Quantitative characterisation of ipRGCs in retinal degeneration using a computation platform for extracting and reconstructing single neurons in 3D from a multi-colour labeled population.

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.

Divergent G-protein selectivity across melanopsins from mice and humans.

Melanopsin is an opsin photopigment and light-activated G-protein-coupled receptor; it is expressed in photoreceptive retinal ganglion cells (mRGCs) and can be employed as an optogenetic tool. Mammalian melanopsins can signal via Gq/11 and Gi/o/t heterotrimeric G proteins, but aspects of the mRGC light response appear incompatible with either mode of signalling. We use live-cell reporter assays in HEK293T cells to show that melanopsins from mice and humans can also signal via Gs. We subsequently show that this mode of signalling is substantially divergent between species. The two established structural isoforms of mouse melanopsin (which differ in the length of their C-terminal tail) both signalled strongly through all three G-protein classes (Gq/11, Gi/o and Gs), whereas human melanopsin showed weaker signalling through Gs. Our data identify Gs as a new mode of signalling for mammalian melanopsins and reveal diversity in G-protein selectivity across mammalian melanopsins.

Burning the candle at both ends: Intraretinal signaling of intrinsically photosensitive retinal ganglion cells.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) are photoreceptors located in the ganglion cell layer. They project to brain regions involved in predominately non-image-forming functions including entrainment of circadian rhythms, control of the pupil light reflex, and modulation of mood and behavior. In addition to possessing intrinsic photosensitivity via the photopigment melanopsin, these cells receive inputs originating in rods and cones. While most research in the last two decades has focused on the downstream influence of ipRGC signaling, recent studies have shown that ipRGCs also act retrogradely within the retina itself as intraretinal signaling neurons. In this article, we review studies examining intraretinal and, in addition, intraocular signaling pathways of ipRGCs. Through these pathways, ipRGCs regulate inner and outer retinal circuitry through both chemical and electrical synapses, modulate the outputs of ganglion cells (both ipRGCs and non-ipRGCs), and influence arrangement of the correct retinal circuitry and vasculature during development. These data suggest that ipRGC function plays a significant role in the processing of image-forming vision at its earliest stage, positioning these photoreceptors to exert a vital role in perceptual vision. This research will have important implications for lighting design to optimize the best chromatic lighting environments for humans, both in adults and potentially even during fetal and postnatal development. Further studies into these unique ipRGC signaling pathways could also lead to a better understanding of the development of ocular dysfunctions such as myopia.

Viral Transduction of Human Rod Opsin or Channelrhodopsin Variants to Mouse ON Bipolar Cells Does Not Impact Retinal Anatomy or Cause Measurable Death in the Targeted Cells.

The viral gene delivery of optogenetic actuators to the surviving inner retina has been proposed as a strategy for restoring vision in advanced retinal degeneration. We investigated the safety of ectopic expression of human rod opsin (hRHO), and two channelrhodopsins (enhanced sensitivity CoChR-3M and red-shifted ReaChR) by viral gene delivery in ON bipolar cells of the mouse retina. Adult Grm6Cre mice were bred to be retinally degenerate or non-retinally degenerate (homozygous and heterozygous for the rd1Pde6b mutation, respectively) and intravitreally injected with recombinant adeno-associated virus AAV2/2(quad Y-F) serotype containing a double-floxed inverted transgene comprising one of the opsins of interest under a CMV promoter. None of the opsins investigated caused changes in retinal thickness; induced apoptosis in the retina or in transgene expressing cells; or reduced expression of PKCα (a specific bipolar cell marker). No increase in retinal inflammation at the level of gene expression (IBA1/AIF1) was found within the treated mice compared to controls. The expression of hRHO, CoChR or ReaChR under a strong constitutive promoter in retinal ON bipolar cells following intravitreal delivery via AAV2 does not cause either gross changes in retinal health, or have a measurable impact on the survival of targeted cells.

A Measure of Concurrent Neural Firing Activity Based on Mutual Information.

