OFF-type Bipolar Cells Research Articles

Glycinergic Inhibition Targets Specific Off Cone Bipolar Cells in Primate Retina.

Adapting between scotopic and photopic illumination involves switching the routing of retinal signals between rod and cone-dominated circuits. In the daytime, cone signals pass through parallel On and Off cone bipolar cells (CBCs), that are sensitive to increments and decrements in luminance, respectively. At night, rod signals are routed into these cone-pathways via a key glycinergic interneuron, the AII amacrine cell (AII-AC). AII-ACs also provide On-pathway-driven crossover inhibition to Off-CBCs under photopic conditions. In primates, it is not known whether all Off-bipolar cell types receive functional inputs from AII-ACs. Here, we show that select Off-CBC types receive significantly higher levels of On-pathway-driven glycinergic input than others. The rise and decay kinetics of the glycinergic events are consistent with involvement of the α1 glycine receptor (GlyR) subunit, a result supported by a higher level of GLRA1 transcript in these cells. The Off-bipolar types that receive glycinergic input have sustained physiological properties and include the flat midget bipolar (FMB) cells, which provide excitatory input to the Off-midget ganglion cells (GCs; parvocellular pathway). Our results suggest that only a subset of Off-bipolar cells have the requisite receptors to respond to AII-AC input. Taken together with results in mouse retina, our findings suggest a conserved motif whereby signal output from AII-ACs is preferentially routed into sustained Off-bipolar signaling pathways.

Two Types of Cl Transporters Contribute to the Regulation of Intracellular Cl Concentrations in ON- and OFF-type Bipolar Cells in the Mouse Retina

In the retina, ON- and OFF-type bipolar cells are classified by subtype-specific center responses, which are attributed to differences in glutamate receptor subtypes. However, the mechanisms by which ON- and OFF-type bipolar cells generate subtype-specific surround responses remain unclear. One hypothesis for surround responses is that intracellular Cl concentrations ([Cl-]i) are set at different levels to achieve opposite polarities for GABA responses in ON- and OFF-type bipolar cells. Although this hypothesis is supported by previous findings obtained from rod (ON-) type bipolar cells, there is currently no information on OFF-type bipolar cells. In the present study, we examined the distribution and function of the Cl transporters, the Na-K-Cl co-transporter (NKCC1) and K-Cl co-transporter (KCC2), in rod (ON-) and OFF-type bipolar cells using immunohistochemical, in situ hybridization, and electrophysiological methods. Rod (ON-) and OFF-type bipolar cells both expressed NKCC1 and KCC2. However, the functional contribution of NKCC1 and KCC2 to the regulation of [Cl−]i differed between rod (ON-) and OFF-type bipolar cells. Strong NKCC1 activity increased [Cl−]i in rod (ON-) type bipolar cells, while that of KCC2 decreased [Cl−]i in OFF-type bipolar cells. We also confirmed the presence of a [Cl−]i gradient between dendrites and axon terminals in rod (ON-type) bipolar cells. Thus, the subtype-specific control of [Cl−]i is achieved by the activity of NKCC1 relative to that of KCC2 and appears to influence the polarity of surround responses.

Cannabinoid Signaling Selectively Modulates GABAergic Inhibitory Input to OFF Bipolar Cells in Rat Retina.

PurposeIn the mammalian retina, cannabinoid type 1 receptors (CB1Rs) are well-positioned to alter inhibitory synaptic function from amacrine cells and, thus, might influence visual signal processing in the inner retina. However, it is not known if CB1R modulates amacrine cells feedback inhibition at retinal bipolar cell (BC) terminals.MethodsUsing whole-cell voltage-clamp recordings, we examined the pharmacological effect of CB1R activation and inhibition on spontaneous inhibitory postsynaptic currents (sIPSCs) and glutamate-evoked IPSCs (gIPSCs) from identified OFF BCs in light-adapted rat retinal slices.ResultsActivation of CB1R with WIN55212-2 selectively increased the frequency of GABAergic, but not glycinergic sIPSC in types 2, 3a, and 3b OFF BCs, and had no effect on inhibitory activity in type 4 OFF BCs. The increase in GABAergic activity was eliminated in axotomized BCs and can be suppressed by blocking CB1R with AM251 or GABAA and GABAρ receptors with SR-95531 and TPMPA, respectively. In all OFF BC types tested, a brief application of glutamate to the outer plexiform layer elicited gIPSCs comprising GABAergic and glycinergic components that were unaffected by CB1R activation. However, blocking CB1R selectively increased GABAergic gIPSCs, supporting a role for endocannabinoid signaling in the regulation of glutamate-evoked GABAergic inhibitory feedback to OFF BCs.ConclusionsCB1R activation shape types 2, 3a, and 3b OFF BC responses by selectively regulate GABAergic feedback inhibition at their axon terminals, thus cannabinoid signaling might play an important role in the fine-tuning of visual signal processing in the mammalian inner retina.

