found.

Depressive Symptom Reduction All seventeen depressed individuals completed the 25 sessions of rTMS treatment. Depressive symptoms reduced by an average of 9.1 points as measured by 24-item Hamilton Rating Scale for Depression.

Connectivity Within the CEN and DMN Post-treatment effect was observed within the DMN but not CEN. For the CEN, reduced functional connectivity in depressed individuals relative to control was observed prior to treatment and remained unaltered after treatment. For the DMN, elevated functional connectivity in depressed individuals relative to control was observed prior to treatment, but mostly normalized after treatment (Figure 1).

Connectivity Between the CEN and DMN Once again, post-treatment effect was observed for the DMN but not CEN. Elevated connectivity between sgACC and CEN in depressed individuals relative to control was observed prior to treatment and remained unaltered after treatment. Reduced connectivity between DLPFC and DMN in depressed individuals relative to control was observed prior to treatment, and connectivity further reduced after treatment.

serotonergic antidepressant medication has modulatory effects on the DMN via serotonin-1A receptors (Hahn et al., 2012), suggesting a common treatment target in the two completely different forms of treatment. However, the study raises more questions than it answers. Particularly, the reason for the difference of treatment effect on the functional connectivity within the CEN and DMN is unclear. Liston et al. provide some possible explanation for this observation. In short, they suggest that the type of neuronal connection (excitatory or inhibitory), the pre-existing activity state of the brain region, and the nature of functional connectivity (hyper- or hypoconnectivity) all contribute to the effect of TMS treatment.

Figure 1. Pre- and post-treatment connectivity within the DMN. Connectivity within the DMN normalizes after treatment. (A) Functional connectivity between sgACC and other parts of DMN is elevated in depressed individuals compared to controls. (B) Treatment normalizes functional connectivity between sgACC and all parts of DMN except the thalamus.

Image adapted from Liston et al. (2014).

Conclusions and Discussion Liston et al. (2014) were one of the first groups of researchers to investigate the effect of TMS on the functional connectivity of resting state networks in depressed individuals. Overall, they found that rTMS treatment relieves depressive symptoms in patients with treatment-resistant major depression and that rTMS treatment modulates functional connectivity of resting state networks. The researchers raise a number of implications resulting from their study. Mainly, their study shows that TMS may treat depressive symptoms by modulating the functional connectivity of resting state networks, both within and between.

Their study also shows that the effects of rTMS treatment are specific, given that different post-treatment effects were observed for the CEN and DMN. The results of their study generate greater understanding about the pathophysiology of major depression and the mechanism of change of TMS. They provide information regarding the role of resting state networks in major depression. Clinically, the results of their study will help improve TMS treatment protocols used for treating major depression.

Conclusions

TMS of the left dorsolateral prefrontal cortex was investigated as a treatment for major depression.

Results of the study suggest that 25 sessions of 10-Hz excitatory rTMS over 5 weeks at the left DLPFC present an overall effective treatment for patients with treatment-resistant major depression.

The study shows that this particular treatment protocol for rTMS relieves depressive symptoms by modulating functional connectivity within the DMN and between the CEN and DMN. The findings provide a preliminary understanding of the mechanism of change of rTMS treatment. Interesting, the implication of the DMN in the study is consistent with evidence that some

Criticisms and Future Directions

Despite the novel findings resulting from the study, it contains a number of issues. Mainly, the small sample size limits the ability to generalize the treatment effect to the specified population of treatment-resistant patients with major depression. Moreover, the lack of a randomized, double-blind experimental design with a placebo group makes it difficult to distinguish the effect of treatment from that of placebo. Future experiments should aim to increase the sample size and employ a randomized, double-blinded placebo group with sham TMS treatments. Nonetheless, findings of the study call for additional questions. Firstly, what are the mechanisms by which rTMS modulates functional connectivity of neural networks? One possible candidate may be the brain derived neurotrophic factor (BDNF). It has been shown that rTMS improves abnormal cortical circuits by upregulating BDNF in mice (Makowiecki, Harvey, Sherrard, & Rodger, 2014). In humans, studies show that rTMS increases BDNF level in depressed patients (Schaller et al., 2013). Future studies may investigate the effectiveness of rTMS treatment on patients carrying different BDNF single nucleotide polymorphisms, comparing the functional Val/Val homozygote group against the less functional Met/Met homozygote group. If the treatment effect is indeed facilitated by an increase in BDNF, it should be hypothesized that rTMS treatment would be more effective in depressive patients in the Val/Val homozygote group. Such experiment may identify possible molecular mechanism of rTMS treatment, genetic contribution to major depression, and provide clinical relevance for predicting treatment effectiveness for potential patients. On the other hand, findings of the study also raise the following question: Are changes in functional connectivity in resting state networks indicative treatment effect? Since clinicians use psychological constructs (i.e. observable behavioural or mood changes) in their assessment of depressive symptoms, it is important to associate changes in functional connectivity in resting state networks to such constructs. Research has shown that pre-treatment connectivity of certain brain regions may predict patientsâ&#x20AC;&#x2122; response to psychotherapy (Crowther et al., 2015), and that there are neurobiological changes after long-term psychotherapy (Buchheim et al., 2012). Future studies may investigate the effectiveness of psychotherapy in modulating functional connectivity in resting state networks.

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References 1. Buchheim, A., Viviani, R., Kessler, H., Kächele, H., Cierpka, M., Roth, G., … Taubner, S. (2012). Changes in Prefrontal-Limbic Function in Major Depression after 15 Months of Long-Term Psychotherapy. PLoS ONE, 7(3), e33745. doi:10.1371/journal.pone.0033745 2. Crowther, A., Smoski, M. J., Minkel, J., Moore, T., Gibbs, D., Petty, C., ... & Dichter, G. S. (2015). Resting-State Connectivity Predictors of Response to Psychotherapy in Major Depressive Disorder. Neuropsychopharmacology: official publication of the American College of Neuropsychopharmacology.

3. Di Simplicio, M., Norbury, R., & Harmer, C. J. (2012).

Short-term antidepressant administration reduces negative self-referential processing in the medial prefrontal cortex in subjects at risk for depression. Molecular Psychiatry, 17(5), 503–10. http://doi.org/10.1038/mp. 2011.16 4. George, M. S., Lisanby, S. H., Avery, D., McDonald, W. M., Durkalski, V., Pavlicova, M., ... & Sackeim, H. A. (2010).

Daily left prefrontal transcranial magnetic stimulation therapy for major depressive disorder: a sham-controlled randomized trial. Archives of General Psychiatry, 67(5), 507-516.

5. Guo, W., Liu, F., Zhang, J., Zhang, Z., Yu, L., Liu, J., ...

Xiao, C. (2014). Abnormal default- mode network homogeneity in first-episode, drug-naive major depressive disorder. PloS One, 9(3), e91102. http://doi.org/10.1371/journal.pone.0091102 6. Hahn, A., Wadsak, W., Windischberger, C., Baldinger, P., Höflich, A. S., Losak, J., ... Lanzenberger, R. (2012). Differential modulation of the default mode network via serotonin-1A receptors. Proceedings of the National Academy of Sciences of the United States of America, 109(7), 2619–24.

7. Kirsch, I., Deacon, B. J., Huedo-Medina, T. B., Scoboria, A., Moore, T. J., & Johnson, B. T. (2008). Initial severity and antidepressant benefits: a meta-analysis of data submitted to the Food and Drug Administration. PLoS medicine, 5(2), e45.

8. Makowiecki, K., Harvey, A. R., Sherrard, R. M., & Rodger, J. (2014). Low-intensity repetitive transcranial magnetic stimulation improves abnormal visual cortical circuit topography and upregulates BDNF in mice. The Journal of Neuroscience : The Official Journal of the Society for Neuroscience, 34(32), 10780–92.

9. Pearson, C., Janz, T., & Ali, J. (2013). Mental and substance use disorders in Canada. Statistics Canada.

10. Schaller, G., Sperling, W., Richter-Schmidinger, T., Mühle, C., Heberlein, A., Maihöfner, C., … Lenz, B. (2013). Serial repetitive transcranial magnetic stimulation (rTMS) decreases BDNF serum levels in healthy male volunteers. Journal of Neural Transmission, 121(3), 307–313.

11. Sheline, Y. I., Barch, D. M., Price, J. L., Rundle, M. M., Vaishnavi, S. N., Snyder, A. Z., ... Raichle, M. E. (2009).

The default mode network and self-referential processes in depression. Proceedings of the National Academy of Sciences of the United States of America, 106(6), 1942–7.

12. Slotema, C. W., Dirk Blom, J., Hoek, H. W., & Sommer, I. E. (2010). Should we expand the toolbox of psychiatric treatment methods to include Repetitive Transcranial Magnetic Stimulation (rTMS)? A meta-analysis of the efficacy of rTMS in psychiatric disorders. Journal of Clinical Psychiatry, 71(7), 873.

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PPARδ: New Target for Alzheimer’s Pharmacotherapy?

Lauren Tessier

Alzheimer’s Disease (AD) is a debilitating, neurodegenerative disorder, progressive in nature and ultimately resulting in death. A major hallmark of Alzheimer’s is the aggregation of soluble beta-amyloid peptides within the brain and the deposition of fibrillary forms of the protein extracellulary. These deposits cause the proinflammatory activation of microglia and astrocytes, which both surround the plaques. However, ineffective clearance of the extracellular beta-amyloid aggregates leads to their chronic production of cytotoxic factors that heighten the pathology of the disease and support neuronal cell death. Peroxisome proliferator-activated receptors (PPARs) are responsible for the regulation of inflammatory processes in microglia and macrophages; they act to suppress the action of cytokines and inflammatory mediators as well to support tissue repair and phagocytosis. The authors sought to investigate the protective mechanism of PPARδ, concentrating on its influence on inflammation and AD-related death of neurons. To achieve these ends, 5XFAD mice, which recapitulate the beta-amyloid pathology, were orally treated with GW0742, a PPARδ agonist, for two weeks. Immunohistochemistry and quantitative PCR were used in conjunction to evaluate the brain beta-amyloid burden, glial activation, and neuronal survival. Further, primary neuronal and microglia cultures and co-cultures were prepared to measure the ability of GW0742 to prevent direct neuronal death as well as inflammation-induced neuron death in vitro. The GW0742-treated transgenic mice displayed decreased levels of brain beta-amyloid levels, increased association between microglia and the amyloid plaques, and suppression of the expression of brain proinflammatory mediators. PPARδ activation was also found to rescue neurons from cell death.

The study demonstrated that GW0742 treatment has a significant anti-inflammatory effect, suggesting that PPARδ agonists may offer viable therapeutic utility in the treatment of Alzheimer’s.

Key words: Alzheimer’s Disease (AD); beta-amyloid; PPARδ; 5XFAD mice; GW0742; neuroprotective Background Peroxisome proliferator-activated receptors are nuclear receptors and ligand-activated transcription factors that bind to sequence-specific DNA elements and act to regulate cellular metabolism. There are three PPAR isoforms (α, δ and γ), and their significant role in the regulation of fatty acid and cholesterol metabolism has been carefully studied. However, in 1998, three groups reported the presence of PPARγ in rodent and human macrophages1-3. The research spawned by these initial studies ultimately led to the establishment of a firm role for PPARγ and PPARδ in the regulation of macrophage lipid metabolism and inflammation4. Macrophages mediate both the initiation and resolution of inflammation when recruited to sites of injury: the proinflammatory response serves to protect the host by targeting, degrading and clearing cellular and foreign debris, whereas inflammatory suppression is critical to preclude the deleterious effects, such as contribution to disease progression, that arise when the inflammatory response is unabated4. Pascual et al. reported the identification of the molecular pathway by which liganded PPARγ represses transcriptional activation of inflammatory response genes in macrophages5. Sumoylation of the PPARγ ligand-binding domain targets PPARγ to nuclear receptor co-repressor (NCoR)/histone deacetylase-3 (HDAC3) complexes on inflammatory gene promoters, thereby precluding the recruitment of the ubiquitylation/19S proteasome machinery that is otherwise responsible for the removal of corepressor complexes necessary for gene activation. In consequence, NCoR complexes are not removed from the gene promoter region and target genes remain in a repressed state5. The mechanism by which PPARδ

mediates inflammation is different than that of PPARγ, and has also been elucidated. In an uncommon manner, PPARδ acts as an inflammatory switch in activated macrophages. Unliganded PPARδ interacts with BCL6, a transcriptional repressor, thus inhibiting its suppressive influence on the expression of target genes. Ligand binding of PPARδ (or, similarly, genetic deletion of PPARδ) thwarts its association with BCL6, accordingly freeing BCL6 for repression of inflammatory genes6. Since such investigations have taken place, the ability of PPARγ activation to suppress inflammatory gene expression has been intensively studied in mouse models of central nervous system diseases4.

For instance, Yamanaka et al. were interested in the compromised microglial clearance functions typical of AD that result from chronic inflammatory activation and wanted to test whether activation of PPARγ would support improved amyloid-beta phagocytosis by microglia. The group found that PPARγ activation enhanced microglial uptake of amyloid-beta in a causative manner7. However, despite understanding of its mode of action, PPARδ has been far less studied in models of brain diseases4. Given its documented neuroprotective effects in Parkinson’s Disease8-9, which is also a neurodegenerative disorder, hypothesizing that PPARδ may have similarly positive effects in models of AD would be reasonable, especially given their comparable pathologic feature of extracellular protein aggregations. Yet, only one other study before that of Malm et al. examined the consequences of PPARδ activation in a mouse model of AD. Kalinin et al. reported that activation of PPARδ with GW742 in 5XFAD mice decreased the subiculum amyloid plaque burden and reduced the activation of astrocytes. The former finding was accompanied by increased expression of amyloid degrading enzymes10. The study 264

currently under review endeavored to substantiate and expand on these conclusions, concentrating particularly on inflammation and AD-related neuronal death11.

Research Overview

Summary of Major Results

The GW0742-treated transgenic mice displayed a notable reduction in the immunoreactivity of 6E10 in the subiculum and hippocampi; as 6E10 is an antibody for beta-amyloid, this conclusion effectively illustrates a decrease in brain beta-amyloid levels, which is a significant finding because, as previously stated, extracellular beta-amyloid accumulation is a pillar of Alzheimer’s Disease pathology. It was also found that GW0742 treatment fostered an increased association between microglia and beta-amyloid deposits; the ratio of microglia surrounding plaques in GW0742-treated mice was significantly higher in comparison to the vehicle treated groups. This was demonstrated by comparing the ratio of Iba-1 and 6E10 immunoreactivity between treated and non-treated mice, Iba-1 being an antibody for microglia/macrophage. The results suggest that PPARδ activation acts to amplify the clearing mechanism. Such was also substantiated by the finding that an overall reduction of Iba-1 positive microglia follows from the clearance of plaques from the brain. Further, it was found that although 5XFAD mice displaced a critical loss in the number of NeuN positive neurons and NeuN immunoreactive areas in their subiculums compared to non-transgenic mice (indicating death of neurons in the subiculum) between 2 and 6 months of age, the two week treatment with GW0742 was shown to have considerably lessened the loss of NeuN immunoreactivity in the subiculum, thus attesting to the neuroprotective actions of PPARδ, a conclusion supported by cell counts of NeuN immunoreactive cells. Despite its effects on the extracellular betaamyloid burden, GW0742 treatment did not reduce the number of neurons with intraneuronal amyloid precursor protein, (APP)/beta-amyloid. Considering that the precise function of beta-amyloid has yet to be determined, this is important, as a decrease in intracellular levels of the protein could thus be deleterious in a multi-faceted way. Neither quantity of intraneuronal 6E10 immunoreactive cells, as measured by cell count, nor the intensity of their expression, as measured by Western analysis, was compromised. Also of note, quantitative PCR revealed that 5XFAD mice displayed significant upregulation in the expression of a number of brain proinflammatory mediators compared to the nontransgenic controls, but treatment with GW0742 largely suppressed the expression of these proinflammatory mediators. GW0742 treatment was equally demonstrated to preclude inflammation-induced neuronal death in vitro. GW0742 exposure was shown to protect against the loss of MAP-2 immunoreactivity compared to vehicle-treated cultures. MAP-2 is a major cytoskeletal protein in neurons and is recognized as having an important role in neurite outgrowth and dendrite development12; it is also a member of the same heat stable microtubule-associated protein family as tau12, which has been comprehensively studied in AD. As 265

previously mentioned, this was only the second study to examine the effects of PPARδ agonism in a model of AD, but it does corroborate the findings of the first study. Though the treatment period differed between the two groups (Kalinin et al. administered GW742 for one month, whereas a two-week drug regime was utilized

by Malm et al.), they did obtain comparable results.

