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Functional Properties of L-Glutamate Regulation in Anesthetized and Freely Moving Mice

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Functional Properties of L-Glutamate Regulation in Anesthetized and Freely Moving Mice University of Kentucky UKnowledge Theses and Dissertations--Neuroscience Neuroscience 2007 FUNCTIONAL PROPERTIES OF L-GLUTAMATE REGULATION IN ANESTHETIZED AND FREELY MOVING MICE Kevin N. Hascup University of Kentucky Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Hascup, Kevin N., "FUNCTIONAL PROPERTIES OF L-GLUTAMATE REGULATION IN ANESTHETIZED AND FREELY MOVING MICE" (2007). Theses and Dissertations--Neuroscience. 16. https://uknowledge.uky.edu/neurobio_etds/16 This Doctoral Dissertation is brought to you for free and open access by the Neuroscience at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Neuroscience by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained needed written permission statement(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine) which will be submitted to UKnowledge as Additional File. I hereby grant to The University of Kentucky and its agents the irrevocable, non-exclusive, and royalty-free license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known. I agree that the document mentioned above may be made available immediately for worldwide access unless an embargo applies. I retain all other ownership rights to the copyright of my work. I also retain the right to use in future works (such as articles or books) all or part of my work. I understand that I am free to register the copyright to my work. REVIEW, APPROVAL AND ACCEPTANCE The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s thesis including all changes required by the advisory committee. The undersigned agree to abide by the statements above. Kevin N. Hascup, Student Dr. Greg Gerhardt, Major Professor Dr. Jane Joseph, Director of Graduate Studies ABSTRACT OF DISSERTATION Kevin Nicholas Hascup The Graduate School University of Kentucky 2007 FUNCTIONAL PROPERTIES OF L-GLUTAMATE REGULATION IN ANESTHETIZED AND FREELY MOVING MICE ____________________________________ 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 Kevin Nicholas Hascup Lexington, Kentucky Director: Dr. Greg Gerhardt, Professor of Anatomy and Neurobiology Lexington, Kentucky 2007 Copyright © Kevin Nicholas Hascup 2007 ABSTRACT OF DISSERTATION FUNCTIONAL PROPERTIES OF L-GLUTAMATE REGULATION IN ANESTHETIZED AND FREELY MOVING MICE L-glutamate (Glu) is the predominant excitatory neurotransmitter in the mammalian central nervous system with involvement encompassing learning and memory, cognition, plasticity, and motor movement. Dysregulation of the glutamatergic system is implicated in several neurological disorders including Parkinson’s disease, Alzheimer’s disease, Huntington’s disease and amyotrophic lateral sclerosis. The mechanisms underlying these neurological disorders are not clear, but evidence suggests that abnormal Glu neurotransmission plays a role. Elevated levels of Glu in the synaptic cleft overstimulate the N-methyl-D- aspartate receptor leading to excitotoxicity, which causes neuronal loss in chronic neurological diseases. What is less understood is the source for the elevated Glu levels. One hypothesis involves alterations in either activity or concentration of Glu metabolizing enzymes. To study this, two transgenic mouse models were developed that increase levels of Glu pyruvate transaminase (GPT), responsible for Glu degradation, and Glu dehydrogenase (GLUD1), responsible for Glu synthesis. Our laboratory is interested in studying stimulus- evoked Glu release and re-uptake dynamics in these mice using an enzyme- based multisite microelectrode array (MEA) capable of subsecond measurements with low detection limits. Our main finding indicates that GLUD1 mice have increased release of Glu. The GLUD1 mice show spontaneous motor neuron degeneration of the hind limbs that could be correlated to an excitotoxic effect from the increased release of Glu. We wanted to study these GLUD1, motor deficient mice without the affects of anesthesia. First, we needed to modify the current MEAs for use in the awake, freely moving mouse. In these studies we measured resting Glu levels as well as MEA viability with local application of 1 mM Glu in both the striatum and prefrontal cortex of C57BL/6 mice. No change in MEA sensitivity for Glu was observed on days 3 through 7 post-implantation. Resting Glu levels are examined in the striatum of the freely moving mouse by locally applying an uptake inhibitor or a sodium-channel blocker. Our studies indicate the resting Glu levels are partially neuronally derived and not from reversal of the high-affinity transporters. This characterization has laid the foundation to study behavioral alterations of Glu in the GLUD1 mice. Keywords: Amperometry, Microelectrode, Sensor, Neurotransmitter, Neurodegeneration Kevin Nicholas Hascup 10/04/07 FUNCTIONAL PROPERTIES OF L-GLUTAMATE REGULATION IN ANESTHETIZED AND FREELY MOVING MICE By Kevin Nicholas Hascup Greg A. Gerhardt Director of Dissertation Jane Joseph Director of Graduate Studies 10/04/07 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 Kevin Nicholas Hascup The Graduate School University of Kentucky 2007 FUNCTIONAL PROPERTIES OF L-GLUTAMATE REGULATION IN ANESTHETIZED AND FREELY MOVING MICE __________________________ 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 Kevin Nicholas Hascup Lexington, Kentucky Director: Dr. Greg A. Gerhardt, Professor of Anatomy and Neurobiology Lexington, Kentucky 2007 Copyright © Kevin Nicholas Hascup Dedicated to my wife, Erin Rutherford Hascup, and my parents, Richard and Christine Hascup. ACKNOWLEDGEMENTS It is difficult to put into words my gratitude towards those who helped me along the road to this presitigious endeavour. First and foremost, I would like to thank my parents, Richard and Christine Hascup. If it were not for their emotional and financial support throughout my collegiate career, I would have been able to reach this point. They taught me the value of hardwork, discipline, and to never stop chasing my dreams. I hope to be as good of a parent to my children that my parents were to me. Next, I would like to thank my mentor, Greg A. Gerhardt. It is truly unique to study under an individual that is performing cutting edge research while taking the time to teach his students how to survive in such a difficult profession. I deeply appreciate his loyalty and support especially during moments when I made life difficult for myself. I look forward to continuing our friendship and scientific endeavours throughout the upcoming years in Stockholm and wherever the winds may carry me. What has made our laboratory great over the years is the willingness of the senior graduate students to take the time to teach the incoming graduate students. Those that guided my graduate career path include Keith Day, Matt Joyce, and Justin Nickell. With their help and support I learned how to properly use our technology, design appropriate experiments, and critically interpret my data. I wish them continued success in their medical as well as academic careers. I would also like to thank the rest of the members of our laboratory including Pete Huettl, François Pomerleau, and Robin Lindsay. They have helped in numerous ways from system development to ordering critical laboratory supplies. Without their help this laboratory would undoubtedly grind to a halt. In addition to the senior laboratory members, fellow graduate students and post- docs have helped make my days in the laboratory more interesting,
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