High-intensity red light suppresses melatonin

Early studies on rodents indicated that the long-wavelength portion of the spectrum (orange- and red-appearing light) could influence circadian and neuroendocrine responses. Since then, both polychromatic and analytic action spectra in various rodent species have demonstrated that long-wavelength light is very weak, if not entirely inactive, for regulating neurobehavioral responses. Since testing of monochromatic light wavelengths above 600 nm is uncommon, many researchers have assumed that there is little to no effect of red light on the neuroendocrine or circadian systems. The aims of the following studies were to test the efficacy of monochromatic light above 600 nm for melatonin suppression in hamsters and humans. Results in hamsters show that 640 nm monochromatic light at 1.1 x 10(17) photons/cm2 can acutely suppress pineal melatonin levels. In normal healthy humans, equal photon density exposures of 1.9 x 10(18) photons/cm2 at 460, 630, and 700 nm monochromatic light elicited a significant melatonin suppression at 460 nm and small reductions of plasma melatonin levels at 630 and 700 nm. These findings are discussed relative to the possible roles of classical visual photoreceptors and the recently discovered intrinsically photosensitive retinal ganglion cells for circadian phototransduction. That physiology, and its potential for responding to red light, has implications for domestic applications involving animal care, the lighting of typical human environments, and advanced applications such as space exploration.     

Photons, clocks, and consciousness

Light profoundly impacts human consciousness through the stimulation of the visual system and powerfully regulates the human circadian system, which, in turn, has a broad regulatory impact on virtually all tissues in the body. For more than 25 years, the techniques of action spectroscopy have yielded insights into the wavelength sensitivity of circadian input in humans and other mammalian species. The seminal discovery of melanopsin, the photopigment in intrinsically photosensitive retinal ganglion cells, has provided a significant turning point for understanding human circadian phototransduction. Action spectra in humans show that the peak wavelength sensitivity for this newly discovered sensory system is within the blue portion of the spectrum. This is fundamentally different from the three-cone photopic visual system, as well as the individual rod and cone photoreceptor peaks. Studies on rodents, nonhuman primates, and humans indicate that despite having a different wavelength fingerprint, these classic visual photoreceptors still provide an element of input to the circadian system. These findings open the door to innovations in light therapy for circadian and affective disorders, as well as possible architectural light applications.     

High‐intensity red light suppresses melatonin

Early studies on rodents indicated that the long‐wavelength portion of the spectrum (orange‐ and red‐appearing light) could influence circadian and neuroendocrine responses. Since then, both polychromatic and analytic action spectra in various rodent species have demonstrated that long‐wavelength light is very weak, if not entirely inactive, for regulating neurobehavioral responses. Since testing of monochromatic light wavelengths above 600 nm is uncommon, many researchers have assumed that there is little to no effect of red light on the neuroendocrine or circadian systems. The aims of the following studies were to test the efficacy of monochromatic light above 600 nm for melatonin suppression in hamsters and humans. Results in hamsters show that 640 nm monochromatic light at 1.1×10¹⁷ photons/cm² can acutely suppress pineal melatonin levels. In normal healthy humans, equal photon density exposures of 1.9×10¹⁸ photons/cm² at 460, 630, and 700 nm monochromatic light elicited a significant melatonin suppression at 460 nm and small reductions of plasma melatonin levels at 630 and 700 nm. These findings are discussed relative to the possible roles of classical visual photoreceptors and the recently discovered intrinsically photosensitive retinal ganglion cells for circadian phototransduction. That physiology, and its potential for responding to red light, has implications for domestic applications involving animal care, the lighting of typical human environments, and advanced applications such as space exploration.     

