Melanopsin regulates visual processing in the mouse retina

The discovery of melanopsin-dependent inner retinal photoreceptors in mammals has precipitated a fundamental reassessment of such non-image forming (NIF) light responses as circadian photoentrainment and the pupil light reflex. By contrast, it remains unclear whether these new photoreceptors also play a role in classical image-forming vision. The retinal ganglion cells that subserve inner retinal photoreception (ipRGCs) project overwhelmingly to brain areas involved in NIF responses, indicating that, in terms of central signaling, their predominant function is non-image forming. However, ipRGCs also exhibit intraretinal communication via gap junction coupling, which could allow them to modulate classical visual pathways within this tissue. Here, we explore this second possibility by using melanopsin knockout (Opn4-/-) mice to examine the role of inner retinal photoreceptors in diurnal regulation of retinal function. By using electroretinography in wild-type mice, we describe diurnal rhythms in both the amplitude and speed of the retinal cone pathway that are a function of both prior light exposure and circadian phase. Unexpectedly, loss of the melanopsin gene abolishes circadian control of these parameters, causing significant attenuation of the diurnal variation in cone vision. Our results demonstrate for the first time a melanopsin-dependent regulation of visual processing within the retina, revealing an important function for inner retinal photoreceptors in optimizing classical visual pathways according to time of day.     

Effect of retinal ischemia on the non-image forming visual system

Retinal ischemic injury is an important cause of visual impairment. The loss of retinal ganglion cells (RGCs) is a key sign of retinal ischemic damage. A subset of RGCs expressing the photopigment melanopsin (mRGCs) regulates non-image-forming visual functions such as the pupillary light reflex (PLR), and circadian rhythms. We studied the effect of retinal ischemia on mRGCs and the non-image-forming visual system function. For this purpose, transient ischemia was induced by raising intraocular pressure to 120?mm Hg for 40?min followed by retinal reperfusion by restoring normal pressure. At 4 weeks post-treatment, animals were subjected to electroretinography and histological analysis. Ischemia induced a significant retinal dysfunction and histological alterations. At this time point, a significant decrease in the number of Brn3a(+) RGCs and in the anterograde transport from the retina to the superior colliculus and lateral geniculate nucleus was observed, whereas no differences in the number of mRGCs, melanopsin levels, and retinal projections to the suprachiasmatic nuclei and the olivary pretectal nucleus were detected. At low light intensity, a decrease in pupil constriction was observed in intact eyes contralateral to ischemic eyes, whereas at high light intensity, retinal ischemia did not affect the consensual PLR. Animals with ischemia in both eyes showed a conserved locomotor activity rhythm and a photoentrainment rate which did not differ from control animals. These results suggest that the non-image forming visual system was protected against retinal ischemic damage.     

Targeted destruction of photosensitive retinal ganglion cells with a saporin conjugate alters the effects of light on mouse circadian rhythms

Non-image related responses to light, such as the synchronization of circadian rhythms to the day/night cycle, are mediated by classical rod/cone photoreceptors and by a small subset of retinal ganglion cells that are intrinsically photosensitive, expressing the photopigment, melanopsin. This raises the possibility that the melanopsin cells may be serving as a conduit for photic information detected by the rods and/or cones. To test this idea, we developed a specific immunotoxin consisting of an anti-melanopsin antibody conjugated to the ribosome-inactivating protein, saporin. Intravitreal injection of this immunotoxin results in targeted destruction of melanopsin cells. We find that the specific loss of these cells in the adult mouse retina alters the effects of light on circadian rhythms. In particular, the photosensitivity of the circadian system is significantly attenuated. A subset of animals becomes non-responsive to the light/dark cycle, a characteristic previously observed in mice lacking rods, cones, and functional melanopsin cells. Mice lacking melanopsin cells are also unable to show light induced negative masking, a phenomenon known to be mediated by such cells, but both visual cliff and light/dark preference responses are normal. These data suggest that cells containing melanopsin do indeed function as a conduit for rod and/or cone information for certain non-image forming visual responses. Furthermore, we have developed a technique to specifically ablate melanopsin cells in the fully developed adult retina. This approach can be applied to any species subject to the existence of appropriate anti-melanopsin antibodies.     

