Neurobiology of Behaviour 2nd Edition

Neurobiology of Behaviour University of Toronto

Fall 2014

2nd Edition

University of Toronto Fall 2014

Table of Contents 1. Alterations in the D1/D2 Medium Spiny Neurons of the Nucleus Accumbens Shell Due to Cocaine Exposure - 1-5 Authors: Cheng Deng, Majid Gasim, Urshita Grover & Kathleen A. Harrison

2. The Venom that Kills Your Pain - 6-8 Authors: Yu (Annie) Feng, Louise Escuban, Elliot Ho, Arun Radhakrishnan

3. D2 Receptors: hold onto your spines! - 9-12 Authors: Dimitar Krastev, Sejin Kwon, Karthik Natarajan, & Darren Tan

4. Neural Basis Behind Episodic Prospection: Preventing Temporal Discounting to Make Better Decisions - 13-17 Authors: Elizabeth Nikovski, Cindy Xin Ma, Eve Yiran Zheng, Faryal Khan

5. Gone “TAI-FISH�ing: A novel way to map emotional valence in the brain - 18-20 Authors: Rachel Oh, Steven Meas, Zara Duquette, Sabina Freiman, & Sarah Peters

6. Adding to the Puzzle: Another Neurotransmitter for the REM Sleep Circuit - 21-23 Authors: Sara Pintwala

7. Cognitive and Emotional Processes Differentially Affect Moral Dilemma Judgments - 24-26 Authors: Meghna G., Mansi P., Saba S., and Sonja S.

8. Examining the role of systemic angiotensin II and exercise-induced neurogenesis in adult rat hippocampus: A preliminary investigation in need of stronger evidence - 27-30 Authors: Romina Nejad, Twayne Pereira and Semih Topbas

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Alterations in the D1/D2 Medium Spiny Neurons of the Nucleus Accumbens Shell Due to Cocaine Exposure

Authors: Cheng Deng, Majid Gasim, Urshita Grover & Kathleen A. Harrison University of Toronto - HMB420 October 28, 2014

The Nucleus Accumbens (NAc) and its associated circuits are integral to the neurobiological mechanisms that underpin motivation, pleasure and reward-related behaviors. Thus, it also serves as a key factor in the structural and functional changes that occur in the brain during drug addiction1.

Repeated exposure to dopaminergic drugs such as cocaine can cause such changes in Medium Spiny Neurons (MSNs) of the NAc, suggesting that a rewiring of NAc circuits occurs, as well as changes in the excitability of these neurons2.

Accordingly, MacAskill, Cassel and Carter (2014) set out to determine changes that occur in the afferents onto the different MSN’s in the NAc, and elucidate the exact structural and functional changes that occur in these neurons3. Using a variety of techniques including optogenetics,

Background MSNs in the NAc have been shown to principally be involved in both the learning and expression of motivation, and rewardrelated behaviors. Previous studies have demonstrated that cocaine exposure in mice induces a behavioral sensitization response, characterized by increased locomotion in response to cocaine administration after a period of withdrawal2. This response has been shown to be the result of alterations in synaptic connections on these MSNs, specifically, an increase in excitatory afferents4. However, the details of how repeated cocaine exposure reorganizes these afferents from different inputs and cell types has yet to be identified.

MSNs in the NAc can be organized into two distinct populations of cells that differ in

pharmacogenetics and two-photon microscopy, they established that cocaine exposure selectively increases excitatory inputs from the basolateral amygdala (BLA) onto D1 dopamine receptors expressed in MSNs. Furthermore, they showed that the BLA was necessary for inducing the behavioral sensitization response.

By elucidating these cell type and input specific changes in the NAc, the researchers provided an explanation for the neurobiological responses that occur to drugs such as cocaine, and hence providing a clear perspective for the rewiring of neural circuitry associated with drug addiction and other neuropsychiatric diseases.

Key words: nucleus accumbens (NAc); drug addiction; cocaine; optogenetics; dopamine receptors; two-photon microscopy; pharmacogenetics; behavioral sensitization

the relative expression of D1 or D2 dopamine receptors. These two types of cells have opposing roles in regulating reward and goalrelated behavior, and hence, reorganization of their excitatory afferents can greatly alter their involved circuitry and its associated functions.

The excitatory afferents examined in the study arose from the basolateral amygdala (BLA), the ventral hippocampus (VH), and the prefrontal cortex (PFC), each carrying their own specific functional signals to the NAc. Hence, MacAskill, Cassel and Carter (2014) examined cocaine’s ability to differentially redistribute these connections onto D1 and D2-MSNs. These changes can greatly alter the excitability of these neurons through synaptic changes and rearrangements.

Recent studies have found that cocaine causes increases in spine density at D1MSNs5. Consequently, the aforementioned

2 researchers set out to determine the exact connections which result in these synaptic changes and rearrangements.

Research Overview Summary of Major Results MacAskill, Cassel and Carter (2014) were able to demonstrate the ways in which repeated cocaine exposure altered medial Nucleus Accumbens (mNAc) circuitry; specifically, medium spiny neurons (MSNs) subtype expressing D1 dopamine receptors.

Figure 1. Cocaine exposure enhances synaptic connectivity. (a) two-photon image of dendrite and spine morphology at D1-MSN and D2-MSNs for mice treated with cocaine or saline. (b) mEPSC frequency (Hz) ratio recorded from mice treated with cocaine or saline. (c) data as in b for mEPSC amplitude (pA).

Sensitization is strongly associated with functional and structural changes in NAc D1-MSNs synaptic connectivity.

Using two-photon microscopy, the researchers found an increased spine density at D1-MSNs due to cocaine exposure but this was not seen at D2-MSNs (Fig. 1a). These structural changes were also reflected in the functional properties at D1-MSNs as cocaine exposure caused an increased mEPSC frequency, with a bias towards D1-MSNs (Fig. 1b). However, there was no change in mEPSC amplitude (Fig. 1c).

Figure 2. Structural alterations at NAc are cell type and input specific. (a) D1MSN/D2-MSN EPSC amplitude (qA) ratios recorded at BLA from mice treated with cocaine (C) or saline (S). (b-c) data as in a for VH and PFC inputs.

Cocaine differentially altered afferents onto D1/D2-MSNs in NAc, with BLA and VH being cell type and input specific.

The researchers also found specific longrange excitatory inputs being involved in the synaptic changes. Firstly, lightevoked EPSCs showed that for inputs from Basolateral Amygdala (BLA), cocaine selectively enhanced EPSCs, showing an altered BLA-NAc connectivity. However, for inputs from ventral hippocampus (VH), the reverse happened in that cocaine selectively dampened EPSCs at D1-MSNs, which was reflective of a suppression of VH connections at D1-MSNs. Alternatively for PFC inputs, EPSCs remained unbiased, so cocaine had no selective effect (Fig.2a-c).

Figure 3. Cocaine enhances input from BLA but suppresses VH input. (a) qEPSC frequency (Hz) ratio recorded at BLA or VH from mice treated with cocaine (C) or saline (S). (b) data as in a for qEPSC amplitude (qA).

3 Repeated exposure to cocaine enhances BLA inputs by more inputs and suppresses VH inputs onto D1-MSNs (postsynaptic changes).

Cocaine increased qEPSC frequency at D1-MSNs but not at D2-MSNs for BLA inputs (Fig. 3a). This qEPSC increase at BLA is consistent with observed whole-cell recordings that indicates increased mEPSC frequency at single synapses. Additionally, this is reflective of an increase in synapses onto D1-MSNs. No significant effect for amplitude was found here (Fig. 3b). For VH inputs, the qEPSC frequency was unaltered following cocaine exposure but the qEPSC amplitude decreased at D1-MSNs (Fig. 3a,b), which is reflective of specificity of postsynaptic effects at D1-MSNs that were not apparent in nonspecific mEPSCs. Repeated cocaine exposure enhances BLA inputs and suppresses VH inputs onto D1-MSN (subcellular changes).

The synaptic density for BLA inputs was initially similar for D1 and D2-MSNs. However, cocaine selectively enhanced synaptic density for D1-MSNs, but no effect was seen on spine volume (Fig. 4a), showing that repeated exposure of cocaine selectively enhances number of BLA connections onto D1-MSNs. For VH inputs, synapse density was similar at D1MSNs and D2-MSNs. But spine volume was initially much larger at D1-MSNs in salinetreated mice, and this difference was abolished after repeated cocaine exposure (Fig. 4b), owing to a reduction in spine volume at D1-MSNs. Combined with qEPSC recordings, this supports the idea that drug exposure suppresses the strength of VH connections onto D1-MSNs.

Figure 4. Cocaine exposure enhances spine density from BLA and suppresses spine volume from VH. (a) synapse density (segment-1) ratio recorded at BLA synapse from mice treated with cocaine (C) or saline (S). (b) data as in a for VH.

NMDAR activation is required for cocaine-induced behavioral sensitization and changes to BLA/ VH connections onto MSNs. MK-801 (NMDA antagonist) in the long term abolished sensitization to cocaine and blocked cocaine-induced changes to both BLA and VH EPSCs (Fig. 5a-c). However, acute pretreatment with MK-801 did not prevent sensitization in mice.

Figure 5. MK-801 abolished behavioural sensitization and synaptic alterations. (a) distance traveled by cocaine treated mice with saline or MK-801 injection. (b) EPSC amplitude (qA) ratio recorded at BLA from cocaine treated mice coupled with saline or MK-801. (c) data as in b for VH.

