University of Kentucky UKnowledge University of Kentucky Doctoral Dissertations Graduate School 2009 GLUTAMATE REGULATION IN THE HIPPOCAMPAL TRISYNAPTIC PATHWAY IN AGING AND STATUS EPILEPTICUS Michelle Lee Stephens University of Kentucky, [email protected] Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Stephens, Michelle Lee, "GLUTAMATE REGULATION IN THE HIPPOCAMPAL TRISYNAPTIC PATHWAY IN AGING AND STATUS EPILEPTICUS" (2009). University of Kentucky Doctoral Dissertations. 736. https://uknowledge.uky.edu/gradschool_diss/736 This Dissertation is brought to you for free and open access by the Graduate School at UKnowledge. It has been accepted for inclusion in University of Kentucky Doctoral Dissertations by an authorized administrator of UKnowledge. For more information, please contact [email protected]. ABSTRACT OF DISSERTATION Michelle Lee Stephens The Graduate School University of Kentucky 2009 GLUTAMATE REGULATION IN THE HIPPOCAMPAL TRISYNAPTIC PATHWAY IN AGING AND STATUS EPILEPTICUS ABSTRACT OF DISSERTATION A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Medicine at the University of Kentucky By Michelle Lee Stephens Lexington, Kentucky Director: Dr. Greg A. Gerhardt, Professor of Anatomy and Neurobiology Lexington, Kentucky 2009 Copyright © Michelle Lee Stephens 2009 ABSTRACT OF DISSERTATION GLUTAMATE REGULATION IN THE HIPPOCAMPAL TRISYNAPTIC PATHWAY IN AGING AND STATUS EPILEPTICUS A positive correlation exists between increasing age and the incidence of hippocampal-associated dysfunction and disease. Normal L-glutamate neurotransmission is absolutely critical for hippocampal function, while abnormal glutamate neurotransmission has been implicated in many neurodegenerative diseases. Previous studies, overwhelmingly utilizing ex vivo methods, have filled the literature with contradicting reports about hippocampal glutamate regulation during aging. For our studies, enzyme-based ceramic microelectrode arrays (MEA) were used for rapid (2 Hz) measurements of extracellular glutamate in the hippocampal trisynaptic pathway of young (3-6 months), late- middle aged (18 mo.) and aged (24 mo.) urethane-anesthetized Fischer 344 rats. Compared to young animals, glutamate terminals in cornu ammonis 3 (CA3) showed diminished potassium-evoked glutamate release in aged rats. In late- middle aged animals, terminals in the dentate gyrus (DG) showed increased evoked release compared to young rats. The aged DG demonstrated an increased glutamate clearance capacity, indicating a possible age-related compensation to deal with the increased glutamate release that occurred in late- middle age. To investigate the impact of changes in glutamate regulation on the expression of a disease process, we modified the MEA technology to allow recordings in unanesthetized rats. These studies permitted us to measure glutamate regulation in the hippocampal formation without anesthetic effects, which showed a significant increase in basal glutamatergic tone during aging. Status epilepticus was induced by local application of 4-aminopyridine. Real- time glutamate measurements allowed us to capture never-before-seen spontaneous and highly rhythmic glutamate release events during status epilepticus. A significant correlation between pre-status tonic glutamate and the quantity of status epilepticus-associated convulsions and glutamate release events was determined. Taken together, this body of work identifies the DG and CA3 as the loci of age-associated glutamate dysregulation in the hippocampus, and establishes elevated levels of glutamate as a key factor controlling severity of status epilepticus in aged animals. Based upon the potential ability to discriminate brain areas experiencing seizure (i.e. synchronized spontaneous glutamate release) from areas not, we have initiated the development of a MEA for human use during temporal lobe resection surgery. The final studies presented here document the development and testing of a human microelectrode array prototype in non-human primates. KEYWORDS: Glutamate, Hippocampus, Aging, Status Epilepticus, Real-time Amperometry Michelle Lee Stephens 07/21/2009 GLUTAMATE REGULATION IN THE HIPPOCAMPAL TRISYNAPTIC PATHWAY IN AGING AND STATUS EPILEPTICUS By Michelle Lee Stephens Greg A. Gerhardt Director of Dissertation Jane Joseph Director of Graduate Studies 07/21/09 RULES FOR THE USE OF DISSERTATIONS Unpublished dissertations submitted for the Doctor’s degree and deposited