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 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. A major reason for this is the complexity of chemical neurotransmission. To understand the computations performed by any part of the brain it is necessary to know the identities of the neurotransmitters and receptor subtypes employed at each synapse. Chemical neurotransmission produces a wide range of effects. Various ion conductances and second-messenger systems can be activated by different receptor subtypes for the same neurotransmitter, depending on the pathway studied (for review see Nicoll, 1988). Receptor activation may produce a fast synaptic potential change, or it may alter, over a longer time span, the properties of voltage-dependent ionic conductances that are involved in control of cell excitability, resulting in such changes as increased action potential duration, changes in firing frequency and firing pattern, and increased Ca2+ entry during an action potential (for review see Kaczmarek and Levitan, 1987). Receptor 2 populations may be up-regulated or down-regulated by their own activation or by the activation of other receptors, and rates of regulation may be modulated by other neurotransmitters (for review see Hollenberg, 1985). Activation of one receptor site may directly modulate or co- activate the effects of a receptor site for a different neurotransmitter (for example, see Thompson et al., 1989). The second-messengers from activation of different receptors by different neurotransmitters may converge on the same ionic conductance, even as each diverges to affect still other conductances (see review by Nicoll, 1988). Long term synaptic strengths may be altered, for example by the action initiated by a voltage-dependent receptor (Artola and Singer, 1987) . Neurotransmission may affect gene expression (for review see Morgan and Curran, 1989), and neurotransmitters may act as inducers during development to affect biochemical (Moran and Patel, 1989) and structural (reviewed by Lipton and Kater, 1989) differentiation. Widespread paracrine effects may even be important in the computations performed by the brain (Herkenham, 1987). Finally, different neurotransmitters can be co-released (for example see Lundberg and Hokfelt, 1983) and receptors may act at axo• axonic and dendrosomatic synapses (Cheramy and Glowinski, 1981) and as presynaptic autoreceptors (see McGeer et al., 1987) . The visual cortex transforms the properties of its thalamic input to generate a wide variety of functional 3 properties required for vision. Each cell in the visual cortex has a receptive field where stimuli of the appropriate orientation, shape, and direction elicit a discharge. Recently it has become possible to make a direct association between individual cortical connections and particular receptive field properties by the pharmacological "microdissection" of cortical circuits using neurotransmitter receptor antagonists. For example, local iontophoresis of bicuculline, an antagonist of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) at GABA^ receptors, abolishes orientation and directional selectivity (reviewed in Bolz, et al., 1988). Here the "chemical circuitry" is beginning to be understood. Experience-dependent synaptic plasticity is crucial to the development of the capacities of the visual system, and receptor activation plays a dominant role (for review see Cynader et al., 1990). For example, competetive binocular interactions occur within a critical period that peaks in kittens at approximately 4 weeks of age (Hubel and Weisel, 1970). At this time, eyelid closure for as brief a period as a few hours produces profound and prolonged effects on both visual capacities and cortical neuronal responses. Axon terminals from dorsal lateral geniculate nucleus relay cells connected to the deprived eye are found to occupy 20 % of the area of the cortex while the undeprived eye has an expanded representation covering 80 % of the thalamic recipient zone. Blockade of neurotransmission by the cholinergic and 4 noradrenergic non-specific afferents to visual cortex substantially reduces this occular dominance shift (Bear and Singer, 1986). Similarly, blockade of N-methyl-D-aspartate (NMDA) receptors on lateral geniculate cells with specific antagonists during the period of sublamina formation prevents retinal afferents from segregating into "On" and "Off" sublaminae (Hahm et al., 1991). Infusion of the GABA^ receptor agonist muscimol in visual cortex during monocular exposure produces an increase in effectiveness of the deprived eye at the expense of the normally viewing eye (Reiter and Stryker, 1988). Occular dominance shifts due to monocular deprivation are associated with an increase in GABA^ receptors in visual cortex (Shaw and Cynader, 1988) . Most neurotransmitter receptors are found to have an idiomorphic laminar distribution in visual cortex, corresponding to specific input/output zones (see below), and changes in these distributions are associated with the critical period for synaptic plasticity (for review see Shaw et al., 1986; Cynader et al, 1990). Figure 1 shows these distributions for selected receptors, and their change in distribution over the critical period. Some of these receptors have been shown to be re-expressed on different neurons (Van Huizen et al., 1988), and from post-synaptic to presynaptic elements (Shaw et al., 1989b). The significance of these changes in receptor distribution is not known, but they emphasize the importance of determining and understanding the chemical circuitry of the visual cortex. 5 To understand computation, plasticity, and development in brain, and in particular in primary visual cortex, a complete mapping of the neurotransmitters in each pathway and their corresponding synaptic effects must be made. The geniculstriate neurotransmitter is a major one of these. 6 Figure 1. A schematic distribution of the laminar binding patterns studied in neonatal kittens and in adult cats. The top row of the figure lists the classes of neurotransmitters whose receptors have been examined. These include acetylcholine (ACh), adrenaline (R), opiates (OP), peptides (PEP) and adenosine (AD) within the broad class of neuromodulatory transmitters. The broad class of amino acid transmitters includes the excitatory amino acids, represented by glutamate (GLUT), and the inhibitory amino acids, represented by gamma-aminobutyric acid (GABA). The various receptor subtypes are listed in the row below. The tritiated ligand employed is listed in the subsequent row. The cortex is divided into six layers and the density of the binding is illustrated using the shading scale given at the bottom of the figure. In neonatal kittens the most common patterns of binding observed are either little or no discernable binding (nicotine, oxytremorine, baclofen), or a pronounced peak in layer 4. In adult animals, the pattern is very different, with relatively few binding sites (nicotine, muscimol, and flunitrazepam) concentrated in layer 4. Most receptors show a distribution emphasizing layers 1 through 3 and 6. (From Cynader et al., 1990, after an earlier version in Shaw et al., 1986) 1 - 3 DAY OLD Neuromodulators AA Transmitters NT/NM ACh 0 OP. PEP. AO. GLUT. GABA Ca4* Subtype N M2 M, Al Q K A BZ B o in < o >< • OJ co m < o < 0. c' (0 N 6 x ^ z I 10 O I Z ID z. Z O a. 2 O Q Z o o o < 2 LL m a ADULT Relative Binding Densities Very high m Moderate No High Light 8 THE GENICULOSTRIATE NEUROTRANSMITTER Area 17, the cortical retinotopic map that receives the majority of the visual sensory input, is commonly referred to as the striate cortex because in primates a white band in upper layer 4, the line of Gennari, gives area 17 a striated appearance (Lund, 1973). The main thalamic input to area 17, which originates from the dorsal lateral geniculate nucleus (DLG), is thus referred to as the geniculostriate projection or pathway (for review see Gilbert, 1983; Stone, 1983). The line of Gennari bears little relationship to the area of termination of geniculate afferents and most probably represents myelinated intrinsic axons (Lund, 1973) . Although cat area 17, which has no line of Gennari (Stone, 1983) is commonly referred to as striate cortex, the primary visual cortex of the rat (Ocl) is not. For simplicity, however, in this thesis the geniculate input to primary visual cortex will be referred to as the geniculostriate projection or pathway in the rat as well. Area 17 is highly organized (for review see Lund, 1973; Gilbert, 1983; Stone, 1983; Sefton and Dreyer, 1986). Neurons are arranged in columns extending from pia to white matter having common receptive field properties within each column. A horizontal organization is also evident corresponding to cortical layering separating simpler monocular responses in layer 4 from more complex binocular responses in layers 2, 3, 5 and 6. The layers also 9 correspond to restricted input and output zones. Figure 2 illustrates this with a schematic diagram that generally holds true for all neocortex. Layer 4 is the primary input layer, where afferents synapse mainly on spiny stellate cells confined to this layer, and on apical dendrites of pyramidal cells of lower layers. Layer 2 and 3 pyramidal cells make cortical associational and commissural projections. Layer 5 pyramidal cells in area 17 project to the superior colliculus (SC) and the pulvinar or its homolog the lateral posterior nucleus (LP) in the rat, and LP/pulvinar complex in the cat. Layer 6 neurons in area 17 project back to the DLG and to the reticular thalamic nucleus (Rt). Other inputs include corticocortical afferents, which generally ramify in the superficial layers; callosal afferents confined to the area 17/18 border; and brainstem modulatory afferents such as those mediated by serotonin or noradrenaline which have their own distinctive laminar distributions in area 17. Other projections exist to various subcortical nuclei from both area 17 and area 18 (secondary visual cortex). The predominant zone for sensory input neurotransmission in visual cortex is layer 4 in area 17 (Ocl), because this is where most of the thalamic terminals synapse, but there are minor exceptions. Figure 3 shows examples of the dense terminal arborations for the two main physiological types of geniculate inputs in the cat. Figure 4 diagrams the layers of terminations of all the various thalamic inputs to area 17 in the cat and monkey. The X and Y geniculate relay 10 NEOCORTEX Afferents Layers Efferents 1 Molecular 2 External granular External V pyramidal 4 Internal granular 5 Internal pyramidal Projection Association and commissural Figure 2. Diagram showing the termination of afferent fibres and the origin of efferent fibres in generalized neocortex. (From Changeux, 1985) 11 Figure 3. Terminations of X-cell and Y-cell efferents in area 17 of the cat. The upper diagram shows the mode of termination in area 17 of an axon of a Y-class geniculate relay cell. The axons spread principally in layer IVab, forming two clusters that may correspond to occular dominance columns. A few small branches extend into layer III, and the axon also gives collaterals to the upper part of layer VI. The lower diagram shows the mode of termination in area 17 of an axon of an X-class geniculate relay cell. The axon terminates principally in layer IVc, although a few small branches extend into layer IVab. This axon also gives collaterals to the upper part of layer VI. (From Ferster and LeVay, 1978) 12 Figure 4. Diagram of the laminar distribution of thalamic afferents in cat and monkey area 17. The C laminae in cat lateral geniculate nucleus contain Y- and W-cells. Parvocellular and magnocellular layers of the monkey lateral geniculate contain Y- and X-cells, respectively. LP in the diagram in cat is the LP/pulvinar complex, homologous to the monkey pulvinar. (From Gilbert, 1983) 13 projections in the cat and the parvocellular and magnocellular geniculate relay projections in the monkey correspond to the physiologically defined retinal ganglion X cell (tonic, small receptive fields, slow) and Y cell (phasic, large fields, fast) afferents. The LP/pulvinar acts as a relay for retinal input but projects to area 17 only sparcely. The DLG and and LP also project to area 18 in rat, cat, and monkey, although much less densely than the geniculostriate projection. Differences exist between species (Figure 4). The primate primary visual cortex is more sharply divided into a series of clearly limited laminae than it is in the cat or rat. W-type geniculate relay cells exist in cat and rat. The DLG shows projections to all layers in the cat, but not so in the monkey. In the rat there are practically no X-like cells, geniculate relay cells are not aggregated into laminae, the DLG does not project to layer 5 as it does in cat, and the LP projects to layers 5 and 6. The neurotransmitters mediating sensory input to primary visual cortex, whether in the rat, cat, or monkey, including the geniculostriate input, are unknown, and what evidence does exist is contradictory. Glutamate is proposed as a neurotransmitter in visual cortex (reviewed by Tsumoto, 1990). Glutamate activates several pharmacologically-distinct receptor subtypes known as excitatory amino acid (EAA) receptors, but so do several other endogenous substances, and the actual neurotransmitters for these receptor subtypes are 14 not determined (see below; for review see Foster and Fagg, 1984; Coyle et al., 1986; Mayer and Westbrook, 1987; Monaghan et al., 1989). Glutamate can excite cells in all layers of visual cortex, including layer 4, the predominant sensory input layer (Krnjevic and Phillis, 1962), and the broad- spectrum EAA receptor antagonist kynurenic acid can block evoked responses in layer 4 of visual cortex (Tsumoto et al., 1986; Hagihara et al., 1988). However, both receptor binding and uptake studies suggest that glutamate may not be the primary geniculostriate neurotransmitter. Receptor binding studies in adult visual cortex show relatively light binding density in layer 4, compared to other layers, for glutamate or for several EAA receptor subtype ligands in the rat (Monaghan and Cotman, 1982, 1985; Monaghan et al., 1984) in the cat (Shaw et al., 1986, 1990; see Figure 1), and in the monkey (Shaw and Cynader, 1986; Shaw et al., 1989, 1990, 1991) (for review see Tsumoto, 1990). Since the dense terminations of geniculostriate axons contribute a large proportion of the synapses in layer 4, this relatively light density of glutamate binding would be unexpected if glutamate is the primary geniculostriate neurotransmitter, unless there is a lower receptor affinity or lower number of receptors per synapse in this layer. The only receptors found to date in relatively high density in adult visual cortex layer 4 besides GABA^ and benzodiazapine receptors in the cat (Shaw et al., 1986; see Figure 1) and presynaptic nicotinic acetylcholine receptors on geniculate terminals in the cat 15 (Prusky et al., 1987; see Figure 1) are for tachykinin peptides in the rat and cat (Danks et al., 1986; Mantyh et al., 1984, 1989; March and Shaw, 1990), which are postsynaptic in the cat (March and Shaw, 1990). Tachykinins, however, are so far known only as neuromodulators or as producing slow excitatory synaptic potentials (reviewed in Jessel and Womack, 1985), which is not consistent with a role as a primary geniculostriate neurotransmitter. Preliminary data suggest that the NMDA subtype of EAA receptor is concentrated in sub-layer 4C6 (see Figure 4) of monkey striate cortex (Shaw et al, 1991), and could therefore conceivably play a role in geniculostriate synaptic transmission, although these receptors do not appear to play such a role in the rat or cat (for review see Tsumoto, 1990). A second form of supporting evidence for the identity of a neurotransmitter in a given pathway is the presence of retrograde uptake and transport of the candidate substance from terminals to cell bodies. Uptake studies with [3H]glutamate in cat have consistently implicated as glutamatergic only the recurrent output projection from cortical layer 6 to the DLG and not the geniculostriate projection (Baughman and Gilbert, 1981), with controversy existing regarding the projection from layer 5 to the pulvinar and SC (Baughman and Gilbert, 1981; Fosse et al., 1984). In the rat, studies employing cortical ablation and measures of endogenous glutamate have implicated as glutamatergic only the cortical projections to the DLG, LP, and SC (Lund-Karlsen and Fonum, 1978; Fosse and Fonnum, 1987). For review see Tsumoto (1990). 