A simulation study on the effect of ionic currents on transmission from cones to retinal OFF type cone bipolar cells

The retinal cone bipolar cells are interneurons which receive inputs from cone photoreceptors and send outputs to retinal ganglion cells. Several subtypes of bipolar cells have been identified by morphology and electrophysiology in the mammalian retina, which convey distinct visual information to higher order neurons in parallel. The neural circuit in the retina not only converts light information to neural . information, but also performs visual information preprocessing that has not yet been fully understood. Recently, it has been revealed that the neural circuits in retinas of higher vertebrates, such as mammals and primates, have various biophysical properties arising from being composed of ionic channels, ionic pumps, and neurotransmitter receptors. Analysis using a mathematical model based on their ionic mechanisms is essential to understand the visual information processing in the retinal neural circuit of the higher vertebrates.
 The cones and the bipolar cells respond to continuous variation of light with a graded potential, in an analog manner. Especially, glutamate is continuously released from a cone synapse in the dark and is decreased by hyperpolarization of the cone that receives the light stimulus. The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate type ionotropic glutamate receptors (iGluRs) of the OFF type bipolar cells (OFF-BCs) exhibit partial or nearly complete desensitization in the sustained presence of glutamate. In the dark, glutamate concentration in the synaptic cleft of the cone pedicle rises to 0.1–0.5 mM.5,6 The baseline glutamate concentration depends on a sustained hyperpolarization of the cone by light. Thus, for understanding the working of the OFF-BCs, it is important to elucidate the mechanisms of synaptic transmission from cones to OFF-BCs via iGluRs, which undergo desensitization in the various background light conditions. Furthermore, there are various kinds of ionic channels in OFF-BCs that mediate membrane potential responses. It is considered that information transmitted from cones to OFF-BCs is modulated by the intrinsic ionic currents. We analyzed how ionic currents of OFF-BCs contribute to the transmission of light responses by developing a mathematical model.

Simulation Analysis of Temporal Coding by Ionotropic Glutamate Receptors of Retinal OFF-type Bipolar Cells

Simulation Analysis of Temporal Coding by Ionotropic Glutamate Receptors of Retinal OFF-type Bipolar Cells

Classification of Mouse Retinal Bipolar Cells: Type-Specific Connectivity with Special Reference to Rod-Driven AII Amacrine Pathways.

We confirmed the classification of 15 morphological types of mouse bipolar cells by serial section transmission electron microscopy and characterized each type by identifying chemical synapses and gap junctions at axon terminals. Although whether the previous type 5 cells consist of two or three types was uncertain, they are here clustered into three types based on the vertical distribution of axonal ribbons. Next, while two groups of rod bipolar (RB) cells, RB1, and RB2, were previously proposed, we clarify that a half of RB1 cells have the intermediate characteristics, suggesting that these two groups comprise a single RB type. After validation of bipolar cell types, we examined their relationship with amacrine cells then particularly with AII amacrine cells. We found a strong correlation between the number of amacrine cell synaptic contacts and the number of bipolar cell axonal ribbons. Formation of bipolar cell output at each ribbon synapse may be effectively regulated by a few nearby inhibitory inputs of amacrine cells which are chosen from among many amacrine cell types. We also found that almost all types of ON cone bipolar cells frequently have a minor group of midway ribbons along the axon passing through the OFF sublamina as well as a major group of terminal ribbons in the ON sublamina. AII amacrine cells are connected to five of six OFF bipolar cell types via conventional chemical synapses and seven of eight ON (cone) bipolar cell types via electrical synapses (gap junctions). However, the number of synapses is dependent on bipolar cell types. Type 2 cells have 69% of the total number of OFF bipolar chemical synaptic contacts with AII amacrine cells and type 6 cells have 46% of the total area of ON bipolar gap junctions with AII amacrine cells. Both type 2 and 6 cells gain the greatest access to AII amacrine cell signals also share those signals with other types of bipolar cells via networked gap junctions. These findings imply that the most sensitive scotopic signal may be conveyed to the center by ganglion cells that have the most numerous synapses with type 2 and 6 cells.

