Light has a profound impact on mammalian physiology and behavior. Intrinsically photosensitive retinal ganglion cells (ipRGCs) express the photopigment melanopsin, rendering them sensitive to light, and are involved in both image-forming vision and non-image forming responses to light such as circadian photo-entrainment and the pupillary light reflex. Following outer photoreceptor degeneration, the death of rod and cone photoreceptors results in global re-modeling of the remnant neural retina. Although ipRGCs can continue signaling light information to the brain even in advanced stages of degeneration, it is unknown if all six morphologically distinct subtypes survive, or how their dendritic architecture may be affected. To answer these questions, we generated a computational platform-BRIAN (Brainbow Analysis of individual Neurons) to analyze Brainbow labeled tissues by allowing objective identification of voxels clusters in Principal Component Space, and their subsequent extraction to produce 3D images of single neurons suitable for analysis with existing tracing technology. We show that BRIAN can efficiently recreate single neurons or individual axonal projections from densely labeled tissue with sufficient anatomical resolution for subtype quantitative classification. We apply this tool to generate quantitative morphological information about ipRGCs in the degenerate retina including soma size, dendritic field size, dendritic complexity, and stratification. Using this information, we were able to identify cells whose characteristics match those reported for all six defined subtypes of ipRGC in the wildtype mouse retina (M1-M6), including the rare and complex M3 and M6 subtypes. This indicates that ipRGCs survive outer retinal degeneration with broadly normal morphology. We additionally describe one cell in the degenerate retina which matches the description of the Gigantic M1 cell in Humans which has not been previously identified in rodent.

Information transfer in the brain relies upon energetically expensive spiking activity of neurons. Rates of information flow should therefore be carefully optimized, but mechanisms to control this parameter are poorly understood. We address this deficit in the visual system, where ambient light (irradiance) is predictive of the amount of information reaching the eye and ask whether a neural measure of irradiance can therefore be used to proactively control information flow along the optic nerve. We first show that firing rates for the retina's output neurons [retinal ganglion cells (RGCs)] scale with irradiance and are positively correlated with rates of information and the gain of visual responses. Irradiance modulates firing in the absence of any other visual signal confirming that this is a genuine response to changing ambient light. Irradiance-driven changes in firing are observed across the population of RGCs (including in both ON and OFF units) but are disrupted in mice lacking melanopsin [the photopigment of irradiance-coding intrinsically photosensitive RGCs (ipRGCs)] and can be induced under steady light exposure by chemogenetic activation of ipRGCs. Artificially elevating firing by chemogenetic excitation of ipRGCs is sufficient to increase information flow by increasing the gain of visual responses, indicating that enhanced firing is a cause of increased information transfer at higher irradiance. Our results establish a retinal circuitry driving changes in RGC firing as an active response to alterations in ambient light to adjust the amount of visual information transmitted to the brain.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) express the photopigment melanopsin and project to central targets, allowing them to contribute to both image-forming and non-image forming vision. Recent studies have highlighted chemical and electrical synapses between ipRGCs and neurons of the inner retina, suggesting a potential influence from the melanopsin-born signal to affect visual processing at an early stage of the visual pathway. We investigated melanopsin responses in ganglion cell layer (GCL) neurons of both intact and dystrophic mouse retinas using 256 channel multi-electrode array (MEA) recordings. A wide 200 μm inter-electrode spacing enabled a pan-retinal visualization of melanopsin's influence upon GCL activity. Upon initial stimulation of dystrophic retinas with a long, bright light pulse, over 37% of units responded with an increase in firing (a far greater fraction than can be expected from the anatomically characterized number of ipRGCs). This relatively widespread response dissipated with repeated stimulation even at a quite long inter-stimulus interval (ISI; 120 s), to leave a smaller fraction of responsive units (<10%; more in tune with the predicted number of ipRGCs). Visually intact retinas appeared to lack such widespread melanopsin responses indicating that it is a feature of dystrophy. Taken together, our data reveal the potential for anomalously widespread melanopsin responses in advanced retinal degeneration. These could be used to probe the functional reorganization of retinal circuits in degeneration and should be taken into account when using retinally degenerate mice as a model of disease.

Retinal dystrophy through outer photoreceptor cell death affects 1 in 2,500 people worldwide with severe impairment of vision in advanced stages of the disease. Optogenetic strategies to restore visual function to animal models of retinal degeneration by introducing photopigments to neurons spared degeneration in the inner retina have been explored, with variable degrees of success. It has recently been shown that the non-steroidal anti-inflammatory and non-selective gap-junction blocker meclofenamic acid (MFA) can enhance the visual responses produced by an optogenetic actuator (channelrhodopsin) expressed in retinal ganglion cells (RGCs) in the degenerate retina. Here, we set out to determine whether MFA could also enhance photoreception by another optogenetic strategy in which ectopic human rod opsin is expressed in ON bipolar cells.