Melanopsin contributions to irradiance coding in the thalamo-cortical visual system

Photoreception in the mammalian retina is not restricted to rods and cones but extends to a subset of retinal ganglion cells expressing the photopigment melanopsin (mRGCs). These mRGCs are known to drive such reflex light responses as circadian photoentrainment and pupillomotor movements. By contrast, until now there has been no direct assessment of their contribution to conventional visual pathways. Here, we address this deficit. Using new reporter lines, we show that mRGC projections are much more extensive than previously thought and extend across the dorsal lateral geniculate nucleus (dLGN), origin of thalamo-cortical projection neurons. We continue to show that this input supports extensive physiological light responses in the dLGN and visual cortex in mice lacking rods+cones (a model of advanced retinal degeneration). Moreover, using chromatic stimuli to isolate melanopsin-derived responses in mice with an intact visual system, we reveal strong melanopsin input to the ～40% of neurons in the LGN that show sustained activation to a light step. We demonstrate that this melanopsin input supports irradiance-dependent increases in the firing rate of these neurons. The implication that melanopsin is required to accurately encode stimulus irradiance is confirmed using melanopsin knockout mice. Our data establish melanopsin-based photoreception as a significant source of sensory input to the thalamo-cortical visual system, providing unique irradiance information and allowing visual responses to be retained even in the absence of rods+cones. These findings identify mRGCs as a potential origin for aspects of visual perception and indicate that they may support vision in people suffering retinal degeneration.     

Melanopsin-dependent photoreception provides earliest light detection in the mammalian retina

BACKGROUND: The visual system is now known to be composed of image-forming and non-image-forming pathways. Photoreception for the image-forming pathway begins at the rods and cones, whereas that for the non-image-forming pathway also involves intrinsically photosensitive retinal ganglion cells (ipRGCs), which express the photopigment melanopsin. In the mouse retina, the rod and cone photoreceptors become light responsive from postnatal day 10 (P10); however, the development of photosensitivity of the ipRGCs remains largely unexplored. RESULTS: Here, we provide direct physiological evidence that the ipRGCs are light responsive from birth (P0) and that this photosensitivity requires melanopsin expression. Interestingly, the number of ipRGCs at P0 is over five times that in the adult retina, reflecting an initial overproduction of melanopsin-expressing cells during development. Even at P0, the ipRGCs form functional connections with the suprachiasmatic nucleus, as assessed by light-induced Fos expression. CONCLUSIONS: The findings suggest that the non-image-forming pathway is functional long before the mainstream image-forming pathway during development.     

Phosphorylation of Mouse Melanopsin by Protein Kinase A

The visual pigment melanopsin is expressed in intrinsically photosensitive retinal ganglion cells (ipRGCs) in the mammalian retina, where it is involved in non-image forming light responses including circadian photoentrainment, pupil constriction, suppression of pineal melatonin synthesis, and direct photic regulation of sleep. It has recently been shown that the melanopsin-based light response in ipRGCs is attenuated by the neurotransmitter dopamine. Here, we use a heterologous expression system to demonstrate that mouse melanopsin can be phosphorylated by protein kinase A, and that phosphorylation can inhibit melanopsin signaling in HEK cells. Site-directed mutagenesis experiments revealed that this inhibitory effect is primarily mediated by phosphorylation of sites T186 and S287 located in the second and third intracellular loops of melanopsin, respectively. Furthermore, we show that this phosphorylation can occur in vivo using an in situ proximity-dependent ligation assay (PLA). Based on these data, we suggest that the attenuation of the melanopsin-based light response by dopamine is mediated by direct PKA phosphorylation of melanopsin, rather than phosphorylation of a downstream component of the signaling cascade.     

