SEX DIFFERENCES IN THE BEHAVIORAL AND NEUROMOLECULAR EFFECTS OF THE RAPID-ACTING ANTIDEPRESSANT DRUG KETAMINE IN MICE Dissertation Submitted to The College of Arts and Sciences of the UNIVERSITY OF DAYTON In Partial Fulfillment of the Requirements for The Degree of Doctor of Philosophy in Biology By Connor Francis Thelen UNIVERSITY OF DAYTON Dayton, OH December 2019 SEX DIFFERENCES IN THE BEHAVIORAL AND NEUROMOLECULAR EFFECTS OF THE RAPID-ACTING ANTIDEPRESSANT DRUG KETAMINE IN MICE Name: Thelen, Connor Francis APPROVED BY: ____________________________________ Pothitos M. Pitychoutis, Ph.D. Faculty Advisor ____________________________________ Mark Nielsen, Ph.D. Committee member ____________________________________ Madhuri Kango-Singh, Ph.D. Committee member ____________________________________ Renu Sah, Ph.D. Committee member ____________________________________ Amit Singh, Ph.D. Committee member ii © Copyright by Connor Francis Thelen All rights reserved 2019 iii ABSTRACT SEX DIFFERENCES IN THE BEHAVIORAL AND NEUROMOLECULAR EFFECTS OF THE RAPID-ACTING ANTIDEPRESSANT DRUG KETAMINE IN MICE Name: Thelen, Connor Francis University of Dayton Advisor: Dr. Pothitos M. Pitychoutis Over 350 million people currently suffer from Major Depressive Disorder (MDD). This debilitating neuropsychiatric disease is the greatest cause of disability worldwide, and available pharmacotherapeutic treatment options are largely ineffective in many depressed patients. Currently, conventional antidepressants (e.g., selective serotonin reuptake inhibitors; SSRIs and tricyclic antidepressants; TCAs) are only partially effective in managing depressive symptoms in MDD patients. Additionally, currently marketed antidepressant drugs require weeks to alleviate symptomology in depressed patients. Notably, women are twice as likely to be diagnosed with MDD as compared to men, yet until recently most clinical and preclinical studies in this field were conducted in the male sex. Ketamine, a non-competitive N-methyl-D-aspartate receptor (NMDAR) antagonist is the first rapid-acting antidepressant agent discovered that has the unique ability to relieve depressive symptoms within hours in both MDD patients and in animal models of depression. While earlier studies have determined that this drug has great promise for treating refractory depression, little is still known as to ketamine’s putative sex- differentiated effects and about its long-term safety. iv In the context of the current dissertation we explored the sex-differentiated behavioral and neuromolecular effects of antidepressant-relevant doses of ketamine following acute and repeated drug treatment in male and female mice. Specifically, we showed that behavioral responsiveness to ketamine in female mice is not accompanied by the neurochemical and synaptogenic effects that are typically observed in the male brain, and we further exposed a brain region that may be implicated in the sex-differentiated response to this drug. Moreover, we provided the first evidence that repeated ketamine dosing induced beneficial antidepressant-like effects in male mice but was associated with adverse anxiety-like and depressive-like effects in females. Taken together, the research findings pertaining to the current dissertation provide novel insights into the sex-dependent effects of the rapid-acting antidepressant drug ketamine and also suggests that the heightened sensitivity of females to this drug may put them at risk for sex-specific adverse effects following chronic treatment. v Dedicated to my parents, lab-mates, and my mentor Dr. Pothitos Pitychoutis, all of whom helped support and guide my research vi ACKNOWLEDGEMENTS My special thanks are in order to my mentor Dr. Pothitos “Takis” Pitychoutis, for providing the time, equipment, and funding necessary to complete the work contained herein, and for patiently providing guidance during the construction of this dissertation. Takis has been the embodiment of what it means to be a graduate advisor. His ability to celebrate our successes and provide steadfast support during periods of difficulty was the driving force that pushed me to completing my research and schooling at the University of Dayton. My appreciation also goes out to the other members of my graduate advisory committee who, alongside Dr. Pitychoutis, lent their knowledge and advice to help me become a better scientist and to conduct ethical and meaningful research that reaches beyond the bounds of our university; in this context I would like to thank, Dr. Amit Singh, Dr. Madhuri Kango-Singh, Dr. Mark Nielsen, Dr. Renu Sah, and the late Dr. Panagiotis Tsonis. Moreover, I would like to thank the current and the past Chairs of the Biology Department, Dr. Karolyn Hansen and Dr. Mark Nielsen for their