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Figure 6. BLA activity is required for structural and functional changes induced by cocaine. (a) distance traveled by cocaine treated mice with hM4D or without (Ctrl). (b) EPSC amplitude (pA) ratio recorded at BLA from cocaine treated mice with hM4D or without (Ctrl). (c-d) data in b for mEPSC frequency (Hz) and spine density (Âľm-1).

BLA and VH EPSCs are dissociable with only BLA inputs causing sensitization, through BLA structural and functional BLA D1-MSNs innervation.

Inhibiting BLA activity abolished behavioral sensitization and abolished changes to BLA EPSCs (Fig. 6a-b), but had no effect on cocaineinduced changes to VH EPSCs. This showed that BLA activity is necessary for cocaineevoked synaptic plasticity in the NAc medial shell. Additionally, inhibiting BLA activity eliminated cocaine-induced increase in mEPSC frequency at D1-MSNs and abolished cocaine-induced increase in spine density at D1-MSNs (Fig. 6c-d) showing that enhanced BLA connectivity is responsible for these. On the other hand, inhibiting VH activity did not affect behavioral sensitization and had no effect on cocaine-induced changes to BLA EPSCs, but it abolished changes to VH EPSCs, showing that VH activity is only required for VH inputs.

Conclusions and Discussion Researchers MacAskill, Cassel and Carter in their 2014 Nature Neuroscience paper investigated the impact of short term cocaine exposure on D1 or D2 expressing MSN of the mNAc. This article illustrated

how cocaine exposure reweighted cortical inputs from the BLA and VH entering the mNAc. Their experiments showed that 5 consecutive days of cocaine exposure, and 3 days of withdrawal, alters synaptic connectivity in the mNAc during behavioral sensitization through increased mEPSC frequency and spine density at D1-MSNs; observed connectivity changes are cell type and input specific as seen in increased BLA D1 EPSC, and dampened VH D1 EPSC; input specific changes are specific to the number and strength of connections, with BLA D1 qESPC frequency increased, and VH D1 qESPC amplitude dampened; subcellular structures are reflectively altered, with BLA D1s having increased spine density, and VH D1 spine volume reduced; connectivity changes are explained by classic LTP NMDAR systems; BLA & VH EPSCs changes depend on BLA and VH activity respectively; and that only BLA activity is needed for behavioral sensitization.

These findings have huge implications for better understanding cortical, cognitive, and behavioral changes, which occur post cocaine exposure. Specifically, this research showed goal-directed and motivated behavior is differentially regulated by activation of D1/ D2-MSNs in the NAc, and that drug exposure rebalances BLA and VH input connectivity. Usually dominant VH inputs become replaced with BLA inputs.

Thus, short-term cocaine exposure shifts motivated behavior from VH memory based inputs to BLA emotional inputs.

However, these findings may not be widely applicable to other timelines, and methods of cocaine exposure. Some of the issues to integrating MacAskill, Cassel and Carter’s (2014) findings with the rest of cocaine literature are different changes which occur in cocaine self administration

5 paradigms, longer withdrawal periods, differences in other areas of the NAc and greater striatum being altered due to cocaine. There is no context of how these findings are integrated with other important areas such as the VTA, and contrasting evidence concerning cell-type and input-specific redistribution in D1-MSNs and D2-MSNs from other experimental models. Hopefully, future work will attempt to consolidate these issues so that there may be a better understanding of neurobiological, and behavioral alterations post cocaine exposure.

References 1. Sesack, S. & Grace, A. (2010). Cortico-Basal Ganglia Reward Network: Microcircuitry.

Neuropsychopharmacology 35, 27-47.

2. Hyman, S., Malenka, R. & Nestler, E. (2006).

Neural Mechanisms of Addiction: The Role of Reward-Related Learning and Memory.

Annu. Rev. Neurosci. 29, 565-598.

3. MacAskill, A., Cassel, J. & Carter, A. (2014).

Cocaine exposure reorganizes cell type– and input-specific connectivity in the nucleus accumbens. Nature Neuroscience 17, 11981207.

4. Russo, S., Dietz, D. M., Dumitiu, D., Morrison, J. H., Malenka, R. C. & Nestler, E. J. (2010). The addicted synapse: mechanisms of synaptic and structural plasticity in nucleus accumbens. Trends in Neurosciences 33, 267276.

5. Kim, J., Park, B., Lee, J., Park, S. & Kim, J. (2011). Cell Type-Specific Alterations in the Nucleus Accumbens by Repeated Exposures to Cocaine. Biological Psychiatry 69, 10261034.

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The Venom that Kills Your Pain Authors: Yu (Annie) Feng, Louise Escuban, Elliot Ho, Arun Radhakrishnan University of Toronto - HMB420 October 28, 2014

Introduction There is a constant need for the development of new pain medications, sometimes to cater to the different medical predispositions of patients, at other times because existing treatment is not sufficiently effective. The authors of this paper looked to a novel source for potential pain-killing substances: the venom of the black mamba (Dendroaspis polylepis) snake. Mambalgin-1 and -2 are newly isolated peptides shown to be potent analgesics in a mouse model, and which appear to take effect by blocking acidsensing ion channels (ASICs). Their effects are comparable to morphine, however they show less tolerance and fewer severe side effects such as respiratory distress.

Mambalgin-induced analgesia also appeared to be naloxone-independent, suggesting it did not kill pain through the opiate pathway.

This article provided a comprehensive initial investigation into the workings of a mysterious new compound, but there is still much to investigate before mambalgins can be considered for clinical trials.

Background Acid-sensing ion channels (ASICs) are a family of proton-gated sodium channels that have been previously implicated in studies of pain and analgesia (4,8). ASICs have four subunits, some with splice variants such as ASIC1a, ASIC1b, ASIC2a, and ASIC2b (5). ASIC channels are composed of subunits assembled into homomultitrimeric or hetermultitrimeric complexes. Studies

have been conducted linking toxins and ASICs previously: the spider toxin PcTx1 was found to induce a naloxone-dependent analgesia through blocking ASIC1a channels (naloxone being an opioid antagonist) (3). ASIC subunits have been localized to sensory receptors, synapses, and all over the neuron, suggesting it may act in multiple locations at once. Emphasizing the importance of this family of proteins, they have been found both in the CNS as well as the PNS, and are remarkably well conserved across species (4). Preliminary studies with Xenopus oocytes found peptides in black mamba venom (mambalgins) to be effective reversible inhibitors of the ASIC1a channel.

Biochemical analysis revealed mambalgins to be a member of the family of three-finger toxins with no sequence homology with either PcTx1, or APETx2 (a sea anemone toxin also implicated in inflammatory pain).

Methods and Results The Analgesic Effect of Mambalgins To test if mambalgins have an analgesic effect, the researchers used a measure called the tail-flick and paw-flick tests, typical anti-nociceptive measures (1). These involve, allowing the rat’s tail or paw to touch a hot surface and observe the latency. A higher latency indicates a larger analgesic effect because the ASIC channels are blocked. Knockout mice, without ASIC channels were used. When mambalgin-1 is injected there was an increase in tail-flick and paw-flick latency in comparison to knockout mice (Figures a and b).

7 Figure a

Figure b

Other ASIC Channels Figure 5. MK801 abolished behavioural sensitization and synaptic alterations. (a) distance traveled by cocaine treated mice with saline or MK-801 injection. (b) EPSC amplitude (qA) ratio recorded at BLA from cocaine treated mice coupled with saline or MK-801. (c) data as in b for VH.

Compared to Morphine It is also important to investigate the effects of mambalgins in comparison to traditional painkillers, such as morphine.

In comparison to morphine, the researchers that mambalgins are resistant to naxolone, an anti-opioid drug, whereas morphine is not resistan. tMorphine is very effective at dampening pain perception, but it comes with many side effects including: headaches, vomiting, addictive behaviours and respiratory problems (7).

However, mice administered mambalgins showed less tolerance to the drug over a period of five days when compared to morphine, as paw-flick latency decreases at a slower rate. Also less respiratory issues were found in mice who were administered mambalgins as opposed to morphine.

Other ASIC channel subtypes like ASIC2a/2b channels also play a role in the perception of algesic inflammatory response since blocking with these channels with siRNA causes an increase in latency.

ASIC channels in the periphery including primary nociceptors and ASIC1b containing channels were tested, but the role of these channels are still not fully known.

Conclusion Overall, Diochot et al had conducted a well-controlled experiment. It is able to demonstrate the analgesic effect of mambalgin that is comparable to morphine, the specificity of mambalgin towards ASIC1a and ASIC2a in central nervous system and ASIC1b in nociceptors, and the analgesic effect of mambalgin that is independent from the opioid pathway. Moreover, since the analgesic effect of mambalgin is independent from the opioid pathway, there were no significant adverse effects recorded in the experiment. However, this promising alternative pain medication is still in its early stage and more research is needed before it reaches potential clinical trials. Since mambalgin is still being studied in the mouse model study, transition from mouse models to primate models might provide a better insight in how mambalgin will interact with a model that is more closely related to human. Moreover, in this reviewed article, animal models were treated with mambalgin for only one week, and we believe that this duration is not long enough to significantly identify any adverse effects from long-term use. This has significant clinical implications because patients suffering from chronic pain are constantly using pain medications, and most

8 likely more than once over their lifetime.

