Cacna1s encodes the α1S subunit (Cav1.1) of voltage-dependent calcium channels, and is required for normal skeletal and cardiac muscle function, where it couples with the ryanodine receptor to regulate muscle contraction. Recently CACNA1S was reported to be expressed on the tips of retinal depolarizing bipolar cells (DBCs) and colocalized with metabotropic glutamate receptor 6 (mGluR6), which is critical to DBC signal transduction. Further, in mGluR6 knockout mice, expression at this location is down regulated. We examined RNAseq data from mouse retina and found expression of a novel isoform of Cacna1s. To determine if CACNA1S was a functional component of the DBC signal transduction cascade, we performed immunohistochemistry to visualize its expression in several mouse lines that lack DBC function. Immunohistochemical staining with antibodies to CACNA1S show punctate labeling at the tips of DBCs in wild type (WT) retinas that are absent in Gpr179 nob5 mutant retinas and decreased in Grm6 −/− mouse retinas. CACNA1S and transient receptor potential cation channel, subfamily M, member 1 (TRPM1) staining also colocalized in WT retinas. Western blot analyses for CACNA1S of either retinal lysates or proteins after immunoprecipitation with the CACNA1S antibody failed to show the presence of bands expected for CACNA1S. Mass spectrometric analysis of CACNA1S immunoprecipitated proteins also failed to detect any peptides matching CACNA1S. Immunohistochemistry and western blotting after expression of GPR179 in HEK293T cells indicate that the CACNA1S antibody used here and in the retinal studies published to date, cross-reacts with GPR179. These data suggest caution should be exercised in conferring a role for CACNA1S in DBC signal transduction based solely on immunohistochemical staining.

Amacrine cells are a morphologically and functionally diverse group of inhibitory interneurons. Morphologically, they have been divided into approximately 30 types. Although this diversity is probably important to the fine structure and function of the retinal circuit, the amacrine cells have been more generally divided into two subclasses. Glycinergic narrow-field amacrine cells have dendrites that ramify close to their somas, cross the sublaminae of the inner plexiform layer, and create cross talk between its parallel ON and OFF pathways. GABAergic wide-field amacrine cells have dendrites that stretch long distances from their soma but ramify narrowly within an inner plexiform layer sublamina. These wide-field cells are thought to mediate inhibition within a sublamina and thus within the ON or OFF pathway. The postsynaptic targets of all amacrine cell types include bipolar, ganglion, and other amacrine cells. Almost all amacrine cells use GABA or glycine as their primary neurotransmitter, and their postsynaptic receptor targets include the most common GABAA, GABAC, and glycine subunit receptor configurations. This review addresses the diversity of amacrine cells, the postsynaptic receptors on their target cells in the inner plexiform layer of the retina, and some of the inhibitory mechanisms that arise as a result. When possible, the effects of GABAergic and glycinergic inputs on the visually evoked responses of their postsynaptic targets are discussed.

The presence of inhibitory bipolar cells in salamander retina was investigated by a comparative analysis of the distribution of glutamate- and GABA-immunoreactivities (GLU-IR; GABA-IR) using a postembedding immunocytochemical method. GLU-IR was found in virtually all photoreceptors, bipolar cells and ganglion cells, neuronal elements that transfer information vertically through the retina. GLU-IR also was found in numerous amacrine cells in the mid and proximal inner nuclear layer as well as in the cytoplasm of horizontal cells, while the nucleus of horizontal cells was either lightly labeled or not labeled at all. GLU-IR was found in the outer plexiform layer and intensely in the inner plexiform layer, in which there was no apparent sublamination. Forty-seven percent of Type IB bipolar cells in the distal inner nuclear layer and 13% of the displaced bipolar cells were GABA-IR. All bipolar cells were also GLU-IR, indicating that GABA-IR bipolar cells were a subset of GLU-IR bipolar cells rather than a separate population. About 12% of the Type IB bipolar cells were moderately GABA-IR and likely comprised a GABAergic subtype. GLU-IR levels in the presumed GABAergic bipolar cells were higher than in other purely GLU-IR bipolar cells suggesting that these GABA-IR bipolar cells are glutamatergic as well. All of the displaced bipolar cells were only lightly GABA-IR, indicating that displaced bipolar cells comprise a more homogeneous class of glutamatergic cell than orthotopic bipolar cells. GAD-IR co-localized with GABA-IR in orthotopic but not displaced bipolar cells, further supporting the idea that some orthotopic bipolar cells are GABAergic. A small proportion of bipolar cells in salamander retina contain relatively high levels of both GABA and glutamate. Co-release of these substances by bipolar cells could contribute to the “push-pull” modulation of ganglion cell responses.

