Visual Neuroscience (1992), 8, 57-64. Printed in the USA. Copyright © 1992 Cambridge University Press 0952-5238/92 $5.00 + .00

Localization of GABAA receptor subtypes in the tiger salamander retina

CHEN-YU YANG, ZHEN-SHI LIN, AND STEPHEN YAZULLA Department of Neurobiology and Behavior, State University of New York, Stony Brook (RECEIVED April 11, 1991; ACCEPTED June 13, 1991)

Abstract Dry autoradiography was used to determine the distribution of GABA A binding sites in tiger salamander retina. High-affinity binding of [3H]-flunitrazepam ([ 3 H]-FNZ) was used to localize benzodiazepine receptors (BZR) and [3H]-muscimol was used to localize the GABA A recognition site. Specific [ 3 H]-FNZ binding was present only in the inner retina, primarily in the inner plexiform layer (IPL). Co-incubation with GABA enhanced [ 3 H]-FNZ binding by 20-50%. [3H]-muscimol binding was found throughout the IPL and in the outer plexiform layer (OPL). Mouse monoclonal antibodies 62-3G1 and BD-17, that recognize the GABAAj32,(33 polypeptides, and BD-24, that recognizes the GABA A a, polypeptide, did not label either the OPL or IPL, despite numerous variations in the fixation and immunoprocessing methods. GABA A receptor location, as revealed by [3H]-muscimol binding, matches the distribution of presumed GABAergic terminals in the OPL and IPL. We suggest that there are at least two subtypes of GABA A receptor in the tiger salamander retina: one type is present only in the inner retina, primarily in the IPL and is functionally coupled to BZRs; the other type is located in the OPL and is not coupled to the BZRs. Furthermore, GABAA receptors in the tiger salamander retina appear to be of a different epitope than GABA A receptors in numerous other preparations that are recognized by mAbs 62-3G1, BD-17, and BD-24. Keywords: GABA A receptor, Benzodiazepine receptor, Dry autoradiography, Tiger salamander retina Introduction

pam (Clz)] bind to the benzodiazepine receptor (BZR). Benzodiazepine binding to the receptor complex allosterically modulates the GABA recognition site and thereby increases the frequency of GABA-mediated Cl~ channel openings (Bormann, 1988; Costa et al., 1989). In addition, the binding of [3H]-FNZ may be potentiated by GABA, indicating reciprocal allosteric interactions (Tallman et al., 1978; Wastek et al., 1978). In retina, the properties of retinal GABA A receptors and BZRs have been studied extensively using binding techniques on tissue homogenates (Yazulla, 1986, for review; Friedman & Redburn, 1990). However, there are relatively few studies dealing with the autoradiographical localization of these receptors in the retina (Young & Kuhar, 1979; Altstein et al., 1981; Yazulla, 1981; Yazulla & Brecha, 1981; Zarbin et al., 1986). In fact, there are no studies relating to the morphological localization of GABA receptors in amphibian retinas. Recently, monoclonal antibodies (mAbs BD-17, BD-24, 62-3G1) against the GABA A receptor/benzodiazepine receptor/Cl~ channel complex (Richards et al., 1987; Vitorica et al., 1988) have been used to localize GABA A receptors in goldfish, chicken, rat, rabbit, cat, and primate retinas (Richards et al., 1987; Hughes et al., 1989, 1991; Yazulla et al., 1989; Brecha & Weigmann, 1990). We therefore have attempted to localize GABA A receptors in tiger salamander retina by a combination of dry autoradiography of [3H]-muscimol and [3H]-FNZ binding along with the immunocytochemical detection of mAb 62-3G1, BD-17, and BD-24. This material has been presented previously in abstract form (Yang et al., 1991).

There is a massive body of data supporting the role of GABA as a neurotransmitter in the retinas of all vertebrates, including urodele amphibians (i.e. tiger salamander and mudpuppy) (Yazulla, 1986, for review). Morphological studies have shown that the major classes of GABAergic neurons in salamander retina include a type of horizontal cell and several subtypes of amacrine cell (Yang & Yazulla, 1988), similar to that reported for other nonmammalian vertebrates (Yazulla, 1986, for review). Consistent with the anatomy, electrophysiological studies have demonstrated pharmacological effects of GABA on the electrical activity of neurons ramifying in both the outer and inner plexiform layers (Miller et al., 19&la,b; Wu, 1986; Lukasiewiez & Werblin, 1990). Involvement of both GABAA- and GABABtype receptors have been reported (Maguire et al., 1989; Slaughter & Bai, 1989; Stockton & Slaughter, 1991). The GABAA receptor is a multimeric transmembrane protein containing a GABA binding site coupled to a Cl~ channel (Bormann, 1988). The GABAA receptor also may contain specific binding sites for benzodiazepines and barbiturates (Olsen et al., 1986). Muscimol, a GABA agonist, binds to the GABAA receptor; benzodiazepines [e.g. flunitrazepam (FNZ); clonaze-

Reprint requests to: Chen-Yu Yang, Department of Neurobiology and Behavior, State University of New York, Stony Brook, NY 117945230, USA.

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C.-Y. Yang, Z.-S. Lin, and S. Yazulla

58 Methods Tissue preparation Choice of fixative Earlier studies of [3H]-muscimol binding in chicken and goldfish retinas used 0.1 % paraformaldehyde as the primary fixative (Yazulla & Brecha, 1981; Yazulla, 1981). However, the morphology of the tiger salamander retina often was poor at this low concentration. Thus, tissues also were fixed in 1% paraformaldehyde to improve histology. [3H]-FNZ binding appears to be unaffected up to 2% paraformaldehyde (Murrin, 1980). Preparation Tiger salamanders (Ambystome tigrinum) were maintained in aerated tanks at 7°C on a 12-h light/12-h dark cycle and fed live goldfish. Tiger salamanders were decapitated, double-pithed, and the eyes were enucleated. Eyecups or isolated retinas, four of each, were fixed in either 0.1% or 1% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4, overnight at 4°C. The fixed tissues were cryoprotected overnight. Frozen sections (10 or 16 f

Localization of GABAA receptor subtypes in the tiger salamander retina.

Dry autoradiography was used to determine the distribution of GABAA binding sites in tiger salamander retina. High-affinity binding of [3H]-flunitraze...
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