Multiple methods have been developed in an attempt to quantify stimulus-induced neural coordination and to understand internal coordination of neuronal responses by examining the synchronization phenomena in neural discharge patterns. In this work we propose a novel approach to estimate the degree of concomitant firing between two neural units, based on a modified form of mutual information (MI) applied to a two-state representation of the firing activity. The binary profile of each single unit unfolds its discharge activity in time by decomposition into the state of neural quiescence/low activity and state of moderate firing/bursting. Then, the MI computed between the two binary streams is normalized by their minimum entropy and is taken as positive or negative depending on the prevalence of identical or opposite concomitant states. The resulting measure, denoted as Concurrent Firing Index based on MI (CFIMI), relies on a single input parameter and is otherwise assumption-free and symmetric. Exhaustive validation was carried out through controlled experiments in three simulation scenarios, showing that CFIMI is independent on firing rate and recording duration, and is sensitive to correlated and anti-correlated firing patterns. Its ability to detect non-correlated activity was assessed using ad-hoc surrogate data. Moreover, the evaluation of CFIMI on experimental recordings of spiking activity in retinal ganglion cells brought insights into the changes of neural synchrony over time. The proposed measure offers a novel perspective on the estimation of neural synchrony, providing information on the co-occurrence of firing states in the two analyzed trains over longer temporal scales compared to existing measures.

Using a bistable animal opsin for switchable and scalable optogenetic inhibition of neurons.

There is no consensus on the best inhibitory optogenetic tool. Since Gi/o signalling is a native mechanism of neuronal inhibition, we asked whether Lamprey Parapinopsin ("Lamplight"), a Gi/o-coupled bistable animal opsin, could be used for optogenetic silencing. We show that short (405 nm) and long (525 nm) wavelength pulses repeatedly switch Lamplight between stable signalling active and inactive states, respectively, and that combining these wavelengths can be used to achieve intermediate levels of activity. These properties can be applied to produce switchable neuronal hyperpolarisation and suppression of spontaneous spike firing in the mouse hypothalamic suprachiasmatic nucleus. Expressing Lamplight in (predominantly) ON bipolar cells can photosensitise retinas following advanced photoreceptor degeneration, with 405 and 525 nm stimuli producing responses of opposite sign in the output neurons of the retina. We conclude that bistable animal opsins can co-opt endogenous signalling mechanisms to allow optogenetic inhibition that is scalable, sustained and reversible.

A Bright Idea for Improving Spatial Memory.

In this issue of Neuron, Huang et al. (2021) reveal a new influence of light on memory. They show that in mice, daily exposure to bright light over several weeks produces lasting increases in spatial memory and assign this effect to a circuit originating in the retina and encompassing the ventral lateral geniculate and reuniens nuclei.

A High-Dimensional Quantification of Mouse Defensive Behaviors Reveals Enhanced Diversity and Stimulus Specificity.

Instinctive defensive behaviors, consisting of stereotyped sequences of movements and postures, are an essential component of the mouse behavioral repertoire. Since defensive behaviors can be reliably triggered by threatening sensory stimuli, the selection of the most appropriate action depends on the stimulus property. However, since the mouse has a wide repertoire of motor actions, it is not clear which set of movements and postures represent the relevant action. So far, this has been empirically identified as a change in locomotion state. However, the extent to which locomotion alone captures the diversity of defensive behaviors and their sensory specificity is unknown. To tackle this problem, we developed a method to obtain a faithful 3D reconstruction of the mouse body that enabled to quantify a wide variety of motor actions. This higher dimensional description revealed that defensive behaviors are more stimulus specific than indicated by locomotion data. Thus, responses to distinct stimuli that were equivalent in terms of locomotion (e.g., freezing induced by looming and sound) could be discriminated along other dimensions. The enhanced stimulus specificity was explained by a surprising diversity. A clustering analysis revealed that distinct combinations of movements and postures, giving rise to at least 7 different behaviors, were required to account for stimulus specificity. Moreover, each stimulus evoked more than one behavior, revealing a robust one-to-many mapping between sensations and behaviors that was not apparent from locomotion data. Our results indicate that diversity and sensory specificity of mouse defensive behaviors unfold in a higher dimensional space, spanning multiple motor actions.

