Melanopsin photoreception in the eye regulates light‐induced skin colour changes through the production of α‐MSH in the pituitary gland

How skin colour adjusts to circadian light/dark cycles is poorly understood. Melanopsin (Opn4) is expressed in melanophores, where in vitro studies suggest it regulates skin pigmentation through a ‘primary colour response’ in which light photosensitivity is translated directly into pigment movement. However, the entrainment of the circadian rhythm is regulated by a population of melanopsin‐expressing retinal ganglion cells (mRGCs) in the eye. Therefore, in vivo, melanopsin may trigger a ‘secondary colour response’ initiated in the eye and controlled by the neuro‐endocrine system. We analysed the expression of opn4m and opn4x and melanin aggregation induced by light (background adaptation) in Xenopus laevis embryos. While opn4m and opn4x are expressed at early developmental times, light‐induced pigment aggregation requires the eye to become functional. Pharmacological inhibition of melanopsin suggests a model whereby mRGC activation lightens skin pigmentation via a secondary response involving negative regulation of alpha‐melanocyte‐stimulating hormone (α‐MSH) secretion by the pituitary.     

Human melanopsin forms a pigment maximally sensitive to blue light (λmax ≈ 479 nm) supporting activation of Gq/11 and Gi/o signalling cascades

A subset of mammalian retinal ganglion cells expresses an opsin photopigment (melanopsin, Opn4) and is intrinsically photosensitive. The human retina contains melanopsin, but the literature lacks a direct investigation of its spectral sensitivity or G-protein selectivity. Here, we address this deficit by studying physiological responses driven by human melanopsin under heterologous expression in HEK293 cells. Luminescent reporters for common second messenger systems revealed that light induces a high amplitude increase in intracellular calcium and a modest reduction in cAMP in cells expressing human melanopsin, implying that this pigment is able to drive responses via both G_q and G_i/o class G-proteins. Melanopsins from mouse and amphioxus had a similar profile of G-protein coupling in HEK293 cells, but chicken Opn4m and Opn4x pigments exhibited some G_s activity in addition to a strong G_q/11 response. An action spectrum for the calcium response in cells expressing human melanopsin had the predicted form for an opsin : vitamin A1 pigment and peaked at 479 nm. The G-protein selectivity and spectral sensitivity of human melanopsin is similar to that previously described for rodents, supporting the utility of such laboratory animals for developing methods of manipulating this system using light or pharmacological agents.     

Two light‐activated neuroendocrine circuits arising in the eye trigger physiological and morphological pigmentation

Two biological processes regulate light‐induced skin colour change. A fast ‘physiological pigmentation change’ (i.e. circadian variations or camouflage) involves alterations in the distribution of pigment containing granules in the cytoplasm of chromatophores, while a slower ‘morphological pigmentation change’ (i.e. seasonal variations) entails changes in the number of pigment cells or pigment type. Although linked processes, the neuroendocrine coordination triggering each response remains largely obscure. By evaluating both events in Xenopus laevis embryos, we show that morphological pigmentation initiates by inhibiting the activity of the classical retinal ganglion cells. Morphological pigmentation is always accompanied by physiological pigmentation, and a melatonin receptor antagonist prevents both responses. Physiological pigmentation also initiates in the eye, but with repression of melanopsin‐expressing retinal ganglion cell activity that leads to secretion of alpha‐melanocyte‐stimulating hormone (α‐MSH). Our findings suggest a model in which eye photoperception links physiological and morphological pigmentation by altering α‐MSH and melatonin production, respectively.     

Pharmacological induction of skin pigmentation unveils the neuroendocrine circuit regulated by light

Light‐regulated skin colour change is an important physiological process in invertebrates and lower vertebrates, and includes daily circadian variation and camouflage (i.e. background adaptation). The photoactivation of melanopsin‐expressing retinal ganglion cells (mRGCs) in the eye initiates an uncharacterized neuroendocrine circuit that regulates melanin dispersion/aggregation through the secretion of alpha‐melanocyte‐stimulating hormone (α‐MSH). We developed experimental models of normal or enucleated Xenopus embryos, as well as in situ cultures of skin of isolated dorsal head and tails, to analyse pharmacological induction of skin pigmentation and α‐MSH synthesis. Both processes are triggered by a melanopsin inhibitor, AA92593, as well as chloride channel modulators. The AA9253 effect is eye‐dependent, while functional data in vivo point to GABA_A receptors expressed on pituitary melanotrope cells as the chloride channel blocker target. Based on the pharmacological data, we suggest a neuroendocrine circuit linking mRGCs with α‐MSH secretion, which is used normally during background adaptation.     

