Neural architecture underlying thirst regulation Thesis by Vineet Augustine In Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy CALIFORNIA INSTITUTE OF TECHNOLOGY Pasadena, California 2019 (Defended 13th February 2019) ii 2019 Vineet Augustine ORCID: 0000-0003-4431-1663 iii ACKNOWLEDGEMENTS The successful completion of any project requires many hands, and this is a PhD. I have numerous people to thank without whose help this would not have been possible. I would like to express my heartfelt gratitude to my advisor, Prof. Yuki Oka, for giving me the opportunity to work in his lab. His enthusiasm for science and the ability to work hard is infectious. More importantly, the freedom which he gave me allowed me to explore areas that in the end turned out to be very rewarding. Being his first graduate student, I got the invaluable opportunity to learn the ropes of setting up a new lab- buying equipment, setting up protocols, and most importantly hiring new members. This is one of the most valuable things I have learned and will forever be grateful. I could not have asked for a more productive graduate school experience. I would like to thank my thesis committee members, Prof. Thanos Siapas, Prof. David Anderson, and Prof. Viviana Gradinaru, for their invaluable input that forced me to go back to the drawing board and think. They were always available to answer my queries and provide advice. I hope to stay in touch and keep on learning from them. A special word of thanks to my program chairs, Profs. Siapas and Pietro Perona, for having faith in me and being supportive throughout. Without their guidance and support, this thesis would not have seen the light of the day. A special shoutout to Prof. K VijayRaghavan who is probably the busiest person in Indian science, but has always made time for me. His humility and passion is something I wish I could emulate. My high school teacher, Madam Sandipa Bhattacharjee, deserves a special mention. It was because of her help that I got through my graduate applications in the first place and has been a constant source of encouragement throughout. I would like to thank other lab members: Sangjun, Brittany, Kutal, Haruka, Takako, Yuan, Bo, and Allan, for being co-travelers along this journey. Though small, the Oka Lab has a very conducive environment for carrying out science and it is largely because of the lab members. Prof. Ralph Adolphs has been one of my greatest inspirations to pursue neuroscience. Since the time I was an undergraduate researcher in his lab till now, he has been there always. He is the reason why I got into Caltech. No words can express the gratitude I feel. One of the greatest gifts that I have received from Caltech are a bunch of friends whom I cherish dearly and will maintain for a lifetime. My cohort: Janis, Ryan, Gabi, Juri, and Mason. We lived together, danced on pool-tables, had crazy parties, did homework, egged each other on, and are now a family. My brothers from different mothers, Sangjun and Dhruv, were my pillars of support at Caltech. To literally ensuring I would have food every day to doing experiments together, from disappearing from lab all together midweek for roadtrips to waiting for hours at the emergency room, we did it all. Their company is what I will miss the most. Thanks folks! iv Pursuing a PhD is a long journey, where failure abounds much more than success. And during these times I always had my safe haven to go back to. My godfather, Prof. Charles Greene, who picked up a small town boy and instilled in him the belief that he can do well, supported him at every stage and gave him the most sage advice ever. This thesis will be incomplete without his mention. Thank you Chuck! Chilly and Chris aunty, Sinobi and Soniya chechi for having their doors always open, for paying a bunch of my fees and for sending me home-cooked food from across the country. Alice and Elizabeth aunty, Joy achan for their support in spirit. Abhishek and Supriya for all the road-trips across the US. Prateek, Lenin, Ved, Sweta, Ajaay, Prakash and Prem for all the fun conversations. Thank you! Lastly, but most importantly, my parents, Rosamma and Augustine, Julie and Joy, and my brother, Ashly, for being the main reason for where I am today. Their love, kindness, and support are my greatest blessings. And finally, a big thank you to my wife, Janet, who gave up everything she had - friends, family, career - to join me in a different country along this journey. She is my biggest cheerleader and my greatest strength. Words are inadequate a vessel to express what I feel for her. This thesis is for them. v ABSTRACT An important aspect of thirst is its quick quenching. When thirsty, you drink a glass of water for a few seconds; the water travels from the