Dopamine is one of the major neurotransmitters in the retina. It is released from amacrine and interplexiform cells into both inner (IPL) and outer (OPL) plexiform layers. Several dopaminergic actions are known to occur through D1 receptors (D1R) but the precise location of these receptors has not been established. An antibody that recognizes the intracytoplasmic C-terminal of the rat D1R was used to detect D1R, immunohistochemically, in rats (Wistar and RCS), mouse, hamster, and macaque monkey retinas. The OPL was heavily stained in each species, consistent with the known actions of dopamine on horizontal cells. Three to five bands were observed in the IPL, depending on species. Three were in the a sublayer, the outermost of which was close to the amacrine cell layer, and may represent the massive dopamine input to the AII rod-amacrine cells. As observed in mice, where bipolar cells are D1-immunoreactive, the band located in sublayer 3 of the IPL may contain cone-bipolar cell terminals. A band of D1R-immunoreactivity in the b sublayer of the IPL contains ON-bipolar cell terminals and a second site of interaction between dopaminergic cells and the AII amacrine cells. This sublayer was absent from the RCS rat retina, suggesting a severe impairment of the rod-driven pathway following rod degeneration in these mutant rats. Cells in the ganglion cell layer exhibited relatively heavy staining, and may be ganglion cells or displaced amacrine cells. Some extrasynaptic localizations of D1R in the retina are suggested.

This paper describes experiments on GABA-activated whole-cell membrane currents in bipolar cells freshly isolated from the adult rat retina. The main goal was to determine whether bipolar cell responses to GABA could be resolved in terms of mediation by the GABAA receptor, the GABAB receptor, or both. Bipolar cells were isolated by gentle enzymatic dissociation and identified by their distinct morphology. GABA agonists and antagonists were applied focally by pressure and the resultant currents were recorded under whole-cell voltage clamp. In all bipolar cells tested, GABA (0.1–100 μM) induced a monophasic response associated with a conductance increase (IGABA). The shift in reversal potential for IGABA as a function of pipet [CI] paralleled that predicted based on the Nernst equation for Cl−. IGABA was mimicked by muscimol (5–20 μM) and antagonized by bicuculline (20–100 μM). Baclofen (0.1–1.0 mM) produced no apparent conductance change. “Hot spots” of sensitivity to GABA which might be associated with regions of synaptic contact were not found; both the soma and processes of all bipolar cells were responsive to focally applied GABA. Furthermore, all bipolar cells tested responded to glycine.

In conclusion, we have established the presence of GABAA receptors on rat retinal bipolar cells. Our data suggest further that these cells lack GABAB receptors. Finally, our observation that bipolar cells in the rat retina are relatively homogeneous in terms of their sensitivity to GABA and glycine lead us to postulate that the functional significance of the presence of receptors and their distribution on a neuron may be dictated more by the topography of the presynaptic inputs than by its inherent chemosensitivity.

The neurotransmitter used by horizontal cells in mammals has not been identified. GABA has been the leading candidate, but doubt has remained because of failure to clearly demonstrate the GABA synthetic enzyme, glutamic acid decarboxylase (GAD) in these cells. Because GAD was recently shown to exist as two isoforms, 65 kDa and 67 kDa, we considered whether there might be a mismatch between the forms of GAD expressed in horizontal cells and the probes used to detect it. Accordingly, we stained sections of mammalian retina with antibodies specific for each isoform. Cat horizontal cells of both types (A and B) were immunoreactive for GAD67 but negative for GAD65; monkey horizontal cells of both types (H1 and H11) were positive for GAD65 and negative for GAD67. The findings reconcile previous, apparently conflicting, observations and strengthen considerably the hypothesis that mammalian horizontal cells are GABAergic.

