Melanopsin Signaling Research Articles

Re-evaluating the Role of Intrinsically Photosensitive Retinal Ganglion Cells: New Roles in Image-Forming Functions.

Recently, researchers identified a novel ganglion cell photoreceptor in vertebrates, called intrinsically photosensitive retinal ganglion cells (ipRGCs). These ipRGCs are photosensitive due to expression of a photopigment, melanopsin. Although ipRGCs were initially thought to be a uniform population of cells involved solely in subconscious, non-image forming behaviors, recent research points to a role for ipRGCs in pattern vision. Here we highlight the emerging evidence for this influence of ipRGCs on pattern vision and discuss important future directions for understanding this newly appreciated contribution of melanopsin signaling to visual processing.

Human Visual Cortex Responses to Rapid Cone and Melanopsin-Directed Flicker.

The retina contains cone photoreceptors and ganglion cells that contain the photopigment melanopsin. Cones provide brightness and color signals to visual cortex. Melanopsin influences circadian rhythm and the pupil, but its contribution to cortex and perception is less clear. We measured the response of human visual cortex with fMRI using spectral modulations tailored to stimulate the cones and melanopsin separately. We found that cortical responses to cone signals vary systematically across visual areas. Differences in temporal sensitivity for achromatic, red-green, and blue-yellow stimuli generally reflect the known perceptual properties of vision. We found that melanopsin signals do not produce a measurable response in visual cortex at temporal frequencies between 0.5 and 64 Hz at daytime light levels.

Retrograde Melanopsin Signaling Increases With Age in Retinal Degenerate Mice Lacking Rods and the Majority of Cones.

Following on from reports of retrograde retinal signaling in mice, we sought to investigate the influence of age and retinal location on this phenomenon using mice that lack rods and the majority of cones. We used functional anatomy for c-fos (Fos) and tyrosine hydroxylase (TH) to measure light-driven activation of dopamine neurons along a dorsal-ventral transect in C3H/He wild-type and rodless-coneless rd/rd cl (rdcl) mice aged 3, 5, and >14 months. A parallel series of retinae from 3-month-old mice was also stained for cone opsins and melanopsin. Analysis by confocal microscopy revealed light-driven Fos activation in TH cells residing in the middorsal retina of the youngest rdcl mice. This region was largely devoid of residual cones but contained a large number of intrinsically photosensitive retinal ganglion cells (ipRGCs) and the highest density of melanopsin neurites. With advancing age, there was a paradoxical increase in retrograde signaling from ∼3% Fos-positive (Fos+) TH cells at 3 months to ∼36% in rdcl mice >14 months. This increased activation occurred in more central and peripheral retinal regions. Our data provide new insights into the anatomy and plasticity of retrograde melanopsin signaling in mice with severe rod/cone dystrophy. The increased retrograde signaling we detect may result from either an increased potency of melanopsin signaling with advancing age and/or postsynaptic modification to dopaminergic neurons.

Comparison of acute non-visual bright light responses in patients with optic nerve disease, glaucoma and healthy controls.

This study examined the effect of optic nerve disease, hence retinal ganglion cell loss, on non-visual functions related to melanopsin signalling. Test subjects were patients with bilateral visual loss and optic atrophy from either hereditary optic neuropathy (n = 11) or glaucoma (n = 11). We measured melatonin suppression, subjective sleepiness and cognitive functions in response to bright light exposure in the evening. We also quantified the post-illumination pupil response to a blue light stimulus. All results were compared to age-matched controls (n = 22). Both groups of patients showed similar melatonin suppression when compared to their controls. Greater melatonin suppression was intra-individually correlated to larger post-illumination pupil response in patients and controls. Only the glaucoma patients demonstrated a relative attenuation of their pupil response. In addition, they were sleepier with slower reaction times during nocturnal light exposure. In conclusion, glaucomatous, but not hereditary, optic neuropathy is associated with reduced acute light effects. At mild to moderate stages of disease, this is detected only in the pupil function and not in responses conveyed via the retinohypothalamic tract such as melatonin suppression.