Kalinin et al. also showed decreased amyloid plaque burden, only in the subiculum (not, too, in the hippocampi). They did, however, demonstrate concomitant increased expression of amyloid degrading enzymes, which Malm et al. were unable to detect. Further comparison between the two paradigms would be useful to determine the distinct mechanisms of action that lead to this discrepancy. Malm et al. postulated that reduction of the amyloid burden was due to enhanced microglial-mediated clearance of beta-amyloid, and the next step would be to quantify this suggestion. This could be accomplished by, for example, fluorescently labeling beta-amyloid, and, following GW0742 treatment, determining whether that same fluorescence can be detected in surrounding microglia. Kalinin et al. additionally demonstrated a causational relationship between treatment with GW742 and reduced astrocytic activation; Malm et al.’s results indicating an overall decrease in microglial presence following treatment are analogous to such. Of note, Malm et al. demonstrated the anti-inflammatory properties of GW0742 treatment suggested by Kalinin et al. thereby increasing the available data in the field.

Figure 1. Reduced 6E10 immunoreactivity in 5XFAD mice with two-week treatment of GW0742 compared to vehicle-treated mice in the subiculum (A) and hippocampus (B).

Conclusions and Discussion PPARδ activation via the agonist GW0742 in a mouse model of Alzheimer’s Disease led to a decrease in beta-amyloid aggregation that is related to an important decrease in inflammation, a critical result seeing as the microglia that accumulate around these amyloid deposits produce various cytotoxic products that are thought to contribute to the death of neurons in Alzheimer’s Disease. Furthermore, results suggested that the effects of PPARδ activation were not attributable to the induction of ApoE lipidation.

There is a significant amount of literature that suggests that ApoE is among the primary mechanisms responsible for clearing beta-amyloid accumulations in the Alzheimer’s Disease brain, so the authors’ finding that GW0742 influenced the environment independently from increased ApoE lipidation could inspire further research into the elucidation of different avenues through which beta-amyloid deposits may be cleared.

Figure 2. Depiction of NeuN positive neurons in WT vehicletreated (A), transgenic vehicle-treated (B), and transgenic GW0742-treated (C). Number of neurons in the subiculum of the mice from each group (D), and the percentage of NeuN immunoreactive areas in the subiculum of each mouse group.

All data suggests that GW0742 treatment acts to rescue neurons from cell death.

Conclusions Based on the preliminary findings, the study provides support for the suggestion that PPARδ agonism may be a viable focus for Alzheimer’s pharmacotherapy.

Criticisms and Future Directions While the findings are very promising, the ultimate suggestion that the data warrants the consideration of PPARδ agonists as a potential therapeutic strategy in the treatment of Alzheimer’s could be substantiated by additional experiments.

First, it would be beneficial to examine whether or not PPARδ activation is able to improve the cognitive deficits associated with learning and memory in AD. Yamanaka et al. have previously shown that in the APP/ PS1 mouse model of Alzheimer’s, treatment with the gamma isoform of PPAR led to, among other promising effects, improved spatial learning7. Malm et al., using the 5XFAD mouse model, could take advantage of the three groups they utilized in the initial study (wild-type control, transgenic control and transgenic GW0742treated) and use one of the well-established tests for hippocampal-dependent memory to investigate whether GW0742 treatment ameliorates spatial memory and facilitates learning. The Morris Water Maze, the Barnes Maze, or the Radial Arm Maze could be used in such an examination. The stressful environment associated with the Morris Maze has led to anxiety being deemed as a possibly conflating factor, and the Radial Maze requires much longer for proper training, so the Barnes Maze ostensibly represents an effective middle ground to test for spatial memory. The maze consists of a center platform exposed to light, with holes lining the periphery of the platform. There is a hidden dropbox under one

of the holes, and for the first few trials, the mice are guided to that hole. They learn to find the dropbox using visual cues also around the periphery of the platform, and the time it takes to find it, how many trials finding it takes, and how many other holes are examined are recorded. Data between the transgenic groups could then be compared and analysis of statistical significance could be carried out to determine whether the differences in timing would be notable enough to be interpreted as a treatment-induced improvement of memory and learning.

Conceivable improvement in the transgenic treated mice could also be compared to the wild-type to evaluate the extent of reversal of symptoms. If the authors could show such cognitive benefits, the suggested allure of PPARδ agonists as therapeutic agents would increase.

Second, Burgess et al. formerly showed that the blood-brain-barrier becomes more permeable with MRI-guided focused ultrasound (FUS), and they used the technique to deliver stem cells to the brain13.

The technique has equally been used to facilitate the delivery of intravenously-administered antibodies to the brain in a mouse model of AD14. To increase the BBB, the focused ultrasound must be at low-intensities, and MRI is conjunctionally used to enable precise targeting before applying the therapeutic levels of ultrasound energy. In regards to PPARδ, it would be of interest to investigate whether coupling GW0742 treatment with FUS enhances the mechanism of action of GW0742; in other words, examining whether the expression levels of brain pro-inflammatory mediators and the beta-amyloid burden were further reduced.

Brain levels of GW0742 with and without FUS could be compared. An antibody and appropriately conjugated secondary antibody for GW0742 would be required for imaging purposes, to enable the visualization of the potential difference in GW0742 brain concentrations between both groups.

Finally, as PPARs are known to endogenously heterodimerize with the retinoid X receptor (RXR)15, it would be of utility to assess whether the conclusions in the Malm et al. study currently under investigation are influenced by the concomitant activation of RXR. In the Yamanaka et al. study already mentioned, as well as one conducted by Cramer et al., the activation of RXR exhibited the ability to drive the reduction of the betaamyloid plaque burden, and the latter was also able to associate it with improved memory15. Incorporating the evaluation of the effects of dual activation of PPARδ and RXR could be achieved rather straightforwardly: in addition to the administration of GW0742, an RXR agonist, such as bexarotene, would be administered to the mice at the same time. The effects of RXR activation could also be measured independently, by introducing another group of transgenic mice, treated only with bexarotene. Any resulting differences in solely activating one type of receptor and the potential additive effects of activating both receptors could then be assessed, and the effects on AD pathology could be measured using the same methods.

Ultimately, the largest flaw in the study by Malm et al. was failure to perform behavioural testing on the mice, as rescuing memory loss is one of the critical focuses in developing treatments targeted to AD. Further, it might be interesting to examine whether the effects 266

of PPARδ agonism are as preventative as they seem to be curative, meaning studying whether GW0742 treatment at an earlier time (modeling a younger age) would delay or preclude the onset of the stereotypic symptoms of AD. Nevertheless, the initial findings may substantially contribute to the field of Alzheimer’s research, and further experiments hold the promise of strengthening such a suggestion.

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2. Tontonoz P, Nagy L, Alvarez JG, Thomazy VA, Evans RM. PPARgamma promotes monocyte/macrophage differentiation and uptake of oxidized LDL. Cell: 93, 241–252 (1998).

3. Jiang C, Ting AT, Seed B. PPAR-gamma agonists inhibit production of monocyte inflammatory cytokines. Nature: 391, 82–86 (1998).

4. Chawla A. Control of macrophage activation and function

by PPARs. Circ Res:106, 1559–69 (2010).

5. Pascual G, Fong AL, Ogawa S, Gamliel A, Li AC, Perissi V, Rose DW, Willson TM, Rosenfeld MG, Glass CK. A SUMOylation-dependent pathway mediates transrepression of inflammatory response genes by PPAR-gamma. Nature: 437, 759–763 (2005).

6. Lee CH, Chawla A, Urbiztondo N, Liao D, Boisvert WA, Evans RM. Transcriptional repression of atherogenic inflammation: modulation by PPARdelta. Science: 302, 453–457 (2003).

7. Yamanaka M, Ishikawa T, Griep A, Axt D, Kummer MP, Heneka MT. PPARgamma/RXRalpha-induced and CD36mediated microglial amyloid- beta phagocytosis results in cognitive improvement in amyloid precursor protein/presenilin 1 mice. J Neurosci: 32, 17321–31 (2012).

8. Martin HL, Mounsey RB, Sathe K, Mustafa S, Nelson MC, Evans RM, et al. A peroxisome proliferator-activated receptor-delta agonist provides neuropro- tection in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson’s disease. Neuroscience: 240, 191–203 (2013).

9. Iwashita A, Muramatsu Y, Yamazaki T, Muramoto M, Kita Y, Yamazaki S, et al. Neuroprotective efficacy of the peroxisome proliferator-activated receptor delta-selective agonists in vitro and in vivo. J Pharmacol Exp Ther. 2007: 320,1087–96 (2007).

10. Kalinin S, Richardson JC, Feinstein DL . A PPARdelta agonist reduces amyloid burden and brain inflammation in a transgenic mouse model of Alzheimer’s disease. Curr Alzheimer Res: 6, 431–7 (2009).

11. Malm, T., Mariani, M., Donovan, L.J., Neilson, L., Landreth, G.E. Activation of the nuclear receptor PPARδ is neuroprotective in a transgenic mouse model of Alzheimer’s disease through inhibition of inflammation. J Neuroinflammation: 12 (2015).

12. Lim, R.W.L, Halpain, S. Regulated Association of Microtubule-associated Protein 2 (MAP2) with Src and Grb2: Evidence for MAP2 as a Scaffolding Protein. JBC: 275, 20578-0587 (2000).

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13. Burgess, A. et al. Targeted Delivery of Neural Stem Cells to the Brain Using MRI-Guided Focused Ultrasound to Disrupt the Blood-Brain Barrier. PLoS One, (2011).

14. Jordão, J.F. et al. Amyloid-β plaque reduction, endogenous antibody delivery and glial activation by brain-targeted, transcranial focused ultrasound. Exp Neurol 248, 16-29 (2013).

15. Cramer, P.E. et al. ApoE-Directed Therapeutics Rapidly Clear β-Amyloid and Reverse Deficits in AD Mouse Models.

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Carmen Tu

Temporal-Spatial Disconnect of Tauopathy and Amyloidopathy in Alzheimer’s Disease

The two major histopathological hallmarks of Alzheimer’s disease are amyloid-β plaques and phosphorylated tau. The accumulations of the two proteins, however, are distinct in their neuroanatomical manifestation. Even if both amyloid-β and tau were present at the same time, there would be no overlap at the cellular level. Using sectioning and immunohistological staining techniques, brain sections of 34 middle-aged subjects were studied for amyloid-β plaques and phosphorylated tau distributions. Findings indicated a disconnect in time and space for these hallmark biomarkers of Alzheimer’s disease, indicating that the accumulation of amyloid-β plaques and phosphorylated tau emerges independently and are distinct to specific regions in the brain.

Key words: Alzheimer’s disease, amyloid-beta, phospho-tau, spatial-temporal disconnect, neurodegeneration Background Alzheimer’s disease is one of the most common dementing disorders in elders, leaving its patients with impaired memory, thinking, and behavior. Eventually, all cortical functions will be obliterated, and ultimately death (Lubin and Gandy, 2010). Pathologically, this neurodegenerative disease is defined by a distinctive loss of hippocampal and cerebrovascular neurons, along with interstitial and cerebrovascular deposition of amyloids and neurofibrillary tangles. This accumulation of structural pathology results in the destruction of cytoskeletons (Lubin and Gandy, 2010). The neurodegenerative disease is caused by the formation of amyloid-β plaques, and neurofibrillary tangles made up of a phosphorylated protein called tau (Fornicola et al., 2014). Amyloid-β plaques occur extracellularly, and are composed of a dense fibrillar core with inflammatory cells and dystrophic neurites encompassed around it (Jack and Holtzman, 2013). The other causative agent of Alzheimer’s disease is neurofibrillary tangles, made up of aggregating hyperphosphorylated tau proteins (Jack and Holtzman, 2013).

Initially, the Amyloid Hypothesis was originally proposed to explain the pathogenesis in Alzheimer’s disease as a result of amyloid-β plaque accumulations in the brain tissue; the formation of neurofibrillary tangles of phosphorylated tau was due to the imbalance of amyloid-β production and clearance (Hardy and Selkoe, 2002). Although there is a positive correlation with age and amyloid-β accumulation (Villemagne et al., 2013), recent studies indicated that neurodegeneration in the medial temporal lobe by tau tangles antecedes cortical amyloid-β accumulation (Jack et al., 2015; Song et al., 2015; Schönheit, Zarski, & Ohm, 2004). This implies that tauopathies may promote neurodegeneration without the presence of amyloid-β (Jack and Holtzman, 2013).

To test against the linear amyloid-β-phospho-tau model, Fornicola et al., (2014) analyzed brain tissues of 34 subjects. The brain tissues were fixed using formalin.

After two weeks of immersion and fixation, the brains were sectioned accordingly; the cerebral hemispheres were sectioned coronally; the brainstem was sectioned at the axial plane; the cerebellum was sectioned in the mid-sagittal and parasagittal plane (Fornicola et

al., 2014). These sections were further processed; immunohistological studies were then performed on the sections that contained the medial temporal lobe with hippocampal formation and frontal premotor cortex.

Since carriers of the ApoE allele suggests a higher risk of developing Alzheimer’s disease, ApoE genotyping was then performed on the sections containing the cerebellum (Fornicola et al., 2014). Brain sections were then analyzed for any associates between ApoE carriers and early accumulation of amyloid-β or phospho-tau (Fornicola et al., 2014).

Major Results From past studies, it is believed that amyloid-β accumulation progresses from the neocortex, to the allocortex, the deep grey matter, and into the cerebellum. However, this accumulation of amyloid-β appeared to correlate with age and through disease (Fornicola et al., 2014). Phospho-tau on the other hand, manifests as early accumulation in the brainstem, then to the limbic structures, the association cortices, and ultimately into the primary neocortical areas; it can and does accumulate in the absence of amyloid-β (Fornicola et al., 2014). Neuronal dysfunction and degeneration of the medial temporal lobe (MTL) has been shown to impair episodic memory, a hallmark symptom of Alzheimer’s disease (Frings et al., 2013).

The MTL is a crucial brain region in the pathogenesis of Alzheimer’s disease. Postmortem studies revealed that before the onset of dementia, tau aggregates deposit initially at the trans-entorhinal cortex, then the entorhinal cortex (Taylor and Probst, 2008). In this study, all of the cases had an accumulation of phospho-tau in the locus ceruleus and forebrain, with no amyloid-β in the forebrain and brainstem. There were neurofibrillary pathology present in the entorhinal cortex in 19/20 of the cases; however, only about half of the cases had amyloid-β. In the frontal cortex, 11/20 subjects possessed amyloid-β deposits, while 7/20 had tau deposits. The subjects that had both amyloid-β and phospho-tau did not demonstrate any colonization. This indicates that the process of tau phosphorylation, which ultimately leads to neurofibrillary degeneration, occurs before the accumulation of amyloid-β in general (Forni268

cola et al., 2014). In addition, there were no significant difference in amyloid-β and phospho-tau as a function of ApoE compared to the frequencies already present in the population (Fornicola et al., 2014).

Fornicola et al. (2014) have indicated that amyloid-β and phosphor-tau accumulates independently of each other; there is a spatial and temporal disconnect between the two, diminishing the prior belief of the linear amyloidβ-phospho-tau model. Data from previous studies have already shown that the phosphorylation of tau occurs predominantly in the medial temporal lobe and brainstem, but appears to be limited in the neocortex, where amyloid-β dominates (Fornicola et al., 2014).

Figure 1. Schematic representation of the spatial and temporal accumulation of amyloid-β plaques and phosphorylated tau.

