Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 2002 Synaptic transmission and plasticity in the spinal cord substantia gelatinosa: the role of GluR2, GluR5 and GluR6 glutamate receptor subunits Dong-ho Youn Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Animal Sciences Commons, Medical Pharmacology Commons, Neuroscience and Neurobiology Commons, Neurosciences Commons, Pharmacology Commons, Physiology Commons, and the Veterinary Physiology Commons Recommended Citation Youn, Dong-ho, "Synaptic transmission and plasticity in the spinal cord substantia gelatinosa: the role of GluR2, GluR5 and GluR6 glutamate receptor subunits " (2002). Retrospective Theses and Dissertations. 555. https://lib.dr.iastate.edu/rtd/555 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. 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. ProQuest Information and Learning 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 800-521-0600 Synaptic transmission and plasticity in the spinal cord substantia gelatinosa: The role of GluR2, GluR5 and GluR6 glutamate receptor subunits by Dong-ho Youn A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Physiology (Pharmacology) Program of Study Committee: Mirjana Randic, Major Professor Charles Drewes Richard J. Martin Vjekoslav Miletic Donald Sakaguchi Esturo Uemura Iowa State University Ames, Iowa 2002 Copyright © Dong-ho Youn, 2002. All rights reserved. UMI Number: 3073490 UMI® UMI Microform 3073490 Copyright 2003 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. 00x1346 Ann Arbor, Ml 48106-1346 11 Graduate College Iowa State University This is to certify that the doctoral dissertation of Dong-ho Youn has met the dissertation requirements of Iowa State University Signature was redacted for privacy. Maj Professor Signature was redacted for privacy. the Major Prog iii TABLE OF CONTENTS LIST OF ABBREVIATIONS iv ABSTRACT vi CHAPTER 1. GENERAL INTRODUCTION 1 CHAPTER 2. MATERIALS AND METHODS 19 CHAPTER 3. AMPA RECEPTOR- 25 CHAPTER 4. KAINATE RECEPTOR 38 CHAPTER 5. GENERAL CONCLUSIONS 102 REFERENCES 105 ACKNOWLEDGMENTS 127 iv LIST OF ABBREVIATIONS AiVlPA a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate ATPA (RS)-2-Amino-3-(3-hydroxy-5-zerr-butylisoxazol-4-yl)propanoic acid CGP55845 (2S)-3[[( l S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl] (phenylmethyl) phosphinic acid CNQX 6-cyano-7-nitroquinoxaline-2,3-dione CNS central nervous system ConA concanavalin A DA domoic acd; (2S,3S,4R,5,R)-2-carboxy-4-(5'-carboxy-l'-methyl-lZ,3E- hexadienyl)-3-pyrrolidineacetic acid DAP5 D-(-)-2-amino-5-phosphonopentanoic acid DH dorsal horn DNQX 6,7-dinitroquinoxaline-2,3-dione DR dorsal root DRG dorsal root ganglia EPSC Excitatory postsynaptic current EPSP fast excitatory postsynaptic potentials ES embryonic stem GluR Glutamate receptors GYKI52466 (l-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine) GYKI53655 ( 1 -(4-aminophenyl)-3-methylcarbamyl-4-methyl-7,8-methylenedioxy-3,4- dihydro-5 H-2,3 -benzodiazepine). HFS high-frequency stimulation HVAC high voltage activated Ca2* (HVAC) channels iGluR lonotropic GluRs I-V current-voltage JSTX Joro Spider toxin KA kainic acid; (2S,3S,4R)-carboxy-4-(l-methylethenyl)-3-pyrrolidineacetic acid LTD long-term depression LTP long-term potentiation LY382884 (3S,4aR,6S,8aR)-6-(4-carboxyphenyl)methyl-l,2,3,4,4a,5,6,7,8,8a- decahydroisoquinoline-3-carboxylic acid V MCPG (S)-a-methyl-4-carboxyphenyl glycine mEPSC miniature excitatory postsynaptic current mGluR metabotropic GluRs MK801 (5S, 10/?)