Cone-Opponent Ganglion Cells in the Primate Fovea Tuned to Noncardinal Color Directions.

A long-standing question in vision science is how the three cone photoreceptor types-long (L), medium (M), and short (S) wavelength sensitive-combine to generate our perception of color. Hue perception can be described along two opponent axes: red-green and blue-yellow. Psychophysical measurements of color appearance indicate that the cone inputs to the red-green and blue-yellow opponent axes are M vs. L + S and L vs. M + S, respectively. However, the "cardinal directions of color space" revealed by psychophysical measurements of color detection thresholds following adaptation are L vs. M and S vs. L + M. These cardinal directions match the most common cone-opponent retinal ganglion cells (RGCs) in the primate retina. Accordingly, the cone opponency necessary for color appearance is thought to be established in the cortex. While neurons with the appropriate M vs. L + S and L vs. M + S opponency have been reported in the retina and lateral geniculate nucleus, their existence continues to be debated. Resolving this long-standing debate is necessary because a complete account of the cone opponency in the retinal output is critical for understanding how downstream neural circuits process color. Here, we performed adaptive optics calcium imaging to noninvasively measure foveal RGC light responses in the living Macaca fascicularis eye. We confirm the presence of L vs. M + S and M vs. L + S neurons with noncardinal cone opponency and demonstrate that cone-opponent signals in the retinal output are more diverse than classically thought.

Cone-Opponent Ganglion Cells in the Primate Fovea Tuned to Non-Cardinal Color Directions.

A long-standing question in vision science is how the three cone photoreceptor types - long (L), medium (M) and short (S) wavelength sensitive - combine to generate our perception of color. Hue perception can be described along two opponent axes: red-green and blue-yellow. Psychophysical measurements of color appearance indicate that the cone inputs to the red-green and blue-yellow opponent axes are M vs. L+S and L vs. M+S, respectively. However, the "cardinal directions of color space" revealed by psychophysical measurements of color detection thresholds are L vs. M and S vs. L+M. The cardinal directions match the most common cone-opponent retinal ganglion cells (RGCs) in the primate retina. Accordingly, the cone opponency necessary for color appearance is thought to be established in cortex. However, small populations with the appropriate M vs. L+S and L vs. M+S cone-opponency have been reported in large surveys of cone inputs to primate RGCs and their projections to the lateral geniculate nucleus (LGN) yet their existence continues to be debated. Resolving this long-standing open question is needed as a complete account of the cone-opponency in the retinal output is critical for efforts to understand how downstream neural circuits process color. Here, we performed adaptive optics calcium imaging to longitudinally and noninvasively measurements of the foveal RGC light responses in the living macaque eye. We confirm the presence of L vs. M+S and M vs. L+S neurons with non-cardinal cone-opponency and demonstrate that cone-opponent signals in the retinal output are substantially more diverse than classically thought.

Optogenetic Stimulation of Single Ganglion Cells in the Living Primate Fovea.

Though the responses of the rich variety of retinal ganglion cells (RGCs) reflect the totality of visual processing in the retina and provide the sole conduit for those processed responses to the brain, we have much to learn about how the brain uses these signals to guide behavior. An impediment to developing a comprehensive understanding of the role of retinal circuits in behavior is the paucity of causal studies in the intact primate visual system. Here we demonstrate the ability to optogenetically activate individual RGCs with flashes of light focused on single RGC somas in vivo , without activation of neighboring cells. The ability to selectively activate specific cells is the first step toward causal experiments that directly link retinal circuits to visual experience and behavior.

In vivo chromatic and spatial tuning of foveolar retinal ganglion cells in Macaca fascicularis.

