Evolution of neuronal cell classes and types in the vertebrate retina.

The basic plan of the retina is conserved across vertebrates, yet species differ profoundly in their visual needs1. Retinal cell types may have evolved to accommodate these varied needs, but this has not been systematically studied. Here we generated and integrated single-cell transcriptomic atlases of the retina from 17 species: humans, two non-human primates, four rodents, three ungulates, opossum, ferret, tree shrew, a bird, a reptile, a teleost fish and a lamprey. We found high molecular conservation of the six retinal cell classes (photoreceptors, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells (RGCs) and Müller glia), with transcriptomic variation across species related to evolutionary distance. Major subclasses were also conserved, whereas variation among cell types within classes or subclasses was more pronounced. However, an integrative analysis revealed that numerous cell types are shared across species, based on conserved gene expression programmes that are likely to trace back to an early ancestral vertebrate. The degree of variation among cell types increased from the outer retina (photoreceptors) to the inner retina (RGCs), suggesting that evolution acts preferentially to shape the retinal output. Finally, we identified rodent orthologues of midget RGCs, which comprise more than 80% of RGCs in the human retina, subserve high-acuity vision, and were previously believed to be restricted to primates2. By contrast, the mouse orthologues have large receptive fields and comprise around 2% of mouse RGCs. Projections of both primate and mouse orthologous types are overrepresented in the thalamus, which supplies the primary visual cortex. We suggest that midget RGCs are not primate innovations, but are descendants of evolutionarily ancient types that decreased in size and increased in number as primates evolved, thereby facilitating high visual acuity and increased cortical processing of visual information.

Comprehensive Classification of Retinal Bipolar Neurons by Single-Cell Transcriptomics.

Patterns of gene expression can be used to characterize and classify neuronal types. It is challenging, however, to generate taxonomies that fulfill the essential criteria of being comprehensive, harmonizing with conventional classification schemes, and lacking superfluous subdivisions of genuine types. To address these challenges, we used massively parallel single-cell RNA profiling and optimized computational methods on a heterogeneous class of neurons, mouse retinal bipolar cells (BCs). From a population of ∼25,000 BCs, we derived a molecular classification that identified 15 types, including all types observed previously and two novel types, one of which has a non-canonical morphology and position. We validated the classification scheme and identified dozens of novel markers using methods that match molecular expression to cell morphology. This work provides a systematic methodology for achieving comprehensive molecular classification of neurons, identifies novel neuronal types, and uncovers transcriptional differences that distinguish types within a class.

Dopamine D1 receptor expression is bipolar cell type-specific in the mouse retina.

In the retina, dopamine is a key molecule for daytime vision. Dopamine is released by retinal dopaminergic amacrine cells and transmits signaling either by conventional synaptic or by volume transmission. By means of volume transmission, dopamine modulates all layers of retinal neurons; however, it is not well understood how dopamine modulates visual signaling pathways in bipolar cells. Here we analyzed Drd1a-tdTomato BAC transgenic mice and found that the dopamine D1 receptor (D1R) is expressed in retinal bipolar cells in a type-dependent manner. Strong tdTomato fluorescence was detected in the inner nuclear layer and localized to type 1, 3b, and 4 OFF bipolar cells and type 5-2, XBC, 6, and 7 ON bipolar cells. In contrast, type 2, 3a, 5-1, 9, and rod bipolar cells did not express Drd1a-tdTomato. Other interneurons were also found to express tdTomato including horizontal cells and a subset (25%) of AII amacrine cells. Diverse visual processing pathways, such as color or motion-coded pathways, are thought to be initiated in retinal bipolar cells. Our results indicate that dopamine sculpts bipolar cell performance in a type-dependent manner to facilitate daytime vision. J. Comp. Neurol. 524:2059-2079, 2016. © 2015 Wiley Periodicals, Inc.

Connectivity between the OFF bipolar type DB3a and six types of ganglion cell in the marmoset retina.

