CONTRIBUTIONS OF GENETICALLY DEFINED CELL POPULATIONS IN THE EXTENDED AMYGDALA TO EMOTIONAL BEHAVIOR Christopher Mazzone A dissertation submitted to the faculty at the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Neuroscience Curriculum. Chapel Hill 2017 Approved by: Fulton T. Crews Todd E. Thiele Garret D. Stuber Spencer L. Smith Thomas L. Kash © 2017 Christopher Mazzone ALL RIGHTS RESERVED ii ABSTRACT Christopher Mazzone: Contributions of Genetically Defined Cell Populations in the Extended Amygdala to Emotional Behavior (Under the direction of Thomas Kash) Emotional disorders, including anxiety, remain pervasive and debilitating conditions throughout the world despite decades of progress in the development of pharmacological treatments. Limitations in treatment efficacy exist, in part, due to our lack of understanding of the precise neural pathways and mechanisms underlying emotional disorders. Here I focus on the extended amygdala, an area of the brain that has long been implicated in the regulation of emotional behaviors. In this dissertation work, I use combinatorial approaches to determine the cellular identity and receptors that contribute to anxiety so that we may better develop more selective drug treatments for emotional disorders. In Chapter 2, I use Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to manipulate G protein-coupled signaling pathways in a population of inhibitory cells of the bed nucleus of the stria terminalis (BNST), a component of the extended amygdala. This approach allowed us to identify that activation of Gq-coupled signaling pathways within inhibitory cells of the BNST is sufficient to induce an anxiety-like state in mice. I then identified endogenous Gq-coupled receptors expressed by inhibitory cells of the BNST, ultimately creating a list of potential candidate receptors for alleviating anxiety. In Chapter 3, I further dissect BNST anxiety circuitry by focusing on the impact of serotonin on BNST corticotropin-releasing factor (CRF) expressing neurons. I show that the serotonergic input from the dorsal raphe (DR) nucleus to the BNST can induce both serotonin release and anxiety-like behavior. Using electrophysiological tools we found that serotonin produces opposing effects on membrane properties within BNST CRF neurons. Interestingly, selective serotonin reuptake inhibitors (SSRIs) are known to exacerbate learned fear responses and we found that inhibition of BNST CRF neurons is able to occlude the SSRI-induced enhancements of fear. These results highlight inhibition of BNST CRF neurons iii as a potential therapeutic strategy for SSRI-induced increases in anxiety seen in patients during the early stages of SSRI-based therapies. Together, these studies untangle part of the complex emotional circuits that may contribute to pathological emotional states and provide new therapeutic targets for the alleviation of both acute and SSRI-induced anxiety. iv ACKNOWLEDGEMENTS First, I’d like to thank the University of North Carolina at Chapel Hill and the Curriculum in Neurobiology for providing an outstanding environment for my graduate studies. The program directors, Dr. Bill Snider, Dr. Aldo Rustioni, and Dr. Garret Stuber, all oversaw and contributed to my progress as a student, and I’m grateful for the support they provided. I’d like to especially thank my graduate mentor, Dr. Tom Kash, for all of his guidance throughout this work. I can unequivocally say I will miss working in his lab and am deeply grateful for his mentorship during the past five years. As a mentor, he could not have been more supportive or understanding of life both within and beyond the lab. His perspective on mentorship is something I will carry with me well beyond graduate school. I’d also like to thank all of the current and former members of the Kash Lab for their help and wealth of support. Chapter 3 of this dissertation would not have been possible without the remarkable contributions of Dr. Catherine Marcinkiewcz, and I’m honored that our work together led to my co-first authorship on that manuscript. Beyond the Kash Lab, I’d like to especially thank Dr. Zoe McElligott who has been an invaluable mentor and friend, and who collected the fast scan cyclic voltammetry work in Chapter 3. I’d like to thank other members of the UNC Neuroscience community, including Dr. Todd Thiele, Dr. Bryan Roth, Dr. Fulton Crews, Dr. Spencer Smith, Dr. Ben Philpot, Dr. Scott Magness, Dr. Robert Currin, and Dr. Vladimir Ghuskasyan who all contributed greatly to my training across a breadth of techniques. Additionally, all of the work in this dissertation would be impossible without the incredibly attentive and compassionate animal care staff at UNC, particularly Ashley