Prior to the onset of clinical symptoms, phospho-tau deposition occurs within the neurons of the locus coeruleus, then the neocortex for the onset of symptoms. Amyloid-β deposition occurs at the neocortex, but does not manifest in the cerebellar cortex until disease is considerably advanced.

From “The complexities of the pathology–pathogenesis relationship in Alzheimer disease”, by Castellani, R. J., & Perry, G. (2014). Biochemical Pharmacology, 88(4), 671–676. http://doi.org/10.1016/j.bcp.2014.01.009

Conclusions and Discussions Alzheimer’s disease is one of the most prevalent neurodegenerative disorders affecting elders.

Caused by an accumulation of amyloid-β plaques and neurofibrillary tangles, this Alzheimer’s disease is not only irreversible, but it progressively diminishes the patient’s memory, cognitive skills, and ability to perform everyday tasks (Fornicola et al., 2014).

Although tauopathy and amyloidopathy may be an independent process, the general cause of their pathogenesis in the brain seem to be a natural agerelated failure to clear misfolded proteins, and failure of protective mechanisms to eliminate soluble forms of the proteins (Clifford et al., 2013).

The accumulation of amyloid-β appears to be slow and lengthy, spanning for more than two decades (Villemagne et al., 2013). This signifies that deposition of amyloid-β occurs before cerebral atrophy and cognitive decline, thus before the onset of clinical symptoms. The deposition of amyloid-β occurs initially in the association cortex, progressing to lower cortical structures, into deep grey matter, the brainstem, and then the cerebellum (Castellani and Perry, 2014).

Studies done by Villemagne et al. (2013) have shown that elderly individuals with high rates of amyloid-β depositions developed atrophies in their grey matter and had a decline in memory notably quicker compared to those with lower rates of amyloid-β deposition.

Neurofibrillary tangles on the other hand, manifest in a stereotypic topographic progressing pattern. They first emerge from the brainstem and trans-entorhinal cortex, and then the hippocampus. As neurofibrillary tangles become more numerous in these regions, it begins to materialize in the paralimbic and adjacent medial-basal temporal cortex, then the cortical association areas, and lastly at the primary sensory, motor, and visual areas (Jack and Holtzman, 2013; Guillozet et al., 2003).

The entorhinal cortex was most abundant in tangles, whereas the primary sensory motor areas contained only a minimal amount. Neurofibrillary tangle density was highest in the fusiform gyrus, temporal pole, and inferior temporal gyrus (Guillozet et al., 2003). Overall, the two proteins manifest in different neuroanatomical areas responsible for different functions, and seem to only appear together when the dementing disease has markedly advanced (Castellani and Perry, 2014). In these cases, deposition of tau tangles in the MTL may appear to overlap with amyloid-β in neocortical areas; however, underlying pathological pathways of the two proteins may still hold to be independent (Jack and Holtzman, 2013).

On histological assays (Fig. 2), tauopathy and amyloidopathy manifest at different neocortical laminar regions. Tauopathy occurs initially in the hippocampal formation and then the neocortex, in the neocortical layers V and VI, closer to the grey matter-white matter boundaries. Amyloidopathy on the other hand, was dispersed throughout the width of the neocortical ribbon (Li et al., 2015).

Figure 2. Tau and Amyloid distributions in the right temporal cortex of an Alzheimer’s disease patient using voxels. (A) MRI of right temporal cortex of an Alzheimer’s disease patient. (B) Tau-positive. (C) Amyloid-positive. (D) Overlap of Tau and Amyloid positive. Tauopathy tend to occur close to grey matter-white matter boundaries, whereas amyloid-β is more uniformly distributed across the cortical ribbon.

From “Cortical Laminar Binding of PET Amyloid and Tau Tracers in Alzheimer Disease” by Li, Y., Tsui, W., Rusinek, H., Butler, T., Mosconi, L., Pirraglia, E., … de Leon, M. J. (2015). Journal of Nuclear Medicine, 56(2), 270–273. http://doi.org/10.2967/jnumed.114.149229 269

Concluions The findings from this study refuted the theory of a linear amyloid-β-phospho-tau model in Alzheimer’s disease; amyloid-β and phospho-tau accumulates at different times and locations in aging brains. Therefore, one can accumulate in the absence of the other, indicating a spatial and temporal disconnect between the two hallmark proteins of Alzheimer’s disease (Fornicola et al., 2014). Amyloid-β accumulation progresses from the neocortex into the cerebellum, while phosphor-tau can accumulate early in the brainstem before amyloid-β appears in the neocortex (Fornicola et al., 2014).

Criticisms and Future Directions In the study, Fornicola et. al. (2014) disproved the prior belief of a linear amyloid-β-phospho-tau model, demonstrating that the accumulation of these proteins are independent of each other temporally and spatially.

In their experiments, brains were first sectioned; immunohistological studies were then used on sections that contained the medial temporal lobe with hippocampal formation and frontal premotor cortex. All of the cases had an accumulation of the neurofibrillary tangle aggregates, phospho-tau, in the locus ceruleus and forebrain; however, amyloid-β was absent in the forebrain and brainstem. The process of tau phosphorylation, which ultimately leads to neurofibrillary degeneration, occur before the accumulation of amyloid-β in these brain areas (Fornicola et al., 2014).

However, their experiment did not consider environmental reasons that can potentially affect this differential accumulation. Another study outlined by Ramphlett and Jew (2014) can be performed to determine if exposure to heavy metals will induce the accumulation of one or both of the Alzheimer’s causative agents, amyloid-β plaques and phosphorylated tau. Neurons in the locus ceruleus are most susceptible to up-taking circulating toxicants.

The experiment would analyze patients who have Alzheimer’s, using AT8 immunostaining, and a healthy control group for heavy metals, using autometallography.

This experiment can determine whether there is a linear correlation with exposure to heavy metals and the onset of Alzheimer’s causative agents. To further these findings, the experiment outlined by Li et al. (2015) would be able to locate the exact location of where amyloid-β and phosphorylated-tau manifests in the brain. The experiment combines the use of PET radiotracers and voxels with image analysis to locate each protein with respect to the grey matter-white matter boundary. The results of these distance tracers can enable manipulation and analysis of the whole brain to see exactly when and where tau and amyloid-β deposition occurs, what can induce this, and what happens clinically when they occur together at different ages.

Although Fornicola et al.’s (2014) experiment disproved the theory that tau formation is a consequence of amyloid formation, it did not consider whether environmental effects could induce the formation of either causative agents. Their study was able to provide insight on where each protein accumulates, but results where not 100% precise. In the entorhinal cortex, neurofibrillary pathology was present in 19/20 of the cases studied. However,

with the experiment outlined by Li et al. (2015), we will be able to determine the exact location of each protein in the brain. In order to attain more confident results, more studies would have to be done on a larger scale of patients. Future studies in why the proteins accumulate in their respective brain areas may also aid in our understanding of this neurodegenerative disease, aiding in preventative and treatment plans.

References 1. Arnold, S. E., Hyman, B. T., Flory, J., Damasio, A. R., & Van Hoesen, G. W. (1991). The Topographical and Neuroanatomical Distribution of Neurofibrillary Tangles and Neuritic Plaques in the Cerebral Cortex of Patients with Alzheimer’s Disease. Cerebral Cortex, 1(1), 103–116. http://doi. org/10.1093/cercor/1.1.103 2. Castellani, R. J., & Perry, G. (2014). The complexities of the pathology–pathogenesis relationship in Alzheimer disease.

Biochemical Pharmacology, 88(4), 671–676. http://doi. org/10.1016/j.bcp.2014.01.009 3. Fornicola, W., Pelcovits, A., Li, B.-X., Heath, J., Perry, G., & Castellani, R. J. (2014). Alzheimer Disease Pathology in Middle Age Reveals a Spatial-Temporal Disconnect Between Amyloid-β and Phosphorylated Tau. The Open Neurology Journal, 8(1), 22–26. http://doi.org/10.2174/1 874205x01408010022 4. Frings, L., Spehl, T. S., Weber, W. A., Hull, M., & Meyer, P. T. (2013). Amyloid- Load Predicts Medial Temporal Lobe Dysfunction in Alzheimer Dementia. Journal of Nuclear Medicine, 54(11), 1909–1914. http://doi.org/10.2967/ jnumed.113.120378 5. Guillozet, A. L., Weintraub, S., Mash, D. C., & Mesulam, M. M. (2003). Neurofibrillary Tangles, Amyloid, and Memory in Aging and Mild Cognitive Impairment. Archives of Neurology, 60(5). http://doi.org/10.1001/archneur.60.5.729 6. Hardy, J., Selkoe, D. J. (2002). The Amyloid Hypothesis of Alzheimer’s Disease: Progress and Problems on the Road to Therapeutics. Science, 297(5580), 353–356. http:// doi.org/10.1126/science.1072994 7. Jack, C., Knopman, D., Jagust, W., Petersen, R., Weiner, M., Aisen, P., … Trojanowski, J. (2013). Update on hypothetical model of Alzheimer’s disease biomarkers.

Alzheimer’s & Dementia, 9(4). http://doi.org/10.1016/j. jalz.2013.04.248 8. Jack, C. R., & Holtzman, D. M. (2013). Biomarker Modeling of Alzheimer’s Disease. Neuron, 80(6), 1347– 1358. http://doi.org/10.1016/j.neuron.2013.12.003 9. Li, Y., Tsui, W., Rusinek, H., Butler, T., Mosconi, L., Pirraglia, E., … de Leon, M. J. (2015). Cortical Laminar Binding of PET Amyloid and Tau Tracers in Alzheimer Disease.

Journal of Nuclear Medicine, 56(2), 270–273. http://doi. org/10.2967/jnumed.114.149229 10. Lublin, A. L., & Gandy, S. (2010). Amyloid-β Oligomers: Possible Roles as Key Neurotoxins in Alzheimer’s Disease.

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11. Pamphlett, R., & Jew, S. K. (2014). Different Populations of Human Locus Ceruleus Neurons Contain Heavy Metals or Hyperphosphorylated Tau: Implications for Amyloid-β and Tau Pathology in Alzheimer’s Disease. Journal of Alzheimer’s Disease.

12. Schönheit, B., Zarski, R., & Ohm, T. G. (2004). Spatial and temporal relationships between plaques and tangles in Alzheimer-pathology. Neurobiology of Aging, 25(6), 697–711. http://doi.org/10.1016/j.neurobiolaging.2003.09.009 13. Song, Z., Insel, P. S., Buckley, S., Yohannes, S., Mezher, A., Simonson, A., … Weiner, M. W. (2015). Brain Amyloid-β Burden Is Associated with Disruption of Intrinsic Functional Connectivity within the Medial Temporal Lobe in Cognitively Normal Elderly. Journal of Neuroscience, 35(7), 3240–3247. http://doi.org/10.1523/jneurosci.2092-14.2015 14. Taylor, K. I., & Probst, A. (2008). Anatomic localization of the transentorhinal region of the perirhinal cortex.

Neurobiology of Aging, 29(10), 1591–1596. http://doi. org/10.1016/j.neurobiolaging.2007.03.024 15. Villemagne, V. L., Burnham, S., Bourgeat, P., Brown, B., Ellis, K. A., Salvado, O., … Masters, C. L. (2013).

Amyloid β deposition, neurodegeneration, and cognitive decline in sporadic Alzheimer’s disease: a prospective cohort study. The Lancet Neurology, 12(4), 357–367. http://doi.org/10.1016/s1474-4422(13)70044-9 Copyright © 2015 Dr. Bill JU, Neurosciences, Human Biology Program

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Neuregulin 1-ErbB4 Signaling and Reduced Activity in NMDA Receptors: A Molecular Pathway for the Development of Schizophrenia and a Potential Target for Future Antipsychotics Madli Vahtra

Schizophrenia is considered to be one of the most severe mental disorders. It is a lifelong mental illness and affects approximately 1% of the world’s population. Symptoms come in three forms and their presence varies among individuals. Positive symptoms include delusions, hallucinations, and paranoid thinking. Negative symptoms comprise of social isolation, flat affect, and catatonia. Cognitive deficits include the general decline in learning, memory, and other cognitive processes throughout the course of the illness (Huang, Pei, Luo, Chen, Chen, & Lai, 2015). Current treatment involves the use of both typical and atypical antipsychotic medications. Typical antipsychotics are the older generation of medication and attempt to reduce positive symptoms. Atypical antipsychotics are newer drugs and their purpose is to reduce positive and negative symptoms. Both antipsychotics block dopaminergic d2 receptors based on the hypothesis that high levels of dopamine release are responsible for the presence of schizophrenic symptoms.

(Bruijnzeel, Suryadevara, & Tandon, 2014). Unfortunately, current antipsychotics only mildly reduce symptoms, if at all. Treatment resistance is common, especially when medication is first prescribed. Patients often need to change medications multiple times in order to find the type that best suits them. In addition, side effects for both typical and atypical antipsychotics are severe. Typical antipsychotics commonly generate extrapyramidal motor symptoms, the most popular being tardive dyskinesia. Atypical antipsychotics have demonstrated side effects that include weight gain and increased risk for developing type two diabetes. Treatment resistance and severe side effects are the primary reasons for low treatment adherence in the schizophrenic population (Liberman et al. 2005). Clearly, the current treatment for schizophrenia is inadequate. Schizophrenia research is now turning its attention to developing new antipsychotics that treat psychotic symptoms more effectively.

High dopamine levels are not the only contribution to the development of schizophrenia. New antipsychotic designs are planning to target other biological mechanisms that influence the pathophysiology of the disease. Many studies are looking at the genetic components of schizophrenia. There are currently few candidate genes that contribute to the development of Schizophrenia. For example, the gene coding for neuregulin 1, NRG1, has been closely examined to determine its role in schizophrenia symptom development. Neuregulin 1 is a neurotrophin that plays crucial role in neural development and maturation. It binds to the ErbB4 receptor tyrosine kinases in order to carry out its actions. NRG1 has many polymorphisms, some of which have been shown to relate to the generation of Schizophrenia (Paterson, Wang, Kleinman, & Law, 2014). In particular, a polymorphism in the promoter region causes over-expression of the NRG1 gene, which is thought to affect pathways that induce hallucinations and cognitive deficits seen in many schizophrenic patients. Graham Pitcher and his colleagues (2011) investigated the biological pathway of the NRG1 polymorphism in order to understand the gene’s effects and provide insight for future treatment possibilities.

Key words: NRG1 gene; Neuregulin1; ErbB4 tyrosine kinase; Schizophrenia; Src-mediated NMDA receptors; hypofunction

Background and Major Findings Graham Pitcher’s study (2011) provided evidence supporting the relationship between the NRG1 gene and a molecular pathway believed to be involved in the generation of schizophrenia. In the study, NRG1 was synthetically over-expressed by injecting rodent hippocampal and prefrontal cortex cells with neuregulin 1 (Nrg1). The high levels of Nrg1 reduced activity of tyrosine kinase-mediated NMDA receptors. There are two types of NMDA receptors. Some activate upon glutamate binding alone, but others display enhanced activity when phosphorylated by tyrosine kinase Src. Nrg1 normally binds to the receptor tyrosine kinase ErbB4 and regulates proper neural development, myelination, and cell signaling. Too much Nrg1-ErbB4 signaling however, can reduce tyrosine kinase- mediated NMDA receptor activity through the inhibition of Src (Harrison & Law, 2006). Thus, the activity levels of Src-mediated NMDA receptors decrease to levels seen in Src-independent NMDA receptors. Results from Pitcher’s experiment illustrating the reduced NMDA receptor activity can be

seen in figure 1. The reduction in receptor activity has many downstream effects. For example, less active NMDA receptors inhibit long–term potentiation in the hippocampal cells. This may be the reason for reduced learning and cognitive abilities seen in schizophrenia patients. Lower activity in the prefrontal cortex NMDA receptors also affects pathways involved in attention and executive function (Pitcher et al. 2011). By understanding this pathway, treatments can be developed to reverse or change the inhibiting effects of NRG1 and allow for proper NMDA receptor functioning.

