Suppression of connexin 43 phosphorylation promotes astrocyte survival and vascular regeneration in proliferative retinopathy.

Degeneration of retinal astrocytes precedes hypoxia-driven pathologic neovascularization and vascular leakage in ischemic retinopathies. However, the molecular events that underlie astrocyte loss remain unclear. Astrocytes abundantly express connexin 43 (Cx43), a transmembrane protein that forms gap junction (GJ) channels and hemichannels. Cx channels can transfer toxic signals from dying cells to healthy neighbors under pathologic conditions. Here we show that Cx43 plays a critical role in astrocyte apoptosis and the resulting preretinal neovascularization in a mouse model of oxygen-induced retinopathy. Opening of Cx43 hemichannels was not observed following hypoxia. In contrast, GJ coupling between astrocytes increased, which could lead to amplification of injury. Accordingly, conditional deletion of Cx43 maintained a higher density of astrocytes in the hypoxic retina. We also identify a role for Cx43 phosphorylation in mediating these processes. Increased coupling in response to hypoxia is due to phosphorylation of Cx43 by casein kinase 1δ (CK1δ). Suppression of this phosphorylation using an inhibitor of CK1δ or in site-specific phosphorylation-deficient mice similarly protected astrocytes from hypoxic damage. Rescue of astrocytes led to restoration of a functional retinal vasculature and lowered the hypoxic burden, thereby curtailing neovascularization and neuroretinal dysfunction. We also find that absence of astrocytic Cx43 does not affect developmental angiogenesis or neuronal function in normoxic retinas. Our in vivo work directly links phosphorylation of Cx43 to astrocytic coupling and apoptosis and ultimately to vascular regeneration in retinal ischemia. This study reveals that targeting Cx43 phosphorylation in astrocytes is a potential direction for the treatment of proliferative retinopathies.

Gap junctional coupling between retinal amacrine and ganglion cells underlies coherent activity integral to global object perception.

Coherent spike activity occurs between widely separated retinal ganglion cells (RGCs) in response to a large, contiguous object, but not to disjointed objects. Since the large spatial separation between the RGCs precludes common excitatory inputs from bipolar cells, the mechanism underlying this long-range coherence remains unclear. Here, we show that electrical coupling between RGCs and polyaxonal amacrine cells in mouse retina forms the synaptic mechanism responsible for long-range coherent activity in the retina. Pharmacological blockade of gap junctions or genetic ablation of connexin 36 (Cx36) subunits eliminates the long-range correlated spiking between RGCs. Moreover, we find that blockade of gap junctions or ablation of Cx36 significantly reduces the ability of mice to discriminate large, global objects from small, disjointed stimuli. Our results indicate that synchronous activity of RGCs, derived from electrical coupling with amacrine cells, encodes information critical to global object perception.

Inhibitory masking controls the threshold sensitivity of retinal ganglion cells.

KEY POINTS: Retinal ganglion cells (RGCs) in dark-adapted retinas show a range of threshold sensitivities spanning ∼3 log units of illuminance. Here, we show that the different threshold sensitivities of RGCs reflect an inhibitory mechanism that masks inputs from certain rod pathways. The masking inhibition is subserved by GABAC receptors, probably on bipolar cell axon terminals. The GABAergic masking inhibition appears independent of dopaminergic circuitry that has been shown also to affect RGC sensitivity. The results indicate a novel mechanism whereby inhibition controls the sensitivity of different cohorts of RGCs. This can limit and thereby ensure that appropriate signals are carried centrally in scotopic conditions when sensitivity rather than acuity is crucial. ABSTRACT: The responses of rod photoreceptors, which subserve dim light vision, are carried through the retina by three independent pathways. These pathways carry signals with largely different sensitivities. Retinal ganglion cells (RGCs), the output neurons of the retina, show a wide range of sensitivities in the same dark-adapted conditions, suggesting a divergence of the rod pathways. However, this organization is not supported by the known synaptic morphology of the retina. Here, we tested an alternative idea that the rod pathways converge onto single RGCs, but inhibitory circuits selectively mask signals so that one pathway predominates. Indeed, we found that application of GABA receptor blockers increased the sensitivity of most RGCs by unmasking rod signals, which were suppressed. Our results indicate that inhibition controls the threshold responses of RGCs under dim ambient light. This mechanism can ensure that appropriate signals cross the bottleneck of the optic nerve in changing stimulus conditions.

