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.

Glycine receptors (GlyRs) and their role in retinal circuitry were analyzed immunocytochemically in the rat retina. Specific antibodies against the alpha 1 subunit of the GlyR and against the GlyR-associated protein gephyrin, respectively, were used. In the inner plexiform layer (IPL), both antibodies produced a punctate label that was shown by electron microscopy to occur at synapses. Gephyrin-like immunoreactivity (-LI) was more widely distributed, indicating that gephyrin might also occur at nonglycinergic synapses. At the ultrastructural level, gephyrin-LI was found at the cytoplasmic face of postsynaptic membranes of amacrine and ganglion cells, but was never detected in bipolar cell axons. Immunoreactivity for the alpha 1 subunit was concentrated in the cleft of conventional synapses made by amacrine cell processes onto ganglion cell dendrites and cone bipolar axons. The latter synapses differ from other glycinergic synapses since they are not labeled by the antibody against gephyrin used in this study. In order to identify the type of bipolar cell involved in these synapses, the distribution of the alpha 1 subunit was compared with that of recoverin-immunoreactive cone bipolar cells and with that of parvalbumin-immunoreactive All-amacrine cells. Double-label immunofluorescence showed that, in the outer part of the IPL, 75% of the alpha 1-immunoreactive puncta were colocalized with recoverin-positive bipolar cell axons and 71% of the alpha 1-immunoreactive puncta were colocalized with parvalbumin-positive All-amacrine processes. Hence, the alpha 1 subunit of the GlyR is present at the chemical synapses established by All-amacrine cells with OFF-cone bipolar cells and OFF-ganglion cells. These synapses play a key role in the transmission of scotopic signals through the OFF-channel of the rod pathway.

Cone pedicles, the synaptic terminals of cone photoreceptors, are connected in the macaque monkey retina to several hundred postsynaptic dendrites. Using light and electron microscopy, we found underneath each cone pedicle a laminated distribution of dendritic processes of bipolar and horizontal cells. Superimposed were three strata of glutamate receptor (GluR) aggregates, including a novel layer of glutamate receptors clustered at desmosome-like junctions. They are, most likely, postsynaptic densities on horizontal cell dendrites. GABA(A) and GABA(C) receptors are aggregated on bipolar cell dendrites in a narrow band underneath the cone pedicle. Glutamate released from cone pedicles and GABA released from horizontal cell dendrites act not only through direct synaptic contacts but also (more so) through diffusion to the appropriate receptors.

Melanopsin is a photopigment expressed in retinal ganglion cells, which are intrinsically photosensitive and are also involved in retinal circuits arising from rod and cone photoreceptors. This circuitry, however, is poorly understood. Here, we studied the morphology, distribution and synaptic input to melanopsin-containing ganglion cells in a New World monkey, the common marmoset (Callithrix jacchus). The dendrites of melanopsin-containing cells in marmoset stratify either close to the inner nuclear layer (outer stratifying), or close to the ganglion cell layer (inner stratifying). The dendritic fields of outer-stratifying cells tile the retina, with little overlap. However, the dendritic fields of outer-stratifying cells largely overlap with the dendritic fields of inner-stratifying cells. Thus, inner-stratifying and outer-stratifying cells may form functionally independent populations. The synaptic input to melanopsin-containing cells was determined using synaptic markers (antibodies to C-terminal binding protein 2, CtBP2, for presumed bipolar synapses, and antibodies to gephyrin for presumed amacrine synapses). Both outer-stratifying and inner-stratifying cells show colocalized immunoreactive puncta across their entire dendritic tree for both markers. The density of CtBP2 puncta on inner dendrites was about 50% higher than that on outer dendrites. The density of gephyrin puncta was comparable for outer and inner dendrites but higher than the density of CtBP2 puncta. The inner-stratifying cells may receive their input from a type of diffuse bipolar cell (DB6). Our results are consistent with the idea that both outer and inner melanopsin cells receive bipolar and amacrine input across their dendritic tree.