Optogenetic Strategies for Vision Restoration.

The loss of photoreceptor cells caused by retinal degenerative diseases leads to blindness. The optogenetic approach for restoring vision involves converting the surviving inner retinal neurons into photosensitive cells, thus imparting light sensitivity to the retina following the loss of photoreceptor cells. Our first demonstration of the feasibility of such an approach involved expressing ChR2 in the retinal ganglion cells of blind mice; since then, optogenetic vision restoration has been demonstrated by using a variety of optogenetic tools, especially microbial channelrhodopsins (ChRs). A ChR-based optogenetic therapy for treating blindness has advanced to clinical trials. In this chapter, we review our early proof-of-concept study of optogenetic vision restoration. We also discuss our studies for developing better ChR tools and for restoring intrinsic visual processing features in retinas with degenerated photoreceptors.

Improved CoChR Variants Restore Visual Acuity and Contrast Sensitivity in a Mouse Model of Blindness under Ambient Light Conditions.

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.

Sex-related differences in the progressive retinal degeneration of the rd10 mouse.

The retinal degeneration 10 (rd10) mouse is a model of autosomal recessive retinitis pigmentosa (RP), a disease that causes blindness through the progressive loss of photoreceptors. This study shows evidence of sex-related differences in RP onset and progression in rd10 retinas. The disease onset was considerably earlier in the female rd10 mice than in the male rd10 mice, as evidenced by a loss of PDE6ß proteins and rod-dominated electroretinogram (ERG) responses at an early age. Single photopic flash and flicker ERG responses and immunolabeling of opsin molecules were analyzed in both genders to assess the sex differences in the degeneration of cones in the RP retinas. The averaged amplitudes of cone-mediated ERG responses obtained from the females were significantly smaller than the amplitudes of the responses from the age-matched males in the late stages of the RP, suggesting that cones might degenerate faster in the female retinas as the disease progressed. The rapid degeneration of cones caused a more substantial decrease in the ERG responses derived from the On-pathway than the Off-pathway in the females. In addition, the male rd10 mice had heavier body weights than their female counterparts aged between postnatal (P)18 and P50 days. In summary, female rd10 mice were more susceptible to retinal degeneration, suggesting that the female sex might be a risk factor for RP. The results have important implications for future studies exploring potential sex-related differences in RP development and progression in the clinic.

Cre-mediated recombination efficiency and transgene expression patterns of three retinal bipolar cell-expressing Cre transgenic mouse lines.

PURPOSE: Retinal bipolar cells, comprising multiple types, play an essential role in segregating visual information into multiple parallel pathways in the retina. The ability to manipulate gene expression in specific bipolar cell type(s) in the retina is important for understanding the molecular basis of their normal physiological functions and diseases/disorders. The Cre/LoxP recombination system has become an important tool for allowing gene manipulation in vivo, especially with the increasing availability of cell- and tissue-specific Cre transgenic mouse lines. Detailed in vivo examination of the Cre/LoxP recombination efficiency and the transgene expression patterns for cell- and tissue-specific Cre transgenic mouse lines is essential for evaluating their utility. In this study, we investigated the Cre-mediated recombination efficiency and transgene expression patterns of retinal bipolar cell-expressing Cre transgenic lines by crossing with a Cre reporter mouse line and through Cre-dependent recombinant adeno-associated virus (rAAV) vector-mediated transgene delivery. METHODS: Three retinal bipolar cell-expressing Cre-transgenic mouse lines, 5-HTR2a-cre, Pcp2-cre, and Chx10-cre, were crossed with a strong Cre reporter mouse line that expresses a red fluorescent protein variant, tdTomato. rAAV2 vectors carrying a double-floxed inverted open-reading frame sequence encoding channelrhodopsin-2-mCherry (ChR2-mCherry) driven by a ubiquitous neuronal EF1α or a ubiquitous CMV promoter with a rAAV2 capsid mutation (Y444F) were injected into the intravitreal space of the eyes. Immunohistochemistry using retinal bipolar cell type-specific markers was performed to examine Cre-mediated recombination efficiency and the transgene expression patterns in bipolar cells in retinal whole mounts and vertical sections. RESULTS: For the 5-HTR2a-cre and Pcp2-cre mouse lines, the expression pattern of the Cre-mediated recombination by crossing the reporter line largely resembled the expression pattern of Cre. The bipolar cells showing Cre-mediated recombination in the 5-HTR2a-cre line and the Pcp2-cre line were predominantly type 4 cone bipolar cells and rod bipolar cells, respectively. For the Chx10-cre mouse line, the expression pattern of the Cre-mediated recombination by crossing the reporter line was different from that of Cre. The Cre-mediated transgene expression in retinal bipolar cells in the Chx10-cre line was not observed by crossing with the reporter mouse line but through Cre-dependent rAAV vector delivery. A rAAV2 vector with the combination of a CMV promoter and the Y444F capsid mutation achieved Cre-dependent transgene expression in retinal bipolar cells. CONCLUSIONS: The retinal bipolar cell-expressing Cre-transgenic lines and the Cre-dependent rAAV vector reported in this study could be valuable tools for gene targeting and manipulation in retinal bipolar cells in mice.

