Bright daytime light enhances circadian amplitude in a diurnal mammal.

Mammalian circadian rhythms are orchestrated by a master pacemaker in the hypothalamic suprachiasmatic nuclei (SCN), which receives information about the 24 h light-dark cycle from the retina. The accepted function of this light signal is to reset circadian phase in order to ensure appropriate synchronization with the celestial day. Here, we ask whether light also impacts another key property of the circadian oscillation, its amplitude. To this end, we measured circadian rhythms in behavioral activity, body temperature, and SCN electrophysiological activity in the diurnal murid rodent Rhabdomys pumilio following stable entrainment to 12:12 light-dark cycles at four different daytime intensities (ranging from 18 to 1,900 lx melanopic equivalent daylight illuminance). R. pumilio showed strongly diurnal activity and body temperature rhythms in all conditions, but measures of rhythm robustness were positively correlated with daytime irradiance under both entrainment and subsequent free run. Whole-cell and extracellular recordings of electrophysiological activity in ex vivo SCN revealed substantial differences in electrophysiological activity between dim and bright light conditions. At lower daytime irradiance, daytime peaks in SCN spontaneous firing rate and membrane depolarization were substantially depressed, leading to an overall marked reduction in the amplitude of circadian rhythms in spontaneous activity. Our data reveal a previously unappreciated impact of daytime light intensity on SCN physiology and the amplitude of circadian rhythms and highlight the potential importance of daytime light exposure for circadian health.

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.

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.

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.

Modulations in irradiance directed at melanopsin, but not cone photoreceptors, reliably alter electrophysiological activity in the suprachiasmatic nucleus and circadian behaviour in mice.

Intrinsically photosensitive retinal ganglion cells convey intrinsic, melanopsin-based, photoreceptive signals alongside those produced by rods and cones to the suprachiasmatic nucleus (SCN) circadian clock. To date, experimental data suggest that melanopsin plays a more significant role in measuring ambient light intensity than cone photoreception. Such studies have overwhelmingly used diffuse light stimuli, whereas light intensity in the world around us varies across space and time. Here, we investigated the extent to which melanopsin or cone signals support circadian irradiance measurements in the presence of naturalistic spatiotemporal variations in light intensity. To address this, we first presented high- and low-contrast movies to anaesthetised mice whilst recording extracellular electrophysiological activity from the SCN. Using a mouse line with altered cone sensitivity (Opn1mwR mice) and multispectral light sources we then selectively varied irradiance of the movies for specific photoreceptor classes. We found that steps in melanopic irradiance largely account for the light induced-changes in SCN activity over a range of starting light intensities and in the presence of spatiotemporal modulation. By contrast, cone-directed changes in irradiance only influenced SCN activity when spatiotemporal contrast was low. Consistent with these findings, under housing conditions where we could independently adjust irradiance for melanopsin versus cones, the period lengthening effects of constant light on circadian rhythms in behaviour were reliably determined by melanopic irradiance, regardless of irradiance for cones. These data add to the growing evidence that modulating effective irradiance for melanopsin is an effective strategy for controlling the circadian impact of light.

Visual responses in the dorsal lateral geniculate nucleus at early stages of retinal degeneration in rd1 PDE6ß mice.