Dendritic stratification differs among retinal OFF bipolar cell types in the absence of rod photoreceptors.

Retinal OFF bipolar cells show distinct connectivity patterns with photoreceptors in the wild-type mouse retina. Some types are cone-specific while others penetrate further through the outer plexiform layer (OPL) to contact rods in addition to cones. To explore dendritic stratification of OFF bipolar cells in the absence of rods, we made use of the ‘cone-full’ Nrl-/- mouse retina in which all photoreceptor precursor cells commit to a cone fate including those which would have become rods in wild-type retinas. The dendritic distribution of OFF bipolar cell types was investigated by confocal and electron microscopic imaging of immunolabeled tissue sections. The cells’ dendrites formed basal contacts with cone terminals and expressed the corresponding glutamate receptor subunits at those sites, indicating putative synapses. All of the four analyzed cell populations showed distinctive patterns of vertical dendritic invasion through the OPL. This disparate behavior of dendritic extension in an environment containing only cone terminals demonstrates type-dependent specificity for dendritic outgrowth in OFF bipolar cells: rod terminals are not required for inducing dendritic extension into distal areas of the OPL.

Differential signalling and glutamate receptor compositions in the OFF bipolar cell types in the mouse retina.

Using whole-cell clamp methods, we characterized the temporal coding in each type of OFF bipolar cell. We found that type 2 and 3a cells are transient, type 1 and 4 cells are sustained, and type 3b cells are intermediate. The light-evoked excitatory postsynaptic potentials in some types were rectified, suggesting that they provide inputs to the non-linear ganglion cells. Visual signalling from the photoreceptors was mediated exclusively through the kainate receptors in the transient OFF bipolar cells, whereas both kainate and AMPA receptors contributed in the other cells. This study demonstrates, for the first time, that parallel visual encoding starts at the OFF bipolar cells in a type-specific manner. The retina is the entrance to the visual system, which receives various kinds of image signals and forms multiple encoding pathways. The second-order retinal neurons, the bipolar cells, are thought to initiate multiple neural streams by encoding various visual signals in different types of cells. However, the functions of each bipolar cell type have not been fully understood. We investigated whether OFF bipolar cells encode visual signals in a type-dependent manner. We recorded the changes in the bipolar cell voltage in response to two input functions: step and sinusoidal light stimuli. Type 1 and 4 OFF bipolar cells were sustained cells and responded to sinusoidal stimuli over a broad range of frequencies. Type 2 and 3a cells were transient and exhibited band-pass filtering. Type 3b cells were in the middle of these two groups. The distinct temporal responses might be attributed to different types of glutamate receptors. We examined the AMPA and kainate glutamate receptor composition in each bipolar cell type. The light responses in the transient OFF bipolar cells were exclusively mediated by kainate receptors. Although the kainate receptors mediated the light responses in the sustained cells, the AMPA receptors also mediated a portion of the responses in sustained cells. Furthermore, we found that some types of cells were rectified more than other types. Taken together, we found that the OFF bipolar cells encode diverse temporal image signals in a type-dependent manner, confirming that each type of OFF bipolar cell initiates diverse temporal visual processing in parallel.

OFF bipolar cells in macaque retina: type-specific connectivity in the outer and inner synaptic layers.