Two cDNA clones for nicotinic acetylcholine receptor (nAChR) subunits sensitive to α-bungarotoxin (α-Bgt) have been isolated, the so-called α-Bgt binding proteins α1 (or α7 nAChR subunit) and α2 (or α8 nAChR subunit). Immunohistochemical experiments have shown that both α7 and α8 subunits, as well as subunits insensitive to α-Bgt (β2 and α3), are present in amacrine and ganglion cells of the chick retina. However, only the α8 subunit was observed in presumptive bipolar cells. The present study investigated in detail the pattern of distribution of the bipolar cells containing the α8 nAChR subunit and its relation to the pattern of distribution of bipolar cells immunoreactive to protein kinase C (PKC). Presumptive α8-and PKC-like immunoreactive (α8-LI and PKC-LI) bipolar cells were observed sending their dendrites to the outer plexiform layers and their axons to the inner plexiform layer. Where as α8-LI bipolar cells corresponded to 40–53% of the whole population of bipolar cells, PKC-LI bipolar cells represented only 6–8% of the same population. The soma sizes of the α8-LI bipolar cells were slightly smaller (mean ± s.d.; 4.9 ± 0.8 μm) than the soma sizes of the PKC-LI bipolar cells (5.4 ± 0.9 μm). Double-labeling experiments indicated that probably all PKC-LI bipolar cells also contain α8-LI. This indicates that two distinct groups of cholinoceptive bipolar cells exist in the chick retina, one that contains PKC-LI, and another one that does not.

The short wavelength-sensitive (blue) cone bipolar cells was found to have a nonrandom distribution by analyzing the nearest neighbors and by calculating the density recovery profile (DRP). Blue cones had been shown previously to have a nonrandom distribution (Curcio et al., 1991). The relationship between the two arrays was then analyzed by calculating the cross-correlational density recovery profile (cDRP), which indicates the local density of blue cones around each blue cone bipolar cell. Although both cell types appeared to be distributed uniformly at the macroscopic level, the cDRP was 1.7 times higher within 15 μm of each bipolar cell perikaryon than in the surrounding area. The area of higher density was approximately the same as that in which the blue cone bipolar cells made synaptic contacts with blue cones. The finding that the blue cones and the blue cone bipolar cells were closer together than expected suggests that the positions of the perikarya of these neurons were influenced by their synaptic connections or other developmental interactions.

The distribution of GABA-like immunoreactivity in the cat retina was studied through the use of preembedding immunocytochemistry for light microscopy and by postembedding immunogold techniques for electron microscopy. Staining was observed in both inner and outer plexiform layers. Approximately 30% of the somata in the amacrine portion of the inner nuclear layer were immunoreactive and included amacrine and interplexiform cells. Horizontal cells and a subpopulation of cone bipolar cells were also stained. In the ganglion cell layer, staining was observed in both small- and medium-sized neurons. GABA-labeled amacrine cells were presynaptic to somata of amacrine cells and to dendrites of amacrine, bipolar, and ganglion cells. Bipolar cells were a major target, receiving more than 60% of all labeled synapses in the inner plexiform layer. Many of these contacts were reciprocal synapses. These findings support a major role for GABA-labeled amacrines in providing feedback inhibition to bipolar cells in the inner retina.

