Wiechmann AF , Sherry DM .

P20RR017703 NCRR NIH HHS , P30EY012190 NEI NIH HHS , P20 RR017703 NCRR NIH HHS , P30 EY012190 NEI NIH HHS

	Figure 1. Mel1a and Mel1b receptors are differentially distributed in the outer retina. A: Mel1a immunoreactivity (red) in the outer plexiform layer (OPL) is present in large processes (arrows) morphologically similar to horizontal cell axons. Mel1b immunoreactivity (green) in the OPL is present in a distinct set of slender processes that form periodic clusters (arrowheads). Mel1b labeling is also present in the cell bodies of bipolar and amacrine cells (BC and AC, respectively) in the inner nuclear layer (INL). Mel1b labeling is also present at the level of the photoreceptor inner segments (PH). Mel1a and Mel1b receptors show differential distribution in the inner plexiform layer (IPL). Mel1b receptors are diffusely distributed throughout the IPL, but Mel1a receptors are found in discrete puncta (small arrows). The box in (A) indicates the area that is shown at higher magnification in (B–D). The confocal image is comprised of 16 optical slices of ≈400 nm. B–D: Examination of single confocal optical planes confirms that Mel1a and Mel1b localize to distinct processes in the OPL. Single optical slice of ≈400 nm is shown in B–D. B: Mel1b is present in slender processes arising from bipolar cells (BC) that form clusters in the distal OPL (large arrowhead). Mel1b labeling also is present in bipolar cell axons (arrow). C: Mel1a labeling is present in relatively large processes (small arrowheads). D: Overlay of panels B,C. Nuclei are counterstained with DAPI (blue) in all panels. Scale bars = 10 μm in all panels.
	Figure 2. Patchy Mel1b receptor distribution in the OPL resembles the distribution of cone terminals. Mel1b receptor labeling in retinal flatmounts confirms the localization of Mel1b to discrete patches of processes (arrows) in the OPL, reminiscent of cone terminal distribution. The patches of Mel1b receptor labeling tend to form annuli surrounding a center region devoid of Mel1b labeling (*). Confocal image stack comprised of 18 optical slices of ≈400 nm each. Scale bar = 10 μm.
	Figure 3. Mel1b receptor-immunoreactive processes contact cone photoreceptor terminals in the outer plexiform layer (OPL). A: Mel1b-immunoreactive (green) processes (arrows) in the OPL specifically contact cone photoreceptor terminals (arrows) labeled for calbindin (red). Punctate Mel1b immunoreactivity is also present at the level of the outer limiting membrane (OLM) and photoreceptor inner segments (arrowheads, PH), just distal to the outer nuclear layer (ONL). Confocal image stack comprised of 22 optical slices of ≈400 nm each. B: Double labeling for Mel1b receptors (green) and XAP-1 (red), a marker for the extracellular matrix surrounding photoreceptor inner and outer segments (PH) and cone terminals (arrowheads) in the OPL, confirms the interaction of Mel1b-positive processes and cone terminals. Confocal image stack comprised of 11 optical slices of ≈400 nm each. Nuclei are counterstained with DAPI (blue) in both panels. INL, inner nuclear layer. Scale bars = 10 μm in both panels.
	Figure 4. Mel1b receptors are expressed by OFF bipolar cells. A: Double labeling for Mel1b receptors (green) and the ON bipolar cell marker Goα (red) shows that Mel1b receptor-immunoreactivity is absent from the cell bodies of ON bipolar cells (ON), identifying the Mel1b receptor-immunoreactive bipolar cells as OFF bipolar cells (OFF). Confocal image stack comprised of seven optical slices of 400 nm each. Apparent colocalization of Mel1b and Goα immunoreactivity in processes in the outer plexiform layer (OPL) is due to their close proximity and the relative thickness of the image stack, and does not represent genuine colocalization (see panels B–D, below). Immunolabeling for both Mel1b and Goα is present in the inner plexiform layer (IPL), with strongly Mel1b-positive processes (small arrows) present along the inner margin of the layer. Mel1b immunoreactive puncta (small arrowheads) are also present at the level of the outer limiting membrane (OLM). The box in (A) indicates area shown in panels B–D. B–D: Examination of a single confocal optical plane confirms that Mel1b labeling does not colocalize with labeling for Goα in ON bipolar cells. Images in panels B–D represent a single optical slice of ≈400 nm. B: Mel1b immunoreactivity in the cell body and primary and secondary dendrites (arrow and large arrowhead respectively) of a bipolar cell (OFF). C: ON bipolar cell dendrites labeled for Goα (small arrowheads). D: Overlay of panels B,C showing that ON bipolar cell dendrites are devoid of Mel1b receptor labeling. Nuclei are counterstained with DAPI (blue) in all panels. INL, inner nuclear layer; PH, photoreceptor inner segments. Scale bars = 10 μm in all panels.
