Selective synaptic connections in the retinal pathway for night vision

The mammalian retina encodes visual information in dim light using rod photoreceptors and a specialized circuit: rods→rod bipolar cells→AII amacrine cell. The AII amacrine cell uses sign-conserving electrical synapses to modulate ON cone bipolar cell terminals and sign-inverting chemical (glycinergic) synapses to modulate OFF cone cell bipolar terminals; these ON and OFF cone bipolar terminals then drive the output neurons, retinal ganglion cells (RGCs), following light increments and decrements, respectively. The AII amacrine cell also makes direct glycinergic synapses with certain RGCs, but it is not well established how many types receive this direct AII input. Here, we investigated functional AII amacrine→RGC synaptic connections in the retina of the guinea pig (Cavia porcellus) by recording inhibitory currents from RGCs in the presence of ionotropic glutamate receptor (iGluR) antagonists. This condition isolates a specific pathway through the AII amacrine cell that does not require iGluRs: cone→ON cone bipolar cell→AII amacrine cell→RGC. These recordings show that AII amacrine cells make direct synapses with OFF Alpha, OFF Delta and a smaller OFF transient RGC type that co-stratifies with OFF Alpha cells. However, AII amacrine cells avoid making synapses with numerous RGC types that co-stratify with the connected RGCs. Selective AII connections ensure that a privileged minority of RGC types receives direct input from the night-vision pathway, independent from OFF bipolar cell activity. Furthermore, these results illustrate the specificity of retinal connections, which cannot be predicted solely by co-stratification of dendrites and axons within the inner plexiform layer.     

Type 4 OFF cone bipolar cells of the mouse retina express calsenilin and contact cones as well as rods

Immunocytochemical discrimination of distinct bipolar cell types in the mouse retina is a prerequisite for analyzing retinal circuitry in wild-type and transgenic mice. Here we demonstrate that among the more than 10 anatomically defined mouse bipolar cell types, type 4 bipolar cells are specifically recognized by anti-calsenilin antibodies. Axon terminals in the inner plexiform layer are not readily identifiable because calsenilin is also expressed in a subset of amacrine and ganglion cells. In contrast, in the outer plexiform layer calsenilin immunoreactivity allows the analysis of photoreceptor to type 4 bipolar cell contacts. A dense plexus of calsenilin-positive dendrites makes several basal contacts at cone pedicles. An individual calsenilin-positive bipolar cell contacts five to seven cones. In addition, some calsenilin-positive dendrites contact rod photoreceptors. On average we counted 10 rod spherule contacts per type 4 bipolar cell, and approximately 10% of rods contacted type 4 bipolar cells. We suggest that type 4 bipolar cells, together with the recently described type 3a and b cells, provide an alternative and direct route from rods to OFF cone bipolar cells. In the Bassoon DeltaEx4/5 mouse, a mouse mutant that shows extensive remodeling of the rod system including sprouting of horizontal and rod bipolar cells into the outer nuclear layer due to impaired synaptic transmission, we found that in addition mixed-input (type 3 and 4) OFF bipolar cells sprout to ectopic sites. In contrast, true cone-selective type 1 and 2 OFF cone bipolar cells did not show sprouting in the Bassoon mouse mutant.     

Morphology and connectivity of the small bistratified A8 amacrine cell in the mouse retina

Amacrine cells comprise ～30 morphological types in the mammalian retina. The synaptic connectivity and function of a few γ‐aminobutyric acid (GABA)ergic wide‐field amacrine cells have recently been studied; however, with the exception of the rod pathway‐specific AII amacrine cell, the connectivity of glycinergic small‐field amacrine cells has not been investigated in the mouse retina. Here, we studied the morphology and connectivity pattern of the small‐field A8 amacrine cell. A8 cells in mouse retina are bistratified with lobular processes in the ON sublamina and arboreal dendrites in the OFF sublamina of the inner plexiform layer. The distinct bistratified morphology was first visible at postnatal day 8, reaching the adult shape at P13, around eye opening. The connectivity of A8 cells to bipolar cells and ganglion cells was studied by double and triple immunolabeling experiments by using various cell markers combined with synaptic markers. Our data suggest that A8 amacrine cells receive glutamatergic input from both OFF and ON cone bipolar cells. Furthermore, A8 cells are coupled to ON cone bipolar cells by gap junctions, and provide inhibitory input via glycine receptor (GlyR) subunit α1 to OFF cone bipolar cells and to ON A‐type ganglion cells. Measurements of spontaneous glycinergic postsynaptic currents and GlyR immunolabeling revealed that A8 cells express GlyRs containing the α2 subunit. The results show that the bistratified A8 cell makes very similar synaptic contacts with cone bipolar cells as the rod pathway‐specific AII amacrine cell. However, unlike AII cells, A8 amacrine cells provide glycinergic input to ON A‐type ganglion cells. J. Comp. Neurol. 523:1529–1547, 2015. © 2015 Wiley Periodicals, Inc.     

