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Characterization and Optimization of Microelectrode Arrays for Glutamate Measurements in the Rat Hippocampus

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Characterization and Optimization of Microelectrode Arrays for Glutamate Measurements in the Rat Hippocampus University of Kentucky UKnowledge University of Kentucky Doctoral Dissertations Graduate School 2010 CHARACTERIZATION AND OPTIMIZATION OF MICROELECTRODE ARRAYS FOR GLUTAMATE MEASUREMENTS IN THE RAT HIPPOCAMPUS Pooja Mahendra Talauliker 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 Talauliker, Pooja Mahendra, "CHARACTERIZATION AND OPTIMIZATION OF MICROELECTRODE ARRAYS FOR GLUTAMATE MEASUREMENTS IN THE RAT HIPPOCAMPUS" (2010). University of Kentucky Doctoral Dissertations. 759. https://uknowledge.uky.edu/gradschool_diss/759 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 Pooja Mahendra Talauliker The Graduate School University of Kentucky 2010 CHARACTERIZATION AND OPTIMIZATION OF MICROELECTRODE ARRAYS FOR GLUTAMATE MEASUREMENTS IN THE RAT HIPPOCAMPUS 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 Pooja Mahendra Talauliker Lexington, Kentucky Director: Dr. Greg A. Gerhardt, Professor of Anatomy and Neurobiology Lexington, Kentucky 2010 Copyright © Pooja Mahendra Talauliker 2010 ABSTRACT OF DISSERTATION CHARACTERIZATION AND OPTIMIZATION OF MICROELECTRODE ARRAYS FOR GLUTAMATE MEASUREMENTS IN THE RAT HIPPOCAMPUS An overarching goal of the Gerhardt laboratory is the development of an implantable neural device that allows for long-term glutamate recordings in the hippocampus. Proper L-glutamate regulation is essential for hippocampal function, while glutamate dysregulation is implicated in many neurodegenerative diseases. Direct evidence for subregional glutamate regulation is lacking in previous in vivo studies because of limitations in the spatio-temporal resolution of conventional experimental techniques. We used novel enzyme-coated microelectrode arrays (MEAs) for rapid measurements (2Hz) of extracellular glutamate in urethane-anesthetized rats. Potassium-evoked glutamate release was highest in the cornu ammonis 1 (CA1) subregion and lowest in the cornu ammonis 3 (CA3). In the dentate gyrus (DG), evoked-glutamate release was diminished at a higher potassium concentration but demonstrated faster release kinetics. These studies are the first to show subregion specific regulation of glutamate release in the hippocampus. To allow for in vivo glutamate measurements in awake rats, we have adapted our MEAs for chronic use. Resting glutamate measurements were obtained up to six days post-implantation but recordings were unreliable at later time points. To determine the cause(s) for recording failure, a detailed investigation of MEA surface characteristics was conducted. Scanning electron microscopy and atomic force microscopy showed that PT sites have unique surface chemistry, a microwell geometry and nanometer-sized features, all of which appear to be favorable for high sensitivity recordings. Accordingly, studies were initiated to improve enzyme coatings using a computer-controlled microprinting system (Microfab Technologies, Plano, TX). Preliminary testing showed that microprinting allowed greater control over the coating process and produced MEAs that met our performance criteria. Our final studies investigated the effects of chronic MEA implantation. Immunohistochemical analysis showed that the MEA produced minimal damage in the hippocampus at all time points from 1 day to 6 months. Additionally, tissue attachment to the MEA surface was minimal. Taken together with previous electrophysiology data supporting that MEAs are functional up to six months, these studies established that our chronic MEAs technology is capable of maintaining a brain-device interface that is both functional and biocompatible for extended periods of time. KEYWORDS: Glutamate, Hippocampus, In Vivo Amperometry, Functional Biocompatibility, Brain-Microelectrode Interface Pooja Mahendra Talauliker 