17 AN INSTRUCTIVE CASE: NEUROTRANSMISSION WITH NAAG The modified dipeptide N-acetyl-aspartyl-glutamate (NAAG) has been recently implicated as a neurotransmitter in several brain pathways. NAAG is found in high levels in brain, regionally distributed in neurons (Coyle et al, 1986). It is epileptogenic in hippocampus, with a high affinity for the 2-amino-4-phosphonobutyrate (AP4) selective subtype of EAA receptors (Zaczeck, 1983). The determination of a neurotransmitter role for NAAG in particular pathways is instructive, not only for the importance of multiple criteria in identifying any substance as a neurotransmitter (see below), but because it shows how an endogenous small peptide can take the stage as the primary candidate for neurotransmitter status in pathways previously thought to be mediated by the ubiquitous EAA neurotransmitter candidate glutamate. Evidence such as release and excitation in pyriform cortex had suggested that glutamate or aspartate were the neurotransmitters of the lateral olfactory tract (LOT) (Bradford and Richards, 1976; Collins, 1978). More recently, the EAA antagonist AP4 was shown to block the electrophysiological response of pyriform cortex pyramidal cells to both the monosynaptic excitation elicited by stimulation of the LOT and to iontophoretically-applied NAAG, while leaving unaffected the responses to applied glutamate and aspartate (ffrench-Mullen et al., 1985). Then 18 immunocytochemical staining localized NAAG, but not glutamate or aspartate, to the olfactory bulb mitral cells (which project as the LOT) (Blakely et al., 1987). Further neurotransmitter criteria are fulfilled by the demonstration of calcium-dependent release in rat brain slices (Zollinger et al., 1988), and a method of rapid inactivaton by a selective dipeptidase, followed by reuptake of the degraded glutamate in synaptosomes (Blakely et al, 1986; Robinson et al., 1987). Release of glutamate and aspartate in the LOT is therefore most likely due to the fact that they are degradation products of released NAAG. Most recently NAAG has been implicated as a neurotransmitter of the septo- hippocampal pathway (Senut et al., 1990) and at all retinal target zones, including the DLG and SC (Moffett et al., 1991). An interesting possibility is suggested by Coyle et al. (1986). In analogy to the cholinergic system, choline itself is known to have agonist actions at both muscarinic and nicotinic cholinergic receptors. Glutamate has broad and uniform excitatory effects on neurons, acting on distinct EAA receptor subtypes. A possible explanation for this broad action is that glutamate or aspartate are common components of a group of small peptides like NAAG which interact with discrete E/AA receptors. Glutamate or aspartate in sufficient concentrations may produce agonist effects by binding to that domain of the EAA receptor that recognizes the glutamate or aspartate present within the endogenous neurotransmitter. 19 Such an interpretation is not inconsistent with the observation that gamma-D-glutamyl-glycine is a potent antagonist of EAA receptors. It is further possible that the degradation products of these small peptides may have a direct physiological role to play in interaction with EAA receptors, as "secondary" neurotransmitters. Small di- and tri-peptides may take part in fast synaptic potential EAA-type neurotransmission at distinct, known EAA receptor types, as well as at possibly novel receptor subtypes specific to that small peptide. The relative paucity of known EAA receptors in layer 4 in primary visual cortex coupled with the excitation of layer 4 by glutamate suggest the possibility of a novel receptor type for a novel neurotransmitter, with some affinity for glutamate and antagonism by kynurenic acid (see above). NAAG itself is present in lowest amounts in neocortex, with its highest binding in upper layers (Coyle, et al., 1986). Glutathione, or gamma-glutamyl-cysteinyl-glycine (GSH), an endogenous tripeptide (structure shown in Figure 5) is a possible alternative candidate for neurotransmission in visual cortex, and for the geniculostriate neurotransmitter in particular. 20 0. Gly H—C—H N—H 0=C y-Glu—C s—Gly Cys H—C—CH2— SH N—H S 0=C y-Glu—Cys—Gly CH Oxidized glutathione CH2 Y-Glu +H,N—C— H Reduced glutathione (y-Glutamylcysteinylglycine) Figure 5. The chemical structure of reduced glutathione (GSH) and oxidized glutathione disulfide (GSSG). (From Stryer, 1988) 21 GLUTATHIONE IN BRAIN . GSH plays many biochemical roles throughout the body, including the brain (for review see Orlowski and Karkowsky, 1976; Meister, 1989). GSH acts to protect cellular thiol groups against oxidative damage by cycling between the predominant reduced form and an oxidized, disulfide form (GSSG, see Figure 5), often in conjunction with enzymes such as glutathione peroxidase. GSH acts as an S-conjugate of toxic substances, in conjunction with various glutathione S- transferases or non-enzymatically, to aid excretion. GSH acts as a coenzyme in hydration, dehydrogenation, isomerization, and dehydrochlorination reactions. Glutathione reductase maintains GSH in its predominant reduced form (GSH:GSSG = 500:1). A membrane-bound, extracellular-acting enzyme, gamma-glutamyl-transpeptidase, which is concentrated in capillaries, choroid plexus, and ependymal cells, degrades GSH into cysteinyl-glycine and into glutamate that is conjugated with free amino acids (gamma- glutamyl-AA). The biochemistry of GSH is illustrated in Figure 6. GSH may play a- role in neurotransmission in addition to its other functions. Although GSH is found in high concentrations in brain (Reichelt and Fonnum, 1969; Orlowski and Karkowsky, 1976), and is shown to be necessary for maintenance of mitochondrial function in brain (Jain et al., 1991), there is little detoxification by GSH conjugation in 22 brain (Orlowski and Karkowsky, 1976) . GSH is concentrated in the neuropil and white matter throughout rodent and primate brain, with the exception of cell bodies of cerebellar Purkinje cells (Slivka et al., 1987; Philbert et al., 1991). In rat brain, GSH peroxidase is confined largely to the nuclei of only some neurons (Ushijima et al., 1986) and the GSH S-transferases are found only in astrocytes (Senjo et al., 1986). GSH binding sites are seen in crude synaptic membrane preparations from rat brain, including cortex, showing regional variation (Ogita et al. 198 6b; Ogita and Yoneda 1987, 1988). These sites include a possible GSH receptor with a K^ value typical of EAA receptors, as well as a putative Na-independent, temperature dependent uptake site. GSH also shows affinity for glutamate binding sites, with selectivity for the AMPA and AP4 receptors (Ogita and Yoneda, 1987; Oja, 1988) and for the NMDA receptor antagonist- preferring site (Ogita and Yoneda, 1990) (see below). Cysteine is released in a calcium-dependent manner from depolarized rat cortical slices, and may thus be a degradation product of a larger released precursor such as GSH (Keller et al., 1989). The above data suggest that GSH could be a neurotransmitter of the type hypothesised in the previous section, and it has now been shown to be excitatory in primary visual cortex. Recently Timothy J. Teyler at N.E. Ohio University College of Medicine (personal communication) demonstrated a strong field-potential source in slices of rat 23 visual cortex in layer 6 in response to iontophoretic application of GSH to layer 4, apparently by activating apical dendrites of layer 6 cells in layer 4. This is consistent with the possibility that GSH is a geniculostriate neurotransmitter. 2 Figure 6. An outline of the metabolism and function of glutathione. Enzymes: (1) gamma-glutamyltranspeptidase; (2) gamma-glutamylcyclotransferase; (3) 5-oxoprolinase; (4) gamma-glutamylcysteine synthetase; (5) glutathione synthetase; (6) dipeptidase; (7) glutathione S-transferase; (8) glutathione peroxidase; (;9) glutathione reductase; (10) transhydrogenases. AA = amino acids; X = compounds that react with glutathione to yield conjugates. (From Meister, 1983) 25 EXCITATORY AMINO ACID RECEPTORS AND TRANSMITTERS As demonstrated by other investigators, GSH may interact with a range of EAA receptor subtypes, in addition to a possible GSH receptor. It may also be enzymatically degraded into a series of amino acids and dipeptides that can themselves interact with EAA receptors. GSH is degraded into cysteinyl-glycine and gamma-glutamyl-amino acids by gamma- glutamyltranspeptidase; cysteinyl-glycine is degraded into cysteine and glycine by cysteinylglycinase; cysteine is metabolized to cysteine sulfinic acid (CSA) by cysteine dioxegenase; and CSA is metabolized to cysteic acid by cysteine sulfinic acid decarboxylase (Meister, 1983; Rassin and Gall, 1987; Hill et al., 1985). The following section briefly describes some of the characteristics of the known EAA receptor subtypes and the processes in which they are involved, and lists their proposed endogenous neurotransmitters. For reviews see Foster and Fagg (1984), Mayer and Westbrook (1987), Monaghan et al. (1989), and Griffiths (1990). The EAA receptor subtypes are distinguished by the specificity of radioligand binding and the physiological effects of agonists and antagonists, each type named after its most preferential or potent agonist (nomenclature taken from Monaghan et al [1989]). The ionotropic NMDA, KAIN (kainate), and AMPA (alpha-amino-3-hydroxy-5-methylisoxazole- 4-propionate) receptor subtypes all increase Na+ and K+ 26 conductances, preferentially activating different conductance states of the same ion channel type (Jar and Stevens, 1987). The NMDA receptor is more complex. It is voltage dependent, permeable to the second messenger Ca2+, contributes both a fast and a second, slow synaptic potential component not seen with the other two channel receptors (Angelo et al., 1990), and contributes to tonic background voltage-dependence and facilitation of action potentials (Sah et al., 1989). The NMDA receptor is co-activated by the neurotransmitter glycine, modulated by Zn2+, modulated by a non-competitive antagonist binding site inside the channel, and modulated by an antagonist-preferring recognition site that may be either a separate site from the agonist site or an alternate state. Activation of NMDA, AMPA, and KAIN receptors can also decrease inositol-tris-phosphate (IP3) production induced by muscarinic, serotonin, or histamine receptor activation (Nobel et al., 1989). A second KAIN receptor subtype is permiable to Ca2+ (lino et al., 1990; Holopainen et al., , 1990). Recent evidence indicates the presence of a common AMPA/KAIN receptor in addition to separate AMPA and KAIN receptor subtypes (reviewed in Barnard and Henley, 1990). The metabotropic ACPD (trans-l-amino-1,3- cyclopentanedicarboxylate) receptor is G-protein linked to an increase in IP3 production, leading to a decrease in Ca2+- dependent voltage-gated K+ conductance in hippocampus, increasing responsivity of the cell (Charpak et al., 1990). The AP4 (L-2-amino-4-phosphonobutyrate) receptor is not yet 27 well characterized; it appears to play a dominant role as a presynaptic receptor attenuating release at glutamatergic terminals, as well as occuring as a postsynaptic receptor (Bridges et al., 1986). Finally, there appears to be a CSA (cysteine sulfinate) receptor (Pullan et al., 1987; Pin et al., 1987), which may stimulate cyclic AMP production in the hippocampus (Baba et al., 1988) and possibly a Na+ conductance (Pullan et al., 1987). Desensitization of EAA receptors occurs in two forms: fast (10 to 100 ms) for NMDA receptors, and slow (seconds) for non-NMDA receptors (Trussel et al., 1987). EAA receptors are implicated in a range of plasticity and developmental processes. NMDA receptors are instrumental in associative long-term potentiation (LTP) in striate cortex (Artola and Singer, 1987). ACPD receptors are associated with LTP in striatum (Dumuis et al., 1990), and with the development of the critical period in striate cortex (Dudek and Singer, 1989). Quisqualate (QUIS) sensitive receptors (i.e., AMPA receptors, ACPD receptors, or both) are implicated in associative long-term depression in cerebellum (Kano and Kato, 1987). Different EAA receptors can have different effects on neuronal development depending on brain region and age, but in general there is a continuum of actions for EAA neurotransmitters, where at low levels they stimulate sprouting, at higher levels halt outgrowth, and at still higher levels prune dendritic morphology (for review see Lipton and Kater, 1989). Biochemical differentiation can 28 also be induced by activation of NMDA receptors: such activation induces expression of the dopaminergic phenotype in cultured cerebellar granule cells (Moran and Patel, 1989). Various endogenous substances have been proposed as the neurotransmitters for the EAA receptors. Glutamate and aspartate are proposed for all subtypes. Quinolinic acid and homocysteic acid are proposed for the NMDA receptor in particular. Kynurenic acid may be a physiological antagonist of NMDA receptors, acting at the glycine site (Swartz et al., 1990). Cysteine is released upon depolarization of rat cortex slices (Keller et al., 1989), reduces production of IP3 induced by noradrenaline (Xi and Jope, 1989), and has the same affinity for the AP4 receptor as does NAAG (about 300 nM K(j) (Pullan et al., 1987; Zaczek et al., 1983), so that either of these substances may be endogenous ligands for the AP4 receptor. Cysteine sulfinate may be the neurotransmitter for the CSA receptor (Iwata et al., 1982a,b; for review see Griffith, 1990). Cysteic acid (CA) has affinity for KAIN and AP4 receptors (Pullan et al., 1987). Gamma- glutamylglutamate is proposed for TAMPA, NMDA, and KAIN receptors, and gamma-glutamylaspartate for KAIN receptors (Varga et al., 1989). Glutathione in the form of GSH and GSSG has been proposed as a modulator of AMPA receptors (Oja et al., 1988; Varga et al., 1989; Ogita et al., 1987), and GSH has been proposed as an endogenous ligand for the competitive antagonist-preferring site of NMDA receptors (Ogita and Yoneda, 1990). 29 NEUROTRANSMITTER CRITERIA In 1966, Werman reviewed the criteria for identification of a neurotransmitter in the central nervous system. He pointed out many problems of interpretation of data and proposed many theoretical objections to the necessity of many of the so-called "criteria" applied in the case of a new transmitter candidate. The only recourse is to conduct multiple tests and develop as large a body of supportive evidence as possible, consistent with the proposition that a given substance is released as a neurotransmitter from terminals of a given neuron. The following outline of tests that should be conducted is adapted from McGeer et al. (1987). The candidate substance should probably demonstrate many of the following: 1. Presence in adequate amounts. 2. Non-homogeneous distribution in brain. 3. Show a drop in concentration following lesions of suspected long-axon pathways. 4. Increase in concentration postnatally, concurrent with synapse proliferation, in brain regions where it is functionally involved. 5. Localization by histochemical or immunohistochemical studies of the substance or its synthesizing enzyme at the cellular level. 6. Release (a) from suitable tissue preparations in vitro by a process that is Ca2+-dependent and stimulated by 30 K or other depolarization; and (b) upon nerve stimulation in vivo (recovered by, for example, brain dialysis). 7. High-affinity uptake of the substance or its metabolites into nerve terminals against a concentration gradient (not demonstrated for peptides). As discussed in the Thesis Rationale, retrograde transport correlates with selective uptake of neurotransmitters or transmitter metabolites and can thus delineate, using exogenous radiolabeled substances, the neuronal pathway employing the transmitter. 