Retinal bipolar cells exhibit a center-surround antagonistic receptive field to a light stimulus (Werblin & Dowling, 1969; Kaneko, 1970), and thus constitute an early stage of spatial information processing. We injected Lucifer Yellow and a small biotinylated tracer, biocytin, into bipolar cells of the teleost retina to examine electrical coupling in these cells. Lucifer-Yellow coupling was observed in one of 55 stained bipolar cells; the coupling pattern was one injected bipolar cell and three surrounding cells. Biocytin coupling was observed in 16 of 55 stained bipolar cells, six of which were ON center and ten OFF center. Although biocytin usually coupled to three to six bipolar cells, some OFF-center bipolar cells showed strong coupling to more than 20 cells. The biocytin-coupled bipolar cells were morphologically homologous. Membrane appositions resembling gap junctions were found between dendrites and between axon terminals of neighboring bipolar cells.

In the strongest biocytin-coupled bipolar cells, the contacts between bipolar cells and cone photoreceptor cells were examined after reconstruction of the dendritic trees of five well-stained, serially sectioned OFF-center bipolar cells. Each of these bipolar cells was in contact with different numbers of cones: 11 to 20 for twin cones and two to four for single cones. This implies that, although these bipolar cells belong to the same category, the signal inputs differ among bipolar cells. Numerical simulation conducted on a hexagonal array network model demonstrated that the electrical coupling of bipolar cells can decrease the difference in input (≈80%) without causing significant loss of spatial resolution. Our results suggest that electrical coupling of bipolar cells has the advantage of decreasing the dispersion of input signals from cones, and permits bipolar cells of the same class to respond to light with similar properties.

The mammalian rod bipolar, for which only one class has been identified, has been described as being hyperpolarizing by some investigators and depolarizing by others. We now report the effects of 2-amino-4-phosphonobutyrate (APB), a potent blocker of depolarizing bipolar cells, on visual behavior in the dark-adapted monkey. While in mesopic and photopic conditions only the monkeys' ability to detect incremental stimuli is impaired, under scotopic conditions all light mediated response in the monkey is eliminated. Assuming APB is acting on rod bipolars in the same fashion as it does on cone bipolars, we conclude that the primate rod bipolars all depolarize to light and that the ON and OFF channels are formed by the amacrine cell network.

The main route of information flow through the vertebrate retina is from the photoreceptors towards the ganglion cells whose axons form the optic nerve. Bipolar cells of the frog have been so far reported to contact mostly amacrine cells and the majority of input to ganglion cells comes from the amacrines. In this study, ganglion cells of frogs from two species (Bufo marinus, Xenopus laevis) were filled retrogradely with horseradish peroxidase. After visualization of the tracer, light-microscopic cross sections showed massive labeling of the somata in the ganglion cell layer as well as their dendrites in the inner plexiform layer. In cross sections, bipolar output and ganglion cell input synapses were counted in the electron microscope. Each synapse was assigned to one of the five equal sublayers (SLs) of the inner plexiform layer. In both species, bipolar cells were most often seen to form their characteristic synaptic dyads with two amacrine cells. In some cases, however, the dyads were directed to one amacrine and one ganglion cell dendrite. This type of synapse was unevenly distributed within the inner plexiform layer with the highest occurrence in SL2 both in Bufo and Xenopus. In addition, SL4 contained also a high number of this type of synapse in Xenopus. In both species, we found no or few bipolar to ganglion cell synapses in the marginal sublayers (SLs 1 and 5). In Xenopus, 22% of the bipolar cell output synapses went onto ganglion cells, whereas in Bufo this was only 10%. We conclude that direct bipolar to ganglion cell information transfer exists also in frogs although its occurrence is not as obvious and regular as in mammals. The characteristic distribution of these synapses, however, suggests that specific type of the bipolar and ganglion cells participate in this process. These contacts may play a role in the formation of simple ganglion cell receptive fields.

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.

The response to spatially focal flicker is enhanced by dim, spatially diffuse, rod-stimulating backgrounds. This effect is called suppressive rod-cone interaction (SRCI) as it reflects a tonic, suppressive influence of dark-adapted rods upon cone pathways which is removed by selective rod-light adaptation. SRCI is observed in amphibian retina with intracellular recordings from most cone-driven cells including the cones themselves, and is most obvious using stimuli flickering at frequencies too rapid for rods to follow. SRCI is blocked by glutamate analogs which selectively block the photic response of horizontal cells (HCs). In the presence of these agents, flicker responses from bipolar cells and cones are enhanced to levels normally seen only with selective rod-light adaptation. In the HCs themselves, SRCI is similarly blocked by lead chloride which blocks rod-, but not cone-related activity.