Meadows and Matt Ehinger. I’ll be forever grateful for their attention to detail and for their patience with all of my numerous requests and questions. To put it bluntly, I cannot imagine working with a better community of scientists and researchers than the team I’ve been fortunate to be a part of at UNC. Beyond the UNC community, I’d like to thank the many collaborators from other institutions that contributed to the work in this dissertation. In particular, I’d like to thank Dr. Yasmin Hurd and Dr. Michael Michaelides for assisting with the DREADD Assisted Metabolic Mapping study in Chapter 2, and Dr. Jom v Hammack, Dr. William Falls, and Dr. Andrew Fox for providing the data assessing 5-HT2CR effects on anxiety in the BNST in Chapter 2. I would like to thank Dr. Francisco Javier Rubio Gallego for providing the protocol used for single-cell dissociations. In work presented in Chapter 3, I’d like to thank Dr. Lindsay Halladay and Dr. Andrew Holmes for collecting the in vivo electrophysiology data, Dr. Giuseppe D’Agostino, Dr. Claudia Cristiano, and Lora Heisler for conducting the 5HT2C-Cre DREADD study, and Dr. Charu Ramakrishnan and Dr. Karl Deisseroth for providing the INTRSECT constructs. Lastly, I’d be remiss to not thank all of the incredible scientists I met at various conferences over the past five years: thank you all for inspiring my own work and providing semi-annual reminders of how fortunate I am to work in a field with great people far beyond the Research Triangle. Prior to graduate school, I worked as a technician in a neuropeptide laboratory run by Dr. Richard Mains and Dr. Betty Eipper at the University of Connecticut Health Center. There are no words that can convey my gratitude for my time in their lab, and for their continued friendship. I’d also like to thank Dr. James Chrobak for allowing me to volunteer in his lab while I was an undergraduate student at the University of Connecticut. His research and teaching ignited my initial interest in neuroscience. Lastly, I’d like to thank my high school AP Biology teacher Jerry Lentz. I have no doubt that the root of my scientific interests can be traced back to his enthusiastic lectures and challenging assignments. The ways our earliest mentors and teachers shape our lives cannot be overstated and I am so grateful to have been raised by great teachers. Lastly, I’d like to thank my family for their unwavering love and support throughout all stages of my life. To my mother, my father, and my sister: thank you for everything. vi TABLE OF CONTENTS LIST OF TABLES ........................................................................................................................................ ix LIST OF FIGURES ....................................................................................................................................... x LIST OF ABBREVIATIONS......................................................................................................................... xii CHAPTER 1 INTRODUCTION .................................................................................................................... 1 1.1 Anxiety in Humans ......................................................................................................................... 1 1.2 Rodent Models of Anxiety- and Fear-Related Behavior ................................................................. 2 1.3 Involvement of the Extended Amygdala in Anxiety and Fear ........................................................ 5 1.4 Anatomical Characterization of the BNST ..................................................................................... 7 1.5 Neuromodulation of BNST Function by Corticotropin-Releasing Factor and Serotonin ................ 8 1.6 Optogenetic and Chemogenetic Tools for Modulating Discrete Neural Circuits .......................... 14 1.7 Dissecting Contributions of Signaling within the BNST to Anxiety and Fear-Related Behavior ....................................................................................................................... 17 1.8 Figures ......................................................................................................................................... 20 CHAPTER 2 CHEMOGENETIC MODULATION OF Gq-COUPLED GPCR SIGNALING IN THE BNST INDUCES ANXIETY-RELATED BEHAVIOR .......................................................................... 22 2.1 Historical Context ......................................................................................................................... 22 2.2 Methods ........................................................................................................................................ 23 2.3 Results