THE EFFECTS OF BMS-204352, AN ACTIVATOR OF VOLTAGE-GATED POTASSIUM CHANNELS, IN THE INFRALIMBIC CORTEX OF THE Fmr1 KNOCKOUT MOUSE, AN ANIMAL MODEL OF FRAGILE X SYNDROME AN ABSTRACT SUBMITTED ON THE TWENTY THIRD OF MAY 2020 TO THE TULANE UNIVERSITY BRAIN INSTITUTE IN PARTIAL FULFILLMENT OF THE REQUIREMENTS OF THE SCHOOL OF SCIENCE AND ENGINEERING OF TULANE UNIVERSITY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY BY Ted Sawyer EDWARD JOSEPH SAWYER APPROVED: _______________________ LAURA SCHRADER, Ph.D. Advisor _______________________ JillJILL M DANIEL, Daniel Ph.D. _______________________ HAI HUANG, Ph.D. _______________________ RICARDO MOSTANY, Ph.D. Abstract Autism spectrum disorders (ASD) are commonly characterized by abnormal social behaviors. Fragile X syndrome (FXS) is the most common inherited intellectual disability in humans and the most common single-gene cause of ASD symptoms. FXS is caused by the loss or malfunction of the fragile X mental retardation protein (FMRP), an mRNA-binding protein that regulates numerous synaptic proteins, both translationally and through direct protein-protein interactions. One direct-binding target is the large- conductance potassium (BK) channel. BK channels have been shown to be hypoactive in FXS, and represent possible targets for treatment in both general ASD and in FXS specifically. Also, two members of the KCNQ class of voltage-activated potassium channels, KV7.2 and KV7.3, have been identified as FMRP translation targets. Finally, a commonly observed abnormality in the ASD brain is an imbalance in the ratio of excitatory to inhibitory signaling (E/I balance) causing general hyperexcitability in numerous brain areas. One area in which altered E/I balance is often observed is the medial prefrontal cortex (mPFC), which is involved with the processing of social information. Therefore, the goal of this dissertation was to determine if stimulating potassium channel function in the mPFC of Fmr1 KO mice would correct abnormal social behavior. In addition, the possible mechanistic determinants and effects on E/I balance were investigated in WT and Fmr1 KO mice. Infusion of the potassium channel activator, BMS-204352, into the mPFC of KO mice had no effect on social approach behavior, but corrected social novelty impairments as measured by a 3-Chamber Test. Whole-cell patch clamp recordings of pyramidal neurons in layer V of the mPFC revealed no differences in mEPSCs between KO and WT mice, but did reveal higher frequency of mIPSCs in KO mice. Treatment with BMS- 204352 resulted in a decrease in mEPSC amplitude in both genotypes, which was blocked by the BK channel antagonist, paxilline. Effects of BMS-204352 treatment on mIPSCs revealed two possible populations of cell types. One population of exhibited a decrease in frequency of mIPSCs, an effect seen in both genotypes. The other population exhibited a slight increase in frequency of mIPSCs, but this was seen only in KO cells. Treatment with paxilline caused a decrease in mIPSC frequency in both genotypes, which was not altered with subsequent BMS-204352 treatment. Pretreatment with the KV7 channel antagonist XE 991 prevented BMS-204352-induced cross-genotype decrease in mIPSC frequency, but did not prevent BMS-204352-induced frequency increase in KO cells. Western blot analyses revealed no changes between genotypes in BK channel expression, but a trend to increased KV7.3 expression in the PFC of KOs compared to WTs. With these data, it was concluded that aberrant activity of potassium channels in the mPFC of KOs mediates some of the social abnormalities observed in the phenotype, that KOs may exhibit increased KV7.3 expression as a potential compensatory mechanism for BK channel dysfunction, and that potassium channels are a promising potential target for future treatment of ASD symptoms. THE EFFECTS OF BMS-204352, AN ACTIVATOR OF VOLTAGE-GATED POTASSIUM CHANNELS, IN THE INFRALIMBIC CORTEX OF THE Fmr1 KNOCKOUT MOUSE, AN ANIMAL MODEL OF FRAGILE X SYNDROME A DISSERTATION SUBMITTED ON THE TWENTY THIRD OF MAY 2020 TO THE TULANE UNIVERSITY BRAIN INSTITUTE IN PARTIAL FULFILLMENT OF THE REQUIREMENTS OF THE SCHOOL OF SCIENCE AND ENGINEERING OF TULANE UNIVERSITY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY BY Ted Sawyer EDWARD JOSEPH SAWYER APPROVED: _______________________ LAURA SCHRADER, Ph.D. Advisor _______________________ JillJILL M DANIEL, Daniel Ph.D. _______________________ HAI HUANG, Ph.D. _______________________ RICARDO MOSTANY, Ph.D. Acknowledgements This dissertation represents over six years of work and it would not have been possible without the support of numerous colleagues, friends, and family. I would like to thank my advisor, Dr. Laura Schrader, for her unflagging support and patience. Though I occasionally questioned