GABA(A) receptor binding and localization in the tiger salamander retina

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the retina and also appears to act as a trophic factor regulating photorece ptor development and regeneration. Although the tiger salamander is a major model system for thp,study of retinal circuitry and regeneration, our unde rstanding of GABA receptors in this species is almost exclusively based on the results of physiological studies. Therefore, we have examined the pharm acological binding properties of GABA(A) receptors and their anatomical loc alization in the tiger salamander retina. Radioligand-binding studies showe d that specific H-3-GABA binding to GABA(A) receptors was dominated by a si ngle high-affinity binding site (K-d = 15.6 +/- 6.9 nM). Specific binding o f H-3-GABA was almost completely eliminated by muscimol (K-i = 105 +/- 62 n M) and bicuculline (K-i = 14.3 +/- 2.2 mu M); however, SR-95531 only displa ced about 40% of specific H-3-GABA binding (K-i = 35.0 +/- 3.8 nM). These d ata indicate that there are at least two subtypes of GABA(A) receptors pres ent 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 probl ematic, GABA(A) receptors were localized by fluorescent ligand binding comb ined with immunocytochemical labeling for cell specific markers. Binding of fluorescently labeled muscimol to GABA(A) receptors was present in both pl exiform layers and on photoreceptor cell bodies. GABA(A) receptors in the o uter plexiform layer were localized to both photoreceptor terminals and hor izontal cell processes.