Action potential generation at an axon initial segment-like process in the axonless retinal AII amacrine cell.

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 with the AIS-targeting motif of sodium channels (Na(v)II-III). The AII processes showed clustering of voltage-gated Na+ channel 1.1 (Na(v)1.1) as well as AIS markers ankyrin-G and neurofascin. Furthermore, Na(v)II-III targeting disrupted Na(v)1.1 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.

Targeted Expression of Retinoschisin by Retinal Bipolar Cells in XLRS Promotes Resolution of Retinoschisis Cysts Sans RS1 From Photoreceptors.

Purpose: Loss of retinoschisin (RS1) function underlies X-linked retinoschisis (XLRS) pathology. In the retina, both photoreceptor inner segments and bipolar cells express RS1. However, the loss of RS1 function causes schisis primarily in the inner retina. To understand these cell type-specific phenotypes, we decoupled RS1 effects in bipolar cells from that in photoreceptors. Methods: Bipolar cell transgene RS1 expression was achieved using two inner retina-specific promoters: (1) a minimal promoter engineered from glutamate receptor, metabotropic glutamate receptor 6 gene (mini-mGluR6/ Grm6) and (2) MiniPromoter (Ple155). Adeno-associated virus vectors encoding RS1 gene under either the mini-mGluR6 or Ple-155 promoter were delivered to the XLRS mouse retina through intravitreal or subretinal injection on postnatal day 14. Retinal structure and function were assessed 5 weeks later: immunohistochemistry for morphological characterization, optical coherence tomography and electroretinography (ERG) for structural and functional evaluation. Results: Immunohistochemical analysis of RS1expression showed that expression with the MiniPromoter (Ple155) was heavily enriched in bipolar cells. Despite variations in vector penetrance and gene transfer efficiency across the injected retinas, those retinal areas with robust bipolar cell RS1 expression showed tightly packed bipolar cells with fewer cavities and marked improvement in inner retinal structure and synaptic function as judged by optical coherence tomography and electroretinography, respectively. Conclusions: These results demonstrate that RS1 gene expression primarily in bipolar cells of the XLRS mouse retina, independent of photoreceptor expression, can ameliorate retinoschisis structural pathology and provide further evidence of RS1 role in cell adhesion.

Optogenetics for light control of biological systems.

Optogenetic techniques have been developed to allow control over the activity of selected cells within a highly heterogeneous tissue, using a combination of genetic engineering and light. Optogenetics employs natural and engineered photoreceptors, mostly of microbial origin, to be genetically introduced into the cells of interest. As a result, cells that are naturally light-insensitive can be made photosensitive and addressable by illumination and precisely controllable in time and space. The selectivity of expression and subcellular targeting in the host is enabled by applying control elements such as promoters, enhancers and specific targeting sequences to the employed photoreceptor-encoding DNA. This powerful approach allows precise characterization and manipulation of cellular functions and has motivated the development of advanced optical methods for patterned photostimulation. Optogenetics has revolutionized neuroscience during the past 15 years and is primed to have a similar impact in other fields, including cardiology, cell biology and plant sciences. In this Primer, we describe the principles of optogenetics, review the most commonly used optogenetic tools, illumination approaches and scientific applications and discuss the possibilities and limitations associated with optogenetic manipulations across a wide variety of optical techniques, cells, circuits and organisms.

Selecting Channelrhodopsin Constructs for Optimal Visual Restoration in Differing Light Conditions.