The primate fovea is specialized for high acuity chromatic vision, with the highest density of cone photoreceptors and a disproportionately large representation in visual cortex. The unique visual properties conferred by the fovea are conveyed to the brain by retinal ganglion cells, the somas of which lie at the margin of the foveal pit. Microelectrode recordings of these centermost retinal ganglion cells have been challenging due to the fragility of the fovea in the excised retina. Here we overcome this challenge by combining high resolution fluorescence adaptive optics ophthalmoscopy with calcium imaging to optically record functional responses of foveal retinal ganglion cells in the living eye. We use this approach to study the chromatic responses and spatial transfer functions of retinal ganglion cells using spatially uniform fields modulated in different directions in color space and monochromatic drifting gratings. We recorded from over 350 cells across three Macaca fascicularis primates over a time period of weeks to months. We find that the majority of the L vs. M cone opponent cells serving the most central foveolar cones have spatial transfer functions that peak at high spatial frequencies (20-40 c/deg), reflecting strong surround inhibition that sacrifices sensitivity at low spatial frequencies but preserves the transmission of fine detail in the retinal image. In addition, we fit to the drifting grating data a detailed model of how ganglion cell responses draw on the cone mosaic to derive receptive field properties of L vs. M cone opponent cells at the very center of the foveola. The fits are consistent with the hypothesis that foveal midget ganglion cells are specialized to preserve information at the resolution of the cone mosaic. By characterizing the functional properties of retinal ganglion cells in vivo through adaptive optics, we characterize the response characteristics of these cells in situ.

Optogenetic therapy restores retinal activity in primate for at least a year following photoreceptor ablation.

All retina-based vision restoration approaches rely on the assumption that photoreceptor loss does not preclude reactivation of the remaining retinal architecture. Whether extended periods of vision loss limit the efficacy of restorative therapies at the retinal level is unknown. We examined long-term changes in optogenetic responsivity of foveal retinal ganglion cells (RGCs) in non-human primates following localized photoreceptor ablation by high-intensity laser exposure. By performing fluorescence adaptive optics scanning light ophthalmoscopy (AOSLO) of RGCs expressing both the calcium indicator GCaMP6s and the optogenetic actuator ChrimsonR, it was possible to track optogenetic-mediated calcium responses in deafferented RGCs over time. Fluorescence fundus photography revealed a 40% reduction in ChrimsonR fluorescence from RGCs lacking photoreceptor input over the 3 weeks following photoreceptor ablation. Despite this, in vivo imaging revealed good cellular preservation of RGCs 3 months after the loss of photoreceptor input, and histology confirmed good structural preservation at 2 years. Optogenetic responses of RGCs in primate persisted for at least 1 year after the loss of photoreceptor input, with a sensitivity index similar to optogenetic responses recorded in intact retina. These results are promising for all potential therapeutic approaches to vision restoration that rely on preservation and reactivation of RGCs.

Localized Photoreceptor Ablation Using Femtosecond Pulses Focused With Adaptive Optics.

Purpose: The development of new approaches to human vision restoration could be greatly accelerated with the use of nonhuman primate models; however, there is a paucity of primate models of outer retina degeneration with good spatial localization. To limit ablation to the photoreceptors, we developed a new approach that uses a near-infrared ultrafast laser, focused using adaptive optics, to concentrate light in a small focal volume within the retina. Methods: In the eyes of eight anesthetized macaques, 187 locations were exposed to laser powers from 50 to 210 mW. Laser exposure locations were monitored for up to 18 months using fluorescein angiography (FA), optical coherence tomography (OCT), scanning laser ophthalmoscopy (SLO), adaptive optics scanning laser ophthalmoscope (AOSLO) reflectance imaging, two-photon excited fluorescence (TPEF) ophthalmoscopy, histology, and calcium responses of retinal ganglion cells. Results: This method produced localized photoreceptor loss with minimal axial spread of damage to other retinal layers, verified by in-vivo structural imaging and histologic examination, although in some cases evidence of altered autofluorescence was found in the adjacent retinal pigment epithelium (RPE). Functional assessment using blood flow imaging of the retinal plexus and calcium imaging of the response of ganglion cells above the photoreceptor loss shows that inner retinal circuitry was preserved. Conclusions: Although different from a genetic model of retinal degeneration, this model of localized photoreceptor loss may provide a useful testbed for vision restoration studies in nonhuman primates. Translational Relevance: With this model, a variety of vision restoration methods can be tested in the non-human primate.