my ability to complete this work, Laura never did, and her support and guidance saw me through some truly difficult times. I would also like to thank the members of my committee, Dr. Jill Daniel, Dr. Ricardo Mostany, and Dr. Hai Huang for their support, encouragement, and input over the years. Thank you to Dr. Gary Dohanich and Dr. Robert Dotson, both of whom I consider honorary committee members and who have dispensed invaluable advice and perspective on numerous occasions. Thank you to Amy Pierce for her assistance with establishing and managing my mouse colony, and to the Uptown vivarium staff for taking care of the mice that were central to my work. Thank you to Sherrie Calogero for her excellent work in keeping both the Brain Institute and the Neuroscience Program running. Thank you to all my lab mates, past and present, who have made the Schrader lab an enjoyable and intellectually engaging environment: Dr. Damek Homiack, Dr. Allen Yu, Dr. Jeremy Hartner, Dr. Matthieu Maroteaux, and Izzy Febo. Thank you to all the colleagues outside of the lab who have collaborated on projects, assisted me with research techniques, and have helped me become a better scientist, particularly Sabrina Kragness, Dr. Grant Weiss, Marco Fisher, Dr. Danny Oseid, Dr. Jonathan Fadok, Lee Munoz, Yowelunh McLester-Davis, Dr. Kevin Pollard, and Dr. Chris Jones. Thank you to all of the undergraduates that have assisted with this work, and without whom it would not have come to fruition: Kacie Sholz, Evan Blair, and Alec Zingale. ii Thank you to Dr. Elizabeth Lanthier, my academic advisor at Northern Virginia Community College. She was integral in my developing an interest in social psychology, which eventually grew into an interest in social neuroscience, and she continues to be a source of support and encouragement. Thank you to my long-time friends Cristin Guinan-Wiley and Leet Wood, who each played a significant role in my decision to try my hand at college after a near-decade hiatus from school. Finally, I would like to both thank my mother, Jean Sawyer, and dedicate this work to her. Raising a child by one’s self is never easy, and raising a child with special needs is even less so. My mother fought a years-long, but ultimately successful battle with my school system to ensure that they provided me with appropriate learning accommodations and a chance at cultivating a genuine education, and without that effort I would never have gone on to do this work. She always encouraged my creativity and curiosity, instilled in me a strong moral compass and sense of duty, and has always supported my choices and autonomy. I also dedicate this work to my brother—Sergeant Kevin Sawyer, USA, Ret.—who began working as soon as he was able in order to help support the family, sacrificing a portion of his childhood so that I would not have to later. They both worked very hard in different ways to help me cultivate the skills and abilities that have allowed me to live the kind of independent life that some of my early doctors and teachers never thought I’d be able to manage. I will always be thankful for what they’ve both done for me. iii Table of Contents Acknowledgements..................................................................................................page ii List of Figures..........................................................................................................page vi Chapter 1: Introduction……………………………..............................................page 1 Autism Spectrum Disorder............................................................................page 1 Fragile X Syndrome......................................................................................page 4 Prefrontal Cortex...........................................................................................page 9 Molecular Interactions of FMRP...................................................................page 11 Animal Models of Fragile X Syndrome........................................................page 11 E/I Balance and Voltage-Gated Channels.....................................................page 13 Current Project..............................................................................................page 17 Chapter 2: Can infusion of BMS-204352 into the infralimbic cortex correct social impairments in Fmr1 KO mice? Introduction…....................................................…………………………...page 19 Methods.........................................................................................................page 20 Results...........................................................................................................page 34 Conclusions...................................................................................................page