An antibody directed against protein kinase C (PKC) was applied to various mammalian retinae. In the cat, rat, rabbit, and macaque monkey we found PKC-like immunoreactivity in bipolar cells which had the morphology of rod bipolar cells; in the rat some amacrine cells were also immunoreactive. In the outer plexiform layer, labeled dendrites were always the central elements of the rod spherule invagination, and in the inner plexiform layer only rod bipolar axons and their axon terminals were immunoreactive. The antibody against PKC thus can be used to distinguish rod bipolar cells from cone bipolar cells. The antibody against PKC was used to determine the densities of rods and rod bipolar cells in the cat retina. In the central retina we found a rod to rod bipolar ratio of 16 to 1, in the periphery the ratio increases to 25 to 1. In freshly dissociated retina, cells with rod bipolar morphology could be identified; these cells were also labeled with the anti-PKC antibody. Hence, PKC-like immunoreactivity can be used to recognize rod bipolar cells in vitro.

Gephyrin is a protein that copurifies with the glycine receptor (GlyR) and is required for the clustering of GlyRs at postsynaptic sites. Previously, it was thought that antibody mAb 7a, directed against gephyrin, was a specific marker for GlyR. However, there is evidence that gephyrin can also be found at nonglycinergic synapses. Here, immunocytochemistry was applied to show this directly for the rat retina. Both gephyrin and different subunits of the gamma-aminobutyric acid (GABA)A receptor were localized to discrete puncta in the inner plexiform layer, and these puncta were shown by electron microscopy to represent synaptic sites. Double immunocytochemistry revealed that GABAA receptors and GlyRs are not colocalized. However, gephyrin and different subunits of GABAA receptors were found to occur at the same synapses. The amount of colocalization varied with the GABAA receptor subunit composition and was most extensive for the alpha 2 subunit, less for the alpha 3 subunit, and minimal for the alpha 1 subunit. The gephyrin present at GABAergic synapses of the retina might also be involved with clustering of receptors at the postsynaptic sites. Hence, localization of gephyrin can no longer be considered as a unique marker of glycinergic synapses.

The distributions of nine different subunits of the gamma-aminobutyric acidA (GABAA) receptor (alpha 1, alpha 2, alpha 3, alpha 5; beta 1, beta 2, beta 3; gamma 2; delta) were investigated in the rat retina using immunocytochemistry and in situ hybridization. With the exception of the alpha 5 subunit, all subunits could be localized. Each subunit was expressed in characteristic strata within the inner plexiform layer (IPL). Some subunits (e.g., gamma 2) showed a ubiquitous distribution, while others (e.g., delta) were restricted to narrow sublayers. Double labeling experiments using different combinations of the subunit-specific antibodies revealed colocalizations of subunits within individual neurons. Additionally, GABAA receptor subunits were mapped to distinct populations of retinal neurons by coapplication of defined immunocytochemical markers and subunit-specific antibodies. Cholinergic amacrine cells were found to express the alpha 2, beta 1, beta 2/3 and delta subunits, while dopaminergic amacrine cells express the alpha 2, alpha 3 and gamma 2 subunits. Dissociated rod bipolar cells express the alpha 1 and gamma 2 subunits. In summary, this study provides evidence for the existence of multiple GABAA receptor subtypes in the retina. The distinct stratification pattern of the subunits in the IPL suggests that different functional circuits involve specific subtypes of GABAA receptors.

Reticular pseudodrusen represent subretinal deposits that extend through the outer nuclear layer, affect photoreceptor integrity, and are associated with retinal gliosis and RPE damage.

Three approaches to study the function of mammalian rod bipolar cells are described. Extracellular recordings from the intact cat eye under light- and dark-adapted conditions showed that in dark-adapted retina all light responses can be blocked by 2-amino-4-phosphonobutyrate (APB). Immunocytochemical staining with an antibody against protein kinase C (PKC) labeled rod bipolar cells in all mammalian retinae tested. When rat retinae were dissociated, PKC immunoreactivity was also found in isolated bipolar cells and could be used for their identification as rod bipolars. Patch-clamp recordings were performed from such dissociated rod bipolar cells and their responses to APB were measured. APB closed a nonselective cation channel in the cell membrane. The actions of GABA and glycine were also tested and both opened chloride channels in dissociated rod bipolar cells. These results suggest that rod bipolar cells are depolarized by a light stimulus and that GABA as well as glycine modulate their light responses.