Channelrhodopsin (ChR)-based optogenetics is one promising approach to restore vision in photoreceptor degenerative diseases such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Currently, a large number of ChRs from different alga species as well as engineered variants are available. They vary with their light response properties like peak sensitive wavelength (λmax), current amplitude, and kinetics. Therefore, it is important to choose an appropriate ChR for practical applications, such as vision restoration. Here we describe a standard laboratory protocol for characterizing properties of ChRs in in vitro in human embryonic kidney (HEK) cells. Based on such characterization, we also discuss the criteria for selecting optimal ChRs for optogenetic vision restoration.

Ectopic expression of a microbial-type rhodopsin restores visual responses in mice with photoreceptor degeneration.

The death of photoreceptor cells caused by retinal degenerative diseases often results in a complete loss of retinal responses to light. We explore the feasibility of converting inner retinal neurons to photosensitive cells as a possible strategy for imparting light sensitivity to retinas lacking rods and cones. Using delivery by an adeno-associated viral vector, here, we show that long-term expression of a microbial-type rhodopsin, channelrhodopsin-2 (ChR2), can be achieved in rodent inner retinal neurons in vivo. Furthermore, we demonstrate that expression of ChR2 in surviving inner retinal neurons of a mouse with photoreceptor degeneration can restore the ability of the retina to encode light signals and transmit the light signals to the visual cortex. Thus, expression of microbial-type channelrhodopsins, such as ChR2, in surviving inner retinal neurons is a potential strategy for the restoration of vision after rod and cone degeneration.

Ectopic expression of multiple microbial rhodopsins restores ON and OFF light responses in retinas with photoreceptor degeneration.

By expressing channel rhodopsin-2 (ChR2) in inner retinal neurons, previous studies have demonstrated restoration of ON responses in the retina after the death of rod and cone photoreceptors. In this study, we report that the expression of halorhodopsin (HaloR), a light-driven chloride pump, can effectively restore OFF responses in inner retinal neurons of mice with retinal degeneration. We show that HaloR-expressing retinal ganglion cells respond to light with rapid hyperpolarization and suppression of spike activity. After termination of the light stimulus, their membrane potential exhibits a rapid rebound overshoot with robust sustained or transient spike firing. Furthermore, we show that coexpression of ChR2/HaloR in retinal ganglion cells can produce ON, OFF, and even ON-OFF responses, depending on the wavelength of the light stimulus. Our results suggest that the expression of multiple microbial rhodopsins such as ChR2 and HaloR is a possible strategy to restore both ON and OFF light responses in the retina after the death of rod and cone photoreceptors.

Retinal channelrhodopsin-2-mediated activity in vivo evaluated with manganese-enhanced magnetic resonance imaging.

PURPOSE: Ectopic expression of light-sensitive proteins, such as channelrhodopsin-2, represent a novel approach for restoring light-detection capabilities to degenerated retina. A noninvasive method that can detect light-mediated activities of such light-sensitive proteins in the retina in vivo would be important for correlating expression patterns and retinal function. In this study, we tested the hypothesis that retinal uptake of manganese, measured noninvasively with manganese-enhanced magnetic resonance imaging (MEMRI), is a biomarker of channelrhodopsin-2-mediated activity in vivo. METHODS: The eyes of 3-month-old rd1/rd1 mice were either untreated ("uninjected," negative control) or injected intravitreally with either saline ("saline," negative control) or adeno-associated virus carrying a fusion construct of channelopsin-2 (Chop2) and green fluorescent protein (GFP; "Chop2-GFP"). MEMRI examination was performed 2 months later on either dark or continuous bright blue light-exposed mice to assess the distribution and extent of manganese uptake in the retina and optic nerve. In separate experiments, MEMRI was used to map laminar accumulation of manganese vertically through the retina. For comparison, Chop2-GFP expression was evaluated in whole mounts and vertical sections of virus-infected retinas and optic nerve. RESULTS: In the two control groups (regardless of lighting exposure) and between the control groups and the dark-exposed virus-treated eyes, retinal and optic nerve uptake of manganese did not differ. In light-exposed virus-treated eyes, manganese uptake in the retina and optic nerve was significantly greater relative to the other groups. In a retinal cross-section, manganese accumulation in light-exposed virus-treated eyes was spatially matched with Chop2-GFP expression in the optic nerve and all remaining retinal layers except the inner nuclear layer. CONCLUSIONS: First-time evidence is presented indicating the usefulness of measuring intraretinal manganese accumulation as a noninvasive biomarker of channelrhodopsin-2-mediated activity in vivo.