	Figure 5. Mel1b receptor-immunoreactive OFF bipolar cell dendrites and ON bipolar cell dendrites are spatially segregated at cone terminals. A: Immunolabeling for Mel1b (green) and Goα (magenta) in a retinal flatmount preparation focused at the level of the outer plexiform layer (OPL). Mel1b-immunoreactive OFF bipolar cell dendrites (arrowheads) and Goα-immunoreactive ON bipolar cell dendrites (arrows) both contact the same cone terminals, but tend to contact cone terminals in discrete locations. Occasionally, cone terminals receiving numerous contacts from Goα-immunoreactive ON bipolar cell dendrites, but little or no contacts from Mel1b-immunoreactive OFF bipolar cell dendrites were observed (circles), suggesting that Mel1b-immunoreactive OFF bipolar cells may not contact all cone types equally. Confocal image stack comprised of nine optical slices of ≈400 nm each. B: Mel1b (green) and Goα (red) immunolabeling in the OPL in vertical frozen sections of retina shows that Mel1b-immunoreactive OFF bipolar cell dendrites (arrowheads) penetrated slightly deeper into the cone terminals than Goα-immunoreactive ON bipolar cell dendrites (arrows). An ON bipolar cell body (BC) labeled for Goα is also visible. Nuclei are counterstained with DAPI (blue). Confocal image stack comprised of seven optical slices of 400 nm each. Scale bars = 10 μm for both panels.
	Figure 6. Mel1a receptors are not expressed in ON bipolar cell dendrites. A: Labeling for Mel1a receptor (green) and Goα (red) localize to different processes in the outer plexiform layer (OPL). Mel1a receptors are expressed in distinctive processes morphologically similar to horizontal cell axons (large arrows). These processes are distinct from the Goα-immunoreactive ON bipolar cell dendrites (arrowheads). Apparent colocalization of Mel1b and Goα immunoreactivity in processes in the outer plexiform layer (OPL) is due to their close proximity and the relative thickness of the image stack, and does not represent genuine colocalization (see panels B–D, below). Mel1a immunoreactivity also is present in puncta throughout the inner plexiform layer (IPL; small arrows) and in ganglion cell axons (long arrows) in the nerve fiber layer (NFL). Confocal image stack comprised of 15 optical slices of ≈400 nm each. The box indicates the area shown in panels B–D. B–D: Examination of thin stacks of confocal optical planes confirms that Mel1a labeling is not localized to ON bipolar cell dendrites. Images in panels B–D are comprised of five optical slices of ≈400 nm each. B: Mel1a immunoreactivity in horizontal cell axons (arrowheads) in the OPL. C: ON bipolar cell dendrites labeled for Goα (small arrow). D: Overlay of panels B,C showing that ON bipolar cell dendrites (Goα-positive) and Mel1a receptor-positive processes are not colocalized. Nuclei are counterstained with DAPI (blue) in all panels. BC, Bipolar cell; PH, photoreceptors; IPL, inner plexiform layer; NFL, nerve fiber layer. Scale bars = 10 μm in all panels.
	Figure 7. Mel1a melatonin receptor-immunoreactive processes in the outer plexiform layer (OPL) contact cone photoreceptor terminals. A: Branches of Mel1a-immunoreactive processes (green; arrows) ascend through the OPL to contact the terminals of calbindin-immunoreactive cone photoreceptors (red; arrowheads) terminals. Confocal image stack comprised of 11 optical slices of ≈400 nm each. B: Mel1a melatonin receptor-immunoreactive processes (green; arrowheads) in the OPL contact XAP-1-immunoreactive cone photoreceptor terminals (red). The extracellular matrix surrounding the inner and outer segments of the photoreceptors is also XAP-1-immunoreactive. Confocal image stack comprised of 10 optical slices of ≈400 nm each. Nuclei are counterstained with DAPI (blue) in both panels. PH, photoreceptor inner segments; ONL, outer nuclear layer; INL, inner nuclear layer. Scale bars = 10 μm in both panels.
	Figure 8. Mel1b receptor-immunoreactive OFF bipolar cell dendrites and Mel1a receptor-immunoreactive horizontal cell axons contact the same cone terminals. Immunolabeling for Mel1b (green) and Mel1a (magenta) in a retinal flatmount focused at the level of the outer plexiform layer (OPL). Mel1b-immunoreactive OFF bipolar cell dendrites (arrowheads) and Mel1a-immunoreactive horizontal cell axons (arrows) selectively contact the same cone terminals. Confocal image stack comprised of seven optical slices of ≈400 nm each. Scale bar = 10 μm.
	Figure 9. Schematic model of the relationship between melatonin receptors and functional organization of connections among Xenopus cones and bipolar and horizontal cell processes. A,C: Schematic summary of the predicted organization of horizontal, OFF bipolar, and ON bipolar cell processes at cone terminals in the Xenopus retina shown in the vertical (A) and horizontal (C) orientation. B,D: Schematic summary of the organization of Mel1a and Mel1b receptors at cone terminals in the Xenopus retina shown in the vertical (B) and horizontal (D) orientation.

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