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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. |
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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. |
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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. |