Gap junction-mediated death of retinal neurons is connexin and insult specific: a potential target for neuroprotection.

Secondary cell death via gap junctions (GJs) plays a role in the propagation of neuronal loss under a number of degenerative disorders. Here, we examined the role of GJs in neuronal death in the retina, which has arguably the most diverse expression of GJs in the CNS. Initially, we induced apoptotic death by injecting single retinal ganglion cells and glia with cytochrome C and found that this resulted in the loss of neighboring cells to which they were coupled via GJs. We next found that pharmacological blockade of GJs eradicated nearly all amacrine cell loss and reduced retinal ganglion cell loss by ∼70% after induction of either excitotoxic or ischemic insult conditions. These data indicate that the GJ-mediated secondary cell death was responsible for the death of most cells. Whereas genetic deletion of the GJ subunit Cx36 increased cell survivability by ∼50% under excitotoxic condition, cell loss in Cx45 knock-out mouse retinas was similar to that seen in wild-type mice. In contrast, ablation of Cx45 reduced neuronal loss by ∼50% under ischemic insult, but ablation of Cx36 offered no protection. Immunolabeling of the connexins showed differential changes in protein expression consistent with their differing roles in propagating death signals under the two insults. These data indicate that secondary cell death is mediated by different cohorts of GJs dependent on the connexins they express and the type of initial insult. Our results suggest that targeting specific connexins offers a novel therapeutic strategy to reduce progressive cell loss under different neurodegenerative conditions.

The diverse functional roles and regulation of neuronal gap junctions in the retina.

Electrical synaptic transmission through gap junctions underlies direct and rapid neuronal communication in the CNS. The diversity of functional roles that electrical synapses have is perhaps best exemplified in the vertebrate retina, in which gap junctions are formed by each of the five major neuron types. These junctions are dynamically regulated by ambient illumination and by circadian rhythms acting through light-activated neuromodulators such as dopamine and nitric oxide, which in turn activate intracellular signalling pathways in the retina.The networks formed by electrically coupled neurons are plastic and reconfigurable, and those in the retina are positioned to play key and diverse parts in the transmission and processing of visual information at every retinal level.

Neuroprotection of Retinal Ganglion Cells Suppresses Microglia Activation in a Mouse Model of Glaucoma.

Purpose: Microglial activation has been implicated in many neurodegenerative eye diseases, but the interrelationship between cell loss and microglia activation remains unclear. In glaucoma, there is no consensus yet whether microglial activation precedes or is a consequence of retinal ganglion cell (RGC) degeneration. We therefore investigated the temporal and spatial appearance of activated microglia in retina and their correspondence to RGC degeneration in glaucoma. Methods: We used an established microbead occlusion model of glaucoma in mouse whereby intraocular pressure (IOP) was elevated. Specific antibodies were used to immunolabel microglia in resting and activated states. To block retinal gap junction (GJ) communication, which has been shown previously to provide significant neuroprotection of RGCs, the GJ blocker meclofenamic acid was administered or connexin36 (Cx36) GJ subunits were ablated genetically. We then studied microglial activation at different time points after microbead injection in control and neuroprotected retinas. Results: Histochemical analysis of flatmount retinas revealed major changes in microglia morphology, density, and immunoreactivity in microbead-injected eyes. An early stage of microglial activation followed IOP elevation, as indicated by changes in morphology and cell density, but preceded RGC death. In contrast, the later stage of microglia activation, associated with upregulation of major histocompatibility complex class II expression, corresponded temporally to the initial loss of RGCs. However, we found that protection of RGCs afforded by GJ blockade or genetic ablation largely suppressed microglial changes at all stages of activation in glaucomatous retinas. Conclusions: Together, our data strongly suggest that microglia activation in glaucoma is a consequence, rather than a cause, of initial RGC degeneration and death.

A Robust Microbead Occlusion Model of Glaucoma for the Common Marmoset.