Rod pathways: the importance of seeing nothing.

Anatomical and physiological studies of the mammalian retina have revealed two primary pathways available for the transmission of rod signals to the ganglion cells: one via ON rod bipolars, amacrine II cells, and ON and OFF cone bipolars, which is exquisitely designed for the transmission of single-photon absorption events; and a second via rod-cone gap junctions, and ON and OFF cone bipolars, which is designed for the transmission of multiple photon-absorption events at higher light levels. Psychophysical and electroretinographic (ERG) studies in normal observers and in two rare types of observer, who are devoid of either rod or cone function, support an analogous duality in the human visual system, the clearest signature of which is a loss of flicker visibility and ERG amplitude at frequencies near 15 Hz that results from destructive interference between sensitive 'slow' and insensitive 'fast' rod signals. The slow rod signal is most probably derived from the ON rod bipolar pathway and the fast signal from the rod-cone gap junction and cone pathways. Evidence has emerged recently for a third, insensitive rod pathway between rods and OFF cone bipolars, but it has so far only been observed clearly in rodents.     

Differential output of the high-sensitivity rod photoreceptor: AII amacrine pathway

In the mammalian retina, the scotopic threshold of ganglion cells is in part dependent on how rod inputs are summed by their presynaptic cone bipolar cells. For ON cone bipolar cells, there are two anatomical routes for rod signals: 1) cone photoreceptors receive inputs via gap junctions with the surrounding, more numerous rods; and 2) ON cone bipolar cells receive highly convergent input via gap junctions with AII amacrine cells, which each receive input from hundreds of rods. Rod-cone coupling is thought to be utilized at higher photon fluxes relative to the AII-ON cone bipolar pathway due to the impedance mismatch of a single small rod driving a larger cone. Furthermore, it is widely held that the convergence of high-gain chemical synapses onto AIIs confers the highest sensitivity to ON cone bipolar cells and ganglion cells. A lack of coupling between one or more types of ON cone bipolar cells and AIIs would obviate this high-sensitivity pathway and explain the existence of ganglion cells with elevated scotopic thresholds. To investigate this possibility, we examined Neurobiotin and glycine diffusion from AIIs to bipolar cells and found that approximately one-fifth of ON cone bipolar cells are not coupled to AIIs. Unlike AII-AII coupling, which changes with ambient background intensity, the fraction of noncoupled ON cone bipolar cells was unaltered by dark or light adaptation. These data suggest that one of five morphologically distinct ON cone bipolar cell types is not coupled to AIIs and suggest that AII-ON cone bipolar coupling is modulated differently from AII-AII coupling.     

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.     

Subcellular distribution of connexin45 in OFF bipolar cells of the mouse retina

In the mouse retina, connexin45 (Cx45) participates in the gap junction between ON cone bipolar cells and AII amacrine cells, which constitutes an essential element of the primary rod pathway. Although it has been shown that Cx45 is also expressed in OFF bipolar cells, its subcellular localization and functional role in these cells are unknown. Here, we analyzed the localization of Cx45 on OFF bipolar cells in the mouse retina. For this, we used wild-type mice and a transgenic mouse line that expressed, in addition to native Cx45, a fusion protein consisting of Cx45 and the enhanced green fluorescent protein (EGFP). Cx45-EGFP expression generates an EGFP signal at gap junctions containing Cx45. Combining immunohistochemistry with intracellular injections, we found that Cx45 was present on dendrites and axon terminals of all OFF bipolar cell types. Cx45 was not found at intersections of two terminal processes of the same type, suggesting that Cx45 might not form gap junctions between axon terminals of the same OFF bipolar cell type but rather might connect OFF bipolar cells to amacrine or ganglion cells. In OFF bipolar cell dendrites, Cx45 was found predominantly in the proximal outer plexiform layer (OPL), well below the cone pedicles. Cx45 did not colocalize with Cx36, which is found predominantly in the distal OPL. We conclude that Cx45 is expressed on OFF bipolar cell dendrites, presumably forming gap junctions with cells of the same type, and on OFF bipolar cell axon terminals, presumably forming heterologous gap junctions with other retinal neurons.     