Comparison of AAV-Mediated Optogenetic Vision Restoration between Retinal Ganglion Cell Expression and ON Bipolar Cell Targeting.

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 adeno-associated 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.

Effect of Proteasome Inhibitors on the AAV-Mediated Transduction Efficiency in Retinal Bipolar Cells.

BACKGROUND: Adeno-associated Virus (AAV) vectors are the most promising vehicles for therapeutic gene delivery to the retina. To develop a practical gene delivery tool, achieving high AAV transduction efficiency in specific cell types is often required. AAV-mediated targeted expression in retinal bipolar cells is needed in certain applications such as optogenetic therapy, however, the transduction efficiency driven by endogenous cell-specific promoters is usually low. Methods that can improve AAV transduction efficiency in bipolar cells need to be developed. OBJECTIVE: The study aimed to examine the effect of proteasome inhibitors on AAV-mediated transduction efficiency in retinal bipolar cells. METHODS: Quantitative analysis of fluorescent reporter protein expression was performed to assess the effect of two proteasome inhibitors, doxorubicin and MG132, on AAV-mediated transduction efficiency in retinal bipolar cells in mice. RESULTS: Our results showed that doxorubicin can increase the AAV transduction efficiency in retinal bipolar cells in a dose-dependent manner. We also observed doxorubicin-mediated cytotoxicity in retinal neurons, but the cytotoxicity could be mitigated by the coapplication of dexrazoxane. Three months after the coapplication of doxorubicin (300 µM) and dexrazoxane, the AAV transduction efficiency in retinal bipolar cells increased by 33.8% and no cytotoxicity was observed in all the layers of the retina. CONCLUSION: Doxorubicin could enhance the AAV transduction efficiency in retinal bipolar cells in vivo. The potential long-term cytotoxicity caused by doxorubicin to retinal neurons could be partially mitigated by dexrazoxane. The coapplication of doxorubicin and dexrazoxane may serve as a potential adjuvant regimen for improving AAV transduction efficiency in retinal bipolar cells.

Evaluation of the adeno-associated virus mediated long-term expression of channelrhodopsin-2 in the mouse retina.

PURPOSE: The conversion of inner retinal neurons to photosensitive cells via viral mediated expression of channelrhodopsin-2 (ChR2) offers a new potential approach for the restoration of vision after photoreceptor degeneration. This study was conducted to evaluate the recombinant adeno-associated virus serotype 2 (rAAV2)-mediated long-term expression and safety of ChR2 in the mouse retina. METHODS: rAAV2 vectors carrying a fusion construct of channelopsin-2 (Chop2) and green fluorescent protein (GFP; Chop2-GFP) under the control of a hybrid cytomegalovirus early enhancer and chicken beta-actin (CAG) promoter were injected at different concentrations into the eyes of wild-type adult mice. The retinas were harvested up to 18 months after virus injection for immunostaining and electrophysiological studies. Injected mice were kept either under normal light conditions, or exposed to a strong blue light. The expression of GFP and the density of the cells in the ganglion cell layer (GCL) were examined. RESULTS: The expression of Chop2-GFP was stable for up to 18 months. Chop-GFP was observed predominantly in retinal ganglion as well as amacrine cells. At the highest virus concentration (6 x 10(12) GC/ml), up to 20% of the cells in the GCL were infected by the virus. At the lowest virus concentration (1 x 10(10) GC/ml), the expression was targeted to AII amacrine cells. The concentration of the virus, the light conditions, and the percentage of Chop2-GFP-positive cells had no effect on the density and, thus, on the survival of the cells in the GCL. Sufficient number of functional ChR2 channels were maintained in ganglion cells to drive robust membrane depolarization and spike firing in response to light. CONCLUSIONS: Expression of Chop2-GFP could be achieved in retinal neurons in vivo for the duration of the lifespan of mice. The expression of Chop2-GFP did not cause any detectable toxicity and cell death to neurons of the ganglion cell layer.

Differential expression of three T-type calcium channels in retinal bipolar cells in rats.