-(+)-5-methyl-10,1 l-dihydro-5//-dibenzo[a,d]cyclohepten-5, 10- imine maleate NBQX 2,3-dioxy-6-nitro-l,2,3,4 tetrahydrobenzero-7- sulphanoylbenzo[f]quinoxaline-2,3-dione NK1 neurokinin I NMDA N-methyl-D-aspartate RJ rectification index RS-PCR reverse transcription polymerase chain reaction SDH superficial spinal dorsal horn SG substantia gelatinosa SYM 2081 ((2S-4/Î) diastereomer of 4-methylglutamate) SYM 2206 ((±)-4-(4-aminophenyl)-1,2-dihydro-1-methyl-2-propylcarbamoyl-6,7- methylenedioxyphthalazine) TTX tetrodotoxin VI ABSTRACT To understand the physiological role of the AMPA-type or kainate-type ionotropic glutamate receptors and their participation in sensory information processing, including pain, it will be necessary to develop a comprehensive description of their actions in the adult mouse spinal cord substantia gelatinosa (SG) region. Without selective antagonists of the AMPA and kainate receptors, however, pharmacology has provided little assistance in this endeavor. In this study, gene-targeted mice lacking GluR2 AMPA subunit and GluR5 or GluR6 kainate receptor subunits were used to identify the receptor subunits that comprise the AMPA and KA receptors responsible for modulation of primary afferent neurotransmission. AMPA receptors are not thought to be involved in the induction of LTP of excitatory synaptic transmission in the SG region, but they may be involved in the expression via several messenger pathways. However, one subunit of the AMPA receptors, GluR2, is known to control Ca2~ influx. To test whether GluR2 plays any role in the induction of LTP, the mice lacking the subunit were used in the present work. In GluR2 mutants, LTP in the SG region of spinal slices was markedly enhanced. These results suggest an important role for GluR2 subunit of AMPA receptors in regulating synaptic plasticity and pain behavior. In this study, gene-targeted mice lacking GluR5 or GluR6 kainate receptor subunits have also been used to identify the receptor subunits that comprise the kainate receptors responsible for presynaptic modulation of primary afferent neurotransmission. In the presence of synaptic inhibition, both GluR5 and GluR6 subunits contribute to the depressant action of kainate at the C-fiber and A5-fiber- activated polysynaptic pathways. In the absence of synaptic inhibition, the GluR6 subunit is critically involved in inhibiting transmission at both AS- and C-fiber monosynaptic pathways, whereas GluR5 plays a lesser role in inhibiting the C-fiber-activated pathway. Both GluR5 and GluR6 KA receptor subunits contribute to the KA receptor-mediated facilitation of excitatory synaptic transmission at synapses on the SG neurons. These results indicate that AMPA and kainate receptors play multiple and complex roles in regulation of excitatory synaptic transmission in the spinal cord SG region with potentially significant implications for pain control. 1 CHAPTER 1. GENERAL INTRODUCTION Thesis Organization This dissertation consists of five chapters (general introduction, materials and methods, results (AMPA receptor and kainate receptor), and general conclusion) and a list of references cited. The chapters, AMPA receptor and kainate receptor, contain research results which were formulated as introduction, results and discussion for future publications. The dissertation contains a large part of the experimental results obtained by the author during the course of his graduate study under the supervision of Dr. Miijana Randic. Research Objective The dorsal horn (DH), especially substantia gelatinosa (SG) of the spinal cord, is known to play an important role in the modulation of nociceptive transmission through fine myelinated and unmyelinated primary afferent fibers arising from the periphery. Glutamate, or a related amino acid, is the major excitatory neurotransmitter at primary afferent fiber-spinal DH neuron synapses (so called glutamatergic synapses). Elucidating the function of glutamate and their receptors has traditionally relied on pharmacological approaches using compounds that act as agonists or antagonists at specific receptors on the pre- and postsynaptic membranes. Recently, a new tool has been added to the armamentarium for studying the synaptic function, namely genetic manipulations (esp. null mutations or 'knockouts') that delete specific subunits comprising glutamate receptors. The objective of this research was to investigate the role(s) of glutamate receptor subunits (GluR2,