Localization of the calcium‐binding protein secretagogin in cone bipolar cells of the mammalian retina

Secretagogin, a recently cloned member of the EF‐hand family of calcium binding proteins, was localized in the mouse, rat, and rabbit retina using immunofluorescence immunohistochemistry. Secretagogin is expressed in subpopulations of ON and OFF cone bipolar cells; however, no immunoreactivity was observed in rod bipolar cells in any of these species. Using subtype‐specific markers and mice expressing green fluorescent protein (GFP) within specific cell classes, we found that secretagogin is expressed in Types 2, 3, 4, 5, 6 and possibly Type 8 cone bipolar cells in the mouse retina. The expression pattern in the rat retina differs slightly with expression in cone bipolar cell Types 2, 5, 6, 7, and 8. Evaluation of secretagogin in the developing mouse retina revealed expression as early as postnatal day 6, with OFF cone bipolar cells showing secretagogin expression prior to the ON cone bipolar cells. Secretagogin is a useful marker of cone bipolar cells for studying alterations in bipolar cell morphology during development and degeneration. Further work will be necessary to elucidate the functional role of this protein in bipolar cells. J. Comp. Neurol. 518:513–525, 2010. © 2009 Wiley‐Liss, 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. J. Comp. Neurol. 524:1839–1858, 2016. © 2015 Wiley Periodicals, Inc.     

Cone bipolar cells in the retina of the microbat Carollia perspicillata

We studied the retinal cone bipolar cells of Carollia perspicillata, a microchiropteran bat of the phyllostomid family. Microchiroptera are strongly nocturnal, with small eyes and rod‐dominated retinae. However, they also possess a significant cone population (2–4%) comprising two spectral types, which are hence the basis for daylight and color vision. We used antibodies against the calcium‐binding protein recoverin and the carbohydrate epitope 15 (CD15) as reliable markers for certain cone bipolar cells. Dye injections of recoverin‐ or CD15‐prelabeled cone bipolar cells in vertical slices revealed the morphology of the axon terminal system of individual bipolar cells. Seven distinct cone bipolar cell types were identified. They differed in the morphology and stratification level of their axon terminal system in the inner plexiform layer and in immunoreactivity for recoverin and/or CD15. Additional immunocytochemical markers were used to assess the functional ON/OFF subdivision of the inner plexiform layer. In line with the extended thickness of the ON sublayer of the inner plexiform layer in the microbat retina, more ON than OFF cone bipolar cell types were found, namely, four versus three. Most likely, in the bats' predominantly dark environment, ON signals have greater importance for contrast perception. We conclude that the microbat retina conforms to the general mammalian blueprint, in which light signals of intensities above rod sensitivity are detected by cones and transmitted to various types of ON and OFF cone bipolar cells. J. Comp. Neurol. 523:963–981, 2015. © 2014 Wiley Periodicals, Inc.     

Synaptic inputs onto small bistratified (blue‐ON/yellow‐OFF) ganglion cells in marmoset retina

The inner plexiform layer of the retina contains functional subdivisions, which segregate ON and OFF type light responses. Here, we studied quantitatively the ON and OFF synaptic input to small bistratified (blue‐ON/yellow‐OFF) ganglion cells in marmosets (Callithrix jacchus). Small bistratified cells display an extensive inner dendritic tier that receives blue‐ON input from short‐wavelength‐sensitive (S) cones via blue cone bipolar cells. The outer dendritic tier is sparse and is thought to receive yellow‐OFF input from medium (M)‐ and long (L)‐wavelength‐sensitive cones via OFF diffuse bipolar cells. In total, 14 small bistratified cells from different eccentricities were analyzed. The cells were retrogradely labeled from the koniocellular layers of the lateral geniculate nucleus and subsequently photofilled. Retinal preparations were processed with antibodies against the C‐terminal binding protein 2, the AMPA receptor subunit GluR4, and/or gephyrin to identify bipolar and/or amacrine input. The results show that the synaptic input is evenly distributed across the dendritic tree, with a density similar to that reported previously for other ganglion cell types. The population of cells showed a consistent pattern, where bipolar input to the inner tier is about fourfold greater than bipolar input to the outer tier. This structural asymmetry of bipolar input may help to balance the weight of cone signals from the sparse S cone array against inputs from the much denser M/L cone array. J. Comp. Neurol. 517:655–669, 2009. © 2009 Wiley‐Liss, Inc.     