OFF bipolar cells in the macaque retina were recently classified into five types: flat midget bipolar (FMB) and diffuse bipolar (DB) 1, 2, 3a, and 3b. We examined all parallel pathways from cone photoreceptors via OFF bipolar cells to parasol and midget ganglion cells by serial section transmission electron microscopy. Basal contacts of OFF bipolar cells to cone pedicles were previously categorized as triad-associated (TA) and non-TA (NTA). The latter was further divided into two groups located in the middle and marginal areas of the pedicle at the present eccentricity of 15°. We then mapped the distributions of all three basal contacts of the five OFF bipolar cell types in the same area of cone pedicles. TA contacts were more numerous than NTA contacts in FMB (93%), DB1 (67%), and DB3a (81%) cells, but less in DB2 (30%) and DB3b (21%) cells. Cluster analysis of these contact parameters reconfirmed five distinct OFF bipolar cell types and showed these positional configurations of basal synapses to be cell type-specific. This architecture is thought to provide a spatial framework for the interstitial diffusion and local uptake of the neurotransmitter (glutamate) that spills over from ribbon synapses. All five OFF bipolar cell types formed ribbon-synaptic contacts to both parasol and midget ganglion cells. DB2 and 3a, DB1 and 3b, and FMB predominantly, moderately, and negligibly contacted parasol ganglion cells, respectively. FMB almost exclusively contacted midget ganglion cells, to which DB1 provided dominant output (58%), and DB2, 3a, and 3b provided between 3% and 10% of their output. Consequently, the cone signal sampling routes of a midget ganglion cell consisted of two substructures: the narrow (mainly 2-3 cones) FMB pathway and the wide (mainly 10 cones) DB pathway, where connection strength was four-fold greater in the FMB than DB pathway. The narrow and strong FMB pathway may confer the highest spatial resolution and sporadically may include blue cone signals.

Some OFF bipolar cell types make contact with both rods and cones in macaque and mouse retinas.

This study compared the types of OFF bipolar cells found in the macaque retina with those found in the mouse retina and determined whether these OFF bipolar cells make direct contacts with both rods and cones by serial section transmission electron microscopy. We performed scatter plots and cluster analysis of the morphological variables of their axon terminals such as the stratification level, the arbor thickness, the arbor area, and the number of ribbons. Five OFF bipolar cell types, including the recently discovered DB3b type, were identified in the macaque retina. The macaque OFF bipolar cell types FMB, DB1, DB2, DB3a, and DB3b corresponded to the mouse OFF bipolar cell types 2, 1, 4, 3a, and 3b, respectively. In addition to contacting rod bipolar cells, ~7% of rods in the macaque retina made basal contacts exclusively with one cell type, DB3b, whereas 18% of rods in the mouse retina made basal contacts with one or two of types, 3a, 3b, and 4. Approximately 3% of mouse rods were divergently connected to two OFF bipolar cells of different types, but macaque rods were solely connected to one OFF bipolar cell. Rod-rod gap junctions were localized at rod cell bodies and axons in the outer nuclear layer in both macaque and mouse retinas. The direct rod-OFF bipolar connection system is slightly more developed in the mouse retina than in the macaque retina, possibly as a fine-tuned adaptation to nocturnal conditions. This one-step direct synaptic pathway from rods to OFF bipolar cells may enhance the response speed to OFF light stimuli compared with more indirect pathways via rod-cone gap junctions (a two-step pathway) and via rod bipolar and AII amacrine cells (a three-step pathway).

Kainate receptors mediate synaptic input to transient and sustained OFF visual pathways in primate retina.

Visual signals are segregated into parallel pathways at the first synapse in the retina between cones and bipolar cells. Within the OFF pathways of mammals, the selective expression of AMPA or kainate-type glutamate receptors in the dendrites of different OFF-bipolar cell types is thought to contribute to formation of distinct temporal channels. AMPA receptors, with rapid recovery from desensitization, are proposed to transmit high temporal frequency signals, whereas kainate receptors (KARs) are presumed to encode lower temporal frequencies. Here we studied the glutamate receptors expressed by OFF-bipolar cells in slice preparations of macaque monkey retina, where the low (midget/parvocellular) and high-frequency (parasol/magnocellular) temporal channels are well characterized. We found that all OFF-bipolar types receive input primarily through KARs and that KAR antagonists block light-evoked input to both OFF-midget and OFF-parasol ganglion cells. KAR subunits were differentially expressed in OFF-bipolar types; the diffuse bipolar (DB) cells, DB2 and DB3b, expressed GluK1 and showed transient responses to glutamate and the KAR agonist, ATPA. In contrast, flat midget bipolar, DB1, and DB3a cells lacked GluK1 and showed relatively sustained responses. Finally, we found that the KAR accessory protein, Neto1, is expressed at the base of cone pedicles but is not colocalized with the GluK1 subunit. In summary, the results indicate that transient signaling in the OFF pathway of macaques is not dependent on AMPA receptors and that heterogeneity of KARs and accessory proteins may contribute to the formation of parallel temporal channels.