Nonvisual photoreceptors of the deep brain, pineal organs and retina

The role of the nonvisual photoreception is to synchronise periodic functions of living organisms to the environmental light periods in order to help survival of various species in different biotopes. In vertebrates, the so-called deep brain (septal and hypothalamic) photoreceptors, the pineal organs (pineal- and parapineal organs, frontal- and parietal eye) and the retina (of the "lateral" eye) are involved in the light-based entrain of endogenous circadian clocks present in various organs. In humans, photoperiodicity was studied in connection with sleep disturbances in shift work, seasonal depression, and in jet-lag of transmeridional travellers. In the present review, experimental and molecular aspects are discussed, focusing on the histological and histochemical basis of the function of nonvisual photoreceptors. We also offer a view about functional changes of these photoreceptors during pre- and postnatal development as well as about its possible evolution. Our scope in some points is different from the generally accepted views on the nonvisual photoreceptive systems. The deep brain photoreceptors are hypothalamic and septal nuclei of the periventricular cerebrospinal fluid (CSF)-contacting neuronal system. Already present in the lancelet and representing the most ancient type of vertebrate nerve cells ("protoneurons"), CSF-contacting neurons are sensory-type cells sitting in the wall of the brain ventricles that send a ciliated dendritic process into the CSF. Various opsins and other members of the phototransduction cascade have been demonstrated in telencephalic and hypothalamic groups of these neurons. In all species examined so far, deep brain photoreceptors play a role in the circadian and circannual regulation of periodic functions. Mainly called pineal "glands" in the last decades, the pineal organs actually represent a differentiated form of encephalic photoreceptors. Supposed to be intra- and extracranially outgrown groups of deep brain photoreceptors, pineal organs also contain neurons and glial elements. Extracranial pineal organs of submammalians are cone-dominated photoreceptors sensitive to different wavelengths of light, while intracranial pineal organs predominantly contain rod-like photoreceptor cells and thus scotopic light receptors. Vitamin B-based light-sensitive cryptochromes localized immunocytochemically in some pineal cells may take part in both the photoreception and the pacemaker function of the pineal organ. In spite of expressing phototransduction cascade molecules and forming outer segment-like cilia in some species, the mammalian pineal is considered by most of the authors as a light-insensitive organ. Expression of phototransduction cascade molecules, predominantly in young animals, is a photoreceptor-like characteristic of pinealocytes in higher vertebrates that may contribute to a light-percepting task in the perinatal entrainment of rhythmic functions. In adult mammals, adrenergic nerves--mediating daily fluctuation of sympathetic activity rather than retinal light information as generally supposed--may sustain circadian periodicity already entrained by light perinatally. Altogether three phases were supposed to exist in pineal entrainment of internal pacemakers: an embryological synchronization by light and in viviparous vertebrates by maternal effects (1); a light-based, postnatal entrainment (2); and in adults, a maintenance of periodicity by daily sympathetic rhythm of the hypothalamus. In addition to its visual function, the lateral eye retina performs a nonvisual task. Nonvisual retinal light perception primarily entrains genetically-determined periodicity, such as rod-cone dominance, EEG rhythms or retinomotor movements. It also influences the suprachiasmatic nucleus, the primary pacemaker of the brain. As neither rods nor cones seem to represent the nonvisual retinal photoreceptors, the presence of additional photoreceptors has been supposed. Cryptochrome 1, a photosensitive molecule identified in retinal nerve cells and in a subpopulation of retinal photoreceptors, is a good candidate for the nonvisual photoreceptor molecule as well as for a member of pacemaker molecules in the retina. When comparing various visual and nonvisual photoreceptors, transitory, "semi visual" (directional) light-perceptive cells can be detected among them, such as those in the parietal eye of reptiles. Measuring diffuse light intensity of the environment, semivisual photoreceptors also possess some directional light perceptive capacity aided by complementary lens-like structures, and screening pigment cells. Semivisual photoreception in aquatic animals may serve for identifying environmental areas of suitable illumination, or in poikilotermic terrestrial species for measuring direct solar irradiation for thermoregulation. As directional photoreceptors were identified among nonvisual light perceptive cells in the lancelet, but eyes are lacking, an early appearance of semivisual function, prior to a visual one (nonvisual --> semivisual --> visual?) in the vertebrate evolution was supposed.     

