Influence of Chrna5 on Habenulopeduncular Neurotransmission by Sanghavy Sivakumaran A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Physiology University of Toronto © Copyright by Sanghavy Sivakumaran 2020 ii Influence of Chrna5 on Habenulopeduncular Neurotransmission Sanghavy Sivakumaran Master of Science Department of Physiology University of Toronto 2020 Abstract The habenulopeduncular pathway is known to modulate aversion behaviours, which are tightly associated with Chrna5 gene expression. Chrna5 encodes the α5 nicotinic receptor subunit, which is highly expressed in the interpeduncular nucleus but remains pharmacologically intractable. Here we investigate the impact of this subunit using wild-type and Chrna5-deleted mice. We optogenetically activated the habenulopeduncular pathway; thought to co-release acetylcholine and glutamate, to probe the electrophysiological role of α5 in interpeduncular neurons in acute brain slice. Optogenetic stimulation of this pathway yielded heterogenous responses in wild-type and Chrna5 deleted mice. Unexpectedly, the absence of the Chrna5 gene led to stronger light evoked glutamatergic and GABAergic neurotransmission. These light evoked glutamatergic responses were acutely attenuated by nicotinic antagonists in Chrna5 deleted mice. Intriguingly, our optogenetic experiments did not evoke a distinct cholinergic current in either genotype. This work provides insight into the action of Chrna5 on habenulopeduncular circuitry and raises new mechanistic questions. iii Acknowledgments I would like to sincerely thank my supervisor Dr. Evelyn Lambe for her incredible support of me and my work in her lab. She continuously provided inquisitive insights and guidance for navigating my research project. Her encouragement, patience, support and advice have proven to be invaluable in helping me learn to approach difficult research questions. I am incredibly appreciative of the encouraging research environment and wonderful mentorship she provided. Evelyn consistently encouraged me to present my research at multiple conferences, allowed for TA opportunities and helped curate a very thorough and rewarding research and learning experience through my master’s program. I am incredibly grateful for my time in the Lambe lab and the valuable skills I have learned from Evelyn and her lab. I would also like to thank my supervisory committee members, Dr. Amy Ramsey and Dr. Junchul Kim, for their feedback, questions and support for my ever-evolving project. Both Dr. Ramsey and Dr. Kim have assessed my work thoroughly and consistently provided novel insights. Thank you to Dr. Steve Prescott, Dr. Bernard Le Foll, Dr. Richard Horner and Dr. Amy Ramsey for their feedback and insights as part of my thesis defense committee. Thank you to the former and current members of the Lambe lab, including: Dr. Daniel Sparks, Sridevi Venkatesan, Saige Power, Katheron Intson, Janice McNabb, Jennifer (Tianhui) Chen, Kevin (Yupeng) Liu and Steve (Ha-Seul) Jeoung for your advice, encouragement, feedback and insights. I am incredibly grateful to have such wonderful lab-mates and have greatly enjoyed working with you all! Finally, thank you to my family and loved ones for their encouragement and support throughout my research journey. iv Table of Contents Acknowledgments.......................................................................................................................... iii Table of Contents ........................................................................................................................... iv List of Tables ................................................................................................................................. vi List of Figures ............................................................................................................................... vii List of Appendices ....................................................................................................................... viii List of Abbreviations ..................................................................................................................... ix Chapter 1 ..........................................................................................................................................1 General Introduction ...................................................................................................................1 1.1 The interpeduncular nucleus ................................................................................................2 1.1.1 Functional role .........................................................................................................2 1.1.2 Anatomy and connectivity .......................................................................................3 1.1.3 Distribution of neurophysiological components ......................................................5 1.2 The habenula and fasciculus retroflexus ..............................................................................7 1.2.1 Functional role .........................................................................................................7 1.2.2 Anatomy and connectivity .......................................................................................8 1.2.3 Distribution of neurophysiological components ....................................................10 1.3 Nicotinic cholinergic receptors ..........................................................................................11 1.3.1 Nicotinic versus muscarinic cholinergic receptors ................................................11 1.3.2 Structure and conductance of nAChRs ..................................................................12 1.3.3 Mechanisms of nAChR characterization ...............................................................13 1.4 Mammalian implications of nAChR α5 .............................................................................15 1.4.1 Function and distribution of nAChR α5 in the human brain .................................15 1.4.2 Function and distribution of nAChR α5 in rodent models.....................................16 1.5 Modulation of habenulopeduncular signalling ..................................................................19 1.5.1 Habenula versus IPN regarding nAChR α5 modulated signalling ........................19 v 1.5.2 Habenulopeduncular co-release mechanisms ........................................................20 1.5.3 Habenulopeduncular physiological relevance .......................................................21 1.6 Thesis Objective.................................................................................................................22 Chapter 2 .......................................................................................................................................24 Methods for opto-physiological characterization of Chrna5 modulation of habenulopeduncular neurotransmission ....................................................................................24 Chapter 3 .......................................................................................................................................28 Results for characterization of habenulopeduncular neurotransmission modulated by Chrna5 ......................................................................................................................................28 Chapter 4 .......................................................................................................................................42 Discussion .................................................................................................................................42 4.1. Summary of findings..........................................................................................................47 4.2. Future Work .......................................................................................................................47 4.3. Conclusions ........................................................................................................................48 Bibliography ..................................................................................................................................50 Appendices .....................................................................................................................................60 Copyright Acknowledgements.......................................................................................................62 vi List of Tables Table 1: Electrophysiological properties of α5WT-ChAT and α5KO-ChAT cells………………30 vii List of Figures Figure 1.1: Summary IPN sub-nuclei and IPN afferent/efferent connections…………………......4 Figure 1.2: Summary of in situ hybridization of the genes encoding the nicotinic cholinergic receptor subunits in coronal rostral IPN sections of an adult mouse brain…………………………6 Figure 1.3: Summary of how the FR and habenular sub-nuclei innervates the IPN as well as the afferent/efferent connections mediating habenular signalling………………………………..……9 Figure 1.4: Summary of in situ hybridization of the genes encoding signalling associated proteins in the habenula of an adult mouse brain………………………………………………………..…11 Figure 1.5: Summary of Chrna5 in situ hybridization in coronal sections of an adult mouse brain………………………………………………………………………………………...……18 Figure 1: Optogenetic stimulation to characterize habenulopeduncular signalling………………29