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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. |
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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. |
