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.

With the ever-growing number of transgenic mice being used in vision research, a precise knowledge of the cellular organization of the mouse retina is required. As with the cat, rabbit, rat, and primate retinae, as many as 10 cone bipolar types and one rod bipolar type can be expected to exist in the mouse retina; however, they still have to be defined. In the current study, several immunocytochemical markers were applied to sections of mouse retina, and the labeling of bipolar cells was studied using confocal microscopy and electron microscopy. By using antibodies against the neurokinin-3 receptor NK3R; the plasma membrane calcium ATPase1 (PMCA1); and the calcium (Ca)-binding proteins CaB1, CaB5, caldendrin, and recoverin, three different OFF-cone bipolar cells could be identified. One type of ON-cone bipolar cell was identified through its immunoreactivity for CaB5 and PMCA1. Rod bipolar cells, comparable in morphology to those of other mammalian retinae, expressed protein kinase Calpha and CaB5. It was also shown that putative OFF-cone bipolar cells receive light signals through flat contacts at the cone pedicle base, whereas ON-cone bipolar signaling involves invaginating contacts. The distribution of the kainate receptor subunit GluR5 was studied by confocal and electron microscopy. GluR5 was expressed at flat bipolar cell contacts; however, it appears to be involved with only certain types of OFF-cone bipolar cells. This suggests that different bipolar cell types receive their light signals through different sets of glutamate receptors.

We studied the morphology of retinal ganglion cells in a diurnal New World primate, the marmoset Callithrix jacchus. This species is of interest as a model for primate vision because it has good behavioural visual acuity, and the retina and subcortical visual pathways are very similar to those of Old World monkeys and humans. Ganglion cells were labelled by placing small crystals of the carbocyanin dye DiI into the optic fibre layer, or by intracellular injection of neurobiotin. Two main classes of ganglion cell were labelled. We call these Group A cells and Group B cells: they are respectively homologous to parasol and midget cell classes. Group A and Group B cells show similar patterns of dye coupling, dendritic stratification and dendritic field size as their counterparts in Old World monkeys and humans. A third group of cells, which we call Group C, is morphologically heterogeneous. Examples corresponding to wide-field ganglion cell types described in Old World primates were encountered. One subgroup of C cells has a morphology very similar to that of the small bistratified (blue-on) cell described in macaque retina, suggesting that this functional pathway is common to all primates. As for other New World monkeys, the marmoset shows a sex-linked polymorphism of cone pigment expression, such that all males are dichromats and the majority of females are trichromats. No systematic differences in Group B cells were seen between male and female retinas, suggesting that trichromacy is not accompanied by specific changes in ganglion cell morphology.

We studied the relationship between the morphology of ganglion cells and the spatial density of photoreceptors in the retina of two Old World primates, human and macaque monkey; the diurnal New World marmoset Callithrix jacchus; and the cat. Ganglion cells in macaque and marmoset were labelled by intracellular injection with Neurobiotin or by DiI diffusion labelling in fixed tissue. Cone photoreceptor densities were measured from the same retinas. Supplemental data for macaque and data for human and cat were taken from published studies. For the primates studied, the central retina is characterised by a constant numerical convergence of cones to ganglion cells. Midget ganglion cells derive their input, via a midget bipolar cell, from a single cone. Parasol cells derive their input from 40-140 cones. Outside the central retina, the convergence increases with eccentricity. The convergence to beta cells in the cat retina is very close to that for parasol cells in primate retina. The convergence of rod photoreceptors to ganglion cells is similar in human, macaque, and marmoset, with parasol cells receiving input from 10-15 times more rods than midget cells. The low convergence of cones to midget cells in human and macaque retinas is associated with distinctive dendritic "clusters" in midget cells' dendritic fields. Convergence in marmoset is higher, and the clusters are absent. We conclude that the complementary changes in photoreceptor density and ganglion cell morphology should be considered when forming linking hypotheses between dendritic field, receptive field, and psychophysical properties of primate vision.

