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Benzodiazepines and the GABA, Receptor: Effecs of C~~UB~Cexposure

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Benzodiazepines and the GABA, Receptor: Effecs of C~~UB~Cexposure The University of Alberta Benzodiazepines and the GABA, Receptor: Effecs of C~~UB~CExposure Michelle Ingird Arnot O A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillrnent of the requirements for the degree of Doctor of Philosophy. Department of Pharmacology Edmonton, Alberta FaIl, 1998 National Library Bibliothèque nationale of Canada du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395, me Wellington OüawaON K1AW ûüawaON K1AON4 Canada canada The author has granted a non- L'auteur a accorde une licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distn'buer ou copies of this thesis in microform, vendre des copies de cette thése sous paper or electronic formats. la forme de microfiche/nlm, de reproduction sur papier ou sur format élecîronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thése ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. A mind once stretched by a new idea, never regains its original dimensions. - Author Unknown ABSTRACT The benzodiazepine pharmacological profile includes sedation. anxiolysis. muscle relaxation and anticonvulsant effects; however. after long term treatment with full agonists tolerancc develops to certain aspects of this profile. These cornpounds produce their overt effects via allosrenc modulation of the GABA, receptor. The GABA, receptor is a oligomeric pentamer surrounding a central chioride ion pore; activation of the receptor normdly results in chloride influx and membrane hyperpolarization. This pentameric cornplex is comprised of different subunits, emh of which are encoded by a separate gene. The experiments outlined in this thesis attempt to increase Our understanding of the response of the GABAergic sy stem to chronic benzodiazepine exposure. An alternative mechanism for dmg delivery was established to ensure continuous diflzepam delivery over an extended period. This paradigm was then utilized for exposure of rats for 7, 14, 28 days continuous infusion of 15 mg/kg/day diazepam. There were temporal alterations in steady-state GABA, receptor mRNA IeveIs which were brain region specific, and either attenuated or rebounded after 3 days dmg cessation. This continuous infusion was compared to a daily injection regime; each 14 day dosing paradigm resulted in equivalent daily doses of 15 mgkg diazepam, yet there were differentiai dterations in cortical GABA, receptor steady-state mRNA levels. Both chronic dosing regimes resulted in reduced GABA enhancement of [3~]-flunitrazepambinding, but animals from the daily diazepam injection regime exhibited an increase in cortical [)H]-Ro 15-45 13 binding and in the proportion of diazepam insensitive binding. Following each diazepam administration there was a similar development of tolerance to sedation, yet there were differences in the developrnent of anxiolytic tolerance. Alterations in the GABA, receptor after 14 days of - daily diazeparn injection were compared to similar treatment with parilal agonists, which do not result in tolerance developrnent. Diazeparn chronic treatment demonstrated changes in steady-state mRNA levels which were distinct from partiai agonist induced alterations. These data are consistent with the hypothesis that benzodiazepine chronic administration results in the concomitant up- and down-rcgulûtion of GABA, receptor subunit gene expression. This "isoform switching" subsequentl y alten benzodiazepine binding profi les and modulation between the GABA and benzodiazepine-sites in an atrempt to maintain homeostatic balance. To my parents John and Linda, my brother Jon, and in loving memory of my grandmother Susie L. Kvamme ACKNOWLEDGEMENTS I would like to thank my supervison Dn. Alan Bateson and Ian Martin for their continued support, advice and intellectud input, which formed the backbone of my graduate education. Their ideas. enthusiasrn, and work ethic will be continually recognized. Funher, my cornmittee rnemben Drs. Susan Dunn and Glen Baker whose doors were always open and opinions and advice always rendered. Everyone in the Martin, Bateson, and Dunn laboratories over the years, primarily Brian Tancowny for ail the help in surgery and al1 the "extra" help that gets forgotten dong the way and Dr. Chein-Tsai Lai for his help with "undesirable" experimental protocol. To Dr. Sandra File and everyone in her Iaboratory in London, especidly Dr. Cathy Fernandes, who taught me more than just the study of animal behavior. To Dn. Joy S teele, Rob