GLUTATHIONE AS A NEUROTRANSMITTER IN PRIMARY VISUAL CORTEX: BINDING SITES AND NEURONAL UPTAKE by STEPHEN BOWLSBY B.Sc, The University of British Columbia, 1977 .F.A., The University of Southern California, 1980 A THESIS SUBMITTED IN PARTIAL FULLFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Physiology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 1991 (c) Stephen Arthur Bowlsby, 1991 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia/ I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives, it is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of ^U*^S7 ft/tfCji^ The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT Understanding the response properties and plasticity of primary visual (striate) cortex depends on the determination of its "chemical circuitry", yet the neurotransmitters that mediate sensory input to striate cortex are not known, and such evidence as exists is contradictory. The geniculpstriate input bears a similarity to glutamatergic neurotransmission, but glutamate is not a good candidate in this pathway. In contrast, glutathione (GSH) has been suggested to be one of the excitatory-amino-acid neurotransmitters in cortex (Ogita and Yoneda, 1987). Criteria for the identification of a neurotransmitter include the demonstration of the presence of receptors for the substance (necessary) and the demonstration of retrograde uptake and transport of the substance (supportive). The present study attempted to characterize GSH binding sites, examine their distribution, and examine possible GSH interaction with excitatory amino acid receptors in rat and cat primary visual cortex using in-vitro radioligand methods on 20-/xm-thick cortex sections. In addition, the present study used in-vivo uptake and transport of radiolabeled tracers to attempt to support the localization of GSH neurotransmission to pathways within the visual system. Saturation binding experiments using radiolabeled GSH in rat primary occipital cortex sections revealed a high- affinity site (K(j = 5.4 nM; Bmax = 235 fmol/mg protein) and a iii denser low-affinity site (K^ = 1.3 /iM; Bmax = 1.3 pmol/mg protein) , the KQ< of which is typical of excitatory amino acid receptors. Kinetic and competition experiments yielded similar K<j values. Competition studies of the low-affinity GSH binding site showed a separate site as well as binding with affinity for the neurotransmitter candidates cysteine, aspartate, and glutamate. Excitatory-amino-acid receptor subtype affinity was shown for AMPA at pH 7.4 and for NMDA at pH 6.9.. Radiolabeled GSH binding in adult rat visual cortex showed a relatively uniform distribution across all cortical layers. Binding distribution studies in cat striate cortex showed densest [35S]GSH binding in layer 4, the geniculostriate input layer, from 13 days postnatal to adult. Distribution of [35S]GSH binding sites also showed a distinct preference for lower layer 4 in monkey striate cortex. Microinjection of [35S]GSH, [3H]GSH, and its constituent amino acids, [3H]glutamate, [35S]cysteine, and [3H]glycine, into primary and secondary visual cortex in the rat produced uptake to visual-system thalamic nuclei and superior colliculus. Possible retrograde uptake to cell bodies was determined for [3H]GSH and [35S]cysteine in the dorsal lateral geniculate and lateral posterior nuclei in the rat. Microinjection of [3H]GSH in cat cortical area 17 produced uptake to the dorsal lateral geniculate nucleus, but possible retrograde uptake to cell bodies could not be determined. These results support the proposition that GSH plays a role as a neurotransmitter in primary visual cortex, in iv particular as a geniculostriate neurotransmitter, and may take part in neurotransmission by interacting with excitatory amino acid receptors in addition to GSH receptors. V TABLE OF CONTENTS page ABSTRACT ii TABLE OF CONTENTS V LIST OF TABLES vii LIST OF FIGURES viii LIST OF ANATOMICAL ABBREVIATIONS X ACKNOWLEDGEMENTS xi INTRODUCTION 1 Chemical Circuitry and Visual Cortex 1 The Geniculostriate Neurotransmitter 8 An Instructive Case — Neurotransmission with NAAG 17 Glutathione in Brain 21 Excitatory Amino Acid Receptors and Transmitters 25 Neurotransmitter Criteria 29 Thesis Rationale 31 MATERIALS AND METHODS 36 Ligands and Tracers 3 6 In Vitro Receptor Binding Procedures 3 6 Binding Site Analysis 41 In Vivo Uptake Procedures 49 RESULTS 52 Parametric Studies 52 Binding Site Characterization 70 Excitatory Amino Acid Receptor Interactions 84 Binding Site Distributions 89 Uptake Experiments In Vivo v 98 vi page DISCUSSION 107 Binding Site Characterization 107 Laminar Distribution 112 Pathways 115 Astrocytes 120 Excitatory Amino Acid Receptor Interactions 122 Speculation — A Novel Form of Neurotransmission? 125 SUMMARY AND CONCLUSIONS 128 REFERENCES 130 vii LIST OF TABLES page Nuclei showing presence of radiolabel from uptake. 