8. Presence of degrading enzymes, particularly for non- peptide transmitters. These may be in dendrites or glial cells or on cell membranes. 9. Binding with high affinity to postsynaptic receptors. Criteria for receptor binding are discussed in the Thesis Rationale. Receptor distibutions should correlate with terminal fields of the proposed pathways utilizing the transmitter. 10. Identity of action: exogenous application of the presumed neurotransmitter should mimic to a large degree the effects of stimulation of the neuronal pathway for which it is believed to be the tranmsitter. 11. Pharmacological agents: it should be possible to find drugs that interfere with or enhance the action of the neurotransmitter at any of the stages of synthesis, storage, release, or receptor activation. A drug that blocks receptor activation should show identity of action as in #10. 31 THESIS RATIONALE Understanding the response properties, development, and plasticity of primary visual cortex depends on the determination of its chemical circuitry. The major neurotransmitters mediating fast transmission are likely to be similar to those proposed as EAA neurotransmitter candidates, which may include di- and tri-peptides. Geniculostriate input bears a similarity to glutamatergic neurotransmission, yet there is a relative paucity of EAA binding in layer 4, the major input layer, and the geniculostriate axons do not take up and retrogradely transport glutamate (see below). It was hypothesized that the acidic glutamyl tripeptide GSH, which shows receptor binding in rat cortical synaptic membranes, is a neurotransmitter in visual cortex, and may be, more specifically, a geniculostriate neurotransmitter. The goal of this study was to use radiolabeled GSH to (1) characterize possible GSH receptor binding in rat Ocl; (2) visualize GSH binding site distribution in rat, cat, and monkey primary visual cortex; (3) determine the change in distribution of GSH binding sites over the postnatal "critical period" in cat area 17; (4) discern possible interactions of GSH with other known EAA receptor subtypes in rat Ocl; and 32 (5) localize putative GSH neurotransmission to specific visual system pathways in rat and cat brain by demonstrating in vivo the presence of retrograde uptake and transport to cell bodies in specific nuclei. Binding experiments in vitro Previous binding studies with radiolabeled GSH were conducted with suspensions of synaptic membrane fractions of brain homogenates (Ogita et al, 1986; Ogita and Yoneda, 1987, 1988, 1989). The study reported here used in-vitro binding of radiolabeled GSH in thin sections of rat and cat primary visual cortex. Young and Kuhar (1979) showed for opioid receptors that binding in vitro to postmortem, previously frozen, mounted tissue sections did not change the receptor characteristics as determined in a homogenate preparation, or the distributions as determined by in-vivo binding. This method allows both autoradiography and quantitative characterization to be performed. The pharmacology of the receptors can be determined first, and endogenous ligand reduced, prior to autoradiography. While no single property uniquely serves to identify a receptor interaction, several criteria taken together can serve to distinguish receptor from nonreceptor interactions (Cautrecasas and Hollenberg, 1976; Burt, 1978; Limbrid, 1986). To demonstrate the possible presence of receptors for GSH, the binding of radiolabeled GSH must show saturation at 33 physiological concentrations, reversibility, relatively high affinity (<10 /iM K Time course, competition, and saturation binding experiments were conducted under conditions that minimized binding to EAA receptors and allowed characterization of a separate binding site for GSH. Further competition experiments with EAA receptor agonists, antagonists, and putative neurotransmitters were conducted under more physiological conditions to indicate possible interactions with EAA receptors. It was predicted that binding would increase in cat cortex with increasing postnatal age, over the critical period, and that, if GSH is the geniculostriate neurotransmitter, the densest binding would be found in layer 4, continuing in the adult, in rat, cat, and monkey primary visual cortex. 34 Uptake experiments in vivo Another form of supporting evidence for neurotransmitter status is the demonstration of a high-affinity uptake system to pump the material or its metabolites back into nerve endings (Iversen, 1971). The feasibility of simultaneously determining connectivity and transmitter specificity by the in-vivo uptake and retrograde axonal transport of radiolabeled neurotransmitters has also been demonstrated (Malthe-Sorenssen et al., 1979; Streit, 1980). Terminal uptake and retrograde transport back to cell bodies is selective for the neurotransmitter of that pathway, but not for its precursors, and does not occur for non-transmitter amino acids (Streit, 1980). High affinity uptake of non- transmitter amino acids is found in most neurons (Hannuniemi and Oja, 1981), but is confined to the cell body, where the amino acids are incorporated into proteins and anterogradely transported exclusively in this form by active axoplasmic transport (Octis et al., 1967), thus demonstrating axonal connections if the amino acids are radiolabeled (Cowan et al., 1972). Anterograde transport of neurotransmitters is much poorer (Streit, 1980). Retrograde transport of the transmitter-candidate amino acid glutamate in cat visual system pathways occurs by free diffusion and is confined to specific pathways (Baughman and Gilbert, 1981). A pathway that shows retrograde uptake of glutamate from distant terminals into cell bodies also shows anterograde transport from cell bodies to terminals, presumably being first taken 35 up retrogradely to the cell body by locally ramifying axon terminals, whereas pathways that showed no retrograde transport of glutamate also showed no anterograde transport (Baughman and Gilbert, 1981). Thus retrograde transport of an amino acid indicates a role as a neurotransmitter or neurotransmitter metabolite, while anterograde transport is equally consistent with a metabolic or a neurotransmission function. Terminal uptake may be assumed to be selective, but not absolutely specific, since, for example, the uptake site for L-glutamate also takes up L-aspartate, D-aspartate, cysteine sulfinate, and cysteic acid (Balcar and Johnson, 1972; Wilson and Pastusko, 1986). In the uptake experiments, radiolabeled GSH and its constituent amino acids cysteine, glycine, and glutamate, as well as the amino acid receptor agonists muscimol and kainate were individually injected into area Ocl of eleven rats. In one cat area 17 was injected with [3H]GSH. After suitable survival, uptake to other brain regions was visualized by autoradiography. It was predicted that, if GSH is a geniculostriate neurotransmitter, there might be uptake of label in cell bodies of the DLG from the injection into primary visual cortex of radiolabeled GSH, or of any of its constituent amino acids, or both. Such a finding would be supportive of a neurotransmitter role, but is not an essential criterion. 36 MATERIALS AND METHODS LIGANDS AND TRACERS For in vitro binding experiments [35S]GSH (31.4—112.5 Ci/mmol), labeled on the cysteine residue, [3H]GSH (1—0.853 Ci/mmol), labeled on the glycine residue, and [3H]glutamate (36—49 Ci/mmol) were obtained from New England Nuclear (NEN, Boston, MA). All other chemicals were obtained from Sigma (St. Louis, MO). Radiolabeled tracers for in vivo uptake experiments were all obtained from New England Nuclear (NEN, Boston, MA) and included [35S]GSH (labeled on the cysteine residue, 57.5 Ci/mmol), [3H]GSH (labeled on the glycine residue, 1-0.85 Ci/mmol), [35S]cysteine (1,113 Ci/mmol), [3H]glycine (53 Ci/mmol), [3H]glutamate (36 Ci/mmol), [3H]kainate (58 Ci/mmol), and [3H]muscimol (20 Ci/mmol). IN VITRO RECEPTOR BINDING PROCEDURES Standard methods of radioligand binding and corresponding data analysis (see following section) are well established (reviewed by Bennett, 1978; Bylund, 1980; Limbrid, 1986; McGonigle and Molinoff, 1989; Yamamura et al., 1990), and are described as follows. Time course experiments 37 are conducted in which the amount bound is determined as a function of time, with free radioligand concentration held constant (for association) or brought to zero (for dissociation). The time required to reach equilibrium (i.e., where the rate of association equals the rate of dissociation, so that the binding has reached steady state) is then used for the other experiments. The dissociation half-time is used to determine optimal rinse times (i.e., removal of free radioligand with minimal reduction of specific binding). Specificity is shown by competition experiments. In these experiments binding is determined as a function of the increase in the concentration of an unlabeled ligand while the concentration of radioligand is held constant. If there is affinity for the binding site by the unlabeled ligand, the binding of labeled ligand will decrease over a concentration range of unlabeled ligand correlated with its affinity for the site. Saturation binding experiments are then performed in which the concentration of free ligand is increased and binding is determined at equilibrium. A value for the density of maximum binding at saturating concentrations is calculated from the saturation binding data. A value for the affinity of binding can be calculated from each of the three types of experiments as a check on the reliability of an affinity value. A difference of a factor of two between values from different assays for the same parameter is considered to be good agreement in characterization studies (Limbrid, 1986). 38 Saturation and time course experiments measure "specific binding". The basic assumption in a binding assay is that the tissue contains a finite amount of specific receptor (recognition) sites which become saturated when the preparation is exposed to increasing concentrations of free radioligand. The binding of the ligand can be envisaged as the sum of at least two processes — "specific" binding to saturable sites and "nonspecific" binding that is nonsaturable, increases linearly with free radioligand concentration, and includes binding to other membrane components and other material. Addition of an excess amount of nonradioactive molecules of the same or related compound will, by competition, displace a portion of bound radioactivity. Nonspecific binding is generally defined as that radiolabeled binding that is not displaced by a concentration of nonradiolabeled competitor at least 100 times the (see below) of the specific binding site, or a concentration taken from the plateau at the bottom of the curve generated by the competition experiments. The specific binding at each concentration or time is then calculated as the difference between total and nonspecific binding. All experiments were performed on rat primary occipital cortex (Ocl) (Paxinos and Watson, 1986) and cat area 17 (Tusa et al., 1981; Snider, 1961). Adult Sprague-Dawley rats weighing 200 to 600 g were anesthetized with halothane and killed by decapitation. Cats from 6 to 230 days postnatal age were sacrificed with an overdose of sodium pentobarbitol 39 and rapidly perfused through the heart with cold phosphate buffer solution (PBS) followed in most cases by a PBS/0.2% formaldehyde solution. The brains were dissected out and frozen in liquid Freon or isopentane for storage at -2 0 to - 60° C. Specific binding of [3H]GSH in rat cortex showed no decrease from storage for up to one year. Coronal sections (20 /xm) were cut on a cryostat (Hacker-Bright) and thaw- mounted onto subbed glass slides (i.e., slides coated with gelatin and chromium potassium sulfate). Sections were pre-incubated in coplin jars in cold (4° C) buffer, followed by two five-minute rinses in the same buffer, with the intent to remove as much endogenous ligand as possible. The slides were then placed face up on a plastic tray, dried in a stream of cool air, and then 500 /ul of incubation solution containing radiolabled ligand was dripped onto each section. Incubations for autoradiography included 0.2% bovine serum albumin in order to reduce binding to nonspecific sites. Some time course and autoradiography incubations were performed at 4° C to eliminate binding to possible Na-independent uptake sites (Ogita and Yoneda, 1989). Incubation was terminated by a single 6-second wash in cold (4° C) buffer and rapid drying in a stream of cool air, except for autoradiography, in which three 5-second washes were used to attempt to further reduce LAHC binding (see Binding Site Analysis — specific binding that is not a receptor). 40 Assays of total binding were run in at least triplicate; non-specific binding was determined in at least triplicate by addition of 50 ul of 10"1 M GSH and 10_1 M GSSG to give a final concentration of 2 x 10~2 M total displacer as indicated from competition experiments (see Results). Twenty jul samples of incubation medium were removed to determine free ligand concentration. The washed and dried sections were scraped onto a small circle of glass microfibre filter paper (Whatman FG/B), and placed directly in counting vials in 4 ml of Formula NEF-963 (NEN). Vials were capped, shaken, left overnight, and the amount of bound receptor was determined in an LS 6000 IC Beckman Scintillation Counter (efficiency 55% for tritium, 95% for [35S]). In the saturation binding experiments alternate sections were used for determination of protein content by the methods of Lowry et al. (1951). Sections were processed for autoradiography by apposition for approximately 24 hours to Amersham Hyperfilm- 3H, which was then conventionally developed and fixed. Original sections were then stained for cytochrome oxidase levels by the method of Wong-Riley (1979), which clearly and specifically delineates layer 4 of area 17 in cat (Wong- Riley, 1979) and in monkey (Carrol and Wong-Riley, 1984), and is suggested to correlate with the density of thalamic terminations (Wong-Riley, 1979). Quantitative analysis of laminar binding density of [35S] on autoradiographic films was done using an Imaging Research Inc. (St. Catherine's, ONT 41 Canada) image analysis system, "MCID", calibrated with [ C] standards (ARC, St. Louis, MD; .002 - 35.0 /^Ci/g) . A density profile was constructed across all cortical layers in one representative section of striate cortex from each animal. BINDING SITE ANALYSIS Mathematical analysis of the binding data, as described by Bylund (1980) and other authors as noted, is as follows. The assumption made is that ligand-receptor binding is a reversible bimolecular reaction that at equilibrium obeys the law of mass action and can be described as follows: [L] + [R] <====> [LR] [1] k-i where [L] is the concentration of free ligand, [R] the concentration of the free receptor, [RL] is the concentration of the ligand/receptor complex, and k+i and k_i are the association and dissociation constants, respectively. The equilibrium dissociation constant (K^) for the ligand- receptor interaction is used as a measure of the affinity of the ligand for the receptor and is defined by the law of mass action: Kd = [L][R]/[LR] = k_!/k+1 [2] 42 Association and dissociation kinetics: The can be determined from k+i and k_! (equation 2). For the determination of k+i, the pseudo-first order method was used, where it is assumed that [L] >> [R], and thus is can be assumed that the concentration of radioligand added approximates a constant [L]. From equation 1 the following integrated rate equation is then derived: ln(Be/(Be-Bt)) = (k+1[L] + k_!)t = kobt . [3] where B-t is the amount bound at any time and Be is the amount bound at equilibrium. A plot of In(Be/(Be-Bt)) versus t will thus have a slope of k^. Obtaining k_! from independent experiments allows calculation of k+i from k0j-, and [L] (equation 3). To determine k_x, the binding reaction is allowed to proceed to equilibrium and then, at t = 0, rebinding of ligand is prevented by an effectively infinite dilution, and the amount bound is determined at various times thereafter. If B = Bg at t = 0, then the integrated dissociation rate equation is derived from equation 1: In (B/B0) = - k_!t [4] such that a plot of In (B/BQ) versus t has a slope of -k_]_. 