Purpose: To establish a robust experimental model of glaucoma in the common marmoset (Callithrix jacchus), a New World primate, using an intracameral microbead injection technique. Methods: Elevated intraocular pressure (IOP) was induced by an injection of polystyrene microbeads. Morphologic changes in the retina and optic nerve of glaucomatous eyes were assessed and electroretinogram (ERG) recordings were performed to evaluate functional changes. Results: Microbead injections induced a sustained IOP elevation for at least 10 weeks in a reproducible manner. At the end of the 10-week experimental period, there was significant loss of retinal ganglion cells (RGCs) in all quadrants and eccentricities, although it was more prominent in the mid-peripheral and peripheral regions. This was consistent with a thinning of the Retinal nerve fiber layer (RNFL) seen in spectral domain optical coherence tomography scans. Surviving RGCs showed marked changes in morphology, including somatic shrinkage and dendritic atrophy. Retinas also showed significant gliosis. The amplitude of the ERG photopic negative response, with subsequent a- and b-wave changes, was reduced in glaucomatous eyes. The optic nerve of glaucomatous eyes showed expanded cupping, disorganization of the astrocytic matrix, axonal loss, and gliosis. Conclusions: We developed a robust and reproducible model of glaucoma in the marmoset. The model exhibits both structural and functional alterations of retina and optic nerve characteristic of glaucoma in humans and animal models. Translational Relevance: The glaucoma model in the marmoset described here forms a robust method to study the disease etiology, progression, and potential therapies in a nonhuman primate, allowing for more effective translation of animal data to humans.

Masked excitatory crosstalk between the ON and OFF visual pathways in the mammalian retina.

A fundamental organizing feature of the visual system is the segregation of ON and OFF responses into parallel streams to signal light increment and decrement. However, we found that blockade of GABAergic inhibition unmasks robust ON responses in OFF α-ganglion cells (α-GCs). These ON responses had the same centre-mediated structure as the classic OFF responses of OFF α-GCs, but were abolished following disruption of the ON pathway with L-AP4. Experiments showed that both GABA(A) and GABA(C) receptors are involved in the masking inhibition of this ON response, located at presynaptic inhibitory synapses on bipolar cell axon terminals and possibly amacrine cell dendrites. Since the dendrites of OFF α-GCs are not positioned to receive excitatory inputs from ON bipolar cell axon terminals in sublamina-b of the inner plexiform layer (IPL), we investigated the possibility that gap junction-mediated electrical synapses made with neighbouring amacrine cells form the avenue for reception of ON signals. We found that the application of gap junction blockers eliminated the unmasked ON responses in OFF α-GCs, while the classic OFF responses remained. Furthermore, we found that amacrine cells coupled to OFF α-GCs display processes in both sublaminae of the IPL, thus forming a plausible substrate for the reception and delivery of ON signals to OFF α-GCs. Finally, using a multielectrode array, we found that masked ON and OFF signals are displayed by over one-third of ganglion cells in the rabbit and mouse retinas, suggesting that masked crossover excitation is a widespread phenomenon in the inner mammalian retina.

Neuroprotection of the Inner Retina Also Prevents Secondary Outer Retinal Pathology in a Mouse Model of Glaucoma.

Purpose: We examined structural and functional changes in the outer retina of a mouse model of glaucoma. We examined whether these changes are a secondary consequence of damage in the inner retina and whether neuroprotection of the inner retina also prevents outer retinal changes. Methods: We used an established microbead occlusion model of glaucoma whereby intraocular pressure (IOP) was elevated. Specific antibodies were used to label rod and cone bipolar cells (BCs), horizontal cells (HCs), and retinal ganglion cells (RGCs), as well as synaptic components in control and glaucomatous eyes, to assess structural damage and cell loss. ERG recordings were made to assess outer retina function. Results: We found structural and functional damage of BCs, including significant cell loss and dendritic/axonal remodeling of HCs, following IOP elevation. The first significant loss of both BCs occurred at 4 to 5 weeks after microbead injection. However, early changes in the dendritic structure of RGCs were observed at 3 weeks, but significant changes in the rod BC axon terminal structure were not seen until 4 weeks. We found that protection of inner retinal neurons in glaucomatous eyes by pharmacological blockade of gap junctions or genetic ablation of connexin 36 largely prevented outer retinal damage. Conclusions: Together, our results indicate that outer retinal impairments in glaucoma are a secondary sequalae of primary damage in the inner retina. The finding that neuroprotection of the inner retina can also prevent outer retinal damage has important implications with regard to the targets for effective neuroprotective therapy.

Light increases the gap junctional coupling of retinal ganglion cells.

