Severe photoreceptor cell death in retinal degenerative diseases leads to partial or complete blindness. Optogenetics is a promising strategy to treat blindness. The feasibility of this strategy has been demonstrated through the ectopic expression of microbial channelrhodopsins (ChRs) and other genetically encoded light sensors in surviving retinal neurons in animal models. A major drawback for ChR-based visual restoration is low light sensitivity. Here, we report the development of highly operational light-sensitive ChRs by optimizing the kinetics of a recently reported ChR variant, Chloromonas oogama (CoChR). In particular, we identified two CoChR mutants, CoChR-L112C and CoChR-H94E/L112C/K264T, with markedly enhanced light sensitivity. The improved light sensitivity of the CoChR mutants was confirmed by ex vivo electrophysiological recordings in the retina. Furthermore, the CoChR mutants restored the vision of a blind mouse model under ambient light conditions with remarkably good contrast sensitivity and visual acuity, as evidenced by the results of behavioral assays. The ability to restore functional vision under normal light conditions with the improved CoChR variants removed a major obstacle for ChR-based optogenetic vision restoration.

In axon-bearing neurons, action potentials conventionally initiate at the axon initial segment (AIS) and are important for neuron excitability and cell-to-cell communication. However in axonless neurons, spike origin has remained unclear. Here we report in the axonless spiking AII amacrine cell of the mouse retina a dendritic process sharing organizational and functional similarities with the AIS. This process was revealed through viral-mediated expression of channelrhodopsin-2-GFP (ChR2-GFP) with the AIS-targeting motif of sodium channels (NavII-III). The AII-processes showed clustering of voltage-gated Na+ channel 1.1 (Nav1.1) as well as AIS markers ankyrin-G and neurofascin. Furthermore, NavII-III targeting disrupted Nav1.1 clustering in the AII-process which drastically decreased Na+ current and abolished the ability of the AII amacrine cell to generate spiking. Our findings indicate that despite lacking an axon, spiking in the axonless neuron can originate at a specialized AIS-like process.

The ectopic expression of microbial opsin-based optogenetic sensors, such as channelrhodopsin-2 (ChR2) in surviving inner retinal neurons, is a promising approach to restoring vision after retinal degeneration. However, a major limitation in using native ChR2 as a light sensor for vision restoration is the low light sensitivity of its expressing cells. Recently, two ChR2 mutations, T159C and L132C, were reported to produce higher photocurrents or have ultra light sensitivity. In this study, we created additional ChR2 mutants at these two sites to search for more light responsive ChR2 forms and evaluate their suitability for vision restoration by examining their light responsive properties in HEK cells and mouse retinal ganglion cells. We found additional ChR2 mutants at these two sites that showed a further increase in current amplitude at low light levels in the cells expressing these mutants, or operational light sensitivity. However, the increase in the operational light sensitivity was correlated with a decrease in temporal kinetics. Therefore, there is a trade-off between operational light sensitivity and temporal resolution for these more light responsive ChR2 mutants. Our results showed that for the two most light responsive mutants, L132C/T159C and L132C/T159S, the required light intensities for generating the threshold spiking activity in retinal ganglion cells were 1.5 and nearly 2 log units lower than wild-type ChR2 (wt-ChR2), respectively. Additionally, their ChR2-mediated spiking activities could follow flicker frequencies up to 20 and 10 Hz, respectively, at light intensities up to 1.5 log units above their threshold levels. Thus, the use of these more light responsive ChR2 mutants could make the optogenetic approach to restoring vision more feasible.

PurposeTo develop an animal behavioral assay for the quantitative assessment of the functional efficacy of optogenetic therapies.MethodsA triple-knockout (TKO) mouse line, Gnat1−/−Cnga3−/−Opn4−/−, and a double-knockout mouse line, Gnat1−/−Cnga3−/−, were employed. The expression of channelrhodopsin-2 (ChR2) and its three more light-sensitive mutants, ChR2-L132C, ChR2-L132C/T159C, and ChR2-132C/T159S, in inner retinal neurons was achieved using rAAV2 vectors via intravitreal delivery. Pupillary constriction was assessed by measuring the pupil diameter. The optomotor response (OMR) was examined using a homemade optomotor system equipped with light-emitting diodes as light stimulation.ResultsA robust OMR was restored in the ChR2-mutant-expressing TKO mice; however, significant pupillary constriction was observed only for the ChR2-L132C/T159S mutant. The ability to evoke an OMR was dependent on both the light intensity and grating frequency. The most light-sensitive frequency for the three ChR2 mutants was approximately 0.042 cycles per degree. Among the three ChR2 mutants, ChR2-L132C/T159S was the most light sensitive, followed by ChR2-L132C/T159C and ChR2-L132C. Melanopsin-mediated pupillary constriction resulted in a substantial reduction in the light sensitivity of the ChR2-mediated OMR.ConclusionsThe OMR assay using TKO mice enabled the quantitative assessment of the efficacy of different optogenetic tools and the properties of optogenetically restored vision. Thus, the assay can serve as a valuable tool for developing effective optogenetic therapies.