Additivity in murine circadian phototransduction

Additivity in the circadian phototransduction system of the mouse has not been tested directly. Because of this, accurate prediction of circadian phase shifts elicited by polychromatic light stimuli cannot be derived from the results of studies using monochromatic light stimuli. This limitation also makes it impossible to deduce the relative contributions of the photoreceptive mechanisms (rods, cones and melanopsin-containing retinal ganglion cells) underlying circadian phototransduction in the mouse. Using nearly monochromatic light stimuli of different spectral composition, and combinations thereof, we demonstrated that murine circadian phototransduction exhibits additivity. Based on the locomotor activity phase shifts elicited by these stimuli, we developed the first quantitative assessment of the relative contributions of conventional and novel photoreceptive mechanisms for circadian functioning in the mouse.     

Melanopsin-dependent nonvisual responses: evidence for photopigment bistability in vivo

In mammals, nonvisual responses to light have been shown to involve intrinsically photosensitive retinal ganglion cells (ipRGC) that express melanopsin and that are modulated by input from both rods and cones. Recent in vitro evidence suggests that melanopsin possesses dual photosensory and photoisomerase functions, previously thought to be a unique feature of invertebrate rhabdomeric photopigments. In cultured cells that normally do not respond to light, heterologous expression of mammalian melanopsin confers light sensitivity that can be restored by prior stimulation with appropriate wavelengths. Using three different physiological and behavioral assays, we show that this in vitro property translates to in vivo, melanopsin-dependent nonvisual responses. We find that prestimulation with long-wavelength light not only restores but enhances single-unit responses of SCN neurons to 480-nm light, whereas the long-wavelength stimulus alone fails to elicit any response. Recordings in Opn4-/- mice confirm that melanopsin provides the main photosensory input to the SCN, and furthermore, demonstrate that melanopsin is required for response enhancement, because this capacity is abolished in the knockout mouse. The efficiency of the light-enhancement effect depends on wavelength, irradiance, and duration. Prior long-wavelength light exposure also enhances short-wavelength-induced phase shifts of locomotor activity and pupillary constriction, consistent with the expression of a photoisomerase-like function in nonvisual responses to light.     

Divergent photic thresholds in the non-image-forming visual system: entrainment, masking and pupillary light reflex

Light is the principal cue that entrains the circadian timing system, but the threshold of entrainment and the relative contributions of the retinal photoreceptors—rods, cones and intrinsically photosensitive retinal ganglion cells—are not known. We measured thresholds of entrainment of wheel-running rhythms at three wavelengths, and compared these to thresholds of two other non-image-forming visual system functions: masking and the pupillary light reflex (PLR). At the entrainment threshold, the relative spectral sensitivity and absolute photon flux suggest that this threshold is determined by rods. Dim light that entrained mice failed to elicit either masking or PLR; in general, circadian entrainment is more sensitive by 1–2 log units than other measures of the non-image-forming visual system. Importantly, the results indicate that dim light can entrain circadian rhythms even when it fails to produce more easily measurable acute responses to light such as phase shifting and melatonin suppression. Photosensitivity to one response, therefore, cannot be generalized to other non-image-forming functions. These results also impact practical problems in selecting appropriate lighting in laboratory animal husbandry.     

Effect of vitamin A depletion on nonvisual phototransduction pathways in cryptochromeless mice