Conclusion The findings that Nrg1 signaling is connected to schizophrenic symptom development are supported by other research done on NRG1. Chang-Gyu Hahn and his colleagues (2006) studied post-mortem brains of schizophrenia patients and their Nrg1-ErbB4 signaling processes. Upon stimulating the brain slices with Nrg1, ErbB4 phosphorylation increased signifi272

cantly and resulted in reduced NMDA receptor activity.

Unlike Pitcher, this study further pieced together the molecular pathway between Nrg1 activity and lower NMDA receptor activity. They found ErbB4 receptors in schizophrenic brains to be associated much more with the postsynaptic density protein, PSD-95, than healthy control brains. The PSD-95 protein is connected to Src-mediated NMDA receptors and regulates their activity. When PSD-95 is phosphorylated

by ErbB4 tyrosine kinase, it binds to Src and inhibits its activity. Therefore, Src can no longer phosphorylate NMDA receptors and their activity decreases. (Kalia, Pitcher, Pelkey, & Salter, 2006). Since there is more ErbB4-PSD-95 coupling in schizophrenia brains, when Nrg1 binds to and activates ErbB4, PSD-95 is also more active and has a greater effect on NMDA receptor activity reduction (Hahn et al. 2006). Figure 2 demonstrates the increased activity of PSD-95 in the brains of post-mortem Schizophrenia patients. Both Pitcher’s and Hahn’s studies support the hypothesis that NRG1 gene overexpression causes NMDA hypofunction, which plays a role in the development of schizophrenia.

Both Pitcher and Hahn’s research investigated the molecular pathway involved in NMDA receptor hypofunction and together, they found very similar results.

First, NRG1 gene over-expression is a key component in inhibiting tyrosine-kinase mediated NMDA receptors. Also, the presence of ErbB4 tyrosine kinase receptors is necessary for Nrg1 inhibitory signals to occur (Pitcher et al. 2011; Hahn et al. 2006). One particularly unique advantage of Pitcher’s study alone (2011) was that his team was able to conclude that Nrg1-ErbB4 signaling only had effects on Src-mediated NMDA receptors. Excess Nrg1 levels did not impact other glutamate receptors such as AMPA receptors and tyrosine kinase-independent NMDA receptors.

Figure 1: This figure demonstrates the activity levels of NMDA receptors over time. Control NMDA receptors are Srcindependent NMDA receptors (grey circles). EPQ(pY)EEIPIA is a protein used to activate Src and represents Src-mediated NMDA receptors. One group of Src-mediated NMDA receptor was treated with excess Nrg1 (black circles) and another group was measured without excess Nrg1 signaling (white circles).

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Discussion Based on the evidence from both studies, future Schizophrenia treatments can be designed to particularly target the Nrg1-NMDA receptor pathway. Current antipsychotics mainly target dopamine d2 receptors, but also have a slight effect on the Nrg1-ErbB4 signaling system.

Long-term use of both typical and atypical antipsychotics has demonstrated lower levels of Nrg1 and ErbB4 expression in both the prefrontal cortex and hippocampal areas (Deng, Pan, Hu, Han, & Huang, 2015). This component of current antipsychotics may be contributing slightly to some of the therapeutic effects.

The importance of proper NMDA receptor activity is evident in new treatment development as well. Many new potential antipsychotic drugs attempt to reverse the symptomatic effects of NMDA receptor hypofunction. For example, one method of treatment being investigated inhibits the glycine transporter, GlyT1.

Glycine is a co-agonist of glutamate and is needed for the activation of NMDA receptors. Glycine transporters normally up glycine to be broken down or recycled.

To counter the low levels of NMDA receptor activity seen in schizophrenia patients, the GlyT1 inhibitor, bitopertin was created. Animal studies show that this drug has positive effects on synaptic plasticity and long-term potentiation, which are inhibited due to the downstream effects of NMDA receptor hypofunction (Dunlop & Brandon, 2015). In preliminary human studies, bitopertin demonstrated improvement in negative and cognitive symptoms without the serious side effect of weight gain often seen in patients treated with atypical antipsychotics (Cioffi, 2013). Studying and understanding the molecular pathway between Nrg1 overexpression, NMDA receptor hypofunction, and the presence of Schizophrenic symptoms is an important step towards finding new treatment methods that outperform current antipsychotics. Much progress is currently being made in the discovery of antipsychotics that counter the effects of low NMDA receptor activity, but even more research and a better understanding of the etiology of schizophrenia is needed.

Figure 2: a) demonstrates higher frequency of PSD-95 in Schizophrenic patients even though ErbB4 levels are roughly the same and b) shows PSD-95 activity levels.

Criticisms and Future Directions There are a few questions that remain unanswered about the relationship between excess Nrg1, reduced NMDA receptor activity, and the development of schizophrenic symptoms. Although the molecular pathway between excess Nrg1 and NMDA receptor

hypofunction has been found, further studies need to confirm that NMDA receptor hypofunction is in fact, a causal factor in schizophrenic symptoms. So far, association studies have been conducted, and can only conclude a correlation between Nrg1 levels and schizophrenic symptoms, not causation. According to a gene association study conducted by Hreinn Stefansson and his colleagues (2002), the NRG1 gene increases the risk of schizophrenia development by about 16%. Although this indicates a significant relationship, causation cannot be concluded.

Future studies should focus on how excess Nrg1 causes schizophrenia through the use of transgenic mice. Producing a mouse that over-expresses the NRG1 gene can be analyzed for physiological abnormalities and behavioral indications of Schizophrenia.

Transgenic studies also reduce potential confounding factors that may interfere with NRG1’s contribution to the development of schizophrenia (Jones, Watson, & Fone, 2011). In addition, further research is needed to progress towards designing a treatment for schizophrenia based on the Nrg1-induced NMDA receptor hypofunction. The initial steps in this direction include finding a way to reverse the effects of excess Nrg1 signaling. Some progress is already being made in this field. One study antagonized the ErbB4 receptors that usually bind to Nrg1 and induce signaling.

Although the antagonist bound and inhibited some of the neuregulin-ErbB4 signaling, the affinity for the ErbB4 receptor was low, and the effects were not significant (Xu et al. 2013). A review article stated some other ways the Nrg1-Erbb4 signaling activity can potentially be reduced. For example, one theory argues that NRG1 activity levels are time-dependent and that high levels in early life have harmful effects and high levels later in life can have beneficial effects.

Based on this idea, future studies may use NRG1 itself as a treatment approach (Deng et al. 2013).

Another hypothesis has been generated based on Pitcher’s study and believes that restoring Src activity can counteract the reduced activity seen in Srcmediated NMDA receptors (Hahn, 2011). All of these hypotheses are based on the results of preliminary studies, but have not been fully tested. By testing these various ideas, the medical community can make great strides in designing and implementing a new, more effective treatment for schizophrenia.

References 1. Bruijnzeel D., Suryadevara U., Tandon R. (2014). Antipsychotic treatment of schizophrenia: an update. Asian Journal of Psychiatry. 11, 3-7.

2. Cioffi C.L. (2013). Modulation of NMDA receptor function as a treatment for schizophrenia. Bioogranic & Medicinal Chemistry Letters. 23, 5034-5044.

3. Deng C., Engel M., Huang X.F. (2013). Neuregulin-1 signalling and antipsychotic treatment. Psychopharmacology.

226, 201- 215.

4. Deng C., Pan B., Hu C.H., Han M., Huang X.F. (2015).

Differential effects of short- and long-term antipsychotic treatment on the expression of neuregulin-1 and erbb4 receptors in

the rat brain. Psychiatry Research. 225, 347-254.

5. Dunlop J., Brandon N.J. (2015). Schizophrenia drug discovery and development in an evolving era: are new drug targets fulfilling expectation? Journal of Psychopharmocology.

29(2), 230-238.

6. Hahn C.G., Wang H.Y., Cho D.S., Talbot K., Gur R.E., Berrettini W.H…Arnold S.E. (2006). Altered neuregulin 1-erbb4 signaling contributes to NMDA receptor hypofunction in schizophrenia. Nature Medicine. 12(7), 824-828.

7. Hahn C.G. (2011). A Src link in schizophrenia. Natural Medicine. 17, 425–427.

8. Harrison P.J., Law A.J. (2006) Neuregulin 1 and schizophrenia: genetics, gene expression, and neurobiology.

Biological Psychiatry. 60, 132-140.

9. Huang C.H., Pei J.C. Luo D.Z., Chen C., Chen .T.W., Lai W.S. (2015). Investigation of gene effects and epistatic interactions between akt1 and neuregulin in the regulation of behavioral phenotypes and social functions in genetic mouse models of schizophrenia. Frontiers in Behavioral Neuroscience. 8, 1-11.

10. Jones, C.A., Watson D.J.G., Fone KCF. (2011) Animal models of schizophrenia. British Journal of Pharmacology.

164, 1162-1194.

11. Kalia L.V., Pitcher G.M., Pelkey K.A., Salter M.W. (2006). PSD-95 is a negative regulator of the tyrosine src in the NMDA receptor complex. The EMBO Journal. 25, 49714982.

12. Liberman J.A., Stroup T.S., McEvoy J.P., Swartz M.S., Rosenheck R.A., Perkins D.O…. Hsia J.K. (2005). Effectiveness of antipsychotic drugs in patients with chronic schizophrenia. The New England Journal of Medicine.

353(12), 1209-1223.

13. Paterson C., Wang Y., Kleinman J.E., Law A.J. (2014).

Effects of schizophrenia risk variation in the nrg1 gene on nrg1-iv splicing during fetal and early postnatal human neocortical development. American Journal of Psychiatry. 171(9), 979-989.

14. Pitcher G.M, Kalia L.V, Ng D, Goodfellow N.M, Yee K.T, Lambe E.K, Salter M.W. (2011). Schizophrenia susceptibility pathway neuregulin 1-erbb4 suppresses Src upregulation of NMDA receptors. Nature Medicine. 17, 470-478.

15. Stefansson H., Sigurdsson E., Steinthorsdottir V., Bjornsdottir S., Sigmundsson T., Ghosh S… Stefansson K. (2002).

Neuregulin 1 and susceptibility to schizophrenia. The American Society of Human Genetics. 71, 877-892.

16. Xu R., Pankratova S., Christiansen S.H., Woldbye D., Hojland A., Bock E., Berezin V. (2013). A peptide antagonist of erbb receptors, inherbin3, induces neutrite outgrowth from rat cerebellar granule neurons through erbb1 inhibition. Neurochemistry Research. 38, 2550 2558.

Copyright © 2015 Dr. Bill JU, Neurosciences, Human Biology Program

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Amygdala Dependent Retroactive Consolidation of Episodic Memories

Chuqi Sandy Wang

The amygdala has long been implicated in fear memories. The current research under review suggests that through an amygdala dependent fear learning mechanism, a weakly encoded memory can be strengthened at a time in the future by a related emotional stimulus. It has been posited that weakly encoded memories act as a stimulus that creates a synaptic “tag” in the brain that indicates the occurrence of stimulation, and if a second stimulus is delivered soon after the first stimulus, it is able to “capture” and strengthen the previously encoded weak memory in a protein synthesis dependent manner. This mechanism is known as synaptic tag and capture. The primary study reviewed in this article recruits human participants to behaviorally investigate the enhancement of a weakly encoded episodic memory. Participants viewed photographs of different categories and then underwent a fear conditioning phase where a foot shot was paired with the presentation of certain photographs. The study found that after a 24 hour delay, previously encoded memories that were related in concept to the conditioned stimulus (the same category of photographs) were selectively strengthened.

This suggests that previously stored memories can be further strengthened by a future event in an amygdala dependent manner. These findings are relevant to the current field of neuroscience as it poses a possible mechanism that may underlie fear and intrusive memory encoding and may be relevant to future research on stress disorders.

Keywords: Synaptic tag and capture; amygdala; memory consolidation; fear conditioning; long term potentiation (LTP) Background The human senses are inundated with information, but only a small percentage of this is encoded into memory.

How memories are stored and retrieved has been a mystery for centuries. Stemming from a neurobiological viewpoint, a mechanism has been proposed where weak memories formed in the past can be strengthened at a later time. The original mechanism, proposed by Frey and Morris (1997), known as synaptic tag and capture, is a model of late phase long term potentiation (LTP).

In this mechanism, it was proposed that a stimulus is able to create a “tag” that indicates that stimulation has occurred, and a later biochemical cascade of events eventually results in protein synthesis. This mechanism is able to turn an early phase LTP produced from weak stimulation to late phase LTP if a second stimulus is delivered soon after the first stimulus and protein synthesis occurs. This mechanism has been shown to work in the hippocampus in spatial tasks conducted with rodents (Wang, Redondo & Morris, 2010). Further, a recently published study testing the transformation of early to late phase LTP in the hippocampus shows that this reinforced LTP comes with certain changes in the receptor levels in the hippocampus (Subramaniyan et al., 2015).

More recent studies have applied this model to memory consolidation models to elucidate the underpinnings of long term memory formation. These theories suggest that an initially weak memory can be strengthened and potentiated by a stronger stimulus at a time in the future (Ballarini et al., 2009). Experiments in rats employed open field tests followed by inhibitory avoidance training showed a LTP dependent memory strengthening in the hippocampus. Specifically, it was found that in the formation of long term memory, a synaptic tag is set that can later be “captured” by a second stimulus and in turn leads to memory strengthening (Ballarini et al., 2009; Moncada et al., 2011). This has been further applied to emotional memories. Emotional events often create strong and long lasting memories (LaBar & Cabeza, 2006). Further, the amygdala complex is found to be a key structure in memory storage and emotional memo275

ries (Cahill & McGaugh, 1998). It would be sensible from both an evolutionary perspective and a motivational view for individuals to store episodic memories of emotional events, because this might help the individual perform more adaptively in a future situation and avoid harmful situations. On the other hand, there are many non-emotional events and minute details that are not necessary for our functioning and are often forgotten.

A problem arises since a person does not always know when an emotionally meaningful situation will arise, it would make sense then, if all memories leave a synaptic “tag” in the brain and if they do become important later on, become strengthened by a future event. This could be an application of the synaptic tag and capture process in the amygdala. The selective enhancement of memory based on future relevance is not a novel idea. This has been shown in sleep research, several studies have concluded that if information is encoded with the knowledge that a person will be tested on the information in the future, sleep acts more to consolidate those relevant memories and leads to better recall of the previously encoded information (Wilhelm et al., 2011; Dongen et al., 2012). Although past research has shown that a previously encoded memory can be modulated, there has not been enough work done in the amygdala and there currently exist a lack of human behavioral studies supporting the synaptic tag and capture model. Therefore the current study under review is especially relevant as it specifically addresses this model behaviorally in human participants and tests if previously encoded memory can be enhanced selectively due to a related event in the future through an amygdala dependent pathway using fear conditioning (Dunsmoore et al., 2015).

Research Overview

Summary of Major Results

It was found that memories that were related to a conditioned stimulus encoded at an earlier time could be selectively enhanced. This was demonstrated by

a three phase experiment. Starting with the preconditioning phase, 138 human participants were shown a series of photographs of two categories, tools or animals, and asked to put them into their respective categories.

Following this, participants underwent amygdala dependent fear conditioning where a shock was paired with the presentation of new photographs, again of different tools or animals. A post conditioning phase was conducted following this, it repeated the procedures in the preconditioning phase using a different set of photographs. Then either immediately after, 6 hours after, or 24 hours after the post conditioning paradigm, the original photographs from the preconditioning phase were presented once again and participants underwent a recognition task (Fig. 1). Only in the 24 hours post conditioning period was a significant result observed. If the shock was paired with the tools category in the conditioning phase, memory was enhanced for the tools that were encoded in the preconditioning phase in the 24 hour condition, but not the animals’ category, and vice versa (Fig. 2). This is consistent behaviorally with the mechanism of synaptic tag and capture, where in the preconditioning phase, a weak memory is encoded of the presented photographs.

Later, with the onset of an emotional event, late LTP occurs, and this emotional event can retroactively enhance previously encoded memories that are conceptually related to the emotional event.

Conclusions and Discussion This pathway supports the previous literature and supports a similar tag and capture pathway behaviorally.