Future experiments should focus on mambalgin’s potential chronic-use adverse effects, such as tolerance, hyperalgesia, drug-drug interaction, addiction, and alteration of the brain chemistry. Tolerance was looked at very briefly in this study, and longer-term observations are definitely an important next-step. In-vivo studies might also shed more light on the substancesystem interactions and how this foreign element interacts with organic processes over a length of time. ASICs involving in pain apparently can be up-regulated and induces hyperalgesia (8). In addition to that, ASIC can also be found in the hippocampus (6), so mambalgin might have a potential effect on memory consolidation and learning, which are waiting to be discovered.

Recent research (5) has shed some light on the biochemical processes of mambalgins, and found that at the protein level, there are in fact, some structures that can be modified in the mambalgins to improve the chemical for analgesia.

References 1. 1. Bannon & Malmberg. 2007. Models of Nociception: Hot-Plate, Tail-Flick, and Formalin Tests in Rodents. Current Protocols in Neuroscience.

2. 2. Salinas, M. et al. 2012. The Binding Site and Inhibitory Mechanism of Mambalgin-2 Pain-Relieving Peptide on of A­­­­cid-Sensing Ion Channel 1a. J. Biol. Chem.43 289 3. 3. Karcsewski J, Spencer RH, Garsky VM, Liang A, Leitl MD, Cato MJ, Cook SP, Kane S, Urban MO. 2010. Reversal of acid-induced and inflammatory pain by the selective ASIC3 inhibitor, APETx2. Br J Pharmacol. 161(4):950960.

4. 4. Wemmie JA, Price MP, Welsh MJ. 2006. Acid-sensing ion channels: advances, questions and therapeutic opportunities.

TRENDS in Neurosciences. 29(10):578-586.

5. 5. Bronstein-Sitton N. 2004. Acid-Sensing Ion Channels: Structure and Function.

Modulator. [accessed 2014 October 26];18:13. http://www.alomone.com/upload/ newsletters/modulator%2018%20papers/ acid-sensing%20ion%20channels.pdf 6. 6. Diochot, S. et al. 2012 Black mamba venom peptieds target acid-sensing ion channels to abolish pain. Nature 490,a aa552555.

7. 7. Bladen, C. 2013. Taking a bite out of pain. Channels (Austin). 7(2): 69-70 8. 8. Duan, B. et al., Upregulation of AcidSensing Ion Channel ASIC1a in Spinal Dorsal Horn Neurons Contributes to Inflammatory Pain Hypersensitivity. The Journal of neuroscince 27 (41), 11139-11148 (2007).

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D2 Receptors: hold onto your spines!

Authors: Dimitar Krastev, Sejin Kwon, Karthik Natarajan, & Darren Tan University of Toronto - HMB420 October 28, 2014

Introduction This study aimed to determine whether D2 receptors affect synaptic connections in hippocampal neurons. It was the first study to find that D2 receptors had a role in the development of dendritic spines, and that this process occurred in an age dependent manner through a specific type of glutamate receptor subunit. This study has a significant impact on the understanding of mental illnesses, particularly schizophrenia. It has previously been found that the levels of D2 receptors are higher in the striatum of schizophrenics than in normal individuals1, and that schizophrenics exhibit a decrease in synaptic connectivity in axons between deep layer three and deep layers five and six2. This study could provide the missing link between these two findings3.

Another important molecule in neuronal plasticity is the NMDA receptor (NMDAR).

This receptor responds to glutamate and naturally occurs as a tetramer of two GluN1 monomers, as well as two monomers of a GluN2 variant7. The developing brain is rich in the GluN2B variant, but will begin to overexpress GluN2A during maturation, eventually leading to a predominance of GluN2A subunits across the adult brain’s synapses8. NMDARs containing GluN2B are known to have different conductance properties, and can stay open longer than receptors containing GluN2A8. The cause of this developmental switch is unknown, but changes in GluN2B abundance have been implicated in the cessation of developmental plasticity8.

Background D2 receptors are inhibitory metabotropic receptors which respond to dopamine in an antagonistic manner compared to the excitatory D1 receptors. Bonci and Hopf reported that when activated, D2 receptors inhibit the activity of adenyl cyclase through a Gi protein4 (Fig. 1). Low adenyl cyclase activity then leads to lower intracellular concentrations of cAMP, and thus decreases the cell’s abundance of active CREB5. CREB has been known to influence the neuronal expression of BDNF, tyrosine hydroxylase6, and many plasticity molecules. It is thus reasonable to suspect that active D2 receptors may contribute to long term depression.

Figure 1. Activated D2 receptors work through an inhibitory G protein to deactivate adenyl cyclase4. This will reduce the amount of plasticity factors, BDNF, and tyrosine hydroxylase in the cell by inactivating CREB5,6. D2 receptors also have an endocytosis pathway through a betaarrestin mediated negative feedback response3.

Results Spinal Methods The authors injected the D2R agonists quinpirole and bromocriptine, or the D2R antagonist, eticlopride into murine hippocampi. Mice that had D2R agonists injected showed lower dendritic spine density, while mice injected with D2R

10 antagonists showed increased spine density (Fig. 2). This demonstrated the negative effects of D2R activity on neural connectivity. To further validate their hypothesis, the authors then preformed a rescue in Sandy mice, a schizophrenia model which is known to have decreased synaptic connectivity and overactive D2 receptors. Spine density was comparable to wild type mice after a siRNA knockout of the D2 receptor gene. A similar experiment using D2R agonists and antagonists was done to test the role of D2R on spine maturity – it was found that D2R agonists decreased thin spine density and increased the density of the immature spines, filopodium3.

Figure 2. Hippocampal neurons were injected with D2R agonists quinpirole or bromocriptine, or the D2R antagonist eticlopride, and slices were prepared.

It can be seen that D2R agonists lowered dendritic spine density, while the D2R antagonist increased density3.

Pathway Elucidation In order to elucidate the potential mechanism of D2R’s action on spine density, NMDAR’s involvement was tested3. Primary hippocampal neurons were treated with either quinpirole, AP5 (NMDAR antagonist), or both. Combined treatment showed that AP5 eliminated quinpirole-induced changes with an increase in the overall spine number. Furthermore, the critical period of D2R function was explored with mice of various ages. Only mice between the ages of 3-6 weeks that were treated with quinpirole had a reduction in spine density, thus suggesting that D2R’s effects on

spine density may be age dependent (Fig. 3). As further evidence of NMDAR’s role, the authors reversed the GluN2B-GluN2A developmental switch by overexpressing GluN2B in mature hippocampal neurons and found treated neurons were once again susceptible to D2R’s effects3.

Figure 3. To determine the critical period for D2R spine regulation, either vehicle or quinpirole was intraperitoneally injected into mice of ages 2-12 weeks. Hippocampal slices were prepared 24h post-injection3.

Memory and Learning Impairment in Adulthood

Figure 4. A cholera toxin retrograde trace on entorhinal connections from CA1. Sdy and Quinpirole treated mice show less connections to the medial entorhinal cortex (MEC)3, which was rescued by eticlopride treatment in adolescence.

The authors wanted to test the lasting effects of adolescent D2R activation on the adult brain. After retrograde tracing with Cholera Toxin subunit B, they found that the ratio of connections between the Medial and Lateral Entorhinal Cortex (Fig. 4) was significantly decreased in Sandy mice and adolescent mice fed quinpirole3. However, feeding eticlopride to Sandy mice during adolescence produced connectivity similar to wild type mice, further supporting that the age dependent activation of D2R impairs the learning pathway.

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Figure 5. Y maze test results. Mice were subjected to a Y maze test, where they normally alternate which arm they explore frequently3.

Adolescent mice fed quinpirole and sandy mice alternated arms less frequently. Eticlopride treatment in adolescence resulted in recovery, but no recovery was seen when treated in adulthood.

Finally, the authors used a Y maze test (Fig. 5) to examine if this altered connectivity had cognitive effects. They observed that sandy mice and mice fed quinpirole in adolescence showed impaired performance3, demonstrating that the altered connectivity of the learning pathway translated into reduced performance on spatial memory tasks.

Conclusions and Future Directions These findings support the authors’ hypothesis that D2R activity has a negative impact on spine density. The authors went beyond that to find that this effect only happens during adolescence, and is dependent on NMDARs3. They brought forth valid evidence that D2R activity can change circuit connectivity, resulting in visible behavioral effects3. These findings support the theorized link between D2R activity and schizophrenia.

Though the results of the article appear to provide a solid foundation for these conclusions, several problems plagued the experiment, raising doubts about the validity of these results. One such problem is the spatial resolution of the confocal microscope.

The authors admit that they were unable to clearly distinguish between the different types of spines due to limited resolution3, calling their observations into question. In addition to imprecise equipment, the authors also present conflicting data within their results. In two instances within the paper, the number of spines on the neurons of mice treated with eticlopride were counted.

In one instance, the authors found that the mice treated with eticlopride showed a significantly higher number of spines than the wild type. Repeating this experiment with spines separated by morphology revealed that no single spine type varied significantly between the wild type and eticlopride treated mice3. These conflicting results may be due to poor experimental controls, which is another flaw in the paper. The authors of the paper failed to control for the type of neurons used in their experiments, stating simply that they largely used undefined hippocampal neurons, despite clear morphological differences (Fig. 6)9. Using different types of neurons may lead to vastly varying results especially in spine density.

Figure 6. Two morphologically different neurons being used in comparison. The wild type neuron resembles a layer II/III cortical neuron, while the Sdy neuron more closely resembles a CA3 neuron9.

