UNIVERSITY OF CALIFORNIA, SAN DIEGO Molecular and Pharmacological Evidence for the Role of Glucocorticoid Receptors in Alcohol Dependence A Thesis submitted in partial satisfaction for the requirements for the degree Master of Science in Biology by Lauren Grace Macshane Committee in charge: Professor George F. Koob, Chair Professor Eduardo R. Macagno, Co-Chair Professor Kathleen A. French 2014 © Lauren Grace Macshane, 2014 All rights reserved. The Thesis of Lauren Grace Macshane is approved, and it is acceptable in quality and form for publication on microfilm and electronically: ______________________________________________________________ ______________________________________________________________ Co-Chair ______________________________________________________________ Chair University of California, San Diego 2014 iii DEDICATION This is dedicated to the following: To my parents for being there and supporting me through all of my schooling. iv TABLE OF CONTENTS Signature Page....................................................................................................... iii Dedication............................................................................................................... iv Table of Contents..................................................................................................... v List of Figures.......................................................................................................... vi List of Tables............................................................................................................ vii Acknowledgements.................................................................................................. viii Abstract.................................................................................................................... ix Introduction.............................................................................................................. 1 Materials and Methods............................................................................................. 8 Results..................................................................................................................... 13 Discussion................................................................................................................ 17 Figures and Tables................................................................................................... 24 References.............................................................................................................. 33 v LIST OF FIGURES Figure 1: Deaths Attributed to Alcohol Abuse World Wide...................................... 24 Figure 2: GR Signaling............................................................................................ 24 Figure 3: Operant Chamber.................................................................................... 25 Figure 4: Vapor Chambers...................................................................................... 25 Figure 5: GR Phosphorylation in Alcohol Dependent and Nondependent Rats..... 26 Figure 6: Alcohol Self-Administration after Intra-CeA Mifepristone Injections........ 26 Figure 7: Systemic Mifepristone Effects on Alcohol Self-Administration................. 27 Figure 8: Systemic CORT113176 Effects on Alcohol Self-Administration.............. 28 Figure 9: Systemic CORT113885 Effects on Alcohol Self-Administration.............. 29 Figure 10: Systemic CORT108297 Effects on Alcohol Self-Administration............ 30 Figure 11: Systemic Mifepristone, CORT113176, and CORT113885 Effects on Saccharin Self-Administration.................................................................................. 30 Figure 12: Conversion Between Active Cortisol and Inactive Cortisone................. 31 Figure 13: CORT Bound GR Complex and Responses........................................... 31 vi LIST OF TABLES Table 1: Mifepristone, CORT113176, CORT118335, and CORT108297 characterization in in vitro assays............................................................................ 