Melanopsin--shedding light on the elusive circadian photopigment

Circadian photoentrainment is the process by which the brain's internal clock becomes synchronized with the daily external cycle of light and dark. In mammals, this process is mediated exclusively by a novel class of retinal ganglion cells that send axonal projections to the suprachiasmatic nuclei (SCN), the region of the brain that houses the circadian pacemaker. In contrast to their counterparts that mediate image-forming vision, SCN-projecting RGCs are intrinsically sensitive to light, independent of synaptic input from rod and cone photoreceptors. The recent discovery of these photosensitive RGCs has challenged the long-standing dogma of retinal physiology that rod and cone photoreceptors are the only retinal cells that respond directly to light and has explained the perplexing finding that mice lacking rod and cone photoreceptors can still reliably entrain their circadian rhythms to light. These SCN-projecting RGCs selectively express melanopsin, a novel opsin-like protein that has been proposed as a likely candidate for the photopigment in these cells. Research in the past three years has revealed that disruption of the melanopsin gene impairs circadian photo- entrainment, as well as other nonvisual responses to light such as the pupillary light reflex. Until recently, however, there was no direct demonstration that melanopsin formed a functional photopigment capable of catalyzing G-protein activation in a light-dependent manner. Our laboratory has recently succeeded in expressing melanopsin in a heterologous tissue culture system and reconstituting a pigment with the 11-cis-retinal chromophore. In a reconstituted biochemical system, the reconstituted melanopsin was capable of activating transducin, the G-protein of rod photoreceptors, in a light-dependent manner. The absorbance spectrum of this heterologously expressed melanopsin, however, does not match that predicted by previous behavioral and electophysiological studies. Although melanopsin is clearly the leading candidate for the elusive photopigment of the circadian system, further research is needed to resolve the mystery posed by its absorbance spectrum and to fully elucidate its role in circadian photoentrainment.     

Contribution of human melanopsin retinal ganglion cells to steady-state pupil responses

The recent discovery of melanopsin-containing retinal ganglion cells (mRGCs) has led to a fundamental reassessment of non-image forming processing, such as circadian photoentrainment and the pupillary light reflex. In the conventional view of retinal physiology, rods and cones were assumed to be the only photoreceptors in the eye and were, therefore, considered responsible for non-image processing. However, signals from mRGCs contribute to this non-image forming processing along with cone-mediated luminance signals; although both signals contribute, it is unclear how these signals are summed. We designed and built a novel multi-primary stimulation system to stimulate mRGCs independently of other photoreceptors using a silent-substitution technique within a bright steady background. The system allows direct measurements of pupillary functions for mRGCs and cones. We observed a significant change in steady-state pupil diameter when we varied the excitation of mRGC alone, with no change in luminance and colour. Furthermore, the change in pupil diameter induced by mRGCs was larger than that induced by a variation in luminance alone: that is, for a bright steady background, the mRGC signals contribute to the pupillary pathway by a factor of three times more than the L- and M-cone signals.     

Unexpected diversity and photoperiod dependence of the zebrafish melanopsin system

Animals have evolved specialized photoreceptors in the retina and in extraocular tissues that allow them to measure light changes in their environment. In mammals, the retina is the only structure that detects light and relays this information to the brain. The classical photoreceptors, rods and cones, are responsible for vision through activation of rhodopsin and cone opsins. Melanopsin, another photopigment first discovered in Xenopus melanophores (Opn4x), is expressed in a small subset of retinal ganglion cells (RGCs) in the mammalian retina, where it mediates non-image forming functions such as circadian photoentrainment and sleep. While mammals have a single melanopsin gene (opn4), zebrafish show remarkable diversity with two opn4x-related and three opn4-related genes expressed in distinct patterns in multiple neuronal cell types of the developing retina, including bipolar interneurons. The intronless opn4.1 gene is transcribed in photoreceptors as well as in horizontal cells and produces functional photopigment. Four genes are also expressed in the zebrafish embryonic brain, but not in the photoreceptive pineal gland. We discovered that photoperiod length influences expression of two of the opn4-related genes in retinal layers involved in signaling light information to RGCs. Moreover, both genes are expressed in a robust diurnal rhythm but with different phases in relation to the light-dark cycle. The results suggest that melanopsin has an expanded role in modulating the retinal circuitry of fish.     