Therefore,withregardtothisshortcoming, future papers should demonstrate the presence of D2R receptors in the cells they analyze through immunohistochemistry10.

Sudden loss of neuronal tissue density

during late adolescence is characteristic of schizophrenia11. Studies have indicated that neuronal death and gliosis is not the cause but rather a loss in neuronal dendrites, particularly in the prefrontal cortex12.

Future studies can investigate whether D2 receptors play a role in this loss within the mesocortical dopaminergic pathway.

Additionally, treatment-resistant Schizophrenic patients were found to respond best to a combined treatment with ECT and antipsychotics13. ECT produces transient seizures that relieves patients’ psychiatric symptoms, but the mechanism of how it provides this benefit is unclear. It is speculated that ECT rewires and reconnects various neuronal circuits, thus we hypothesize that ECT may be functioning to undo the connectivity damage produced by D2R during adolescence14.

References 1. 1. Howes, O.D. & Kapur, S. The dopamine hypothesis of schizophrenia: version III— the final common pathway. Schizophr. Bull.

2009;35: 549–562.

2. 2. Kolluri, N., Sun, Z., Sampson, A.R. & Lewis, D.A. Lamina–specific reductions in dendritic spine density in the prefrontal cortex of subjects with schizophrenia. Am. J. Psychiatry. 2005;162: 1200–1202.

3. 3. Jia JM, Zhao J, Hu Z, Lindberg D, Li Z. Age-dependent regulation of synaptic connections by dopamine D2 receptors. Nat Neurosci. 2013;16(11):1627-36.

4. 4. Bonci A, Hopf FW. The dopamine D2 receptor: new surprises from an old friend.

Neuron. 2005;47(3):335-8.

5. 5. Barco A, Bailey CH, Kandel ER. Common molecular mechanisms in explicit and implicit

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memory. J Neurochem. 2006;97(6):1520-33.

6. 6. Hong SJ, Huh Y, Chae H, Hong S, Lardaro T, Kim KS. GATA-3 regulates the transcriptional activity of tyrosine hydroxylase by interacting with CREB. J Neurochem. 2006;98(3):773-81.

7. 7. Traynelis SF, Wollmuth LP, McBain CJ, Menniti FS, Vance KM, Ogden KK, et al.

Glutamate receptor ion channels: structure, regulation, and function. Pharmacol Rev. 2010;62(3):405-96.

8. 8. Liu XB, Murray KD, Jones EG. Switching of NMDA receptor 2A and 2B subunits at thalamic and cortical synapses during early postnatal development. J Neurosci. 2004;24(40):888595.

9. 9. Spruston, N. Pyramidal neurons: dendritic structure and synaptic integration.

Nature. 2008; 9: 206 - 221.

10. 10. Iwanaga T, Hozumi Y, TakahashiIwanaga H. Immunohistochemical demonstration of dopamine receptor D2R in the primary cilia of the mouse pituitary gland.

Biomed Res. 2011;32(3): 225-235.

11. 11. Jacobsen LK. et al. Cerebral glucose metabolism in childhood onset schizophrenia.

Psychiatry Res. 1997;75(3): 131-144.

12. 12. Ferrara M, Freda F, Massa R, Carratelliv TJ. Frontal lobe syndrome or adolescent-onset schizophrenia? A case report. Acta Psychiatr Scand. 2005;114(5): 375377.

13. 13. Raphael, B. & Georgios, P. The combined use of electroconvulsive therapy and antipsychotics in patients with schizophrenia. J. ECT. 2005;21(2): 75-83.

14. 14. Hustig, H., Onilov, R. ECT rekindles pharmacological response in schizophrenia.

Euro Psychiatry. 2009;24(8): 521-525.

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Neural Basis Behind Episodic Prospection: Preventing Temporal Discounting to Make Better Decisions Authors: Elizabeth Nikovski, Cindy Xin Ma, Eve Yiran Zheng, Faryal Khan University of Toronto - HMB420 October 27, 2014

Introduction & Background In decision-making, humans often favor immediate gratification over choices that would bring them greater, long-term benefits. This is explained by temporal discounting (TD) - the tendency to hyperbolically decrease one’s subjective value of rewards that are further away in time1 (Fig. 1). This results in choosing a smaller but immediate reward over a larger future reward. However, the mental process of episodic prospection (EP) counteracts TD2.

The act of envisioning possible future events allows subjects to immediately experience them, which raises their subjective reward value to the value at time 0.

Past studies have suggested the prefrontal cortex as a potential neural correlate for these processes since it has been long associated with decision-making3.

Specifically, the medial rostral prefrontal cortex (mrPFC) may be the main region associated with the features of EP events4 and the valuation process5. The mrPFC is also thought to mediate the use of important neural circuits and regions that contribute to the vivid construction of EP events, such as the hippocampus (HC)2. However, there had been no direct evidence supporting the involvement of mrPFC in representing the reward value of envisioned events and whether diminished discounting is reflected in the representations6.

To address these questions, Benoit et al. (2011) recorded fMRI BOLD signal to look for a link between the brain activation for envisioned rewards and the attenuation of TD by EP. It was hypothesized that mrPFC may be the neural correlate that mediates the effect of EP by holding the reward value of envisioned episodes. This study validated that EP effectively diminishes TD and found that mrPFC activation and mrPFCHC coupling are implicated in this effect.

The study’s findings are significant because they enable further research by directing attention to possible targets of study such as functional connectivity of mrPFC with other brain regions. They also contribute to the understanding of dysfunctional decision-making and potential therapeutic approaches.

Materials and Methods 12 participants (4 males, 8 females; mean age = 27.3 years, range = 20.6-36.3 years) took part in the fMRI study involving monetary rewards. A 1.5T MRI scanner was used. For each trial, participants were prompted to perform either an Imagine task or an Estimate task. In the Imagine task, they vividly imagined spending a certain amount of money in a given scenario whereas in the Estimate task, they merely estimated what could be purchased (Fig.2).

14 Participants were then asked to choose either the delayed reward that they had encountered, or an immediate but smaller reward. After each trial, participants rated their emotional intensity as a marker for their engagement in EP and their feeling of mental experience during the task performance. The tasks were performed with a combination of 20 different scenarios, five reward magnitudes and four delays.

Figure 1. Hyperbolic discounting of subjective value due to temporal discounting. Retrieved from: <http://en.wikipedia.org/ wiki/File:Temporal_Discounting_Graph.png>.

Figure 2. Structure of fMRI trials. Trials each began with a brief cue that indicated the type of task (Imagine or Estimate). A scenario was then presented, followed by a delayed reward choice. Subjects either imagined spending the money (Imagine task) or estimated what it could purchase (Estimate task) in the given scenario. Then, they were to choose between the delayed reward or a smaller immediate reward. Finally, they rated their emotional intensity during the earlier Imagine or Estimate task and their feeling of vividly experiencing the event.

Major Results Phenomenological ratings The participants rated their experience of the Imagine task as having significantly greater emotional intensity than the Estimate task (Fig. 3A), showing that the Imagine task employed EP more than the Estimate task.

The Imagine task yielded far more future-oriented choices and greater reward accumulated over trials (reward index) (Fig. 3B). This provided evidence for the effect of EP in diminishing TD i.e. envisioning the larger reward can increase

the likelihood of choosing delayed rewards.

In fact, participants were more likely to choose the delayed reward when they experienced greater emotional intensity.

Furthermore, in order to discern the effects of EP on individual inclination of the participants to consider the future, they were assessed by the Consideration of Future Consequences (CFC) scale7. The CFC ratings were negatively correlated with the reward index of the Imagine Task, suggesting that directed EP was most effective for those who were less likely to consider the future (Fig. 3C).

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Figure 3. Phenomenological ratings. (A) Ratings of feeling of experience & its emotional intensity during Imagine & Estimate tasks. (B) Measure of discounting behavior as indicated by future-oriented choices and the accumulation of reward. (C) Impact of Episodic Prospection as measured by correlation of CFC with the reward index.

Neural correlates based on fMRI BOLD Greater fMRI BOLD signal was seen in mrPFC during the Imagine task than during the Estimate task (Fig. 4A, 4D). There was also stronger correlation between mrPFC BOLD and reward magnitude during the Imagine task (Fig. 4B, 4E). In both cases, the same region of the mrPFC was most strongly activated (Fig. 4C), suggesting that this area is involved in representing the undiscounted reward value due to EP. A greater reward index in the Imagine task than the Estimate task correlated with greater reward sensitivity of the mrPFC, which was measured by the ratio of BOLD signal for the highest versus the lowest reward. In addition, mrPFC BOLD correlated in time with signal in the hippocampus. Thus, the effect of EP on discounting was reflected in mrPFC activation and in mrPFC-HC coupling.

Furthermore, mrPFC recruitment during the Imagine task predicted choice of the delayed reward. On the other hand,

mrPFC activation during the Estimate task did not predict the choice as indicated by an equal tendency to choose between the delayed and the immediate rewards.

Figure 4. fMRI results. A-C, greater BOLD signal, during the Imagine task than the Estimate task (A), when reward magnitude was increased (B), and the overlap of the two results (C). D, E, BOLD signal, in the activated region of the conjunction image (D) and when reward magnitude was increased in the Imagine task (E).

Conclusion Based on the results, the researchers concluded that EP indeed attenuates TD and that mrPFC is involved in mediating this effect by representing the undiminished reward value of envisioned events.