Many retinal dystrophies result in photoreceptor loss, but the inner retinal neurons can survive, making them potentially amenable to emerging optogenetic therapies. Here, we show that ectopically expressed human rod opsin, driven by either a non-selective or ON-bipolar cell-specific promoter, can function outside native photoreceptors and restore visual function in a mouse model of advanced retinal degeneration. Electrophysiological recordings from retinal explants and the visual thalamus revealed changes in firing (increases and decreases) induced by simple light pulses, luminance increases, and naturalistic movies in treated mice. These responses could be elicited at light intensities within the physiological range and substantially below those required by other optogenetic strategies. Mice with rod opsin expression driven by the ON-bipolar specific promoter displayed behavioral responses to increases in luminance, flicker, coarse spatial patterns, and elements of a natural movie at levels of contrast and illuminance (≈50-100 lux) typical of natural indoor environments. These data reveal that virally mediated ectopic expression of human rod opsin can restore vision under natural viewing conditions and at moderate light intensities. Given the inherent advantages in employing a human protein, the simplicity of this intervention, and the quality of vision restored, we suggest that rod opsin merits consideration as an optogenetic actuator for treating patients with advanced retinal degeneration.

Color vision, originating with opponent processing of spectrally distinct photoreceptor signals, plays important roles in animal behavior.1-4 Surprisingly, however, comparatively little is understood about color processing in the brain, including in widely used laboratory mammals such as mice. The retinal gradient in S- and M-cone opsin (co-)expression has traditionally been considered an impediment to mouse color vision.5-8 However, recent data indicate that mice exhibit robust chromatic discrimination within the central-upper visual field.9 Retinal color opponency has been reported to emerge from superimposing inhibitory surround receptive fields on the cone opsin expression gradient, and by introducing opponent rod signals in retinal regions with sparse M-cone opsin expression.10-13 The relative importance of these proposed mechanisms in determining the properties of neurons at higher visual processing stages remains unknown. We address these questions using multielectrode recordings from the lateral geniculate nucleus (LGN) in mice with altered M-cone spectral sensitivity (Opn1mwR) and multispectral stimuli that allow selective modulation of signaling by individual opsin classes. Remarkably, we find many (∼25%) LGN cells are color opponent, that such cells are localized to a distinct medial LGN zone and that their properties cannot simply be explained by the proposed retinal opponent mechanisms. Opponent responses in LGN can be driven solely by cones, independent of cone-opsin expression gradients and rod input, with many cells exhibiting spatially congruent antagonistic receptive fields. Our data therefore suggest previously unidentified mechanisms may support extensive and sophisticated color processing in the mouse LGN.

Photoreceptor degeneration sufficient to produce severe visual loss often spares the inner retina. This raises hope for vision restoration treatments using optogenetics or electrical stimulation, which generate a replacement light input signal in surviving neurons. The success of these approaches is dependent on the capacity of surviving circuits of the visual system to generate and propagate an appropriate visual code in the face of neuroanatomical remodeling. To determine whether retinally degenerate animals possess this capacity, we generated a transgenic mouse model expressing the optogenetic actuator ReaChR in ON bipolar cells (second-order neurons in the visual projection). After crossing this with the rd1 model of photoreceptor degeneration, we compared ReaChR-derived responses with photoreceptor-driven responses in wild-type (WT) mice at the level of retinal ganglion cells and the visual thalamus. The ReaChR-driven responses in rd1 animals showed low photosensitivity, but in other respects generated a visual code that was very similar to the WT. ReaChR rd1 responses had high trial-to-trial reproducibility and showed sensitivity normalization to code contrast across background intensities. At the single unit level, ReaChR-derived responses exhibited broadly similar variations in response polarity, contrast sensitivity, and temporal frequency tuning as the WT. Units from the WT and ReaChR rd1 mice clustered together when subjected to unsupervised community detection based on stimulus-response properties. Our data reveal an impressive ability for surviving circuitry to recreate a rich visual code following advanced retinal degeneration and are promising for regenerative medicine in the central nervous system.

Introduction: Intrinsically photosensitive retinal ganglion cells (ipRGCs) integrate melanopsin and rod/cone-mediated inputs to signal to the brain. Whilst originally identified as a cell type specialised for encoding ambient illumination, several lines of evidence indicate a strong association between colour discrimination and ipRGC-driven responses. Thus, cone-mediated colour opponent responses have been widely found across ipRGC target regions in the mouse brain and influence a key ipRGC-dependent function, circadian photoentrainment. Although ipRGCs exhibiting spectrally opponent responses have also been identified, the prevalence of such properties have not been systematically evaluated across the mouse retina or yet been found in ipRGC subtypes known to influence the circadian system. Indeed, there is still uncertainty around the overall prevalence of cone-dependent colour opponency across the mouse retina, given the strong retinal gradient in S and M-cone opsin (co)-expression and overlapping spectral sensitivities of most mouse opsins. Methods: To address this, we use photoreceptor isolating stimuli in multielectrode recordings from human red cone opsin knock-in mouse (Opn1mwR) retinas to systematically survey cone mediated responses and the occurrence of colour opponency across ganglion cell layer (GCL) neurons and identify ipRGCs based on spectral comparisons and/or the persistence of light responses under synaptic blockade. Results: Despite detecting robust cone-mediated responses across the retina, we find cone opponency is rare, especially outside of the central retina (overall ~3% of GCL neurons). In keeping with previous suggestions we also see some evidence of rod-cone opponency (albeit even more rare under our experimental conditions), but find no evidence for any enrichment of cone (or rod) opponent responses among functionally identified ipRGCs. Conclusion: In summary, these data suggest the widespread appearance of cone-opponency across the mouse early visual system and ipRGC-related responses may be an emergent feature of central visual processing mechanisms.