Synaptic inputs to two types of koniocellular pathway ganglion cells in marmoset retina

The retinal connectivity of the diverse group of cells contributing to koniocellular visual pathways (widefield ganglion cells) is largely unexplored. Here we examined the synaptic inputs onto two koniocellular-projecting ganglion cell types named large sparse and broad thorny cells. Ganglion cells were labeled by retrograde tracer injections targeted to koniocellular layer K3 in the lateral geniculate nucleus in marmosets (Callithrix jacchus) and subsequently photofilled. Retinal preparations were processed with antibodies against the C-terminal binding protein 2, the AMPA receptor subunit GluR4, and against CD15 to identify bipolar (excitatory) and/or antibodies against gephyrin to identify amacrine (inhibitory) input. Large sparse cells are narrowly stratified close to the ganglion cell layer. Broad thorny ganglion cells are broadly stratified in the center of the inner plexiform layer. Bipolar input to large sparse cells derives from DB6 and maybe other ON bipolar types, whereas that to broad thorny cells derives from ON and OFF bipolar cell types. The total number of putative synapses on broad thorny cells is higher than the number on large sparse cells but the density of inputs (between 2 and 5 synapses per 100 μm(2) dendritic area) is similar for the two cell types, indicating that the larger number of synapses on broad thorny cells is attributable to the larger membrane surface area of this cell type. Synaptic input density is comparable to previous values for midget-parvocellular and parasol-magnocellular pathway cells. This suggests functional differences between koniocellular, parvocellular, and magnocellular pathways do not arise from variation in synaptic input densities.

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.

Modeling convergent ON and OFF pathways in the early visual system

For understanding the computation and function of single neurons in sensory systems, one needs to investigate how sensory stimuli are related to a neuron’s response and which biological mechanisms underlie this relationship. Mathematical models of the stimulus–response relationship have proved very useful in approaching these issues in a systematic, quantitative way. A starting point for many such analyses has been provided by phenomenological “linear–nonlinear” (LN) models, which comprise a linear filter followed by a static nonlinear transformation. The linear filter is often associated with the neuron’s receptive field. However, the structure of the receptive field is generally a result of inputs from many presynaptic neurons, which may form parallel signal processing pathways. In the retina, for example, certain ganglion cells receive excitatory inputs from ON-type as well as OFF-type bipolar cells. Recent experiments have shown that the convergence of these pathways leads to intriguing response characteristics that cannot be captured by a single linear filter. One approach to adjust the LN model to the biological circuit structure is to use multiple parallel filters that capture ON and OFF bipolar inputs. Here, we review these new developments in modeling neuronal responses in the early visual system and provide details about one particular technique for obtaining the required sets of parallel filters from experimental data.

Modulation by Brain Natriuretic Peptide of GABA Receptors on Rat Retinal ON-Type Bipolar Cells