It was suggested that mrPFC utilizes information from the hippocampus for the representation, and that the immediate experience of the delayed reward facilitated

by EP may promote future-oriented decisions.

This study reliably tested the cognitive function of interest since the fMRI analysis focused on the period of EP before the reward choice was made, unlike a previous study which obtained data during the choice8. Although rewards were not actually given to the subjects, past studies have shown that hypothetical and actual rewards exhibit similar discounting trends9 and that similar brain regions are involved in evaluating both types of rewards10.

Thus, the hypothetical rewards used in the present study were close to real-life situations so the findings could potentially be applied.

However, the authors themselves admitted that they could not rule out the possibility that subjects may have performed EP during the estimate task, since emotional intensity was measured by subjective self-reporting. The decisionmaking process may also be oversimplified because monetary decision-making in real life involves factors other than time delays such as risks, urgency, or personal interests.

In addition, fMRI BOLD is not a direct measure of neural activity and the level of oxyhemoglobin can be affected by other factors such as breathing and heart rate.

Thus, results from fMRI are correlational and a causal relationship between mrPFC activity and the EP effect cannot be concluded. Furthermore, since functional connectivity was merely measured by correlated activation of voxels in mrPFC and the hippocampus, the two areas could be receiving common input from a third region instead of communicating with each other.

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Future Directions

This study is significant because it identified a candidate for the neural correlate of the EP effect, allowing future research to focus on obtaining causal evidence for its role, and elucidating neural connections with other regions critical for this process. A more recent study of a patient with hippocampal damage questioned the importance of functional connectivity between mrPFC and the hippocampus in the valuation process. Because the patient was able to value future rewards despite not being able to imagine them, it suggested that the mrPFC-HC coupling could play a role but it may not be essential11. To resolve these questions, future experiments could use TMS targeted at mrPFC or the hippocampus to determine whether activity in the areas and the communication between them are critical in the EP effect. Because converging operations would increase the validity of the findings, lesion patients may also be studied. A multiple case-study approach would best examine both the average and variability of data among several patients to see if generalizations could be made.

Seeing how mrPFC-HC coupling may not be required, research can be conducted to understand other neural circuits that may attenuate TD. The present study had suggested an association between greater emotional intensity and greater EP effect.

More recently, emotion was found to affect the direction of TD, where positive emotion led people to prefer a delayed larger reward and negative emotion led them to choose an immediate smaller reward12.

Therefore, neuroimaging studies can be done to investigate any crosstalk that may occur between mrPFC and emotional areas of the limbic system which could potentially mediate the EP effect on TD.

Finally, the findings of this study have

17 significant implications in psychological, medical, and social realms, particularly by contributing to the current understanding of impulse disorders such as substance abuse and gambling. Lower performance in the CFC scale which was used in this study predicts more frequent smoking and alcohol consumption13. CFC ratings have also been implicated in financial behavior with long-term interests in mind14. Because the present study found that EP was most effective for individuals who were less likely to consider the future, EP could potentially mitigate impulsive behaviors as a form of therapy. Neuroimaging studies can be done to compare mrPFC activation in individuals with disorders to the activation in healthy individuals. The effect of EP on activation and behavior can also be observed and compared. After establishing a causal link between the underlying brain region and the cognitive and behavioral effects of EP, it may eventually be possible to use exogenous stimulation of mrPFC to treat severe cases.

References 1. Green, L. & Myerson, J. A discounting framework for choice with delayed and probabilistic rewards.

Psychol. Bull. 130, 769 (2004).

2. Hassabis, D. & Maguire, E.A. Deconstructing episodic memory with construction. Trends.

Cogn. Sci. 11, 299-306 (2007).

3. Lee, D., Rushworth, M. F. S., Walton, M. E., Watanabe, M. & Sakagami, M. Functional specialization of the primate frontal cortex during decision making. J. Neurosci. 27, 81708173 (2007).

4. Addis, D. R., Wong, A. T. & Schacter, D. L. Remembering the past and imagining the future: common and distinct neural substrates during event construction and elaboration.

Neuropsychologia. 45, 1363-1377 (2007).

5. Plassmann, H., Oâ&#x20AC;&#x2122;Doherty, J. & Rangel, A. Orbitofrontal cortex encodes willingness to pay in everyday economic transactions. J. Neurosci. 27, 9984-9988 (2007).

6. Benoit, G. B., Gilbert, S. J. & Burgess, P. W. A neural mechanism mediating the impact of episodic prospection on farsighted decisions. J. Neurosci. 31, 6771-6779 (2011).

7. Strathman,A.,Gleicher,F.,Boninger,D.S.&Edwards, C. S. The Consideration of Future Consequences: Weighing Immediate and Distant Outcomes of Behavior. J. Pers. Soc. Psychol. 66, 742-752 (1994).

8. Peters, J. & Buchel, C. Episodic future thinking reduces reward delay discounting through an enhancement of prefrontal-mediotemporal interactions. Neuron. 66, 138-148 (2010).

9. Johnson, M. W. & Bickel, W. K. Within-subject comparison of real and hypothetical money rewards in delay discounting. J. Exp. Anal.

Behav. 77,129-146 (2002).

10. Bickel, W. K., Pitcock, J. A., Yi, R. & Angtuaco, E. J. Congruence of BOLD response across intertemporal choice conditions: fictive and real money gains and losses. J. Neurosci. 29, 8839-8846 (2009).

11. Kwan, D., Craver, C. F., Green, L., Myerson, J., Boyer, P. & Rosenbaum, R. S. Future decisionâ&#x20AC;? making without episodic mental time travel.

Hippocampus. 22, 1215-1219 (2012).

12. Liu, L., Feng, T., Chen, J. & Li, H. The value of emotion: how does episodic prospection modulate delay discounting?. PloS one. 8, e81717 (2013).

13. Beenstock, J., Adams, J. & White, M. The association between time perspective and alcohol consumption in university students: Cross-sectional study. Eur. J. Public Health. 21, 438-443 (2011).

14. Joireman, J., Sprott, D. E. & Spangenberg, E. R. Fiscal responsibility and the consideration of future consequences. Pers. Indiv. Differ. 39, 1159-1168 (2005).

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Gone â&#x20AC;&#x153;TAI-FISHâ&#x20AC;?ing: A novel way to map emotional valence in the brain Authors: Rachel Oh, Steven Meas, Zara Duquette, Sabina Freiman, & Sarah Peters University of Toronto - HMB420 October 27, 2014

Background Emotions are an integral part of the human experience, helping us navigate the world by motivating our actions. Previous studies have often turned to functional magnetic resonance imaging (fMRI) to compare activation patterns between different emotions. A meta-analysis by Vytal and Hamann1 confirmed that there are indeed unique and consistent activation patterns for discrete emotions such as happiness, fear, and disgust; however, it was the patterns and not distinct regions that made emotions unique. For example, happiness, anger, fear, and disgust all involved the anterior cingulate cortex (ACC), while activation of both the ACC activation and the right superior temporal gyrus differentiated happiness from other mood states1. However, researchers were unable to determine if the same specific ACC neuronal networks were responsible for processing such a range of emotions.

A study by Shabel and Janak2 determined that the amygdala is critical for the processing of both aversive and appetitive stimuli, and that each stimulus type activates the same cell populations. Due to limitations of fMRI and other neuroimaging techniques, researchers were unable to determine if subpopulations within these larger populations of cells were being differentially activated. Another study conducted by Taha and Fields3 found that neurons in the nucleus accumbens (NAc) might also be important

in appetitive behavior: some NAc neurons fired for appetitive behavior, while other populations inhibited appetitive behavior.

In the current study, Xiu et al.4 sought to create a technique that would allow them to view activation patterns for emotions of different valence, focusing on aversive and appetitive stimuli. Authors focused on elucidating whether the same neurons, or different neurons within the same region, were responsible for each valence. Solving which neuronal populations activate for particular valences creates the opportunity to construct a valence map to show how the human brain processes emotion.

Results The combination of immunohistochemistry (IHC) and in-situ hybridization (ISH) takes advantage of the fact that mRNA and protein levels within cells are expressed in differential time courses, such that mRNA levels will reach a maximum and subsequently return to a minimum before protein levels peak. This means that if an organism is exposed to two sequential stimuli, there will be a clear separation of mRNA and protein signals when staining for IHC and ISH in acute brain slices. One difficulty with this technique was finding a gene that was comparable in all cell types being studied. Researchers solved this problem by using c-fos, an immediate

early gene (IEG) that is rapidly activated in response to cell activity. Using this technique along with the administration of positive valence stimuli (morphine, chocolate) and negative valence stimuli (foot-shock, restraint), authors identified three principal patterns in a preliminary emotional valence map: segregated, convergent and intermingled.

The segregated pattern in the central amygdala (CEA) and bed nucleus of the stria terminalis involved discrete anatomical areas with high fidelity activation to positive or negative valence stimuli. In particular, authors revealed that morphine-induced c-fos+ neurons in the centro-lateral region of the amygdala (CEl) were the CEl-off type (which inhibit central medial amygdala neurons) as seen by double labeling for PKC-delta. These results suggested that the two distinct valence stimuli activate distinct subpopulations of neurons in the amygdala that have opposing roles in fear conditioning. The convergent pattern in the paraventricular nucleus consisted of neurons that were indiscriminately activated by either type of stimulus and seemed to respond in a valence-independent manner.

