Reconfiguration of the visual code and retinal cell type complement in closely related diurnal and nocturnal mice.

How does evolution act on neuronal populations to match computational characteristics to functional demands? We address this problem by comparing visual code and retinal cell composition in closely related murid species with different behaviours. Rhabdomys pumilio are diurnal and have substantially thicker inner retina and larger visual thalamus than nocturnal Mus musculus. High-density electrophysiological recordings of visual response features in the dorsal lateral geniculate nucleus (dLGN) reveals that Rhabdomys attains higher spatiotemporal acuity both by denser coverage of the visual scene and a selective expansion of elements of the code characterised by non-linear spatiotemporal summation. Comparative analysis of single cell transcriptomic cell atlases reveals that realignment of the visual code is associated with increased relative abundance of bipolar and ganglion cell types supporting OFF and ON-OFF responses. These findings demonstrate how changes in retinal cell complement can reconfigure the coding of visual information to match changes in visual needs.

Modulating signalling lifetime to optimise a prototypical animal opsin for optogenetic applications.

Animal opsins are light activated G-protein-coupled receptors, capable of optogenetic control of G-protein signalling for research or therapeutic applications. Animal opsins offer excellent photosensitivity, but their temporal resolution can be limited by long photoresponse duration when expressed outside their native cellular environment. Here, we explore methods for addressing this limitation for a prototypical animal opsin (human rod opsin) in HEK293T cells. We find that the application of the canonical rhodopsin kinase (GRK1)/visual arrestin signal termination mechanism to this problem is complicated by a generalised suppressive effect of GRK1 expression. This attenuation can be overcome using phosphorylation-independent mutants of arrestin, especially when these are tethered to the opsin protein. We further show that point mutations targeting the Schiff base stability of the opsin can also reduce signalling lifetime. Finally, we apply one such mutation (E122Q) to improve the temporal fidelity of restored visual responses following ectopic opsin expression in the inner retina of a mouse model of retinal degeneration (rd1). Our results reveal that these two strategies (targeting either arrestin binding or Schiff-base hydrolysis) can produce more time-delimited opsin signalling under heterologous expression and establish the potential of this approach to improve optogenetic performance.

The mouse suprachiasmatic nucleus encodes irradiance via a diverse population of neurons monotonically tuned to different ranges of intensity.

Many neurons of the mammalian master circadian oscillator in the suprachiasmatic nuclei (SCN) respond to light pulses with irradiance-dependent changes in firing. Here, we set out to better understand this irradiance coding ability by considering how the SCN tracks more continuous changes in irradiance at both population and single unit level. To this end, we recorded extracellular activity in the SCN of anaesthetised mice presented with up + down irradiance staircase stimuli covering moonlight to daylight conditions and incorporating epochs with steady light or superimposed higher frequency modulations (temporal white noise (WN) and frequency/contrast chirps). Single unit activity was extracted by spike sorting. The population response of SCN units to this stimulus was a progressive increase in firing rate at higher irradiances. This relationship was symmetrical for up vs. down phases of the ramp in the presence of white noise or chirps but exhibited hysteresis for steady light, with firing systematically higher during increasing irradiance. Single units also showed a monotonic relationship between firing and irradiance but exhibited diversity not only in response polarity (increases vs. decreases in firing), but also in the sensitivity (EC50 ) and slope of fitted functions. These data show that individual SCN neurons exhibit monotonic relationships between irradiance and firing rate but differ in the irradiance range over which they respond. This property may help the SCN to encode the large differences in irradiance found in nature using neurons with a constrained range of firing rates. KEY POINTS: Daily changes in environmental light (irradiance) entrain the suprachiasmatic nucleus (SCN) circadian clock. The mouse SCN shows graded increases in neurophysiological activity with light pulses of increasing irradiance. We show that this monotonic relationship between firing rate and irradiance is retained at population and single unit level when probed with more naturalistic staircase increases and decreases in irradiance. The irradiance response is more reliable in the presence of ongoing higher temporal frequency modulations in light intensity than under steady light. Single units varied in sensitivity allowing the population to cover a wide range of irradiances. Irradiance coding in the SCN has characteristics of a sparse code with individual neurons tracking different portions of the natural irradiance range. This property may address the challenge of encoding a 109 -fold day:night difference in irradiance within the constrained range of firing rates available to individual neurons.