Further, this study employs a human model and shows specificity in the retroactive enhancement of memory in an amygdala dependent fear conditioning pathway.

Interestingly, the enhancement of memory didn’t occur immediately after the post conditioning phase or even in the 6 hour delay condition. This result would suggest that the retroactive memory enhancement with an emotional event requires some time after the emotional event has occurred to be consolidated. This is again consistent with the synaptic tag and capture mechanism, and the authors suggest that fear conditioning is able to modulate the “tag” from the weakly encoded memory formed during the preconditioning phase, and through a protein synthesis cascade enhance the previously encoded memory, the need for protein synthesis could explain the 24 hour time delay for the enhancement of memory to occur. Nonetheless, this is one of the first studies conducted in humans to suggest that previously encoded weak memories can be consolidated by a future experience that is related in context. The specificity of

Figure 2. Experimental results. a) shows memory after 24 hours after the post conditioning phase, memory for preconditioning stimuli that were of the same context as the fear conditioning stimuli were enhanced. This did not occur in b) 6 hour delay period or c) immediately after the post conditioning phase (Dunsmoore et al., 2015).

this is demonstrated as a past memory that is enhanced is related to the future emotional experience and other memories that were encoded together with the past memory are not at all enhanced.

Conclusions

This research has interesting implications for the adaptive capacities of memory, it suggests that we are monitoring our environment and encoding weak memories of these details, although most of this will be forgotten, a future event with meaning can selectively enhance the weak previously encoded memories and strengthen them retroactively. In this way, seemingly useless information at the time of encoding can be later consolidated if it becomes important. Especially in the event of an emotional experience, it strongly enhances a related memory encoded sometime in the past, this may have larger implications for

Figure1. Experimental procedure. The pre-conditioning phase, fear conditioning phase and post conditioning phases are demonstrated. Presentation stimuli appeared on a computer screen and fit into the categories of tools or animals (Dunsmoore et al., 2015).

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the underlying mechanisms behind intrusive memories and trauma related disorders. Overall, this study contributes to an existing literature of retroactive memory consolidation and provides convincing evidence in humans that an emotional experience in the future can selectively enhance a related memory encoded weakly in the past.

Criticisms and Future Directions

Although this is one of the first studies to show that a future event can strengthen a previously encoded memory through fear conditioning in humans, several issues still need to be addressed with the current study.

The behavioral paradigm employed in this study seems to correspond to the underlying synaptic tag and capture mechanism, but this is yet to be elucidated clearly. To begin investigating this in humans, an fMRI imaging design would be a good place to start (Dimitrova et al., 2004). Underlying brain activity can be measured while this retroactive consolidation is occurring. The current experiment should be repeated with participants undergoing fMRI imaging during the paradigm. A greater sample size should also be used to generate more support and validity for the current theory. Activation patterns in the fMRI can be noted in the hippocampus and amygdala during all phases of the experiment. Further, although a synaptic tag and capture model has been shown in rodent species with spatial learning tasks involving the hippocampus, an amygdala fear conditioning dependent pathway should also be investigated in animal models in the future to uncover the specific mechanisms underlying this paradigm (Burman et al., 2014). Mice can be put into a chamber where they become acclimatized to the surrounding environments and are allowed to learn about the room, this can be done with photographs of different categories (tools or animals) on the walls of the room.

Then rats can be placed in a fear conditioning chamber where a foot shock is delivered at regular intervals, and that chamber would contain a specific category of photographs. Following this, by bringing the rat back to the original chamber after different time periods have passed, memory can be tested for the categories of photographs by measuring the time the rat spends examining both categories of photos. From an object novelty stand point, if a rat has remembered something, they are less likely to be interested in it and will not spend as much time examining it. This can provide behavioral evidence that memory of the previously encoded memory was enhanced by fear conditioning. Following this, the synaptic tag and capture mechanisms can be investigated and LTP can be tested.

The hippocampus can be sliced and tested by inserting measurement electrodes into the CA1 neurons and LTP can be measured after the retroactive memory enhancement (Shires et al., 2012). Further, the authors in the current experiment exclusively measured enhancement of previously encoded memories immediately after the post conditioning phase, after a 6 hour delay, or a 24 hour delay, although this suggests a long term memory alteration, persistence of the retroactive consolidation of this memory for times longer than 24 hours should be tested.

Follow-ups should be conducted up to several weeks to test if this retroactive enhancement of memory is indeed a long term memory effect. Lastly, with the potential implications of this study to stress related neurological disorders, and the ties between fear memories and stress disorders 277

shown by previous studies (Rabinak et al., 2011; Sripada et al., 2012; Morey et al., 2012).it may be beneficial to replicate this study in patients with stress related disorders such as post-traumatic stress disorder, and recording if the retroactive enhancement in memory will still occur.

This can have important implications for the workings of these traumatic disorders and how exactly memory is consolidated adaptively or maladaptively under stressful situations. Despite these shortcomings, the current experiment reviewed greatly contributes to the field of memory research and may have many future implications for how memories are consolidated and the retroactive processes that may sometimes accompany it.

References 1. Ballarini F , Moncada D, Martinez M, Alen N, Viola H (2009) Behavioral tagging is a general mechanism of long-term memory formation. P Natl Acad 34:14599-14604.

2. Burman M, Simmons C, Hughes M, Lei L (2014) Developing and validating trace fear conditioning protocols in C57BL/6 mice. J Neurosci Meth 222: 111-117.

3. Cahill L, McGaugh J (1998) Mechanisms of emotional arousal and lasting declarative memory. Trends Neurosci 7: 294-299.

4. Dimitrova A, Kolb F, Elles H, Maschke M, Gerwig M, Gizewski E, Timmann D (2004) Cerebellar activation during leg withdrawal reflex conditioning: An fMRI study. Clin Neurophysio 4: 849-857.

5. Dongen E, Thielen J, Takashima A, Barth M, FernĂĄndez G, Felmingham K (2012) Sleep Supports Selective Retention of Associative Memories Based on Relevance for Future Utilization. PLoS 8: E43426-E43426.

6. Dunsmoore J, Murty V, Davachi L, Phelps E (2015) Emotional learning selectively and retroactively strengthens memories for related events. Nature: 1-13.

7. Frey U, Morris R (1997) Synaptic Tagging And Long-term Potentiation. Nature 6616: 533-536.

8. LaBar K, Cabeza R (2006) Cognitive Neuroscience Of Emotional Memory. Nature Rev Neurosci: 54-64.

9. Moncada D, Ballarini F, Martinez M, Frey J, Viola H (2011) Identification of transmitter systems and learning tag molecules involved in behavioral tagging during memory formation. P Natl Acad 31:12931-12936.

10. Morey R, Gold A, Labar K, Beall S, Brown V, Haswell C (2012) Amygdala Volume Changes in Posttraumatic Stress Disorder in a Large Case-Controlled Veterans Group. Arch Gen Psychiat 11: 1169-1169.

11. Rabinak C, Angstadt M, Welsh R, Kenndy A, Lyubkin M, Martis B, Phan K (2011) Altered Amygdala Resting-State Functional Connectivity in Post-Traumatic Stress Disorder. Front Psychiat.

12. Shires K, Silva B, Hawthorne J, Morris R, Martin S (2012) Synaptic tagging and capture in the living rat. Nature Communications 3: 1246-1246.

13. Sripada R, King A, Garfinkel S, Wang X, Sripada C, Welsh R, Liberzon I (2012) Altered resting-state amygdala functional connectivity in men with posttraumatic stress disorder. Journal of Psychiatry & Neuroscience 4: 241-249.

14. Subramaniyan S, Hajali V, Scherf T, Sase S, Sialana F, GrĂśger M, Lubec G (2015) Hippocampal receptor complexes paralleling LTP reinforcement in the spatial memory holeboard test in the rat. Behav Brain Res 283: 162-174.

15. Wang S, Redondo R, Morris R (2010) Relevance of synaptic tagging and capture to the persistence of long-term potentiation and everyday spatial memory. P Natl Acad 45: 19537-19542.

16. Wilhelm I, Diekelmann S, Molzow I, Ayoub A, Molle M, Born J (2011) Sleep Selectively Enhances Memory Expected to Be of Future Relevance. J Neurosci 5: 1563-1569.

Received February, 02, 2015; revised March, 03, 2015; accepted April, 02, 2015.

This work was supported by the Human Biology Program at the University of Toronto

Address correspondence to: Sandy Wang, Email: sandy.wang@mail.utoronto.ca Copyright Š 2015 Dr. Bill JU, Neurosciences, Human Biology Program

Gene Down-Regulations and Neuronal Implications of Adderall Induction of the Developing Brain

Ting Ting Wang

Cognitive enhancers, such as amphetamines, commonly marketed as Adderall and methylphenidate, commonly marketed as Ritalin, are government-regulated drugs used to treat Attention Deficit Disorder (ADHD). The following article focuses on the amphetamine, Adderall, which act upon the brain’s monoamines, specifically in the nucleus accumbens, prefrontal cortex and locus coerulus. When amphetamine enters a neuron, the VMAT2 is targeted and reverses its direction of transport where by the stored monoamines in the synaptic vesicular stores and nerve terminals released. ADD will provide increased dopamine release over prolonged time. (Fleckenstein and Hanson, 2003), ADD treatment have shown relatively high success rates in treating youth and adult with ADHD, however, the potential side effects of this drug’s long term and short use requires further elucidation and research. With the increasing prescription rate of ADD, longer treatment time and to younger children, some as young as the age 3, it is more crucial than ever to understand the neurological implications of ADD. There have been physiological side effects associated with the use of Adderall, such as constriction of blood vessels, decrease appetite as well as increased risk of heart attacks. In addition to physiological factors, the short-term and long-term neurological implication, especially within the developing neural system, associated with ADD use is currently undergoing research. There has been some associations between long-term ADD use and increased risks of depression, psychosis as well as anxiety, however, further research needs to be conducted to confirm these findings (Surles et al., 2002) This article focuses on the effects that prolonged ADD induction have on the immediate early gene expression of developing brain using infantile and prepubescent rat models. The key finding of this experiment was the significant down regulation of cfos gene, a gene vital to neuron development, in repeated induction of minimum dosage of Adderall in infantile and prepubertal rat brains. This experiment provides crucial results to understanding the safety of Adderall use especially in young children and infants, a period of time of significant brain development.

Key words: Adderall XR (ADD); c-fos gene expression; infantile (PD10); prepubescent (P24);striatum; liquid chromatography; FOs immunoreactivity(iFOs)

Background In the Article, the researchers concentrates on understanding the effects of repeated exposure to Adderall on infantile and prepubertal brains, using P10 (infantile) and P24 (prepubertal) rats as animal models. Previous to this study, these researchers had conducted a similar study also on infantile and prepubertal brains with methylphenidate, another cognitive enhancer drugs used to treat ADHD and commercially sold as Ritalin. Throughout this study, Adderall is compared to the results of methylphenidate, which gives us a fuller perspective of the effects ADD and its effects on the neural system in comparison to another cognitive enhancer. The experimenters wanted to measure the effect of consistent low dosage of ADD in the rats’ brains and they found this optimal level using liquid chromatography of the rats’ blood. Determining this low level of ADD would make the animal models more relevant to the therapeutic treatment of children. A shortcoming that most experiments have is the use of high doses of neurochemicals, which could alter the results. Another control is the measuring of blood levels of amphetamine in immature rats to ensure that they are similar to the levels found in children treated with ADD. They then quantified the level of c-Fos gene expression in P10 and P24 pups with the initial dose of ADD in striatum, frontal/cingulate parietal and piriform cortex by FOs immunoreactivity. They then quantified the level of c-Fos gene expression again after 14 days of repeated optimal dose of ADD induction. (Figure 2) In the P24 pups, the experimenter found significant reduction of c-fos in the dorsal striatum and in cerebral cortex, cingulate cortex after 14 days, however there was

a significant increase in c-fos 24 hours after the initial ADD dosage. In P10 pups, there was also a significant reduction of c-fos in the striatal and moderate reduction in the cingulate cortex. Unlike the P24 pups, in P10 pups, there was no accumulation of c-fos 24 hours after initial ADD dosage. This study showed the downregulation on c-fos gene expression with the continuous ADD induction in various areas of the brain especially in infantile (P10) rats. These results could potentially indicate the danger of prescribing ADD to children, some as young as age 3. However further studies need to be conducted to understand the full extent of ADD’s effect on the developmental brain and all the genes affected.

Research Overview

Summary of Major Results

This study’s objective was to determine whether the cognitive stimulant Adderall XR (ADD) induced any changes in the cfos, fosB and arc gene expression in different areas of the developing brain. This research article provides crucial information due to the recent increasing rates of prescribed ADD to more and younger patients, some as young as 3 years old, for a longer duration of time. This precarious treatment trend is proceeding in absence of long-term safety studies and many dangers of long-term ADD use to the developing brain could be overlooked.

First the experimenters determined an optimal dosage of ADD, which was identified as the minimal effective dosage of ADD. Wakefulness and increase dopamine and 278

norepinephrine efflux PFC constitutes an effective dose of ADD, studied in the works of (Berridge and Stalnaker, 2002). The optimum dosage of ADD for each rat model is determined through the blood levels of amphetamine determined by liquid chromatography-mass spectrometry.

Then two groups of rat models, infantile (10 days postnatal; PD10) and prepubertal mice (24 days postnatal) were induced with this optimal dosage of ADD. From article (Andersen SL, 2003) the experimenters have established that rats at PD10 should be developmentally relevant to children aged 3, which the earliest age children are prescribed ADD. The expression of immediate early genes in different areas of the brain tissue is examined in these rat models to localize the effects of ADD. The main genes particularly sensitive to repeated 14 days of induction of low doses of ADD in immature rat brain is the cfos gene expression. There was a significant c-fos gene down regulation found in both infantile and prepubescent rats, but with infantile (PD10) rats striatum and cortex affected more. In previous research conducted by these experimenters (Renthal et al., 2008) found similar effects in adult rat models however, to a lesser extent, which suggests that the immature rat brain is more sensitive to ADD stimulation.

This result is notable for two main reason, first reason being the significant effects on gene expression of amphetamine in the rat models are comparable to the therapeutic range reported in children and second, the clearance of amphetamine is much faster in rats than in children. Therefore, the possible adverse effects of repeated ADD treatment in rats are relevant to the ADD effects in children. However, there were some differences between the rat’s reaction to ADD vs. the children’s key differences in the levels of ADD was much higher and lasted longer in the blood of children compared to rats, and there was also a less down regulation of the gene cfos in children in comparison to infantile rats.

Figure 1. ADD induced FOS-ir in PD 24 and PD10 in the striatal region of the rat brain. Significant difference is distribution of striatal FOS-ir for c-fos in 2 ages of rats. The diagram on the right is of saline control to show no increase in FOS-ir of c-fos in the brain.

Conclusions and Discussion The results found in this experiment suggested that clinically relevant blood levels of ADD could be potent enough to induce a down-regulation of the c-fos gene expression in children after a brief period of repeated exposure. This seems especially relevant to the developmental brain due to the numerous and high gene expression at this time. Therefore, a disruption in the gene expression during this critical developmental period 279

Figure2. ADD induction and cfos expression downregulation in cerebral cortex of P24 rats. A) C) & E) illustrates the response of cfos with a single stimulation of ADD in parietal, cingulate and piriform cortex. B), D) & F) shows a reduction in FOS-ir imaging following 13 days of oral ADD

could lead to alterations in cellular responsiveness and synaptic connectivity.( Robinson TE and Kolb B, 2004) The main concern surrounding the ADD treatment in children is the likelihood of predisposing them to later substance abuse, (Berman SM et al., 2008) however, not enough uniform concrete evidence to make this statement indisputable. The rodent model showed that there is a high chance of ADD treatment impacting the degree of brain maturation in children, which may dictate behavioral alterations in later stages of life. (Carrey N et al., 2009) The data from this experiment implies that such changes may occur in immature human brain, however, there are no scientific data of such gene down-regulation in humans due to ethical reason. Therefore, the application of this experiment’s results is inferential and not definite. However, additional study on adult primates reported that low doses of amphetamine appear to be neurotoxic and striatal levels of dopamine transporters and VMAT2 were all weakened. Also in a study by Diaz Heijtz et al. (2003) we see that low doses of amphetamine induced increase prefontal cortical neurons branching and length, however, the behavioral and brain’s structural implications of these results need further research. This suggests that the adverse neural effects of ADD extend beyond the developmental brain and gene expression.