Lastly, the NAc showed an intermingled interaction pattern where different neurons responded only to certain types of valence information, but existed within similar spatial locations. Furthermore, authors found a correlation between the two distinct subpopulations of NAc neurons—D1- and D2- medium spiny neurons (MSNs)—and their activation of specific neural ensembles, suggesting that the D1- and D2-MSNs differentially code appetitive and aversive information through the Go and No-Go pathways, respectively.

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Conclusions The authors successfully implemented an updated method to generate neural maps of emotional valence in the NAc and proposed a correlation with the function of distinct neuronal subpopulations. In the CEA, CEl-off neurons and co-localization with PKC-delta staining showed that these neurons responded specifically to morphine (and other similar aversive stimuli). In the NAc, D1- and D2-MSNs leading to the Go and No-Go pathways whose balance is important in the context of certain diseases, like Parkinson’s seemed to be activated by specifically aversive or appetitive stimuli.

TAI-FISH allows for visualization of responses to stimulus valence at the neuronal level by enhancing c-fos protein signals and creating images with distinct, salient patterns of activation. If the maps are indeed accurate, it is critical to consider how they could be further investigated and elaborated upon in future research. First, valence maps could be used to identify animal responses to stimuli based on characteristic neural network activation. If ‘trademark’ neural network patterns that characterize emotion across the valence spectrum are elucidated, it could be possible to ‘read’ animals’ characterization of emotionally valent stimuli (i.e., whether they perceive a stimulus to be positive or negatively valenced) based on these patterns. At the same time, this application could be difficult: authors explained the networks in a way that made them seem localized, but it would likely take many experiments and trials to accurately localize these neural networks. The fact that some neurons express both D1- and D2-receptors, and that some D1-MSNs can respond to both appetitive and aversive

stimuli (possibly in a competitive process of inhibition, similar to retinal cells which exhibit competition to encode visual stimuli), adds to the challenge.

Although TAI-FISH is novel as a molecular technique, it is limited as a stand-alone tool; however, it can be useful if used in combination with, for instance, catFISH, to provide a more complete map. A study

by Nieh et al.5 employed an optogenetic technique to establish a causal relationship between neural activity, emotional valence, and behavior by specifically manipulating individual neurons within the amygdala and striatum in direct and indirect pathways to modulate their distinct functions for aversion, fear, salience, and reward processing. The current valence map provided by TAI-FISH could be used in future research as a guide for both measuring and manipulating emotional behavior. Presently, however, the next steps should focus on successful replication of Xie et al.â&#x20AC;&#x2122;s proposed map in order to firmly establish TAI-FISH as a functional method as well as to provide stronger, more detailed evidence to further delineate the details of NAc function.

References 1. Vytal K & Hamann S. Neuroimaging support for discrete neural correlates of basic motions: a voxel-based meta-analysis. J Cogn Neurosci. 2010; 22: 2864â&#x20AC;&#x201C;2885.

2. Shabel S & Janak P. Substantial similarity in amygdala neuronal activity during conditioned appetitive and aversive emotional arousal.

Proc Natl Acad Sci. 2009; 106: 15031-15036.

3. Taha S & Fields H. Encoding of palatability and appetitive behaviors by distinct neuronal populations in the nucleus accumbens.

Journal of Neuroscience. 2005; 25: 1193-1202.

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4. Xiu J, Zhang Qi, Zhou T, Zhou T, Chen Y, Hu H. Visualizing an emotional valence map in the limbic forebrain by TAI-FISH. Nature. 2014; doi:10.1038/nn.3813.

5. Nieh EH, Kim SY, Namburi P & Tye KM. Optogenetic dissection of neural circuits underlying emotional valence and motivated behaviors. Brain Research. 2014; 1511: 73-92.

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Adding to the Puzzle: Another Neurotransmitter for the REM Sleep Circuit Author: Sara Pintwala University of Toronto - HMB420 October 27, 2014 A review based on the paper: Optogenetic identification of a rapid eye movement sleep modulatory circuit in the hypothalamus. Nature neuroscience 16, 1637â&#x20AC;&#x201C;43 (2013). By: Jego S, Glasgow SD, Herrera CG, Ekstrand M, Reed SJ, Boyce R, Freidman J, Burdakov D and Adamantis A

Introduction The exact circuitry in the brain which generates Rapid Eye Movement (REM) sleep has yet to be fully identified. It has long been known that reciprocal inhibition between cholinergic, serotonergic and noradrenergic neurons within the pons contribute to this network1. Later, GABA and glutamate were found to be related to the circuitry as activators of REM sleep onset2.

Background The purpose of this paper was to build upon previous literature3,4. Of particular interest were neurons in the lateral hypothalamus (LH) expressing the peptide neurotransmitter Melanin Concentrating Hormone (MCH). In these studies, the stimulation of MCH neurons produced increases in both REM and Non-REM (NREM) sleep behaviours5. These and other experiments further implicate this cell group for important role in REM sleep function6.

However, more causal evidence was needed.

As a result, in the current paper optogenetic manipulation of MCH neurons in the LH in vivo was performed to further explore their role in REM sleep.

Results:

REM sleep is Maintained with Stimulation of MCH Neurons Cells of the LH were successfully targeted with a Cre-inducible AAVdj making MCH neurons express the channerhodopsin ChETA. Using blue light stimulation of MCH neurons at both 1 and 20 Hz no effect on NREM sleep duration was found.

However, REM sleep duration increased by 47%. Activation of these cells at the onset of NREM sleep also produced an increased probability of shift from NREM to REM sleep by 71% and 80%. To confirm the efficacy of these results, the excitatory channelrhodopsin 2-derived opsin SSFO was used. Activation of SSFO+ MCH neurons also extended REM sleep duration and had no result on NREM sleep.

Results:

Theta Rhythms Slow with Inhibition of MCH neurons The halorhodopsin eNpHR3.0 to hyperpolarizeMCHneuronssloweroscillations in theta waves to waves of lower frequency were produced. The authors then used archaerhodopsin and were able to confirm the shift towards slow theta oscillations. It was also noted that because slower theta rhythms occur naturally during REM sleep episodes these results were authentic.

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Figure 1: (a) The effect on REM sleep duration in Mchr1+ and -/- mice at 1 and 20Hz (n=6). (b) The effect of bicuculine application of IPSC amplitudes in Mchr1+ and -/- mice (n=6). (c) The effect on IPSC frequency in (c) Mchr1 + mice (n=6), and, (d) Mchr1-/- mice (n=6) at 1 and 20 Hz.

The article then further explored the genetic profile of MCH neurons. MCH neurons were characterized as expressing glutamate decarboxylase (Gad67), indicating the expression of GABAA. These cells were found to project to the tuberomamillary nucleus (TMN) where they mediated inhibitory post-synaptic currents (IPSCs) sensitive to bicuculline, a competitive GABAA antagonist. In MCHR1+ mice, stimulation of ChETA+ MCH neurons at 20 Hz increased the frequency of IPSCs, but lost significance in MCHR1 -/- mice. The deletion of this receptor and its effect on IPSCs suggests a presynaptic mechanism of the MCHR on GABA transmission.

Results:

REM sleep is Maintained with Stimulation of MCH Neurons

Figure 2: (a) An illustration of potential MCH neuronal projections in the mouse brain. (b) The effect on REM sleep duration with optogenetic activation of receptors in control, LH ChETA+, MS ChETA+, TMN ChETA+ and DR ChETA+ mice (n=6).

To elucidate the role of these neurons in REM sleep, the locations of the projections of these cells were examined. Three connections were explored: the LH to medial septum (MS), the LH to TMN, and the LH to the dorsal raphĂŠ. The former two made significant, functional projections while the latter did not.

Activating terminals of LH-TMN and LH-MS neurons produced increased durations of REM sleep, identical to the activation of MCH cell somas7.

Novelties, Conclusions and Caveats The purpose of this experiment was to extend the current knowledge of the neural network regulating REM sleep.

Activation of MCH expressing neurons of the LH extends the duration of REM sleep, as is consistent with previous literature5.

A novelty of this paper was the functional classification of these neurons. In addition to expression of MCH these cells were characterized to express GABAA. The significance of this is seen when considering the projections of these neurons. LH cells were found to make critical connections to the TMN and the MS. Histaminergic neurons of the TMN are responsible for the stabilization of wakefulness and cortical

arousal. The MS is known for the production of theta rhythms, which dominate the hippocampus during REM sleep8. These findings gave the paper its strength. The authors were able to provide experimental and functional evidence of the role of MCH- GABAA-expressing neurons of the LH as a cell group external to the brain stem responsible for stabilizing REM sleep.

A shortcoming of the paper was in the nature of the connections from the LH to the TMN and the MS. It was suggested that the connections were of monosynaptic morphology, but then was never addressed7. The authors spent much time reproducing the experiments from previous studies before reaching the most significant finding of their study. If the authors had chosen to focus more on this for their paper, it would have given it more novelty.

Future Directions The use of halorhodopsin was used to optogenetically silence MCH neurons.

The result of this was a decreased latency of REM sleep, and not the complete suppression of it. This reinforces the idea presented in the introduction of this report. The regulation of sleep is not from one centre alone, it comprises a network of neurons whose interconnected activity results in this behavior. For the full sleep circuit to be identified there is required many more experiments, potentially with the inhibition of multiple brain areas both within in outside the brainstem.

References 1. Pace-Schott, E. F. & Hobson, J. A. The neurobiology of sleep: genetics, cellular

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physiology and subcortical networks.Nat Rev Neuro 3, 591–605 (2002).