Three-dimensional unsupervised probabilistic pose reconstruction (3D-UPPER) for freely moving animals.

A key step in understanding animal behaviour relies in the ability to quantify poses and movements. Methods to track body landmarks in 2D have made great progress over the last few years but accurate 3D reconstruction of freely moving animals still represents a challenge. To address this challenge here we develop the 3D-UPPER algorithm, which is fully automated, requires no a priori knowledge of the properties of the body and can also be applied to 2D data. We find that 3D-UPPER reduces by [Formula: see text] fold the error in 3D reconstruction of mouse body during freely moving behaviour compared with the traditional triangulation of 2D data. To achieve that, 3D-UPPER performs an unsupervised estimation of a Statistical Shape Model (SSM) and uses this model to constrain the viable 3D coordinates. We show, by using simulated data, that our SSM estimator is robust even in datasets containing up to 50% of poses with outliers and/or missing data. In simulated and real data SSM estimation converges rapidly, capturing behaviourally relevant changes in body shape associated with exploratory behaviours (e.g. with rearing and changes in body orientation). Altogether 3D-UPPER represents a simple tool to minimise errors in 3D reconstruction while capturing meaningful behavioural parameters.

Functional integrity of visual coding following advanced photoreceptor degeneration.

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.

Look-up and look-down neurons in the mouse visual thalamus during freely moving exploration.

Visual information reaches cortex via the thalamic dorsal lateral geniculate nucleus (dLGN). dLGN activity is modulated by global sleep/wake states and arousal, indicating that it is not simply a passive relay station. However, its potential for more specific visuomotor integration is largely unexplored. We addressed this question by developing robust 3D video reconstruction of mouse head and body during spontaneous exploration paired with simultaneous neuronal recordings from dLGN. Unbiased evaluation of a wide range of postures and movements revealed a widespread coupling between neuronal activity and few behavioral parameters. In particular, postures associated with the animal looking up/down correlated with activity in >50% neurons, and the extent of this effect was comparable with that induced by full-body movements (typically locomotion). By contrast, thalamic activity was minimally correlated with other postures or movements (e.g., left/right head and body torsions). Importantly, up/down postures and full-body movements were largely independent and jointly coupled to neuronal activity. Thus, although most units were excited during full-body movements, some expressed highest firing when the animal was looking up ("look-up" neurons), whereas others expressed highest firing when the animal was looking down ("look-down" neurons). These results were observed in the dark, thus representing a genuine behavioral modulation, and were amplified in a lit arena. Our results demonstrate that the primary visual thalamus, beyond global modulations by sleep/awake states, is potentially involved in specific visuomotor integration and reveal two distinct couplings between up/down postures and neuronal activity.

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.

Infra-slow modulation of fast beta/gamma oscillations in the mouse visual system.

KEY POINTS: Neurophysiological activity in the subcortical visual system fluctuates in both infra-slow and fast oscillatory ranges, but the level of co-occurrence and potential functional interaction of these rhythms is unknown. Analysing dark-adapted spontaneous activity in the mouse subcortical visual system, we find that these two types of oscillation interact uniquely through a population of neurons expressing both rhythms. Genetic ablation of rod/cone signalling potentiates infra-slow and abolishes fast beta/gamma oscillations while genetic ablation of melanopsin substantially diminishes the interaction between these two rhythms. Our results indicate that in an intact visual system the phase of infra-slow modulates fast beta/gamma oscillations. Thus one possible impact of infra-slow oscillations in vision is to guide visual processing by interacting with fast narrowband oscillations. ABSTRACT: Infra-slow (<0.02 Hz) and fast beta/gamma (20-100 Hz) oscillations in neurophysiological activity have been widely found in the subcortical visual system. While it is well established that fast beta/gamma oscillations are involved in visual processing, the role (if any) of infra-slow oscillations is currently unknown. One possibility is that infra-slow oscillations exert influence by modulating the amplitude of fast oscillations, yet the extent to which these different oscillations arise independently and interact remains unknown. We addressed these questions by recording in vivo spontaneous activity from the subcortical visual system of visually intact mice, and animals whose retinal network was disrupted by advanced rod/cone degeneration (rd/rd cl) or melanopsin loss (Opn4-/- ). We found many neurons expressing only one type of oscillation, and indeed fast oscillations were absent in rd/rd cl. Conversely, neurons co-expressing the two oscillations were also common, and were encountered more often than expected by chance in visually intact but not Opn4-/- mice. Finally, where they co-occurred we found that beta/gamma amplitude was modulated by the infra-slow rhythm. Our data thus reveal that: (1) infra-slow and beta-gamma oscillations are separable phenomena; and (2) that they actively co-occur in a subset of neurones in which the phase of infra-slow oscillations defines beta-gamma oscillations amplitude. These findings suggest that infra-slow oscillations could influence vision by modulating beta-gamma oscillations, and raise the possibility that disruptions in these oscillatory behaviours contribute to vision dysfunction in retinal dystrophy.