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.     

Age-related changes in photosensitive melanopsin-expressing retinal ganglion cells correlate with circadian rhythm impairments in sighted and blind rats

The melanopsin system consists of intrinsically photosensitive retinal ganglion cells containing the photopigment melanopsin (mRGCs). These mRGCs mediate several non-image-forming visual functions, including light entrainment of circadian rhythms. Here we evaluate age-related alterations of the melanopsin system and circadian rhythms in P23H line 1 (P23H-1) rats, a rodent model of retinitis pigmentosa (RP). In homozygous P23H-1 rats and wild-type control rats from the same genetic background (Sprague–Dawley), body temperature and locomotor activity were continuously monitored at 10-min intervals for 7 days, once every 4–5 weeks, between 2 and 24 months of age, using a telemetry transmitter. The distribution and number of mRGCs were assessed in control rats at 12, 18, and 24 months of age and in P23H-1 rats aged 12, 18, 24, and 30 months by immunostaining whole-mount retinas with antibodies against melanopsin. The mean density of mRGCs in control rats showed no significant variations when evaluated at 12 and 18 months of age, and fell by approximately 56% between 18 and 24 months of age. Meanwhile, a significant decrease in the mean number of mRGCs was found in 18-month-old P23H-1 rats as compared to 18-month-old control rats (81% decrease). Parametric and non-parametric analyses of the records showed a gradual age-dependent weakening of body temperature and locomotor activity circadian rhythms robustness in both control and P23H-1 rats from 2 to 24 months of age. However, body temperature and locomotor activity circadian patterns were less robust throughout the experiment in P23H-1 as compared to control rats, with lower amplitude, weaker coupling strength to environmental zeitgebers and higher fragmentation of the rhythms. The present study shows that the degeneration of photoreceptors and inner retinal neurons, characteristic of RP, has age-related degenerative effects on the melanopsin system and is associated with weaker circadian patterns.     

A "melanopic" spectral efficiency function predicts the sensitivity of melanopsin photoreceptors to polychromatic lights

Photoreception in the mammalian retina is not restricted to rods and cones but extends to a small number of intrinsically photosensitive retinal ganglion cells expressing the photopigment melanopsin. These mRGCs are especially important contributors to circadian entrainment, the pupil light reflex, and other so-called nonimage-forming (NIF) responses. The spectral sensitivity of melanopsin phototransduction has been addressed in several species by comparing responses to a range of monochromatic stimuli. The resultant action spectra match the predicted profile of an opsin:vitamin A-based photopigment (nomogram) with a peak sensitivity (λ(max)) around 480 nm. It would be most useful to be able to use this spectral sensitivity function to predict melanopsin's sensitivity to broad-spectrum, including "white," lights. However, evidence that melanopsin is a bistable pigment with an intrinsic light-dependent bleach recovery mechanism raises the possibility of a more complex relationship between spectral quality and photoreceptor response. Here, we set out to empirically determine whether simply weighting optical power at each wavelength according to the 480-nm nomogram and integrating across the spectrum could predict melanopsin sensitivity to a variety of polychromatic stimuli. We show that pupillomotor and circadian responses of mice relying solely on melanopsin for their photosensitivity (rd/rd cl) can indeed be accurately predicted using this methodology. Our data therefore suggest that the 480-nm nomogram may be employed as the basis for a new photometric measure of light intensity (which we term "melanopic") relevant for melanopsin photoreception. They further show that measuring light in these terms predicts the melanopsin response to light of divergent spectral composition much more reliably than other methods for quantifying irradiance or illuminance currently in widespread use.     

