Faculty Bibliography

Purpose/UNASSIGNED: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/UNASSIGNED: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/UNASSIGNED: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/UNASSIGNED: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.

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., 2016. © 2016 Wiley Periodicals, Inc.

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.

KEY POINTS: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.

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 approximately 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 approximately 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 approximately 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.

Purpose: Secondary cell death via gap junctions plays a critical role in the cell loss associated with neurodegenerative diseases (Belousov and Fontes 2013). In the retina we reported that secondary cell death accounts for most cell loss that occurs under excitotoxic and ischemic conditions (ARVO, 2011). Secondary cell death in CNS may be connexin specific and connexins may be up- or down-regulated under different pathological conditions (Rouach et al., 2002). Here we examined whether secondary cell death of retinal ganglion cells (RGCs) under excitotoxic and ischemic conditions is connexin-specific and whether the expression of Cx36 and Cx45 in inner retina is differentially effected. Methods: Excitotoxicity was induced in vitro by incubation of mouse retinas in NMDA. Transient retinal ischemia was induced in vivo by elevation of IOP. Levels of cell death were assayed histologically and antibodies against Cx36 and Cx45 were used to assess their levels in the IPL. Results: Consistent with our earlier work, we found that excitotoxic and ischemic conditions produced a significant loss of RGCs. Ablation of Cx36 in the Cx36 knockout (KO) mouse retina resulted in an ~70% decrease in RGC loss under excitotoxic conditions, whereas RGC loss in the Cx45 KO retina was not statistically different than that seen in WT mice. In contrast, RGC loss with ischemia was significantly reduced in Cx45 KO retinas, whereas the loss in Cx36 KO retinas was similar to that in the WT. In WT retinas the expression of Cx36 and Cx45 in the IPL followed a punctuate pattern typical for gap junctions. Under excitotoxic conditions the expression of Cx45 was down-regulated, whereas there were no detectable changes in Cx36 expression. In contrast, induction of ischemic conditions produced a dramatic change in Cx36 expression, which appeared as dense clusters around nuclei rather than as puncta. We found no change in the control punctate labeling pattern of Cx45 expression in ischemic retinas. Conclusions: Secondary cell death of RGCs is connexin specific where Cx36 gap junctions play a role under excitotoxic conditions and Cx45 gap junctions play a role during ischemia. These results are consistent with changes in connexin expression seen under these two conditions. These results suggest that targeting of specific connexins can be a novel therapeutic strategy for reducing RGC loss under different pathological conditions

Neurons throughout the brain show spike activity that is temporally correlated to that expressed by their neighbors, yet the generating mechanism(s) remains unclear. In the retina, ganglion cells (GCs) show robust, concerted spiking that shapes the information transmitted to central targets. Here we report the synaptic circuits responsible for generating the different types of concerted spiking of GC neighbors in the mouse retina. The most precise concerted spiking was generated by reciprocal electrical coupling of GC neighbors via gap junctions, whereas indirect electrical coupling to a common cohort of amacrine cells generated the correlated activity with medium precision. In contrast, the correlated spiking with the lowest temporal precision was produced by shared synaptic inputs carrying photoreceptor noise. Overall, our results demonstrate that different synaptic circuits generate the discrete types of GC correlated activity. Moreover, our findings expand our understanding of the roles of gap junctions in the retina, showing that they are essential for generating all forms of concerted GC activity transmitted to central brain targets.

Abstract 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 alpha-ganglion cells (alpha-GCs). These ON responses had the same centre-mediated structure as the classic OFF responses of OFF alpha-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 alpha-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 alpha-GCs, while the classic OFF responses remained. Furthermore, we found that amacrine cells coupled to OFF alpha-GCs display processes in both sublaminae of the IPL, thus forming a plausible substrate for the reception and delivery of ON signals to OFF alpha-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

Neural circuits consist of highly precise connections among specific types of neurons that serve a common functional goal. How neurons distinguish among different synaptic targets to form functionally precise circuits remains largely unknown. Here, we show that during development, the adhesion molecule cadherin-6 (Cdh6) is expressed by a subset of retinal ganglion cells (RGCs) and also by their targets in the brain. All of the Cdh6-expressing retinorecipient nuclei mediate non-image-forming visual functions. A screen of mice expressing GFP in specific subsets of RGCs revealed that Cdh3-RGCs which also express Cdh6 selectively innervate Cdh6-expressing retinorecipient targets. Moreover, in Cdh6-deficient mice, the axons of Cdh3-RGCs fail to properly innervate their targets and instead project to other visual nuclei. These findings provide functional evidence that classical cadherins promote mammalian CNS circuit development by ensuring that axons of specific cell types connect to their appropriate synaptic targets

We examined the effect of light adaptation on the gap junctional coupling of alpha-ganglion cells (alpha-GCs) in rabbit and mouse retinas. We assayed changes in coupling by measuring parameters of tracer coupling following injection of alpha-GCs with Neurobiotin and the concerted spike activity of alpha-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 alpha-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 alpha-GC neighbours, it did significantly increase correlated light-evoked spiking. This was seen as an increase in the number of correlated spikes for alpha-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 alpha-GCs, thereby increasing the concerted activity believed to strengthen the capacity and efficiency of information flow across the optic nerve