Melanopsin-derived visual responses under light adapted conditions in the mouse dLGN.

A direct projection from melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) reaches the primary visual thalamus (dorsal lateral geniculate nucleus; dLGN). The significance of this melanopsin input to the visual system is only recently being investigated. One unresolved question is the degree to which neurons in the dLGN could use melanopsin to track dynamic changes in light intensity under light adapted conditions. Here we set out to address this question. We were able to present full field steps visible only to melanopsin by switching between rod-isoluminant ‘yellow’ and ‘blue’ lights in a mouse lacking cone function (Cnga3-/-). In the retina these stimuli elicited melanopsin-like responses from a subset of ganglion cells. When presented to anaesthetised mice, we found that ~25-30% of visually responsive neurones in the contralateral dLGN responded to these melanopsin-isolating steps with small increases in firing rate. Such responses could be elicited even with fairly modest increases in effective irradiance (32% Michelson contrast for melanopsin). These melanopsin-driven responses were apparent at bright backgrounds (corresponding to twilight-daylight conditions), but their threshold irradiance was strongly dependent upon prior light exposure when stimuli were superimposed on a spectrally neutral ramping background light. While both onset and offset latencies were long for melanopsin-derived responses compared to those evoked by rods, there was great variability in these parameters with some cells responding to melanopsin steps in <1 s. These data indicate that a subset of dLGN units can employ melanopsin signals to detect modest changes in irradiance under photopic conditions.

β-Arrestin-dependent deactivation of mouse melanopsin.

In mammals, the expression of the unusual visual pigment, melanopsin, is restricted to a small subset of intrinsically photosensitive retinal ganglion cells (ipRGCs), whose signaling regulate numerous non-visual functions including sleep, circadian photoentrainment and pupillary constriction. IpRGCs exhibit attenuated electrical responses following sequential and prolonged light exposures indicative of an adaptational response. The molecular mechanisms underlying deactivation and adaptation in ipRGCs however, have yet to be fully elucidated. The role of melanopsin phosphorylation and β-arrestin binding in this adaptive process is suggested by the phosphorylation-dependent reduction of melanopsin signaling in vitro and the ubiquitous expression of β-arrestin in the retina. These observations, along with the conspicuous absence of visual arrestin in ipRGCs, suggest that a β-arrestin terminates melanopsin signaling. Here, we describe a light- and phosphorylation- dependent reduction in melanopsin signaling mediated by both β-arrestin 1 and β-arrestin 2. Using an in vitro calcium imaging assay, we demonstrate that increasing the cellular concentration of β-arrestin 1 and β-arrestin 2 significantly increases the rate of deactivation of light-activated melanopsin in HEK293 cells. Furthermore, we show that this response is dependent on melanopsin carboxyl-tail phosphorylation. Crosslinking and co-immunoprecipitation experiments confirm β-arrestin 1 and β-arrestin 2 bind to melanopsin in a light- and phosphorylation- dependent manner. These data are further supported by proximity ligation assays (PLA), which demonstrate a melanopsin/β-arrestin interaction in HEK293 cells and ipRGCs. Together, these results suggest that melanopsin signaling is terminated in a light- and phosphorylation-dependent manner through the binding of a β-arrestin within the retina.

Opponent melanopsin and S-cone signals in the human pupillary light response.

