ALTERTATIONS IN EXCITATORY / INHIBITORY CIRCUITRY IN THE ADULT RAT BRAIN FOLLOWING POSTNATAL GLUTAMATERGIC SYSTEM ACTIVATION BY DAPHNE A. GILL A Thesis Submitted to the Graduate Faculty in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Department of Biomedical Sciences Faculty of Veterinary Medicine University of Prince Edward Island ©2010 D.A. Gill. 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Canada CONDITIONS FOR THE USE OF THE THESIS The author has agreed that the Library, University of Prince Edward Island, may make this thesis freely available for inspection. Moreover, the author has agreed that permission for extensive copying of this thesis for scholarly purposes may be granted by the professor or professors who supervised the thesis work recorded herein, or, in their absence, by the Chair of the Department or the Dean of the Faculty in which the thesis work was done. It is understood that due recognition will be given to the author of this thesis and to the University of Prince Edward Island in any use of the material in this thesis. Copying or publication or any other use of the thesis for financial gain without approval by the University of Prince Edward Island and the author’s written permission is prohibited. Requests for permission to copy or to make any other use of material in this thesis in whole or in part should be addressed to: Chair of the Department of Biomedical Sciences Faculty of Veterinary Medicine University of Prince Edward Island Charlottetown, P.E.I. Canada C1A4P3 11 SIGNATURE PAGE Cniï±Jjv) REMOVED ABSTRACT A critical period for maturation and final organization of excitatory and inhibitory function in the brain occurs during perinatal development when network connections are being formed and refined. In the rat hippocampus, many of these connections reach adult levels by the end of the second postnatal week. Subsequently, insult or injury to the brain at this time may have far-reaching consequences that manifest as changes in behaviour resulting from modifications in underlying neuronal structure and function. It has been previously demonstrated that low daily doses of the glutamate agonist domoic acid (DOM), administered to rat pups throughout the second postnatal week of life, can result in seizure-like behaviours in adulthood similar to temporal lobe epilepsy (TLE) in humans, as well as produce cellular changes in the dentate gyrus and area CA3 of the hippocampus. Now known as the novelty-induced, seizure-like (NIS-L) rat, these animals represent a new developmental model for TLE. The objective of this dissertation was to further characterize the NIS-L model, thus providing additional information regarding potential alterations in neurologic function and underlying mechanistic properties that may result from early-life glutamate system activation. Subsequently, in vivo studies were performed to provide an electrophysiological assessment of electroencephalogram (EEG) in NIS-L rats. This investigation revealed a distinct EEG waveform pattern during NIS-L manifestation as well as subtle alterations in wave band expression, along with decreased seizure threshold levels and increased mossy fibre sprouting, but no difference in seizure propagation to the rest of the brain. In addition, altered sleep patterns were also reported, similar to what is seen in other animal models of TLE. To assess possible underlying mechanisms responsible for these changes, an immunohistochemical study of inhibitory cells in the hippocampus and amygdala showed region and sex-dependent changes in specific interneuronal subpopulations in the model, as well as decreases in a population of susceptible excitatory neurons, while an in vitro evaluation of field post- synaptic potentials in the dentate gyrus and area CA3 of the hippocampus revealed no differences in baseline activity, but did show a concentration-dependent alteration in response to acute DOM perfusion. A final study investigating possible glutamatergic pathways for DOM-mediated effects indicated no involvement for the A-methyl-D- aspartate (NMDA) receptor, but appeared to implicate a-amino-3 -hydroxy-5-methyl-4- isoazolepropionic acid (AMPA)-sensitive mechanisms. Additionally, a dramatic and unexpected response to early-life glutamate antagonism was discovered. These data provide a demonstration of some of the possible changes that may occur in excitatory and inhibitory circuitry in the brain as a result of interference with early developmental processes, and have revealed several underlying mechanisms that may be responsible for these changes. Future studies assessing spontaneity of behavioural seizure expression and evaluating sex-dependent differences in the model, as well as an investigation of the role of GABAergic mechanisms may help further elucidate the possible consequences of interference in the intricate interplay between glutamate and GAB A systems during critical developmental stages. ACKNOWLEDGEMENTS One of the most important facets of research is an unswerving dedication to the search for knowledge and the subsequent sharing of that knowledge with others. During the course of my studies, I have been blessed with the support and guidance of many talented and enthusiastic individuals committed to research excellence. Without their assistance, this thesis and the work contained within would not have been possible. First and foremost, I would like to thank my supervisor. Dr. Andrew Tasker, whose advice and constant encouragement has been invaluable. I would also like to thank the members of my supervisory committee (past and present) for their assistance during this work, as well as express my sincere thanks and appreciation to Dr. William Watson, Dr. David Reynolds, Dr. Tine Stensbol, Dr. Jesper Bastlund, Dr. Neil Anderson and Kasper Larsen for support and supervision during my studies at H. Lundbeck A/S in Copenhagen, Denmark. In addition, a special thank you goes to my earliest mentor. Dr. Catherine Ryan, for inspiring me to continue in the research field. Financial support from the Atlantic Innovation Fund (AIF), the Atlantic Centre for Comparative Biomedical Research (ACCBR), the Natural Sciences and Engineering Research Council (NSERC), and H. Lundbeck A/S is also acknowledged and greatly appreciated. Due to space constraints, it is impossible for me to list all of the individuals who have contributed to the completion of this work, but please know that your help and encouragement has not gone unnoticed. And last, but not least, I would like to thank my family for enduring all the long hours and missed weekends without complaint, and for always being there when I needed you. VI DEDICATION I would like to dedicate this thesis to my parents, Don and Hazel Sears whose passion for learning and knowledge has been my inspiration and to my husband Ron Gill for giving me the courage to pursue my dreams YU TABLE OF CONTENTS CONDITIONS OF U SE...................................................................................................... ii PERMISSION TO USE POSTGRADUATE THESIS................................................. iii CERTIFICATION OF THESIS WORK............................... iv ABSTRACT........................................................................................................................... v ACKNOWLEDGEMENTS................................................................................................vi LIST OF TABLES..............................................................................................................xv LIST OF FIGURES........................................................................................................