Environmental illumination profoundly influences mammalian physiology and behaviour through actions on a master circadian oscillator in the suprachiasmatic nuclei (SCN) and other hypothalamic nuclei. The retinal and central mechanisms that shape daily patterns of light-evoked and spontaneous activity in this network of hypothalamic cells are still largely unclear. Similarly, the exact nature of the sensory information conveyed by such cells is unresolved. Here we set out to address these issues, through multielectrode recordings from the hypothalamus of red cone knockin mice (Opn1mwR). With this powerful mouse model, the photoreceptive origins of any response can be readily identified on the basis of their relative sensitivity to short and long wavelength light. Our experiments revealed that the firing pattern of many hypothalamic cells was influenced by changes in light levels and/or according to the steady state level of illumination. These ‘contrast' and ‘irradiance' responses were driven primarily by cone and melanopsin photoreceptors respectively, with rods exhibiting a much more subtle influence. Individual hypothalamic neurons differentially sampled from these information streams, giving rise to four distinct response types. The most common response phenotype in the SCN itself was sustained activation. Cells with this behaviour responded to all three photoreceptor classes in a manner consistent with their distinct contributions to circadian photoentrainment. These ‘sustained' cells were also unique in our sample in expressing circadian firing patterns with highest activity during the mid projected day. Surprisingly, we also found a minority of SCN neurons that lacked the melanopsin-derived irradiance signal and responded only to light transitions, allowing for the possibility that rod–cone contrast signals may be routed to SCN output targets without influencing neighbouring circadian oscillators. Finally, an array of cells extending throughout the periventricular hypothalamus and ventral thalamus were excited or inhibited solely according to the activity of melanopsin. These cells appeared to convey a filtered version of the visual signal, suitable for modulating physiology/behaviour purely according to environmental irradiance. In summary, these findings reveal unexpectedly widespread hypothalamic cell populations encoding distinct qualities of visual information.

A direct retinal projection targets the suprachiasmatic nucleus (SCN) (an important hypothalamic control center). The accepted function of this projection is to convey information about ambient light (irradiance) to synchronize the SCN's endogenous circadian clock with local time and drive the diurnal variations in physiology and behavior [1-4]. Here, we report that it also renders the SCN responsive to visual images. We map spatial receptive fields (RFs) for SCN neurons and find that only a minority are excited (or inhibited) by light from across the scene as expected for irradiance detectors. The most commonly encountered units have RFs with small excitatory centers, combined with very extensive inhibitory surrounds that reduce their sensitivity to global changes in light in favor of responses to spatial patterns. Other units have larger excitatory RF centers, but these always cover a coherent region of visual space, implying visuotopic order at the single-unit level. Approximately 75% of light-responsive SCN units modulate their firing according to simple spatial patterns (drifting or inverting gratings) without changes in irradiance. The time-averaged firing rate of the SCN is modestly increased under these conditions, but including spatial contrast did not significantly alter the circadian phase resetting efficiency of light. Our data indicate that the SCN contains information about irradiance and spatial patterns. This newly appreciated sensory capacity provides a mechanism by which behavioral and physiological systems downstream of the SCN could respond to visual images [5].

A direct projection from melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) reaches the primary visual thalamus (dorsal lateral geniculate nucleus; dLGN). The significance of this melanopsin input to the visual system is only recently being investigated. One unresolved question is the degree to which neurons in the dLGN could use melanopsin to track dynamic changes in light intensity under light adapted conditions. Here we set out to address this question. We were able to present full field steps visible only to melanopsin by switching between rod-isoluminant 'yellow' and 'blue' lights in a mouse lacking cone function (Cnga3-/-). In the retina these stimuli elicited melanopsin-like responses from a subset of ganglion cells. When presented to anaesthetised mice, we found that ~25-30% of visually responsive neurones in the contralateral dLGN responded to these melanopsin-isolating steps with small increases in firing rate. Such responses could be elicited even with fairly modest increases in effective irradiance (32% Michelson contrast for melanopsin). These melanopsin-driven responses were apparent at bright backgrounds (corresponding to twilight-daylight conditions), but their threshold irradiance was strongly dependent upon prior light exposure when stimuli were superimposed on a spectrally neutral ramping background light. While both onset and offset latencies were long for melanopsin-derived responses compared to those evoked by rods, there was great variability in these parameters with some cells responding to melanopsin steps in <1 s. These data indicate that a subset of dLGN units can employ melanopsin signals to detect modest changes in irradiance under photopic conditions.