Imaging Transplanted Photoreceptors in Living Nonhuman Primates with Single-Cell Resolution.

Stem cell-based transplantation therapies offer hope for currently untreatable retinal degenerations; however, preclinical progress has been largely confined to rodent models. Here, we describe an experimental platform for accelerating photoreceptor replacement therapy in the nonhuman primate, which has a visual system much more similar to the human. We deployed fluorescence adaptive optics scanning light ophthalmoscopy (FAOSLO) to noninvasively track transplanted photoreceptor precursors over time at cellular resolution in the living macaque. Fluorescently labeled photoreceptors generated from a CRX+/tdTomato human embryonic stem cell (hESC) reporter line were delivered subretinally to macaques with normal retinas and following selective ablation of host photoreceptors using an ultrafast laser. The fluorescent reporter together with FAOSLO allowed transplanted photoreceptor precursor survival, migration, and neurite formation to be monitored over time in vivo. Histological examination suggested migration of photoreceptor precursors to the outer plexiform layer and potential synapse formation in ablated areas in the macaque eye.

In vivo-directed evolution of adeno-associated virus in the primate retina.

Efficient adeno-associated virus-mediated (AAV-mediated) gene delivery remains a significant obstacle to effective retinal gene therapies. Here, we apply directed evolution - guided by deep sequencing and followed by direct in vivo secondary selection of high-performing vectors with a GFP-barcoded library - to create AAV viral capsids with the capability to deliver genes to the outer retina in primates. A replication-incompetent library, produced via providing rep in trans, was created to mitigate risk of AAV propagation. Six rounds of in vivo selection with this library in primates - involving intravitreal library administration, recovery of genomes from outer retina, and extensive next-generation sequencing of each round - resulted in vectors with redirected tropism to the outer retina and increased gene delivery efficiency to retinal cells. These viral vectors expand the toolbox of vectors available for primate retina, and they may enable less invasive delivery of therapeutic genes to patients, potentially offering retina-wide infection at a similar dosage to vectors currently in clinical use.

Optogenetic restoration of retinal ganglion cell activity in the living primate.

Optogenetic therapies for vision restoration aim to confer intrinsic light sensitivity to retinal ganglion cells when photoreceptors have degenerated and light sensitivity has been irreversibly lost. We combine adaptive optics ophthalmoscopy with calcium imaging to optically record optogenetically restored retinal ganglion cell activity in the fovea of the living primate. Recording from the intact eye of a living animal, we compare the patterns of activity evoked by the optogenetic actuator ChrimsonR with natural photoreceptor mediated stimulation in the same retinal ganglion cells. Optogenetic responses are recorded more than one year following administration of the therapy and two weeks after acute loss of photoreceptor input in the living animal. This in vivo imaging approach could be paired with any therapy to minimize the number of primates required to evaluate restored activity on the retinal level, while maximizing translational benefit by using an appropriate pre-clinical model of the human visual system.

Functional Assessment of Vision Restoration.

Despite the many promising therapeutic approaches identified in the laboratory, it has proven extremely challenging to translate basic science advances into the eye clinic. There are many recent examples of clinical trials (e.g., Holz FG, Sadda SR, Busbee B, JAMA Ophthalmology 136:666-677, 2018) failing at the most expensive phase three stage, unable to demonstrate efficacy in the patient population. As a community we must think carefully about how we select what goes into that pipeline. Translating vision restoration therapies from the bench to the bedside involves selecting the most appropriate animal models of retinal degeneration and then moving beyond morphology to deploy appropriate functional tests in vitro, in vivo, and in the clinic. In this review we summarize the functional assays available to researchers, future prospects, and highlight areas in need of further development.

Imaging Retinal Activity in the Living Eye.

Retinal function has long been studied with psychophysical methods in humans, whereas detailed functional studies of vision have been conducted mostly in animals owing to the invasive nature of physiological approaches. There are exceptions to this generalization, for example, the electroretinogram. This review examines exciting recent advances using in vivo retinal imaging to understand the function of retinal neurons. In some cases, the methods have existed for years and are still being optimized. In others, new methods such as optophysiology are revealing novel patterns of retinal function in animal models that have the potential to change our understanding of the functional capacity of the retina. Together, the advances in retinal imaging mark an important milestone that shifts attention away from anatomy alone and begins to probe the function of healthy and diseased eyes.