In the human, cone photoreceptors (L, M, and S) and the melanopsin-containing, intrinsically photosensitive retinal ganglion cells (ipRGCs) are active at daytime light intensities. Signals from cones are combined both additively and in opposition to create the perception of overall light and color. Similar mechanisms seem to be at work in the control of the pupil's response to light. Uncharacterized however, is the relative contribution of melanopsin and S cones, with their overlapping, short-wavelength spectral sensitivities. We measured the response of the human pupil to the separate stimulation of the cones and melanopsin at a range of temporal frequencies under photopic conditions. The S-cone and melanopsin photoreceptor channels were found to be low-pass, in contrast to a band-pass response of the pupil to L- and M-cone signals. An examination of the phase relationships of the evoked responses revealed that melanopsin signals add with signals from L and M cones but are opposed by signals from S cones in control of the pupil. The opposition of the S cones is revealed in a seemingly paradoxical dilation of the pupil to greater S-cone photon capture. This surprising result is explained by the neurophysiological properties of ipRGCs found in animal studies.

The post illumination pupil response is reduced in seasonal affective disorder

Individuals with seasonal affective disorder (SAD) may have a decreased retinal sensitivity in the non-image forming light-input pathway. We examined the post illumination pupil response (PIPR) among individuals with SAD and healthy controls to identify possible differences in the melanopsin signaling pathway. We also investigated whether melanopsin gene (OPN4) variations would predict variability in the PIPR. Fifteen SAD and 15 control participants (80% women, mean age 36.7 years, S.D.=14.5) were assessed in the fall/winter. Participants were diagnosed based on DSM-IV-TR criteria. Infrared pupillometry was used to measure pupil diameter prior to, during, and after red and blue stimuli. In response to blue light, the SAD group had a reduced PIPR and a lower PIPR percent change relative to controls. The PIPR after the blue stimulus also varied on the basis of OPN4 I394T genotype, but not OPN4 P10L genotype. These findings may indicate that individuals with SAD have a less sensitive light input pathway as measured by the PIPR, leading to differences in neurobiological and behavioral responses such as alertness, circadian photoentrainment, and melatonin release. In addition, this sensitivity may vary based on sequence variations in OPN4, although a larger sample and replication is needed.

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.

Understanding the Interaction Between Melanopsin and Arrestin using FRET

A small subset of retinal ganglion cells known as the ipRGCs regulate several non-visual processes including pupillary light reflex, circadian rhythmicity, and sleep. These processes are mediated by the photopigment, melanopsin, expressed in the ipRGCs. Upon illumination, melanopsin initiates a signaling transduction cascade within the cell. This signal induces a depolarization resulting in the firing of action potentials that carry light information to higher order processing centers in the brain. Like most G-protein coupled receptors (GPCRs), melanopsin signaling is attenuated by G-protein coupled receptor kinase (GRK) phosphorylation. This phosphorylation is a cue for arrestin binding which terminates the signal. However, it is unknown if arrestin deactivates melanopsin. In mammals, three isoforms of arrestin are expressed: visual arrestin, βarrestin 1 and βarrestin 2. Studies have shown that βarrestin 1 and 2 are co-expressed with melanopsin, while visual arrestin localizes exclusively in the rods and cones. Therefore, we hypothesize that melanopsin is deactivated by either βarrestin 1 and/or 2. Using Forester Resonance Energy Transfer (FRET) we will determine the degree with which melanopsin and arrestin interact. To date, we have successfully constructed melanopsin-eGFP, βarrestin 1-eRFP, and βarrestin 2-eRFP expression vectors. We are currently attempting to express these constructs in HEK-293 cells and verify their expression and localization by Western blot assay and confocal microscopy.

Melanopsin signalling in mammalian iris and retina

Lower vertebrates have an intrinsically-photosensitive iris and thus a local pupillary light reflex (PLR). In contrast, it has been a dogma that the PLR in mammals generally requires neuronal circuitry connecting the eye and the brain. We report here that an intrinsic component of the PLR is actually widespread in nocturnal and crepuscular mammals. In mouse, this intrinsic PLR requires the visual pigment, melanopsin. It also requires PLCβ4, the vertebrate homolog of the Drosophila NorpA phospholipase C mediating rhabdomeric phototransduction. The Plcβ4−/− genotype, besides removing the intrinsic PLR, also essentially eliminates the intrinsic light response of the M1-subtype of melanopsin-expressing, intrinsically-photosensitive retinal ganglion cells (M1-ipRGCs), by far the most photosensitive ipRGCs and with the largest responses. Ablating in mouse the expression of both TRPC6 and TRPC7, members of the TRP channel superfamily, likewise essentially eliminated the M1-ipRGC light response, but spared the intrinsic PLR. Thus, melanopsin signaling exists in both iris and retina, involving a PLCβ4-mediated pathway that nonetheless diverges in the two locations.