meaningful support throughout my Ph.D. candidacy. I would also like to thank the undergraduate research assistants whose daily commitment to this research was invaluable. Special thanks goes to: Emily Flaherty, who helped with a variety of projects, including the quantification and analysis of dendritic spine densities as well as the in vivo microdialysis experiments; Joey Saurine for his help in Golgi staining; Jonathon Sens and Sara Mohamed for their help in western blotting and protein analysis, as well as Anthony Franceschelli, M.Sc., whose work I continued, for his support in the behavioral experiments. Moreover, I would like to thank all the vivarium vii assistants and vivarium director, Dr. Jeffrey Kavanaugh, who maintained a clean and orderly space to conduct research. I would also like to thank the other past and present members of the Pitychoutis lab for their help completing the long list of tasks needed to maintain a highly productive research environment. Graduate students Aikaterini Britzolaki and Benjamin Klocke, as well as all the undergraduate students I had the pleasure to mentor in the lab, including: Patrick Flaherty, Claire Cronin, Madison Schulze, John Coffey, Sean Koeller, Joseph Mauch, Jake Michalakes, Allison Noe, Jonathan Bode, Nicklaus Halloy, Sam Herchick, Evan Birmingham, Katie Fasoli, Anna Schaffstein, Radhika Pandit and Eric Schneider as they all played integral roles in helping me complete the research presented in this dissertation. This research could not have been conducted without the financial support provided by the University of Dayton Graduate School, the University of Dayton honors program, and awarded grants and fellowships. My sincere thanks also go to Cathy Wolfe, Janice Bertke, Rita McGinn, Casey Hanley, Grover Allen, and Susan Trainum for all their help and support, as well as to the Graduate Directors of the Biology Department, Dr. Madhuri Kango-Singh and Dr. Amit Singh. viii TABLE OF CONTENTS ABSTRACT…………………………………………………………………………...…iv DEDICATION……………………………………………………………………………vi ACKNOWLEDGEMENTS…………………………………………………………..….vii LIST OF FIGURES………………………………………………………………..……xiii LIST OF TABLES……………………………………………………………………….xv LIST OF ABBREVIATIONS AND NOTATIONS……………..……………..…….…xvi CHAPTER 1 ON THE SEX-DIFFERENTIATED EFFECTS OF THE RAPID-ACTING ANTIDEPRESSANT DRUG KETAMINE: AN INTRODUCTION…1 1.1 A Short History of Antidepressant Drug Discovery: from Imipramine to Ketamine…………………………………………………………………………..1 1.2 Antidepressant Effects of Ketamine: Clinical Evidence………………………4 1.3 Ketamine: The Prototype Rapid-acting Antidepressant Drug with a Novel Synaptogenic Mechanism of Action………………………………………………7 1.4 Sex Differences in Depression and Response to Conventional Monoaminergic Antidepressants…………………………………………….…..12 1.5 Sex Differences in the Antidepressant Effects of Ketamine: Insights from the Clinic and Animal Models……………………………...……..15 1.6 Focus of the Dissertation…………………………………………………….18 1.7 Figures………………………………………………………………………..20 ix CHAPTER 2 SEX DIFFERENCES IN THE TEMPORAL NEUROMOLECULAR AND SYNAPTOGENIC EFFECTS OF THE RAPID ACTING ANTIDEPRESSANT DRUG KETAMINE IN THE MOUSE BRAIN…………………………………………22 2.1 Introduction…………………………………………………………………..22 2.2 Experimental Procedures…………………………………………………….26 2.2.1 Animals…………………………………………………………….26 2.2.2 Experimental design and drug treatments………………………….26 2.2.3 Experiment #1: sex differences in ketamine-induced glutamate release in the mPFC of male and female mice…………………………...27 2.2.4 Experiment #2: sex differences in the synaptic molecular effects of ketamine………………………………………………………………….28 2.2.5 Experiment #3: sex differences in the time course of spine formation…………………………………………………………………28 2.2.6 In vivo brain microdialysis………………………………………....29 2.2.7 Glutamate analysis with high-performance liquid chromatography (HPLC)…………………………………………………………………...30 2.2.8 Synaptoneurosome preparation and immunoblotting……………...30 2.2.9 Golgi staining and spine analysis…………………………………..31 2.2.10 Statistical analysis………………………………………………...32 2.3 Results………………………………………………………………………..33 2.3.1 Experiment #1……………………………………………………...33 2.3.2 Experiment #2……………………………………………………...33 2.3.3 Experiment #3……………………………………………………...35 x 2.4 Discussion……………………………………………………………………36 2.5 Figures………………………………………………………………………..45 CHAPTER 3 REPEATED KETAMINE TREATMENT INDUCES SEX-SPECIFIC BEHAVIORAL AND NEUROCHEMICAL EFFECTS IN MICE……………………..51 3.1 Introduction…………………………………………………………………..51 3.2 Materials and Methods……………………………………………………….54 3.2.1 Animals…………………………………………………………….54 3.2.2 Experimental design and drug treatments………………………….55 3.2.3 Spontaneous locomotor activity in the open field test (OFT)…...…56 3.2.4 Forced swim test (FST)…………………………………………….56 3.2.5 Neurochemical analysis……………………………………………57 3.2.6