Inherited retinal degenerations encompass a wide range of diseases that result in the death of rod and cone photoreceptors, eventually leading to irreversible blindness. Low vision survives at early stages of degeneration, at which point it could rely on residual populations of rod/cone photoreceptors as well as the inner retinal photoreceptor, melanopsin. To date, the impact of partial retinal degeneration on visual responses in the primary visual thalamus (dorsal lateral geniculate nucleus, dLGN) remains unknown, as does their relative reliance on surviving rod and cone photoreceptors vs. melanopsin. To answer these questions, we recorded visually evoked responses in the dLGN of anesthetized rd1 mice using in vivo electrophysiology at an age (3-5 wk) at which cones are partially degenerate and rods are absent. We found that excitatory (ON) responses to light had lower amplitude and longer latency in rd1 mice compared with age-matched visually intact controls; however, contrast sensitivity and spatial receptive field size were largely unaffected at this early stage of degeneration. Responses were retained when those wavelengths to which melanopsin is most sensitive were depleted, indicating that they were driven primarily by surviving cones. Inhibitory responses appeared absent in the rd1 thalamus, as did light-evoked gamma oscillations in firing. This description of fundamental features of the dLGN visual response at this intermediate stage of retinal degeneration provides a context for emerging attempts to restore vision by introducing ectopic photoreception to the degenerate retina.NEW & NOTEWORTHY This study provides new therapeutically relevant insights to visual responses in the dorsal lateral geniculate nucleus during progressive retinal degeneration. Using in vivo electrophysiology, we demonstrate that visual responses have lower amplitude and longer latency during degeneration, but contrast sensitivity and spatial receptive fields remain unaffected. Such visual responses are driven predominantly by surviving cones rather than melanopsin photoreceptors. The functional integrity of this visual pathway is encouraging for emerging attempts at visual restoration.

A live cell assay of GPCR coupling allows identification of optogenetic tools for controlling Go and Gi signaling.

BACKGROUND: Animal opsins are light-sensitive G-protein-coupled receptors (GPCRs) that enable optogenetic control over the major heterotrimeric G-protein signaling pathways in animal cells. As such, opsins have potential applications in both biomedical research and therapy. Selecting the opsin with the best balance of activity and selectivity for a given application requires knowing their ability to couple to a full range of relevant Gα subunits. We present the GsX assay, a set of tools based on chimeric Gs subunits that transduce coupling of opsins to diverse G proteins into increases in cAMP levels,  measured with a real-time reporter in living cells. We use this assay to compare coupling to Gi/o/t across a panel of natural and chimeric opsins selected for potential application in gene therapy for retinal degeneration. RESULTS: Of the opsins tested, wild-type human rod opsin had the highest activity for chimeric Gs proxies for Gi and Gt (Gsi and Gst) and was matched in Go proxy (Gso) activity only by a human rod opsin/scallop opsin chimera. Rod opsin drove roughly equivalent responses via Gsi, Gso, and Gst, while cone opsins showed much lower activities with Gso than Gsi or Gst, and a human rod opsin/amphioxus opsin chimera demonstrated higher activity with Gso than with Gsi or Gst. We failed to detect activity for opsin chimeras bearing three intracellular fragments of mGluR6, and observed unexpectedly complex response profiles for scallop and amphioxus opsins thought to be specialized for Go. CONCLUSIONS: These results identify rod opsin as the most potent non-selective Gi/o/t-coupled opsin, long-wave sensitive cone opsin as the best for selectively activating Gi/t over Go, and a rod opsin/amphioxus opsin chimera as the best choice for selectively activating Go over Gi/t.

Colour as a signal for entraining the mammalian circadian clock.

Twilight is characterised by changes in both quantity ("irradiance") and quality ("colour") of light. Animals use the variation in irradiance to adjust their internal circadian clocks, aligning their behaviour and physiology with the solar cycle. However, it is currently unknown whether changes in colour also contribute to this entrainment process. Using environmental measurements, we show here that mammalian blue-yellow colour discrimination provides a more reliable method of tracking twilight progression than simply measuring irradiance. We next use electrophysiological recordings to demonstrate that neurons in the mouse suprachiasmatic circadian clock display the cone-dependent spectral opponency required to make use of this information. Thus, our data show that some clock neurons are highly sensitive to changes in spectral composition occurring over twilight and that this input dictates their response to changes in irradiance. Finally, using mice housed under photoperiods with simulated dawn/dusk transitions, we confirm that spectral changes occurring during twilight are required for appropriate circadian alignment under natural conditions. Together, these data reveal a new sensory mechanism for telling time of day that would be available to any mammalian species capable of chromatic vision.

