Interplay between peripheral signals, behaviour and the central clock Claire Gizowski Integrated Program in Neuroscience, McGill University October 2019 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Doctor of Philosophy © Claire Gizowski 2019 TABLE OF CONTENTS SECTION PAGE ABSTRACT ................................................................................................................................... 3 ACKNOWLEDGMENTS ............................................................................................................. 5 PEER-REVIEWED PUBLICATIONS ARISING FROM THIS WORK ............................... 6 CONTRIBUTION TO ORIGINAL KNOWLEDGE ................................................................. 7 CONTRIBUTION OF AUTHORS .............................................................................................. 9 LIST OF ABBREVIATIONS ..................................................................................................... 10 CHAPTERS .................................................................................................................................... CHAPTER 1 – Introduction ............................................................................................. 13 CHAPTER 1.0 – Foreword ................................................................................. 13 CHAPTER 1.1 – General introduction ............................................................... 13 CHAPTER 1.1.1 –The clock ............................................................................... 14 CHAPTER 1.1.2 – Anatomy and organization ................................................... 14 CHAPTER 1.1.3 – Circadian oscillators ............................................................ 16 CHAPTER 1.1.4 – Synchronization of clock time ............................................. 17 CHAPTER 1.1.5 – Clock inputs - photic ............................................................ 18 CHAPTER 1.1.6 – Clock inputs – non-photic .................................................... 19 CHAPTER 1.1.7 – Clock outputs ....................................................................... 20 CHAPTER 1.2 – Regulation of thirst ................................................................. 21 CHAPTER 1.3 – Aims of thesis ......................................................................... 22 CHAPTER 2 – The neural basis of homeostatic and anticipatory thirst ......................... 23 CHAPTER 3 – Clock-driven vasopressin neurotransmission mediates anticipatory Thirst prior to sleep ................................................................................ 69 CHAPTER 4 – Salt regulates clock time and output via an excitatory GABAergic neural circuit ......................................................................................... 109 CHAPTER 5 – Discussion ............................................................................................. 142 CHAPTER 6 – Conclusion ............................................................................................ 152 REFERENCES .......................................................................................................................... 154 APPENDIX A ............................................................................................................................ 179 1 Abstract Almost all living organisms exhibit circadian rhythms, ranging from complex humans to simple cyanobacteria. Circadian rhythms are critical to adapt an organism’s physiology and behaviour to the constraints of earth’s 24h light-dark cycle. In mammals, the suprachiasmatic nucleus (SCN) serves as the body’s master circadian clock and provides anticipatory homeostatic benefits. For example, the SCN opposes overnight adipsia by driving anti-diuretic hormone secretion and lowering body temperature to reduce water loss during sleep. Maintaining optimal body fluid balance is essential to sustain life and such rhythms are indispensable to prevent life-threatening pathologies. However, very little is known about how changes in the electrical activity of clock neurons actually mediate central rhythms. Likewise, we know almost nothing about how their activity can be altered by unanticipated changes in an organism’s physiology. Throughout my PhD, I investigated the bidirectional interactions between the central clock and central osmosensors. Specifically, how (i) the clock can regulate water intake prior to sleep to prevent overnight dehydration and (ii) how an acute rise in osmolality can alter clock time and its output-networks to drive homeostatic responses. Collectively, the findings of this thesis significantly advance our understanding of how the central clock functions. Résumé Presque tous