101 Vlll LIST OF FIGURES page 1. A schematic distribution of the laminar binding patterns in neonatal kittens and adult cats. 6 2. Diagram showing the termination of afferent fibres and the origin of efferent fibres in generalized neocortex. 10 3. Terminations of X-cell and Y-cell efferents in area 17 of the cat. 11 4. Diagram of the laminar distribution of thalamic afferents to cat and monkey area 17. 12 5. The chemical structure of reduced glutathione and oxidized glutathione disulfide. 20 6. An outline of the metabolism and function of glutathione. 24 7. Binding of [ H]GSH in rat visual cortex as a function of concentration of buffer. 54 8. Binding of [ H]GSH in rat visual cortex as a function of pH. 54 9. Binding of [3H]GSH in rat visual cortex as a function buffer type. 56 10. Binding of [3H]GSH in rat visual cortex as a function of ion concentration. 57 11. Binding of [ H]GSH in rat visual cortex as a function of fixation. 61 12. Binding of [3H]GSH in rat visual cortex as a function of the concentration of various biochemical agents. 62 13. Separation of disulfide bonds by the reducing action of dithiothreitol, during which the dithiothreitol is inactivated. 68 14. Binding of L-[3H]glutamate and [3H]CPP in rat brain membranes as a function of pH. 69 15. Association and dissociation rates of radiolabeled GSH binding in rat visual cortex. 71 16. Competition curves for [3H]GSH binding in adult rat and cat visual cortex. 76 ix 17. Competition of GSH with 18 nM [35S]GSH, and its derived "B versus B*I" plot to reveal the separate values. 78 18. Saturation binding curves and Eadie-Hofstee plots of [3H]GSH and [35S]GSH binding sites in rat visual cortex. 82 19. Competition by GSH with [3H]GSH at 20 minutes incubation (pH 7.4) to reduce binding to the dense, nonreceptor, LAHC site, the presence of which would obscure displacements. 86 20. Percent control binding of L-[3H]glutamate in rat visual cortex at pH 8.0 compared to binding at pH 6.9 and at pH 6.9 with various 1 mM competitors. 87 21. Percent control binding of [3H]GSH in rat visual cortex with significant displacement by various l mM competitors at pH 7.4 and at pH 6.9. 88 22. Autoradiographic distributions of [35S]GSH binding in rat, cat, and monkey visual cortex. 91 23. Quantitative analysis of laminar binding density on autoradiographic film across cat area 17 cortex at various ages. 96 24. Autoradiographic distribution of radiolabeled tracer uptake to the thalamus from injection in visual cortex in rats. 102 25. Photomicrographs of combined pyronin Y staining for cell bodies and autoradiographic distribution of silver grains following uptake of [3H]GSH from rat visual cortex to the DLG. 106 X LIST OF ANATOMICAL ABBREVIATIONS ATPD anterior pretectal nucleus, dorsal division CP cerebral peduncle DLG dorsal lateral geniculate nucleus IGL intergeniculate leaf IMA intramedullary thalamic area LP lateral posterior nucleus LPLR lateral posterior nucleus, lateral rostral division LPMC lateral posterior nucleus, medial caudal division LPMR lateral posterior nucleus, medial rostral division Ocl primary occipital cortex Oc2 secondary occipital cortex Oc2L secondary occipital cortex, lateral OC2ML secondary occipital cortex, mediolateral OC2MM secondary occipital cortex, mediomedial Pari parietal cortex, area 1 RSA retrosplenial agranular cortex RSG retrosplenial granular cortex Rt reticular thalamic nucleus sc superior colliculus, superior and inferior grey SubG subgeniculate nucleus VLG ventral lateral geniculate nucleus VLGMC ventral lateral geniculate nucleus, magnocellular VLGPC ventral lateral geniculate nucleus, parvocellular ZI zona incerta ZID zona incerta, dorsal ZIV zona incerta, ventral xi ACKNOWLEDGEMENTS I would like to extend my thanks and appreciation to: Dr. Chris Shaw for providing the opportunity of working in his lab and finding out what research is all about; for his assistance and support in completing this thesis; and for providing the costly radiolabeled chemicals. Dr. Steven Kehl, Derrick March, Mala Glenwright, and John Sanker for technical assistance. Dr. Ken Baimbridge and Dr. William Ovale for use of their cryostat microtomes. Dr. Steven Vincent for use of his computer image analysis system. Dr. Ken Curry for helpful comments. Dr. Ray Pederson for his support as graduate advisor. My wife Joyce for financial support when the student loans unexpectedly dried up. 1 INTRODUCTION CHEMICAL CIRCUITRY AND VISUAL CORTEX To understand the brain, and to be able to intervene on its behalf, it is necessary to model the brain's activity (McNaughton and Morris, 1987; Zipser and Andersen, 1988; Ambros-Ingerson and Lynch, 1990; Servan-Schreiber et al., 1990). Knowledge of anatomical connectivity is insufficient to do this, since neurons are not simple threshold devices.