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.

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.

A Wrist-Worn Internet of Things Sensor Node for Wearable Equivalent Daylight Illuminance Monitoring.

Light exposure is a vital regulator of physiology and behavior in humans. However, monitoring of light exposure is not included in current wearable Internet of Things (IoT) devices, and only recently have international standards defined [Formula: see text] -optic equivalent daylight illuminance (EDI) measures for how the eye responds to light. This article reports a wearable light sensor node that can be incorporated into the IoT to provide monitoring of EDI exposure in real-world settings. We present the system design, electronic performance testing, and accuracy of EDI measurements when compared to a calibrated spectral source. This includes consideration of the directional response of the sensor, and a comparison of performance when placed on different parts of the body, and a demonstration of practical use over 7 days. Our device operates for 3.5 days between charges, with a sampling period of 30 s. It has 10 channels of measurement, over the range 415-910 nm, balancing accuracy and cost considerations. Measured [Formula: see text]-opic EDI results for 13 devices show a mean absolute error of less than 0.07 log lx, and a minimum between device correlation of 0.99. These findings demonstrate that accurate light sensing is feasible, including at wrist worn locations. We provide an experimental platform for use in future investigations in real-world light exposure monitoring and IoT-based lighting control.

Colour and melanopsin mediated responses in the murine retina.

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.

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.

Optogenetic manipulation of BMP signaling to drive chondrogenic differentiation of hPSCs.

Optogenetics is a rapidly advancing technology combining photochemical, optical, and synthetic biology to control cellular behavior. Together, sensitive light-responsive optogenetic tools and human pluripotent stem cell differentiation models have the potential to fine-tune differentiation and unpick the processes by which cell specification and tissue patterning are controlled by morphogens. We used an optogenetic bone morphogenetic protein (BMP) signaling system (optoBMP) to drive chondrogenic differentiation of human embryonic stem cells (hESCs). We engineered light-sensitive hESCs through CRISPR-Cas9-mediated integration of the optoBMP system into the AAVS1 locus. The activation of optoBMP with blue light, in lieu of BMP growth factors, resulted in the activation of BMP signaling mechanisms and upregulation of a chondrogenic phenotype, with significant transcriptional differences compared to cells in the dark. Furthermore, cells differentiated with light could form chondrogenic pellets consisting of a hyaline-like cartilaginous matrix. Our findings indicate the applicability of optogenetics for understanding human development and tissue engineering.

Melanopsin Driven Light Responses Across a Large Fraction of Retinal Ganglion Cells in a Dystrophic Retina.

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.

Form vision from melanopsin in humans.

Detection and discrimination of spatial patterns is thought to originate with photoreception by rods and cones. Here, we investigated whether the inner-retinal photoreceptor melanopsin could represent a third origin for form vision. We developed a 4-primary visual display capable of presenting patterns differing in contrast for melanopsin vs cones, and generated spectrally distinct stimuli that were indistinguishable for cones (metamers) but presented contrast for melanopsin. Healthy observers could detect sinusoidal gratings formed by these metamers when presented in the peripheral retina at low spatial (≤0.8 cpd) and temporal (≤0.45 Hz) frequencies, and Michelson contrasts ≥14% for melanopsin. Metameric gratings became invisible at lower light levels (<1013 melanopsin photons cm-2 sr-1 s-1) when rods are more active. The addition of metameric increases in melanopsin contrast altered appearance of greyscale representations of coarse gratings and a range of everyday images. These data identify melanopsin as a new potential origin for aspects of spatial vision in humans.

Pupil responses to hidden photoreceptor-specific modulations in movies.