Conclusions

In conclusion, the experimenters’ focus was to create an ADD drug induction model that would provide relevant biological responses, such as immediate early gene expression, to children response to ADD. With the induction of these clinically relevant blood levels of stimulants, the experimenters found that the expression of cfos was very sensitive to repeated low doses of ADD. The use low, minimally effective, doses of ADD in this experiments, along with the ensuring the blood levels of amphetamine remains clinically relevant, ensures that this data would simulate the possible effects of extended ADD treatment in children.

Criticisms and Future Directions

Further elucidations about the behavioral and neural implications of c-fos gene down regulation would be useful in this study. Therefore, further extensions of this experiment could include the behavioral observations as well brain analysis of adult mice models who had extended ADD induction during their developmental period. Also, perhaps the use of rats as the animal model doesn’t provide the best relevancy to humans, in comparison to primates, used in (Diaz Heijtz et al., 2003) experiment. Also further insight on the behavioral implications of ADD could be gained from meta-analysis studies on behavioral differences between long-term ADD users, starting from various ages, versus non-ADD users, aka. Controls. There is also a high rate of Adderall abused for recreational as well as study purposes, particularly among college students, which arises the question of ADD’s affect among healthy individuals. (Franke et al., 2014) The brain (Criticism and Future Directions continued…) morphology and functioning of these different participant groups can be analyzed through various techniques such as fMRI, CT, or various other brain scans, to provide a more complete picture.

The experimenters had provided us with proof of c-fos gene down-regulation in this experiment, but did not clarify the effects of this down-regulation and what makes it significant. Additional evidence needs to clarify the role of cfos in developmental brain and how its down-regulation could make the brain more prone to problems like neuron toxicity. Finally, from the results of this paper should be analyzed and compared to other papers such as (Diaz Heijtz et al., 2003) to determine if there’s any relations between the ADD-induced downregulation of cfos and other ADD induced changes, such as the structural changes in neurons. Also whether the ADD-induced down-regulation of cfos expression and other ADD-induced changes are permanent or reversible, remains to be determined.

6. Surles, L.K., May, H.J. & Garry, J.P.Adderall-Induced Psychosis in an Adolescent. JABFM 15, 498-500 (2002) 7. Lakhan,S.E.& Kirchgessner A. Prescription stimulants in individuals with and without attention deficit hyperactivity disorder: misuse, cognitive impact, and adverse effects. Brain and Behavior 2(5), 661-667 (2012) 8. Robinson TE, Kolb B (2004) Structural plasticity associated with exposure to drugs of abuse. Neuropharmacology 47(Suppl 1):33–46.

9. Hughes P, Dragunow M (1995) Induction of immediate-early genes and the control of neurotransmitter-regulated gene expression within the nervous system. Pharmacol Rev 47:133–178.

10. Andersen SL (2003) Trajectories of brain development: point of vulnerability or window of opportunity? Neurosci Biobehav Rev 27:3–18.

11. Carrey N, Chase T, Allen J, Wilkinson M (2009) Psychostimulant- induced developmental neuroadaptation: implications for the treat- ment of ADHD. In: Attention deficit hyperactivity disorder (ADHD). New York, NY: Nova Science Publishers.

12. Berman SM, Kuczenski R, McCracken JT, London ED (2008) Potential adverse effects of amphetamine treatment on brain and behavior: a review. Mol Psychiatry 14:123–142.

13. Fleckenstein AE, Hanson GR (2003) Impact of psychostimulants on vesicular monoamine transporter function. Eur J Pharmacol 479: 283–289.

References 1. Berridge CW, Stalnaker TA (2002) Relationship between low-dose amphetamine-induced arousal and extracellular norepinephrine and dopamine levels within prefrontal cortex.

Synapse 46:140–149.

2. Renthal W, Carle TL, Maze I, Covington HE, Truong HT, Alibhai I, Kumar A, Montgomery RL, Olson EL, Nestler EJ (2008) 􏰂FosB mediates epigenetic desensitization of the c-fos gene after chronic amphetamine exposure. J Neurosci 28:7344–7349.

3. Diaz Heijtz R, Kolb B, Forssberg H (2003) Can a therapeutic dose of amphetamine during pre-adolescence modify the pattern of syn- aptic organization in the brain? Eur J Neurosci 18:3394–3399.

4. Franke, A.G., Bagusat, C., Sebastian, R., Engel, A. & Lieb, K. Substances used and prevalence rates of pharmacological cognitive enhancement among healthy subjects.

Springer 264, 83-90 (2014) 5. Steiner, H. & Waes, V. Addiction-Related Gene Regulation: Risks of Exposure to Cognitive Enhancers vs. Other Psychostimulants. Prog.Neurobiol 100, 60-80 (2012)

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Chronic Coffee and Caffeine Ingestion Effects on the Cognitive Function and Antioxidant System of Rat Brains

Vonny Wong

Due to its worldwide consumption, many seek to determine whether brewed coffee has beneficial or adverse effects on the human body. More specifically, researchers seek to understand its effects on the brain as it’s one of the most vital organs for human survival. The effects of caffeine on cognitive function has been wellcharacterized to be protective in nature as it makes the endogenous antioxidant system work more efficiently to prevent brain tissue from oxidative damage. Renata and colleagues aim to determine whether brewed coffee show similar advantages with respect to cognitive functioning in the aging population as coffee tends to be ingested in a chronic-fashion, over the course of a person’s lifespan. The authors also believe it is important to ensure that there are no adverse effects accompanying the advantages, hence other physiological and behavioral features were tested also.

Key words: endogenous antioxidant system, reactive oxygen species (ROS), glutathione, caffeine, coffee, long-term memory (LTM), cognitive functioning, adenosine receptors The Known Effects of Caffeine and what is to be Learned About Coffee Being one of the most vital organs for human survival, the brain intakes approximately a fifth of the body’s total oxygen supply. Despite this astounding fact, it receives relatively low levels of endogenous antioxidants as compared to other tissues. These two factors in junction allow for increasing numbers of reactive oxygen species (ROS) to take up residence among neurons. This ultimately leads to the gradual accumulation of oxidative damage with age, making the brain highly susceptible to deterioration of cells and therefore decreases in cognitive performance.

Coffee contains numerous substances and many of their biological effects have been examined independently. Without a doubt, the most widely examined component of brewed coffee is caffeine. In studies such as that conducted by Arendash et al. (2006), caffeine was found to benefit long-term memory of test subjects without causing unwanted side effects in sensory and motor functions. Despite the overwhelming amount of knowledge that is known about caffeine and other constituents of coffee, little is known about coffee’s overall effects on the body as interactions between components can cause fundamentally different outcomes. As a result, this study seeks to understand the consequences of chronic coffee and caffeine consumption on the endogenous antioxidant system of the brain and in turn, their effects on cognitive performance. By raising and sacrificing rats solely for the purpose of the experiment, the study is able to more accurately depict the effects of coffee consumption over the experimental life of an animal, thereby better reflecting human’s experience with chronic coffee consumption. If the effects of chronic brewed coffee consumption and caffeine consumption coincide, it is believed that little change resulted from the interactions of the components, hence allowing benefits elicited from brewed coffee to occur as a result of similar pathways as that of caffeine in the body. On the basis of previous studies, the authors hypothesized and sought to support the idea that coffee and caffeine would act as a protective food for 281

the brain and thereby slow cognitive decline. It is also important to note that the researchers placed effort into testing body weight, sensory and motor functions in order to ensure that no adverse side effects were paired with coffee’s advantages.

Though not examined in detail in the paper, it was alluded to that caffeine has a tendency to compete for the binding of adenosine receptors. As a consequence of these antagonistic effects, adenosine is prevented from binding to its receptors and therefore altering the genesis of free radicals in neurons. With the minimized production of free radicals, the brain is rescued from oxidative damage and freed from neuronal degeneration. To make the paper in review more complete, the authors could replicate studies done by other groups, such as those of Nobre Jr. and colleagues, to demonstrate that coffee’s benefits are by way of the adenosine A2 receptors and their interactions with the body’s antioxidant system.

Research Overview

Experimental Outcomes

To better depict the effects of prolonged coffee consumption over the course of a human’s lifespan, rats were breed and weaned solely for the purpose of this study. These newborn rats were then randomly selected and divided into five groups, each subjected to different diets: a control diet, a 3% brewed coffee diet, a 6% brewed coffee diet, a 0.04% caffeine diet and a 0.08% caffeine diet.

The rats’ food consumption was recorded every few days, whereas body-weight was recorded weekly. These assessment were done so throughout the rats’ experimental life and comparisons between groups showed that the diets had no effects on how much the rats ate, nor did they have significant changes in weight. A previous study that was conducted by Silva-Oliverira et al. (2010) also confirms the results found in this study.

When the rats were 90 days of age, they partook in two behavior tasks: (1) an open field task to observe their exploratory behavior and (2) a novel object preference task to observe their short-term and long-term memory.

It was shown that coffee and caffeine supplemented diets had no effect on their exploratory behavior as it did not increase nor decrease the number of crossings and rearings in the open field test. Additionally, rats in all conditions exhibited a decrease in exploration on the second day of testing, a phenomenon accounted for by habituation. As illustrated in Figure 1, neither coffee nor caffeine had an effect during the novel performance task administered 90 minutes after, but coffee/caffeine conditions did have better discrimination during the test given 24 hours after. Despite the difference in opinion about the mechanisms by which the effects are caused, Costa et al. (2008) found similar results in regards to caffeine’s effects on rats’ performance during recognition memory tasks (but believed it was through increases in BDNF and TrkB immunocontents in the brain). Costa and colleagues injected rats with daily doses of caffeine that were equivalent to about 3 cups a day and subjected them to recognition tasks 15 minutes, 90 minutes and 24 hours post-injections. In all the tests given, rats improved in terms of their discrimination indexes.

Other studies that were performed by Costa et al.

(2008) also validate the major findings of the review paper as aging populations of mice which received caffeine supplements perform similar to adult mice of younger ages and less like their aging counterparts of whom did not receive treatment.

10 days after the administration of the behavioral tasks, the rats’ lives were terminated and their brains were tested for indicators of oxidative damage. Analysis of brain matter revealed enhancement changes to the antioxidant system as depicted in Table 1, such as increased concentration of potent endogenous antioxidants (mainly glutathione) and increased activity of antioxidant enzymes (glutathione reductase and superoxide dismutase). Ferrari C KB et al. (2003) comprised a list of foods they believed to have beneficial effects for the human body, one of which was caffeine/coffee. They listed caffeine/coffee as a positive substance for consumption as it provides protection from DNA oxidation by way of boosting the antioxidant system, hence corroborating with the data found by Renata and colleagues. With respect to the effects of caffeine on the antioxidant system, Noschang et al. (2009) and Desvasagayam T.P.A et al (1996) found that including caffeine to the rats’ diets did cause an increase in the activity levels of antioxidant enzymes and reduced lipid peroxidation,

Figure 1. Data from the novel object preference task of the different groups (where CD represents the control diet, 3%Co represents the group that received a 3% brewed coffee supplement, 6%Co represents the group that received a 6% brewed coffee supplement, 0.04%Ca represents the group that received a 0.04% caffeine supplement and 0.08%Ca represents the group that received a 0.08% caffeine supplement). Graph A) displays the results of the sample phase, graph B) displays the results of the test phase after 90 minutes and graph C) displays the results from the test phase that was administered 24 hours after. The discrimination index was derived by dividing the time spent by each animal exploring the novel object by the total time spent on exploring both objects.

respectively, just as shown by the authors. However, another important finding was that this increase was only observed in non-stressed animal models and not in stressed animal models, hence alluding to the idea of environment also playing a role in caffeine’s beneficial effects (for the scope of this review, this point will not be elaborated on further).

Interpretation of the Results As with all experiments, authors aim to gain observable data that can be generalized to other populations outside of those used in the study itself. In addition to breeding rats exclusively for the purpose of this study to mimic the patterns of chronic coffee consumption in a human’s lifespan, the researchers also attempted to match coffee and caffeine dosage levels with a typical human’s daily intake. The experimenters based their estimates on the findings of Van Gelder et al. (2007) of whom concluded that drinking approximately three

Table 1. Measurements from the antioxidant system of the brain obtained from the sacrificed test rats. The levels of the following endogenous antioxidant system components were examined: lipid peroxidation and reduced glutathione content (TBARS), glutathione (GSH), glutathione peroxidase (GPx), glutathione reductase (GR) and superoxide dismutase (SOD). The labels used to represent the different groups are the same as those used for Figure 1: CD represents the control diet, 3%Co represents the group that received a 3% brewed coffee supplement, 6%Co represents the group that received a 6% brewed coffee supplement, 0.04%Ca represents the group that received a 0.04% caffeine supplement and 0.08%Ca represents the group that received a 0.08% caffeine supplement.

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cups of coffee per day was the best suited protection from elderly cognitive decline. However, as explained in the review paper, several other factors needed to be considered as the test subjects used were rats whereas the population of interest for extrapolation are humans. Firstly, the prescribed amounts were within the limited boundaries in which physiological and metabolic effects could be observed without causing toxic effects in the rats. The second consideration revolved around the idea that metabolism occurs faster in rats than in humans. After these considerations, alterations were made which resulted in the following five conditions: a control diet, a 3% brewed coffee diet, a 6% brewed coffee diet, a 0.04% caffeine diet and a 0.08% caffeine diet.

Records gathered from the observations of food intake and body weight exhibited no significant changes. Moreover, through the open field task, no modifications to sensory and motor functions were seen and habituation was present during the second day of testing. The occurrence of habituation indicates normal non-associative processes. All these findings confirmed the notion that there appears to be no adverse effects of coffee in these aspects that accompanies its benefits.

Through the novel object preference task, it was witnessed that neither caffeine nor coffee had an effect during the test completed 90 minutes after, but did allow for better discrimination during the test performed 24 hours later. These test results specify that though there were no enhancements in shortterm memory, long-term memory was improved.

Through fine examination of the brains extracted from the test subjects, considerable changes were made to the antioxidant system. Measurements from the study displayed a decrease in lipid peroxidation of the membranes, increase concentration of antioxidants like glutathione, and increased activity levels of the associated enzymes (glutathione reductase and superoxide dismutase). These alterations are believed to be the fundamental basis for reductions in cognitive decline as brain matter is protected from oxidation. Due to the fact that both the caffeine and coffee supplemented groups showed similar results, the authors were led to believe that little interactions between coffee’s individual constituents occurred, hence allowing the integrity of caffeine’s beneficial effects to work in brewed coffee.

Significance of the Author’s Research

This area of research is most definitely not new but the authors of the review paper used a more holistic approach, aiming to understand coffee’s overall effects on the body as reactions between components may result in adjustments of the typical downstream cascades. With this perspective, the paper’s significance relies on it being a source of incremental advancement on the topic because the conclusions drawn from their experiments were not novel discoveries, but built upon previous research in the view of practical applications (as coffee is the beverage that is consumed, not its individual components). In looking for aids to slow the declines in function associated with aging, it is without a doubt more practice to 283

encourage seniors to consume a certain quantity of brewed coffee per day, as compared to providing them with caffeine injection which would be ineffective in a cost and efficiency aspect. As a result, through the lens of practical application, we can see the profound insight this paper provides in correlating the effects of caffeine and coffee in the human body with respect to cognitive function.

Future Developments

Overall, the work presented by the authors were very well documented as they took into account several aspects of the subject’s health and results were validated using previous research from other groups.

Renata and colleagues, the authors of this review paper were extensive in their research as they not only demonstrated the correlation of effects between caffeine and coffee on cognitive functioning, but also, provided others tests to demonstrate the lack of associated adverse effects (whether it be in terms of physiology like weight or behavior such as exploration). Nevertheless, it is believed that the authors would have benefited from elaborating on the role of adenosine receptors in this system as mentioned in the paper, but not further explain or supported. As briefly mentioned, A2A adenosine receptors, not A1 receptors were the key players in involved.