43 Saturation binding experiments: For receptor binding, a plot of [RL] versus [L] yields a rectangular hyperbole, the equation of which is derived from equation 2: [RL] = [Rip] [L]/(Kd + [L]) [5] where the total receptor concentration [RT] = [R] + [RL]. It can be seen that for the point on the curve where [L] = Kd, [RL] = 1/2 RT (equation 7) , thus the Kd is equal to the concentration of L which occupies one half the total binding sites. Equation 7 can be transformed into the linear Eadie- Hofstee relationship: [RL] = [RT] - Kd([RL]/[L]) [6] These parameters are more commonly represented by the following symbols: B = Bmax - Kd(B/F) [7] In a plot of B versus (B/F) the Bmax is thus the intercept on the Y-axis and the Kd is the negative slope. Analysis of Eadie-Hofstee plots is somewhat more complicated. For single site Eadie-Hofstee plots, Zivin and Waud (1982) show that the values for Bmax and Kd are systematically underestimated, and they provide a means of correcting for this bias, based on an estimate of background 44 error, Erad. The quality of saturation-binding data can be assessed by inspection of the standard deviation of the background error, the SD(Erad). If the SD(Erad) is more than 0.2, the data will be of little value. For Eadie-Hofstee plots of dual binding sites the plot is a curve with two slope components that must be separated. Hunston (1975) provides a method of directly calculating the four parameters (two each of Bmax and Kd) from an initial estimate of the limiting slopes at each end of the curve. Analysis of saturation binding with radiolabeled GSH was performed using a computer program for the single-site Eadie-Hofstee method described by Zivin and Waud (1982) with the following modification. For dual binding sites, the corrected Bmax and Kd values calculated from the two isolated components of the curve were used to determine intercept values for recalculation of Bmax and Kd values by the dual-site method of Hunston (1975). Another parameter derived from saturation binding data is the Hill coefficient. The Hill coefficient is an indication of cooperativity: a value of 1.0 indicates that ligand-receptor interaction occurs via a reaction obeying a simple mass action law. Higher or lower values suggest positive or negative cooperativity, respectively. The Hill coefficient is taken from the generalized form of equation 5, where the binding of each ligand molecule facilitates the binding of the next one: 45 n n B = Bmax-F /(Kd + F ) [8] which can be logarithmically transformed thus: log(B/(B 'max - B)) = n log F - log [9] so that a plot of log(B/(B-'max - B)) versus log F yields a straight line with a slope n, the Hill coefficient. Corrected Hill coefficients were calculated for each component by the program based on the method of Zivin and Waud (1982), and for the whole curve by taking the slope of the plot of equation 9. Competition experiments: The affinity of a compound for the GSH binding site or sites can be determined by its IC50/ the concentration of the unlabeled compound that displaces half of the binding of radiolabeled GSH, which depends on the concentration of radiolabeled GSH added, [L]. "K^" is the term given to refer to the equilibrium constant of the competing compound (or "inhibitor"), which is derived from equation 1: K i = IC50/(1 + [L]/Kd) [10] A competition curve for a competing ligand with significantly different affinities for two or more binding-site subtypes shows a clearly biphasic shape and deviates from the single- 46 site rule. The single site rule states that when the concentration of competitor is increased from 0.1 to 10 times the IC50, the amount of binding decreases from 91% to 9%. When GSH itself is used as the competing ligand, the Kd can be calculated. For multiple sites the most accurate method of determining the separate Kd values was proposed by Bylund (1986), as follows. The equation describing competitive inhibition is B = Bmax'L/(Kd(l + I/Ki) + L [11] where I is the concentration of free "inhibitor" (competitor) and L is the free radioligand concentration. When the radioligand and competitor have the same affinity for the receptor (Kd = K^), then B = Bmax'L/(Kd + I + L) [12] which can be rearranged to produce -1/ [13] B = -B'I (Kd + L) + B-'max •L/(Kd + L) If we define BQ as the value of B when 1=0, then B = -B'I-1/(Kd + L) + B0 [14] Thus a plot of B versus B*I has a slope equal to -l/(Kd + L). 47 A dual site competition plot yields a curved B versus B'l plot analogous to an Eadie-Hofstee plot composed of two components from which the two independent slopes can be extracted. Specific binding that is not a receptor: Unfortunately, receptor binding criteria can be "mimicked" by other phenomena (Cuatrecasas and Hollenberg, 1976). For example, specific binding of [125I]glucagon to millipore filters in the absence of tissue shows high affinity, saturability, reversibility, and specificity in its displacement by native glucagon, growth hormone, and vasopressin. The importance of such phenomena for data analysis is emphasized by this quote from Cuatrecasas and Hollenberg (1976): "Since biological tissues will surely also exhibit some of the "specific" types of interactions described here for simple nonreceptor systems, awareness of such interactions should encourage greater scrutiny, particularly for those interactions observed with high concentrations of hormone, where the results are frequently interpreted to indicate "second" or "multiple" classes of receptors. In view of the ubiquity with which nonreceptor, but "specific", binding of moderate — ft o affinity (apparent Kg-, 10 ° to 10 M) can be demonstrated in a variety of simple systems that do not contain tissue, it would indeed be surprising if complex biological tissues did not also exhibit such properties. In this respect, it is pertinent that virtually all peptide and other hormones 48 (e.g., catecholamines, acetylcholine, steroid) can be demonstrated, under proper conditions, to exhibit a "second" low-affinity and high-capacity binding component when data are analyzed by Scatchard [a variant of Eadie-Hofstee] plots. Unfortunately, it is most difficult to study these low- affinity sites properly in complex tissues. However, special care should be exercised in interpreting the "second" site as a single, unique binding structure or class of receptors rather than as the sum of a complex mixture of components unrelated to receptors." Such low affinity, high capacity (LAHC) nonreceptor specific binding can produce anomalous behaviour in kinetic analyses. Mass action assumptions may no longer hold. There may also be a violation of the assumption of constant [L] in pseudo-first order analysis of association binding conducted at low [L], due to the large Bmax of LAHC binding. (See Results.) An alternative interpretation of LAHC binding is that it represents binding to an uptake site (see Discussion). Plotting and curve stripping: Plots of the data were generated as follows. Standard errors were calculated for each point and used to aid drawing of curves for untransformed data and to calculate significance by Student's t-test. Linear plots for transformed data were determined by a linear regression program (SIGMAPLOT). Each line drawn for a single component of a dual component plot was determined by 49 linear regression on the subset of points attributable to that component only, with the following exceptions. In the case of the dual-component kinetic association curve (Figure 15A), new points were first derived for the early component by mathematical stripping of the slower exponential component by a modification of the method of Lawson (1986). The fitting of the lines for the dual-site B versus B*I plot (Figure 17B) was performed by the graphic method of Rosenthal (1967). The fitting of the line for the dual-site Eadie- Hofstee plot was determined from the values derived from the Hunston (1975) method as described above. IN VIVO UPTAKE PROCEDURES Male Sprague-Dawley rats weighing from 250 to 650 grams were used for each of the radioactive tracers listed, and one adult female cat (100 days old) was used to test [3H]GSH uptake. For surgery, rats were deeply anesthetized with Somnitol (sodium pentobarbitol) (0.1 ml/100 g, i.p.). Atropine sulfate (10 mg/kg, i.m.) was given to prevent suffocation. Animals were held in a stereotaxic apparatus, where holes were made in the skull overlying area Ocl on one side using a "Dremmel" hand drill. A 50 ixl Hamilton syringe with a 33-gauge, blunt-ended needle was placed in the stereotaxic apparatus for injections. Injections were made into primary visual cortex of eleven rats (Ocl) (Paxinos and 50 Watson, 1986), and one cat (area 17) (Tusa et al., 1981). Most injection sites consisted of a series of injections at one minute intervals spanning the depth of the cortex from approximately 750 to 2 00 fxra. from the surface. In several cases the tritiated tracers were used as supplied (1 juCi/jul) , so that at two injection sites 2 ill of tracer was pressure- injected at several cortical levels for a total of 10 fil (10 /LtCi) . In other cases 20 jul of tritiated tracer was first evaporation-condensed to approximately 4 jul and injected in one site at two levels for a total of 20 fxCi in a smaller volume. For the higher-energy beta emmitter, [ S], a 2 ul injection in one site was sufficient. Most rats were allowed to survive for two days to maximize retrograde transport by passive diffusion (Baughman and Gilbert, 1981) while restricting it to the completed fast phase of axoplasmic transport in order to minimize labeling of fibres of passage h. (Cowan et al., *72; Hendrickson, 1972; Swanson et al. 1974). The cat and one rat ([3H]GSH) were sacrificed after one day to minimize transsynaptic transport and loss of terminal label (Hendrickson, 1972). Rats were sacrificed with an overdose of sodium pentobarbitol and rapidly perfused with cold phosphate buffer solution (PBS) followed by either a PBS/2% paraformaldehyde/ 1% glutaraldehyde solution, or a PBS/4% paraformaldehyde solution. The cat was first anesthetized with halothane, then sacrificed with an overdose of sodium pentobarbitol and rapidly perfused through the heart with cold phosphate buffer solution (PBS) followed by a 51 PBS/4% paraformaldehyde solution. The brains were dissected out and frozen in liquid isopentane at -60 °C. The cat brain was first placed in a saturated 3 0% sucrose solution. Coronal sections (20 /im) were cut on a cryostat, thaw-mounted onto subbed glass slides (i.e., slides coated with gelatin and chromium potassium sulfate), and processed for autoradiography by apposition to Amersham Hyperfilm-[ H] for from one to 90 days. Following autoradiography on film, some of the sectioned test brains were stained for Nissl substance with cresyl violet to identify thalamic nuclei and cortical laminae. Other sectioned test brains for all radioligands except [35S]GSH were then used to determine °C for 37 days for [35S] and for 4 months for tritium. Following development of the exposed emulsion coated slides, these sections were stained for cell bodies with pyronin Y. Thalamic nuclei were identified from the cresyl-violet and pyronin Y stained sections in reference to Takahashi (1985) and Paxinos and Watson (1986) for the rats and Snider (1969) for the cat. 52 RESULTS PARAMETRIC STUDIES Experiments were performed with rat visual cortex to define fixed values for incubation parameters across all characterization experiments, in which the dependent variable, binding, would be determined as a function of various factors (see Methods), and for the binding distribution experiments. The criteria for determining these incubation parameters was taken to be a maximization of specific binding of [3H]GSH. Binding of [3H]glutamate and interactions between glutamate and GSH binding were also tested under the conditions so defined, to determine whether GSH was binding to a site exclusive of glutamate binding sites. The parametric experiments were performed with 500 nM [3H]GSH because, due to the low specific activity supplied by the manufacturer, this was the minimum concentration that would allow dpm values significantly above background in low binding conditions. This concentration of [ H]GSH should have resulted in roughly equal proportions of binding to the three independent binding sites found, as estimated from the Kd and Bmax values obtained in the subsequent binding site characterization experiments. The incubation time required to reach equilibrium (steady state binding, see Methods) was determined in preliminary time course experiments with 5 nM 53 [J3S]GSH and 355 nM [JH]GSH in 50 mM Tris-acetate buffer, pH 7.4, at room temperature. Incubations were run for 12 0 minutes, the time required to reach equilibrium at the lowest concentration, ensuring that all conditions were assayed at equilibrium. The dissociation half-time from the aforementioned 355 nM time course experiment allowed calculation of optimal rinse times (for removal of free radioligand) to maximize loss of nonspecific binding and minimize loss of specific binding (Bennett, 1978). Non• specific binding defined by the addition of 10~4 M GSH was up to 50 percent of total binding for the parametric experiments. For the subsequent binding site characterization experiments, the non-specific binding was reduced by the addition of 10"2 M GSH plus 10"2 M GSSG. The need for this relatively large amount of displacer was determined by competition experiments (see Results - Binding Site Characterization; Figure 16), due to the presence of LAHC binding (see Methods). Various conditions were tested for maximum specific binding. Figure 7 indicates optimal buffer osmolarity tested in Tris-acetate. The subsequent incubations were performed at 100 mM buffer. Specific binding showed a marked peak at pH 8.0 (Figure 8), at which the subsequent incubations were performed. (Variations in binding between assays at points representing the same conditions are due to variation in sample size from one assay to another.) Specific binding was significantly reduced by inclusion of ions and buffers 54 Figure 7. Binding of [JH]GSH in rat visual cortex as a function of concentration of buffer. Cryostat sections of adult rat Ocl were incubated in various concentrations of Tris-acetate at pH 7.4 for 120 minutes at room temperature with 500 nM [ HIGSH. Nonspecific binding was defined by the addition of 10 M GSH. The graph represents one experiment conducted in quadruplicate. Figure 8. Binding of [JH]GSH in rat visual cortex as a function of pH. Cryostat sections of adult rat Ocl were incubated in 100 mM Tris-acetate for 120 minutes at room temperature with 500 nM [ H]GSH at various pH values as indicated. Nonspecific binding was defined by the addition of 10~4 M GSH. The graph represents combined data from two separate experiments conducted in quadruplicate. 600 5.900 6.900 7.900 8.900 PH O—Ototal A—A nonspecific •—•specific 56 700.0 Tris-acetate Tris—citrate Tris—HCI Figure 9. Binding of [ H]GSH in rat visual cortex as a function of buffer type. Cryostat sections of adult rat Ocl were incubated in 100 mM Tris-acetate, Tris-citrate, or Tris- HC1 at pH 8.0 for 120 minutes at room temperature with 500 nM [HIGSH. Nonspecific binding was defined by the addition of 10 M GSH. Data is from one experiment conducted in quadruplicate. 57 Figure 10. Binding of [JH]GSH in rat visual cortex as a function of ion concentration. Cryostat sections of adult rat Ocl were incubated in 100 mM Tris-acetate at pH 8.0 for 12 0 minutes at room temperature with 500 nM [3H]GSH and various concentrations of ions as indicated. Nonspecific binding was defined by the addition of 10~4 M GSH. The graph represents combined data from two separate experiments conducted in quadruplicate. (A) NaCl. (B) CaCl2. (C) MgCl2. (D) MgS04. (E) KC1. (F) KH2P04. 