A melanopsin ganglion cell subtype forms a dorsal retinal mosaic projecting to the supraoptic nucleus.

Visual input to the hypothalamus from intrinsically photosensitive retinal ganglion cells (ipRGCs) influences several functions including circadian entrainment, body temperature, and sleep. ipRGCs also project to nuclei such as the supraoptic nucleus (SON), which is involved in systemic fluid homeostasis, maternal behavior, social behaviors, and appetite. However, little is known about the SON-projecting ipRGCs or their relationship to well-characterized ipRGC subtypes. Using a GlyT2Cre mouse line, we show a subtype of ipRGCs restricted to the dorsal retina that selectively projects to the SON. These ipRGCs tile a dorsal region of the retina, forming a substrate for encoding ground luminance. Optogenetic activation of their axons demonstrates they release the neurotransmitter glutamate in multiple regions, including the suprachiasmatic nucleus (SCN) and SON. Our results challenge the idea that ipRGC dendrites overlap to optimize photon capture and suggests non-image forming vision operates to sample local regions of the visual field to influence diverse behaviors.

Functional subtypes of rodent melanopsin ganglion cells switch roles between night and day illumination.

Intrinsically photosensitive retinal ganglion cells (ipRGCs), contain the photopigment melanopsin, and influence both image and non-image forming behaviors. Despite being categorized into multiple types (M1-M6), physiological variability within these types suggests our current understanding of ipRGCs is incomplete. We used multi-electrode array (MEA) recordings and unbiased cluster analysis under synaptic blockade to identify 8 functional clusters of ipRGCs, each with distinct photosensitivity and response timing. We used Cre mice to drive the expression of channelrhodopsin in SON-ipRGCs, enabling the localization of distinct ipRGCs in the dorsal retina. Additionally, we conducted a retrospective unbiased cluster analysis of ipRGC photoresponses to light stimuli across scotopic, mesopic, and photopic intensities, aimed at activating both rod and cone inputs to ipRGCs. Our results revealed shared and distinct synaptic inputs to the identified functional clusters, demonstrating that ipRGCs encode visual information with high fidelity at low light intensities, but poorly at photopic light intensities, when melanopsin activation is highest. Collectively, our findings support a framework with at least 8 functional subtypes of ipRGCs, each encoding luminance with distinct spike outputs, highlighting the inherent functional diversity and complexity of ipRGCs and suggesting a reevaluation of their contributions to retinal function and visual perception under varying light conditions.

Photopharmacology for vision restoration.

Blinding diseases that are caused by degeneration of rod and cone photoreceptor cells often spare the rest of the retinal circuit, from bipolar cells, which are directly innervated by photoreceptor cells, to the output ganglion cells that project axons to the brain. A strategy for restoring vision is to introduce light sensitivity to the surviving cells of the retina. One approach is optogenetics, in which surviving cells are virally transfected with a gene encoding a signaling protein that becomes sensitive to light by binding to the biologically available chromophore retinal, the same chromophore that is used by the opsin photo-detectors of rods and cones. A second approach uses photopharmacology, in which a synthetic photoswitch associates with a native or engineered ion channel or receptor. We review these approaches and look ahead to the next generation of advances that could reconstitute core aspects of natural vision.

Restoration of high-sensitivity and adapting vision with a cone opsin.

Inherited and age-related retinal degenerative diseases cause progressive loss of rod and cone photoreceptors, leading to blindness, but spare downstream retinal neurons, which can be targeted for optogenetic therapy. However, optogenetic approaches have been limited by either low light sensitivity or slow kinetics, and lack adaptation to changes in ambient light, and not been shown to restore object vision. We find that the vertebrate medium wavelength cone opsin (MW-opsin) overcomes these limitations and supports vision in dim light. MW-opsin enables an otherwise blind retinitis pigmenotosa mouse to discriminate temporal and spatial light patterns displayed on a standard LCD computer tablet, displays adaption to changes in ambient light, and restores open-field novel object exploration under incidental room light. By contrast, rhodopsin, which is similar in sensitivity but slower in light response and has greater rundown, fails these tests. Thus, MW-opsin provides the speed, sensitivity and adaptation needed to restore patterned vision.

Author Correction: Restoration of patterned vision with an engineered photoactivatable G protein-coupled receptor.

Kevin J. Cao and Richard H. Kramer, who developed extended release with beta cyclodextrin, were inadvertently omitted from the author list and author contributions section of this Article. These errors have now been corrected in both the PDF and HTML versions of the Article.

Optogenetic Retinal Gene Therapy with the Light Gated GPCR Vertebrate Rhodopsin.