Can We See with Melanopsin?

A small fraction of mammalian retinal ganglion cells are directly photoreceptive thanks to their expression of the photopigment melanopsin. These intrinsically photosensitive retinal ganglion cells (ipRGCs) have well-established roles in a variety of reflex responses to changes in ambient light intensity, including circadian photoentrainment. In this article, we review the growing evidence, obtained primarily from laboratory mice and humans, that the ability to sense light via melanopsin is also an important component of perceptual and form vision. Melanopsin photoreception has low temporal resolution, making it fundamentally biased toward detecting changes in ambient light and coarse patterns rather than fine details. Nevertheless, melanopsin can indirectly impact high-acuity vision by driving aspects of light adaptation ranging from pupil constriction to changes in visual circuit performance. Melanopsin also contributes directly to perceptions of brightness, and recent data suggest that this influences the appearance not only of overall scene brightness, but also of low-frequency patterns.

Rod Photoreceptor Activation Alone Defines the Release of Dopamine in the Retina.

Retinal dopamine is released by a specialized subset of amacrine cells in response to light and has a potent influence on how the retina responds to, and encodes, visual information. Here, we address the critical question of which retinal photoreceptor is responsible for coordinating the release of this neuromodulator. Although all three photoreceptor classes-rods, cones, and melanopsin-containing retinal ganglion cells (mRGCs)-have been shown to provide electrophysiological inputs to dopaminergic amacrine cells (DACs), we show here that the release of dopamine is defined only by rod photoreceptors. Remarkably, this rod signal coordinates both a suppressive signal at low intensities and drives dopamine release at very bright light intensities. These data further reveal that dopamine release does not necessarily correlate with electrophysiological activity of DACs and add to a growing body of evidence that rods define aspects of retinal function at very bright light levels.

How Does Light Regulate Mood and Behavioral State?

The idea that light affects mood and behavioral state is not new. However, not much is known about the particular mechanisms and circuits involved. To fully understand these, we need to know what properties of light are important for mediating changes in mood as well as what photoreceptors and pathways are responsible. Increasing evidence from both human and animal studies imply that a specialized class of retinal ganglion cells, intrinsically photosensitive retinal ganglion cells (ipRGCs), plays an important role in the light-regulated effects on mood and behavioral state, which is in line with their well-established roles in other non-visual responses (pupillary light reflex and circadian photoentrainment). This paper reviews our current understanding on the mechanisms and paths by which the light information modulates behavioral state and mood.

Photoreceptive retinal ganglion cells control the information rate of the optic nerve.

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.

Intensity-dependent nonlinear refraction of antimonene dispersions in the visible and near-infrared region.

Antimonene is a stable 2D allotrope of antimony that is predicted to have a direct bandgap, high third-order optical nonlinear susceptibility, and high electron mobility. These properties give it huge potential applications in photonics and optoelectronics. However, the nonlinear refractive response of antimonene dispersions, which is the key to nonlinear refraction-based devices, has not been fully investigated. In this work, we investigated the optical nonlinearities of the antimonene dispersions by spatial self-phase modulation (SSPM) at 405, 785, and 1064 nm wavelengths. The SSPM rings were observed at 405, 785, and 1064 nm, implying the broadband nonlinear optical response of antimonene dispersions from visible to near-infrared. The effective nonlinear refractive index, n2, and the third-order susceptibility, χ(3), of the antimonene dispersion were measured to be ∼10-5 cm2 W-1 and ∼10-8 esu, respectively. Furthermore, the nonlinearity of antimonene was demonstrated to be tuneable by the laser intensities. The relative change of the nonlinear refractive index, Δn2e/n2e, was observed to range from 14% to 63% for different intensities. Our results will be helpful for the photonic applications of antimonene in a broadband wavelength range, such as optical modulators and switchers.

Efficacy and Safety of Glycosidic Enzymes for Improved Gene Delivery to the Retina following Intravitreal Injection in Mice.