Parallel visual pathways originate at the first synapse in the retina, where cones make connections with cone bipolar cells that in turn contact ganglion cells. There are more ganglion cell types than bipolar types, suggesting that there must be divergence from bipolar to ganglion cells. Here we analyze the contacts between an OFF bipolar type (DB3a) and six ganglion cell types in the retina of the marmoset monkey (Callithrix jacchus). Ganglion cells were transfected via particle-mediated gene transfer of an expression plasmid for the postsynaptic density 95-green fluorescent protein (PSD95-GFP), and DB3a cells were labeled via immunohistochemistry. Ganglion cell types that fully or partially costratified with DB3a cells included OFF parasol, OFF midget, broad thorny, recursive bistratified, small bistratified, and large bistratified cells. On average, the number of DB3a contacts to parasol cells (18 contacts per axon terminal) is higher than that to other ganglion cell types (between four and seven contacts). We estimate that the DB3a output to OFF parasol cells accounts for at least 30% of the total DB3a output. Furthermore, we found that OFF parasol cells receive approximately 20% of their total bipolar input from DB3a cells, suggesting that other diffuse bipolar types also provide input to OFF parasol cells. We conclude that DB3a cells preferentially contact OFF parasol cells but also provide input to other ganglion cell types.

The projective field of retinal bipolar cells and its modulation by visual context.

The receptive field of a sensory neuron spells out all the receptor inputs it receives. To understand a neuron's role in the circuit, one also needs to know its projective field, namely the outputs it sends to all downstream cells. Here we present the projective fields of the primary excitatory neurons in a sensory circuit. We stimulated single bipolar cells of the salamander retina and recorded simultaneously from a population of ganglion cells. Individual bipolar cell signals diverge through polysynaptic pathways into ganglion cells of many different types and over surprisingly large distance. However, the strength and polarity of the projection depend on the cell types involved. Furthermore, visual stimulation strongly modulates the bipolar cell projective field, in opposite direction for different cell types. In this way, the context from distant parts of the visual field can control the routing of signals in the inner retina.

Sensory experience shapes the development of the visual system's first synapse.

Specific connectivity patterns among neurons create the basic architecture underlying parallel processing in our nervous system. Here we focus on the visual system's first synapse to examine the structural and functional consequences of sensory deprivation on the establishment of parallel circuits. Dark rearing reduces synaptic strength between cones and cone bipolar cells, a previously unappreciated effect of sensory deprivation. In contrast, rod bipolar cells, which utilize the same glutamate receptor to contact rods, are unaffected by dark rearing. Underlying the physiological changes, we find the localization of metabotropic glutamate receptors within cone bipolar, but not rod bipolar, cell dendrites is a light-dependent process. Furthermore, although cone bipolar cells share common cone partners, each bipolar cell type that we examined depends differentially on sensory input to achieve mature connectivity. Thus, visual experience differentially affects maturation of rod versus cone pathways and of cell types within the cone pathway.

The first stage of cardinal direction selectivity is localized to the dendrites of retinal ganglion cells.

Inferring the direction of image motion is a fundamental component of visual computation and essential for visually guided behavior. In the retina, the direction of image motion is computed in four cardinal directions, but it is not known at which circuit location along the flow of visual information the cardinal direction selectivity first appears. We recorded the concerted activity of the neuronal circuit elements of single direction-selective (DS) retinal ganglion cells at subcellular resolution by combining GCaMP3-functionalized transsynaptic viral tracing and two-photon imaging. While the visually evoked activity of the dendritic segments of the DS cells were direction selective, direction-selective activity was absent in the axon terminals of bipolar cells. Furthermore, the glutamate input to DS cells, recorded using a genetically encoded glutamate sensor, also lacked direction selectivity. Therefore, the first stage in which extraction of a cardinal motion direction occurs is the dendrites of DS cells.

