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Information to Users INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9” black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. University Microfilms International A Bell & Howell Information Company 300 North Zeet) Road Ann Arbor Ml 40106-1346 USA 313761-4700 000 521-0600 Order Number 9421038 The effects of acute and chronic ethanol administration on the expression ofGABA a /BZ receptor subunit mRNA in the mouse cerebellum Wu, Chieh-Hsi, Ph.D. The Ohio State University, 1994 Copyright ©1994 by Wu, Chi eh-Hal. All rights reserved. UMI 300 N. Zeeb Rd. Ann Arbor. M I 48106 THE EFFECTS OF ACUTE AND CHRONIC ETHANOL ADMINISTRATION ON THE EXPRESSION OF GABAa /B Z RECEPTOR SUBUNIT mRNA IN THE MOUSE CEREBELLUM DISSERTATION Presented In Partial Fulfillment of the Requirements for the degree Doctor of Philosophy in the Graduate School of The Ohio State University By Chieh-Hsi Wu, B.S. The Ohio State University 1994 Dissertation Committee: Approved by Andrej Rotter Adrienne Frostholm Richard Fertel Thomas Boyd Department of Pharmacology ACKNOWLEDGMENTS I would like to express my sincere appreciation to my advisor, Dr. Andrej Rotter for his guidance and instruction throughout my entire graduate career. With his patience and understanding, I was able to conquer many academic difficulties and gradually build up my confidence in designing experiments, thus accomplishing my dissertation work. Thanks also go to Dr. Adrienne Frostholm for her conceptual and technical assistance on my experiments. I thank her for letting me mature in science. Without her help, patience and enthusiasm, the writing of this dissertation would not have been possible. Like a friend, supervisor, and lecturer, Dr. Frostholm gave a lot of critical advice and her efforts on my behalf will be appreciated for ever. My appreciation is extended to my dissertation committee members, Drs. Richard Fertel and Tom Boyd, for their encouragement and valuable evaluation of this document. To my colleagues in the laboratory, Dr. Vera Luntz-Leybman, Dr. Jim Evans, and Anne Chang, thank you for giving me a great deal of help and an enjoyable time together. Gratitude and love is also extended to my family and friends, my mother Chaieshe, my siblings, Meeiling, Meeibi, Meeifang, Cheihchi, and Anning. Thank you all for the endless spiritual support. VITA September 11, 1964 Born - Taiwan, R.O.C. 1986 B.S., Taipei Medical College Taiwan, R.O.C. Major: Pharmacy 1 989-present The Ohio State University Department of Pharmacology College of Medicine Columbus, Ohio PUBLICATIONS 1. Varecka L., Wu C. H., Rotter A. and Frostholm A. "GABAA/Benzodiazepine receptor a6 subunit mRNA in granule cells of the cerebellar cortex and cochlear nuclei: expression in developing and mutant mice" J. Comp. Neurol.383 (1993) 1-12. 2. Wu C. H., Frostholm A. and Rotter A. "Effect of acute and chronic ethanol exposure on gene expression for GABAA/Benzodiazepine receptor subunits in mouse cerebellum" Soc. Neurosci. Abstr. 19 (1993) 595. FIELDS OF STUDY Major Field: Pharmacology Studies in: Neuroscience TABLE OF CONTENTS Page ACKNOWLEDGMENTS............................................................................................ ii VITA..........................................................................................................................in LIST OF TABLES.....................................................................................................ix LIST OF FIGURES...................................................................................................xi CHAPTER I: INTRODUCTION.................................................................................1 A. Introduction ................................................................................................1 B. The pharmacokinetics of ethanol consumption ................................3 C. The physiological effects of ethanol consumption ........................5 D. Structure and function of the GABAA/BZ receptor ........................8 E. The pharmacological properties of GABAa /BZ receptor subunit variants ........................................................................................... 17 F. The relationship between ethanol and the GABAa /BZ receptor ........................................................................................................ 18 1. Acute ethanol effects .................................................................. 21 2. Chronic ethanol effects .............................................................. 22 3. Molecular studies ........................................................................ 24 G. Ethanol and the cerebellum ..................................................................25 1. An overview of cerebellar anatomy ...................................... 25 i v 2. The effect of ethanol on adult and developing cerebellar neurons ........................................................................... 30 H. GABAa/BZ receptor subunit localization in the cerebellum ...................................................................................................33 I. Hypothesis ................................................................................................ 36 CHAPTER II: PREPARATION OF RIBONUCLEOTIDE PROBES AND GENERAL METHODOLOGY........................................................................38 A. Ethanol treatment ...................................................................................38 1. Animals........................................................................................ 38 2. Acute ethanol treatment .............................................................38 3. Chronic ethanol treatment ..........................................................39 4. Prenatal Ethanol Treatment .......................................................40 5. Blood ethanol concentration measurement ........................ 40 B. Riboprobe synthesis ............................................................................... 41 1. mRNA isolation by oligo (dT) chromatography ..................41 2. Reverse transcription ..................................................................43 3. Polymerase chain reaction (PCR) .............................................44 4. Ligation of amplified fragments into pBluescript II SK+ phagemid vector .......................................46 5. Small-scale preparation of pBluescript SK+ plasmid (Miniprep).............................................................. 49 6. Large-scale plasmid isolation (Maxiprep) ...........................52 7. Linearization of the insert-containing plasmids for transcription.......................................................... 54 C. Sequencing double-stranded plasmid template .............................54 v 1. Alkali denaturation of supercoiled plasmid DNA ............ 54 2. Probe labeling .............................................................................. 55 3. Gel preparation and electrophoresis ...................................... 56 4. Autoradiography .......................................................................... 59 D. Northern blot analysis ............................................................................59 1. Labeling riboprobes with p2p] UTP ........................................ 59 2. RNA electrophoresis ................................................................... 60 3. Northern blot .................................................................................61 4. Hybridization ................................................................................62 E. In vitro transcription of linearized template ...................................63 F. In situ hybridization ................................................................................ 64 1. Tissue preparation ...................................................................... 64 2. Prehybridization wash .................................................................65 3. Hybridization ................................................................................66 4. Posthybridization washing .........................................................67 G. Immunocytochemistry ........................................................................... 68 H. Radioligand binding ................................................................................70
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