To gather some possible future experiments that can be performed to fill this gap in knowledge, the authors can look to other research groups and replicate their experiments to validate their findings and support the idea that A2A receptors are the pathway which dictate the rescue of the brain from declines in function.

In an experiment performed by Cunha and colleagues, a group of rats mimicking an aging population was created using beta-amyloid injections which caused synaptic deterioration and hence, neuronal death.

After some time, rats were treated with selective A2A receptor antagonists such as SCH58261 or KW6002.

These particular antagonists were chosen because they exhibited no peripheral effects. Using these techniques, it was shown that the blocking of A2A receptors was indeed beneficial in reducing cognitive disruption that was caused by the induced neuronal death. It can be noted that the same experimental method was used by Dall’Igna and colleagues with the exception of the task they chose to administer to test for memory abilities.

The last procedure I recommend to be the next step for the authors of the chosen review paper is one that follows suit to that of Nobre Jr. and colleagues. This group of researchers decided to inject caffeine variants, of which were known to be A2A receptor antagonists, into cytotoxic rat mesencephalic cells. Through careful monitoring, these variants were observed to substantially increase the number of viable cells by way of returning nitrate levels to homeostatic levels, thereby decreasing oxidative stress and free radical production. In my personal option, this technique is of more value to Renata and colleagues because it makes reference to both the A2A receptor antagonists and the antioxidant system in the body, giving a more complete representation of the interconnections between all the systems involved.

References 1. Arendash G.W. et al. (2006) Caffeine protects Alzheimer’s mice against cognitive impairment and reduces brain ß-amyloid production. Neuroscience 142:941-952.

2. Costa M.S. et al. (2008) Caffeine improves adult mice performance in the object recognition task and increases BDNF and TrkB independent on phospho-CREB immunocontent in the hippocampus. Neurochem Int 53:89-94.

3. Costa M.S. et al. (2008) Caffeine prevents age-associated recognition memory decline and changes brain-derived neurotrophic factor and tirosine kinase receptor (TrkB) content in mice. Neuroscience 153:1071-1078.

4. Cunha G.M.A. et al. (2008) Adenosine A 2A receptor blockade prevents memory dysfunction caused by β-amyloid peptides but not by scopolamine or MK-801. Exp Neurol 210:776-781.

5. Dall’Igna O.P. et al. (2007) Caffeine and adenosine A 2a receptor antagonists prevent β-amyloid (25-35)-induced cognitive deficits in mice. Experim Neurol 203:241-245.

6. Devasagayam T. PA. (1996) Caffeine as an antioxidant: inhibition of lipid peroxidation induced by reactive oxygen species. Biochim Biophys Acta 1282:63-70.

7. Ferrari C KB et al. (2003) Biochemical pharmacology of functional foods and prevention of chronic diseases of aging.

Biomed Pharmacother 57:251-260.

8. Nobre Jr. H.V. et al. (2010) Caffeine and CSC, adenosine A2A antagonists, offer neuroprotection against 6-OHDAinduced neurotoxicity in rat mesencephalic cells. Nerochem Int 56:51-58.

9. Noschang et al. (2009) Interactions Between Chronic Stress and Chronic Consumption of Caffeine on the Enzymatic Antioxidant System. Neurochem Res 34:1568-1574.

10. Silva-Oliverira et al. (2010) Effect of Coffee on Chemical Hepatocarcinogenesis in Rats. Nutr Cancer 62:336-342.

11. Van Gelder B.M. et al. (2007) Coffee consumption is inversely associated with cognitive decline in elderly European men: the FINE StudyCoffee consumption and cognitive decline. Eur J Clin Nutr 61:226.

Received February 8, 2011; revised May 10, 2011; accepted June 8, 2011.

*Corresponding author at: Faculdade de Farmacia, Universidade Federal de Minas Gerais. Departamento de Alimentos-Sala 2099-B3, Av. Antonio Carlos, 6627, Pampulha, 31270-901-Belo Horizonte, MG, Brazil. Tel.: +55 31 3409 6917; fax: +55 31 3409 6989. Email address: tmoraes@ufmg.br (T. Moraes-Santos).

Copyright © 2015 Vonny Wong, Neurosciences, Human Biology Program Submission date: 06-Apr-2015 2:10 AM EDT

284

Engraftment of Stem Cell Derived Dopamine Neurons offers a Possible Regenerative Treatment for Parkinson’s disease

Jiawei Zhang

Kriks, et al (2011) investigated the use of human pluripotent stem cells in regenerative cell therapy as a potential treatment for Parkinson’s disease. The floor-plate (FP) and rosette procedures were used to differentiate stem cells into specialized dopamine neurons. The in vivo performance of these neurons was assessed in three different animal models. Molecular profiling techniques were also used to monitor overgrowth and neuronal development. Results indicate high survival rates and low overgrowth in all three models.

The major findings and basic procedures of this study will be summarized in this literature review. The significance of the conclusions along with criticisms of the study will be discussed further. Possible future directions and alternative methods derived from other studies will also be examined in terms of improving the efficacy of the experimental procedure.

Key words: Parkinson’s disease; Dopamine; pluripotent stem cells; engraftment; midbrain; neurodegeneration Background In 1817, James Parkinson published the first documented cases of Parkinson’s disease (PD) in his “essay on the shaking palsy”. (Jankovic, 2008) It was later discovered that patients who were diagnosed with this disorder suffered from a loss of dopaminergic (DA) neurons in the substantia nigra. The results of several recent studies have shown that both genetic and environmental factors play a role in causing neuronal death. 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) models have been important in elucidating the cellular pathways involved in the degeneration of DA neurons. (Dauer, Przedborski, 2003) More specifically, oxidative stress, an accumulation of mutated proteins and a deficit in the autophagy–lysosomal pathway has been suggested as major contributors to the degeneration of these neurons. (Pan, Kondo, Le, & Jankovic, 2008; Jankovic, 2008) Patients diagnosed with Parkinson’s disease can be characterized by several deficits that impact motor function. Cardinal signs of PD include tremors at rest, rigidity/stiffness, Bradykinesia, hypokenesia, akinesia and also a loss of postural reflexes. (Jankovic, 2008) In addition, secondary motor symptoms and nonmotor symptoms may also appear. Everyday functions can be affected to variable degrees depending on the stage of the progressive disorder.

Current therapies for PD are useful in alleviating symptoms rather than preventing DA neuron degradation.

(Li & Zhou, 2013) Neuroprotective treatments are inadequate due to limited research. In a recent study conducted by Kriks et al. (2011), human pluripotent stem cells (PSCs) were examined as a possible regenerative treatment for PD. The significance of PSCs in cell therapy was investigated by evaluating the regenerative performance of PSCs in several animal models.

Both the floor-plate (FP) and rosette protocols were used to derive DA neurons from human PSCs cells in the first part of the experiment. (Kriks et al., 2011) The expression of FP precursors is important to stem cell specialization/ differentiation. The expression of CHIR precursors was used to activate canonical Wnt signalling. (Kriks et al., 2011) The activation of the Wnt pathway then allowed for the induction of roof285

plate (RP) marker LMX1A to promote the specialization of PSCs into DA neurons in vitro. (Joksimovic et al., 2009) PSCs-derived DA neurons were available for engraftment by day 25 after the FP/rosette procedures.

For part two of the study, the PSCs-derived DA neurons were inserted into the midbrains of three host animal populations, each of which was a model for Parkinson’s. The control group was composed of nonlesioned mature rats. The two experimental groups were composed of 6-hydroxy-dopamine (6-OHDA) lesioned parkinsonian rats and MPTP lesioned adult rhesus monkeys. PSCs-derived DA neurons experienced high survival rates after engraftment in all three animal models with no signs of abnormal overgrowth.

Further analysis also illustrated a rescue of amphetamineinduced rotation behaviour in Parkinsonian rats through akinesia and forelimb tests (i.e. stepping test and cylinder test). The study showed promising results for future applications of stem cell based therapies for treating other neurodegenerative disorders in addition to PD. Research Overview

Summary of Major Results

Section 1: Derivation of DA neurons from Human PSCs The floor-plate procedure (a modified dual-SMAD inhibition protocol) was used to induce the expression of DA neuron specific FP and RP markers in developing PSCs through the activation of the canonical Wnt signalling pathway via CHIR precursors. (Kriks et al., 2011) Wnt activation induces the expression of FOXA2 (FP marker) and LMX1A (RP marker) important for the conversion of PSCs to DA neurons. (Ferri et al., 2007) There were three treatment groups: LSB/S/ F8/CHIR (midbrain DA), LSB/S/F8 (hypothalamic) and forebrain LSB cell cultures.

The results indicated that out of the three treatment conditions, only the marker profiles for Tyrosine Hydroxylase positive (TH+) cells in the LSB/S/F8/ CHIR group matched midbrain DA neurons fates.

The in vitro and in vivo properties of the PSC-derived DA

neurons were also examined by comparing the FP and the rosette protocols. The results indicated that the FP method produced a higher percentage of TH+ neurons (expressing FOXA2, LMX1A and NURR1) consistent with the findings of the Cossette et al. (2004) study.

These cells were capable of long-term survival in vitro compared to the rosette method (refer to Figure 1). In addition, the FP derived neurons exhibited significantly higher levels of DA, DOPAC and HVA compared to the cells derived by the rosette method. (Kriks et al., 2011) These FP derived DA neurons also showed a higher expression of neuronal markers such as synapsin, DA transporters, GIRK2 channels, etc. (Kriks et al., 2011)

Figure 1. FP-derived TH+ cells exhibit significantly higher percentages of FOXA2, LMX1A and NURR1 relative to the Rosette-derived cells. Both protocols co-express NURR1 with TH, whereas only FP-derived cells co-express FOXA2 and LMX1A with TH. Source: (Kriks et al., 2011)

Conclusions and Discussion

Conclusion Figure 1. FP-derived TH+ cells exhibit significantly higher percentages of FOXA2, LMX1A and NURR1 relative to the Rosette-derived cells. Both protocols co-express NURR1 with TH, whereas only FP-derived cells co-express FOXA2 and LMX1A with TH. Source: (Kriks et al., 2011)

Section 2: Viability of PSC-derived DA neurons in vivo The differentiated PSC-derived DA neurons were inserted into the midbrains of three model animal populations to test for neuronal sustainability in vivo.

The engraftment of day 25 PSC-derived DA neurons into non-lesioned mature rats (control group) showed high survival rates even six weeks. No abnormal overgrowth was observed. (Kriks et al., 2011) In addition, the results also indicated high survival rates without overgrowth after the cells were injected into NOD-SCID IL2Rgc null, 6-OHDA lesioned parkinsonian hosts. (Kriks et al., 2011) The development of PSC-derived DA neurons showed high sustainability even four and a half months after transplantation.

(Kriks et al., 2011) The results also indicated a full rescue of amphetamine-induced rotation behavior in parkinsonian rats transplanted with FP derived DA neurons in comparison to rats with the rosette-derived neurons (refer to Figure 2). Parkinsonian hosts with FP derived neurons showed improved scores on motor tasks. Unexpectedly the results indicated that there was excessive overgrowth with rosette-derived DA grafts.

Lastly as a representative model for the human brain, day 25 FP derived DA neurons were engrafted into two adult MPTP lesioned rhesus monkeys. Cells were injected with DA precursors into the caudate and putamen within the basal ganglia on both sides.

(Kriks et al., 2011) One side of the brain was also injected with Green fluorescent protein (GFP) to track the development of the FP derived neurons. (Kriks et al., 2011) The results showed no overgrowth and high DA neuron survival rates based on GFP expression just after one month. (Kriks et al., 2011)

In conclusion, all three animal models showed high PSC-derived DA neuron survival in vivo. Based on the major findings of the study, FP derived cells have higher efficacy in comparison to rosette derived cells.

The experimenters presented a novel protocol to derive midbrain DA neurons from human pluripotent stem cells. The floor-plate method was proven to be an efficient technique in terms of producing a viable source of DA neurons in vivo without overgrowth.

More importantly, the results of the study established a basis for developing sustainable supplies of TH+ neurons that can be used for engraftment in human models. Stem cell research holds a promising future for finding treatments for Parkinsonâ&#x20AC;&#x2122;s as well as other neurodegenerative disorders.

Discussion

Overgrowth is a major concern in the field of stem cell research. Cellular differentiation needs to be tightly controlled to avoid abnormal proliferation and inappropriate development. (Ferrari, Sanchez-Pernaute, Lee, Studer, & Isacson, 2006 ) In this case, excessive overgrowth of PSC-derived DA neurons in vivo will have major consequences on the efficacy and effectiveness of PSC-based treatments of Parkinsonâ&#x20AC;&#x2122;s disease.

To combat the issue of overgrowth, the experimenters made sure only cells at an appropriate stage were chosen for engraftment based on previous transplantation studies such as the one conducted by Olanow et al. (1996). The experimenters proposed that only PSC-derived cells at the exit stage, expressing the NURR1 marker would be acceptable for grafting.

The results of the study indicated a full recovery of amphetamine-induced rotation behavior in FP-derived grafts. However, the rosette-derived grafts on the other hand did not show any improvements, substantial overgrowth was seen instead.

Consistent with the result of a previous study, extensive overgrowth associated with the rosette protocol was likely induced by long periods of proliferation (4.5 months). (Hargus et al., 2010) Other factors 286

considered by the experimenters include impure cell samples with excess precursors. In agreement with the hypothesis that anterior neuroectodermal cells promoted overgrowth, the rosette-derived grafts had high levels of FOXG1, which induced the expression of astroglial cells. (Kriks et al., 2011) Criticism and Future Directions One major problem associated with the engraftment of PSCs would be the difficulty in determining a highly viable cell culture for tissue transplantation into the host. Although the use of the NURR1 markers in determining the appropriate stage of engraftment in this study was proven to be effective, we should consider other possible methods.

An alternative method that can be incorporated into the current experiment would be to implement Sundberg et al., (2013)’s experimental design. PSC-derived DA neurons rich in NCAM(+)/CD29(low) factors can be used for tissue engraftments. DA neurons rich in NCAM(+)/CD29(low) expresses more LMX1A and FOXA2 mRNA. (Sundberg et al., 2013) The finding of this study ensured that NCAM(+)/CD29(low) neurons were responsible for the recovery of motor functions in parkinsonian rodents, specifically in 6-OHDA lesioned rats. (Sundberg et al., 2013) DA neurons with these specific factors were found to have even higher survival rates (up to a year) in the brains of the hosts. Incorporating this method with the NURR1 labeling technique will ensure a viable population of cells for engraftment.

Predicting the efficacy of PSC-derived DA neurons was another challenge presented in this study. In 2013, Salti et al. established a new protocol for neuronal cell labelling. Cellular profile markers (i.e. markers for populations of cells abundant in neuronal progenitors, floor-plate and Wnt signalling factors) can be labelled to increase precision. (Salti et al., 2013) This method can be implemented into the current study. Discrete profiles of specific cell cultures can be obtained to predict their DA neuronal fate development in vitro. The progress of these cells can also be tracked after tissue transplantation in vivo. Any abnormal changes (i.e. malignant outgrowth or differentiation of these cells into non-neuronal cells) in the cell’s development can be tracked and the overall efficacy of the cells can also be predicted based on these profiles. (Salti et al., 2013) One way to further examine the variability in efficiency of human embryonic and pluripotent stem cells (ESC/iPSC) is to assess their potency to differentiate.

(Boulting et al., 2011) In a 2011 study conducted by Boulting, et al., the researchers tested different sets of ESCs and iPSCs to evaluate the differences in cellular potency. (Boulting et al., 2011) Results indicated differences in efficacy, capacity and potential for neuronal differentiation between the various cell lines. (Boulting et al., 2011) By implicating this test in the current study, we can examine which strains of human-PSC are most efficient at differentiating into DA neurons.

