ACUTE MICROGLIAL RESPONSES TO SINGLE NON-EPILEPTOGENIC VS. EPILEPTOGENIC SEIZURES IN MOUSE HIPPOCAMPUS A Dissertation submitted to the Faculty of the Graduate School of Arts and Sciences of Georgetown University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Neuroscience By Alberto Sepulveda-Rodriguez, B.S. Washington, D.C. June 5, 2019 Copyright 2019 by Alberto Sepulveda-Rodriguez All Rights Reserved ii ACUTE MICROGLIAL RESPONSES TO SINGLE NON-EPILEPTOGENIC VS. EPILEPTOGENIC SEIZURES IN MOUSE HIPPOCAMPUS Alberto Sepulveda-Rodriguez, B.S. Thesis Advisor: Stefano Vicini, Ph.D. ABSTRACT As the resident macrophage of the central nervous system, microglia are in a uniquely privileged position to both affect and be affected by neuroinflammation, neuronal activity and injury, which are all hallmarks of seizures and the epilepsies. In one of the prototypical rodent models of seizure-induced epilepsy, hippocampal microglia become activated after prolonged, damaging seizures known as Status Epilepticus (SE). However, since SE comprises both neuronal hyperactivity and injury, the specific mechanisms triggering this microglial activation remain unclear, as does its relevance to the ensuing epileptogenic processes. The present studies employed another well-established seizure model, electroconvulsive shock (ECS), to study the effect of paroxysmal/ictal neuronal hyperactivity on mouse hippocampal microglia, in the absence of concomitant neuronal degeneration. Unlike SE, ECS did not cause neuronal injury and did not alter hippocampal CA1 microglial and astrocytic density, morphology nor baseline process motility. In contrast, both ECS and SE produced a similar increase in ATP-directed microglial process motility in acute slices and similarly upregulated expression of the iii chemokine CCL2. As opposed to the general pro-inflammatory environment produced after SE (where induction of CCL2, TNF, IL-1 and IL-6 signaling among others have been reported), the acute microglial response to ECS was more selective: I found upregulated expression of Ccl2 message but unchanged transcript levels of the pro- inflammatory cytokine Tnf. Whole-cell patch-clamp recordings of hippocampal CA1sr microglia showed that ECS enhanced purinergic currents mediated by P2X7 receptors, in the absence of changes in passive properties or voltage-gated currents or changes in receptor expression. This differs from previously described alterations in intrinsic characteristics and purinoreceptor upregulation which coincided with enhanced purinergic currents following SE. These ECS-induced effects point to a common “seizure signature” in hippocampal microglia, characterized by functional alterations to purinergic signaling. The data herein demonstrate that ictal activity per se can drive changes in microglial physiology. These described physiological changes (which up until now have been exclusively associated with prolonged and damaging seizures) are of added interest as they may be relevant to electroconvulsive therapy which remains a gold-standard treatment for depression. iv ACKNOWLEDGEMENTS “O Lord, that lends me life, lend me a heart replete with thankfulness!” - William Shakespeare, Henry VI The research and writing of this thesis are dedicated to: Karen Gale, who picked my application and called me from the hospital to propose that we submit the CONACyT fellowship that supported my work these past 5 years. Nothing would make me feel prouder than when others would comment on how she would be proud of my progress and project. My mother Cecilia and my father Alberto, their spouses and all my siblings, but especially Gua for the constant inspiration. I honestly feel that she deserved a CONACyT award more than me, and am certain she would have received one if they still existed. The rest of my family, who have set me up for success no matter where or what I choose to dedicate myself to. My talented, intelligent and beautiful partner Meghan, and her entire family that has welcome me with open arms from the very start. My caring mentors Stefano Vicini and Patrick Forcelli, who have taught me PLENTY of physiology, pharmacology and other biosciences, but even more v about life. You continue to inspire me with your paradoxical youth and wisdom respectively. My amazingly diligent thesis committee (chaired by John Partridge and featuring Lindsay DeBiase, Kathleen Maguire-Zeiss and Patrick Forcelli) who have been instrumental for this project. I could not have asked for anything more than what they gave me. Their time, effort and energy collectively made this a more rigorous and impactful study, as well as a better story. Other faculty that helped me and cared about me and my project without any obligation to do so, namely Bob Yasuda, Kathy