Viral gene delivery is showing great promise for treating retinal disease. Although subretinal vector delivery has mainly been used to date, intravitreal delivery has potential advantages if low retinal transduction efficiency can be overcome. To this end, we investigated the effects of co-injection of glycosaminoglycan-degrading enzymes, singly or in combination, with AAV2 as a method of increasing retinal transduction. Experiments using healthy mice demonstrated that these enzymes enhance retinal transduction. We found that heparinase III produced the greatest individual effect, and this was enhanced further by combination with hyaluronan lyase. In addition, this optimized AAV2-enzyme combination led to a marked improvement in transduction in retinas with advanced retinal degeneration compared with AAV2 alone. Safety studies measuring retinal function by flash electroretinography indicated that retinal function was unaffected in the acute period and at least 12 months after enzyme treatment, whereas pupillometry confirmed that retinal ganglion cell activity was unaffected. Retinal morphology was not altered by the enzyme injection. Collectively these data confirm the efficacy and safety of this intravitreal approach in enhancing retinal transduction efficiency by AAV in rodents. Translating this method into other species, such as non-human primates, or for clinical applications will have challenges and require further studies.

Meclofenamic acid improves the signal to noise ratio for visual responses produced by ectopic expression of human rod opsin.

PURPOSE: 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. METHODS: We used in vitro multielectrode array (MEA) recordings to characterize the light responses of RGCs in the rd1 mouse model of advanced retinal degeneration following intravitreal injection of an adenoassociated virus (AAV2) driving the expression of human rod opsin under a minimal grm6 promoter active in ON bipolar cells. RESULTS: We found treated retinas were light responsive over five decades of irradiance (from 1011 to 1015 photons/cm2/s) with individual RGCs covering up to four decades. Application of MFA reduced the spontaneous firing rate of the visually responsive neurons under light- and dark-adapted conditions. The change in the firing rate produced by the 2 s light pulses was increased across all intensities following MFA treatment, and there was a concomitant increase in the signal to noise ratio for the visual response. Restored light responses were abolished by agents inhibiting glutamatergic or gamma-aminobutyric acid (GABA)ergic signaling in the MFA-treated preparation. CONCLUSIONS: These results confirm the potential of MFA to inhibit spontaneous activity and enhance the signal to noise ratio of visual responses in optogenetic therapies to restore sight.

Optogenetic interrogation reveals separable G-protein-dependent and -independent signalling linking G-protein-coupled receptors to the circadian oscillator.

BACKGROUND: Endogenous circadian oscillators distributed across the mammalian body are synchronised among themselves and with external time via a variety of signalling molecules, some of which interact with G-protein-coupled receptors (GPCRs). GPCRs can regulate cell physiology via pathways originating with heterotrimeric G-proteins or ß-arrestins. We applied an optogenetic approach to determine the contribution of these two signalling modes on circadian phase. RESULTS: We employed a photopigment (JellyOp) that activates Gαs signalling with better selectivity and higher sensitivity than available alternatives, and a point mutant of this pigment (F112A) biased towards ß-arrestin signalling. When expressed in fibroblasts, both native JellyOp and the F112A arrestin-biased mutant drove light-dependent phase resetting in the circadian clock. Shifts induced by the two opsins differed in their circadian phase dependence and the degree to which they were associated with clock gene induction. CONCLUSIONS: Our data imply separable G-protein and arrestin inputs to the mammalian circadian clock and establish a pair of optogenetic tools suitable for manipulating Gαs- and ß-arrestin-biased signalling in live cells.

Chemogenetic Activation of ipRGCs Drives Changes in Dark-Adapted (Scotopic) Electroretinogram.