Distribution of Mammalian-Like Melanopsin in Cyclostome Retinas Exhibiting a Different Extent of Visual Functions

Mammals contain 1 melanopsin (Opn4) gene that is expressed in a subset of retinal ganglion cells to serve as a photopigment involved in non-image-forming vision such as photoentrainment of circadian rhythms. In contrast, most nonmammalian vertebrates possess multiple melanopsins that are distributed in various types of retinal cells; however, their functions remain unclear. We previously found that the lamprey has only 1 type of mammalian-like melanopsin gene, which is similar to that observed in mammals. Here we investigated the molecular properties and localization of melanopsin in the lamprey and other cyclostome hagfish retinas, which contribute to visual functions including image-forming vision and mainly to non-image-forming vision, respectively. We isolated 1 type of mammalian-like melanopsin cDNA from the eyes of each species. We showed that the recombinant lamprey melanopsin was a blue light-sensitive pigment and that both the lamprey and hagfish melanopsins caused light-dependent increases in calcium ion concentration in cultured cells in a manner that was similar to that observed for mammalian melanopsins. We observed that melanopsin was distributed in several types of retinal cells, including horizontal cells and ganglion cells, in the lamprey retina, despite the existence of only 1 melanopsin gene in the lamprey. In contrast, melanopsin was almost specifically distributed to retinal ganglion cells in the hagfish retina. Furthermore, we found that the melanopsin-expressing horizontal cells connected to the rhodopsin-containing short photoreceptor cells in the lamprey. Taken together, our findings suggest that in cyclostomes, the global distribution of melanopsin in retinal cells might not be related to the melanopsin gene number but to the extent of retinal contribution to visual function.     

Glutamatergic Neurotransmission from Melanopsin Retinal Ganglion Cells Is Required for Neonatal Photoaversion but Not Adult Pupillary Light Reflex

Melanopsin-expressing retinal ganglion cells (mRGCs) in the eye play an important role in many light-activated non-image-forming functions including neonatal photoaversion and the adult pupillary light reflex (PLR). MRGCs rely on glutamate and possibly PACAP (pituitary adenylate cyclase-activating polypeptide) to relay visual signals to the brain. However, the role of these neurotransmitters for individual non-image-forming responses remains poorly understood. To clarify the role of glutamatergic signaling from mRGCs in neonatal aversion to light and in adult PLR, we conditionally deleted vesicular glutamate transporter (VGLUT2) selectively from mRGCs in mice. We found that deletion of VGLUT2 in mRGCs abolished negative phototaxis and light-induced distress vocalizations in neonatal mice, underscoring a necessary role for glutamatergic signaling. In adult mice, loss of VGLUT2 in mRGCs resulted in a slow and an incomplete PLR. We conclude that glutamatergic neurotransmission from mRGCs is required for neonatal photoaversion but is complemented by another non-glutamatergic signaling mechanism for the pupillary light reflex in adult mice. We speculate that this complementary signaling might be due to PACAP neurotransmission from mRGCs.     

Chemistry and biology of vision

Visual perception in humans occurs through absorption of electromagnetic radiation from 400 to 780 nm by photoreceptors in the retina. A photon of visible light carries a sufficient amount of energy to cause, when absorbed, a cis,trans-geometric isomerization of the 11-cis-retinal chromophore, a vitamin A derivative bound to rhodopsin and cone opsins of retinal photoreceptors. The unique biochemistry of these complexes allows us to reliably and reproducibly collect continuous visual information about our environment. Moreover, other nonconventional retinal opsins such as the circadian rhythm regulator melanopsin also initiate light-activated signaling based on similar photochemistry.     

Circadian photoreception in humans and mice

Circadian rhythms are the endogenous oscillations, occurring with a periodicity of approximately twenty-four hours, in the biochemical and behavioral functions of organisms. In mammals, the phase and period of the rhythm are synchronized to the daily light-dark cycle by light input through the eye. Certain retinal degenerative diseases affecting the photoreceptor cells, both rods and cones, in the outer retina reveal that classical opsins (i.e., rhodopsin and color opsins located in these cells) are essential for vision, but are not required for circadian photoreception. The mammalian cryptochromes and melanopsin (and possibly other opsin family pigments) have been proposed as circadian photoreceptor pigments that exist in the inner retina. Genetic analysis indicates that the cryptochromes, which contain flavin and folate as the light-absorbing cofactors, are the primary circadian photoreceptors. The classical photoreceptors in the outer retina, and melanopsin or other minor opsins in the inner retina, may perform redundant functions in circadian rhythmicity.     