Natriuretic peptides (NPs) may work as neuromodulators through their associated receptors [NP receptors (NPRs)]. By immunocytochemistry, we showed that NPR-A and NPR-B were expressed abundantly on both ON-type and OFF-type bipolar cells (BCs) in rat retina, including the dendrites, somata, and axon terminals. Whole-cell recordings made from isolated ON-type BCs further showed that brain natriuretic peptide (BNP) suppressed GABAA receptor-, but not GABAC receptor-, mediated currents of the BCs, which was blocked by the NPR-A antagonist anantin. The NPR-C agonist c-ANF [des(Gln18, Ser19, Gln20, Leu21, Gly22)ANF(4-23)-NH2] did not suppress GABAA currents. The BNP effect on GABAA currents was abolished with preincubation with the pGC-A/B antagonist HS-142-1 but mimicked by application of 8-bromoguanosine-3',5'-cyclomonophosphate. These results suggest that elevated levels of intracellular cGMP caused by activation of NPR-A may mediate the BNP effect. Internal infusion of the cGMP-dependent protein kinase G (PKG) inhibitor KT5823 essentially blocked the BNP-induced reduction of GABAA currents. Moreover, calcium imaging showed that BNP caused a significant elevation of intracellular calcium that could be caused by increased calcium release from intracellular stores by PKG. The BNP effect was blocked by the ryanodine receptor modulators caffeine, ryanodine, and ruthenium red but not by the IP3 receptor antagonists heparin and xestospongin-C. Furthermore, the BNP effect was abolished after application of the blocker of endoplasmic reticulum Ca2+-ATPase thapsigargin and greatly reduced by the calmodulin inhibitors W-7 and calmidazolium. We therefore conclude that the increased calcium release from ryanodine-sensitive calcium stores by BNP may be responsible for the BNP-caused GABAA response suppression in ON-type BCs through stimulating calmodulin.

Uncoupling of horizontal cells alters the receptive fields of retinal bipolar cells.

Effects of uncoupling of horizontal cells by 1-octanol, a non-specific gap junction uncoupling agent, on the receptive field organization of cone-dominant bipolar cells were investigated in isolated, superfused carp retina, using intracellular recording techniques. At 1 mM, 1-octanol increased responses of cone driven horizontal cells to light spots, but decreased those to light annuli, indicating a reduction of the receptive field size of these cells by uncoupling. Furthermore, 1-octanol eliminated the surround response of OFF type bipolar cells and increased their center response. Similar effects of 1-octanol on the center and surround responses were observed in ON type bipolar cells. These results suggest that uncoupling of horizontal cells can significantly alter the receptive field organization of retinal bipolar cells.

Expression of AMPA-type glutamate receptor subunit (GluR2) in ON-bipolar neurons in the rat retina.

The role of glutamate receptors (GluR) in the signal pathways of the retina is widely recognized. Photoreceptors make synaptic contact with functionally different classes of bipolar cells. The OFF-type bipolar cells mediate light offset-evoked responses and use ionotropic alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA)- or kainate-type GluRs, whereas bipolars involved in the ON-pathway use the metabotropic GluR6. This dichotomy predicts a defined expression pattern of AMPA-type GluRs and mGluR6 in bipolar cell classes. This hypothesis was tested by performing immunocytochemical double labeling studies combining GluR-specific antibodies with markers specific for the diverse bipolar cell populations in the rat retina. AMPA-type receptors are composed of combinations of four types of subunits, GluR1-4. GluR1 is expressed by a few somata in the outer part of the inner nuclear layer (INL). Sparse colocalization with any of the bipolar markers used could be established. In contrast, GluR2 is expressed by many of the somata in the outer zone of the INL. At the transcript level, in situ hybridizations demonstrated abundant GluR2 expression over the complete width of the INL. In contrast to our expectations, approximately 70% of the somata labeled by the rod ON-bipolar markers protein kinase C (PKC) or Goalpha, colocalized with GluR2. Approximately 90% of the OFF-type bipolar cells, identified as recoverin-positive, showed GluR2 immunoreactivity. At least 40% of the somata that were mGluR6-immunoreactive, a both rod and cone ON-type bipolar marker, were GluR2-immunopositive. Ultrastructurally, examples were observed of GluR2 localization in bipolar processes with labeling outside the actual compartment associated with the synaptic complex of the rod terminal. No specific antibody was available against GluR3, but 74% of the PKC-positive cells were GluR2/3-positive. GluR4 did not show a somatic localization making double labeling impossible. On the basis of these results, we conclude that ionotropic GluRs are expressed by rod ON-type bipolar cells (PKC- or Goalpha-immunoreactive), and by cone ON- and OFF-type bipolars based on a colocalization with nearly all of the present recoverin-positive somata. Our observations show that the functional dichotomy in ON- and OFF-type bipolars is not reflected in a matching expression pattern of ionotropic and metabotropic GluRs. This finding raises the intriguing possibility that the AMPA-type GluRs are, in an as yet unclear manner, involved in the ON signaling pathways of rods and cones.