Mice exhibit multiple nonvisual responses to light, including 1) photoentrainment of circadian rhythm; 2) "masking," which refers to the acute effect of light on behavior, either negative (activity suppressing) or positive (activity inducing); and 3) pupillary constriction. In mammals, the eye is the sole photosensory organ for these responses, and it contains only 2 known classes of pigments: opsins and cryptochromes. No individual opsin or cryptochrome gene is essential for circadian photoreception, gene photoinduction, or masking. Previously, the authors found that mice lacking retinol-binding protein, in which dietary depletion of ocular retinaldehyde can be achieved, had normal light signaling to the SCN, as determined by per gene photoinduction. In the present study, the authors analyzed phototransduction to the SCN in vitamin A-replete and vitamin A-depleted rbp-/- and rbp-/-cry1-/-cry2-/- mice using molecular and behavioral end points. They found that vitamin A-depleted rbp-/- mice exhibit either normal photoentrainment or become diurnal. In contrast, while vitamin A-replete rbp-/-cry1-/-cry2-/- mice are light responsive (with reduced sensitivity), vitamin A-depleted rbp-/-cry1-/-cry2-/- mice, which presumably lack functional opsins and cryptochromes, lose most behavioral and molecular responses to light. These data demonstrate that both cryptochromes and opsins regulate nonvisual photoresponses.     

Changes in the colour of light cue circadian activity

The discovery of melanopsin, the non-visual opsin present in intrinsically photosensitive retinal ganglion cells (ipRGCs), has created great excitement in the field of circadian biology. Now, researchers have emphasized melanopsin as the main photopigment governing circadian activity in vertebrates. Circadian biologists have tested this idea under standard laboratory, 12h Light: 12h Dark, lighting conditions that lack the dramatic daily colour changes of natural skylight. Here we used a stimulus paradigm in which the colour of the illumination changed throughout the day, thus mimicking natural skylight, but luminance, sensed intrinsically by melanopsin containing ganglion cells, was kept constant. We show in two species of cichlid, Aequidens pulcher and Labeotropheus fuelleborni, that changes in light colour, not intensity, are the primary determinants of natural circadian activity. Moreover, opponent-cone photoreceptor inputs to ipRGCs mediate the sensation of wavelength change, and not the intrinsic photopigment, melanopsin. These results have implications for understanding the evolutionary biology of non-visual photosensory pathways and answer long-standing questions about the nature and distribution of photopigments in organisms, including providing a solution to the mystery of why nocturnal animals routinely have mutations that interrupt the function of their short wavelength sensitive photopigment gene.     

Recommendations for daytime,evening, and nighttime indoor light exposure to best support physiology,sleep, and wakefulness in healthy adults

Ocular light exposure has important influences on human health and well-being through modulation of circadian rhythms and sleep, as well as neuroendocrine and cognitive functions. Prevailing patterns of light exposure do not optimally engage these actions for many individuals, but advances in our understanding of the underpinning mechanisms and emerging lighting technologies now present opportunities to adjust lighting to promote optimal physical and mental health and performance. A newly developed, international standard provides a SI-compliant way of quantifying the influence of light on the intrinsically photosensitive, melanopsin-expressing, retinal neurons that mediate these effects. The present report provides recommendations for lighting, based on an expert scientific consensus and expressed in an easily measured quantity (melanopic equivalent daylight illuminance (melaponic EDI)) defined within this standard. The recommendations are supported by detailed analysis of the sensitivity of human circadian, neuroendocrine, and alerting responses to ocular light and provide a straightforward framework to inform lighting design and practice.

Light exposure influences human health and wellbeing by modulating circadian rhythms and sleep. This Consensus View outlines the first expert scientific consensus recommendations for appropriate daily patterns of light exposure to promote health and wellbeing and inform lighting design and practice.     

Cephalochordate melanopsin: evolutionary linkage between invertebrate visual cells and vertebrate photosensitive retinal ganglion cells

