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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 Mel1a and Mel1c melatonin receptor types have been reported, but the localization of the Mel1b receptor, which is the most highly expressed melatonin receptor type in the retina, is unknown. To determine the cellular distribution of Mel1b melatonin receptor protein in the Xenopus laevis retina and other ocular tissues, polyclonal antibodies were raised against a peptide fragment of the X. laevis Mel1b 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 Mel1a 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). Mel1b immunoreactivity was localized to the apical membranes of RPE cells, and punctate Mel1b 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 Mel1b, and were exclusive of the Mel1a immunoreactivity present in the OPL. Neither TOH nor GAD co-localized with the Mel1b immunoreactivity that was present in the inner plexiform layer (IPL), suggesting that Mel1b is not expressed in dopaminergic or GABA-ergic amacrine cells. Mel1b 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.
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15381042
???displayArticle.link???Exp Eye Res ???displayArticle.grants???[+]
Fig. 1.
Western blot of Xenopus ocular tissues with Mel1b receptor antibody. Membrane preparations of sclera (S), cornea (C), retinal pigment epithelium/choroid (RPE), neural retina (Ret) or lens were separated by SDS-PAGE, blotted to nitrocellulose, and labeled with an antibody against the Mel1b receptor. Molecular weight standards are indicated to the left of the blot.
Fig. 2.
Confocal image of immunocytochemistry of Xenopus laevis retina with Mel1b melatonin receptor antibody. Fixed cryostat sections of Xenopus retina were incubated with the Mel1b receptor antibody or with normal rabbit immunoglobulin (control). The primary antibody was labeled with a green fluorescent dye-antibody conjugate, and counter-stained with a blue nuclear dye. (A) Specific Mel1b immunolabeling is observed in the retinal pigment epithelium (RPE), proximal portion of the photoreceptor inner segments (IS), outer plexiform layer (OPL), inner plexiform layer (IPL), and ganglion cell layer (GCL). The photoreceptor outer segments (OS) do not demonstrate specific immunolabeling. (B) Specific immunoreactivity is not observed in sections treated with normal rabbit immunoglobulin. Scale bar represents 50 μm.
Fig. 3.
Confocal image of immunocytochemistry of Xenopus laevis retina with Mel1b melatonin receptor antibody. Fixed cryostat sections of Xenopus retina were incubated with the Mel1b receptor antibody (Mel1b), with normal rabbit immunoglobulin (control), or with Mel1b receptor antibody following an incubation with the antigenic peptide (Mel1b+Peptide B). The primary antibody was labeled with a green fluorescent dye-antibody conjugate, and counter-stained with a blue nuclear dye. (A) Higher magnification of an area of image 2A. Specific Mel1b immunolabeling is observed in the proximal portion of the photoreceptor inner segments (IS; arrowheads) and outer plexiform layer (OPL; arrows). The photoreceptor outer segments (OS) do not demonstrate specific immunolabeling. (B) Specific immunoreactivity is not observed in sections treated with normal rabbit immunoglobulin. (C) Specific immunoreactivity is not observed in sections treated with the Mel1b receptor antibody that had been pre-incubated with the antigenic peptide. Scale bar represents 100 μm.
Fig. 4.
Confocal image of immunocytochemistry of Xenopus laevis retina with opsin antibody and Mel1b melatonin receptor antibody. Fixed cryostat sections of Xenopus retina were incubated with the Mel1b receptor antibody followed by incubated with an antibody against opsin. The Mel1b receptor antibody was labeled with a green fluorescent dye-antibody conjugate, and the opsin antibody was labeled with a red fluorescent dye-antibody conjugate, and then counter-stained with a blue nuclear dye. (A) Specific Mel1b immunolabeling is observed in the proximal portion of the photoreceptor inner segments (IS) and inner retina layers. The photoreceptor outer segments (OS) are immunoreactive for opsin. (B) Higher magnification of an area of image (A). Specific opsin immunoreactivity is present in the rod outer segments, as well as in the inner segments located below. The red opsin immunoreactivity in the inner segments is located in the same cells as the punctate Mel1b immunolabeling. Note the presence of punctate Mel1b immunolabeling in cone photoreceptor inner segments that lack opsin immunoreactivity. The oil droplets of the cone inner segments are denoted by an asterisk (*). Scale bars represent 50 μm.