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. J. Comp. Neurol. 521:977–1000, 2013. © 2012 Wiley Periodicals, Inc.     

Ectopic retinal ON bipolar cell synapses in the OFF inner plexiform layer: Contacts with dopaminergic amacrine cells and melanopsin ganglion cells

A key principle of retinal organization is that distinct ON and OFF channels are relayed by separate populations of bipolar cells to different sublaminae of the inner plexiform layer (IPL). ON bipolar cell axons have been thought to synapse exclusively in the inner IPL (the ON sublamina) onto dendrites of ON‐type amacrine and ganglion cells. However, M1 melanopsin‐expressing ganglion cells and dopaminergic amacrine (DA) cells apparently violate this dogma. Both are driven by ON bipolar cells, but their dendrites stratify in the outermost IPL, within the OFF sublamina. Here, in the mouse retina, we show that some ON cone bipolar cells make ribbon synapses in the outermost OFF sublayer, where they costratify with and contact the dendrites of M1 and DA cells. Whole‐cell recording and dye filling in retinal slices indicate that type 6 ON cone bipolars provide some of this ectopic ON channel input. Imaging studies in dissociated bipolar cells show that these ectopic ribbon synapses are capable of vesicular release. There is thus an accessory ON sublayer in the outer IPL. J. Comp. Neurol. 517:226‐244, 2009. © 2009 Wiley‐Liss, Inc.     

Expression of vesicular glutamate transporter 1 in the mouse retina reveals temporal ordering in development of rod vs. cone and ON vs. OFF circuits

Glutamatergic transmission is crucial to the segregation of ON and OFF pathways in the developing retina. The temporal sequence of maturation of vesicular glutamatergic transmission in rod and cone photoreceptor and ON and OFF bipolar cell terminals is currently unknown. Vesicular glutamate transporters (VGLUTs) that load glutamate into synaptic vesicles are necessary for vesicular glutamatergic transmission. To understand better the formation and maturation of glutamatergic transmission in the rod vs. cone and ON vs. OFF pathways of the retina, we examined the developmental expression of VGLUT1 and VGLUT2 immunocytochemically in the mouse retina. Photoreceptor and bipolar cell terminals showed only VGLUT1-immunoreactivity (-IR); no VGLUT2-IR was present in any synapses of the developing or adult retina. VGLUT1-IR was first detected in cone photoreceptor terminals at postnatal day 2 (P2), several days before initiation of ribbon synapse formation at P4-P5. Rod terminals showed VGLUT1-IR by P8, when they invade the outer plexiform layer (OPL) and initiate synaptogenesis. Developing OFF bipolar cell terminals showed VGLUT1-IR around P8, 2-3 days after bipolar terminals were first identified in the inner plexiform layer (IPL) by labeling for the photoreceptor and bipolar cell terminal marker, synaptic vesicle protein 2B. Although terminals of ON bipolar cells were present in the IPL by P6-P8, most did not show VGLUT1-IR until P8-P10 and increased dramatically from P12. These data suggest a hierarchical development of glutamatergic transmission in which cone circuits form prior to rod circuits in both the OPL and IPL, and OFF circuits form prior to ON circuits in the IPL.     