In amphibian and cat HCs and in human observers, SRCI is limited by a space constant of very similar value (between 100 and 150 μm). We suggest that SRCI in all three species is mediated by HCs: in amphibians, SRCI must at least partially reflect rod-modulation of HC feedback onto cones.

Polyclonal (7493 and 7317) and monoclonal (mAb3) antibodies, generated to the α subunit of the voltage-gated sodium channel (αNaCh), were employed to assess the cell types containing αNaCh-like immunoreactivity in the mature cat and monkey retina. Immunoblot analyses of retinal proteins in the cat revealed that the polyclonal and monoclonal antibodies we employed labeled a band in the 260–kDa region which corresponds to the molecular mass of the α subunit of the NaCh. In both the cat and monkey, these antibodies immunolabeled several distinct types of retinal cells. With the polyclonal antibodies immunoreactivity was observed in ganglion cells and their intraretinal axons, in horizontal cells, and unexpectedly, in cones. In addition, in both species, a limited number of heavily labeled profiles, presumed to be bipolar cells, were seen in the inner nuclear layer. In cat and monkey the monoclonal antibody labeled axons in the fiber layer, ganglion cell somata, and a continuous band of immunoreactive cell bodies (presumed bipolar cells) situated in the outer half of the inner nuclear layer. By immunolabeling isolated cells dissociated from the cat retina, it was possible to demonstrate unequivocally that a population of bipolar cells was labeled by the monoclonal and the polyclonal antibodies we employed. The differences in the labeling observed with the monoclonal antibody as compared to the polyclonal antibodies were interpreted as reflecting the presence of different α-subunit subtypes in the mammalian retina. Collectively, our findings suggest that αNaCh-like proteins are expressed by a more diverse population of retinal cells than expected on the basis of previous physiological and immunohistochemical studies.

There is some evidence that the mammalian rod bipolar cell expresses ionotropic glutamate receptors. This is surprising in light of the strong evidence that the glutamate released by the rod photoreceptor acts upon a metabotropic glutamate receptor-mGluRo-present in the dendrites of the rod bipolar cell. To reexamine the issue of which glutamate receptor subunits may be present on the rod bipolar cell, an immunohistochemical study of acutely dissociated retinal cells was undertaken. Two monoclonal antibodies provided some evidence that GluR2 and/or GluR4, as well as NMDAR1 subunit, are present on the rod bipolar cell. A monoclonal antibody directed against the N-terminus of GluR2 labeled the rod bipolar cells, but two antisera directed against the C-terminus of the same subunit did not. One possible explanation for this discrepancy could be that the rare splice variant GluR2-long, which is endowed with a different C-terminus, could be expressed by the rod bipolar cell. To explore this possibility, RT-PCR was used to amplify the transcripts encoding GluR2 in the neural retina. This revealed that GluR2-long transcripts, with the flop exon, are present.

Some neurochemical features of the neuronal circuitry regulating dopamine release were examined in the retina of the turtle, Pseudemys scripta elegans. Glutamate antagonists that block hyperpolarizing bipolar cells, such as 2,3 piperidine dicarboxylic acid (PDA), produced dose-dependent dopamine release. In contrast, the glutamate agonist 2-amino-4-phosphonobutyric acid (APB), which blocks depolarizing bipolar cell responses with high specificity, had no effect on the release of dopamine. The γ-aminobutyric acid (GABA) antagonist, bicuculline, also produced potent dose-dependent release of dopamine. The release of dopamine produced by PDA was blocked by exogenous GABA and muscimol, suggesting that the PDA-mediated release process was polysynaptic and involved a GABAergic synapse interposed between the bipolar and dopaminergic amacrine cells. The only other agents that produced dopamine release were chloride-free media and high extracellular K+; in particular, kainic acid and glutamate itself were ineffective. These results suggest that the primary neuronal chain mediating dopamine release in the turtle retina is: cone → hyperpolarizing bipolar cell → GABAergic amacrine cell → dopaminergic amacrine cell.