Intrinsic phototransduction persists in melanopsin-expressing ganglion cells lacking diacylglycerol-sensitive TRPC subunits

In mammals, intrinsically photosensitive retinal ganglion cells (ipRGCs) mediate various non-image-forming photic responses, such as circadian photoentrainment, pupillary light reflex and pineal melatonin suppression. ipRGCs directly respond to environmental light by activation of the photopigment melanopsin followed by the opening of an unidentified cation-selective channel. Studies in heterologous expression systems and in the native retina have strongly implicated diacylglycerol-sensitive transient receptor potential channels containing TRPC3, TRPC6 and TRPC7 subunits in melanopsin-evoked depolarization. Here we show that melanopsin-evoked electrical responses largely persist in ipRGCs recorded from early postnatal (P6-P8) and adult (P22-P50) mice lacking expression of functional TRPC3, TRPC6 or TRPC7 subunits. Multielectrode array (MEA) recordings performed at P6-P8 stages under conditions that prevent influences from rod/cone photoreceptors show comparable light sensitivity for the melanopsin-evoked responses in these mutant mouse lines in comparison to wild-type (WT) mice. Patch-clamp recordings from adult mouse ipRGCs lacking TRPC3 or TRPC7 subunits show intrinsic light-evoked responses equivalent to those recorded in WT mice. Persistence of intrinsic light-evoked responses was also noted in ipRGCs lacking TRPC6 subunits, although with significantly smaller magnitudes. These results demonstrate that the melanopsin-evoked depolarization in ipRGCs is not mediated by either TRPC3, TRPC6 or TRPC7 channel subunits alone. They also suggest that the melanopsin signaling pathway includes TRPC6-containing heteromeric channels in mature retinas.

Dissecting a Role for Melanopsin in Behavioural Light Aversion Reveals a Response Independent of Conventional Photoreception

Melanopsin photoreception plays a vital role in irradiance detection for non-image forming responses to light. However, little is known about the involvement of melanopsin in emotional processing of luminance. When confronted with a gradient in light, organisms exhibit spatial movements relative to this stimulus. In rodents, behavioural light aversion (BLA) is a well-documented but poorly understood phenomenon during which animals attribute salience to light and remove themselves from it. Here, using genetically modified mice and an open field behavioural paradigm, we investigate the role of melanopsin in BLA. While wildtype (WT), melanopsin knockout (Opn4−/−) and rd/rd cl (melanopsin only (MO)) mice all exhibit BLA, our novel methodology reveals that isolated melanopsin photoreception produces a slow, potentiating response to light. In order to control for the involvement of pupillary constriction in BLA we eliminated this variable with topical atropine application. This manipulation enhanced BLA in WT and MO mice, but most remarkably, revealed light aversion in triple knockout (TKO) mice, lacking three elements deemed essential for conventional photoreception (Opn4−/− Gnat1−/− Cnga3−/−). Using a number of complementary strategies, we determined this response to be generated at the level of the retina. Our findings have significant implications for the understanding of how melanopsin signalling may modulate aversive responses to light in mice and humans. In addition, we also reveal a clear potential for light perception in TKO mice.