58 O O total A A nonspecific • • specific OH 1 1 1 1 0 0.1 1 10 100 mM CaCI o O — O total A—A nonspecific • — •specific 59 0-| 1 1 1 1 0 0.01 0.1 1 10 mM MgCI 2 O-—Ototal A—-A nonspecific • — • specific 60 .0 0.01 0.1 E mM KCI O O total A- •A nonspecific specific 400$ 0 0.01 0.1 mM KHJ^0 F 4 O—O total A—A nonspecific >— # specific 61 1000.0 % formalin Figure 11. Binding of [JH]GSH in rat visual cortex as a function of fixation. Cryostat sections of adult rat Ocl were preincubated in cold (4°C) Tris-acetate (pH 8, 100 mM) with 0.6%, 0.2%, or no formalin for ten minutes and then incubated in 100 mM Tris-acetate at pH 8.0 for 120 minutes at room temperature with 500 nM :[3H]GSH. Data is from one experiment conducted in quadruplicate. Nonspecific binding was defined by the addition of 10~4 M GSH. 62 Figure 12. Binding of [JH]GSH in rat visual cortex as a function of the concentration of various biochemical agents. Cryostat sections of adult rat Ocl were incubated in 100 mM Tris-acetate at pH 8.0 for 120 minutes at room temperature with 500 nM [3H]GSH and various concentrations of these agents as indicated. The graph represents combined data from two separate experiments conducted in quadruplicate. Nonspecific binding was defined by the addition of 10 M GSH. (A) Bacitracin. (B) Acivicin. (C) Dithiotheitol. 63 400-F 0.1 1 100 A mM bacitracin O- O total A A nonspecific specific 400-F 0 0.1 1 10 100 B mM acivicin O Ototal A A nonspecific #—-< specific 64 O O total A A nonspecific • •specific 65 commonly used in studies of EAA or peptide receptors (Blakely et al., 1986; Luini et al., 1984; Pin et al., 1987; Mantyh et al., 1989; Pullan et al., 1987). These included NaCl, CaCl2, MgCl, MgS04, or KCl, KH2P04, Tris-hydrochloride, and Tris- citrate (Figures 9 and 10). Fixation with 0.2% formalin included in the first step of the preincubaton showed no significant reduction of specific binding (Figure 11), and was included in the following incubations. The phosphatase inhibitor bacitracin, commonly included in receptor binding incubations, produced a slight reduction of specific binding (Figure 12A). The inclusion of acivicin to block the possible degrading action of endogenous plasma-membrane-bound gamma-glutamyl transpeptidase (GGTP) (Hill et al., 1985) was omitted from the subsequent characterization experiments because it was determinined that up to 10 mM acivicin showed no effect on specific binding under these in-vitro conditions (Figure 12B). The reducing agent dithiothreitol (DTT) (see Figure 13) had a negative impact on GSH binding. A parametric assay (Figure 12C) showed a small reduction of specific binding and an anomalous effect on nonspecific binding with concentration. However, preliminary competition studies of radiolabeled GSH binding by nonlabeled GSH showed a marked reduction in affinity due to the presence of the DTT included by the manufacturer in the radioligand stock solution (10 mM), a phenomemon that was determined as follows. 3 Competition of 2 jiiM [ H]GSH with GSH showed an IC50 value two 66 orders of magnitude higher than competition under the same 35 conditions with 18 nM [ S]GSH. The shift in IC50 due to the higher radioligand concentration should have been only a factor of two (see equation 10, Methods). The concentration of DTT in the incubation medium at 2 JUM [3H]GSH was calculated to be 10 /uM. This concentration of DTT has been shown by Oja et al. (1988) to reduce glutamate binding and to shift the competition curve of glutamate by GSH to the right, suggesting that DTT affects a sulfhydryl group on the receptor binding site. Because of this, 100 mM diethyl disulfide was added to the incubation buffer in all experiments at high concentrations of radiolabeled GSH, in order to neutralize the DTT that was included in the stock solution by the manufacturer (as shown in Figure 13). This proved to return the GSH IC50 values to their expected range. Under these conditions (100 mM Tris-acetate, pH 8, 120 minutes, room temperature) there was no displacement of [ H]GSH binding by glutamate, and no displacement of Na- independent [3H]glutamate binding by GSH, as shown in single — 7 —T competition experiments ranging from 10 to 10 M competitor, at free radioligand concentrations of 40 nM and 2 JUM for [3H]glutamate and in two experiments at 2 /iM for T .... [ H]GSH (data not shown). This is consistent with the known reduction in glutamate binding at pH 8, as shown in Figure 14 (Fagg et al., 1988). Under these conditions, [3H]glutamate binding was 14 percent of [3H]GSH binding, and there was no displacement of [ H]glutamate by glutamate itself, or by 67 aspartate, homocysteic acid, or quinolinic acid at 10" M (two experiments), indicating lowered binding affinity for EAA receptors. These conditions therefore allow characterization of binding sites separate from other EAA receptors. R—CH2—S—S—CH2—R Disulfide-linked chains H OH I I HS—CH,—C—C—CH,—SH I I OH H Dithiothreitol (Added in excess) R—CH2—SH + HS—CH2—R' Separated reduced chains Figure 13. Separation of disulfide bonds by the reducing action of dithiothreitol, during which the dithiothreitol inactivated. (From Stryer, 1988) 69 Figure 14. Binding of L-f-^H] glutamate and [JH]3-(2- carboxypiperazin-4-yl)propyl-l-phosphonate (CPP) in rat brain membranes as a function of pH (different y-axis scales for each ligand) . The curve for CPP is similar to that for the ACPD receptor antagonist 2-amino-3-phosphonopropionic acid (AP3), for AP4, and for the competitive NMDA receptor antagonist AP5 (2-amino-5-phosphonovalerate). (From Fagg et al., 1988) 70 BINDING SITE CHARACTERIZATION Incubations were carried out at room temperature in 100 mM Tris-acetate buffer at pH 8.0 for 120 minutes (see above), except where noted below. Non-specific binding defined by displacement using 10~2 M GSH and 10~2 M GSSG ranged from 10 to 25% of total, increasing linearly with concentration of radioligand. In rat visual cortex, high- and low-concentration time course and saturation binding experiments were conducted in order to reveal high- and low-affinity receptor subtypes, respectively. The low concentration experiments used [35S]GSH, which had a high specific activity sufficient to resolve low amounts of binding . Competition experiments were conducted in both rat and cat visual cortex. Two binding sites indicative of receptor subtypes were revealed, as well as an LAHC site (see Discussion). Such "specific" but non• receptor LAHC binding is likely to appear as a second binding-site component in data from binding experiments conducted at high concentrations (see Methods - Binding Site Analysis). A competition assay for crossover with other EAA receptors or neurotransmitters in rat Ocl was conducted at 20-minutes incubation at pH 7.4, which minimized the LAHC binding. Figure 15 shows data from time course experiments in rat Ocl that revealed a low-affinity possible GSH-receptor 71 Figure 15. Association and dissociation rates of radiolabeled GSH binding in cryostat sections of rat visual cortex. Data is transformed to calculate K0ks from the association slope and K_^ from the dissociation slope. On the ordinate, Be is binding at equilibrium, while B is binding at the time indicated. Sections were incubated in radiolabled GSH for various times up to 120 minutes. Remaining sections were then introduced into an 11 infinite dilution" of buffer and incubated for further times up to 40 minutes. Nonspecific binding was defined by the addition of 10~2 M GSH and 10"2 M GSSG. (A) Data from specific binding at a free ligand concentration of 5 /uM [3H]GSH (room temperature) to reveal low-affinity receptor kinetics. The curves represent normalized data from two separate experiments conducted in quadruplicate. Regression lines are drawn only for the possible receptor components. The late component of the association curve and the early component of the dissociation curve show binding to the LAHC non-receptor site (see Results). For the first component of the association plot, linear regression was performed on points derived from mathematically stripping off the slower component by a modification of the method of Lawson (1986). Linear regression was performed on the last 8 points of the dissociation. (B) Data from specific binding at free ligand concentration of 5 nM [35S]GSH (4° C) to reveal low-affinity receptor kinetics. Regression lines are drawn only for the possible receptor components. The late component of the association curve shows non-mass-action kinetics, while the early component of the dissociation curve reflects non• receptor (LAHC) kinetics (see Results). The curves represent normalized data from three separate experiments conducted in triplicate. Linear regression was performed on the first 5 points of the association and last 4 points of the dissociation. 72 Time (mins.) Time (mins.) 73 binding site (Figure 15A) and a high affinity possible GSH- receptor binding site (Figure 15B). In the high- concentration time course experiments (5 /xM) specific binding reached equilibrium by 90 minutes; dissociation half-time was 1.5 minutes. Figure 15A shows association and dissociation experiments at high concentration (see equations 3 and 4). There were two components of each of the two curves shown, representing possible receptor and non-receptor binding (see saturation experiments, below). The first component of the observed rate constant (k0ks) was used with the second component of the dissociation rate constant (k_i) to calculate the association rate constant (k+i), while the remaining components, which did not represent binding to a "real" receptor site, could not be used to calculate kinetic constants (see saturation and competition experiments; Methods - Binding Site Analysis; Discussion; and below). The value for koj-, (after exponential stripping, see Figure 15) was 0.067 +/- 0.012 min-1, and the value for k_i was 0.010 +/- 0.003 min-1 (mean and S.E. from two separate experiments -1 -1 each). The k+1 value was calculated to be 0.0114 /LtM min (equation 3, Methods). Since Kd = k_i/k+i, these rate constants resulted in a Kd of 0.88 /xM. The choice of the second component of the dissociation plot for k_i is indicated by an alternative method of independently deriving k+i and k_i from the association data alone, as follows. Because k^ = k+^[L] + k_i (from equation 3) , a graph of k0j-, versus [L] will yield the k+i as the slope 74 and the k_! as the y-axis intercept. Using the kOD from the preliminary time course experiment performed at 355 nM [3H]GSH, two points were available to define a line which had a slope of 0.01 /LiM-1min-1 (K+i) and a y-axis intercept of 0.017 min"1 (k_i). This value for k_i is very close to the value of 0.01 min-1 obtained from the second component of the dissociation plot, and very far from the value of 0.15 min-1 for the first component. In order for the y-axis intercept to yield a k_^ value of 0.15, the slope (k+i) would have to, be negative, an impossibility. The behaviour of the first component presumably results from non-receptor LAHC binding. In the low-concentration, time course experiments at 5 nM at 4° C, specific binding reached equilibrium by 120 minutes; dissociation half-time was 1 minute (Figure 15B). Values for kOD and k_i were determined as described above: 0.014 +/- 0.002 min-1 and 0.009 +/- 0.001 min-1 respectively (mean and S.E. from three separate experiments each), yielding a K+1 of -1 .001 nM'-'-min and a Kd of 9 nM. The remaining components of each of the two curves could not be used to calculate kinetic constants (as above). The second component of the association curve violates the pseudo-first order assumptions of a constant free ligand concentration [L], which requires that [L] be much larger than the receptor concentration in that incubation volume [R] (see Methods - Binding Site Analysis). Since this component represented the more slowly- associating low-affinity binding site and LAHC site (see saturation and competition experiments), the corresponding 75 ratios of [L]/[R], as calculated from Bmax values obtained in the saturation experiments (described below), were 2.2 for the low-affinity site and 0.1 for the LAHC site (while the high-affinity site ratio was 12). The two low ratios would have produced large deviations in [L]. A constant [L] is assumed in order to calculate kinetic values (Bylund, 1980), and thus this component of the association curve would yield anomalous results if used to determine kinetic constants. Competition with 2 juM [3H]GSH by non-radiolabled GSH in rat (Figure 16A) showed a marked deviation from a single-site profile (91-9% rule) with the extension of the curve to the right showing the LAHC site. Because of the high amount of binding to the LAHC site at 2 MM [3H]GSH, a two-site analysis of the Kd values was performed on data from competition of 18 35 nM [ S]GSH with GSH, as shown in Figure 17. The Kd was determined to be 1.3 +/- 0.5 JUM for the possible receptor component and 92 +/- 2 /xM for the LAHC site (mean and S.E. of three separate experiments). Specificity was shown by other competitors. For GSSG the possible receptor binding site IC5o was the same as for GSH, with a slightly higher affinity for the LAHC binding (Figure 16A). Three other substances showed affinity for [3H]GSH binding at 1 mM (Figure 16A), in the following order: cysteine, S-methyl-GSH, and CSA. Under these conditions (pH 8, 120 minutes) many substances failed to show displacement of [3H]GSH binding from 1 /xM to 1 mM. These were L-glutamate, L-aspartate, D/L-homocysteic acid, quinolinic acid, D/L-2- 76 Figure 16. Competition curves for [JH]GSH binding in adult rat (A) and cat (B) cryostat sections of visual cortex. Sections were incubated for 12 0 minutes at room temperature (pH 8) in 2 jiM [3H]GSH and various concentrations of competitors as indicated. The curves in (A) represent normalized data from two separate experiments conducted in at least triplicate. The curves in (B) are from one experiment conducted in at least triplicate. Note that the extended section of the curves at high competitor concentrations represents the LAHC non-receptor binding site. 77 120.0- % 100.0* i—o i n. 80.o- c 60.0- C • GSH £ 40.0 • GSSG o A Cysteine 20.0- oS-methyl-GSH 0.0 •H- I 1 1 1 1 ' 1 1—| r- A -8 -7 -6 -5 -4 -3 -2 120.0 1 A 100.0 i—o i ^ 80.0- C7> C 60.0- c lo •GSH "5 40.0- • GSSG -M o ^ Cysteine •+-» 20.0- 0.0 i • i—•—i—i—i—i—i—i—|— 8 -7 -6 -5 -4 -3 -2 B Log M 78 Figure 17. Competition of GSH with 18 nM [JDS]GSH and its derived "B versus B'l" plot to reveal the separate Kd values for the possible receptor binding site and nonreceptor LAHC site. Cryostat sections of adult rat visual cortex were incubated for 120 minutes at room temperature (pH 8) in 18 nM [35S]GSH and various concentrations of nonlabeled GSH as indicated. The graphs represent normalized data from three separate experiments conducted in triplicate. (A) Competition curve. (B) "B versus B*I" plot, where the negative inverse of the slopes of the first and second components yield Kd values of 1.3 /zM and 92 /uM, respectively. Extraction of the lines for the two separate components was performed by the method of Rosenthal (1967). 80 amino-3-phosphonopropionic acid (AP3), D/L-AP4, D/L-2-amino- 5-phosphonopentanoic acid (AP5), gamma-D-glutamylglycine (gamma-DGG), AMPA, and QUIS. NMDA showed no significant displacement at 1 mM. A pH of 8 is optimal for binding of AP3, AP4, and AP5 to EAA receptors (see Figure 14). Figure 18 shows data from saturation experiments that reveal the low-affinity, possible GSH receptor binding site (Figure 18A) and the high-affinity possible GSH receptor binding site (Figure 18B). Eadie-Hofstee analysis of the high-concentration saturation binding experiment (Figure 18A; see Methods) yielded a Kd of 1.3 +/-0.3 iiM and a BTOax of 1.3 +/-0.2 pmoles/mg protein (mean and S.E. from two separate experiments). The second component shown in the Eadie- Hofstee plot in Figure 18A is the non-receptor LAHC binding site invariably found at high concentrations (see Methods) with a very high Kd of 17.2 +/-7.8 /nM and a very large Bmax of 31.4 +/-11.4 pmoles/mg protein (see Discussion). The low- concentration saturation experiment (Figure 18B) was performed at 45 minutes incubation to minimize binding to the low-affinity binding site and the LAHC site, in order to maintain mass-action assumptions, for the reason described above in the time course experiments. Eadie-Hofstee analysis (Figure 18B) yielded a single component with a low Kd of 5.4 +/-0.8 nM and a smaller Bmax of 0.24 +/-0.02 pmoles/mg protein (mean and S.E. of three separate experiments). The quality of saturation-binding data can be assessed by inspection of the Hill coefficients and SD(Eracj) values. 