Animal photoreceptor cells can be classified into two distinct types, depending on whether the photopigment is borne on the membrane of a modified cilium (ciliary type) or apical microvilli (rhabdomeric type) [1]. Ciliary photoreceptors are well known as vertebrate rods and cones and are also found in several invertebrates. The rhabdomeric photoreceptor, in contrast, is a predominant type of invertebrate visual cell, but morphologically identifiable rhabdomeric photoreceptors have never been found in vertebrates. It is hypothesized that the rhabdomeric photoreceptor cell had evolved to be the photosensitive retinal ganglion cell for the vertebrate circadian photoentrainment [2, 3 and 4] owing to the fact that some molecules involved in cell differentiation are common among them [5]. We focused on the cephalochordate amphioxus because it is the closest living invertebrate to the vertebrates, and interestingly, it has rhabdomeric photoreceptor cells for putative nonvisual functions [6]. Here, we show that the amphioxus homolog of melanopsin [7, 8 and 9], the circadian photopigment in the photosensitive retinal ganglion cells of vertebrates, is expressed in the rhabdomeric photoreceptor cells of the amphioxus and that its biochemical and photochemical properties, not just its primary structure, are considerably similar to those of the visual rhodopsins in the rhabdomeric photoreceptor cells of higher invertebrates. The cephalochordate rhabdomeric photoreceptor represents an evolutionary link between the invertebrate visual photoreceptor and the vertebrate circadian photoreceptor.     

The retina-attached SCN slice preparation: an in vitro mammalian circadian visual system

The suprachiasmatic nucleus (SCN), the mammalian circadian pacemaker, receives information about ambient light levels through the retinohypothalamic tract. This information resets the molecular clock of SCN neurons, thereby entraining overt animal behavior and physiology to the solar cycle. Progress toward functional characterization of retinal influences on the SCN has been hampered by limitations of established experimental paradigms. To overcome this hurdle, the authors have developed a novel in vitro preparation of the rat retinohypothalamic circuit that maintains functional connectivity between the retinas and the SCN. This method permits whole-cell patch-clamp recordings from visually identified, light-responsive SCN neurons. Using this preparation, the authors have found that in the SCN, light-evoked responses are partly driven by the melanopsin photosensory system of the intrinsically photosensitive retinal ganglion cells and that SCN neurons exhibit light adaptation. The authors have also been able to generate this preparation from mice, demonstrating the feasibility of applying this method to transgenic mice.     

Inducible ablation of melanopsin-expressing retinal ganglion cells reveals their central role in non-image forming visual responses

Rod/cone photoreceptors of the outer retina and the melanopsin-expressing retinal ganglion cells (mRGCs) of the inner retina mediate non-image forming visual responses including entrainment of the circadian clock to the ambient light, the pupillary light reflex (PLR), and light modulation of activity. Targeted deletion of the melanopsin gene attenuates these adaptive responses with no apparent change in the development and morphology of the mRGCs. Comprehensive identification of mRGCs and knowledge of their specific roles in image-forming and non-image forming photoresponses are currently lacking. We used a Cre-dependent GFP expression strategy in mice to genetically label the mRGCs. This revealed that only a subset of mRGCs express enough immunocytochemically detectable levels of melanopsin. We also used a Cre-inducible diphtheria toxin receptor (iDTR) expression approach to express the DTR in mRGCs. mRGCs develop normally, but can be acutely ablated upon diphtheria toxin administration. The mRGC-ablated mice exhibited normal outer retinal function. However, they completely lacked non-image forming visual responses such as circadian photoentrainment, light modulation of activity, and PLR. These results point to the mRGCs as the site of functional integration of the rod/cone and melanopsin phototransduction pathways and as the primary anatomical site for the divergence of image-forming and non-image forming photoresponses in mammals.     

Does pupil constriction under blue and green monochromatic light exposure change with age?