Cellular-scale evaluation of induced photoreceptor degeneration in the living primate eye.

Progress is needed in developing animal models of photoreceptor degeneration and evaluating such models with longitudinal, noninvasive techniques. We employ confocal scanning laser ophthalmoscopy, optical coherence tomography (OCT) and high-resolution retinal imaging to noninvasively observe the retina of non-human primates with induced photoreceptor degeneration. Photoreceptors were imaged at the single-cell scale in three modalities of adaptive optics scanning light ophthalmoscopy: traditional confocal reflectance, indicative of waveguiding; a non-confocal offset aperture technique visualizing scattered light; and two-photon excited fluorescence, the time-varying signal of which, at 730 nm excitation, is representative of visual cycle function. Assessment of photoreceptor structure and function using these imaging modalities revealed a reduction in retinoid production in cone photoreceptor outer segments while inner segments appeared to remain present. Histology of one retina confirmed loss of outer segments and the presence of intact inner segments. This unique combination of imaging modalities can provide essential, clinically-relevant information on both the structural integrity and function of photoreceptors to not only validate models of photoreceptor degeneration but potentially evaluate the efficacy of future cell and gene-based therapies for vision restoration.

Functional architecture of the foveola revealed in the living primate.

The primate foveola, with its high cone density and magnified cortical representation, is exquisitely specialized for high-resolution spatial vision. However, uncovering the wiring of retinal circuitry responsible for this performance has been challenging due to the difficulty in recording receptive fields of foveal retinal ganglion cells (RGCs) in vivo. In this study, we use adaptive optics scanning laser ophthalmoscopy (AOSLO) to image the calcium responses of RGCs in the living primate, with a stable, high precision visual stimulus that allowed us to localize the receptive fields of hundreds of foveal ganglion cells. This approach revealed a precisely radial organization of foveal RGCs, despite the many distortions possible during the extended developmental migration of foveal cells. By back projecting the line connecting RGC somas to their receptive fields, we have been able to define the 'physiological center' of the foveola, locating the vertical meridian separating left and right hemifields in vivo.

In Vivo Functional Imaging of Retinal Neurons Using Red and Green Fluorescent Calcium Indicators.

Adaptive optics retinal imaging of fluorescent calcium indicators is a minimally invasive method used to study retinal physiology over extended periods of time. It has potential for discovering novel retinal circuits, tracking retinal function in animal models of retinal disease, and assessing vision restoration therapy. We previously demonstrated functional adaptive optics imaging of retinal neurons in the living eye using green fluorescent calcium indicators; however, the use of green fluorescent indicators presents challenges that stem from the fact that they are excited by short-wavelength light. Using red fluorescent calcium indicators such as jRGECO1a, which is excited with longer-wavelength light (~560 nm), makes imaging approximately five times safer than using short-wavelength light (~500 nm) used to excite green fluorescent calcium indicators such as GCaMP6s. Red fluorescent indicators also provide alternative wavelength imaging regimes to overcome cross talk with the sensitivities of intrinsic photoreceptors and blue light-activated channelrhodopsins. Here we evaluate jRGECO1a for in vivo functional adaptive optics imaging of retinal neurons using single-photon excitation in mice. We find that jRGECO1a provides similar fidelity as the established green indicator GCaMP6s.

All-optical recording and stimulation of retinal neurons in vivo in retinal degeneration mice.

Here we demonstrate the application of a method that could accelerate the development of novel therapies by allowing direct and repeatable visualization of cellular function in the living eye, to study loss of vision in animal models of retinal disease, as well as evaluate the time course of retinal function following therapeutic intervention. We use high-resolution adaptive optics scanning light ophthalmoscopy to image fluorescence from the calcium sensor GCaMP6s. In mice with photoreceptor degeneration (rd10), we measured restored visual responses in ganglion cell layer neurons expressing the red-shifted channelrhodopsin ChrimsonR over a six-week period following significant loss of visual responses. Combining a fluorescent calcium sensor, a channelrhodopsin, and adaptive optics enables all-optical stimulation and recording of retinal neurons in the living eye. Because the retina is an accessible portal to the central nervous system, our method also provides a novel non-invasive method of dissecting neuronal processing in the brain.