Intracellular chloride concentration is higher in rod bipolar cells than in cone bipolar cells of the mouse retina

Bipolar cells (BCs) have antagonistic center-surround receptive field. Surround illumination evokes depolarization in the OFF-type cone BC, and hyperpolarization in the rod BC and the ON-type cone BC. Surround illumination reduces gamma-aminobutyric acid (GABA) release from horizontal cells. If GABA hyperpolarize BCs, the polarity of the GABA-induced effect agrees with the light-evoked surround response in the OFF-type BC, but contradicts in the rod BC and the ON-type cone BC. Immunohistochemical study of the Cl − transporter of BCs has suggested that the intracellular Cl − concentration is different among BC subtypes. We examined the reversal potential of GABA-induced current of BCs using gramicidin-perforated patch clamp technique in the mouse retina, and found that GABA depolarizes rod BC and hyperpolarizes cone BCs. Our results are consistent with the GABAergic input to rod BC dendrite.

An alternative pathway for rod signals in the rodent retina: rod photoreceptors, cone bipolar cells, and the localization of glutamate receptors.

In the mammalian retina, extensive processing of spatiotemporal and chromatic information occurs. One key principle in signal transfer through the retina is parallel processing. Two of these parallel pathways are the ON- and OFF-channels transmitting light and dark signals. This dual system is created in the outer plexiform layer, the first relay station in retinal signal transfer. Photoreceptors release glutamate onto ON- and OFF-type bipolar cells, which are functionally distinguished by their postsynaptic expression of different types of glutamate receptors, namely ionotropic and metabotropic glutamate receptors. In the current concept, rod photoreceptors connect only to rod bipolar cells (ON-type) and cone photoreceptors connect only to cone bipolar cells (ON- and OFF-type). We have studied the distribution of (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor subunits at the synapses in the outer plexiform layer of the rodent retina by immunoelectron microscopy and serial section reconstruction. We report a non-classical synaptic contact and an alternative pathway for rod signals in the retina. Rod photoreceptors made synaptic contact with putative OFF-cone bipolar cells that expressed the AMPA glutamate receptor subunits GluR1 and GluR2 on their dendrites. Thus, in the retina of mouse and rat, an alternative pathway for rod signals exists, where rod photoreceptors bypass the rod bipolar cell and directly excite OFF-cone bipolar cells through an ionotropic sign-conserving AMPA glutamate receptor.

Metabotropic Glutamate Receptor-Mediated Suppression of an Inward Rectifier Current Is Linked via a cGMP Cascade.

Glutamate, the neurotransmitter released by photoreceptors, excites horizontal cells and OFF-type bipolar cells by activating ionotropic receptors. This study investigated an additional action of glutamate in which it modulates a voltage-gated ion channel in horizontal cells. We find that glutamate and APB (2-amino-4-phosphonobutyrate) produce a delayed and moderately prolonged suppression of an inward rectifier current (IRK+). This effect is proposed to occur via an APB-sensitive metabotropic glutamate receptor (mGluR) because common agonists for the ionotropic or APB-insensitive mGluRs are ineffective and the APB-insensitive receptor antagonist alpha-methyl-4-carboxyphenylglycine (MCPG) does not block the actions of glutamate or APB. 8-Br-cGMP, 1-methyl-3-isobutylxanthine (IBMX), and atrial natriuretic peptide (ANP) but not 8-Br-cAMP mimic the suppression of IRK+. The effects of glutamate and APB are blocked by protein kinase inhibitors including Rp-8-pCPT-cGMPS, H-8, and H-7 as well as by ATPgammaS. We hypothesize that the APB receptor suppresses IRK+ via upregulation of cGMP and subsequent activation of a cGMP-dependent protein kinase. This pathway is likely regulated by an ATP-dependent phosphorylation. This is a novel signaling pathway for mGluRs and indicates that at least two distinct APB-activated pathways exist in the retina. Functionally, this APB receptor-mediated action found in horizontal cells would provide a means by which spatially restricted changes of glutamate, produced by local illumination of photoreceptors, could regulate IRK+ and consequently the response properties of these neurons. This would serve to adapt selectively retinal regions stimulated by small regions of the visual world.