An uncommon neuronal class conveys visual signals from rods and cones to retinal ganglion cells.

Neurons in the central nervous system (CNS) are distinguished by the neurotransmitter types they release, their synaptic connections, morphology, and genetic profiles. To fully understand how the CNS works, it is critical to identify all neuronal classes and reveal their synaptic connections. The retina has been extensively used to study neuronal development and circuit formation. Here, we describe a previously unidentified interneuron in mammalian retina. This interneuron shares some morphological, physiological, and molecular features with retinal bipolar cells, such as receiving input from photoreceptors and relaying visual signals to retinal ganglion cells. It also shares some features with amacrine cells (ACs), particularly Aii-ACs, such as their neurite morphology in the inner plexiform layer, the expression of some AC-specific markers, and possibly the release of the inhibitory neurotransmitter glycine. Thus, we unveil an uncommon interneuron, which may play an atypical role in vision.

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.

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.

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.

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.

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.

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.

ChR2 mutants at L132 and T159 with improved operational light sensitivity for vision restoration.

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.