Vision science and adaptive optics, the state of the field.

Adaptive optics is a relatively new field, yet it is spreading rapidly and allows new questions to be asked about how the visual system is organized. The editors of this feature issue have posed a series of question to scientists involved in using adaptive optics in vision science. The questions are focused on three main areas. In the first we investigate the use of adaptive optics for psychophysical measurements of visual system function and for improving the optics of the eye. In the second, we look at the applications and impact of adaptive optics on retinal imaging and its promise for basic and applied research. In the third, we explore how adaptive optics is being used to improve our understanding of the neurophysiology of the visual system.

Calibration-free sinusoidal rectification and uniform retinal irradiance in scanning light ophthalmoscopy.

Sinusoidal rectification (i.e., desinusoiding) is necessary for scanning imaging systems and is typically achieved by calculating a rectification transform from a calibration image such as a regular grid. This approach is susceptible to error due to electronic or mechanical instability that can alter the phase of the imaging window with respect to the calibration transform. Here, we show a calibration-free rectification method implemented from live video of a scanning light ophthalmoscope (SLO) with or without adaptive optics (AO). This approach, which capitalizes on positional differences in the images obtained in the forward and backward scan directions, dynamically keeps the imaging window in phase with the motion of the sinusoidal resonant scanner, preventing errors from signal drift over time. A benefit of this approach is that it allows the light power across the field-of-view (FOV) to be modulated inversely to achieve uniform irradiance on the retina, a feature desirable for functional imaging methods and light safety in SLOs.

Imaging light responses of foveal ganglion cells in the living macaque eye.

The fovea dominates primate vision, and its anatomy and perceptual abilities are well studied, but its physiology has been little explored because of limitations of current physiological methods. In this study, we adapted a novel in vivo imaging method, originally developed in mouse retina, to explore foveal physiology in the macaque, which permits the repeated imaging of the functional response of many retinal ganglion cells (RGCs) simultaneously. A genetically encoded calcium indicator, G-CaMP5, was inserted into foveal RGCs, followed by calcium imaging of the displacement of foveal RGCs from their receptive fields, and their intensity-response functions. The spatial offset of foveal RGCs from their cone inputs makes this method especially appropriate for fovea by permitting imaging of RGC responses without excessive light adaptation of cones. This new method will permit the tracking of visual development, progression of retinal disease, or therapeutic interventions, such as insertion of visual prostheses.

Focal damage to macaque photoreceptors produces persistent visual loss.

Insertion of light-gated channels into inner retina neurons restores neural light responses, light evoked potentials, visual optomotor responses and visually-guided maze behavior in mice blinded by retinal degeneration. This method of vision restoration bypasses damaged outer retina, providing stimulation directly to retinal ganglion cells in inner retina. The approach is similar to that of electronic visual protheses, but may offer some advantages, such as avoidance of complex surgery and direct targeting of many thousands of neurons. However, the promise of this technique for restoring human vision remains uncertain because rodent animal models, in which it has been largely developed, are not ideal for evaluating visual perception. On the other hand, psychophysical vision studies in macaque can be used to evaluate different approaches to vision restoration in humans. Furthermore, it has not been possible to test vision restoration in macaques, the optimal model for human-like vision, because there has been no macaque model of outer retina degeneration. In this study, we describe development of a macaque model of photoreceptor degeneration that can in future studies be used to test restoration of perception by visual prostheses. Our results show that perceptual deficits caused by focal light damage are restricted to locations at which photoreceptors are damaged, that optical coherence tomography (OCT) can be used to track such lesions, and that adaptive optics retinal imaging, which we recently used for in vivo recording of ganglion cell function, can be used in future studies to examine these lesions.