Localization of Mel 1b melatonin receptor-like immunoreactivity in ocular tissues of Xenopus laevis

Abstract

The circadian signaling molecule, melatonin, is produced by pinealocytes and retinal photoreceptors. In the retina, melatonin is thought to diffuse into the inner retina to act as a paracrine signal of darkness by binding to specific receptors in retinal neurons. The retinal cell locations of the Mel 1a and Mel 1c melatonin receptor types have been reported, but the localization of the Mel 1b receptor, which is the most highly expressed melatonin receptor type in the retina, is unknown. To determine the cellular distribution of Mel 1b melatonin receptor protein in the Xenopus laevis retina and other ocular tissues, polyclonal antibodies were raised against a peptide fragment of the X. laevis Mel 1b receptor. Western blot analysis of several ocular tissues revealed the presence of one or more immunoreactive bands in the sclera, cornea, lens, retinal pigment epithelium (RPE)/choroid, and neural retina. In the neural retina, the major immunoreactive bands displayed electrophoretic mobilities corresponding to approximately 35, 42, 45, and 80 Kd. Sections of X. laevis eyes were analyzed by immunocytochemistry and confocal microscopy, in combination with antibodies against the Mel 1a melatonin receptor, a rod photoreceptor-specific protein, opsin, and two amacrine cell-specific markers, tyrosine hydroxylase (TOH; dopaminergic cells) and glutamic acid decarboxylase (GAD; GABA-ergic cells). Mel 1b immunoreactivity was localized to the apical membranes of RPE cells, and punctate Mel 1b immunoreactivity was observed in both rod and cone photoreceptor inner segments. Presumptive horizontal cells that ramify in the outer plexiform layer (OPL) were immunoreactive for Mel 1b, and were exclusive of the Mel 1a immunoreactivity present in the OPL. Neither TOH nor GAD co-localized with the Mel 1b immunoreactivity that was present in the inner plexiform layer (IPL), suggesting that Mel 1b is not expressed in dopaminergic or GABA-ergic amacrine cells. Mel 1b immunoreactivity was observed in ganglion cells of the retina, a population of cells covering the outer surface of the outer fibrous layer of the sclera, and in lens fibers located in the outer regions of the lens. These results suggest that melatonin may influence retinal function by binding to receptors on RPE and photoreceptor cells, and by acting on neurons of the inner retina that do not use dopamine or GABA as a neurotransmitter. Furthermore, melatonin may bind to receptors on cells located in the sclera and lens, perhaps to modify the growth or function of these ocular tissues.