Melanopsin-based brightness discrimination in mice and humans

Photoreception in the mammalian retina is not restricted to rods and cones but extends to a small number of intrinsically photoreceptive retinal ganglion cells (ipRGCs), expressing the photopigment melanopsin. ipRGCs are known to support various accessory visual functions including circadian photoentrainment and pupillary reflexes. However, despite anatomical and physiological evidence that they contribute to the thalamocortical visual projection, no aspect of visual discrimination has been shown to rely upon ipRGCs. Based on their currently known roles, we hypothesized that ipRGCs may contribute to distinguishing brightness. This percept is related to an object's luminance-a photometric measure of light intensity relevant for cone photoreceptors. However, the perceived brightness of different sources is not always predicted by their respective luminance. Here, we used parallel behavioral and electrophysiological experiments to first show that melanopsin contributes to brightness discrimination in both retinally degenerate and fully sighted mice. We continued to use comparable paradigms in psychophysical experiments to provide evidence for a similar role in healthy human subjects. These data represent the first direct evidence that an aspect of visual discrimination in normally sighted subjects can be supported by inner retinal photoreceptors.     

Melanopsin forms a functional short-wavelength photopigment

Recently, melanopsin has emerged as the leading candidate for the elusive photopigment of the mammalian circadian system. This novel opsin-like protein is expressed in retinal ganglion cells that form the retinohypothalamic tract, a neuronal connection between the retina and the suprachiasmatic nucleus. These hypothalamic structures contain the circadian pacemaker, which generates daily rhythms in physiology and behavior. In mammals, proper synchronization of these rhythms to the environmental light-dark cycle requires retinal input. Surprisingly, rod and cone photoreceptors are not required. Instead, the melanopsin-containing ganglion cells are intrinsically sensitive to light, perhaps responding via a melanopsin-based signaling pathway. To test this hypothesis, we have characterized melanopsin following heterologous expression in COS cells. We found that melanopsin absorbed maximally at 424 nm after reconstitution with 11-cis-retinal. Furthermore, melanopsin activated the photoreceptor G-protein, transducin, in a light-dependent manner. In agreement with the measured absorbance spectrum, melanopsin was most efficiently excited by blue light (420-440 nm). In contrast, published action spectra suggest that the photopigment underlying the intrinsic light sensitivity of SCN-projecting RGCs has an absorption maximum near 484 nm. In summary, our experiments constitute the first direct demonstration that melanopsin forms a photopigment capable of activating a G-protein, but its spectral properties are not consistent with the action spectrum for circadian entrainment.     

Visual responses in mice lacking critical components of all known retinal phototransduction cascades

The mammalian visual system relies upon light detection by outer-retinal rod/cone photoreceptors and melanopsin-expressing retinal ganglion cells. Gnat1(-/-);Cnga3(-/-);Opn4(-/-) mice lack critical elements of each of these photoreceptive mechanisms via targeted disruption of genes encoding rod α transducin (Gnat1); the cone-specific α3 cyclic nucleotide gated channel subunit (Cnga3); and melanopsin (Opn4). Although assumed blind, we show here that these mice retain sufficiently widespread retinal photoreception to drive a reproducible flash electroretinogram (ERG). The threshold sensitivity of this ERG is similar to that of cone-based responses, however it is lost under light adapted conditions. Its spectral efficiency is consistent with that of rod opsin, but not cone opsins or melanopsin, indicating that it originates with light absorption by the rod pigment. The TKO light response survives intravitreal injection of U73122 (a phospholipase C antagonist), but is inhibited by a missense mutation of cone α transducin (Gnat2(cpfl3)), suggesting Gnat2-dependence. Visual responses in TKO mice extend beyond the retina to encompass the lateral margins of the lateral geniculate nucleus and components of the visual cortex. Our data thus suggest that a Gnat1-independent phototransduction mechanism downstream of rod opsin can support relatively widespread responses in the mammalian visual system. This anomalous rod opsin-based vision should be considered in experiments relying upon Gnat1 knockout to silence rod phototransduction.