Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1991 Release of endogenous amino acids with putative neurotransmitter function from the rat spinal dorsal horn in vitro--modulation by neuropeptides Ivan Kangrga Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Medical Pharmacology Commons, Neuroscience and Neurobiology Commons, Neurosciences Commons, and the Pharmacology Commons Recommended Citation Kangrga, Ivan, "Release of endogenous amino acids with putative neurotransmitter function from the rat spinal dorsal horn in vitro-- modulation by neuropeptides " (1991). Retrospective Theses and Dissertations. 9535. https://lib.dr.iastate.edu/rtd/9535 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. 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Ann Arbor, MI 48106 Release of endogenous amino acids with putative neurotransmitter function from the rat spinal dorsal horn in vitro-- modulation by neuropeptides by Ivan Kangrga A Dissertation Submitted to the Graduate Faculty in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Department: Veterinary Physiology and Pharmacology Major: Physiology (Pharmacology) Approved : Signature was redacted for privacy. In Charge of Major Work Signature was redacted for privacy. Signature was redacted for privacy. For the Graduate College Iowa State University Ames, Iowa 1991 11 TABLE OF CONTENTS Page LIST OF ABBREVIATIONS v GENERAL INTRODUCTION 1 Explanation of Dissertation Format 1 Research Objective 1 Background and Literature Review 2 The spinal dorsal horn--structural and functional organization 2 Cytoarchitectonic of the dorsal horn: The Rexed's scheme 11 Organization of dorsal horn neurotransmitter systems 15 Release of Putative Endogenous Amino Acid Neurotransmitters in the Rat Spinal Dorsal Horn 40 The release and uptake of excitatory amino acids 40 The presynaptic Ca^* channel 45 Release of glutamate and aspartate in the spinal dorsal horn 47 Coexistence of excitatory amino acids and neuropeptides in the primary sensory neurons: physiological implications 50 Rationale 51 SECTION I. TACHYKININS AND CALCITONIN GENE-RELATED PEPTIDE ENHANCE RELEASE OF ENDOGENOUS GLUTAMATE AND ASPARTATE FROM THE RAT SPINAL DORSAL HORN SLICE 55 SUMMARY 56 INTRODUCTION 58 METHODS 62 RESULTS 66 DISCUSSION 93 ACKNOWLEDGEMENTS 103 REFERENCES 104 ill SECTION II. THE EFFECTS OF SUBSTANCE P AND CALCITONIN GENE- RELATED PEPTIDE ON THE EFFLUX OF ENDOGENOUS GLUTAMATE AND ASPARTATE FROM THE RAT SPINAL DORSAL HORN IN VITRO 114 SUMMARY 115 INTRODUCTION 116 METHODS 117 RESULTS 118 DISCUSSION 124 ACKNOWLEDGEMENTS 126 REFERENCES 127 SECTION III. OUTFLOW OF ENDOGENOUS AMINO ACIDS FROM THE RAT SPINAL DORSAL.HORN IN VITRO BY ACTIVATION OF LOW- AND HIGH-THRESHOLD PRIMARY AFFERENT FIBERS- -MODULATION BY OPIOIDS 129 SUMMARY 130 INTRODUCTION 131 METHODS 133 RESULTS 127 DISCUSSION 142 ACKNOWLEDGEMENTS 144 REFERENCES 145 APPENDIX. EFFECTS OF S- AND «-OPIOID RECEPTOR AGONISTS ON THE RELEASE OF ENDOGENOUS GLUTAMATE AND ASPARTATE FROM THE RAT SPINAL DORSAL HORN 147 Introduction 147 Methods 149 Results 149 Iv SECTION IV. EFFECTS OF PHORBOL ESTERS ON THE BASAL AND EVOKED RELEASE OF PUTATIVE ENDOGENOUS AMINO ACID NEUROTRANSMITTERS FROM THE RAT SPINAL DORSAL HORN 154 INTRODUCTION 155 METHODS 157 RESULTS 159 DISCUSSION 162 ACKNOWLEDGEMENTS 165 REFERENCES 166 SECTION V. ACTIONS OF (-)-BACLOFEN ON RAT DORSAL HORN NEURONS 171 SUMMARY 172 INTRODUCTION 174 METHODS 178 RESULTS 181 DISCUSSION 207 ACKNOWLEDGEMENTS 212 REFERENCES 213 DISCUSSION 221 REFERENCES 227 ACKNOWLEDGEMENTS 266 V LIST OF ABBREVIATIONS AHP Afterhyperpolarization Ala Alanine AP Action potential Asp Aspartate Asn Asparagine CCK Cholecys tokinin