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DELETION OF GLUTAMATE RECEPTOR TRAFFICKING PROTEINS IN THE MEDIAL PREFRONTAL CORTEX AND THEIR SEX-SPECIFIC EFFECTS ON COCAINE ADDICTION A Dissertation Submitted to the Temple University Graduate Board In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY by Megan Marie Wickens May 2020 Examining Committee Members: Dr. Lisa Briand, Advisory Chair, Temple University Psychology Department Dr. Debra Bangasser, Temple University Psychology Department Dr. Vinay Parikh, Temple University Psychology Department Dr. Mathieu Wimmer, Temple University Psychology Department Dr. Vishnu Murty, Temple University Psychology Department Dr. Scott Rawls, Temple University Pharmacology Department ABSTRACT Dysregulation of glutamatergic signaling mechanisms is a component of many psychiatric diseases. A number of these diseases exhibit a bias toward one sex, yet the ways in which glutamate is affected by or modulates this bias is poorly understood. In cocaine addiction, women progress from initial use of the drug to substance use disorder faster than men, and have more difficulty remaining abstinent. The same is true in female rodents. We used a mouse model of cocaine self-administration to study the role of glutamate receptor trafficking proteins in cocaine addiction-like behavior in males and females. In the first set of experiments, mice received a conditional knockout of glutamate receptor interacting protein 1 (GRIP1) in the medial prefrontal cortex (mPFC). This led to an increase in motivation for cocaine as well as enhanced likelihood of relapse behavior, as measured by a progressive ratio schedule and cue-induced reinstatement, respectively. No sex differences were seen after prefrontal deletion of GRIP1. The next set of experiments used the same behavioral paradigm, but mice received a conditional knockout of protein interacting with C kinase 1 (PICK1) in the mPFC. PICK1 and GRIP1 are both involved in the activity dependent trafficking of the GluA2-containing AMPA receptor, but while GRIP1 maintains these receptors in the synapse, PICK1 internalizes them in response to a stimulus such as drug experience. The prefrontal deletion of PICK1 was predicted to decrease cue-reinstatement responding, and this was observed in the male mice. The female mice displayed an increase in cue-induced reinstatement responding, similar to the effects seen by prefrontal GRIP1 deletion. Sex differences in PICK1 have not previously been described in the literature. Our results suggest that PICK1 is involved in different baseline processes in females, and merit further study. The final set of experiments ii considered the interaction of gonadal hormones and PICK1 in males. Bilateral gonadectomy or sham surgery was combined with prefrontal PICK1 knockout to determine if circulating gonadal hormones could explain the results in males. After gonadectomy or sham surgery, there was no significant effect of prefrontal PICK1 deletion on cue-induced reinstatement. These results do not fully explain the sex difference observed in intact mice. Together, these studies suggest that baseline sex differences exist in PICK1-mediated mechanisms of cocaine reinstatement and that these differences are not due to the influence of gonadal hormones alone. iii ACKNOWLEDGMENTS This dissertation would not have been possible without the enormous and continued support of many people. First and foremost, I would like to thank my advisor Dr. Lisa Briand. I have learned so much over the past five years and I do not think I would be where I am today if it were not for her continued support. Dr. Briand encourages us to strive for the highest scientific standards, not only in the lab, but at conferences, seminars, and outreach events as well. It is thanks to her that I can say I truly feel like a scientist. I would also like to thank my committee, Dr. Debra Bangasser, Dr. Vinay Parikh, Dr. Mathieu Wimmer, Dr. Scott Rawls, and Dr. Vishnu Murty. You have provided invaluable feedback and advice and I am grateful for the time you took to help me progress through graduate school. To the current and former members of Briand lab, I cannot thank you enough. I truly could not have done this without you. And to Sam, Evie, and Corey, thanks for keeping me in good spirits as I made these final edits to the document. Of course, I cannot forget to thank my family for their support over the last few years. Whether it was Dad driving 1000 miles in one weekend to help me move, or Mom listening to my practice poster presentations, or Aunt Kim inviting me over to dinner and even paying the toll over the bridge for me – I really cannot thank you enough. Lastly, I would be remiss if I did not thank Jim, Meg, and Rachel of Breaking Point Fitness. Your welcoming community became my “home away from home” and I can’t believe how lucky I am to have been able to join you all for the last three years. iv TABLE OF CONTENTS Page ABSTRACT ........................................................................................................................ ii ACKNOWLEDGMENTS ................................................................................................. iv LIST OF TABLES………………………………………………………………………..ix LIST OF FIGURES ........................................................................................................... x CHAPTER 1 1. SEX DIFFERENCES IN PSYCHIATRIC DISEASE: A FOCUS ON THE GLUTAMATE SYSTEM ............................................................................................. 1 The Glutamate System ............................................................................................ 2 Sex Differences in the Glutamate System ............................................................... 3 Baseline Differences ................................................................................... 3 Changes Across the Lifespan ...................................................................... 5 Sex Differences in Glutamate System in Disease ................................................... 7 Alzheimer’s Disease ................................................................................... 7 Major Depressive Disorder ....................................................................... 10 Schizophrenia ............................................................................................ 13 Autism Spectrum Disorder........................................................................ 18 Attention Deficit Hyperactivity Disorder (ADHD) .................................. 20 Conclusion ............................................................................................................ 22 v CHAPTER 2 2. GLUTAMATE RECEPTOR INTERACTING PROTEIN ACTS WITHIN THE PREFRONTAL CORTEX TO BLUNT COCAINE SEEKING ........................ 27 Methods................................................................................................................. 29 Subjects ..................................................................................................... 29 Prefrontal Microinjections and Adeno-Associated Virus Constructs ....... 30 Operant Food Training .............................................................................. 30 Jugular Catheterization Surgery ................................................................ 31 Cocaine Self-Administration..................................................................... 31 Operant Set Shifting Task ......................................................................... 32 Western Blot ............................................................................................. 33 Electrophysiology ..................................................................................... 34 Statistical Analysis .................................................................................... 36 Results ................................................................................................................... 36 Viral Mediated Deletion of GRIP1 in the Medial Prefrontal Cortex ........ 36 Prefrontal GRIP1 Knockout Does Not Affect Fixed Ratio Self- Administration of Sucrose or Cocaine ................................................ 37 Prefrontal GRIP1 Knockout Enhances Responding for Cocaine but not Sucrose on a Progressive Ratio Schedule ..................................... 41 Prefrontal GRIP1 Knockout Enhances Responding for Cocaine but not Sucrose During Cue-Reinstatement .............................................. 41 Prefrontal GRIP1 Knockout Does Not Lead to Deficits in Cognitive Flexibility ........................................................................... 42 vi Prefrontal GRIP1 Knockout Alters Glutamate Transmission in the PFC and the NAc ................................................................................ 45 Discussion ............................................................................................................. 46 GRIP1 Knockout in the Prefrontal Cortex Increases Motivation for Cocaine and Potentiates Cocaine Seeking in Both Males and Females ............................................................................................... 46 Prefrontal GRIP1 Does Not Play a Role in Natural Reward Taking or Seeking ........................................................................................... 48 Knocking Out GRIP1 in the PFC Does Not Alter Cognitive Function .............................................................................................. 49 Altering AMPA Trafficking in the PFC has Downstream

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