ON cone bipolar cell axonal synapses in the OFF inner plexiform layer of the rabbit retina.

Analysis of the rabbit retinal connectome RC1 reveals that the division between the ON and the OFF inner plexiform layer (IPL) is not structurally absolute. ON cone bipolar cells make noncanonical axonal synapses onto specific targets and receive amacrine cell synapses in the nominal OFF layer, creating novel motifs, including inhibitory crossover networks. Automated transmission electron microscopic imaging, molecular tagging, tracing, and rendering of ~400 bipolar cells reveals axonal ribbons in 36% of ON cone bipolar cells, throughout the OFF IPL. The targets include γ-aminobutyrate (GABA)-positive amacrine cells (γACs), glycine-positive amacrine cells (GACs), and ganglion cells. Most ON cone bipolar cell axonal contacts target GACs driven by OFF cone bipolar cells, forming new architectures for generating ON-OFF amacrine cells. Many of these ON-OFF GACs target ON cone bipolar cell axons, ON γACs, and/or ON-OFF ganglion cells, representing widespread mechanisms for OFF to ON crossover inhibition. Other targets include OFF γACs presynaptic to OFF bipolar cells, forming γAC-mediated crossover motifs. ON cone bipolar cell axonal ribbons drive bistratified ON-OFF ganglion cells in the OFF layer and provide ON drive to polarity-appropriate targets such as bistratified diving ganglion cells (bsdGCs). The targeting precision of ON cone bipolar cell axonal synapses shows that this drive incidence is necessarily a joint distribution of cone bipolar cell axonal frequency and target cell trajectories through a given volume of the OFF layer. Such joint distribution sampling is likely common when targets are sparser than sources and when sources are coupled, as are ON cone bipolar cells.

Paired-pulse plasticity in the strength and latency of light-evoked lateral inhibition to retinal bipolar cell terminals.

Synapses in the inner plexiform layer of the retina undergo short-term plasticity that may mediate different forms of adaptation to regularities in light stimuli. Using patch-clamp recordings from axotomized goldfish Mb bipolar cell (BC) terminals with paired-pulse light stimulation, we isolated and quantified the short-term plasticity of GABAergic lateral IPSCs (L-IPSCs). Bright light stimulation evoked ON and OFF L-IPSCs in axotomized BCs, which had distinct onset latencies (∼50-80 and ∼70-150 ms, respectively) that depended on background light adaptation. We observed plasticity in both the synaptic strength and latency of the L-IPSCs. With paired light stimulation, latencies of ON L-IPSCs increased at paired-pulse intervals (PPIs) of 50 and 300 ms, whereas OFF L-IPSC latencies decreased at the 300 ms PPI. ON L-IPSCs showed paired-pulse depression at intervals <1 s, whereas OFF L-IPSCs showed depression at intervals ≤1 s and amplitude facilitation at longer intervals (1-2 s). This biphasic form of L-IPSC plasticity may underlie adaptation and sensitization to surround temporal contrast over multiple timescales. Block of retinal signaling at GABA(A)Rs and AMPARs differentially affected ON and OFF L-IPSCs, confirming that these two types of feedback inhibition are mediated by distinct and convergent retinal pathways with different mechanisms of plasticity. We propose that these plastic changes in the strength and timing of L-IPSCs help to dynamically shape the time course of glutamate release from ON-type BC terminals. Short-term plasticity of L-IPSCs may thus influence the strength, timing, and spatial extent of amacrine and ganglion cell inhibitory surrounds.