Immunocytochemical analysis of bipolar cells in the macaque monkey retina

Transfer of visual information from photoreceptors to ganglion cells within the retina is mediated by specialized groups of bipolar cells. At least 10 different morphological types of bipolar cells have been distinguished in Golgi studies of primate retina. In the present study, bipolar cell populations in the macaque monkey retina were identified by their differential immunoreactivity to a spectrum of antibody markers. This enabled their spatial density and photoreceptor connections to be analysed. An antibody against the β isozyme of protein kinase C (PKCA_β) labelled many cone bipolar cells. Invaginating (presumed ON) cone bipolar cells and rod bipolar cells were prefentially labelled with a monoclonal antibody raised against rabbit olfactory bulb. Flat (presumed OFF) bipolar cells were labelled with an antiserum against the glutamate transporter protein (GLT-1). Different populations of diffuse cone bipolar cells, which contact 5–10 cones, could be distinguished. The GLT-1 artiserum preferentially labelled the flat diffuse bipolar cell type DB2 (Boycott and Wässle, 1991, Eur. J. Neurosci. 3:1069–1088) as well as flat midget bipolar cells. Antibodies to calbindin (CaBP D-28K) labelled the flat diffuse bipolar cell type DB3 and (possibly) the invaginating diffuse bipolar cell type DB5. An antibody against the α isozyme of PKC labelled an invaginating diffuse bipolar cell type (DB4) as well as rod bipolar cells. Comparison of the spatial density of cone bipolar cell populations with that of photoreceptors suggests that each bipolar cell class provides a complete coverage of the cone array (each cone is contacted by at least one member of every bipolar cell class). These results support the classification scheme of Boycott and Wässle (1991) by showing that different diffuse bipolar cell classes express different patterns of immunoreactivity, and they reinforce the view that different spatial and temporal components of the signal from the photoreceptor array are processed in parallel within the primate retina. © 1994 Wiley-Liss, Inc.     

Agonist-stimulated cobalt uptake provides selective visualization of neurons expressing AMPA- or kainate-type glutamate receptors in the retina

Fast-acting excitatory neurotransmission in the retina is mediated primarily by glutamate, acting at alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) -selective and kainate-selective receptors. To localize these sites of action, cat retinas were stimulated with either AMPA or kainate and processed for histochemical visualization of cobalt uptake through calcium-permeable channels. Treatment with both agonists resulted in staining of A- and B-type horizontal cells and several types of OFF cone bipolar cells; there was no evidence for staining of ON cone bipolar cells or rod bipolar cells. The subpopulations of OFF cone bipolar cells differed in their responses with two distinct types that stained heavily with cobalt after exposure to AMPA and three different types that were preferentially labeled after exposure to kainate. Although many amacrine and ganglion cells appeared to respond to both agonists, AII amacrine cells were stained after stimulation by AMPA but not by kainate. The OFF cone bipolar cells that exhibit AMPA-stimulated cobalt uptake were found to have a high level of correspondence with cells that show immunocytochemical staining for the AMPA-selective glutamate receptor subunits GluR1 and GluR2/3. Similarly, the cone bipolar cells exhibiting kainate-stimulated cobalt uptake resemble those that are immunoreactive for the kainate subunit GluR5. The results indicate that, whereas many retinal neurons express both AMPA and kainate receptors, AII amacrine cells and subpopulations of OFF cone bipolar cells are limited to the expression of either AMPA or kainate receptors. This differential expression may contribute to the unique character of transmission by these cell types.     

Localization of heterotypic gap junctions composed of connexin45 and connexin36 in the rod pathway of the mouse retina

The primary rod pathway in mammals contains gap junctions between AII amacrine cells and ON cone bipolar cells which relay the rod signal into the cone pathway under scotopic conditions. Two gap junctional proteins, connexin36 (Cx36) and connexin45 (Cx45), appear to play a pivotal role in this pathway because lack of either protein leads to an impairment of visual transmission under scotopic conditions. To investigate whether these connexins form heterotypic gap junctions between ON cone bipolar and AII amacrine cells, we used newly developed Cx45 antibodies and studied the cellular and subcellular distribution of this protein in the mouse retina. Specificity of the Cx45 antibodies was determined, among others, by Western blot and immunostaining of mouse heart, where Cx45 is abundantly expressed. In mouse retina, Cx45 immunosignals were detected in both plexiform layers and the ganglion cell layer. Double staining for Cx45 and Cx36 revealed a partial overlap in the punctate patterns in the ON sublamina of the inner plexiform layer of the retina. We quantified the distributions of these two connexins in the ON sublamina, and detected 30% of the Cx45 signals to be co-localized with or in close apposition to Cx36 signals. Combining immunostaining and intracellular dye injection revealed an overlap or tight association of Cx36 and Cx45 signals on the terminals of injected AII amacrine and two types of ON cone bipolar cells. Our results provide direct evidence for heterotypic gap junctions composed of Cx36 and Cx45 between AII amacrine and certain types of ON cone bipolar cells.     