81 An SD(Erad) value more than 0.2 means the data is of little value. A Hill coefficient higher or lower than one suggests positive or negative cooperativity, respectively (see Methods - Binding Site Analysis). Hill coefficient and SD(Erad) values for each separate component of the radiolabeled GSH saturation binding described above are as follows: 0.99 +/- 0.05 and 0.07, respectively, for the high-affinity receptor; 0.98 +/- 0.09 and 0.09 for the low-affinity receptor; 0.98 +/- 0.17 and 0.11 for the LAHC site; and a Hill coefficient of 0.92 +/- 0.11 for the high-concentration saturation experiment as a whole. A single competition experiment was carried out for cat visual cortex in order to ascertain that affinity and specificity under these incubation conditions were not radically different from those in rat brain, since autoradiographic distribution studies in cat cortex were expected to be more revealing of laminar binding (based on previous receptor binding studies [see Introduction] and preliminary autoradiography). In sections from adult cat area 17, competition with 2 /iM [3H]GSH (pH 8, 120 min) showed similar affinity, specificity, and LAHC binding as that in rat Ocl (Figure 16B). The cat data is not sufficient for determining binding parameters. A possible difference from rat was displacement in cat area 17 by 1 mM glutamate at pH 8 (p < 0.02). 82 Fig. 18. Saturation binding curves and Eadie-Hofstee plots of [3H]GSH and [35S]GSH binding sites on cryostat sections of adult rat visual cortex. Nonspecific binding was defined by the addition of 10~2 M GSH and 10~2 M GSSG. (A) High free- ligand concentration range ([3H]GSH) to reveal parameters for low-affinity receptors. The graphs represent combined data from two separate experiments conducted in quadruplicate at room temperature, 12 0 minutes incubation, pH 8. In the Eadie- Hof stee plot the component with steeper slope (no line drawn) represents the LAHC non-receptor site. The line for the possible receptor component was determined by the method of Hunston (1975). (B) Low free-ligand concentration range ([35S]GSH), to reveal parameters for high-affinity receptors, incubated for 45 minutes (pH 8, room temp.) to minimize binding to the much denser LAHC sites to retain mass action assumptions (see Results). The graphs represent combined data from three separate experiments conducted in triplicate. The Eadie-Hofstee line is drawn by linear regression. Bound (pM) 84 EXCITATORY AMINO ACID RECEPTOR INTERACTIONS To further characterize possible interactions between GSH and EAA neurotransmission under physiological conditions, competition experiments were conducted at 20 minutes incubation at pH 7.4, in which there was a reduction of the LAHC binding as shown by the competition curve in Figure 19, and at pH 6.9 (also 20 min), which is optimal for glutamate receptor binding (Fagg et al., 1988). Control specific binding of [3H]GSH with 2 0-minute incubations, reducing the LAHC component, showed ratios relative to binding at pH 7.4 of 3.6 at pH 6.9 and 2.5 at pH 8.0, which differs from the binding with pH seen at 12 0 minutes incubation (Figure 8). In addition, binding and competition experiments with [3H]glutamate were conducted at pH 6.9, the optimum pH for glutamate binding (Figure 14) . As shown in Figure 20, [3H]glutamate binding was significantly (p < 0.05) greater at pH 6.9. At pH 6.9, as opposed to at pH 8, there was significant displacement (p < 0.05) of [3H]glutamate binding by both glutamate and GSH at 1 mM GSH. There was, however, no displacement by 1 mM AMPA at this pH. At pH 7.4 there were significant displacements of [3H]GSH by substances at 1 mM as shown in Figure 21. There was no significant displacement by D/L-AP3, D/L-AP4, NMDA, KAIN, or CSA. At pH 6.9 there was significant displacement of [JH]GSH as shown in Figure 21, with no displacement by glutamate, L-aspartate, D/L-AP3, D/L-AP4, or KAIN. 86 120.0- 0.0-1 • 1 • , , . , . , . , . r- 0 -8 -7 -6 -5 -4 -3 -2 log M Figure 19. Competition by GSH with [JH]GSH at 20 minutes incubation (pH 7.4) to reduce binding to the dense, nonreceptor, LAHC site, the presence of which would obscure displacements. The curve is compared to the previous data from competition at 120 minutes at pH 8.0 (dotted line) to show that there is a reduction in the righthand, low affinity component. These conditions are optimal for examining interactions with EAAs. Cryostat sections of adult rat visual cortex were incubated at room temperature (pH 8) in 2.6 p,K [ H]GSH and various concentrations of nonlabeled GSH as indicated. Data is from one experiment where six sections were used at each data point. 87 CD 200.0 -t-> o pH 8 K8pH6.9 E -t-D> TTcnI 150.0 <+- o 100.0- cn c TD ~~5 50.0- c o o 0.0 control control GSH AMPA Figure 20. Percent control binding of L-[ H]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. Cryostat sections of adult rat Ocl were incubated in Tris-acetate for 45 minutes at room temperature with 3 /nM [3H]glutamate and various competitors as indicated. Data is combined from two separate experiments conducted in triplicate. The difference between binding without competitors at pH 8.0 and pH 6.9 is significant (p <0.05). 88 Figure. 21. Percent control binding of [JH]GSH in rat visual cortex with significant displacement (p <.05) by various 1 mM competitors at pH 7.4 and at pH 6.9. Control binding is relative to binding at pH 7.4 without competitors. Cryostat sections of adult rat Ocl were incubated in Tris-acetate with 2 /xM [3H]GSH and competitors for 2 0 minutes (room temp.) to minimize LAHC binding. The graph represents normalized data from three separate experiments conducted in triplicate. Only significant displacement is shown. 89 BINDING SITE DISTRIBUTIONS Quantitation of specific binding of [3H]GSH in rat brain showed regional variation with significantly less binding in more lateral, non-visual cortex, brainstem, and white matter (p < .01). In cat brain, non-specific binding of [35S]GSH , determined by the addition of 10~2 M non-radiolabeled GSH, showed densest binding in white matter and brainstem. The laminar distribution of GSH total-binding sites in visual cortex was determined by autoradiography for adult rats, for cats at postnatal ages of 6, 13, 30, 46, 61, 90, and 2 30 days, and for one adult monkey. These incubations were performed with 10 nM [35S]GSH at pH 7.4 for 3 0 minutes to reduce L/AHC binding. Rat cortex showed relatively uniform binding across all layers (Figure 22A). Cat visual cortex showed the highest density of binding in layer 4, confined to area 17, from after 6 days postnatal age. Figure 22 shows representative autoradiographs and Figure 23 shows quantitative densitometry of layers at each age studied. In 6-day old cat area 17 no distinct laminar pattern of [JJS]GSH binding was seen (Figure 23A). At all later ages, densest binding was shown in middle layers of area 17. Cytochrome oxidase staining for layer 4 was obtained in the 46, 61, and 90 day old cats, which shows that the band of densest [35S]GSH binding corresponded consistently with layer 4 along its entire length in area 17 (see for example Figure 22 D and E, F and G). Quantitative analysis of binding density, shown 90 in Figure 23, showed a peak in middle layers after 6 days of age, with binding showing an increase from 6 to 61 days of age and then a reduction at 90 and 230 days of age, with layer 4 binding density becoming more discrete and slightly less prominant at the latter two ages. The region of maximum binding density for the 46, 61, and 90 day old sections corresponded with the cytochrome oxidase band. Adult monkey area 17 showed a distinct [35S]GSH laminar binding preference for layer 4C, as shown by the corresponding cytochrome oxidase stained section (Figure 221,J). 91 Fig. 22. Autoradiographic distribution of [JDS]GSH binding in rat, cat, and monkey visual cortex. Slide-mounted coronal sections (20 izm) from frozen tissue were incubated for 3 0 minutes at 8-10 nM [35S]GSH at room temperature, pH 7.4. Bar = 1 mm. In cat and monkey sections successfully stained for cytochrome oxidase levels, as shown, the position of the band of densest staining (layer 4, circle) corresponds to the position of densest binding of [35S]GSH as shown in the autoradiographs. (A) Adult rat brain autoradiograph from a coronal section at the level of the SC and medial geniculate, lines indicating approximate borders of Ocl. (B) 13-day old cat visual cortex autoradiograph, with area 17 delineated by the band of denser binding at the top righthand side. (C) 3 0-day old cat visual cortex autoradiograph, with area 17 delineated by the band of densest binding along the length of the lefthand side of the section, continuing ventrally into the large sulcus. (D,E) 46-day old cat visual cortex autoradiograph (D) and corresponding cytochrome oxidase stained section (E). (F,G) 61-day old cat visual cortex autoradiograph (F) and corresponding cytochrome oxidase stained section (G). (H) Adult (230 day old) cat visual cortex autoradiograph with area 17 comprising the two top lefthand gyri. (I/J) Adult monkey area 17 autoradiograph (I) and corresponding cytochrome oxidase stained section (J) with arrows indicating, from top to bottom, layers 4A, 4Cot, and 4CB. 92 93 95 I J 96 Fig. 23. Quantitative analysis of laminar binding density on autoradiographic film across cat area 17 cortex radially from white matter to pia at various postnatal ages. Optical density was calibrated using ARC [ C] standards (0.002 - 35.0 ixCi/g) . The graph is a histogram with each point showing the mean value for a bin dimension of 0.145 mm radially and approximately 0.460 mm tangentally. The histograms for all ages are aligned with the center of layer 4, as determined by cytochrome oxidase and Nissl staining. The approximate width of layer 4 is indicated by the horizontal bar. The pia (right side) and the boundary of the white matter (left side) are indicated by the vertical bars crossing the histogram for each age. Width in mm on the ordinate is provided for scaling, with its origin at an arbitrary point in the white matter. Variations in the thickness of cortex and positioning of anatomical layers, depends on the particular sample taken at any given age. All samples are taken from the medial bank, with ages 6, 30, 46, and 61 days taken from the ventral gyrus, age 13 days from the dorsal gyrus, and ages 9 0 and 230 days from the sulcus. A peak in binding density is seen in layer 4 at all ages except day 6. (A) Density profile at postnatal ages of 6, 13, and 30 days. (B) Density profile at postnatal ages 46, 61, 90, and 230 days. 97 10-- b Z3 4 O — O 6 day old 2 A A 13 day old • • 30 day old A A 0 • i i i • i i i i 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 A mm 10 8 en 6 r A1 A b A 4 + • «46 day old O—061 day old 2 A—A 90 day old A — A 230 day old 0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 B mm 98 UPTAKE EXPERIMENTS IN VIVO Using injections of radioactive neurotransmitters or neurotransmitter metabolites, one can label, in principle, distant cell bodies having projections to cortex by making injections in their efferent target area. This retrograde uptake correlates with a neurotransmitter role in the delineated pathway (Streit, 1980; Baughman and Gilbert, 1981). One can at the same time retrogradely label local cells via locally ramifying axons, which can then show uptake and anterograde transport to projected terminals, thus also indicating a neurotransmitter function (Baughman and Gilbert, 1981). For amino acids, however, there may also be uptake by cell bodies for metabolic purposes, with anterograde transport of the label in proteins to efferent target area terminals (Cowan et al., 1972). Although our injections were made exclusively in primary visual cortex in both rat and cat (Ocl and area 17, respectively), in rat cortex there was spread of the tracers into secondary visual cortex (0c2) and sometimes into adjacent non-visual cortex and across the corpus callosum; in the cat the injected tracer remained confined within a portion of area 17. Thalamic and brainstem nuclei showing the presence of radiolabel after injection into visual cortex are summarized in Table I. Examples of autoradiographs are shown in Figure 24. All labeled tracers except [ H]muscimol and [ HJkainate showed uptake and transport. All labeled nuclei (Table I) had direct 99 connection with visual cortex as follows (for abbreviations see List of Anatomical Abbreviations). In rat brain, the following nuclei are not known to project to visual cortex but they do receive projections from both Ocl and 0c2 : the VLG, APTD, Rt, and SC (Sefton and Dreyer, 1985; Swanson et al.,1974; Takahashi, 1985), except for the inferior grey of the SC, the parvocellular division of the VLG, and the IMA, which receive projections only from 0c2. The DLG and ZI (Lin et al., 1990) have reciprocal connections with Ocl and area 17 in the rat and cat (Sefton and Dreyer, 1985; Takahashi, 1985). The SubG is an acetylcholine esterase-staining area of the ZI, as determined by Paxinos and Watson (1985). The LP projection to Ocl in the rat (including LPMC) is not reciprocal except to the lateral rostral division (LPLR) (Sefton and Dreyer, 1985; Takahashi, 1985). The projections and functions of the IGL have not been elucidated separately from the DLG and VLG (Sefton and Dreyer, 1985). [35S]Cysteine transport in the dorsal CP in one rat presumably resulted from diffusion into motor cortex, and callosal transport seen in many cases in the rats may be due to penetration of the white matter. Cat [3H]GSH uptake, restricted to area 17 only, was transported exclusively to the DLG. Registration of silver grains for cellular resolution was performed by the emulsion-dipping method for all test animals except the [35S]GSH-injected rats. Images were obtained in one of the [35S]cysteine-injected and three of 100 the [^HJGSH-injected rats. The higher-energy beta emission in sections from the [35S]cysteine-injected rat produced a dense silver grain distribution over cell bodies and neuropil in the nuclei listed (Table I). In emulsion-coated sections from the [3H]GSH-injected rats silver grains were seen over cell bodies and neuropil in the DLG and LPLR, indicative of retrograde transport, as shown in Figure 25, but the same distribution was seen in the VLG and SC (see Discussion). No silver grains could be detected after four months exposure time in the emulsion-dipped [3H]GSH-injected cat sections or in the [3H]glycine- and [3H]glutamate-injected rat sections. Due to time limits for the present study, it was not possible to wait up to a year or more to obtain silver grain distributions in these sections. It was therefore impossible to determine whether uptake and transport in these brains was retrograde. 101 TABLE I. Nuclei showing presence of radiolabel Animal/tracer N nuclei Rat: [:?H] GSH 3 DLG VLG LPLR ZI/SubG SC IMA IGL ["S]GSH 2 DLG LPLR it S]cysteine 2 DLG VLG LPLR APTD ZI/SubG SC IGL Rt CP [?H]glutamate 1 DLG VLG LPMC ZI/SubG IMA [3H]glycine 1 DLG VLG LPLR APTD ZI/SubG SC IMA • [^HJmuscimol 1 [3H]kainate 1 Cat: [3H]GSH 1 DLG 102 Fig. 24. Autoradiographic distributions of radiolabeled tracer uptake to thalamus from in vivo injection in visual cortex in rats. Bar = 1 mm. (A) Atlas of relevant structures at the same level as the autoradiograph in (B) (Bregma minus 3.8 mm, from Paxinos and Watson, 1986). (B) [3H]GSH uptake to DLG, IGL, and VLG. (C) Atlas of relevant structures at the same level as the autoradiographs in (D), (E), and (F) (Bregma minus 4.3 mm, from Paxinos and Watson, 1986). (D) [35S]Cysteine uptake to LPLR, DLG, ILG, VLG, and ZI/SubG. (E) [3H]Glutamate uptake to LPLR, DLG, VLG, and ZI/SubG. (F) [3H]Glycine uptake to APTD, LPLR, IMA, DLG, VLG, and ZI/SubG. 103 104 105 106 Fig. 25. Bright field photomicrograph of combined pyronin Y staining for cell bodies and autoradiographic distribution of silver grains in the rat DLG following uptake of [3H]GSH from visual cortex. In these 20 /urn thick sections, silver grains should not be seen over most cell bodies unless retrograde transport into the cell bodies had taken place. 