Synaptic vesicle cycling is not impaired in a glutamatergic and a cholinergic synapse that exhibit deficits in acidification and filling

The purpose of the present work was to investigate synaptic vesicle trafficking when vesicles exhibit alterations in filling and acidification in two different synapses: a cholinergic frog neuromuscular junction and a glutamatergic ribbon-type nerve terminal in the retina. These synapses display remarkable structural and functional differences, and the mechanisms regulating synaptic vesicle cycling might also differ between them. The lipophilic styryl dye FM1-43 was used to monitor vesicle trafficking. Both preparations were exposed to pharmacological agents that collapse ΔpH (NH4Cl and methylamine) or the whole ΔµH+ (bafilomycin), a necessary situation to provide the driving force for neurotransmitter accumulation into synaptic vesicles. The results showed that FM1-43 loading and unloading in neuromuscular junctions did not differ statistically between control and experimental conditions (P > 0.05). Also, FM1-43 labeling in bipolar cell terminals proved highly similar under all conditions tested. Despite remarkable differences in both experimental models, the present findings show that acidification and filling are not required for normal vesicle trafficking in either synapse.

O objetivo do presente trabalho foi investigar o tráfego de vesículas sinápticas quando estas apresentam alterações no armazenamento de neurotransmissores e acidificação em duas distintas sinapses: a junção neuromuscular colinérgica de rãs versus o terminal nervoso glutamatérgico do tipo ribbon em céulas bipolares da retina. Essas sinapses exibem notáveis diferenças estruturais e funcionais e os mecanismos de regulação de ciclo das vesículas sinápticas podem ser diferentes entre eles. Para monitorar o tráfego de vesícula, foi utilizado o marcador lipofílico FM1-43. Ambas as preparações foram expostas a agentes farmacológicos que provocam o colapso de ΔpH (NH4Cl e metilamina) ou de todo ΔµH+ (bafilomicina), gradientes necessários para o acúmulo de neurotransmissores em vesículas sinápticas. Nossos resultados demonstram que a marcação e desmarcação de FM1-43 nas junções neuromusculares não foi estatisticamente diferente entre as diversas condições experimentais (P > 0,05). Além disso, a marcação de FM1-43 em terminais sinápticos de células bipolares foram bastante semelhantes em todas as condições testadas. Apesar das diferenças marcantes em ambos os modelos experimentais, nossos achados demonstram que a acidificação e o preenchimento de vesículas sinápticas não são necessários para o tráfico normal da vesícula nas sinapses estudadas.

Light-evoked lateral GABAergic inhibition at single bipolar cell synaptic terminals is driven by distinct retinal microcircuits.

Inhibitory amacrine cells (ACs) filter visual signals crossing the retina by modulating the excitatory, glutamatergic output of bipolar cells (BCs) on multiple temporal and spatial scales. Reciprocal feedback from ACs provides focal inhibition that is temporally locked to the activity of presynaptic BC activity, whereas lateral feedback originates from ACs excited by distant BCs. These distinct feedback mechanisms permit temporal and spatial computation at BC terminals. Here, we used a unique preparation to study light-evoked IPSCs recorded from axotomized terminals of ON-type mixed rod/cone BCs (Mb) in goldfish retinal slices. In this preparation, light-evoked IPSCs could only reach axotomized BC terminals via the lateral feedback pathway, allowing us to study lateral feedback in the absence of overlapping reciprocal feedback components. We found that light evokes ON and OFF lateral IPSCs (L-IPSCs) in Mb terminals having different temporal patterns and conveyed via distinct retinal pathways. The relative contribution of rods versus cones to ON and OFF L-IPSCs was light intensity dependent. ACs presynaptic to Mb BC terminals received inputs via AMPA/KA- and NMDA-type receptors in both the ON and OFF pathways, and used TTX-sensitive sodium channels to boost signal transfer along their processes. ON and OFF L-IPSCs, like reciprocal feedback IPSCs, were mediated by both GABA(A) and GABA(C) receptors. However, our results suggest that lateral and reciprocal feedback do not cross-depress each other, and are therefore mediated by distinct populations of ACs. These findings demonstrate that retinal inhibitory circuits are highly specialized to modulate BC output at different light intensities.

In vivo evidence that retinal bipolar cells generate spikes modulated by light.