We examined the effect of light adaptation on the gap junctional coupling of α-ganglion cells (α-GCs) in rabbit and mouse retinas. We assayed changes in coupling by measuring parameters of tracer coupling following injection of α-GCs with Neurobiotin and the concerted spike activity of α-GC neighbours under dark- and light-adapted conditions. We found that light adaptation using mesopic or photopic background lights resulted in a dramatic increase in the labelling intensity, number, and spatial extent of ganglion and amacrine cells coupled to OFF α-GCs when compared to levels seen under dark adaptation. While this augmentation of coupling by light did not produce an increase in the concerted spontaneous activity of OFF α-GC neighbours, it did significantly increase correlated light-evoked spiking. This was seen as an increase in the number of correlated spikes for α-GC neighbours and an extension of correlations to second-tier neighbours that was not seen under dark-adapted conditions. Pharmacological studies in the rabbit retina indicated that dopamine mediates the observed changes in coupling by differentially activating D1 and D2 receptors under different adaptation states. In this scheme, activation of dopamine D1 receptors following light exposure triggers cAMP-mediated intracellular pathways resulting in an increase in gap junctional conductance. Overall, our results indicate that as we move from night to day there is an enhanced electrical coupling between α-GCs, thereby increasing the concerted activity believed to strengthen the capacity and efficiency of information flow across the optic nerve.

GABA blockade unmasks an OFF response in ON direction selective ganglion cells in the mammalian retina.

One unique subtype of retinal ganglion cell is the direction selective (DS) cell, which responds vigorously to stimulus movement in a preferred direction, but weakly to movement in the opposite or null direction. Here we show that the application of the GABA receptor blocker picrotoxin unmasks a robust excitatory OFF response in ON DS ganglion cells. Similar to the characteristic ON response of ON DS cells, the masked OFF response is also direction selective, but its preferred direction is opposite to that of the ON component. Given that the OFF response is unmasked with picrotoxin, its direction selectivity cannot be generated by a GABAergic mechanism. Alternatively, we find that the direction selectivity of the OFF response is blocked by cholinergic drugs, suggesting that acetylcholine release from presynaptic starburst amacrine cells is crucial for its generation. Finally, we find that the OFF response is abolished by application of a gap junction blocker, suggesting that it arises from electrical synapses between ON DS and polyaxonal amacrine cells. Our results suggest a novel role for gap junctions in mixing excitatory ON and OFF signals at the ganglion cell level. We propose that OFF inputs to ON DS cells are normally masked by a GABAergic inhibition, but are unmasked under certain stimulus conditions to mediate optokinetic signals in the brain.

Amacrine cells coupled to ganglion cells via gap junctions are highly vulnerable in glaucomatous mouse retinas.

We determined whether the structural and functional integrity of amacrine cells (ACs), the largest cohort of neurons in the mammalian retina, are affected in glaucoma. Intraocular injection of microbeads was made in mouse eyes to elevate intraocular pressure as a model of experimental glaucoma. Specific immunocytochemical markers were used to identify AC and displaced (d)ACs subpopulations in both the inner nuclear and ganglion cell layers, respectively, and to distinguish them from retinal ganglion cells (RGCs). Calretinin- and γ-aminobutyric acid (GABA)-immunoreactive (IR) cells were highly vulnerable to glaucomatous damage, whereas choline acetyltransferase (ChAT)-positive and glycinergic AC subtypes were unaffected. The AC loss began 4 weeks after initial microbead injection, corresponding to the time course of RGC loss. Recordings of electroretinogram (ERG) oscillatory potentials and scotopic threshold responses, which reflect AC and RGC activity, were significantly attenuated in glaucomatous eyes following a time course that matched that of the AC and RGC loss. Moreover, we found that it was the ACs coupled to RGCs via gap junctions that were lost in glaucoma, whereas uncoupled ACs were largely unaffected. Our results suggest that AC loss in glaucoma occurs secondary to RGC death through the gap junction-mediated bystander effect. J. Comp. Neurol. 527:159-173, 2019. © 2016 Wiley Periodicals, Inc.

Connexin36 is essential for transmission of rod-mediated visual signals in the mammalian retina.

To examine the functions of electrical synapses in the transmission of signals from rod photoreceptors to ganglion cells, we generated connexin36 knockout mice. Reporter expression indicated that connexin36 was present in multiple retinal neurons including rod photoreceptors, cone bipolar cells, and AII amacrine cells. Disruption of electrical synapses between adjacent AIIs and between AIIs and ON cone bipolars was demonstrated by intracellular injection of Neurobiotin. In addition, extracellular recording in the knockout revealed the complete elimination of rod-mediated, on-center responses at the ganglion cell level. These data represent direct proof that electrical synapses are critical for the propagation of rod signals across the mammalian retina, and they demonstrate the existence of multiple rod pathways, each of which is dependent on electrical synapses.