Melanopsin Signalling: Low Pigment Density, Large Single-Photon Response, and High-Efficiency Transmission

A subset of retinal ganglion cells has recently been discovered to be intrinsically photosensitive. These cells use melanopsin as the visual pigment and are the only photoreceptors known in mammals besides rods and cones. They project primarily to brain centers that serve non-image-forming visual functions such as the pupillary light reflex and circadian photoentrainment. How well these cells signal intrinsic light absorption to drive such behaviors remains unclear. We have used perforated-patch and loose-patch recordings to study mouse ipRGCs that are genetically labeled by a fluorescent protein, tdTomato. Here we report fundamental parameters that govern their intrinsic light responses, and the associated spike generation. They express melanopsin at a membrane density that is four orders of magnitude lower than that of rod and cone pigments (∼3 μm2), giving rise to a low photon-catch and thus a phototransducing role in relatively bright light. Nonetheless, the single-photon response is even larger than that of rods, suggesting high amplification in melanopsin phototransduction. It is also extraordinarily prolonged, with an integration time that is ∼20-fold longer than that of rods, and has a unique shape among known photoreceptors. Remarkably, ipRGCs are capable of signalling single-photon absorption to the brain. Moreover, a flash resulting in a few hundred isomerized melanopsin molecules in a retina is sufficient for reaching the threshold of the pupillary light reflex.

Rods-cones and melanopsin detect light and dark to modulate sleep independent of image formation

Light detected in the retina modulates several physiological processes including circadian photo-entrainment and pupillary light reflex. Intrinsically photosensitive retinal ganglion cells (ipRGCs) convey rod-cone and melanopsin-driven light input to the brain. Using EEGs and electromyograms, we show that acute light induces sleep in mice during their nocturnal active phase whereas acute dark awakens mice during their diurnal sleep phase. We used retinal mutant mouse lines that lack (i) the ipRGCs, (ii) the photo-transduction pathways of rods and cones, or (iii) the melanopsin protein and showed that the influence of light and dark on sleep requires both rod-cone and melanopsin signaling through ipRGCs and is independent of image formation. We further show that, although acute light pulses overcome circadian and homeostatic drives for sleep, upon repeated light exposures using a 3.5 h/3.5 h light/dark cycle, the circadian and homeostatic drives override the light input. Thus, in addition to their known role in aligning circadian physiology with day and night, ipRGCs also relay light and dark information from both rod-cone and melanopsin-based pathways to modulate sleep and wakefulness.

Expected and Unexpected Properties of Melanopsin Signaling

Expected and Unexpected Properties of Melanopsin Signaling

Functional Expression, Targeting and Ca2+ Signaling of a Mouse Melanopsin‐eYFP Fusion Protein in a Retinal Pigment Epithelium Cell Line†

Melanopsin, first discovered in Xenopus melanophores, is now established as a functional sensory photopigment of the intrinsically photosensitive retinal ganglion cells. These ganglion cells drive circadian rhythm and pupillary adjustments through projection to the brain. Melanopsin shares structural similarities with all known opsins. Comprehensive characterization of melanopsin with respect to its spectral properties, photochemical cascade and signaling partners requires a suitable recombinant system and high expression levels. This combination has not yet been described. To address this issue, we have expressed recombinant mouse melanopsin in several cell lines. Using enhanced yellow fluorescent protein (eYFP) as a visualization tag, expression was observed in all cell lines. Confocal microscopy revealed that melanopsin was properly routed to the plasma membrane only in retinal pigment epithelium (RPE)-derived D407 cells and in human embryonic kidney (HEK) cells. Further, we performed intracellular calcium measurements in order to probe the melanopsin signaling activity of this fusion protein. Transfected cells were loaded with the calcium indicator Fura2-AM. Upon illumination, an immediate but transient calcium response was observed in HEK as well as in D407 cells, while mock-transfected cells showed no calcium response under identical conditions. Supplementation with 11-cis retinal or all-trans retinal enhanced the response. After prolonged illumination the cells became desensitized. Thus, RPE-derived cells expressing recombinant melanopsin may constitute a suitable system for the study of the structural and functional characteristics of melanopsin.