Under typical daytime light levels, the human pupillary light response (PLR) is driven by the activity of the L, M, and S cones, and melanopsin expressed in the so-called intrinsically photosensitive retinal ganglion cells (ipRGCs). However, the importance of each of these photoreceptive mechanisms in defining pupil size under real-world viewing conditions remains to be established. To address this question, we embedded photoreceptor-specific modulations in a movie displayed using a novel projector-based five-primary spatial stimulation system, which allowed for the precise control of photoreceptor activations in time and space. We measured the pupillary light response in eleven observers, who viewed short cartoon movies which contained hidden low-frequency (0.25 Hz) silent-substitution modulations of the L, M and S cones (no stimulation of melanopsin), melanopsin (no stimulation of L, M and S cones), both L, M, and S cones and melanopsin or no modulation at all. We find that all photoreceptors active at photopic light levels regulate pupil size under this condition. Our data imply that embedding modulations in photoreceptor contrast could provide a method to manipulate key adaptive aspects of the human visual system in everyday, real-world activities such as watching a movie.

Cones Support Alignment to an Inconsistent World by Suppressing Mouse Circadian Responses to the Blue Colors Associated with Twilight.

In humans, short-wavelength light evokes larger circadian responses than longer wavelengths [1-3]. This reflects the fact that melanopsin, a key contributor to circadian assessments of light intensity, most efficiently captures photons around 480 nm [4-8] and gives rise to the popular view that "blue" light exerts the strongest effects on the clock. However, in the natural world, there is often no direct correlation between perceived color (as reported by the cone-based visual system) and melanopsin excitation. Accordingly, although the mammalian clock does receive cone-based chromatic signals [9], the influence of color on circadian responses to light remains unclear. Here, we define the nature and functional significance of chromatic influences on the mouse circadian system. Using polychromatic lighting and mice with altered cone spectral sensitivity (Opn1mwR), we generate conditions that differ in color (i.e., ratio of L- to S-cone opsin activation) while providing identical melanopsin and rod activation. When biased toward S-opsin activation (appearing "blue"), these stimuli reliably produce weaker circadian behavioral responses than those favoring L-opsin ("yellow"). This influence of color (which is absent in animals lacking cone phototransduction; Cnga3-/-) aligns with natural changes in spectral composition over twilight, where decreasing solar angle is accompanied by a strong blue shift [9-11]. Accordingly, we find that naturalistic color changes support circadian alignment when environmental conditions render diurnal variations in light intensity weak/ambiguous sources of timing information. Our data thus establish how color contributes to circadian entrainment in mammals and provide important new insight to inform the design of lighting environments that benefit health.

Exploiting metamerism to regulate the impact of a visual display on alertness and melatonin suppression independent of visual appearance.

Objectives: Artificial light sources such as visual display units (VDUs) elicit a range of subconscious and reflex light responses, including increases in alertness and suppression of pineal melatonin. Such responses employ dedicated retinal circuits encompassing melanopsin photoreceptors. Here, we aimed to determine whether this arrangement can be exploited to modulate the impact of VDUs on melatonin onset and alertness without altering visual appearance. Methods: We generated a five-primary VDU capable of presenting metameric movies (matched for color and luminance) but varying in melanopic-irradiance. Healthy human participants (n = 11) were exposed to the VDU from 18:00 to 23:00 hours at high- or low-melanopic setting in a randomized cross-over design and measured salivary melatonin and self-reported sleepiness at 30-minute intervals. Results: Our VDU presented a 3× adjustment in melanopic-irradiance for images matched photometrically for color and luminance. Participants reported no significant difference in visual appearance (color and glare) between conditions. During the time in which the VDU was viewed, self-reported sleepiness and salivary melatonin levels increased significantly, as would be expected in this phase of the diurnal cycle. The magnitude of the increase in both parameters was significantly enhanced when melanopic-irradiance was reduced. Conclusions: Our data demonstrate that melatonin onset and self-reported sleepiness can be modulated independent of photometric parameters (color and luminance) under a commonly encountered light exposure scenario (evening use of a VDU). They provide the first demonstration that the impact of light on alertness and melatonin production can be controlled independently of visual experience, and establish a VDU capable of achieving this objective.

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.