Plasticity of Retinal Gap Junctions: Roles in Synaptic Physiology and Disease.

Electrical synaptic transmission via gap junctions underlies direct and rapid neuronal communication in the central nervous system. The diversity of functional roles played by electrical synapses is perhaps best exemplified in the vertebrate retina, in which gap junctions are expressed by each of the five major neuronal types. These junctions are highly plastic; they are dynamically regulated by ambient illumination and circadian rhythms acting through light-activated neuromodulators. The networks formed by electrically coupled neurons provide plastic, reconfigurable circuits positioned to play key and diverse roles in the transmission and processing of visual information at every retinal level. Recent work indicates gap junctions also play a role in the progressive cell death and aberrant activity seen in various pathological conditions of the retina. Gap junctions thus form potential targets for novel neuroprotective therapies in the treatment of neurodegenerative retinal diseases such as glaucoma and ischemic retinopathies.

Light-induced changes in spike synchronization between coupled ON direction selective ganglion cells in the mammalian retina.

Although electrical coupling via gap junctions is prevalent among ganglion cells in the vertebrate retina, there have been few direct studies of their influence on the light-evoked signaling of these cells. Here, we describe the pattern and function of coupling between the ON direction selective (DS) ganglion cells, a unique subtype whose signals are transmitted to the accessory optic system (AOS) where they initiate the optokinetic response. ON DS cells are coupled indirectly via gap junctions made with a subtype of polyaxonal amacrine cell. This coupling underlies synchronization of the spontaneous and light-evoked spike activity of neighboring ON DS cells. However, we find that ON DS cell pairs show robust synchrony for all directions of stimulus movement, except for the null direction. Null stimulus movement evokes a GABAergic inhibition that temporally shifts firing of ON DS cell neighbors, resulting in a desynchronization of spike activity. Thus, detection of null stimulus movement appears key to the direction selectivity of ON DS cells, evoking both an attenuation of spike frequency and a desynchronization of neighbors. We posit that active desynchronization reduces summation of synaptic potentials at target AOS cells and thus provides a secondary mechanism by which ON DS cell ensembles can signal direction of stimulus motion to the brain.

Targeting neuronal gap junctions in mouse retina offers neuroprotection in glaucoma.

The progressive death of retinal ganglion cells and resulting visual deficits are hallmarks of glaucoma, but the underlying mechanisms remain unclear. In many neurodegenerative diseases, cell death induced by primary insult is followed by a wave of secondary loss. Gap junctions (GJs), intercellular channels composed of subunit connexins, can play a major role in secondary cell death by forming conduits through which toxic molecules from dying cells pass to and injure coupled neighbors. Here we have shown that pharmacological blockade of GJs or genetic ablation of connexin 36 (Cx36) subunits, which are highly expressed by retinal neurons, markedly reduced loss of neurons and optic nerve axons in a mouse model of glaucoma. Further, functional parameters that are negatively affected in glaucoma, including the electroretinogram, visual evoked potential, visual spatial acuity, and contrast sensitivity, were maintained at control levels when Cx36 was ablated. Neuronal GJs may thus represent potential therapeutic targets to prevent the progressive neurodegeneration and visual impairment associated with glaucoma.

Gap junctional coupling underlies the short-latency spike synchrony of retinal alpha ganglion cells.

We examined whether coupling between neighboringalpha-type ganglion cells (alpha-GCs) in the rabbit retina underlies their synchronous spike activity. Simultaneous recordings were made from arrays of alpha-GCs to determine the synchrony of both spontaneous and light-evoked spike activity. One cell within each array was then injected with the biotinylated tracer Neurobiotin to determine which of the cells were coupled via gap junctions. Cross-correlation analyses indicated that neighboring off-center alpha-GCs maintain short-latency (approximately 2.5 msec) synchronous spiking, whereas the spontaneous spike activities of on-centeralpha-GC neighbors are not correlated. Without exception, those off-centeralpha-GCs showing synchronous spiking were found to be tracer coupled to both amacrine cells and neighboring off-centeralpha-GCs. In contrast, on-center alpha-GCs were never tracer coupled. Furthermore, whereas spikes initiated in an off-center alpha-GC with extrinsic current injection resulted in short-latency synchronized spiking in neighboring off-center alpha-GCs, this was never seen between on-center alpha-GCs. These results indicate that electrical coupling via gap junctions underlies the short-latency concerted spike activity of neighboring alpha-GCs.