CGRP Calcitonin gene-related peptide CGRP-LI Calcitonin gene-related peptide like immunoreactlvlty CNS Central nervous sytem DC Direct current (d.c) DH Dorsal horn DR Dorsal root(s) DRG Dorsal root ganglion DYN Dynorphine EAA Excitatory amino acids EPSP Excitatory postsynaptic potential GABA 7-Aminobutyric acid GAD Glutamic acid decarboxylase Glu Glutamate Gin Glutamine Gly Glycine h.p. Holding potential HPLC High performance liquid chromatography HRP Horseradish peroxidase IPSP Inhibitory postsynaptic potential I-V Current-voltage m Mean NKA Neurokinin A NKB Neurokinin B OPA o-phthaldialdehyde PSP Postsynaptic potential PSC Postsynaptic current Rn Neuronal input resistance S.E.M. Standard error of the means SD Standard deviation SEPSP Slow excitatory postsynaptic potential Ser Serine SP Substance P SS Somatostatin TEA Tetraethylammonium Thr Threonine TTX Tetrodotoxin VASO Arginine-vasopress ine Vh Holding potential VIP Vasoactive intestinal polypeptide Vm Membrane potential 1 GENERAL INTRODUCTION Explanation of Dissertation Format This dissertation is written in an alternate thesis format, as permitted by the Graduate College. It Includes experimental objective, a background and literature review, a rationale, an experimental part, a discussion, a summary, a list of references cited in the background and literature review and in the discussion. The experimental part has five sections. Sections I and II represent two research papers already published, sections III and V represent two manuscripts submitted for publication in the Brain Research, and section IV is a part of a published research paper. 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. Mirjana Randié. Research Objective Anatomical and physiological data have provided a detailed description of the organization of afferent projections to the spinal cord and of the second order neurons in the dorsal horn. However, the identity of the neurotransmitter and neuromodulator substances at primary afferent synapses, and cellular mechanisms of their pre- and postsynaptic actions in the dorsal horn are still largely unknown. The objective of this research was to examine basal and depolarization-induced release of nine endogenous 2 amino acids, Including glutamate and aspartate, from the spinal dorsal horn. The specific purpose of the conducted experiments was to study the characteristics of amino acid release in response to selective activation (electrical, chemical) of either primary afferent A- or C- fibers, the origin of the released compounds by using capsaicin as a probe of primary afferent C-fiber function, and the regulation of their basal and evoked release by peptides. The possibility of modulation of endogenous amino acids release by tachykinins (substance P and neurokinin A), calcitonin gene-related peptide, opioid peptides, and by the activation of B subtype of 7-aminobutyric receptors, has been Investigated. The experiments used spinal cord slice-dorsal root ganglion in vitro preparation, high performance liquid chromatography with fluorlmetrlc detection and intracellular recording from dorsal horn neurons. Background and Literature Review This section briefly outlines the projections of the primary afferent fibers, the cytoarchitectonic organization and the principal neurotransmitter systems in the spinal dorsal horn. The objective is to provide background information for the study of chemical neurotransmission in the spinal dorsal horn. The spinal dorsal horn - structural and functional organization Primary sensory neurons, primary afferent fibers and sensory receptors Primary sensory neurons, located in the dorsal root ganglia (DRG), mediate