Purpose: The purpose of this study was to investigate the impact of activating melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) on dark-adapted (scotopic) electroretinograms (ERG). Methods: We used mice (Opn4Cre/+) expressing cre recombinase in melanopsin-expressing cells for a targeted gene delivery of a chemogenetic Gq-coupled receptor, hM3Dq, to ipRGCs. Intraperitoneal injection of clozapine N-oxide (CNO) at 5 mg/kg was used for acute activation of hM3Dq and thus excitation of ipRGCs in darkness. Dark-adapted flash ERGs were recorded across a 9-fold range of irradiances from hM3Dq Opn4Cre/+ and control Opn4Cre/+ mice before and after intraperitoneal injection of CNO. A- and b-wave amplitudes and implicit times and oscillatory potentials (OPs) were analyzed. Paired-flash stimuli were used to isolate cone-driven responses. Results: Clozapine N-oxide application suppressed a- and b-wave amplitudes of the dark-adapted ERG across the flash intensity range in hM3Dq Opn4Cre/+ mice compared to control mice. Examination of the normalized irradiance-response functions revealed a shift in b-wave but not a-wave sensitivity. No changes in a- and b-wave implicit times were detected. Total OP amplitudes were also reduced in hM3Dq Opn4Cre/+ mice compared to controls following CNO administration. The paired-flash method revealed reduction in both the first (rods and cones) and second (cones only) flash response. Conclusions: Acute and selective activation of ipRGCs modulates the amplitude of both a- and b-waves of the scotopic ERG, indicating that the influence of this ganglion cell class on the retinal physiology extends to the photoreceptors as well as their downstream pathways.

Chemogenetic Activation of Melanopsin Retinal Ganglion Cells Induces Signatures of Arousal and/or Anxiety in Mice.

Functional imaging and psychometric assessments indicate that bright light can enhance mood, attention, and cognitive performance in humans. Indirect evidence links these events to light detection by intrinsically photosensitive melanopsin-expressing retinal ganglion cells (mRGCs) [1-9]. However, there is currently no direct demonstration that mRGCs can have such an immediate effect on mood or behavioral state in any species. We addressed this deficit by using chemogenetics to selectively activate mRGCs, simulating the excitatory effects of bright light on this cell type in dark-housed mice. This specific manipulation evoked circadian phase resetting and pupil constriction (known consequences of mRGC activation). It also induced c-Fos (a marker of neuronal activation) in multiple nuclei in the hypothalamus (paraventricular, dorsomedial, and lateral hypothalamus), thalamus (paraventricular and centromedian thalamus), and limbic system (amygdala and nucleus accumbens). These regions influence numerous aspects of autonomic and neuroendocrine activity and are typically active during periods of wakefulness or arousal. By contrast, c-Fos was absent from the ventrolateral preoptic area (active during sleep). In standard behavioral tests (open field and elevated plus maze), mRGC activation induced behaviors commonly interpreted as anxiety like or as signs of increased alertness. Similar changes in behavior could be induced by bright light in wild-type and rodless and coneless mice, but not melanopsin knockout mice. These data demonstrate that mRGCs drive a light-dependent switch in behavioral motivation toward a more alert, risk-averse state. They also highlight the ability of this small fraction of retinal ganglion cells to realign activity in brain regions defining widespread aspects of physiology and behavior.

Melanopsin-driven increases in maintained activity enhance thalamic visual response reliability across a simulated dawn.

Twice a day, at dawn and dusk, we experience gradual but very high amplitude changes in background light intensity (irradiance). Although we perceive the associated change in environmental brightness, the representation of such very slow alterations in irradiance by the early visual system has been little studied. Here, we addressed this deficit by recording electrophysiological activity in the mouse dorsal lateral geniculate nucleus under exposure to a simulated dawn. As irradiance increased we found a widespread enhancement in baseline firing that extended to units with ON as well as OFF responses to fast luminance increments. This change in baseline firing was equally apparent when the slow irradiance ramp appeared alone or when a variety of higher-frequency artificial or natural visual stimuli were superimposed upon it. Using a combination of conventional knockout, chemogenetic, and receptor-silent substitution manipulations, we continued to show that, over higher irradiances, this increase in firing originates with inner-retinal melanopsin photoreception. At the single-unit level, irradiance-dependent increases in baseline firing were strongly correlated with improvements in the amplitude of responses to higher-frequency visual stimuli. This in turn results in an up to threefold increase in single-trial reliability of fast visual responses. In this way, our data indicate that melanopsin drives a generalized increase in dorsal lateral geniculate nucleus excitability as dawn progresses that both conveys information about changing background light intensity and increases the signal:noise for fast visual responses.

Restoration of Vision with Ectopic Expression of Human Rod Opsin.

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.