Fig. 5.
Confocal image of immunocytochemistry of Xenopus laevis retinal pigment epithelium with and Mel1b melatonin receptor antibody with or without opsin antibody. Fixed cryostat sections of Xenopus retinal pigment epithelium (RPE) were incubated with the Mel1b receptor antibody followed by incubation with an antibody against opsin. The Mel1b receptor antibody was labeled with a green fluorescent dye-antibody conjugate, and the opsin antibody was labeled with a red fluorescent dye-antibody conjugate, and then counter-stained with a blue nuclear dye. (A) Specific Mel1b immunolabeling is observed in the apical membrane of the retinal RPE, but is lacking in the basal membrane (BM). (B) Specific Mel1b immunolabeling is observed in the apical membrane of the retinal pigment epithelium (RPE), and are in direct contact with the red opsin-immunolabled rod outer segments (ROS). (C) Specific immunoreactivity is not observed in sections of RPE treated with normal rabbit immunoglobulin. Scale bar represents 50 μm.
Fig. 6.
Differential distribution of Mel1a and Mel1b receptor immunoreactivity in Xenopus laevis retina. Sections of Xenopus retina were incubated first with the Mel1b receptor antibody and labeled with a red fluorescent dye-antibody conjugate then incubated with the Mel1a antibody labeled with a green fluorescent dye-antibody conjugate. Mel1b and Mel1a immunolabeling appears in the outer plexiform layers (OPL), and appear to be located in separate populations of cells. Scale bar represents 50 μm.
Fig. 7.
Double label immunocytochemistry of Mel1b melatonin receptor with GAD and TOH in Xenopus laevis retina. The melatonin receptor labeling is red, the GAD and TOH labeling is green, and the nuclear stain is blue. (A) Dopaminergic cell processes are labeled with an antibody to tyrosine hydroxylase (TOH, arrows) located in sublamina 1 of the inner plexiform layer (IPL), and some punctate labeling in sublamina 5. (D) The red labeling of the Mel1b receptor and the green labeling of the TOH does not result in any yellow fluorescence in the IPL, suggestive of a lack of co-localization between Mel1b receptor and TOH-expression cells. (B) GABA-ergic neuronal cell processes are labeled with an antibody to glutamate decarboxylase (GAD), in the distal and proximal layers of the IPL. (B) The lack of yellow fluorescence in the IPL is suggestive of a lack of co-localization for the Mel1b receptor and GAD, although there may be some degree of co-localization of the Mel1b receptor and GAD in the proximal layers of the IPL. Scale bar represents 50 μm.
Fig. 8.
Confocal image of immunocytochemistry of Xenopus laevis sclera with Mel1b melatonin receptor antibody. Fixed cryostat sections of Xenopus sclera were incubated with the Mel1b receptor antibody or with normal rabbit immunoglobulin (control). The primary antibody was labeled with a green fluorescent dye-antibody conjugate, and counter-stained with a blue nuclear dye. (A, B) Specific Mel1b immunolabeling is observed in cells positioned distally to the outer fibrous layer (OFL) of the sclera. (C) Same magnification the area distal to the OFL of the sclera, illustrating the clusters of cells that are Mel1b-immunoreactive. (D) Same magnification as in (C), of a sclera section treated with normal rabbit immunoglobulin. Specific immunoreactivity is not observed in sections treated with normal rabbit immunoglobulin. Scale bar represents 50 μm.
Fig. 9.
Confocal image of immunocytochemistry of Xenopus laevis lens with Mel1b melatonin receptor antibody. Fixed cryostat sections of Xenopus lens were incubated with the Mel1b receptor antibody or with normal rabbit immunoglobulin (control). The primary antibody was labeled with a green fluorescent dye-antibody conjugate, and counter-stained with a blue nuclear dye. (A) Specific Mel1b immunolabeling is observed in membranes of the lens fibers, and some punctate immunolabeling is also present. (B) Specific immunoreactivity is not observed in sections treated with normal rabbit immunoglobulin. Scale bar represents 50 μm.