mouth to the stomach and you are satiated. The water has not yet been absorbed into the blood, so the brain needs to have mechanisms to signal stopping of drinking. It cannot simply depend on the body, as the body takes a good 15 - 30 minutes to even start absorption. In this dissertation, I describe dynamic thirst circuits that integrate the homeostatic- instinctive requirement for fluids, the consequent drinking behavior, and reward processing to maintain internal water balance. In Chapter 1, I show how neural populations in the lamina terminalis, a forebrain structure, form a hierarchical circuit architecture to regulate thirst. Among them, excitatory neurons in the median preoptic nucleus (MnPO) are essential for the integration of signals from the thirst-driving neurons of the subfornical organ (SFO). Thirst-driving neurons in the SFO receive temporarily distinct preabsorptive inhibition by drinking action and gastrointestinal osmolality sensing. A distinct inhibitory circuit, involving MnPO GABAergic neurons that express glucagon-like peptide 1 receptor (GLP1R), is activated immediately upon drinking and monosynaptically inhibits SFO thirst neurons. These responses are induced by the ingestion of fluids but not solids, and are time-locked to the onset and offset of drinking. Furthermore, loss-of-function manipulations of these neurons lead to a polydipsic, overdrinking phenotype. These neurons therefore facilitate rapid satiety of thirst by monitoring real-time fluid ingestion. In Chapters 2 and 3, I talk about how thirst triggers a strong motivational state that drives animals toward drinking behavior. The consequent fluid intake provides both satiation and pleasure of drinking to animals. However, how these two factors are processed and represented by the brain remains poorly understood. Here I will use in vivo optical recording, genetics, and intragastric infusion approaches to dissect thirst satiation circuits and their contribution to reward signals. Thirst-driving neurons in the subfornical organ (SFO) receive multiple temporally-distinct satiation signals prior to the homeostatic vi recovery: oropharyngeal stimuli induced by drinking action and gastrointestinal sensing of osmolality changes. In chapter 1, I have shown that drinking action is represented by inhibitory neurons in the median preoptic nucleus (MnPO). Here, I demonstrate that gut osmolality signals are mediated by specific GABAergic neurons in the SFO. These neurons were selectively activated by hypo-osmotic stimuli in the gut independent of drinking action. Optogenetic gain- and loss-of-function of this inhibitory population suppressed and increased water intake in thirsty animals, respectively. These results indicate that oropharyngeal- and gastrointestinal-driven satiation signals are transmitted to thirst neurons through different neural pathways. Furthermore, I investigated the contribution of thirst satiation signals to the reward circuit using a genetically-encoded ultrafast dopamine (DA) sensor. Interestingly, oral ingestion but not gut osmolality changes triggered robust DA release. Importantly, chemogenetic activation of thirst-quenching neurons did not induce DA release in water-deprived animals. Together, this dissected genetically-defined thirst satiation circuits, the activity of which are functionally separable from reward-related brain activity. Taken together, these finding provide answers to some longstanding questions in the neural control of fluid intake, and appetite in general. vii PUBLISHED CONTENT AND CONTRIBUTIONS Augustine, V. et al. (2018). “Hierarchical neural architecture underlying thirst regulation”. In: Nature 555, pp. 204-209. doi: 10.1038/nature25488. V.A. conceived the research program, designed and carried out experiments, analyzed data and wrote the paper. Augustine, V. et al. (2018). “Peripheral and Central Nutrient Sensing Underlying Appetite Regulation”. In: Trends in Neurosciences 41, pp. 526–539. doi: 10.1016/j.tins.2018.05.003 V.A. conceived the manuscript outline, designed and made figures, and wrote the review. Augustine, V. et al. (2019). “Temporally-and spatially-distinct thirst satiation signals”. In: Neuron. doi:10.1016/ j.neuron.2019.04.039 V.A. conceived the research program, designed and carried out experiments, analyzed data and wrote the paper. Lee, S., Augustine, V. et al. (2019). “Chemosensory modulation of neural circuits for sodium appetite”. In: Nature 41, pp. 93–97. doi: 10.1038/s41586-019-1053-2. V.A. designed and carried out experiments and analyzed data Ichiki, T., Augustine, V. et al. (2019). “Neural populations for maintaining body fluid balance”. In: Current Opinion of Neurobiology 57, pp. 134–140. doi: 10.1016/j.conb.2019.01.014 V.A. conceived the manuscript