Retinal bipolar cells convey visual information from photoreceptors to retinal third-order neurons, amacrine and ganglion cells, with graded potentials through diversified cell types. To understand the possible role of voltage-dependent T-type Ca2+ currents in retinal bipolar cells, we investigated the pharmacological and biophysical properties of T-type Ca2+ currents in acutely dissociated retinal cone bipolar cells from rats using whole-cell patch-clamp recordings. We observed a broad group of cone bipolar cells with prominent T-type Ca2+ currents (T-rich) and another group with prominent L-type Ca2+ currents (L-rich). Based on the pharmacological and biophysical properties of the T-type Ca2+ currents, T-rich cone bipolar cells could be divided into three subgroups. Each subgroup appeared to express a single dominant T-type Ca2+ channel subunit. The T-type calcium currents could generate low-threshold regenerative potentials or spikes. Our results suggest that T-type Ca2+ channels may play an active and distinct signaling role in second-order neurons of the visual system, in contrast to the common signaling by L-rich bipolar cells.

Two types of cone bipolar cells express voltage-gated Na+ channels in the rat retina.

Two groups of retinal cone bipolar cells (CBCs) in rats were found to express voltage-gated Na+ channels. The axon terminals of the first group stratify in sublamina 2 of the inner plexiform layer (IPL) and partially overlap with the OFF-cholinergic band. This group was identified as type 3 CBCs. The axon terminals of the second group stratify in sublamina 3 of the IPL, slightly distal to the ON-cholinergic band. Cells of this second group resemble type 5 CBCs. In addition, we observed another group of ON-type CBCs with terminal stratification similar to that of the second group. However, this latter group did not show any Na+ current, instead exhibiting a large hyperpolarization-activated cyclic nucleotide-gated cation current, suggesting the existence of two subclasses of physiologically distinct type 5 CBCs. Both groups of Na+-expressing bipolar cells were capable of generating a rapid tetrodotoxin-sensitive action potential as revealed by current injection. Multiple spike-like potentials were also observed in some of these cells. Results of this study provide valuable insights into the function of voltage-gated Na+ channels of retinal bipolar cells in retinal processing.

A Robust Optomotor Assay for Assessing the Efficacy of Optogenetic Tools for Vision Restoration.

Purpose: To develop an animal behavioral assay for the quantitative assessment of the functional efficacy of optogenetic therapies. Methods: A 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. Results: A 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. Conclusions: The 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.

Immunohistochemical Procedures for Characterizing the Retinal Expression Patterns of Cre Driver Mouse Lines.

The retina is a thin neural tissue sitting on the backside of the eye, composed of light-sensing cells, interneurons, and output ganglion neurons. The latter send electrical signals to higher visual centers in the brain. Transgenic mouse lines are becoming one of the most valuable mammalian animal models for the study of visual signal processing within the retina. Especially, the generation of Cre recombinase transgenic mouse lines provides a powerful tool for genetic manipulation. A key step for the utilization of transgenic lines is the characterization of their transgene expression patterns in the retina. Here we describe a standard protocol for characterizing the expression pattern of the Cre recombinase or fluorescent proteins in the retina with an immunohistochemical approach.

Morphological evaluation of retinal ganglion cells expressing the L132C/T159C ChR2 mutant transgene in young adult cynomolgus monkeys.

To characterize recombinant AAV2 (rAAV2)-mediated expression of L132C/T159C ChR2 mutant in retinal ganglion cells (RGCs) of young adult cynomolgus monkeys. rAAV2 vectors carrying a fusion construct of the ChR2 mutant and GFP (ChR2-GFP) were delivered to the vitreous chamber by intravitreal injection. Expression patterns of the ChR2 mutant in RGCs were examined by immunohistochemical methods three months after injection. The RNA-binding protein with multiple splicing (RBPMS) was used as an RGC specific marker to differentiate RGCs from other retinal neurons and non-neuronal cells. The numbers of RBPMS+ and GFP+ double-labeled RGCs in the central foveal varied with the eccentricity. The expression peaked within 100 µm from the edge of the foveola and drastically decreased to a single superficial RGC layer approximately 300 µm from the edge. On average, the ratio of the double-labeled RGCs versus RBPMS+ RGCs approached 0.32±0.15 (n=14 fields) at the central foveal region (0.1 to 0.53 mm). We observed that the ratio reached 0.78±0.16 (n=21 fields) at peripheral retinal locations (eccentricity >7 mm). This investigation demonstrates that RBPMS could serve as a valuable RGC specific marker for future investigations in this field.

Targeted Expression of Channelrhodopsin-2 to the Axon Initial Segment Alters the Temporal Firing Properties of Retinal Ganglion Cells.

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.