Retinal bipolar cells have been assumed to generate purely graded responses to light. To test this idea we imaged the presynaptic calcium transient in live zebrafish. We found that ON, OFF, transient and sustained bipolar cells are all capable of generating fast 'all-or-none' calcium transients modulated by visual stimulation.

Chromatic bipolar cell pathways in the mouse retina.

Like most mammals, mice feature dichromatic color vision based on short (S) and middle (M) wavelength-sensitive cone types. It is thought that mammals share a retinal circuit that in dichromats compares S- and M-cone output to generate blue/green opponent signals, with bipolar cells (BCs) providing separate chromatic channels. Although S-cone-selective ON-BCs (type 9 in mouse) have been anatomically identified, little is known about their counterparts, the M-cone-selective OFF-BCs. Here, we characterized cone connectivity and light responses of selected mouse BC types using immunohistochemistry and electrophysiology. Our anatomical data indicate that four (types 2, 3a/b, and 4) of the five mouse OFF-BCs indiscriminately contact both cone types, whereas type 1 BCs avoid S-cones. Light responses showed that the chromatic tuning of the BCs strongly depended on their position along the dorsoventral axis because of the coexpression gradient of M- and S-opsin found in mice. In dorsal retina, where coexpression is low, most type 2 cells were green biased, with a fraction of cells (≈ 14%) displaying strongly blue-biased responses, likely reflecting S-cone input. Type 1 cells were also green biased but did not comprise blue-biased "outliers," consistent with type 1 BCs avoiding S-cones. We therefore suggest that type 1 represents the green OFF pathway in mouse. In addition, we confirmed that type 9 BCs display blue-ON responses. In ventral retina, all BC types studied here displayed similar blue-biased responses, suggesting that color vision is hampered in ventral retina. In conclusion, our data support an antagonistically organized blue/green circuit as the common basis for mammalian dichromatic color vision.

Bipolar cells of the ground squirrel retina.

Parallel processing of an image projected onto the retina starts at the first synapse, the cone pedicle, and each cone feeds its light signal into a minimum of eight different bipolar cell types. Hence, the morphological classification of bipolar cells is a prerequisite for analyzing retinal circuitry. Here we applied common bipolar cell markers to the cone-dominated ground squirrel retina, studied the labeling by confocal microscopy and electron microscopy, and compared the resulting bipolar cell types with those of the mouse (rod dominated) and primate retina. Eight different cone bipolar cell types (three OFF and five ON) and one rod bipolar cell were distinguished. The major criteria for classifying the cells were their immunocytochemical identity, their dendritic branching pattern, and the shape and stratification level of their axons in the inner plexiform layer (IPL). Immunostaining with antibodies against Gγ13, a marker for ON bipolar cells, made it possible to separate OFF and ON bipolars. Recoverin-positive OFF bipolar cells partly overlapped with ON bipolar axon terminals at the ON/OFF border of the IPL. Antibodies against HCN4 labeled the S-cone selective (bb) bipolar cell. The calcium-binding protein CaB5 was expressed in two OFF and two ON cone bipolar cell types, and CD15 labeled a widefield ON cone bipolar cell comparable to the DB6 in primate.

Ret-PCP2 colocalizes with protein kinase C in a subset of primate ON cone bipolar cells.

