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Manipulation of Metabotropic and AMPA Glutamate Receptors in the Brain

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Manipulation of Metabotropic and AMPA Glutamate Receptors in the Brain Manipulation of Metabotropic and AMPA Glutamate Receptors in the Brain ©Amy G. M. Lam, B.Sc. Hons (University of London) A thesis submitted for the Degree of Doctor of Philosophy to the Faculty of Medicine, University of Glasgow Wellcome Surgical Institute & Hugh Fraser Neuroscience Laboratories, University of Glasgow, Garscube Estate, Bearsden Road, Glasgow G61 1QH March 1999 ProQuest Number: 11007789 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 11007789 Published by ProQuest LLC(2018). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 O SGOW b DIVERSITY LIBRARY Co| Declaration I declare that this thesis comprises my own original work and has not been accepted in any previous application for a degree. The work, of which it is a record, has been carried out by myself, except as acknowledged and indicated in the thesis. All sources of information have been specifically referenced. Amy Lam Acknowledgements This thesis would not have been possible without the assistance and support of many people. First and foremost, my greatest thanks go to Professor James McCulloch for his never-failing support over the past few years. Thank you for everything. I extend my sincerest gratitude and appreciation to my Industrial supervisor, Dr. David Lodge, and all those at Eli Lilly who made me feel very much at home during my two month stay. Especial thanks go to Dr. Michael J. O’Neill, for the countless times he has been called upon, and most of all for his constant good humour when faced with my demands; and to Dr. Ann Kingston for her invaluable assistance, guidance and time. My thanks also to Mrs. Caroline Hicks, Mr. Mark Ward and Miss. Tracey Murray, and to Mrs. Rosemarie Tomlinson, Miss. Non Evans and Mr. Roger Moore. Thank you for all your help and allowing me to share your bench space. I would like to thank Professor Richard Morris, Dr. Gemot Riedel and Dr. Jacques Micheau from the University of Edinburgh and Universite de Bordeaux I for their part in this thesis. It was a privilege to have had the opportunity to collaborate with them; their exuberance and dedication has made a lasting impression. Many thanks also to Dr. Marc Soriano for his involvement in this thesis, but in particular for his friendship and infectious enthusiasm for all things scientific. I would also like to extend my thanks to Mr Alan May of the Department of photography, who produced some of the photographic work in this thesis, and to Miss. Rosalyn Stanners of Medical Illustration, Southern General Hospital, for all her help with some of the figures in this thesis. Much appreciation and thanks to Dr. Christine Thomson of the Department of small animal clinical studies. I really am indebted to you for all your time and your willingness to help. Lastly, I would like to thank all the staff and fellow students at the Wellcome Surgical Institute. In particular, thanks to Dr. Omar Touzani and Dr. Karen Horsburgh for all their sound knowledge and impartial advice, and to those of you who never needed too much persuading to go for the odd drink or two on mutually ‘bad days’. LIST OF CONTENTS Declaration.................................................................................................................................. i Acknowledgements...................................................................................................................ii List of Contents......................................................... ............................................................... iv List of Figures...........................................................................................................................ix List of Tables............................................................................................................................xii List of Publications................................................................................................................ xiv Summary ..................................................................................................................................xvi CHAPTER 1 INTRODUCTION 1.1 Glutamate in the CNS.........................................................................................................1 1.2 Ionotropic glutamate receptor classification...................................................................1 1.2.1 The NMD A receptor.....................................................................................................2 1.2.2 The non-NMDA receptors............................................................................................ 2 1.2.3 Current concepts of ionotropic glutamate receptor subunit identification.............. 14 1.3 Metabotropic glutamate receptor classification........................................................... 15 1.3.1 Group I receptors.........................................................................................................19 1.3.2 Group II receptors....................................................................................................... 20 1.3.3 Group HI receptors.....................................................................................................22 1.4 Distribution and physiological roles of glutamate receptors..................................... 25 1.4.1 Ionotropic glutamate receptors...................................................................................25 1.4.2 Metabotropic glutamate receptors............................................................................. 31 1.5 Glutamate and neurodegeneration.................................................................................43 1.5.1 Ionotropic glutamate receptors and mechanisms of excitotoxicity..........................46 1.5.2 Existing neuroprotective strategies for glutamate-induced toxicity......................... 53 1.6 Emerging roles of metabotropic glutamate receptors in brain pathology............... 57 1.7 Imaging cerebral function with [14C]2-deoxyglucose autoradiography................ 61 1.8 Aims of thesis .....................................................................................................................67 CHAPTER 2 2.1 In vivo [14C]2-deoxyglucose autoradiography............................................................ 69 2.1.1 Preparation of animals for [14C]2-deoxyglucose measurement.................................69 2.1.2 Cannulae preparation................................................................................................... 69 2.1.3 The [14C]2-deoxyglucose procedure .......................................................................... 70 2.1.4 Liquid scintillation analysis ......................................................................................... 70 2.1.5 Preparation of autoradiograms ....................................................................................71 2.1.6 Quantitative densitometric image analysis ..................................................................71 2.2 Hippocampal stereotaxic surgery................................................................................... 72 2.2.1 Intrahippocampal stereotaxic placement of cannulae ................................................ 72 2.2.2 Avertin preparation ..................................................................................................... 75 2.3 Focal cerebral ischaemia in the rat.................................................................................75 2.3.1 Preparation of animals .................................................................................................75 2.3.2 Middle cerebral artery occlusion ...... 76 2.3.3 Tissue processing .........................................................................................................78 2.3.4 Measurement of the volume of infarction ...................................................................78 2.4 In vitro models of neurotoxicity...................................................................................... 80 2.4.1 Primary cortical astrocyte culture ...............................................................................80 2.4.2 Primary cortical neuronal cultures ...............................................................................84 2.4.3 Induction of hypoxia and glucose deprivation ........................................................... 85 2.4.4 Induction of staurosporine induced neurotoxicity ......................................................86 2.5 Immunohistochemistry.................................................................................................... 86 2.5.1 In vivo material............................................................................................................86 2.5.2 In vitro material............................................................................................................92 2.6 Assessment of neuronal damage..................................................................................... 93 2.6.1 Lactate dehydrogenase
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