Conant, Robin Tucker, Ken Kellar, Tom Coate, Brent Harris, Ludise Malkova, Barry Wolfe, Bill Rebeck, JY Wu and Mark Burns among many others. My close friends and labmates Nour, Adam, Selena, Lorenza, Irene and David, and obviously Steph too because ephys is hard. Also, the Forcelli- Malkova lab for being so welcoming and making me feel like I had a 2nd home there. Megan Huizenga, Pinggan Li, Tahiyana Khan, James Ma, Colby Carlone, Lorenzo Bozzelli, Carissa Winland, Yan Li and the rest of my coauthors. The students and fellows that allowed me to learn even more by training them, namely Tahi, Serena, Colby, Rebecca and Pinggan. vi My happy hour/venting/dreaming support group, who helped me cope: Erin, Megan, Kaela, Gabby, Lorenzo, Jeremiah, Steph D, Edith, Steph S, Evan, Thien, Saf, and many others. The animals that were used in my experiments, and the DCM and GUACUC staff devoted to protecting them while enabling our research. All the other faculty, staff and students that supported me and kept me sane through this endeavor. It really does take a village, and there are entirely too many people to list. My best friends David and Martin. I would do anything for you guys, and I know you would and indeed have done the same for me. The brothers of my fraternity Delta Upsilon, particularly my gigantic and raunchy Alpha Upsilon pledge class, the classes of 2011 and 2014 seniors who like me are mostly overachievers while always having a good time, Trevor and Kyle, the presidents that preceded me and were always available to guide me, and the officers that served on my executive board during those trying times for DU Rochester. Dikaia Upotheke indeed. The faculty and staff at my alma mater, particularly Ben Hayden who introduced me to neuroscience research, Renee Miller who awoke in me a latent fascination with neurochemistry and pharmacology in me, and Anne Shields for her continued mentorship. The IPN and BGE for funding my last 5 years here together with CONACyT. vii GU for providing me a place to not only learn and research my own science, but also become exposed to a never-ending flux of interesting people, from amazing scientists and clinicians to policymakers, world leaders and even Nobel Prize laureates. It will truly be an honor and a privilege to call myself a Georgetown alum soon. Eternally grateful, ALBERTO viii TABLE OF CONTENTS 1. INTRODUCTION ........................................................................................................................... 1 A. Seizures and epilepsy ................................................................................................................... 3 i. Treatment approaches against the epilepsies .............................................................. 4 ii. Status epilepticus .................................................................................................................... 7 B. Sequelae of damaging seizures have potential links to epileptogenesis.................. 9 i. Neural plasticity, neurogenesis and structural changes ........................................ 10 ii. Neuronal and blood brain brain barrier damage, metabolic or oxidative stress and inflammation ........................................................................................................ 11 C. Microglia: A labile cell type enabled by unique physiology ....................................... 14 i. Roles in development, physiology and pathology .................................................... 15 ii. Microglia become “activated” in response to a wide array of insults.............. 16 iii. Morphology and motility: “Scouting” microglia from box scores vs. game film ................................................................................................................................................. 18 iv. Physiology of microglia .................................................................................................... 22 v. P2 purinoreceptors underlie many basic functions of microglia ...................... 25 vi. Focusing on P2X7R as a key mediator of microglial function in health and disease .......................................................................................................................................... 28 D. Microglial responses to seizures and potential roles in epilepsy ............................ 32 i. A survey of studies on acute microglial responses to status epilepticus ........ 34 ii. Experimental status epilepticus induces a particular activation state in murine hippocampal microglia ........................................................................................... 35 ix E. A gap in our knowledge: “Benign” seizures and microglia ........................................