Many nonvisual functions are regulated by light through a photoreceptive system involving melanopsin-expressing retinal ganglion cells that are maximally sensitive to blue light. Several studies have suggested that the ability of light to modulate circadian entrainment and to induce acute effects on melatonin secretion, subjective alertness, and gene expression decreases during aging, particularly for blue light. This could contribute to the documented changes in sleep and circadian regulatory processes with aging. However, age-related modification in the impact of light on steady-state pupil constriction, which regulates the amount of light reaching the retina, is not demonstrated. We measured pupil size in 16 young (22.8±4 years) and 14 older (61±4.4 years) healthy subjects during 45-second exposures to blue (480 nm) and green (550 nm) monochromatic lights at low (7×10(12) photons/cm2/s), medium (3×10(13) photons/cm2/s), and high (10(14) photons/cm2/s) irradiance levels. Results showed that young subjects had consistently larger pupils than older subjects for dark adaptation and during all light exposures. Steady-state pupil constriction was greater under blue than green light exposure in both age groups and increased with increasing irradiance. Surprisingly, when expressed in relation to baseline pupil size, no significant age-related differences were observed in pupil constriction. The observed reduction in pupil size in older individuals, both in darkness and during light exposure, may reduce retinal illumination and consequently affect nonvisual responses to light. The absence of a significant difference between age groups for relative steady-state pupil constriction suggests that other factors such as tonic, sympathetic control of pupil dilation, rather than light sensitivity per se, account for the observed age difference in pupil size regulation. Compared to other nonvisual functions, the light sensitivity of steady-state pupil constriction appears to remain relatively intact and is not profoundly altered by age.     

Short-wavelength light sensitivity of circadian, pupillary, and visual awareness in humans lacking an outer retina

As the ear has dual functions for audition and balance, the eye has a dual role in detecting light for a wide range of behavioral and physiological functions separate from sight. These responses are driven primarily by stimulation of photosensitive retinal ganglion cells (pRGCs) that are most sensitive to short-wavelength ( approximately 480 nm) blue light and remain functional in the absence of rods and cones. We examined the spectral sensitivity of non-image-forming responses in two profoundly blind subjects lacking functional rods and cones (one male, 56 yr old; one female, 87 yr old). In the male subject, we found that short-wavelength light preferentially suppressed melatonin, reset the circadian pacemaker, and directly enhanced alertness compared to 555 nm exposure, which is the peak sensitivity of the photopic visual system. In an action spectrum for pupillary constriction, the female subject exhibited a peak spectral sensitivity (lambda(max)) of 480 nm, matching that of the pRGCs but not that of the rods and cones. This subject was also able to correctly report a threshold short-wavelength stimulus ( approximately 480 nm) but not other wavelengths. Collectively these data show that pRGCs contribute to both circadian physiology and rudimentary visual awareness in humans and challenge the assumption that rod- and cone-based photoreception mediate all "visual" responses to light.     

Circadian phototransduction and the regulation of biological rhythms

The vertebrate circadian system that controls most biological rhythms is composed of multiple oscillators with varied hierarchies and complex levels of organization and interaction. The retina plays a key role in the regulation of daily rhythms and light is the main synchronizer of the circadian system. To date, the identity of photoreceptors/photopigments responsible for the entrainment of biological rhythms is still uncertain; however, it is known that phototransduction must occur in the eye because light entrainment is lost with eye removal. The retina is also rhythmic in physiological and metabolic activities as well as in gene expression. Retinal oscillators may act like clocks to induce changes in the visual system according to the phase of the day by predicting environmental changes. These oscillatory and photoreceptive capacities are likely to converge all together on selected retinal cells. The aim of this overview is to present the current knowledge of retinal physiology in relation to the circadian timing system.     

Cell death and the creation of regional differences in neuronal numbers.

Regional variations in cell death are ubiquitous in the nervous system. In the retina, cell death in retinal ganglion cells is elevated in the retinal periphery and may be important in setting up the initial conditions that produce central retinal specializations such as an area centralis or visual streak. In central visual system structures, pronounced spatial and spatiotemporal inhomogeneities in cell death are seen both in layers and regions of the lateral geniculate nucleus and superior colliculus; similar indications of inhomogeneities are seen in those nonvisual structures that have been examined. Cell death in the cortex is highly nonuniform, by layer and by cortical area. A variety of possible functions for these regional losses are proposed, in the context of a uniform mechanism for cell death that allows it to assume multiple functions.     