2. Lu, J., Sherman, D., Devor, M. & Saper, C. B. A putative flip-flop switch for control of REM sleep. Nature 441, 589–94 (2006).

3. Verret, L. et al. A role of melaninconcentrating hormone producing neurons in the central regulation of paradoxical sleep.

BMC Neuro. 4, 19 (2003).

4. Schöne, C. et al. Optogenetic probing of fast glutamatergic transmission from hypocretin/ orexin to histamine neurons in situ. J Neuro.

32,12437–43 (2012).

5. Konadhode, R. R. et al. Optogenetic stimulation of MCH neurons increases sleep.

The Journal of neuroscience : the official journal of the Society for Neuroscience 33,10257–63 (2013).

6. Hassani, O. K., Lee, M. G. & Jones, B. E. Melanin-concentrating hormone neurons discharge in a reciprocal manner to orexin neurons across the sleep-wake cycle. Proc the Nat Acad Sci. 106, 2418–22 (2009).

7. Jego, S. et al. Optogenetic identification of a rapid eye movement sleep modulatory circuit in the hypothalamus. Nature neuroscience 16, 1637–43 (2013).

8. Adamantidis, A. et al. Disrupting the melanin-concentrating hormone receptor 1 in mice leads to cognitive deficits and alterations of NMDA receptor function.The European journal of neuroscience 21, 2837–44 (2005).

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Cognitive and Emotional Processes Differentially Affect Moral Dilemma Judgments Authors: Meghna G., Mansi P., Saba S., and Sonja S. University of Toronto - HMB420 October 27, 2014 Moral dilemmas are situations in which decisions need to be made where each decision conforms to certain moral values but contravenes others resulting in a conflict. There are two types of moral dilemmas: personal dilemmas and impersonal dilemmas1. There are 3 criteria for a situation to be categorized as a moral personal dilemma1. Furthermore, moral impersonal dilemmas will fulfill only 1 or 2 of the 3 criteria, while moral personal dilemmas will fulfill all 31. The mechanism underlying judgment formation during moral dilemmas is not yet fully understood.

As such, there are two main opposing theories that attempt to explain the neural underpinnings for the decision-making process during a moral dilemma; the single process theory2 and the dual-process theory1. The single-process theory posits that emotional and cognitive processes in the brain work together to influence the resulting moral judgment2. The dualprocess theory, on the other hand, suggests that the cognitive processes and emotional processes in the brain work independently and compete with each other to form moral judgments1,3.

The right dorsolateral prefrontal cortex (rDLPFC) has been associated with the cognitive processes involved during moral decision-making and the right temporalparietal region (rTPJ) is associated with the emotional processes as the rTPJ plays a role in social cognition, theory of mind, and empathy4,5.

All previous studies investigating the interplay of brain regions during moral dilemmas have either been correlational studies or have been conducted on patients with brain damage and lesions6,7,8.

The objective of this study was to use transcranial magnetic stimulation (TMS) to establish a causal link between rDLPFC and rTPJ activity in the brain, and judgments made during a moral dilemma, to see if these processes differentially affect different types of moral dilemmas.

In support of the dual-process theory, the authors of the paper hypothesized that there will be a double dissociation between the activity of the rTPJ and the rDLPFC. The authors hypothesized that disrupting rTPJ activity will affect impersonal dilemma judgments and disrupting rDLPFC activity will affect personal dilemma judgments.

To test the hypotheses, the authors used TMS to disrupt the function of the rDLPFC and rTPJ in a time dependent manner.

Three pulses of TMS were applied 1.5, 2, 2.5, or 3 seconds after the presentation of the dilemma. Subsequently, participants were required to judge the appropriateness of the action presented in the moral dilemma and rate the following: desire to change their judgment, confidence in their decision, feeling of responsibility, and feelings of regret after making the judgment.

The results indicate that disruption of activity in both brain regions 2.5 seconds after stimulus presentation led to an increase in the proportion of participants that indicated that the action performed in the moral dilemma was inappropriate.

However rDLPFC stimulation only affected moral personal dilemma judgments (Figure 1A) and rTPJ stimulation only affected moral impersonal dilemma judgments (Figure 1B). Furthermore, ratings of regret were influenced by disruption of the rDLPFC activity yielding significantly lower ratings of regret in all types of dilemmas compared to TMS at the rTPJ.

Figure 1 Proportion of inappropriate responses at 4 times of TMS application over the rDLPFC and rTPJ for 3 types of moral dilemma.

Figure 2 Rating of regret for all 3 types of moral dilemmas after the application of TMS over rDLPFC and rTPJ.

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This study addresses the differences in current theories of moral decision making through disruption of key brain regions associated with emotional and utilitarian decision making. As hypothesized, TMS revealed a double dissociation between the rTPJ and moral impersonal decisions, and the rDLPFC and moral personal decisions, thus supporting a dual process theory of morality. This is the first controlled study to show a causal link between the rTPJ, rDLPFC, and moral judgments via the use of TMS. This s tudy has an advantage over previous studies6,7,8 investigating moral judgments as TMS provides much better control than lesions and brain damage, resulting in a better understanding of the brain areas that are implicated in moral judgments.

This study, however, has some limitations. A major disadvantage is the lack of information providing regarding the effect of TMS on these regions. Results suggest an inhibitory effect on the rDLPFC and an excitatory effect on the rTPJ. While the prototypical switch and footbridge dilemma are contextually similar, other dilemmas used as stimuli in this study were not contextually similar9. Hence, some of the differences between moral personal and impersonal decisions could be the result of unmatched personal and impersonal scenarios. Additionally, while the participantsâ&#x20AC;&#x2122; age ranged from 18-54, the mean age is 23.7. This is problematic because corticostriatal loops which play a critical role in decision making are not completely developed until the ages of 212510.

Despite its weaknesses, this study is crucial in enhancing our understanding of the functional significance of these regions in moral decision making. Current studies have only investigated how the

brain responds when making decisions in hypothetical scenarios. Research suggests that there is greater activity in medial orbitofrontal cortex and ventral striatum when making a real choice compared to decisions made in hypothetical scenarios11.

Therefore, future studies need to test the role of these brain regions using more realistic circumstances to capture results that are most similar to judgments made during real-life dilemmas. Follow up studies should explore how individuals from other cultures respond to moral dilemmas.

Individualistic cultures tend to emphasize the individual, while collectivist cultures focus on the greater good of the group.

Differences in beliefs may arise due to these cultural differences. For instance, people from collectivist cultures may choose to save the group over the individual more often than people from individualistic cultures. Finally, these findings can be applied to clinical populations. As a future study, activity and interactions between rTPJ and rDLPFC in patients with antisocial personality disorder can be investigated and repetitive-TMS to these regions could be considered a possible treatment option to treat the impairment in morality judgments caused by these disorders.

References 1. Greene, J. D., Sommerville, R. B., Nystrom, L. E., Darley, J. M., & Cohen, J. D. An fMRI investigation of emotional engagement in moral judgment. Science. 293, 2105-2108 (2001) 2. Bluhm, R. No Need for Alarm: A Critical Analysis of Greene’s Dual-Process Theory of Moral Decision-Making. Neuroethics. 1-18 (2014).

3. Greene, J. D., Nystrom, L. E., Engell, A. D.,

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Darley, J. M., & Cohen, J. D. The neural bases of cognitive conflict and control in moral judgment. Neuron. 44, 389-400 (2004) 4. Saxe, R., & Kanwisher, N. People thinking about thinking people: the role of the temporo-parietal junction in “theory of mind”. Neuroimage. 19, 1835-1842 (2003) 5. Carter, R. M., Bowling, D. L., Reeck, C., & Huettel, S. A. A distinct role of the temporalparietal junction in predicting socially guided decisions. Science. 337, 109-111 (2012) 6. Mendez, M. F., Anderson, E., & Shapira, J. S. An investigation of moral judgement in frontotemporal dementia. Cogn. behav. neurol. 18, 193-197 (2005) 7. Ciaramelli, E., Muccioli, M., Ladavas, E., & di Pellegrino, G. Selective deficit in personal moral judgment following damage to ventromedial prefrontal cortex. Soc. Cogn.

Affect Neurosci. 2, 84-92 (2007) 8. Koenigs, M., Young, L., Adolphs, R., Tranel, D., Cushman, F., Hauser, M., & Damasio, A. Damage to the prefrontal cortex increases utilitarian moral judgements. Nature, 446, 908-911 (2007) 9. McGuire, J., Langdon, R., Coltheart, M., & Mackenzie, C. A reanalysis of the personal/ impersonal distinction in moral psychology research. J. Exp. Soc, 45, 577-580 (2009) 10. Fareri D, Martin L, Delgado M. Rewardrelated processing in the human brain: developmental considerations.

Dev. Psychopathol. 20, 1191 – 1211 (2008) 11. Kang M, Rangel A, Camus M, Camerer C. Hypothetical and real choice differentially activate common valuation areas. J. Neurosci.

31, 461-468 (2011)

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Examining the role of systemic angiotensin II and exercise-induced neurogenesis in adult rat hippocampus: A preliminary investigation in need of stronger evidence Author: Romina Nejad, Twayne Pereira and Semih Topbas University of Toronto - HMB420 October 27, 2014 BACKGROUND Neurogenesis in the adult brain takes place in two distinct regions, the sub-ventricular zone of the lateral ventricle and the sub-granular zone (SGZ) of the hippocampus. Environmental enrichment and exercise have been shown to promote neurogenesis in the SGZ3. Here we review the role of angiotensin II (Ang II) as a mediator of exercise-induced neurogenesis.