We have studied the components of the short wavelength-sensitive (SWS or "blue") cone pathway in the retina of a New World primate, the marmoset Callithrix jacchus. Of particular interest was the small bistratified ganglion cell, which has been identified in macaque monkey to be the morphological substrate of the blue-ON cell (Dacey and Lee [1994] Nature 367:731-735). Small bistratified cells were intracellularly filled with Neurobiotin in an in vitro retinal wholemount preparation. Their morphology, size, and level of dendritic stratification were similar to their homologues in macaque and human retina. A number of different antibodies were applied to vertical cryostat sections, some of which were cut through the processes of injected small bistratified or parasol ganglion cells. We used antibodies against cholecystokinin (CCK) to label blue cone bipolar cells, and antibodies against the human SWS cone photopigment to label SWS cones. Double-labelled preparations showed that blue cone bipolar cell dendrites contact SWS cone pedicles, and the inner dendrites of the small bistratified cell are costratified with the axon terminals of blue cone bipolar cells. A monoclonal antibody against calbindin was used to label a subpopulation of bipolar cells that stratifies in the outer half of the inner plexiform layer. The axon terminals of these bipolar cells occasionally cross the outer dendrites of small bistratified cells and show extensive costratification with the dendrites of OFF parasol cells. We conclude that an SWS cone pathway with similar connectivity is a preserved feature of the primate visual system.

Rod bipolar (RB) cells of the mammalian retina release glutamate in a graded, light-dependent fashion from 20 to 40 ribbon synapses (dyads). At the dyads, two classes of amacrine cells, the AI and AII cells, are the postsynaptic partners. We examined the glutamate receptors (GluRs) that are expressed by AI and AII cells using immunocytochemistry with specific antibodies against GluR subunits. Sections of macaque monkey and rabbit retina were examined by confocal microscopy. AII amacrine cells were selectively labeled for calretinin, and AI cells in rabbits were labeled for 5-HT uptake. Thus, double- and triple-labeling for these markers and GluR subunits was possible. Electron microscopy using postembedding immunocytochemistry and double-labeling was applied to show the synaptic expression of GluRs. We also studied the synaptic localization of the two postsynaptic density proteins PSD-95 and glutamate receptor-interacting protein (GRIP). We found that AII amacrine cells express the AMPA receptor subunits GluR2/3 and GluR4 at the RB cell dyads, and they are clustered together with PSD-95. In contrast, AI amacrine cells express the delta1/2 subunits that appear to be associated with kainate receptor subunits and to be clustered together with GRIP. The RB cell dyad is therefore a synapse that initiates two functionally and molecularly distinct pathways: a "through conducting" pathway based on AMPA receptors and a modulatory pathway mediated by a combination of delta1/2 subunits and kainate receptors.

Two types of cone bipolar cells, the blue cone bipolar cell and the diffuse bipolar cell (DB3), were labelled immunohistochemically and investigated in the retina of a New World monkey, the marmoset. Blue cone bipolar cells were labelled with an antiserum against cholecystokinin. Short-wavelength-sensitive (SWS) cones were labelled with an antiserum against the SWS cone opsin. The DB3 cells were labelled with antibodies to calbindin. Blue cone bipolar cells in marmoset do not form a regular mosaic but instead follow the random distribution of the SWS cones. Nevertheless, the SWS cone to blue cone bipolar cell connectivity in marmoset is very similar to that previously described for macaque. In contrast to the blue cone bipolar cells, the DB3 cells form a regular mosaic. The synaptic connectivity of DB3 cells in the inner plexiform layer was analyzed. They make output synapses onto ganglion cells and amacrine cells, and gap junctions with each other. Our results provide further evidence for the existence of parallel bipolar cell pathways in the primate retina and support the view that the retinae of Old World and New World primates have common neuronal connectivity. The random distribution of SWS cones and blue cone bipolar cells is an exception to the general rule of a regular mosaic distribution of cell populations in the retina.

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