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The hormone melatonin is an output signal of an endogenous circadian clock in retinal photoreceptors. Melatonin may act as a paracrine and/or intracrine neurohormone by binding to specific receptors in the eye. The distribution of Mel(1a) and Mel(1c) melatonin receptors in the Xenopus laevis retina was examined by immunocytochemistry, using antibodies prepared against specific sequences of the Xenopus receptor proteins. Antibodies that label dopaminergic and GABA-ergic amacrine cells were used in double-label experiments with the melatonin receptor antibodies. The distribution of Mel(1a) and Mel(1c) receptor immunoreactivity was similar insofar as the two receptors were localized in the inner plexiform layer. However, the Mel(1c) receptor displayed some immunoreactivity in the photoreceptor cells, whereas the Mel(1a) receptor displayed little if any photoreceptor labelling. The Mel(1c) antibody, but not the Mel(1a), labelled a population of ganglion cells. While both receptors were localized to the outer plexiform layer, they did not appear to localize to the identical cell types. These results demonstrate that the Mel(1a) and Mel(1c) receptor proteins are present in cells of the X. laevis retina, and their distribution in the photoreceptors and inner retina is very similar to that reported in the human retina. The differential pattern of expression of the melatonin receptors suggests that melatonin may convey differential effects on various target cells in the retina.
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12589779
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Fig. 1.
Immunocytochemistry of the X. laevis retina with Mel1a and Mel1c melatonin receptor antibodies. Fixed cryostat sections of Xenopus retina were incubated with the Mel1a receptor antibody or with antibody plus excess Mel1a or Mel1c peptide (controls). The primary antibody was labelled with a green fluorescent dye–antibody conjugate, and counter stained with a blue nuclear dye. (A) Specific Mel1a immunolabelling is observed in the OPL (arrowheads), IPL, and GCL. The photoreceptor outer segments (OS) do not demonstrate specific immunolabelling. (B) Mel1a antibody pre-incubation with Mel1a antigen peptide blocks all specific immunoreactivity. (C) Mel1a antibody pre-incubation with Mel1c antigen peptide does not diminish Mel1a immunoreactivity. Scale bar represents 50 μm.
Fig. 2.
Differential distribution and co-localization of Mel1a and Mel1c receptor immunoreactivity in the X. laevis retina. Sections of Xenopus retina were incubated first with the Mel1c receptor antibody and labelled with a green fluorescent dyeâantibody conjugate then incubated with the Mel1a antibody labelled with a red fluorescent dyeâantibody conjugate. (A) Mel1a immunolabelling appears in OPL and IPL. (B) Mel1c immunolabelling appears primarily in the OPL and IPL, but also in the photoreceptor inner segments (IS). (C) Merged images of (A) and (B) show that Mel1c and Mel1a appear to have some co-localization in the OPL (white arrows) and IPL, and indicated by the yellow fluorescence. There is also some differential expression, where the red fluorescence representing Mel1a immunoreactivity is apparent in the OPL (arrowheads). Also, the Mel1c green fluorescence is located in the area of the ganglion cell soma in the GCL, but the red fluorescence of the Mel1a is not as prevalent in the GCL. Although Mel1c is apparent in the photoreceptor IS as indicated by the green fluorescence, red fluorescence in the IS is very low. Scale bar represents 50 μm.
Fig. 3.
High magnification light micrograph merged image of the X. laevis retina double-labelled with Mel1a (red) and Mel1c (grn) antibodies. Mel1c immunoreactivity is present alone in the photoreceptor IS (white arrows), but not in the outer segments (OS). Also, Mel1c immunoreactivity is present alone in proximal regions of the OPL (black arrows), and in the ganglion cell soma (black arrows). Mel1a immunoreactivity is present alone in the distal portion of the OPL (large arrowheads). Co-localization of Mel1a and Mel1c immunoreactivity, as indicated by the yellow fluorescence, is present as a broad band in the IPL. There is also a small amount of yellow fluorescence in the distal portion of the OPL (small arrowheads), which may represent the close approximation of the photoreceptor synaptic terminals (green), and some horizontal cell processes (red). Scale bar represents 100 μm.
Fig. 4.
Double label immunocytochemistry of Mel1a melatonin receptor with GAD and TOH in the X. laevis retina. The Mel1a melatonin receptor antibody appears to label both GABA-ergic and dopaminergic cell processes in the IPL. The melatonin receptor labelling is green, the GAD and TOH labelling is red, and the nuclear stain is blue. (A) GABA-ergic neuronal cell processes are labelled with an antibody to glutamate decarboxylase (GAD, arrows), in the distal and proximal layers of the IPL. (B) Some degree of co-localization of the Mel1a receptor and GAD is suggested by the yellow and orange fluorescence. (C) Dopaminergic cell processes are labelled with an antibody to tyrosine hydroxylase (TOH, arrows) located in sublamina 1 of the IPL, and some punctate labelling in sublamina 5. (D) The green labelling of the Mel1c receptor and the red labelling of the TOH results in a yellow fluorescence in the proximal portion of sublamina 1 (black arrows), which is suggestive of co-localization. However, in the distal portion of sublamina 1, only TOH immunolabelling is observed (white arrows). Scale bar represents 50 μm.
Fig. 5.
Confocal image of Mel1c immunoreactivity in the photoreceptor layers. Mel1c immunoreactivity is present in the photoreceptor inner segments (IS; arrows), but not in the outer segments (OS; the axoneme labelling is also seen in control sections). Also, Mel1c immunoreactivity is present in distal regions of the OPL directly adjacent to photoreceptor nuclei in the outer nuclear layer (ONL), and may represent photoreceptor synaptic terminals. The oblique orientation and cross sections of photoreceptor IS reveal the plasma membrane localization of the Mel1c receptor (arrows). In the IS, Mel1c labelling is exclusively present on the plasma membrane (arrows). The illusion of Mel1c immunoreactivity filling the IS is due to the thickness of the tissue sections, showing the immunoreactivity of the cell membrane as it spans the width of the IS. Scale bars represent 50 μm.