Photochemical properties of Mammalian melanopsin

Melanopsin is the photoreceptor molecule of intrinsically photosensitive retinal ganglion cells, which serve as the input for various nonvisual behavior and physiological functions fundamental to organisms. The retina, therefore, possess a melanopsin-based nonvisual system in addition to the visual system based on the classical visual photoreceptor molecules. To elucidate the molecular properties of melanopsin, we have exogenously expressed mouse melanopsin in cultured cells. We were able to obtain large amounts of purified mouse melanopsin and conducted a comprehensive spectroscopic study of its photochemical properties. Melanopsin has an absorption maximum at 467 nm, and it converts to a meta intermediate having an absorption maximum at 476 nm. The melanopsin photoreaction is similar to that of squid rhodopsin, exhibiting bistability that results in a photosteady mixture of a resting state (melanopsin containing 11-cis-retinal) and an excited state (metamelanopsin containing all-trans-retinal) upon sustained irradiation. The absorption coefficient of melanopsin is 33000 ± 1000 M(-1) cm(-1), and its quantum yield of isomerization is 0.52; these values are also typical of invertebrate bistable pigments. Thus, the nonvisual system in the retina relies on a type of photoreceptor molecule different from that of the visual system. Additionally, we found a new state of melanopsin, containing 7-cis-retinal (extramelanopsin), which forms readily upon long-wavelength irradiation (yellow to red light) and photoconverts to metamelanopsin with short-wavelength (blue light) irradiation. Although it is unclear whether extramelanopsin would have any physiological role, it could potentially allow wavelength-dependent regulation of melanopsin functions.     

The circadian response of intrinsically photosensitive retinal ganglion cells

Intrinsically photosensitive retinal ganglion cells (ipRGC) signal environmental light level to the central circadian clock and contribute to the pupil light reflex. It is unknown if ipRGC activity is subject to extrinsic (central) or intrinsic (retinal) network-mediated circadian modulation during light entrainment and phase shifting. Eleven younger persons (18-30 years) with no ophthalmological, medical or sleep disorders participated. The activity of the inner (ipRGC) and outer retina (cone photoreceptors) was assessed hourly using the pupil light reflex during a 24 h period of constant environmental illumination (10 lux). Exogenous circadian cues of activity, sleep, posture, caffeine, ambient temperature, caloric intake and ambient illumination were controlled. Dim-light melatonin onset (DLMO) was determined from salivary melatonin assay at hourly intervals, and participant melatonin onset values were set to 14 h to adjust clock time to circadian time. Here we demonstrate in humans that the ipRGC controlled post-illumination pupil response has a circadian rhythm independent of external light cues. This circadian variation precedes melatonin onset and the minimum ipRGC driven pupil response occurs post melatonin onset. Outer retinal photoreceptor contributions to the inner retinal ipRGC driven post-illumination pupil response also show circadian variation whereas direct outer retinal cone inputs to the pupil light reflex do not, indicating that intrinsically photosensitive (melanopsin) retinal ganglion cells mediate this circadian variation.     

The visual input stage of the mammalian circadian pacemaking system: II. The effect of light and drugs on retinal function.

Acute light pulses as well as long-term light exposure may not only modulate photoreceptive properties, but also induce reversible or irreversible damage to the retina, depending on exposure conditions. Illuminance levels in laboratory animal colonies and manipulations of lighting regimens in circadian rhythm research can threaten retinal structure and physiology, and may therefore modify zeitgeber input to the central circadian system. Given the opportunity to escape light at any time, the nocturnal rat self-selects a seasonally varying "naturalistic skeleton photoperiod" that protects the eyes from potential damage by nonphysiological light exposures. Both rod rod-segment disk shedding and behavioral circadian phase shifts are elicited by low levels of twilight stimulation. From this vantage point, we hypothesize that certain basic properties of circadian rhythms (e.g., Aschoff's rule and splitting) may reflect modulation of retinal physiology by light. Pharmacological manipulations with or without the addition of lighting strategies have been used to analyze the neurochemistry of circadian timekeeping. Drug modulation of light input at the level of the retina may add to or interact with direct drug modulation of the central circadian pacemaking system.