32 vii ACKNOWLEDGMENTS I would like to acknowledge Dr. George F. Koob and Dr. Olivier George for giving me the opportunity to work in Koob Lab and earn a Master's degree. I would also like to acknowledge Dr. Leandro Vendruscolo for guiding me through pharmacological experiments, answering all of my questions, helping me revise my thesis, and for his incredible patience and kindness. He ran the statistical analysis of the intra-CeA and effects of Mifepristone, CORT113176, CORT118335, and CORT108297 on alcohol self-administration. I would also like to acknowledge Dr. Scott Edwards for his input on the GR phosphorylation study and helping to edit my thesis. I would also like to acknowledge Dr. Carrie Wade for listening to and helping me clarify my thesis presentation. I would also like to acknowledge Dr. Timothy Whitfield for guiding me through rat addiction experimentation. I also would like to acknowledge Yanabel Grant for guiding me periodically with behavioral testing, and generally how to handle rats. I also would like to acknowledge Dr. Joel E. Schlosburg for helping me with dilutions and molecular binding assays. I would also like to acknowledge Eva Zamora-Martinez for teaching and introducing me to Western blot analysis, among other things. The Materials and Methods in full, Results in full, and Figures 5 – 11are being prepared for submission for publication of the material. Leandro F. Vendruscolo, Scott Edwards, Lauren G. Macshane, Joel E. Scholsburg, M. Adrienne McGinn, Eva R. Zamora-Martinez, Vez Rapute-Canonigo, Marian L. Logrip, Joseph K. Belanoff, Hazel J. viii Hunt, Pietro P. Sanna, and George F. Koob. The dissertation author is an author for this paper. Figure 5 was produced by Dr. Scott Edwards. Figures 6, 7, 8, 9, 10, and 11 were produced by Dr. Leandro Vendruscolo. Table 1 was produced by Dr. Hazel Hunt. ix ABSTRACT OF THE THESIS Molecular and Pharmacological Evidence for the Role of Glucocorticoid Receptors in Alcohol Dependence by Lauren Grace Macshane Master of Science in Biology University of California, San Diego, 2014 Professor George F. Koob, Chair Professor Eduardo R. Macagno, Co-Chair Alcoholism or alcohol dependence is characterized by a compulsion to seek and ingest alcohol, uncontrolled intake, and emergence of a negative emotional state (e.g., dysphoria, anxiety) during withdrawal. Similar to stress, we hypothesize that chronic x xi alcohol exposure and withdrawal sensitize brain stress systems. Glucocorticoid receptors (GR), activated by stress hormones (cortisol in humans; corticosterone in rodents), have been shown to be dysregulated in stress/reward-related brain regions. We report increased GR activation (indexed by GR phosphorylation) in the central amygdala (CeA), a stress-related brain region, in alcohol-dependent compared with nondependent rats. Consistently, GR antagonism with mifepristone injected directly into the CeA reduces alcohol self-administration in dependent rats compared with nondependent rats. Systemic GR antagonism with mifepristone also reduces alcohol self-administration specifically in dependent rats. Mifepristone, however, also inhibits progesterone receptors, which may participate in alcohol-related behaviors. Therefore, more selective drugs (CORT113176, CORT118335, and CORT108297) that do not block progesterone receptors but do bind GR were tested on alcohol self-administration. CORT113176 significantly reduced alcohol self-administration in dependent rats, providing unambiguous evidence for a functional role of GR in alcohol dependence. Finally, the GR antagonist CORT118335 reduced self-administration in both dependent and nondependent rats, whereas CORT108297 had no effect on alcohol self- administration in either dependent or nondependent groups. These findings suggest that behavioral outcomes depend on distinct conformational and signaling changes produced by each antagonist when associated with GR. The present results provide compelling evidence for GR dysregulation in alcohol dependence and suggest that GR constitutes a viable pharmacological target for alcohol dependence. xi Introduction Alcoholism or alcohol dependence is characterized by a compulsion to seek and ingest alcohol, uncontrolled intake, and emergence of a negative emotional state (e.g., dysphoria, anhedonia, anxiety) during withdrawal (Koob et al., 2014). Initially, drug intake in a nondependent state (not addicted) provides a positive, or pleasurable/rewarding emotional response (positive reinforcement) characterized by a general “high.” Positive reinforcement is defined as adding an appetitive or pleasant stimulus (e.g., alcohol) to increase the probability of a certain behavior or response to reoccur. As addiction (dependence) develops, the drug’s pleasant effect decreases and the brain stress system becomes sensitized (Koob and Le Moal, 1997, 2008; Koob et al., 2014). Drug intake becomes compulsive and is needed regularly to reach a normal emotional state and reduce/prevent negative emotions associated with drug withdrawal. In this case, negative reinforcement comes to predominate, i.e., the removal/relief of an unpleasant emotional state increases the likelihood of drug use. Among all drugs of abuse, alcohol is one of the most harmful and lethal. Fig. 1 illustrates the burden of disease caused by alcohol. Harmful use of alcohol leads to 2.5 million deaths globally each year (WHO, 2011) and about 80,000 deaths per year in the U.S. (National