Holt, Manin Davies, Bill Colmers and Rory iMcQuiston, I enjoyed "picking your brains" and bouncing ideas from you; your intellectual input in more than the scientific side of my life was appreciated. To my many fiends who support and believed in me, and were involved in sides of my life other than science. And findiy to my family, my brother Jon and my parents John and Linda whose continued beleif, support, and love 1 could not have done this without ... the Iessons of perseverance have paid off. TABLE OF CONTENTS PAGE CHAPTER 1 - Introduction Generai overview The history and development of the benzodiazepines GABA, receptor structure and function The a-su bunit The P-subunit The y-subunit Other subunits Developmental subunit expression Benzodiazepine pharmacology Benzodiazepine dependence: tolerance and withdrawal Specific Aims CHAPTER 2 - Dimethyl sulfoxide/propylene glycol is a suitable solvent for the delivery of diazepam from osmotic rninipumps htroduc tion Materiais and methods Results Discussion CHAPTER 3 - GABA, receptor isoform gene expression: time course effects of chronic diazeparn exposure via continuous infusion Introduction Materids and methods Drug treatment Quantification of CNS diazepam levels by reveaed phase HPLC RNA isolation Solution hybridization Densitometry Statistical anaiysis Results Drug levek Steady-state rnRNA changes Cortex Cerebellum Hippocampus Discussion CEIAPTER 4 - GABA, receptor isoform expression in rat cortex: effects of chronic diazepam exposure via two different treatment regimes Introduction Materials and methods Drug treatment Diazepam quantification, RNA isolation. solution hybridization and densitometric analysis Binding analysis Bcnzodiazcpine receptor subtvpc definition GABA enhancement Total specific binding and diazepam insensitive binding Statistical analysis Results Dmg levels Changes in steady-state mRNA levels Differences between drug and vehicle treatment Differences between diazeparn treatment regimes Binding analysis Discussion CHAPTER 5 - Behavioral and anticonvukant tolerance following chronic diazepam administration by daily injection or continuous infusion htroduc tion PART 1 - Development of anficonvulsant tolerance Materials and methods Dmg treatment Penty lenetetrazole chalienge Statistical analysis Results Discussion PART 2 - Development of sedative and anxiolytic tolerance Materials and methods Drug treatment Quantification of drug lcvels by revened-phase HPLC Behavioral tests Holeboard Social Interaction Elevated plus-maze Procedure Statistics ResuIts Drug Ievels Holeboard test Sociai interaction Elevated pIus-maze Discussion CHAPTER 6 - Differential effects of chronic full and partial agonist benzodiazepine treatment: alterations in GABA, receptor mRNA levels and binding characteristics in cortex, cerebellum, and hippocampus Introduction Materials and methods Drug treatment Quantification of drug Ievels by reversed-phase HPLC Diazepam and bretrizenil RP 60503 RNA isolation and soiution hybridization Densitometry and statistical andysis of mRNA steady-state changes Binding analysis Drug levels Changes in steady-state mRNA levels Cortex Cerebeilum Hi ppocarnpus GABA, receptor -benzodiazepine binding analysis Benzodiazepine subtype ratios GABA enhancement of [3~-flunitnzeparnbinding Totd [3w-Ro 15 45 1 3 binding and diazeparn insensitive binding Discussion CHAPTER 7 - General Discussion Future Directions Bibliography Appendix I S 1 Nuclease htra-assay and Inter-assay Variability Phosphorimaging venus Autoradiographic Cornputer Scanning Linearity and Sensitivity of Phosphorimaging LIST OF TABLES PAGE Table 3.1 The sequences and positions of the oligonucleotide 46 probes which were used in the S 1 Nuclease assay to quantify GABA, receptor steady-state mRNA Ievels Table 3.2 The mean cortical GABA, receptor steady-state 47 mRNA levels as a percentage of vehicle treated controls foliowing 15 rng/kg/day diazepam infusion for 7. 14.28 days and 28 days plus 3 days withdrawal Table 3.3 The mean cerebellar GABA, receptor steady-state mRNA levels as a percentage of vehicle treated controls following 15 rng/kg/day diazeparn infusion for 7, 14.28 days and 28 days plus 3 days withdrawal Table 3.4 The mean hippocampal GABA, receptor steady-state mRNA levels as a percentage of vehicle treated controls following 15 mg/~g/daydiazepam infusion for 7, 14,28 days and 28 days plus 3 days withdrawal Table 4.1 Mean GABA, receptor a-,P-, y-subunit mRNA sieady-state levels as a percentage of vehicle controls afier 14 days chronic treatment with 15 mgkg diazepam via daily injection or pump infusion Table 4.2 The mean percentage
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