D2-like dopamine receptors in amphibian retina: localization with fluorescent ligands

Dopamine induces several light adaptive changes in amphibian retina via receptors with D2-like pharmacology, but the identity of the primary target cells has not been determined. Using a fluorescent probe consisting of a selective D2 antagonist, N-(p-aminophenethyl)-spiperone (NAPS), derivatized with the fluorophore Bodipy (NAPS-Bodipy), we identified the distribution of dopamine binding sites in the retina of two amphibians, post-metamorphic Xenopus laevis and larval Ambystoma tigrinum. Specific labeling was defined as staining that was displaced by D2 selective ligands (eticlopride or sulpiride), but insensitive to D1 selective drugs (SCH 23390), adrenergic catecholamines (epinephrine or norepinephrine), or serotoninergic analogues (ketanserin). Both rod and cone cells showed specific dopamine D2-like binding sites arranged in clustered arrays on discrete membrane domains of the inner segment. Labeling of photoreceptor outer segments was continuous and was not displaced by competition with D2 selective ligands; this labeling was considered nonspecific. In addition, in both species, clustered binding of the D2-probe was found on Müller cells and on a subset of inner retinal cells with the morphology of amacrine/interplexiform cells. Our data provide direct evidence for D2 receptors on both rods and cones, and suggest that the receptors may be clustered into patches within a discrete cellular domain, the inner segment.