107 DISCUSSION BINDING SITE CHARACTERIZATION The present results give the first demonstration of glutathione binding sites in whole brain. Three sites were revealed, with Kd values of 5.4 to 9 nM, 0.9 to 1.3 /^M, and 17 to 92 /iM. The highest values reported in the literature for receptor binding parameters are 8.3 /uM for Kd and 6.9 pmol/mg protein for Bmax, and these are exceptionally high (reviewed in Foster and Fagg, 1984). The LAHC binding site values are significantly higher than these, particularly the Bmax (31 pmol/mg protein), indicating nonreceptor binding. This interpretation is supported by the deviations from mass action and pseudo-first order assumption seen in kinetic analysis. For the low affinity, possible GSH receptor binding site the Kd values and Bmax (1.3 pmol/mg protein) are typical of EAA receptors (Foster and Fagg, 1984). The Kd values and Bmax (0.24 pmol/mg protein) of the high-affinity GSH binding site are typical of peptide and neuromodulator receptors (Shaw et al., 1986). An explanation for the marked decrease in [ H]GSH specific binding seen at physiological concentrations of Na+ and Mg2+ is suggested as follows. For opioid receptor binding in brain, Na+ decreases agonist binding to 37% at 50 mM and abolishes binding by 2 00 mM, due to a decrease in affinity (with a corresponding increase in antagonist 108 affinity), and there is also a general reduction in binding caused by cations (Simon et al., 1975; Young and Kuhar, 1979). For tachykinin peptides in rat cortex, receptor binding is reduced to 60% at 150 mM NaCl, KCl, and NH4C1 (Cascieri et al., 1985). Even binding of glutamate to the Na-dependent high affinity glutamate uptake site in rat cortex synaptosomes is steadily decreased to 50% over the Na+ concentration range of 20 to 200 mM (Bennett et al., 1973). Thus Na+ and other cations can reduce receptor and uptake site binding at physiological concentrations and pH. It is conceivable that effects such as these may be dramatically enhanced at non-physiological pH (for example, pH affects binding to glutamate uptake sites [Bennett et al., 1973]). Such a situation is suggested for GSH binding at pH 8.0 in the present study. The experiments of Ogita and Yoneda (1987) support the idea that GSH receptors are not Na+ sensitive at pH 7.4, since [ H]GSH binding was not decreased by 100 mM CU^COONa in synaptic membranes, a preparation which may exclude metabolic uptake sites and LAHC binding. The alternative possibility that reduction by NaCl is due to Cl~ is also suggested by the reduction of [3H]GSH binding _ consistently found with Cl salts (NaCl, CaCl2, MgCl2, and KCl) and with Tris-HCl. It is unlikely that the possible GSH receptor binding sites described here reflect binding of GSH to its metabolizing enzymes or uptake sites, for the following reasons. (1) The Kd values for GSH binding were in the low 109 nM and low /xM range, while Km values for the various GSH S- transferases are 0.2 to 2 mM in rat liver (Habig et al., 1974) and their activity is low in brain (Orlowski and Karkowsky, 197 6) ; the Km for GSH peroxidase is 18 izM in rat liver (Flohe et al., 1972) and is largely confined to neuronal nuclei (Ushijima, 1986); the Km for the mitochondrial high-affinity GSH transporter is 60 ;uM in liver (Martensson et al., 1990); and the only extracellular enzyme, gamma-glutamyl transpeptidase, which may also be the blood- brain and blood-CSF transport site, may have a Km of about 6 mM in rat brain (Kannan et al., 1990; Jain et al., 1991), and acivicin had no affect on binding. (2) GSSG had a similar affinity for the low-affinity GSH binding site, as it does for the high-affinity site in astrocytes (Guo et al., personal communicaton, see below). (3) There was no necessity for the -SH moiety in the binding, nor is there necessity for the gamma-glutamyl structure in synaptic membrane binding (Ogita and Yoneda, 1987). Likewise the possible receptor binding sites are unlikely to represent degradation of [ S]GSH into [35S]cysteine or of [3H]GSH into [3H]glycine and subsequent binding to cysteine or glycine uptake sites because (1) the brain cysteine uptake site is Na-dependent (Hwang and Segal, 1979), and (2) the Km for the low-affinity Na-independent cortical glycine uptake site was originally found to be 300 /xM (Johnston and Iversen, 1971) , and is found to be 8 /xM in isolated cortical astrocytes and 10 /xM in isolated cortical 110 neurons (Hannuniemi and Oja, 1981). The binding is also unlikely to represent [3H]glycine binding to the strychnine- insensitive NMDA receptor glycine binding site because the Kd for this site is 80 nM in rat cortex sections (Miyoshi et al., 1990). In addition, GSH is not degraded in synaptic membrane preparations in Tris-acetate buffer (Ogita et al., 1986a), and opioid peptides are not degraded in lightly fixed, whole brain section incubations in Tris-HCl (Young and Kuhar, 1979). A caveat is expressed, however, by Limbrid (1986): "Before a ligand binding site can be demonstrated to be a receptor of physiological interest, there must be a biological effect elicited by this ligand to which the properties of radioligand binding can be compared", such that "the potency of unlabeled agents in competing for binding to the receptor should parallel the order of potency of these agents in promoting (agonists) or blocking (antagonists) the physiological effect(s) mediated via the putative receptor." This binding data by itself, therefore, cannot be taken to unequivocably demonstrate the presence of GSH receptors. The low-affinity possible receptor binding site may correspond to the higher-affinity site of the two GSH binding sites previously reported for rat synaptic membranes, with published values (from the same laboratory) of 1.9, 0.6, and 0.8 /xM for the Kd and 9.6, 2.5, and 4.0 pmoles/mg protein for the Bmax (Ogita et al., 1986b; Ogita and Yoneda, 1987, 1988, respectively), and which is similarly displaced by cysteine. Ill The LAHC site may or may not correspond to the previously reported lower-affinity, temperature-dependent, Na- independent putative uptake site because, although the Kd and B max values are similar to the GSH LAHC site (Kd: 5.9, 12.6, 11.0 JUM; Bmax: 21.4, 28.5, 27.6 pmoles/mg protein; Ogita et al., 1986b; Ogita and Yoneda 1987, 1988), the LAHC binding was not potentiated by cysteine as is this reported putative uptake site (Ogita and Yoneda, 1989). From the present experiments it cannot be discerned whether the LAHC binding or the low-affinity possible-receptor binding is temperature dependent because the non-mass-action component of the 4° C time courses could be due to either or both of these sites. It is suggested that synaptic membrane preparations may eliminate the LAHC binding. Such a preparation would also eliminate the high-affinity GSH binding site if this site is a hormone receptor or is confined to astrocytes (see below), but in any case the high concentration range covered by the saturation binding analysis in these previous reports would miss this high-affinity binding site, as demonstrated in the present study. It is possible that the LAHC binding revealed in the present study includes binding to one or more of the following: (1) GSH peroxidase in neuronal nuclei (Km = 18 /xM in liver [Flohe et al., 1972]); (2) a mitochondrial uptake site (Km = 60 /xM for the high-affinity component in liver [Martensson et al., 1990]); (3) the possible neuronal plasma membrane uptake site implicated in the present uptake studies 112 in vivo, since uptake sites generally have high Kd and Bmax values, except that the higher affinity terminal uptake systems appear to be universally Na-dependent, including those for neurotransmitter amino acids (Iversen, 1971; Bennett et al., 1973; Lajtha and Shershen, 1975), while the general metabolic uptake systems have much higher Km values (mM) than the Kd for the LAHC site shown in the present study (Johnston and Iversen, 1971; Bennett et al., 1973); and (4) degradation into [3H]glycine (by reactions not involving gamma-glutamyl transpeptidase) and subsequent binding to the low affinity glycine uptake site, although this also is unlikely (see above). LAMINAR DISTRIBUTION Most studies of the distributions of receptor types, including EAA subtypes, in more highly-developed visual cortex such as in the cat (Shaw et al., 1986) and particularly the monkey (Shaw and Cynader, 1986; Shaw et al., 1991) show greater densities in specific layers. Rat visual cortex shows less distinct (Monaghan et al., 1984; Monaghan and Cotman, 1982, 1985; Shaw, unpublished) or uniform (Miyoshi et al., 1990) lamination for many radioligands, including those for EAA receptors. The [35S]GSH autoradiographic distributions in visual cortex sections are consistent with these trends. 113 In adult monkey area 17, cytochrome oxidase staining is highest in the thin sublayer 4A, in sublayer 4C.ct, and in the lower part of sublayer 4CB (Carrol and Wong-Riley, 1984) (as indicated in Figure 22J; see also Figure 4). The [35S]GSH binding in comparison, then, showed a preference for sublayer 4C (Figure 221), which is exclusively stellate cells receiving geniculate terminations, and this binding is not found in sublayer 4B, a layer that receives no DLG terminations (see Figure 4) and contains the line of Gennari (Lund, 1973; Gilbert, 1983). This pattern is consistent with a role as a geniculostriate neurotransmitter. Cat area 17 layer 4 [35S]GSH binding was somewhat more diffuse than in the monkey, which is consistent with the presence in the cat of DLG afferents to layers immediately above and below layer 4, unlike in the monkey (see Figure 4), and the lack of a line of Gennari (Gilbert, 1983). The general increase in binding from 6 to 61 days of age is coincident with the major synaptogenesis that occurs during this period (Winfield, 1981), while the reduction in binding at 90 and 230 days of age is coincident with the succeeding synapse elimination (for review see Payne et al., 1988). The lack of a binding peak in 6-day old cat area 17 occurs at a time when geniculate innervation is much more uniform, and synapses are fewer (Kato et al., 1983; Payne et al., 1988). At 32 days of age the most distinctive [35S]GSH binding density in layer 4 was seen, at which time in the cat the adult pattern of geniculate innervation has been reached 114 (Kato et al., 1983; Payne et al., 1988), and the critical period for plasticity is peaking (Hubel and Weisel, 1970; for review see Cynader et al., 1990). If GSH does act as a neurotransmitter at the sensory- input synapses to layer 4 in area 17, then the uniform distribution in the rat must be explained, as well as the less distinct layer 4 binding seen in area 17 following the peak of the critical period in the cat. Perhaps the most obvious explanation is that GSH receptor binding is "overshadowed" by binding to the much denser LAHC site, since binding to the latter is still present in 20 minute incubations at high radioligand concentration (Figure 19), and may be present at low concentration as well. An alternative explanation is the following. Layer 4 is the predominant input zone for specific afferents from the DLG, but in the rat there is also geniculate input to at least layers 1, 3, and 6 (for review see Sefton and Dreher, 1985), while in the cat all other layers also receive direct, monosynaptic geniculate input (for review see Stone, 1983), and such terminals may also have GSH receptors. There are also LP projections to at least layers 5 and 6 in the rat (Sefton and Dreyer, 1985), and possibly projections for the LP/pulvinar to layers 1 and 5 in the cat (Gilbert, 1983). It has been suggested that extrageniculate visual input may represent a phylogenetically older and less specifically organized pathway (Diamond and Hall, 1969). In the rat, then, extrageniculate visual input from the LP may rival the 115 geniculate input in terms of number of receptors. Since the in vivo uptake experiments in rats showed possible retrograde uptake of radiolabeled GSH from Ocl to the LP, there is support for the notion that such extrageniculate cortical synapses also have GSH receptors. In the case of cat striate cortex, after 32 days of age there may occur an increase in the ratio of the number, affinity, or subtype of GSH receptors, relative to layer 4, at extrinsic or intrinsic inputs (including other geniculate inputs) to other layers, coincident with ongoing synaptogenesis up to about day 60 (Winfield, 1981). Conversely, or additionally, there could be a down-regulation, affinity decrease, or subtype change of GSH receptors at layer 4 geniculate-terminal synapses. It therefore seems possible to speculate that glutathione may be a geniculostriate neurotransmitter in the adult monkey, cat, and rat. PATHWAYS Uptake showed selectivity since [ H]muscimol and [3H]kainate were not taken up. Possible excitotoxic lesioning by kainate or glutamate would not affect terminal uptake (Coyle, 1987), so retrograde transport would not be affected, and since transport takes place solely by passive diffusion (Baughman and Gilbert, 1981), and the somal degeneration phase of excitotoxicity is delayed up to a day 116 (Choi, 1988; Coyle, 1987), therefore anterograde uptake and transport should not have been disrupted either. The possible presence of retrograde transport suggested by cellular resolution indicates possible neurotransmission roles for GSH and cysteine in the geniculostriate and lateral posterior-cortical projections in rat, where cysteine may be a metabolite of released GSH. The lack of LP/pulvinar and other uptake in the cat is consistent with the known anatomy in that extrageniculate projections to area 17 in the postnatal cat are very sparce (for review see Gilbert, 1983; Stone, 1983; Payne, 1988; Swanson et al., 1974). Probable anterograde transport shown by silver grains in the neuropil in all rat nuclei that showed uptake may represent uptake for metabolic purposes (Ochs et al., 1967; Hannuniemi and Oja, 1981), especially if GSH had been metabolized in vivo into amino acids (see below). Cellular resolution for [3H]glutamate and [3H]glycine uptake in rat was not obtained, so it was not possible to determine whether these are implicated in neurotransmission (retrograde), possibly as metabolites of released GSH, or may simply be participating in protein synthesis (anterograde). For the VLG, APTD, Rt, and SC their known anatomical connectivity precludes retrograde transport, so presence in these nuclei of [3H]glutamate or [3H]glycine does not indicate neurotransmitter-related roles. Presence of [3H]glutamate or [3H]glycine in the DLG and LP is not inconsistent with these 117 amino acids being metabolites of GSH released by cortical terminals of projections from these nuclei. A possible confounding factor for interpretation was observed in the rats. Retrograde uptake was determined by the presence of silver grains over cell bodies. In 2 0 /xm thick sections, most cell bodies are sectioned. It therefore follows that if uptake of radiolabel were solely anterograde and confined to terminals in the neuropil, an area free of silver grains should correspond to many of the stained cell bodies, as demonstrated by Baughman and Gilbert (1981). In conflict with this assumption was the observation in rat of silver grains over many cell bodies in the VLG and SC, which have no projections to visual cortex. To address this issue, the following possibilities are suggested. The one-day survival time for the cat was chosen to minimize trans- synaptic transport (Hendrickson 1972), and it is therefore possible that some of the labeling in thalamic nuclei from rats with two-day survival times represents transsynaptic transport. Since the possibility of trans-synaptic transport cannot be completely ruled out in these rats, it should be noted that many of the uptake nuclei interconnect: SC <-> DLG/VLG; Rt <-> ZI/SubG,LP,DLG; ZI/SubG<->VLG; VLG -> APTD; APTD ->Rt. Retrograde uptake in the VLG may therefore be via its projections to the SC, ZI/SubG, and APTD, all of which showed uptake. Another possibility is noted by Young and Kuhar (1979). The tissue section was coated with wet emulsion to obtain a close register of exposed silver grains. 