GABA_A receptor binding and localization in the tiger salamander retina

Abstract

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the retina and also appears to act as a trophic factor regulating photoreceptor development and regeneration. Although the tiger salamander is a major model system for the study of retinal circuitry and regeneration, our understanding of GABA receptors in this species is almost exclusively based on the results of physiological studies. Therefore, we have examined the pharmacological binding properties of GABA_A receptors and their anatomical localization in the tiger salamander retina. Radioligand-binding studies showed that specific ³H-GABA binding to GABA_A receptors was dominated by a single high-affinity binding site (K_d = 15.6 ± 6.9 nM). Specific binding of ³H-GABA was almost completely eliminated by muscimol (K_i = 105 ± 62 nM) and bicuculline (K_i = 14.3 ± 2.2 μM); however, SR-95531 only displaced about 40% of specific ³H-GABA binding (K_i = 35.0 ± 3.8 nM). These data indicate that there are at least two subtypes of GABA_A receptors present in the salamander retina that can be distinguished by their antagonist binding properties: one sensitive to both bicuculline and SR-95531, and one sensitive to bicuculline but insensitive to SR-95531. Because localization of GABA receptors in the salamander retina by immunocytochemistry is problematic, GABA_A receptors were localized by fluorescent ligand binding combined with immunocytochemical labeling for cell specific markers. Binding of fluorescently labeled muscimol to GABA_A receptors was present in both plexiform layers and on photoreceptor cell bodies. GABA_A receptors in the outer plexiform layer were localized to both photoreceptor terminals and horizontal cell processes.