Abstract
Abstract: :
Purpose:There are many examples of neuronal coupling via gap junctions in the retina. Here we report the distribution of Connexin36 in the outer plexiform layer of the rabbit retina. Methods:Rabbit retina was immunostained with antibodies against Cx36, synaptophysin, GluR5, calretinin, protein kinase C and mGluR6. Monoclonal and polyclonal Cx36 antibodies were compared for labeling of rabbit retina. In situ hybridization was performed with Cx36 probes on retinal sections. Individual bipolar cells were impaled and filled with Neurobiotin. Structures of particular interest, such connexin plaques near photoreceptors, were extracted, aligned and signal averaged to obtain the spatial distribution of other labels at these points. Results:We previously showed a Cx36 polyclonal antibody displayed strong labeling of the AII matrix in the IPL with very faint staining in the OPL. However, a monoclonal Cx36 antibody shows intense labeling in the IPL and OPL. Monoclonal Cx36 antibody labels plaques along the AII matrix similar to the polyclonal antibody. Cx36 labeling in the OPL is approximately 3-10 fold weaker than that in the IPL. However, when polyclonal and monoclonal antibodies are incubated together, the monoclonal antibody displaces the polyclonal antibody from Cx36 sites. This indicates the monoclonal Cx36 antibody has a higher affinity for the antigen site since the polyclonal antibody displays non-specific labeling of rod bipolar cells and microglia. Cx36 plaques occur at the base of rod spherules and cone pedicles. There are 2.5 0.9 and 8.5 1.8 Cx36 plaques per rod spherule and cone pedicle, respectively. Frequently, the Cx36 plaques are near the basal-lateral edges of the rod spherules. The Cx36 labeling is basal and lateral to mGluR6 labeling of ON-cone and rod bipolar cells. Occasionally, below the cone pedicles, large Cx36 plaques are observed which may represent gap junctions between cone telodendria. In situ hybridization reveals intense Cx36 labeling basal to the myoids of cone and rod photoreceptors, sparse labeling in the ONL (photoreceptor somas), dense labeling in the INL (amacrine cell somas), and occasionally in some ganglion cells. Additionally, Cx36 plaques are not colocalized with GluR5 labeled or injected OFF bipolar dendrites. Conclusion:These results place connexin 36 at the rod spherule and cone pedicle bases, suggesting that cone-cone and rod-cone coupling may utilize Cx36.
Keywords: 416 gap junctions/coupling • 517 photoreceptors • 555 retina: distal(photoreceptors, horizontal cells, bipolar cells)