Localization of the AMPA subunit GluR2 in the outer plexiform layer of goldfish retina

Abstract

L-glutamate, the photoreceptor neurotransmitter, depolarizes horizontal cells and OFF bipolar cells by ionotropic AMPA-glutamate receptors. The AMPA-receptor subunit (GluR4) is localized to dendrites of OFF bipolar cells in goldfish retina. Here, we used immunohistochemical techniques to identify AMPA-receptor subunits on horizontal cell dendrites. A monoclonal antibody against rat GluR2, with high sequence homology to the recently cloned goldfish GluR2a receptor, was used for light- and electron-microscopical immunocytochemistry. Light- and dark-adapted retinas were analyzed, with no major difference in results. GluR2-immunoreactivity (IR) was restricted to a narrow band in the outer plexiform layer, in which it appeared as bright dome-shaped structures amidst numerous puncta. At the ultrastructural level, GluR2-IR was found in horizontal cell dendrites that invaginated cones and rods. Dendrites of OFF bipolar cells were not labeled. GluR2-IR was present mostly in horizontal cell dendrites that were the lateral elements of the triad, rather than in dendrites that were the central elements. In light-adapted retinas, GluR2-IR was found in many horizontal cell spinules. GluR2-IR was observed, on occasion, in a mixed rod/cone (Mb) ON bipolar cell process that innervated rod spherules. Verification of the Mb ON bipolar cell was made by protein kinase C and metabotropic mGluR1alpha immunolabeling. The presence of GluR2-IR in lateral elements suggests that lateral horizontal cell dendrites are postsynaptic to cones rather than only sites of feedback inhibition. All horizontal cell types express the GluR2 subunit, uniquely differentiating themselves from OFF bipolar cells that express the GluR4 subunit. This differentiation most likely has a major influence on the glutamate pharmacology and response kinetics of these cell types to glutamate.

Neurochemical anatomy of the zebrafish retina as determined by immunocytochemistry

“…Note the punctate appearance of GAD67-IR in the horizontal cell dendrites, indicating a concentration of reaction product in parts of the dendrite rather than a uniform distribution. This finding is consistent with our EM studies in goldfish showing that GluR2-IR is found in horizontal cell dendrites (Klooster et al, 2001). Thus, we are able to use GluR2-IR as a marker to identify structures as HI dendrites and by exclusion, those that were not.…”

“…The GluR2-IR structures in goldfish were identified, by pre-embedding electron microscopic immunocytochemistry (EM-ICC), as belonging to the distal tips of rod and cone horizontal cells. All cones were innervated by GluR2-IR dendrites (Klooster et al, 2001). Preliminary EM-ICC in this laboratory (Klooster & Yazulla, unpublished) confirms that GluR2-IR is localized to cone horizontal cell dendrites in the zebrafish as well.…”

“…This is the identical pattern, including the co-localization with PKC-IR in only the ON mixed bipolar cells that was observed in goldfish . Klooster et al (2001) also showed by EM-ICC that mGluR1a-IR was localized to the dendrites of ON bipolar cells, innervating both rods and cones. It is highly likely that the same would be found in zebrafish.…”

“…GluR2-IR appeared as regularly spaced, dome-shaped structures in vertical section and as small rosettes when viewed in cross section. This is the same pattern reported in goldfish (VandenBranden et al, 2000a;Klooster et al, 2001). The GluR2-IR structures in goldfish were identified, by pre-embedding electron microscopic immunocytochemistry (EM-ICC), as belonging to the distal tips of rod and cone horizontal cells.…”

Expression of the eight AMPA receptor subunit genes in the developing central nervous system and sensory organs of zebrafish

“…Expression of AMPA receptor subunits 3 and 4 in the horizontal cells was observed in rat and cat (Hamassaki-Britto et al, 1993). In contrast to these results, immunohistochemical analysis in adult zebrafish also revealed expression of gria2 genes in horizontal cells , as also observed in goldfish (Vandenbranden et al, 2000;Klooster et al, 2001) and expression of gria4 in Mueller and bipolar cells but not in horizontal cells ). Those differences can be explained with a change in the expression pattern later during development.…”