The goals of these experiments were to describe the morphology and synaptic connections of amacrine cells in the baboon retina that contain immunoreactive vesicular glutamate transporter 3 (vGluT3). These amacrine cells had the morphology characteristic of knotty bistratified type 1 cells, and their dendrites formed two plexuses on either side of the center of the inner plexiform layer. The primary dendrites received large synapses from amacrine cells, and the higher-order dendrites were both pre- and postsynaptic to other amacrine cells. Based on light microscopic immunolabeling results, these include AII cells and starburst cells, but not the polyaxonal amacrine cells tracer-coupled to ON parasol ganglion cells. The vGluT3 cells received input from ON bipolar cells at ribbon synapses and made synapses onto OFF bipolar cells, including the diffuse DB3a type. Many synapses from vGluT3 cells onto retinal ganglion cells were observed in both plexuses. At synapses where vGluT3 cells were presynaptic, two types of postsynaptic densities were observed; there were relatively thin ones characteristic of inhibitory synapses and relatively thick ones characteristic of excitatory synapses. In the light microscopic experiments with Neurobiotin-injected ganglion cells, vGluT3 cells made contacts with midget and parasol ganglion cells, including both ON and OFF types. Puncta containing immunoreactive gephyrin, an inhibitory synapse marker, were found at appositions between vGluT3 cells and each of the four types of labeled ganglion cells. The vGluT3 cells did not have detectable levels of immunoreactive γ-aminobutyric acid (GABA) or immunoreactive glycine transporter 1. Thus, the vGluT3 cells would be expected to have ON responses to light and make synapses onto neurons in both the ON and the OFF pathways. Taken with previous results, these findings suggest that vGluT3 cells release glycine at some of their output synapses and glutamate at others.

We studied the expression of glycine receptor (GlyR) subunits and gephyrin in the adult rat retina. Reverse transcribed RNA was amplified by polymerase chain reaction (RT-PCR) with primers designed to recognize GlyR α1, α2, α3, β subunits, and gephyrin. Using RNA isolated from the whole retina, signals for all four GlyR subunits and gephyrin could be observed. In rod bipolar cells, in contrast, we detected a subset of GlyR subunits, α1 and β, and no gephyrin. Patch-clamp recording employing two subtype-specific blockers of the GlyR, picrotoxinin and cyanotriphenylborate (CTB), indicated that the GlyR in rod bipolar cells is a heteromeric protein composed of the α1 and β subunit. Moreover, the absence of detectable amounts of gephyrin mRNA suggests that the anchor protein is not required for the function of GlyRs in rod bipolar cells.

Vertical Slices of postnatal day 6 (P6) rat retina were cut and cultured using the roller-tube technique. The organotypic differentiation during a culture period of up to 30 days has been described in a previous study (Feigenspan et al., 1993a). Here we concentrated on the synaptic organization in the retinal slice culture. Electron microscopy revealed the presence of ribbon synapses in the outer plexiform layer and conventional and ribbon syanpses in the inner plexiform layer. Immunofluroscence with antibodies that recognize specific subunits of GABAA or glycine receptors revealed a punctuate distribution of the receptors. They were aggregated in “hot spots” that correspond to a concentration of receptors at postsynaptic sites. Different isoforms of GABAA and glycine receptors occured in the slice cultures. The experiments show that there is a differentiation of synapses and a diversity of transmitter receptors in the slice cultures that is comparable to the in vivo retina.

Being utilized by over 40% of the amacrine cells, glycine is considered to be a major inhibitory neurotransmitter in the retinas of all vertebrate species examined. Localization of gephyrin, which is a 93-kD peripheral membrane glycine receptor-associated anchoring protein, has been used in several studies to identify the sites of glycinergic interactions in the retina and other regions of the central nervous system. Recent studies have shown that gephyrin colocalizes with GABAA receptors which, like those for glycine, are also inhibitory amino acid receptors usually associated with a chloride channel. In the present study, we have used two antibodies which recognize either gephyrin (mAb7a), or the α and β subunits of the glycine receptor (mAb4a) in order to determine to what extent gephyrin is associated with glycine receptors in the mammalian retina. Single-label studies showed extensive punctate staining throughout most of the inner plexiform layer with each antibody. Double labeling showed that nearly 90% of the glycine receptor sites were also immunoreactive for gephyrin. However, nearly 60% of the total punctae immunoreactive for gephyrin were not stained for glycine receptors. This distinction was most pronounced in the most proximal inner plexiform layer where only 24% of the gephyrin-immunoreactive sites were glycine receptor positive. This study suggests that although most glycine receptors in the rabbit retina colocalize with the anchoring protein gephyrin, a significant proportion of the gephyrin-labeled sites are not associated with glycine receptors. In light of studies showing gephyrin association with GABAA receptor subunits, the localization of gephyrin may be indicative of chloride-mediated inhibitory amino acid transmission in general and not solely that of glycinergic. Given several studies which show that bipolar cells express glycine receptors and respond to glycine but do not express gephyrin, the 10% of glycine receptors not colocalized with gephyrin shown in the present study may represent a subtype of glycine receptors found on bipolar cells which do not require gephyrin for the functional clustering of receptor subunits.