We studied the morphology of bipolar cells in fixed vertical tissue sections (slices) of the mouse retina by injecting the cells with Lucifer Yellow and Neurobiotin. Nine different cone bipolar cell types and one rod bipolar cell type were distinguished. The major criteria for classifying the cells were the branching pattern and stratification level of their axon terminals in the inner plexiform layer (IPL). To assess this, the IPL was subdivided into five strata of equal width. The slices were immunostained for calretinin, which labels three horizontal bands serving as a standard measure for the precise localization of the axon terminals. Immunostaining the retina with antibodies against the G-protein Ggamma13, a marker for ON-bipolar cells, made it possible to separate OFF- and ON-bipolar cells. At least two OFF-cone bipolar cells (Types 1 and 2) were immunolabeled with antibodies against the neurokinin 3 receptors (NK3R). A further OFF- and an ON-cone bipolar cell (Types 3 and 5) were immunostained with antibodies against the calcium-binding protein CaB5. The bipolar cell types described here were compared with previous schemes of rat and primate bipolar cells. Homologous types between the three species are discussed.

gamma-Aminobutyric acid (GABA), a major inhibitory neurotransmitter in the mammalian nervous system, is known to operate bicuculline-sensitive Cl- channels through GABAA receptors and bicuculline-insensitive cation channels through GABAB receptors. Recent observations indicate that the retina may contain GABA receptors with unusual pharmacological properties. Here we report that GABA gates bicuculline-insensitive Cl- channels in rod bipolar cells of the rat retina, which were not modulated by flunitrazepam, pentobarbital and alphaxalone and were only slightly blocked by picrotoxinin. Moreover, the GABAB receptor agonist baclofen, and the antagonist 2-hydroxysaclofen had no effect. The underlying single-channel conductance was 7 pS and the open time 150 ms. These values are clearly different from those obtained for GABAA receptor channels recorded in other neurons of the same preparation, and in other parts of the brain. The bicuculline- and baclofen-insensitive GABA receptors were activated selectively by the GABA analogue cis-4-aminocrotonic acid (CACA). Hence they may be similar to those receptors termed GABAC receptors.

In the mammalian retina, rods feed into the cone pathway through electrotonic coupling, and recent histological data suggest the involvement of connexin36 (Cx36) in this pathway. We therefore generated Cx36 null mice and monitored the functional consequences of this deficiency on early visual transmission. The homozygous mutant mice had a normally developed retina and showed no changes in the cellular organization of the rod pathway. In contrast, the functional coupling between AII amacrine cells and bipolar cells was impaired. Recordings of electroretinograms revealed a significant decrease of the scotopic b-wave in mutant animals and an increased cone threshold that is compatible with a distorted, gap junctional transmission between AII amacrine cells and cone bipolar cells. Recordings of visual evoked potentials showed extended latency in mutant mice but unaffected ON and OFF components. Our results demonstrate that Cx36-containing gap junctions are essential for normal synaptic transmission within the rod pathway.

Dopaminergic, interplexiform amacrines (DA cells) were labeled in transgenic mice with human placental alkaline phosphatase, an enzyme that resides on the outer surface of the cell membrane. It was therefore possible to investigate their activity in vitro after dissociation of the retina with whole-cell current and voltage clamp, as well as their connections in the intact retina with the electron microscope. DA cells generate action potentials even in the absence of synaptic inputs. This activity is abolished by the amacrine cell transmitters GABA and glycine, which induce an inward current carried by chloride ions, and is stimulated by kainate, an agonist at the receptor for the bipolar cell transmitter glutamate, which opens nonselective cation channels. Since DA cells are postsynaptic to amacrine and bipolar cells, we suggest that the spontaneous discharge of DA cells is inhibited in the dark by GABAergic amacrines that receive their input from off-bipolars. Upon illumination, the GABA-inhibition is removed, DA cells generate action potentials, and their firing is modulated by the excitation received from on-bipolars.

We have studied the expression pattern of neuronal connexin36 (Cx36) in the mouse and rat retina. In vertical sections of both retinas, a polyclonal antibody directed against Cx36 produced punctate labeling in the inner plexiform layer (IPL). Intense immunoreactivity was localized to the entire OFF sublamina of the IPL, and much weaker staining could be observed in the ON sublamina. Double-labeling experiments in the rat retina with antibodies directed against parvalbumin indicate that Cx36 is expressed on dendrites of AII amacrine cells. Cx36-like immunoreactivity in sublamina a of the IPL did not overlap with lobular appendages or cell bodies of AII amacrine cells. In a mouse retinal slice preparation, AII amacrine and ON cone bipolar cells were intracellularly injected with Neurobiotin and counterstained with antibody against Cx36. Punctate labeling appeared to be in register with dendritic arborization of AII amacrines and cone bipolar cells in the ON sublamina of the IPL. Whereas AII amacrine cells isolated from the rat retina clearly displayed Cx36-like immunoreactivity, isolated ON cone bipolar cells were negative for Cx36. Axon terminals of rod bipolar cells were decorated with Cx36-positive contacts but did not express Cx36 themselves.These results indicate that Cx36 is expressed by AII amacrine cells in homologous and heterologous gap junctions made with AII amacrines and cone bipolar cells, respectively. The heterologous gap junctions appear to be heterotypic, because ON cone bipolar cells do not express Cx36.