RESULTS The authors show that both endogenous and exogenous increases in systemic Ang II leads to increased 5’-bromo-2’-deoxyuridine (BrdU) positive and doublecortin (Dcx) positive cells within the adult hippocampus. They further show that these effects are completely abolished with the use of Losartan, an Ang II receptor antagonist.

During development there are drastic anatomical and physiological changes that occur in a tightly regulated manner. Take the brain for example; it expands in size during development owing to the birth of new neurons, glia and various other support cells. Once we have entered adulthood the brain does not undergo major anatomical changes in a normal individuals. However, neurogenesis still occurs giving rise to astrocytes, neurons and parenchyma.

Neurogenesis in the adult brain takes place in two distinct regions, the sub-ventricular zone of the lateral ventricle and the sub-granular zone (SGZ) of the hippocampus – our focus will be on neurogenesis in the hippocampus.

CONCLUSIONS The findings show enhanced proliferation in the hippocampus to be accompanied by elevated plasma Ang II suggesting Ang II as the upstream signaling molecule for exercise-induced neurogenesis.

CRITICAL ANALYSIS Mukuda et al (2014) provide good evidence for angiotensin-mediated neurogenesis shown by an increase in BrdU positive cells in the granular cortical layer. However, the researchers do not provide a mechanism by which Ang II promotes neurogenesis. This is the first study that uses Ang II in an animal model to show neurogenesis.

Keywords: Neurogenesis; angiotensin II (Ang II); hippocampus; aerobic exercise; 5’-bromo-2’deoxyuridine (BrdU)

Within the SGZ neural progenitor cells will proliferate and differentiate into dentate granule cells (DGCs) and glia.

There are a multitude of factors such as signaling and environmental stimuli that govern this process. DGCs undergo a lengthy maturation process involving GABA and other factors before they become integrated with the local neuronal network1,2. Environmental enrichment and exercise have been shown to promote neurogenesis in the SGZ3. Plasma angiotensin levels increase during exercise and are known to increase trophic factors such as BDNF and VEGF4,5. Angiotensin II (AngII) acts through ATR1 and ATR2 with very different cellular outcomes. AngII via ATR1 mediates endothelial cell proliferation and

growth factor expression whereas ATR2 is involved in an apoptotic pathway6,7. Taken together, we review the role of AngII in hippocampal neurogenesis.

Research Overview Summary of Major Results

Effect of aerobic exercise on blood Ang II levels and cell proliferation in the adult rat hippocampus To examine endogenous increase of systemic Ang II levels, Mukuda et al. (2014) trained adult rats to run on the treadmill for 30 minutes a day for 7 consecutive days. They found an increase in blood Ang II levels in aerobic-exercise rats (n=4) compared to control non-exercising rats (n= 5) (Figure not shown). To investigate the effect of increased Ang II levels on cell proliferation, rats were intravascularly injected with 5â&#x20AC;&#x2122;-bromo-2â&#x20AC;&#x2122;-deoxyuridine (BrdU) 48h and 24h pre-mortem. Aerobic-exercise rats were seen to have higher increases in BrdU (+) cells in the hippocampal DG compared to nonexercising rats (Figure 1).

Effect of intra-atrial administration of Ang II on cell proliferation and neurogenesis To see whether the endogenous Ang II effects on cell proliferation in the adult hippocampal DG could be replicated without the use of exercise, Mukuda et al. (2014) administered 10 -5 M of exogenous Ang II to adults rats (n=8). Ang II was administered intraatrially. Control rats (n=8) were injected with saline using the same protocol. A blood sample was taken from rats post Ang I administration to ensure increased systemic Ang II levels. The authors found that Ang II injected rats show increases in BrdU (+) cells in the SGZ of the DG compared to controls (Figure 2, Panel A; A and B). Further, to test for neurogenesis, the authors did an immuno assay for doublecortin (Dcx). Ang II administered rats show higher

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Dcx (+) cells in the SGZ of the DG compared to control rats (Figure 2, Panel B; A, B and D).

To test whether it was the Ang II that mediates this increase in hippocampal cell proliferation and neurogenesis, the authors administer Ang II + losartan, an AT1R antagonist, to a third group of adult rats (n=6). Losartan injection abolishes increases in cell proliferation (Figure 2, Panel A; C) and neurogenesis in the hippocampal DG (Figure 2, Panel B; C). The number of BrdU (+) cells and Dcx (+) cells increase with exogenous Ang II administration, but show no effect when losartan is administered (Figure 2, Panel C).

Panel A

Panel B

Figure 1. Photomicrographs of the adult rat hippocampal DG. Panel A: (A) Depicts immunostaining for BrdU (+) cells in non-exercising rats- as indicated by arrow heads. (B) Depicts immunostaining for BrdU(+) cells in exercising rats. Panel B: represents the BrdU (+) cells in the SGZ of the DG of non-run (n=5) and run rats (n=4). Horizontal bars represent mean values. **P<0.01. Figure was taken from Mukuda et al. (2014).

Conclusions and Future Directions Taken together, the authors confirm that exercise induces neurogenesis and show a strong correlation between enhanced cell proliferation in the hippocampus and elevated plasma Ang II levels.

Theyfindthatbothendogenousandexogenous elevations in plasma Ang II concentrations result

29 Panel A

Panel B

Panel C

Figure 2. Photomicrographs of the adult rat hippocampal DG post-exogenous intra-atrial administration of Ang II. Panel A: depicts immunostaining for BrdU (+) cells in (A) control rats injected with saline (B) Ang II injected rats (C) Ang II + Losartan injected rats. Panel B: depicts immunostaining for Dcx (+) cells in (A) control rats injected with saline (B and D) Ang II injected rats and (C) Ang II + Losartan injected rats. Dcx (+) cells are depicted by the arrow heads (fluorescent red). Nuclei are depicted in blue using DAPI as a counterstain. Panel C: The upper graph represents the number of BrdU (+) cells in the SGZ of DG. The bottom graph represents the number of Dcx (+) cells in the SGZ of the DG. Mean values are represented by horizontal bars. This figure was taken from Mukuda et al. (2014). GCL- granule cell layer.

in a significant increase in Brd-U incorporating cells in the SGZ when compared with controls.

In addition, the proliferative effects of elevated plasma AngII are also shown to be blocked completely by losartan. Furthermore, elevated plasma AngII are shown to result in the increase of DCX expressing cells in the SGZ. Because DCX is a strong marker for immature neurons, the authors therefore conclude that, through the action of AT1R, elevated plasma AngII induces neurogenesis in the hippocampus by enchancing cell proliferation in the SGZ. However, the actions of AngII cannot be direct as the neural stems cells do not express AT1R to

induce the proliferative effects. Instead, AT1R is expressedontheluminalsurfaceoftheendothelial cells in brain microvessels. AT1R activation here promotes the expression and secretions of growth factors such as VEGF and BDNF which are thought to be the downstream mediators of the cascade initiated by elevated plasma AngII. Because neural stem cells do express receptors for the mentioned growth factors, the authors suggest VEGF and BDNF to directly bind to the neural stem cells here to induce cell proliferation.

Peripheral blockade of VEGF can abolish exercise induced neurogenesis10, suggesting the validity of the proposed mechanism.

In summary, the authors suggest that elevated plasma AngII following a treadmill exercise leads to neurogenesis indirectly by inducing the expression of VEGF and BDNF. Because the authors only suggest the role of VEGF and BDNF and fail to show and direct evidence for this, future studies can be performed to measure peripheral VEGF and BDNF levels in the adult rat following similar endogenous and exogenous elevations in plasma AngII. Furthermore, eventhough both VEGF and AngII are known to open the endothelial fenestrate of the microvessels, VEGF is known to be impermeable to the blood-brain-barrier. Thus, more research needs to be conducted to provide solid proof that increased VEGF expression induced by AT1R activation can indeed act directly on neural stems cells of the SGZ.

Critical Analysis Mukuda et al (2014) provide good evidence for angiotensin-mediated neurogenesis shown by an increase in BrdU positive cells in the granular cortical layer. Additionally, this is the first time where Ang II has been used in an animal model to show neurogenesis. It was shown in previous studies that exercise promotes neurogenesis but the research group identifies a molecular correlate that may be involved in this process.

However, the researchers do not provide a mechanism by which Ang II promotes neurogenesis. The authors suggest proliferation occurs through the Ang II ATR1 receptor, however other research groups have shown ATR2, an alternate receptor, may be involved in Ang II neurogenesis8. Moreover, they cite specific trophic factors, VEGF and BDNF involved in the Ang II pathway but do not provide evidence for this mechanism.

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6. Kang, Y.S. et al. Angiotensin II stimulates the synthesis of vascular endothelial growth factor through the p38 mitogen activated protein kinase pathway in cultured mouse podocytes. J. Mol. Endorcinol. 36, 377-388 (2006).

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Neurotherapeutics 11, 665-678 (2014).

9. Mukuda et al. (2014) Systemic angiotensin II and exercise-induced neurogenesis in adult rat hippocampus. Brain Research, [article in press pp.1-12].

10. Fabel et al. (2003) VEGF is necessary for exercise induced adult hippocampal neurogenesis. Eur. J. Neurosci. 18, 2809-2812

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