Purkinje cell protein 2 (PCP2), a member of the family of guanine dissociation inhibitors and a strong interactor with the G-protein subunit G alpha(o), localizes to retinal ON bipolar cells. The retina-specific splice variant of PCP2, Ret-PCP2, accelerates the light response of rod bipolar cells by modulating the mGluR6 transduction cascade. All ON cone bipolar cells express mGluR6 and G alpha(o), but only a subset expresses Ret-PCP2. Here we test the hypothesis that Ret-PCP2 contributes to shaping the various temporal bandwidths of ON cone bipolar cells in monkey retina. We found that the retinal splice variants in monkey and mouse are similar and longer than the cerebellar variants. Ret-PCP2 is strongly expressed by diffuse cone bipolar type 4 cells (DB4; marked with anti-PKCalpha) and weakly expressed by midget bipolar dendrites (labeled by antibodies against G alpha(o), G gamma 13, or mGluR6). Ret-PCP2 is absent from diffuse cone bipolar type 6 (DB6; marked with anti-CD15) and blue cone bipolar cells (marked with anti-CCK precursor). Thus, cone bipolar cells that terminate in stratum 3 of the inner plexiform layer (DB4) express more Ret-PCP2 than those that terminate in strata 3 + 4 (midget bipolar cells), and these in turn express more than those that terminate in stratum 5 (DB6 and blue cone bipolar cells). This expression pattern approximates the arborization of ganglion cells (GC) with different temporal bandwidths: parasol GCs stratifying near stratum 3 are faster than midget GCs stratifying in strata 3 + 4, and these are probably faster than the sluggish GCs that arborize in stratum 5.

ON inputs to the OFF layer: bipolar cells that break the stratification rules of the retina.

The vertebrate retina is a distinctly laminar structure. Functionally, the inner plexiform layer, in which bipolar cells synapse onto amacrine and ganglion cells, is subdivided into two sublaminae. Cells that depolarize at light offset ramify in sublamina a; those that depolarize at light onset ramify in sublamina b. The separation of ON and OFF pathways appears to be a fundamental principle of retinal organization that is reflected throughout the entire visual system. We show three clear exceptions to this rule, in which the axons of calbindin-positive ON cone bipolar cells make ribbon synapses as they pass through the OFF layers with three separate cell types: (1) dopaminergic amacrine cells, (2) intrinsically photosensitive ganglion cells, and (3) bistratified diving ganglion cells. The postsynaptic location of the AMPA receptor GluR4 at these sites suggests that ON bipolar cells can make functional synapses as their axons pass through the OFF layers of the inner plexiform layer. These findings resolve a long-standing question regarding the anomalous ON inputs to dopaminergic amacrine cells and suggest that certain ON bipolar cell axons can break the stratification rules of the inner plexiform layer by providing significant synaptic output before their terminal specializations. These outputs are not only to dopaminergic amacrine cells but also to at least two ON ganglion cell types that have dendrites that arborize in sublamina a.

Cone contacts, mosaics, and territories of bipolar cells in the mouse retina.

We report a quantitative analysis of the different bipolar cell types of the mouse retina. They were identified in wild-type mice by specific antibodies or in transgenic mouse lines by specific expression of green fluorescent protein or Clomeleon. The bipolar cell densities, their cone contacts, their dendritic coverage, and their axonal tiling were measured in retinal whole mounts. The results show that each and all cones are contacted by at least one member of any given type of bipolar cell (not considering genuine blue cones). Consequently, each cone feeds its light signals into a minimum of 10 different bipolar cells. Parallel processing of an image projected onto the retina, therefore, starts at the first synapse of the retina, the cone pedicle. The quantitative analysis suggests that our proposed catalog of 11 cone bipolar cells and one rod bipolar cell is complete, and all major bipolar cell types of the mouse retina appear to have been discovered.

Modulation by BNP of GABA receptors on ON-type rod bipolar cells is dependent on subcellular sites.