les organismes vivants démontrent des rythmes circadiens, aussi bien les humains dans toute leur complexité que les cyanobactéries simples. Les rythmes circadiens sont primordiaux pour adapter la physiologie et les comportements d’un organisme au cycle de 24h sur terre. Chez les mammifères, le noyau suprachiasmatique (NSC) est reconnu comme étant l’horloge centrale responsable du contrôle des rythmes circadiens fournissant des avantages homéostatiques anticipatifs. Par exemple, le NSC contrecarre la déshydratation survenant durant la nuit en stimulant la sécrétion de l’hormone antidiurétique et en réduisant la température corporelle afin de réduire la perte d’eau durant le sommeil. Maintenir une balance des fluides corporels optimale est essentielle à la vie et de tels rythmes sont indispensables pour prévenir des pathologies mortelles. Toutefois, on n’en connaît que très peu sur la façon dont les changements dans l’activité électrique des 2 neurones-horloge modèrent les rythmes centraux. Pareillement, on ne connaît presque rien sur la façon dont leur activité peut être altérée par des changements inattendus de la physiologie d’un organisme. Tout au long de mon doctorat, j’ai étudié les interactions bidirectionnelles entre l’horloge centrale et les osmosenseurs centraux. Plus précisément, comment (i) l’horloge peut régulariser la prise d’eau avant le sommeil pour empêcher la déshydratation pendant la nuit et (ii) comment une augmentation aiguë en osmolalité peut altérer l’horloge et entraîner des réponses homéostatiques de ses réseaux de sortie. Ensemble, les résultats de cette thèse amènent des progrès significatifs sur notre compréhension du fonctionnement de l’horloge centrale. 3 Acknowledgements First, and foremost, I want to thank my supervisor, Dr. Charles Bourque, for helping me along this wild adventure. Without your unwavering support, none of this would have been possible. I want to thank you for always believing in me and giving me the confidence to ask bold questions and tackle problems from head-to-toe. Anyone who is lucky enough to have you as a mentor and friend will undoubtedly become a better scientist and person. Next, I would like to thank my parents, Nicole Régis and Jim Gizowski. Even though you do not entirely understand what I do, you have always supported my dreams. Without your love, sense of humour and support, I would not be where I am and who I am today. Merci à Mathieu Dionne pour ton aide avec la traduction de mon résumé scientifique en résumé poétique, car la science c’est d’la poésie! I also want to thank my current and former lab mates, Josh, Cristian, Zahra, Eric, Daniel, Nick, Nate, Masha, David and Katrina for your kindness and support throughout the years. We can only truly grow as scientists when we surround ourselves with people who challenge us to think differently. I want to thank my advisory committee members Drs. Keith Murai and Jesper Sjöström, the CRN, BRaIN program and the MNG for the thoughtful discussions and support offered to me throughout my degree. In addition, I want to thank CIHR and the RIMUHC for generously funding this work. Lastly, I want to thank the animal technicians and vets for taking care of my animals, giving me a circadian room, letting me modify cages and use their equipment to complete my experiments. Even though one of you did make me cry, you still helped me a lot. 4 Peer-reviewed publications arising from this work *GIZOWSKI C, and BOURQUE CW (2019) Salt regulates clock time and output via an excitatory GABAergic neural circuit. Submitted. *GIZOWSKI C, and BOURQUE CW (2018) Hypothalamic neurons controlling water homeostasis: it’s about time. Current Opinion in Physiology. 5: 45-50 ZAEZLER C, GIZOWSKI C, SALMON C, MURAI, KK, & BOURQUE, CW (2018) Detection of activity-dependent vasopressin release from neuronal dendrites and axon terminals using sniffer cells. Journal of Neurophysiology. 120: 1386-1396 *GIZOWSKI C, ZAELZER C, and BOURQUE CW (2018) Activation of organum vasculosum neurons and water intake in mice by vasopressin neurons in the suprachiasmatic nucleus. Journal of Neuroendocrinology, (Epub ahead of print) *GIZOWSKI C and BOURQUE CW (2018) The neural basis of homeostatic and anticipatory thirst. Nature Reviews Nephrology 14, 11-25 *GIZOWSKI C, TRUDEL E, and BOURQUE CW (2017) Central and peripheral roles of vasopressin in the circadian defense of body hydration. Best Practice & Research Clinical Endocrinology & Metabolism, 31, 535-546 *GIZOWSKI C and BOURQUE CW (2017) Neurons that drive and quench thirst. Science 357, 1092-1093 *GIZOWSKI C, ZAELZER C and BOURQUE CW (2016) Clock-driven vasopressin neurotransmission mediates anticipatory thirst prior to sleep. Nature 537, 685-688. 5 Contribution to original knowledge