in the University of Kentucky Library are as a rule open for inspection, but are to be used only with due regard to the rights of the authors. Bibliographical references may be noted, but quotations or summaries of parts may be published only with the permission of the author, and with the usual scholarly acknowledgments. Extensive copying or publication of the dissertation in whole or in part also requires the consent of the Dean of the Graduate School of the University of Kentucky. A library that borrows this dissertation for use by its patrons is expected to secure the signature of each user. Name Date ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ DISSERTATION Michelle Lee Stephens The Graduate School University of Kentucky 2009 GLUTAMATE REGULATION IN THE HIPPOCAMPAL TRISYNAPTIC PATHWAY IN AGING AND STATUS EPILEPTICUS DISSERTATION A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Medicine at the University of Kentucky By Michelle Lee Stephens Lexington, Kentucky Director: Dr. Greg A. Gerhardt, Professor of Anatomy and Neurobiology Lexington, Kentucky 2009 Copyright © Michelle Lee Stephens 2009 Dedicated to Maw and Kate, no guts no glory! ACKNOWLEDGEMENTS The completion of my dissertation would not have been possible without the support and guidance of many individuals. First, I would like to thank my Dissertation Advisor, Dr. Greg A. Gerhardt, for providing an exciting research environment and for his mentorship. The unwavering support of Jorge Quintero both academically and personally was absolutely critical to my success as a graduate student. I am also grateful to François Pomerleau and Pete Huettl for their constructive criticism and guidance. Robin Lindsay was always willing to provide assistance that made the extensive traveling I was so fortunate to do as a student, possible. I also wish to thank Anne Williamson for graciously hosting me and teaching me about rodent epilepsy. My undergraduate assistants, Chris Troxell, Jasamine Fegley and Megan Elizabeth Deel, were essential to my productivity as a graduate student. Thanks also to Charlie Snow, Sue Quinlivin, Todd Cheever, Angel Schumacher, Keith Day, the College of Medicine Student Affairs and the M.D./Ph.D. Program for the opportunity to study at the University of Kentucky and for helping me navigate the sometimes difficult path of a MD/PhD student. Finally, I would like to thank my Dissertation Advisory Committee and outside reader: Drs. Nada Porter, James Geddes, Meriem Bensalem-Owen, Paul Glaser, and Chris Norris. Their advice has facilitated my research planning, execution and interpretation, and I am grateful for the growth I have experienced under their guidance. The constant support of my parents, Mark and Melody Stephens, and my family (Maw, Paw and Clotine) has always given me the confidence to pursue my dreams. I would like to thank Justin for his committed friendship and support, and also the small group of peers that have been by my side through my professional and personal pursuits: Omar, Khoa, Theresa, Brittanie, Melissa, Michael and Chris. Thanks to the Cap Family, especially Drs. Shields and Ney, for demanding my best efforts, and more importantly for giving me theirs. Last, but not least, thanks to Verda for the encouragement and endless laughs. iii TABLE OF CONTENTS Acknowledgments ................................................................................................ iii List of Tables ...................................................................................................... viii List of Figures ....................................................................................................... ix Chapter One: Introduction .................................................................................... 1 Anatomy and Neurophysiology of the Trisynaptic Pathway .............................. 1 Anatomic and Neurophysiologic Changes in the Aged Hippocampus .............. 4 Glutamate Neurotransmission in the Young and Aged Hippocampus .............. 7 Animal Models of Status Epilepticus ................................................................. 9 Glutamate and Seizures.................................................................................. 10 Thesis Outline ................................................................................................. 13 Chapter Two: Materials and Methods ................................................................