Brain natriuretic peptide (BNP) suppresses GABA(A) receptor-mediated current of ON-type rod-dominant bipolar cells (RBCs) in the rat retina. Here we report that such BNP-induced modulation is dependent on subcellular sites. Whole-cell currents could be induced by GABA focally applied to both dendrites/somata and axon terminals of isolated ON-type RBCs. Whilst the GABA currents induced at the axon terminal were significantly suppressed by BNP (50 nM), those at the dendrites/somata were hardly changed or slightly suppressed. Similar results were obtained when such experiments were performed in rat retinal slices. Calcium imaging showed that application of BNP (50 nM) caused a prominent increase in intracellular calcium concentrations ([Ca(2+)](i)) at the axon terminal, and the increase monotonically decayed when the acting site of BNP was moved away from the axon terminal along the cell: more distant, less significant. No detectable increase in [Ca(2+)](i) was found at the dendrites. Such increase in [Ca(2+)](i) could be completely blocked by pre-incubation with anantin, an antagonist of the NP-receptor-A (NPR-A). On the other hand, caffeine, an agonist of the ryanodine receptor, caused a similar subcellular site-dependent changes in [Ca(2+)](i), thus mimicking the BNP effect. All these results suggest that BNP-induced modulation of the activity of GABA receptors may be largely restricted to the inner retina.

Development of presynaptic inhibition onto retinal bipolar cell axon terminals is subclass-specific.

Synaptic integration is modulated by inhibition onto the dendrites of postsynaptic cells. However, presynaptic inhibition at axonal terminals also plays a critical role in the regulation of neurotransmission. In contrast to the development of inhibitory synapses onto dendrites, GABAergic/glycinergic synaptogenesis onto axon terminals has not been widely studied. Because retinal bipolar cells receive subclass-specific patterns of GABAergic and glycinergic presynaptic inhibition, they are a good model for studying the development of inhibition at axon terminals. Here, using whole cell recording methods and transgenic mice in which subclasses of retinal bipolar cells are labeled, we determined the temporal sequence and patterning of functional GABAergic and glycinergic input onto the major subclasses of bipolar cells. We found that the maturation of GABAergic and glycinergic synapses onto the axons of rod bipolar cells (RBCs), on-cone bipolar cells (ON-CBCs) and off-cone bipolar cells (OFF-CBCs) were temporally distinct: spontaneous chloride-mediated currents are present in RBCs earlier in development compared with ON- and OFF-CBC, and RBCs receive GABAergic and glycinergic input simultaneously, whereas in OFF-CBCs, glycinergic transmission emerges before GABAergic transmission. Because on-CBCs show little inhibitory activity, GABAergic and glycinergic events could not be pharmacologically distinguished for these bipolar cells. The balance of GABAergic and glycinergic input that is unique to RBCs and OFF-CBCs is established shortly after the onset of synapse formation and precedes visual experience. Our data suggest that presynaptic modulation of glutamate transmission from bipolar cells matures rapidly and is differentially coordinated for GABAergic and glycinergic synapses onto distinct bipolar cell subclasses.

Bipolar cells in the "grouped retina" of the elephantnose fish (Gnathonemus petersii).

To elucidate the specific properties of retinae with grouped photoreceptors the neural pathways in the outer and inner plexiform layer were studied. Photoreceptor bundles in this species consist of more than 100 rods and up to 50 cones, and are usually regarded as functional units. Golgi impregnation in thick and thin sections and light microscopy were used to identify bipolar cell types linking photoreceptors to amacrine and/or ganglion cells. Nine different types were distinguished based on their dendritic morphology and the position of the axon terminal in the inner plexiform layer. Small cells have dendritic fields smaller than the diameter of a photoreceptor bundle and are contacted mostly by cones. The dendritic field size of bushy cells matches that of a photoreceptor bundle; they are contacted mainly by rods. Flat cells receive about equal input from rods and cones; their dendritic field size exceeds the bundle diameter. Within the three major classes there are subtypes addressing three sublaminae of the inner plexiform layer, the proximal On-centre region (sl b), the distal Off-centre region (sl a) and a central sublayer (sl c) probably with transient activity. These observations suggest that cone vision has a spatial acuity better than the "bundle grain". In rod dominated vision the resolution matches that of the bundles; for this pathway, the hypothesis of the bundle as a functional unit is confirmed. The mesopic flat cell pathway has a resolution inferior to the "bundle grain"; it may therefore be dedicated to movement detection.