Spectral-sensitivity curves were derived from the a−, b−, and d−waves of rhesus monkey ERGs after injection of bicuculline, strychnine, or no drug. Without drug injection, the a− and d−wave curves were well-fit by an additive model of weighted photoreceptor absorption spectra, while the b−wave curve requires inhibitory terms to produce an adequate fit. Bicuculline, but not strychnine, reduced the weight assigned to the inhibitory terms in a dose-dependent fashion, to the point that no inhibition was evident. The results suggest that GABAergic synapses are required for the expression of red/green color opponency in primate bipolar cells.

Subtypes of bipolar cells were identified by protein kinase C (PKC)-like immunoreactivity in the goldfish retina. The PKC-like immunoreactivity was visualized by either the avidin/biotin peroxidase method or immunofluorescence method. In frozen cross sections and in wholemounts, the monoclonal antibody against α species of PKC reacted with ON-type bipolar cells, identified by the location of axon terminals in sublamina b of the inner plexiform layer. OFF-type bipolar cells (identified by the location of the axon terminal in sublamina a of the inner plexiform layer) were not immunoreactive. The immunoreactive cells consisted of two subtypes; the rod-dominant ON-type with a large soma and a large bulbous axon terminal, and the cone-dominant ON-type with a small soma and small axon terminal. Antibodies against β and γ species of PKC did not react with any bipolar cells. Of the isolated bipolar cells, enzymatically dissociated from the goldfish retina, 59% were immunoreactive to the monoclonal antibody against α species of PKC. The immunoreactive isolated cells also consisted of two morphological types. Each of them had a morphology typical either to rod-dominant ON-type or to cone-dominant ON-type.

We have used monolayer cultures prepared from early postnatal rabbit retinae (days 2–5) by the sandwich technique to study the capacity of immature neurons to express specific neuronal phenotypes in a homogeneous in vitro environment. Applying morphological, immunocytochemical, and autoradiographic criteria, we demonstrate that a variety of phenotypes could be distinguished after 7–14 days in vitro, and correlated with known retinal cell types. Bipolar cell-like neurons (approximately 4% of total cell number) were identified by cell type-specific monoclonal antibodies (115A10) and their characteristic bipolar morphology. Small subpopulations (about 1%) of GABA-immunoreactive neurons acquired elaborate morphologies strikingly similar to those of A- and B-type horizontal cells. Amongst putative amacrine cells several different subpopulations could be classified. GABA-immunoreactive amacrine-like neurons (6.5%), which also showed high affinity [3H]-GABA uptake, comprised cells of varying size and shape and could be subdivided into subpopulations with respect to their response to different glutamate receptor agonists (NMDA, kainic acid, quisqualic acid). In addition, a small percentage of [3H]-GABA accumulating cells with large dendritic fields showed tyrosine-hydroxylase immunoreactivity. Presumptive glycinergic amacrine cells (18.5%) were rather uniform in shape and had small dendritic fields. Release of [3H]-glycine from these neurons was evoked by kainic and quisqualic acid but not by NMDA. Small [3H]-glutamate accumulating neurons with few short processes were the most frequent cell type (73%). This cell type also exhibited opsin immunoreactivity and probably represented incompletely differentiated photoreceptor cells. Summing the numbers of characterized cells indicated that we were able to attribute a defined retinal phenotype to most, if not all of the cultured neurons. Thus, we have demonstrated that immature neuronal cells growing in monolayer cultures, in the absence of a structured environment, are capable of maintaining or producing specific morphological and functional properties corresponding to those expressed in vivo. These results stress the importance of intrinsic factors for the regulation of neuronal differentiation. On the other hand, morphological differentiation was far from perfect indicating the requirement for regulatory factors.