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.

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.

Rods progressively escape saturation to drive visual responses in daylight conditions.

Rod and cone photoreceptors support vision across large light intensity ranges. Rods, active under dim illumination, are thought to saturate at higher (photopic) irradiances. The extent of rod saturation is not well defined; some studies report rod activity well into the photopic range. Using electrophysiological recordings from retina and dorsal lateral geniculate nucleus of cone-deficient and visually intact mice, we describe stimulus and physiological factors that influence photopic rod-driven responses. We find that rod contrast sensitivity is initially strongly reduced at high irradiances, but progressively recovers to allow responses to moderate contrast stimuli. Surprisingly, rods recover faster at higher light levels. A model of rod phototransduction suggests that phototransduction gain adjustments and bleaching adaptation underlie rod recovery. Consistently, exogenous chromophore reduces rod responses at bright background. Thus, bleaching adaptation renders mouse rods responsive to modest contrast at any irradiance. Paradoxically, raising irradiance across the photopic range increases the robustness of rod responses.

The impact of temporal modulations in irradiance under light adapted conditions on the mouse suprachiasmatic nuclei (SCN).

Electrophysiological responses of SCN neurons to light steps are well established, but responses to more natural modulations in irradiance have been much less studied. We address this deficit first by showing that variations in irradiance for human subjects are biased towards low temporal frequencies and small magnitudes. Using extracellular recordings we show that neurons in the mouse SCN are responsive to stimuli with these characteristics, tracking sinusoidal modulations in irradiance best at lower temporal frequencies and responding to abrupt changes in irradiance over a range of commonly encountered contrasts. The spectral sensitivity of these light adapted responses indicates that they are driven primarily by cones, but with melanopsin (and/or rods) contributing under more gradual changes. Higher frequency modulations in irradiance increased time averaged firing of SCN neurons (typically considered to encode background light intensity) modestly over that encountered during steady exposure, but did not have a detectable effect on the circadian phase resetting efficiency of light. Our findings highlight the SCN's ability to encode naturalistic temporal modulations in irradiance, while revealing that the circadian system can effectively integrate such signals over time such that phase-resetting responses remain proportional to the mean light exposure.

Melanopsin Contributions to the Representation of Images in the Early Visual System.

Melanopsin photoreception enhances retinal responses to variations in ambient light (irradiance) and drives non-image-forming visual reflexes such as circadian entrainment [1-6]. Melanopsin signals also reach brain regions responsible for form vision [7-9], but melanopsin's contribution, if any, to encoding visual images remains unclear. We addressed this deficit using principles of receptor silent substitution to present images in which visibility for melanopsin versus rods+cones was independently modulated, and we recorded evoked responses in the mouse dorsal lateral geniculate nucleus (dLGN; thalamic relay for cortical vision). Approximately 20% of dLGN units responded to patterns visible only to melanopsin, revealing that melanopsin signals alone can convey spatial information. Spatial receptive fields (RFs) mapped using melanopsin-isolating stimuli had ON centers with diameters ∼13°. Melanopsin and rod+cone responses differed in the temporal domain, and responses to slow changes in radiance (<0.9 Hz) and stationary images were deficient when stimuli were rendered invisible for melanopsin. We employed these data to devise and test a mathematical model of melanopsin's involvement in form vision and applied it, along with further experimental recordings, to explore melanopsin signals under simulated active view of natural scenes. Our findings reveal that melanopsin enhances the thalamic representation of scenes containing local correlations in radiance, compensating for the high temporal frequency bias of cone vision and the negative correlation between magnitude and frequency for changes in direction of view. Together, these data reveal a distinct melanopsin contribution to encoding visual images, predicting that, under natural view, melanopsin augments the early visual system's ability to encode patterns over moderate spatial scales.