Microarray Analysis and Functional Genomics Identify Novel Components of Melanopsin Signaling

Within the mammalian retina, there exists a third photoreceptive system based upon a population of melanopsin (Opn4) expressing photosensitive retinal ganglion cells (pRGCs; also termed ipRGCs or intrinsically photosensitive RGCs). Here, we use a microarray-based approach, which we term transcriptional recalibration, coupled with functional genomics to identify downstream targets of melanopsin signaling. In a mouse with genetically ablated rods and cones (rd/rd cl), approximately 30% of the ocular transcriptome is transiently regulated in response to nocturnal light exposure (3112 genes). A total of 163 of these genes were associated with the "intracellular signaling" gene ontology term. On the basis of their similarity to invertebrate phototransduction genes, 14 were selected for further study. Laser capture microdissection demonstrated that eight of these genes (Gnas, Gnb2l1, Gnaq, Prkcz, Pik3r1, Inadl, Slc9a3r1, and Drd1a) colocalized with melanopsin. The impact of genetic ablation of one of these genes, protein kinase C zeta (Prkcz), was assessed. Prkcz-/- animals show attenuated phase-shifting responses to light, reduced period lengthening under constant light, and attenuated pupillary responses at high irradiances, as well as impaired light-induced gene expression in the suprachiasmatic nuclei (SCN). These attenuated responses are indistinguishable from the deficits observed in melanopsin knockout mice. Here, we show that (1) Prkcz plays an as yet unidentified role in melanopsin signaling, (2) the proteins of seven further light-regulated genes emerge as strong candidates in melanopsin signaling, and (3) transcriptional recalibration may provide a powerful new approach for dissecting unmapped signaling pathways.

Melanopsin Triggers the Release of Internal Calcium Stores in Response to Light†

Melanopsin is the photopigment that confers photosensitivity upon intrinsically photosensitive retinal ganglion cells (ipRGCs). This subset of retinal ganglion cells comprises less than 2% of all RGCs in the mammalian retina. The paucity of melanopsin-positive cells has made studies on melanopsin signaling difficult to pursue in ipRGCs. To address this issue, we have established several cell lines consisting of a transformed human embryonic kidney cell line (HEK293) stably expressing human melanopsin. With these cell lines, we have investigated the intracellular rise in calcium triggered upon light activation of melanopsin. Our human melanopsin-expressing cells exhibit an irradiance-dependent increase in intracellular calcium. Control cells expressing human melanopsin, where the Schiff-base lysine has been mutated to alanine, show no responses to light. Chelating extracellular calcium has no effect on the light-induced increase in intracellular calcium suggesting that calcium is mobilized from intracellular stores. This involvement of intracellular stores has been confirmed through their depletion by thapsigargin, which inhibits a subsequent light-induced increase in intracellular calcium. Addition of the nonselective cation channel blocker lanthanum does not alter light-induced rises in intracellular calcium, further supporting that melanopsin triggers a release of internal calcium from internal stores. HEK293 cells stably expressing melanopsin have proven to be a useful tool to study melanopsin-initiated signaling.

Not So Different After All

Melanopsin is an atypical opsin protein required in vertebrates to mediate nonvisual responses to light, including regulation of circadian behavior and pupil constriction. Panda et al. report that, when expressed in Xenopus oocytes, melanopsin activates heterotrimeric G protein signaling pathways characteristic of invertebrate opsins in response to light. Melanopsin could also activate mammalian TRPC channels, whose paralogs in Drosophila serve as phototransduction channels. Thus, an invertebrate signaling system appears to be preserved in the mammalian retina. S. Panda, S. K. Nayak, B. Campo, J. R. Walker, J. B. Hogenesch, T. Jegla, Illumination of the melanopsin signaling pathway. Science 307 , 600-604 (2005). [Abstract] [Full Text]