Convergence and segregation of the multiple rod pathways in mammalian retina.

Using a multidisciplinary approach, we demonstrate that three different pathways are responsible for the transmission of rod signals across the mouse retina. Each pathway serves a primarily nonoverlapping range of stimulus intensities, with ganglion cells receiving either segregated or convergent inputs. For both on-center (ON) and off-center (OFF) ganglion cells, the primary rod pathway carries signals with the lowest threshold, whereas the secondary rod pathway is less sensitive by approximately 1 log unit. In addition, OFF signaling uses a tertiary rod pathway that is approximately 1 log unit less sensitive than the secondary. Although some ganglion cells received rod inputs exclusively from one of the pathways, others showed convergent inputs. Using pharmacological and genetic approaches, we defined classes of ON and OFF ganglion cells for which the scotopic inputs derive only from the primary pathway or from both primary and secondary pathways. In addition, we observed a class of OFF ganglion cell receiving mixed input from primary and tertiary pathways. Interestingly, OFF ganglion cells receiving convergent inputs from all three rod pathways or from the secondary and tertiary pathways together were never observed. Overall, our data show a complex arrangement of convergence and segregation of rod inputs to ganglion cells in the mammalian retina.

A unique role for Kv3 voltage-gated potassium channels in starburst amacrine cell signaling in mouse retina.

Direction-selective retinal ganglion cells show an increased activity evoked by light stimuli moving in the preferred direction. This selectivity is governed by direction-selective inhibition from starburst amacrine cells occurring during stimulus movement in the opposite or null direction. To understand the intrinsic membrane properties of starburst cells responsible for direction-selective GABA release, we performed whole-cell recordings from starburst cells in mouse retina. Voltage-clamp recordings revealed prominent voltage-dependent K(+) currents. The currents were mostly blocked by 1 mm TEA, activated rapidly at voltages more positive than -20 mV, and deactivated quickly, properties reminiscent of the currents carried by the Kv3 subfamily of K+ channels. Immunoblots confirmed the presence of Kv3.1 and Kv3.2 proteins in retina and immunohistochemistry revealed their expression in starburst cell somata and dendrites. The Kv3-like current in starburst cells was absent in Kv3.1-Kv3.2 knock-out mice. Current-clamp recordings showed that the fast activation of the Kv3 channels provides a voltage-dependent shunt that limits depolarization of the soma to potentials more positive than -20 mV. This provides a mechanism likely to contribute to the electrical isolation of individual starburst cell dendrites, a property thought essential for direction selectivity. This function of Kv3 channels differs from that in other neurons where they facilitate high-frequency repetitive firing. Moreover, we found a gradient in the intensity of Kv3.1b immunolabeling favoring proximal regions of starburst cells. We hypothesize that this Kv3 channel gradient contributes to the preference for centrifugal signal flow in dendrites underlying direction-selective GABA release from starburst amacrine cells

Morphology and tracer coupling pattern of alpha ganglion cells in the mouse retina.

Alpha cells are a type of ganglion cell whose morphology appears to be conserved across a number of mammalian retinas. In particular, alpha cells display the largest somata and dendritic arbors at a given eccentricity and tile the retina as independent on- (ON) and off-center (OFF) subtypes. Mammalian alpha cells also express a variable tracer coupling pattern, which often includes homologous (same cell type) coupling to a few neighboring alpha cells and extensive heterologous (different cell type) coupling to two to three amacrine cell types. Here, we use the gap junction-permeant tracer Neurobiotin to determine the architecture and coupling pattern of alpha cells in the mouse retina. We find that alpha cells show the same somatic and dendritic architecture described previously in the mammal. However, alpha cells show varied tracer coupling patterns related to their ON and OFF physiologies. ON alpha cells show no evidence of homologous tracer coupling but are coupled heterologously to at least two types of amacrine cell whose somata lie within the ganglion cell layer. In contrast, OFF alpha cells are coupled to one another in circumscribed arrays as well as to two to three types of amacrine cell with somata occupying the inner nuclear layer. We find that homologous coupling between OFF alpha cells is unaltered in the connexin36 (Cx36) knockout (KO) mouse retina, indicating that it is not dependent on Cx36. However, a subset of the heterologous coupling of ON alpha cells and all the heterologous coupling of OFF alpha cells are eliminated in the KO retina, suggesting that Cx36 comprises most of the junctions made with amacrine cells.