Modulation of Fast Narrowband Oscillations in the Mouse Retina and dLGN According to Background Light Intensity.

Background light intensity (irradiance) substantially impacts the visual code in the early visual system at synaptic and single-neuron levels, but its influence on population activity is largely unexplored. We show that fast narrowband oscillations, an important feature of population activity, systematically increase in amplitude as a function of irradiance in both anesthetized and awake, freely moving mice and at the level of the retina and dorsal lateral geniculate nucleus (dLGN). Narrowband coherence increases with irradiance across large areas of the dLGN, but especially for neighboring units. The spectral sensitivity of these effects and their substantial reduction in melanopsin knockout animals indicate a contribution from inner retinal photoreceptors. At bright backgrounds, narrowband coherence allows pooling of single-unit responses to become a viable strategy for enhancing visual signals within its frequency range.

Melanopsin supports irradiance-driven changes in maintained activity in the superior colliculus of the mouse.

Melanopsin phototransduction allows intrinsically photosensitive retinal ganglion cells (ipRGCs) to maintain firing under sustained illumination and to encode irradiance. ipRGCs project to different parts of the visual system, including the superficial superior colliculus (sSC), but to date there is no description of melanopsin contributions to the activity of that nucleus. We sought to fill that gap using extracellular recordings to describe light response in the sSC. We failed to observe light responses in the sSC of mice lacking rod and cone function, in which melanopsin provides the only photoreception. Nor did the sSC of intact animals track very gradual ramps in irradiance, a stimulus encoded by melanopsin for other brain regions. However, in visually intact mice we did find maintained responses to extended light steps (30 s) and to an irradiance ramp upon which a high frequency (20 Hz) temporal white noise was superimposed. Both of these responses were deficient when the spectral composition of the stimulus was changed to selectively reduce its effective irradiance for melanopsin. Such maintained activity was also impaired in mice lacking melanopsin, and this effect was specific, as responses of this genotype to higher spatiotemporal frequency stimuli were normal. We conclude that ipRGCs contribute to irradiance-dependent modulations in maintained activity in the sSC, but that this effect is less robust than for other brain regions receiving ipRGC input.

The contribution of inner and outer retinal photoreceptors to infra-slow oscillations in the rat olivary pretectal nucleus.

A subpopulation of olivary pretectal nucleus (OPN) neurons discharges action potentials in an oscillatory manner, with a period of approximately two minutes. This 'infra-slow' oscillatory activity depends on synaptic excitation originating in the retina. Signals from rod-cone photoreceptors reach the OPN via the axons of either classic retinal ganglion cells or intrinsically photosensitive retinal ganglion cells (ipRGCs), which use melanopsin for photon capturing. Although both cell types convey light information, their physiological functions differ considerably. The aim of the present study was to disentangle how rod-cone and melanopsin photoresponses contribute to generation of oscillatory activity. Pharmacological manipulations of specific phototransduction cascades were used whilst recording extracellular single-unit activity in the OPN of anaesthetized rats. The results show that under photopic conditions (bright light), ipRGCs play a major role in driving infra-slow oscillations, as blocking melanopsin phototransmission abolishes or transiently disturbs oscillatory firing of the OPN neurons. On the other hand, blocking rod-cone phototransmission does not change firing patterns in photopic conditions. However, under mesopic conditions (moderate light), when melanopsin phototransmission is absent, blocking rod-cone signalling causes disturbances or even the disappearance of oscillations implying that classic photoreceptors are of greater importance under moderate light. Evidence is provided that all photoreceptors are required for the generation of oscillations in the OPN, although their roles in driving the rhythm are determined by the lighting conditions, consistent with their relative sensitivities. The results further suggest that maintained retinal activity is crucial to observe infra-slow oscillatory activity in the OPN.

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.