The loss of photoreceptors in individuals with retinal degenerative diseases leads to partial or complete blindness. Optogenetic therapy is a promising approach for restoring vision to the blind. Multiple strategies have been employed by targeting genetically encoded light sensors, particularly channelrhodopsins, to surviving retinal neurons in animal models. In particular, the strategy of targeting retinal bipolar cells has commonly been expected to result in better vision than ubiquitous expression in retinal ganglion cells. However, a direct comparison of the channelrhodopsin-restored vision between these two strategies has not been performed. Here, we compared the restored visual functions achieved by adenoassociated virus (AAV)-mediated expression of a channelrhodopsin in ON-type bipolar cells and retinal ganglion cells driven by an improved mGluR6 promoter and a CAG promoter, respectively, in a blind mouse model by performing electrophysiological recordings and behavioral assessments. Unexpectedly, the efficacy of the restored vision based on light sensitivity and visual acuity was much higher following ubiquitous retinal ganglion cell expression than that of the strategy targeting ON-type bipolar cells. Our study suggests that, at least based on currently available gene delivery techniques, the expression of genetically encoded light sensors in retinal ganglion cells is likely a practical and advantageous strategy for optogenetic vision restoration.

The axon initial segment (AIS) is essential for initiating action potentials and maintaining neuronal excitability in axon-bearing neurons in the CNS. There is increasing interest in the targeting of optogenetic tools to subcellular compartments, including the AIS, to gain precise control of neuronal activity for basic research and clinical applications. In particular, targeted expression of optogenetic tools in retinal ganglion cells (RGCs) has been explored as an approach for restoring vision after photoreceptor degeneration. Thus, understanding the effects of such targeting on spiking abilities and/or patterns is important. Here, we examined the effects of recombinant adeno-associated virus (rAAV)-mediated targeted expression of channelrhodopsin-2 (ChR2)-GFP with a NaV channel motif in mouse RGCs. We found that this targeted expression disrupted NaV channel clustering at the AIS and converted the spike firing patterns of RGCs from sustained to transient. Our results suggest that the clustering of membrane channels, including NaV channels, at the AIS is important for the ability of RGCs to generate sustained spike firing. Additionally, the targeting of optogenetic tools to the AIS with the NaV channel motif may offer a way to create transient light responses in RGCs for vision restoration.

Retinal bipolar cells relay visual information from photoreceptors to third-order retinal neurons. Bipolar cells, comprising multiple types, play an essential role in segregating visual information into multiple parallel pathways in the retina. The identification of molecular markers that can label specific retinal bipolar cells could facilitate the investigation of bipolar cell functions in the retina. Transgenic mice with specific cell type(s) labeled with green fluorescent protein (GFP) have become a powerful tool for morphological and functional studies of neurons in the CNS, including the retina. In this study, we report a 5-hydroxytryptamine receptor 2a (5-HTR2a) transgenic mouse line in which expression of GFP was observed in two populations of bipolar cells in the retina. Based on the terminal stratification and immunostaining, all the strongly GFP-labeled bipolar cells were found to be type 4 cone bipolar cells. A small population of weakly labeled bipolar cells was also observed, which may represent type 8 or 9 cone bipolar cells. GFP expression in retinal cone bipolar cells was seen as early as postnatal day 5. In addition, despite severe retinal degeneration due to an rd1 (or Pde6brd1) gene in this transgenic line, the density of GFP-labeled cone bipolar cells remained stable up to at least 6 months of the age. This transgenic mouse line will be a useful tool for the study of type 4 cone bipolar cells in the retina under both normal and disease conditions.

Retinal bipolar cells and ganglion cells are known to possess voltage-gated T-type Ca2+ channels. Previous electrophysiological recording studies suggested that there is differential expression of different T-type Ca2+ channel α1 subunits among bipolar cells. The detailed expression patterns of the individual T-type Ca2+ channel subunits in the retina, however, remain unknown. In this study, we examined the expression of the Cav3.2 Ca2+ channel α1 subunit in the mouse retina using immunohistochemical analysis and patch-clamp recordings together with a Cav3.2 knock out (KO) mouse line. The specificity of a Cav3.2 Ca2+ channel antibody was first confirmed in recombinant T-type Ca2+ channels expressed in HEK (human embryonic kidney) cells and in Cav3.2 KO mice. Our immunohistochemical analysis indicates that the Cav3.2 antibody labels a subgroup of type-3 cone bipolar cells (CBCs), the PKAβII-immunopositive type-3 CBCs. The labeling was observed throughout the cell including dendrites and axon terminals. Our patch-clamp recording results further demonstrate that Cav3.2 Ca2+ channels contribute to the T-type Ca2+ current in a subpopulation of type-3 CBCs. The findings of this study provide new insights into understanding the functional roles of T-type Ca2+ channels in retinal processing.