Lastly, the findings of the current study indicated that DA neurons in the substantia nigra pars compacta (SNPC) display a different electrophysiological phenotype compared to other neurons. These neurons fire 287

spontaneously at a lower range of frequencies. Based on past research, it is evident that the lower firing rates are indicative of sub-threshold potentials. (Guzman, Sanchez-Padilla, Chan & Surmeier, 2009) The FP-derived DA neurons had the same electrophysiological phenotype. Further research is necessary to determine if whether these FP-derived DA neurons will exhibit all features of SNPC DA neurons or differentiate into populations with distinctive properties.

References 1. Boulting GL, et al. A functionally characterized test set of human induced pluripotent stem cells. Nat Biotechnol. 29, 279-86 (2011) 2. Cossette, M., Parent, A., Lévesque, D. Tyrosine hydroxylase-positive neurons intrinsic to the human striatum express the transcription factor Nurr1. Eur J Neurosci. 20, 2089-95 (2004) 3. Dauer, W., Przedborski, S. Parkinson’s disease: mechanisms and models. Neuron. 39, 889-909 (2003) 4. Ferrari, D., Sanchez-Pernaute, R., Lee, H., Studer, L. & Isacson, O. Transplanted dopamine neurons derived from primate ES cells preferentially innervate DARPP-32 striatal progenitors within the graft. Eur. J. Neurosci. 24, 1885–1896 (2006) 5. Ferri, A. L. et al. Foxa1 and Foxa2 regulate multiple phases of midbrain dopaminergic neuron development in a dosagedependent manner. Development 134, 2761–2769 (2007) 6. Guzman, J. N., Sanchez-Padilla, J., Chan, C. S. & Surmeier, D. J. Robust pacemaking in substantia nigra dopaminergic neurons. J. Neurosci. 29, 11011–11019 (2009) 7. Hargus, G. et al. Differentiated Parkinson patient-derived induced pluripotent stem cells grow in the adult rodent brain and reduce motor asymmetry in Parkinsonian rats. Proc. Natl Acad. Sci. USA 107, 15921–15926 (2010) 8. Jankovic, J. Parkinson’s disease: clinical features and diagnosis. J Neurol Neurosurg Psychiatry. 79, 368-376 (2008) 9. Joksimovic, M. et al. Wnt antagonism of Shh facilitates midbrain floor plate neurogenesis. Nature Neurosci. 12, 125–131 (2009) 10. Kriks S, et al. Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease. Nature. 480, 547–551 (2011) 11. Li, L., Zhou, F.M. Parallel dopamine D1 receptor activity dependence of L-dopa-induced normal movement and dyskinesia in mice. Neuroscience. 236, 68-76 (2013) 12. Olanow, C. W., Kordower, J. H. & Freeman, T. B. Fetal nigral transplantation as a therapy for Parkinson’s disease.

Trends Neurosci. 19, 102–109 (1996) 13. Pan, T., Kondo, S., Le, W., and Jankovic, J. The role of autophagy-lysosome pathway in neurodegeneration associated with Parkinson’s disease. Brain. 131, 1969-1978 (2008) 14. Salti A, et al. Expression of early developmental markers predicts the efficiency of embryonic stem cell differentiation into midbrain dopaminergic neurons. Stem Cells. 22, 397-411 (2013) 15. Sundberg M, et al. Improved cell therapy protocols for Parkinson’s disease based on differentiation efficiency and safety of hESC-, hiPSC-, and non-human primate iPSC-derived dopaminergic neurons. Stem Cells. 31, 1548-62 (2013)

BDNF overexpression rescues symptoms of Huntington’s disease by ameliorating neuronal loss in the striatum.

Yidong Zhan

Huntington’s Disease is mutation of the hungtington gene that leads to motor impairment and cognitive function, such as memory. The hungtington gene inhibits normal production of brain-derived neurotrophic factor, which is crucial for neuronal development, differentiation and survival. BDNF deficiency affects long term potential which decreases the ability to encode for long term memory. Moreover, it creates an imbalance between direct and indirect pathway of movement. Overexpression of BDNF in the brain shows significant restoration of cognitive functions and motor coordination by rescuing dendritic spine atrophy and preventing striatal neuronal loss. BDNF overexpression is upregulated by transgenic gene, the trkB receptor signal pathway or by ampakine. This review paper explores cellular changes in the striatum as a result of Huntington’s disease and how BDNF can reverse or prevent progressive neuronal changes.

Key words: Brain-derived neurotrophic factor (BDNF), Huntington’s Disease (HD), TrkB receptor, straitum, neuronal growth

Background Huntington’s disease (HD) is neurodegenerative disease that affects motor impairment and cognition.

HD arises from an expansion of CAG trinucleotide repeat on the Huntington gene (htt). (Zuccato et al., 2005) Mutations on the htt can cause excitotoxicity and metabolic toxicity. (Ferrer et al., 2000) Htt mutations directly cause brain-derived neurotrophic factor (BDNF) gene alternations, specifically producing lower levels of BDNF by inhibition BDNF gene expression.

(Canals et al., 2004; Zuccato et al., 2005) BDNF promotes neuronal survival and cell growth, especially striatal neurons, during development and following brain lesions. (Ferrer et al., 2000) In HD patients, decreased BDNF protein expression were observed in the caudate and putamen, though expression levels remain unchanged in the hippocampal region. (Ferrer et al., 2000) This was evident through western blots performed on cerebral cortex brain slices. (Ma et al., 2010) HD mice models with reduced BDNF expression also observes more severe symptoms and earlier motor impairment. (Canals et al., 2004) BDNF protein expression is reduced from axonal transport inhibition or deafferentation. (Ferrer et al., 2000) Immunoreactivity of BDNF of corticol neurons in the paritetal cortex was observed via immunohistochemistry. (Ferrer et al., 2000) BDNF deficiency can cause dendritic and neuronal loss in the cerebral cortex and striatum. (Xie et al., 2010) Medium sized spiny neurons (MSN) in the striatum undergo neurodegeneration. In advanced cases of HD, neurodegeneration may extend to the cerebral cortex.

(Canals et al., 2004) To observe neuronal changes, researchers often perform in situ hybridization on brain slices and immunoblotting. (Xie et al., 2010) The hallmark change in HD patients is motor impairment. (Samadi et al., 2013) Motor dysfunction is caused primarily from striatal projection losses to the cerebral cortex. (Canals et al., 2004) GABAergic neurons from MSN project to external globus pallidum forms the indirect pathway of movement while projections to substantia nigra pars reticular and internal globus pallidum forms the direct pathway. MSN neurodegeneration affects the

indirect pathway, causing choreic movements. (Canals et al., 2004) HD patients also experience cognitive changes such as memory impairment due to inability to induce LTP. (Kramar et al., 2012) There is no definitely treatment for HD but studies show BDNF to be a promising treatment that rescues HD symptoms in mice models. (Xie et al., 2010) Xie et al suggests that BDNF overexpression in HD transgenic line YAC128 mice improve major HD symptoms such as motor dysfunction and cognitive changes. BDNF binds to TrkB receptors to mediate neurotrophic signaling to promote cell survival, increase synaptic plasticity (such as LTP) and encourage neuronal differentiation.

(Ferrer et al., 2000; Simmons et al., 2009) BDNF overexpression can also help in dendritic atrophy and upregulate dopamine receptors, which is important in the indirect pathway of movement. (Xie et al., 2010) BDNF is difficult to administer into site of interest in the brain as it can’t cross the blood brain barrier when administered from the periphery. (Giralt et al., 2011) As such, researchers are attempting BDNF overexpression endogenously. (Simmons et al., 2009) BDNF endogenous overexpression is enhanced through upregulation of TrkB receptor pathways or injection of ampakine. (Baydyuk et al., 2011; Kramar et al., 2012) Research Overview

Summary of Major Results

BDNF in forebrain increases striatum BDNF It was shown that there was refdced levels of BDNF and trkB receptors in the forebrain. (Simmons et al., 2009) BDNF overexpression in the forebrain via transgenic gene is shown to increase BDNF in the striatum. Western blots and in situ hybridization analysis confirmed these findings. (Xie et al., 2010) BDNF upregulation via ampakine also increase BDNF mRNA production in mice forebrains. (Canals et al., 2004) BDNF activates TrkB receptor BDNF activates TrkB signaling pathways to mediate the effects of neuronal growth, differentiation and soma 288

size. (Xie et al., 2010). BDNF release ameliorates soma size and restores striatal volume. (Giralt et al., 2011). Activation of TrkB receptors is important for developmental growth of the brain, particularly dendritic growth in striatal neurons. TrkB receptor activation promotes medium-sized spinal neuron development and survival in the striatum. (Baydyuk et al., 2011).

BDNF prevents atrophy and neuronal loss BDNF overexpression rescues soma size and increase brain weight. It reverses striatal neurons and somatic atrophy in striatal neurons. (Xie et al., 2010) Striatal neuronal atrophy and neuronal loss is correlated with motor impairment on HD mice. (Samadi et al., 2013) BDNF overexpression can prevent neuronal loss suggesting it can reverse motor dysfunction as well as protect brain atrophy as a whole. (Xie et al., 2010) Increased BDNF expression can reverse MSN neuronal loss in the striatum. (Ferrer et al., 2000) It normalizes MSN dendritic morphology and rescued atrophied spinal changes in the striatum. (Xie et al., 2010) BDNF rescues motor impairment Endogenous BDNF determines the onset and severity of motor impairments in HD patients. It shows walking coordination is affected via footprint pattern analysis. (Kramar et al., 2012) HD transgenic mice lines with BDNF overexpression performed similar scores compared to wild type mouse on motor tasks such as on the rotarod test. (Xie et al., 2010) BNDF rescued motor coordination as seen on the beam walk test. HD mice with BDNF upregulation had weaker grip strength than the wild type but performed better than vehicle mice. This suggest while BDNF may not be able to rescue all motor impairment, it can still improve motor coordination. (Xie et al., 2010) BDNF restores LTP BDNF can restore spinal and synaptic morphology changes in the hippocampus. The reorganization of dendritic cytoskeleton is important for restoration to encode for long term memory. (Kramar et al., 2012).

BDNF promotes actin network activity that was previously disrupted by HD to encourage synaptic plasticity. (ampakinep) Continuous BNDF upregulation stabilizes LTP and reduces long term memory deficits in HD mice. (ampakinep) BDNF activates cofilin signaling pathways that induces actin polymerization which in turns produces stable LTP. (BDNF URSP)

Figure 1.

In situ hybridization showing levels of BDNF in the cortex and striatum in wild type mice (WT), HD induced mice (YAC128) and HD mice with BDNF overexpression(YAC;BTg).

BDNF level is significally reduced in the striatum of vehicle mice.

(Xie et al.,)

289

Discussion and Conclusion BNDF is a neurotrophic factor involved in brain processes important for cell survival and neuronal differentiation in the striatum. (Xie et al., 2010) Htt inhibits BDNF expression in the striatum and downregulates BDNF. (Canals et al., 2004) BDNF deficiency in the striatum causes neuronal alterations such as neuronal loss and dendritic abnormalities that affects HD pathogenesis. As a result, severe motor dysfunction and cognitive developmental abnormalities occur in HD patients. (Xie et al., 2010) TrkB receptor binds to BDNF and this signal cascade facilitates neuronal differentiation and cell survival in the striatum. TrkB receptor deletion is associated with large neuronal striatum in the striatum. (Baydyuk et al., 2011). BDNF overexpression was able to rescue these effects and reversed cognitive deficiencies and motor dysfuntion. BDNF protected striatal neurons from further atrophy and normalized striatal neuronal loss. It was able to reverse MSN spine and dendritic atrophy in the striatum. (Xie et al., 2010) Upregulation of BDNF restores normal actin polymerization activity in dendritic spines that affects LTP. (Kramar et al., 2012) Even at low concentrations of BDNF, it was able to induce long lasting LTP by theta burst stimulation in the hippocampus. (Simmons et al., 2009) BDNF can facilitate long term memory encoding by induce LTP. (Xie et al., 2010) BDNF overexpression increased dopamine receptor D2 levels important in the indirect pathway of movement. This will alleviate the symptoms for choreic movement that mostly defines HD. (Baydyuk et al., 2011) Motor impairment is also due to the loss of striatal projection neurons. (Canals et al., 2004) Increased BDNF expression restores movement by increasing inputs to MSN and increase output projections to the striatum. (Cepeda et al., 2004) Significant improvement in motor coordination was observed in standard motor tests given to HD mice with BDNF overexpression. (Xie et al., 2010) BDNF protects the cortical motor regions of the brain for further accelerated striatal degeneration leading to more severe motor incoordination. (Samadi et al., 2013) Increasing BDNF endogenously via ampakine and TrkB receptors signal cascade showed similar neuronal rescue compared to transgenic mice with BDNF gene. (Simmons et al., 2013; Baydyuk et al., 2011) BNDF binds to TrkB receptors to rescue MSN dendritic atrophy in the striatum which normalizes motor coordination in HD patients. (Xie et al., 2010). It also enhances actin polymerization activity in dendritic spines to produce long lasting LTP for memory encoding. (Kramar et al., 2012) BDNF overexpression increases dopamine receptors and restores MSN projections to the striatum which helps balance the indirect pathway of movement. (Canals et al., 2004; Cepeda et al., 2004) Through various pathways, BDNF overexpression reverses cognitive deficiency and motor dysfunction by affecting MSN in the striatum and preventing subsequent neuronal loss.

Figure 2. BDNF overexpression improves motor co-ordination and muscle strength in HD mice. HD mice with BDNF overexpression (YAC;BTg) has scores that can match wild type mice (WT). HD mice (YAC128) shows increased changes of falling and freezing on various motor co-ordination tests compared to wild type and YAC;BTg mice. (Xie et al.,)

Criticisms and Future Directions Xie et al., showed discrepancies in certain lack of findings compared with other literatures. This can be attributed to the fact they used R6/1 transgenic mice instead of R6/2. R6/1 shows more aggressive symptoms and displays earlier onset than R6/2 transgenic mice. (Brooks et al., 2012) It is helpful if Xie et al., can reproduce these results in the R6/2 mice line.

Xie et al., noted seizure activities in entorhinal cortex and hippocampus when BDNF is overexpressed in these areas. (Xie et al., 2010) Xie et al., can consider inducing localized BDNF overexpression in the brain instead of inducing global BDNF upregulating.

BNDF shows promise by rescuing molecular changes caused by HD. However, it remains a problem administering BDNF into the brain. BDNF does not cross the blood brain barrier when injected into the periphery.

BDNF overexpression via gene transplantation remains too experimental to be performed on humans.

It is crucial to find treatment that can endogenously upregulate BDNF in HD patients.

One of the mutation of htt is the inhibition of phosphorylation at serine 421 on the hungtinton gene.

Phosphorylation of the htt at S421 location restores the huntington gene original function to transport and regulate wild type BDNF expression. It is observed that HD mice has increased expression of calcineurin, which is a phosphatase that prevents S421 phsorphylation. Inhibition of calcineurin restores transportation of BDNF both anterogradely and retrogradely into neurons. It is also shown that calcineurin is involved in selective striatal neuronal death. (Pineda et al., 2009) Shifting focus to inhibition calcineurin as a way to endogenously increase BDNF could be considered as a plausible treatment.

Ampakine is a modulator of AMPA receptors.

Ampakine can increase endogenous BDNF expres-

sion allowing LTP restoration. Ampakine is able to normalize long term memory impairments of HD mice.

(Simmons et al., 2009) Ampakine encourages excitatory synpases in the forebrain and upregulates BDNF proteins by AMPA receptors. (Kramar et al., 2012) It is suggested astrogliosis can release conditional endogenous BDNF in the brain. Astrogliosis can also upregulates BDNF in the striatum close to wild type levels. It can be observed that these mice model improved in anxiety tasks, clasping tasks and other striatal dependent behavior. This also improved in synaptic plasticity and astrocytes projected fine excitatory synapses. (Giralt et al., 2011) Activating TrkB pathways, inhibition of calcineurin and inducing ampakine may be easier to invasively (for example, pharmacologically) activate endogenous BDNF expression the brain to alleviate HD symptoms and rescues neuronal loss in the striatum. This poses as an alternative solution to gene therapy.

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