Localization of D2 dopamine receptors in vertebrate retinae with anti‐peptide antibodies

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Abstract
Dopamine plays an important role in modulating various aspects of retinal signal processing. The morphology of dopaminergic neurons and its physiological effects are well characterized. Two classes of receptor molecules (D1 and D2) were shown pharmacologically to mediate specific actions, with differences between individual groups of vertebrates. In an attempt to better understand dopaminergic mechanisms at the cellular level, we used antisera against D2 receptors and investigated the localization of the dopamine D2 receptor in the retinae of rat, rabbit, cow, chick, turtle, frog, and two fish species with immunofluorescence techniques. Antisera were raised in rabbits to two oligopeptides predicted from rat D2 receptor cDNA; one specific for the splice-variant insertion in the third cytoplasmic loop and the other directed towards the extracellular amino terminal region shared by both short and long isoforms. Preadsorption with the synthetic peptide resulted in a significant reduction of label, indicating the presence of specific binding in all species except turtle and goldfish. The pattern of labelling produced by the two antisera was essentially identical; however, the staining obtained with antiserum to the extracellular motif was always more intense. Specific staining was present in photoreceptor inner and outer segments, and in the outer and inner plexiform layers of all species. In mammals and chick, strongly fluorescent perikarya were observed in the ganglion cell layer and at the proximal margin of the inner nuclear layer. Label may be present in the pigment epithelium but could not be established beyond doubt. This pattern of labelling is in accordance with previous observations on D2 receptor localization by means of radioactive ligand binding and in situ hybridization techniques. It suggests that retinal dopamine acts as a neuromodulator as well as a transmitter. In the distal retina, it may reach its targets via diffusion over considerable distances, even crossing the outer limiting membrane; in the inner and outer plexiform layers, conventional synaptic transmission seems to coexist with paracrine addressing of more distant targets, and D2 receptors are expressed by both amacrine and ganglion cells.

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