118 This moisture can sometimes cause a dispersal of the radiolabel from its original position. Thus the distinction between retrograde and anterograde uptake may have been obscured in all nuclei in the rats. It is also possible that exogenous, radiolabeled GSH is catabolized in vivo to cysteinyl-glycine and gamma-glutamy1- amino acids by gamma-glutamyl-transpeptidase, followed by the action of cysteinyl-glycinase to produce cysteine and glycine (reviewed by Meister, 1983, 1989). The [35S]cysteine from the breakdown of [35S]GSH, as well as the [35S]cysteine applied directly, may be taken up by the high-affinity Na- dependent or low affinity cysteine uptake sites found in rat cortical synaptosomes (Hwang and Segal, 1979). Alternatively, [35S]cysteine may be oxidized to cysteine sulfinate (Olney, et al., 1971; Misra, 1983) and then be taken up by the single axon-terminal transporter that takes up glutamate, aspartate, cysteine sulfinate, and cysteate (Balcar and Johnson, 1972; Wilson and Pastuszko, 1986). The breakdown of [3H]GSH (labeled on the glycine residue) would likewise produce [H]glycine, for which has been found a low level of both low-affinity, non-specific (neutral amino acids) cortical synaptosomal uptake (Johnston and Iversen, 1971) and high-affinity cortical neuronal uptake (Hannuniemi, 1981). There are several possible interpretations of the radiolabel uptake reported here. Uptake of [3H]glutamate could represent glutamatergic pathways, especially since it 119 is not necessary for each metabolite of a small peptide neurotransmitter to be taken up by the terminal, as shown for NAAG release (Blakely et al., 1986; Robinson et al., 1897; Coyle et al., 1986), and since the known GSH-degrading enzyme, gamma-glutamyl-transpeptidase, would produce mostly gamma-glutamyl-amino acids rather than free glutamate, although this enzyme may be restricted to capillaries and choroid plexus (Orlowski and Karkowsky, 1976). Likewise [35S] uptake from [35S]cysteine in the form of cysteine sulfinate, as discussed above, could also represent glutamatergic pathways or cysteine sulfinate-mediated pathways (Pin et al., 1987; Iwata et al., 1982a,b). [35S]Cysteine uptake could also indicate cysteinergic pathways (Keller et al., 1989; Li and Jope, 1989). The uptake of [3H]GSH, labeled on the glycine residue, is perhaps the best indicator that we may be observing uptake of neurotransmitter metabolites of GSH or of GSH itself, since glycine neurotransmission is rare in forebrain (reviewed in McGeer et al., 1987), and since glycine is not likely to be the geniculocortical neurotransmitter, having no independent excitatory effect (Thompson et al., 1989; Bonhaus et al., 1989) . Finally, in the mammalian visual system in particular, labeling may sometimes represent fibres of passage, even at short survival times (Swanson et al., 1974; Hendrickson et al., 1972) . 120 A possible similarity to the LOT NAAG-transmission story (see Introduction) occurs here. There is no uptake and transport of glutamate in either direction between the SC and area 17 in cat (Baughman and Gilbert, 1981), but cortical ablation in rat decreases endogenous glutamate release in the SC (Lund-Karlsen and Fonnum, 1978). One might conjecture that if rat and cat share the same neurotransmitter in these pathways the true neurotransmitter could conceivably be GSH, releasing glutamate as a metabolite, similar to NAAG ,neurotransmission. ASTROCYTES Guo et al. (1991) have demonstrated GSH binding sites on cultured astrocytes and in white matter in rat brain. Using low-concentration [35S]GSH saturation binding they find a high-affinity binding site on both fibrous and protoplasmic cortical astrocytes in culture (Kd = 9 nM). The additional presence of the low affinity binding site and LAHC binding site on astrocytes was supported by these authors' competition data (Guo et al., personal communication), which showed an apparent IC50 of 10 /uM, with two-site analysis (Bylund and Yamamura, 1990) yielding separate values of 2.3 JUM and 46 /xM. Cellular resolution obtained with colloidal gold decoration of biotinyl-GSH binding (Guo and Shaw, 1991) showed very high density binding on astrocytes. 121 This high density is in keeping with the Bmax values reported here, where the low-affinity site and LAHC site constitute 99% of the total binding (11% and 88%, respectively). More recent radioactive binding experiments in cortical astrocytes by Guo and Shaw (personal communication) differentiated the high affinity GSH binding site into two separate binding sites (Kd = 2 and 12 nM), so it may be possible that the high affinity binding site reported here for whole brain is resolvable into two subtypes as well. The presence of a low- affinity binding site in synaptic membranes (Ogita et al., 1986) makes it unlikely that the low-affinity receptor is confined to astocytes, but the possibility that the high- affinity receptor is confined to astrocytes cannot be ruled out. Since GSH is found in high concentrations in astrocytes but not in neurons, both in culture and in vivo (Raps et al., 1989; Slivka et al., 1987) and since GSH S-transferase is confined to astrocytes as well (Senjo et al., 1986), one interpretation might be that the high-affinity GSH receptor subtype (or types) is a glial uptake site. Another interpretation is that GSH is involved in a novel form of neuron-glial signalling (Marrero et al., 1989; Usowicz et al., 1989; Lieberman et al., 1989; for reviews see Barres, 1989, and Kimelberg, 1988) in addition to neuronal signalling. Guo et al. (1991) also obtain whole rat brain section distributions of specific binding of biotinylated GSH confined largely to white matter, and a lesser amount in brainstem. This contrasts with autoradiographs from this 122 study, which show densest specific [ DS]GSH binding in cortex and LGN and densest non-specific binding in white matter and brainstem. Why biotinylated GSH has a preference for white matter is not understood. EXCITATORY AMINO ACID RECEPTOR INTERACTIONS None of the neurotransmitters for any of the EAA receptor subtypes have been determined with certainty, and displacement of [3H]GSH binding by subtype-specific ligands or by putative transmitters may indicate a physiological role for GSH through interaction with EAA receptors (Coyle et al., 1986). For EAA reviews see Foster and Fagg (1984) , Mayer and Westbrook (1987), Monaghan et al. (1989), Griffith (1990), and Barnard and Henley (1990). At pH 7.4, the neurotransmitter candidates glutamate, aspartate, and cysteine (Keller et al., 1989; Li and Jope, 1989) partially displaced [3H]GSH binding. Although cysteine displaced GSH, there was no need for the -SH moiety since S-methyl-GSH, GSSG, and CSA also show affinity for the GSH binding site revealed at pH 8.0, where there was no crossover with glutamate binding. Of the six different EAA subtype-specific ligands tested, only AMPA partially displaced [3H]GSH at pH 7.4, suggesting a possible AMPA receptor affinity. This suggestion is supported by the observation that AMPA was a more effective competitor than glutamate, because the 123 affinity of glutamate for AMPA receptors is four times lower than the affinity of AMPA itself (IC50s = 0.3 /xM and 1.3 /xM, respectively) (Foster and Fagg, 1984). Cysteine is a candidate neurotransmitter for the AP4 receptor (Pullan et al., 1987), and although L-AP4 itself did not show competition with [3H]GSH, freezing of the tissue and chlorine-free incubation would have abolished the predominant AP4 uptake site and levels of postsynaptic AP4 receptor would be too low to detect (Fagg et al., 1983; Bridges et al., 1986), thus GSH affinity for AP4 receptors is not ruled out. Failure of L-AP3 to displace [3H]GSH indicates a lack of affinity for the ACPD receptor (Schoepp and Johnson, 1989) . Failure of CSA indicates lack of affinity for the CSA receptor (Pin et al., 1987; Iwata et al., 1982a,b). To compare the results of this study to the literature, Oja et al. (1988) and Varga et al. (1989) found displacement of [3H]AMPA by GSH, and Ogita and Yoneda (1987) found displacement of [3H]GSH by AMPA and by AP4 (1987) and affinity of GSH for the NMDA receptor antagonist-preferring site (Fagg et al., 1988; Monaghan et al., 1988) by its displacement of [3H]3-(2-carboxypiperazin-4-yl)propyl-1- phosphonic acid (CPP) binding (1990). At pH 6.9 the increased binding of [3H]GSH may have been due to an increase of GSH receptor binding, of LAHC binding, or of EAA receptor binding, although glutamate and aspartate did not displace this binding. At this pH [3H]GSH binding is displaced by NMDA as well as by two other ligands with 124 affinity for the NMDA receptor agonist-preferring site: QUIS and CSA (Foster and Fagg, 1984; Pullan et ali( 1987). These displacements may indicate an affinity for the NMDA agonist- preferring site. More likely, these displacements may indicate a structural similarity of the recognition site of the GSH molecule to cysteine (which displaced [3H]GSH at pH 6.9, 7.4, and 8), and therefore AP4 receptor affinity, as well as affinity for AMPA receptors and for the NMDA competitive antagonist-preferring site. This latter possibility is suggested because (1) NMDA showed no displacement of [3H]GSH at pH 7.4; (2) NMDA has a lower affinity for NMDA receptors than does glutamate (Foster and Fagg, 1987), which did not displace the GSH binding at pH 6.9; (3) Ogita and Yoneda (1990) show GSH affinity for only the competitive antagonist-preferring site of the NMDA receptor; (4) QUIS has greater affinity for the NMDA receptor antagonist-preferring site (Ferkany and Coyle, 1983), for the AMPA receptor (for which GSH affinity at pH 7.4 was shown), and for AP4 receptors (Ogita and Yoneda, 1986); and (5) CSA also has affinity for the AP4 receptor (Pullan et al., 1987). In reference to the reported abolishment of visually- and electrically-evoked responses of neurons of layer 4 in cat striate cortex by the excitatory amino acid receptor antagonist kynurenic acid (Tsumoto, 1986; Hagihara et al., 1988), it is possible that this broad-spectrum antagonist (Monaghan et al., 1989) is actually acting in this pathway as an antagonist of glutathione receptors, blocking the effect 125 of endogenous release of GSH from geniculostriate terminals via GSH receptors. This idea is supported by the relative paucity of EAA receptors found in layer 4 (see Introduction), although kynurenic acid could have blocked the effect of GSH on AMPA receptors. Kynurenic acid should be tested as a possible antagonist of GSH receptors. SPECULATION - A NOVEL FORM OF NEUROTRANSMISSION? Binding, uptake, and electrophysiology (Teyler, personal communication) of GSH in visual cortex suggest that GSH may be a neurotransmitter in this system, particularly for the geniculostriate projection. It is also possible that GSH may be rapidly metabolized by known brain enzymes into a series of seven substances, all of them neurotransmitter/modulator candidates — gamma-glutamyl-amino acids and glutamate; cysteine and glycine; cysteine sulfinate; cysteic acid; and taurine, in that order (Meister, 1983; Rassin and Gall, 1987; Hill et al., 1985) — all possibly within the period of a single postsynaptic potential (Jain et al., 1991; Kozak and Tate, 1982; Legay et al., 1987; Misra, 1983). It is therefore interesting to speculate that GSH neurotransmission could be of a novel and highly complex form. GSH receptor activation itself may, in some systems, be metabotropic, since no sodium flux was found upon application to rat striatum (Luini, et al., 1984). GSH may also activate AMPA 126 and AP4 receptors, while antagonizing NMDA receptors (Ogita and Yoneda, 1987, 1990; Varga et al., 1989). NMDA receptor antagonism would produce a decrease in tonic background voltage dependence and reduction of facilitation of action potentials (Sah et al., 1989), and a decrease in glycine-site affinity (Hood et al., 1990). NMDA receptor antagonism has also been suggested to reduce induction of long-term potentiation and developmental plasticity (reviewed in Monaghan et al., 1989; Bear, 1988). Next, gamma-glutamyl amino acids and some free glutamate could activate the full range of glutamate receptors (Varga et al., 1989), and reverse all antagonist effects on NMDA receptors (Hood et al., 1990). Cysteine may then activate AP4 receptors, which may include presynaptic autoreceptors that reduce release of glutamate (Monaghan et al., 1989). Glycine may then increase NMDA-receptor mediated responses (Thompson et al., 1989; Bonhaus et al., 1989), including switching the receptor toward the agonist-preferring state (Monaghan et al., 1988), facilitating action potentials (Sah et al., 1989), and lowering the threshold for induction of long-term potentiation (Oliver et al., 1990) and of developmental synaptic plasticity (reviewed by Monaghan, 1989). Then CSA may activate the CSA receptor (Pin et al., 1987; Iwata et al., 1982a,b; for review see Griffith, 1990), which may stimulate cyclic AMP production (Baba et al., 1988). Excitation may be produced by CA, possibly via KA and AP4 receptors (Pullan et al., 1987). Finally taurine, produced 127 last, may have an inhibitory effect (reviewed in McGeer et al., 1987). Glutathione neurotransmission may even be modulated in a novel manner: the initial enzyme in the metabolic cascade, gamma-glutamyl-transpeptidase, is modulated by somatostatin and LHRH in rat brain (Vali and Vijayan, 1990). Such a hypothesized neurotransmission cascade could allow a wide range of modulation and of regional variation by the modulation of the enzymes involved and by the differential expression of sets of enzymes and receptors by a given neuron or synapse. If true, some neurotransmitter candidates, in some pathways, may turn out to be secondary to glutathione release. 128 SUMMARY AND CONCLUSIONS 1. Glutathione binding sites in rat primary visual cortex in vitro satisfied criteria for receptor binding, including reversibility, saturability, specificity, high affinity, and heterogeneous distribution. 2. Glutathione binding sites characterized in this study (pH 8) were separate from EAA receptors, since there was no displacement of GSH by EAA transmitter candidates or EAA subtype-specific ligands, and no displacement of glutamate binding by GSH. 3. Two possible receptor binding sites were present, one with a low Kd typical of neuropeptide and neuromodulator receptors, and one with a higher Kd typical of EAA receptors. A low affinity, high capacity binding site was also present, typical of nonreceptor binding found commonly in experiments at high concentration of ligand. 4. Glutathione binding sites are present in cat area 17 in highest density in layer 4, and in monkey area 17 in lower layer 4, consitent with a role as receptors for neurotransmission by the geniculostriate projection. Rat Ocl binding sites are more uniformly distributed, but nevertheless do exist in layer 4, which is not inconsitent with a role in the geniculostriate projection. 129 5. Results of uptake experiments in vivo in rat and cat primary visual cortex are consistent with the presence of selective terminal uptake of GSH and/or its constituent amino acids, which indicates that GSH may be a neurotransmitter at such terminals. The pathways involved are the geniculostriate projection in cat and the geniculostriate and lateral-posterior-occipital projections in rat. , 6. Competition experiments at pH 7.4 indicate that GSH could play a role in EAA neurotransmission by interacting with EAA receptors such as the AMPA receptor. 7. These results are important because, in addition to helping determine the chemical circuitry of important pathways in the visual system, they suggest that major neurotransmitters in the brain have yet to be identified, and that the neurotransmitter candidates aspartate and glutamate may not play as extensive a role as previously thought. 8. 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