In retinal disease, despite the loss of light sensitivity as photoreceptors die, many retinal interneurons survive in a physiologically and metabolically functional state for long periods. This provides an opportunity for treatment by genetically adding a light sensitive function to these cells. Optogenetic therapies are in development, but, to date, they have suffered from low light sensitivity and narrow dynamic response range of microbial opsins. Expression of light-sensitive G protein coupled receptors (GPCRs), such as vertebrate rhodopsin , can increase sensitivity by signal amplification , as shown by several groups. Here, we describe the methods to (1) express light gated GPCRs in retinal neurons, (2) record light responses in retinal explants in vitro, (3) record cortical light responses in vivo, and (4) test visually guided behavior in treated mice.

Restoration of patterned vision with an engineered photoactivatable G protein-coupled receptor.

Retinitis pigmentosa results in blindness due to degeneration of photoreceptors, but spares other retinal cells, leading to the hope that expression of light-activated signaling proteins in the surviving cells could restore vision. We used a retinal G protein-coupled receptor, mGluR2, which we chemically engineered to respond to light. In retinal ganglion cells (RGCs) of blind rd1 mice, photoswitch-charged mGluR2 ("SNAG-mGluR2") evoked robust OFF responses to light, but not in wild-type retinas, revealing selectivity for RGCs that have lost photoreceptor input. SNAG-mGluR2 enabled animals to discriminate parallel from perpendicular lines and parallel lines at varying spacing. Simultaneous viral delivery of the inhibitory SNAG-mGluR2 and excitatory light-activated ionotropic glutamate receptor LiGluR yielded a distribution of expression ratios, restoration of ON, OFF and ON-OFF light responses and improved visual acuity. Thus, SNAG-mGluR2 restores patterned vision and combinatorial light response diversity provides a new logic for enhanced-acuity retinal prosthetics.

Optogenetic Vision Restoration Using Rhodopsin for Enhanced Sensitivity.

Retinal disease is one of the most active areas of gene therapy, with clinical trials ongoing in the United States for five diseases. There are currently no treatments for patients with late-stage disease in which photoreceptors have been lost. Optogenetic gene therapies are in development, but, to date, have suffered from the low light sensitivity of microbial opsins, such as channelrhodopsin and halorhodopsin, and azobenzene-based photoswitches. Several groups have shown that photoreceptive G-protein-coupled receptors (GPCRs) can be expressed heterologously, and photoactivate endogenous Gi/o signaling. We hypothesized such a GPCR could increase sensitivity due to endogenous signal amplification. We targeted vertebrate rhodopsin to retinal ON-bipolar cells of blind rd1 mice and observed restoration of: (i) light responses in retinal explants, (ii) visually-evoked potentials in visual cortex in vivo, and (iii) two forms of visually-guided behavior: innate light avoidance and discrimination of temporal light patterns in the context of fear conditioning. Importantly, both the light responses of the retinal explants and the visually-guided behavior occurred reliably at light levels that were two to three orders of magnitude dimmer than required for channelrhodopsin. Thus, gene therapy with native light-gated GPCRs presents a novel approach to impart light sensitivity for visual restoration in a useful range of illumination.

Restoration of visual function by expression of a light-gated mammalian ion channel in retinal ganglion cells or ON-bipolar cells.

Most inherited forms of blindness are caused by mutations that lead to photoreceptor cell death but spare second- and third-order retinal neurons. Expression of the light-gated excitatory mammalian ion channel light-gated ionotropic glutamate receptor (LiGluR) in retinal ganglion cells (RGCs) of the retina degeneration (rd1) mouse model of blindness was previously shown to restore some visual functions when stimulated by UV light. Here, we report restored retinal function in visible light in rodent and canine models of blindness through the use of a second-generation photoswitch for LiGluR, maleimide-azobenzene-glutamate 0 with peak efficiency at 460 nm (MAG0(460)). In the blind rd1 mouse, multielectrode array recordings of retinal explants revealed robust and uniform light-evoked firing when LiGluR-MAG0(460) was targeted to RGCs and robust but diverse activity patterns in RGCs when LiGluR-MAG0(460) was targeted to ON-bipolar cells (ON-BCs). LiGluR-MAG0(460) in either RGCs or ON-BCs of the rd1 mouse reinstated innate light-avoidance behavior and enabled mice to distinguish between different temporal patterns of light in an associative learning task. In the rod-cone dystrophy dog model of blindness, LiGluR-MAG0(460) in RGCs restored robust light responses to retinal explants and intravitreal delivery of LiGluR and MAG0(460) was well tolerated in vivo. The results in both large and small animal models of photoreceptor degeneration provide a path to clinical translation.