01/28/2010 CHARACTERIZATION AND OPTIMIZATION OF MICROELECTRODE ARRAYS FOR GLUTAMATE MEASUREMENTS IN THE RAT HIPPOCAMPUS By Pooja Mahendra Talauliker Greg A. Gerhardt Director of Dissertation Jane Joseph Director of Graduate Studies 01/28/10 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 Pooja Mahendra Talauliker The Graduate School University of Kentucky 2010 CHARACTERIZATION AND OPTIMIZATION OF MICROELECTRODE ARRAYS GLUTAMATE MEASUREMENTS IN THE RAT HIPPOCAMPUS 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 Pooja Mahendra Talauliker Lexington, Kentucky Director: Dr. Greg A. Gerhardt, Professor of Anatomy and Neurobiology Lexington, Kentucky 2010 Copyright © Pooja Mahendra Talauliker 2010 "There are two lasting bequests we can give our children. One is roots, the other is wings." ~ Hodding Carter Dedicated to my parents, Usha and Mahendra Talauliker ACKNOWLEDGEMENTS कायेन वाचा मनसेिन्य्वाै बुदयायात् वा पकर केत सववभावा त। करो�म यद्यत्स परसम् गुरुवरायेक समपर्या� ॥ All I have accomplished, I offer to you, the One who guides us all. I take this opportunity to recognize the support and guidance of several individuals who made the completion of my dissertation a reality. First, I would like to thank my Dissertation Advisor, Dr. Greg Gerhardt for providing guidance and the opportunity to pursue my graduate education in a stimulating research environment. I am also thankful for the honest feedback I received throughout my graduate training from Theresa Thomas and for her unswerving faith in me. Peter Huettl, Francois Pomerleau and Jorge Quintero were always willing to facilitate my learning experience and their constructive input has helped me develop my scientific writing skills. I thank Dr. Ingrid Strömberg for the opportunity to conduct exciting collaborative research. I also thank Drs. Bruce Maley, Randy Hunter, Chris Bjornsson and William Shain for taking the time to teach me about immunohistochemical techniques. The team of scientists at Microfab: David Silva, Patrick Cooley, Mark Christison, Hans-Jochen Trost and Paul Watson, provided much-needed technical support and were always committed to advancing my work with the Jetlab microprinter. Thanks to Larry Rice for providing training on electron microscopy and atomic force microscopy. My students, Edwin Apenbrinck, Janelle Geddes, Emily Cottrell, Kawthar Suleiman and Jacob Mason contributed to my research productivity. Finally, I thank my iii Dissertation Advisory Committee and Outside Examiner: Drs. Wayne Cass, James Geddes, Paul Glaser, Kurt Hauser and Todd Hastings. Their mentorship facilitated the development of my research projects, my professional growth and the quality of this dissertation. The unwavering support of my parents has always given me the confidence to dream big and pursue my ambitions, even if they take me far away from home. I thank my husband Prasad for his understanding, and emotional and intellectual support. I also thank my brother Raaj and the close friends who have shown support for my professional and personal endeavors over the years: Parul, Preeti, Aashish and Maitreyi. Lastly, I thank Drs. Gerry Chader, Richard Rovinelli and Jennifer Brueckner for their mentorship and for expecting the best of me in all my pursuits. iv TABLE OF CONTENTS ACKNOWLEDGEMENTS……………………………………………………………...iii LIST OF TABLES……………………………………………………………………….x LIST OF FIGURES…………………………………………………………………....xi Chapter One: Introduction……………………………………………………………...1 1.1 Neuroanatomy and Physiology of the Hippocampus…………………….…...1 1.2 Glutamate Regulation and Hippocampal Function…………………….……...5 1.3 Glutamate Neurotransmission in the Aging Hippocampus……….…………..8 1.4 Glutamate Dysregulation in Hippocampal Disease…………….……….......10 1.4.1 Alzheimer’s disease…………….………………………………………….....10 1.4.2 Schizophrenia………………………………………………………………….12 1.5 Rapid Glutamate Measurements using Microelectrode Arrays coupled with In Vivo Amperometry…………………………………………………………..........13 1.6 Thesis Outline……………………………………………………………………16
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