THE INTERACTION OF PROGESTERONE AND ALLOPREGNANOLONE WITH FEAR MEMORIES A Dissertation by GILLIAN M. ACCA Submitted to the Office of Graduate and Professional Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Chair of Committee, Stephen Maren Co-Chair of Committee, Naomi Nagaya Committee Members, James W. Grau Farida Sohrabji Intercollegiate Faculty Chair, Jane Welsh August 2017 Major Subject: Neuroscience Copyright 2017 Gillian M. Acca ABSTRACT Sex differences in stress and anxiety disorders are well reported in the clinical population. For instance, women are twice as likely to be diagnosed with post-traumatic stress disorder (PTSD) compared to men. This difference is most profound during the reproductive years suggesting a role for sex hormones and their metabolites in regulating fear and anxiety. Previous studies suggest sex differences also occur in rodents, which is modulated by gonadal hormones. Despite the majority of clinical cases occurring in women, most rodent studies only include male subjects. An understanding of what contributes to this disparity is necessary for sex-specific therapies and interventions. Here, we use Pavlovian fear conditioning, a learned task in which male rats display higher fear levels compared to females, to understand the neurobiological basis for sex differences in fear and anxiety. Specifically, we focus on allopregnanolone (ALLO), a metabolite of progesterone and potent allosteric modulator of GABAA receptors and its effects in the bed nucleus of the stria terminalis (BNST) in male and female rats. The BNST is a sexually dimorphic brain region and site of hormonal modulation that has been implicated in contextual fear. Raising ALLO levels in the BNST of male rats successfully impaired contextual fear and blocking ALLO in the BNST of female rats successfully increased contextual fear. However, impairing contextual fear via ALLO in male rats proved to be the result of state-dependent learning, which was unique to the BNST as global impairments in both contextual and cued fear to ALLO were found in the basolateral amygdala. In females, hormonal changes across the rodent estrous cycle ii follow similar patterns to that of the menstrual cycle in women and provide a naturalistic model to observe changes in fear responses at the cellular level when ALLO levels are high or low due to progesterone fluctuations. Results suggest naturally circulating progesterone may also contribute to state-dependent contextual fear; however, ovariectomized rats administered progesterone do not. The work presented here aims to elucidate the mechanisms underlying ALLO’s influence on sex differences in fear and anxiety and contribute to the growing body of literature on women’s health. iii ACKNOWLEDGEMENTS I would like to thank my graduate school advisors, Drs. Stephen Maren and Naomi Nagaya for their guidance, support, and patience throughout my time at Texas A&M. I would also like to thank my committee members, Drs. Jim Grau and Farida Sohrabji for their valuable insights and support both academically and outside the University. This would not have been possible without the support of TAMIN and for that, I thank Dr. Jane Welsh and Sylvia Bernal Jones. To all of my fellow lab members, past and present, I sincerely thank you for keeping me sane during long hours and for always lending a hand on experiments when needed, no matter how tedious the job. In addition, I would like to thank the undergraduate research assistants for always helping out, even on the less than desirable tasks. I owe immense gratitude to my fellow students in TAMIN who have provided friendship and laughs over the past five years. Thank you all for being a source of support in the tough times and always willing to celebrate the good, no matter how small. None of this would have been possible without my biggest champions: my family. To my mother, for teaching me what it means to persevere, to my father, for always pushing me to be my best, to Donna for never saying no, and to my sister and best friend, Amanda, for always pushing me forward and checking up on me. Most importantly, I would like to thank my partner in all things, Ryan, who always reminded iv me of the big picture and kept me going each and every day. I always said if I ever finished this degree, it is as much yours as it is mine, so here you go. v CONTRIBUTORS AND FUNDING SOURCES This work was supported by a dissertation committee consisting of Professors Stephen Maren (chair) and Naomi Nagaya (co-chair) and James Grau of the Department of Psychology and Professor Farida Sohrabji of the Department of Neuroscience and Experimental Therapeutics. All work for the dissertation was completed by Gillian Acca with guidance and support from Drs. Stephen Maren and Naomi Nagaya. Graduate study was supported by a Heep Fellowship from Texas A&M University to Gillian Acca. This research was also supported by funding from the College of Liberal Arts to Drs. Stephen Maren and Naomi Nagaya, a grant from the National Institutes of Health (R01MH065961) to Dr. Stephen Maren, and a McKnight Memory and Cognitive Disorders Award to Dr. Stephen Maren. vi TABLE OF CONTENTS Page ABSTRACT ....................................................................................................................... ii ACKNOWLEDGEMENTS .............................................................................................. iv CONTRIBUTORS AND FUNDING SOURCES ............................................................. vi TABLE OF CONTENTS ................................................................................................. vii LIST OF FIGURES ........................................................................................................... ix LIST OF TABLES ............................................................................................................. x CHAPTER I INTRODUCTION ....................................................................................... 1 Sex Differences in Fear and Anxiety ............................................................................. 1 Hormonal Control of Fear .............................................................................................. 3 Neuroanatomical Substrates for Sex Differences in Fear and Anxiety ........................ 22 State Dependence: A Potential Mechanism of Hormone Action ................................. 31 Specific Aims ............................................................................................................... 31 CHAPTER II ALLOPREGNANOLONE IN THE BED NUCLEUS OF THE STRIA TERMINALIS MODULATES CONTEXTUAL FEAR IN RATS ................................ 33 Overview ...................................................................................................................... 33 Introduction .................................................................................................................. 34 Materials and Methods ................................................................................................. 37 Results .......................................................................................................................... 45 Discussion .................................................................................................................... 50 CHAPTER III ALLOPREGNANOLONE INDUCES STATE-DEPENDENT FEAR VIA THE BED NUCLEUS OF THE STRIA TERMINALIS ......................................... 56 Overview ...................................................................................................................... 56 Introduction .................................................................................................................. 57 Materials and Methods ................................................................................................. 59 Results .......................................................................................................................... 66 Discussion .................................................................................................................... 74 vii Page CHAPTER IV DIFFERENTIAL EFFECTS OF PROGESTERONE ON CONDITIONED FEAR IN CYCLING AND OVARIECTOMIZED FEMALE RATS ................................................................................................................................ 82 Overview ...................................................................................................................... 82 Introduction .................................................................................................................. 83 Materials and Methods ................................................................................................. 85 Results .......................................................................................................................... 93 Discussion .................................................................................................................... 99 CHAPTER V CONCLUSIONS ..................................................................................... 106 Summary of Results ................................................................................................... 106 BNST and Contextual Fear, Expanded ...................................................................... 109 Hormones, Metabolites, and State-Dependent Learning ........................................... 111 Incorporating Sex