Microcircuitry of bipolar cells in cat retina

We have studied 15 bipolar neurons from a small patch (14 X 120 micron) of adult cat retina located within the area centralis. From electron micrographs of 189 serial ultrathin sections, the axon of each bipolar cell was substantially reconstructed with its synaptic inputs and outputs by means of a computer-controlled reconstruction system. Based on differences in stratification, cytology, and synaptic connections, we identified eight different cell types among the group of 15 neurons: one type of rod bipolar and seven types of cone bipolar neurons. These types correspond to those identified by the Golgi method and by intracellular recording. Those bipolar cell types for which we reconstructed three or four examples were extremely regular in form, size, and cytology, and also in the quantitative details of their synaptic connections. They appeared quite as specific in these respects as invertebrate "identified" neurons. The synaptic patterns observed for each type of bipolar neuron were complex but may be summarized as follows: the rod bipolar axon ended in sublamina b of the inner plexiform layer and provided major input to the AII amacrine cell. The axons of three types of cone bipolar cells also terminated in sublamina b and provided contacts to dendrites of on-beta and other ganglion cells. All three types, but especially the Cb1, received gap junction contacts from the AII amacrine cell. Axons of four types of cone bipolar cells terminated in sublamina a of the inner plexiform layer and contacted dendrites of off-beta and other ganglion cells. One of these cone bipolar cell types, CBa1, made reciprocal chemical contacts with the lobular appendage of the AII amacrine cell. These results show that the pattern of cone bipolar cell input to beta (X) and probably alpha (Y) ganglion cells is substantially more complex than had been suspected. At least two types of cone bipolar contribute to each type of ganglion cell where only a single type had been anticipated. In addition, many of the cone bipolar cell pathways in the inner plexiform layer are available to the rod system, since at least four types of cone bipolar receive electrical or chemical inputs from the AII amacrine cell. This may help to explain why, in a retina where rods far outnumber the cones, there should be so many types of cone bipolar cells.     

G protein subunit G gamma 13 is coexpressed with G alpha o,G beta 3, and G beta 4 in retinal ON bipolar cells

We investigated the expression of Ggamma13, a recently discovered G protein subunit, and a selection of Gbeta subunits in retinal bipolar cells, by using a transgenic mouse strain in which green fluorescent protein is strongly expressed in a single type of cone bipolar cell. The cells have ON morphology, and patch-clamp recordings in slices confirmed that they are of the physiological ON type. Immunohistochemistry showed that Ggamma13 is expressed in rod bipolar cells and ON cone bipolar cells, where it is colocalized in the dendrites with Galphaomicron. ON and OFF cone bipolar cells and rod bipolar cells were identified among dissociated cells by their green fluorescence and/or distinct morphology. Hybridization of single-cell polymerase chain reaction products with cDNA probes for G protein subunits Gbeta1 to 5 showed that Gbeta3, Gbeta4, and Ggamma13 are coexpressed in ON bipolar cells but not present in OFF bipolar cells. Gbeta1, 2, and 5 are expressed in partially overlapping subpopulations of cone bipolar cells. Ggamma13 and Gbeta3 and/or Gbeta4, thus, seem selectively to participate in signal transduction by ON bipolar cells.     

Identification of a cone bipolar cell in cat retina which has input from both rod and cone photoreceptors

It has been generally accepted that rod photoreceptor cells in the mammalian retina make synaptic contact with only a single population of rod bipolar cells, whereas cone photoreceptors contact a variety of cone bipolar cells. This assumption has been challenged in rodents by reports of a type of cone bipolar cell which receives input from both rods and cones. Questions remained as to whether similar pathways are present in other mammals. We have used an antiserum against the glutamate transporter GLT1-B to visualize a population of cone bipolar cells in the cat retina which make flat contacts with axon terminals of both rod and cone photoreceptor cells. These cells are identified as OFF-cone bipolar cells and correspond morphologically to type cb1 (CBa2) cone bipolar cells which are a major source of input to OFF-beta ganglion cells in the cat retina. The GLT1-B transporter was also localized to processes making flat contacts with photoreceptor terminals in rat and rabbit retinas. Examination of tissue processed for the GluR1 glutamate receptor subunit showed that cb1 cone bipolar cells, like their rodent counterparts, express this alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-selective receptor at their contacts with rod spherules. Thus, a direct excitatory pathway from rod photoreceptors to OFF-cone bipolar cells appears to be a common feature of mammalian retinas.