Interaction and developmental activation of two neuroendocrine systems that regulate light‐mediated skin pigmentation

Lower vertebrates use rapid light‐regulated changes in skin colour for camouflage (background adaptation) or during circadian variation in irradiance levels. Two neuroendocrine systems, the eye/alpha‐melanocyte‐stimulating hormone (α‐MSH) and the pineal complex/melatonin circuits, regulate the process through their respective dispersion and aggregation of pigment granules (melanosomes) in skin melanophores. During development, Xenopus laevis tadpoles raised on a black background or in the dark perceive less light sensed by the eye and darken in response to increased α‐MSH secretion. As embryogenesis proceeds, the pineal complex/melatonin circuit becomes the dominant regulator in the dark and induces lightening of the skin of larvae. The eye/α‐MSH circuit continues to mediate darkening of embryos on a black background, but we propose the circuit is shut down in complete darkness in part by melatonin acting on receptors expressed by pituitary cells to inhibit the expression of pomc, the precursor of α‐MSH.     

Isolation and characterization of melanopsin (Opn4) from the Australian marsupial Sminthopsis crassicaudata (fat-tailed dunnart)

Melanopsin confers photosensitivity to a subset of retinal ganglion cells and is responsible for many non-image-forming tasks, like the detection of light for circadian entrainment. Recently, two melanopsin genes, Opn4m and Opn4x, were described in non-mammalian vertebrates. However, only one form, Opn4m, has been described in the mammals, although studies to date have been limited to the placentals and have not included the marsupials. We report here the isolation and characterization of an Opn4 gene from an Australian marsupial, the fat-tailed dunnart (Sminthopsis crassicaudata), and present evidence which suggests that the Opn4x gene was lost before the placental/marsupial split. In situ hybridization shows that the expression of Opn4 in the dunnart eye is restricted to a subset of ganglion cells, a pattern previously reported for rodents and primates. These Opn4-positive cells are randomly distributed across the dunnart retina. We also undertook a comparative analysis with the South American marsupial, the grey short-tailed opossum (Monodelphis domestica), and two placental mammals, mouse and human. This approach reveals that the two marsupials show a higher sequence identity than that seen between rodents and primates, despite separating at approximately the same point in time, some 65-85 Myr ago.     

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.     

Regulation of Melanopsin Expression

Circadian rhythms in mammals are adjusted daily to the environmental day/night cycle by photic input via the retinohypothalamic tract (RHT). Retinal ganglion cells (RGCs) of the RHT constitute a separate light‐detecting system in the mammalian retina used for irradiance detection and for transmission to the circadian system and other non‐imaging forming processes in the brain. The RGCs of the RHT are intrinsically photosensitive due to the expression of melanopsin, an opsin‐like photopigment. This notion is based on anatomical and functional data and on studies of mice lacking melanopsin. Furthermore, heterologous expression of melanopsin in non‐neuronal mammalian cell lines was found sufficient to render these cells photosensitive. Even though solid evidence regarding the function of melanopsin exists, little is known about the regulation of melanopsin gene expression. Studies in albino Wistar rats showed that the expression of melanopsin is diurnal at both the mRNA and protein levels. The diurnal changes in melanopsin expression seem, however, to be overridden by prolonged exposure to light or darkness. Significant increase in melanopsin expression was observed from the first day in constant darkness and the expression continued to increase during prolonged exposure in constant darkness. Prolonged exposure to constant light, on the other hand, decreased melanopsin expression to an almost undetectable level after 5 days of constant light. The induction of melanopsin by darkness was even more pronounced if darkness was preceded by light suppression for 5 days. These observations show that dual mechanisms regulate melanopsin gene expression and that the intrinsic light‐responsive RGCs in the albino Wistar rat adapt their expression of melanopsin to environmental light and darkness.     

Differential enhancement of neural and photoreceptor cell differentiation of cultured pineal cells by FGF-1, IGF-1, and EGF

There are several common features between the pineal organ and the lateral eye in their developmental and evolutionary aspects. The avian pineal is a photoendocrine organ that originates from the diencephalon roof and represents a transitional type between the photosensory organ of lower vertebrates and the endocrine gland of mammals. Previous cell culture studies have shown that embryonic avian pineal cells retain a wide spectrum of differentiative capacities, although little is known about the mechanisms involved in their fate determination. In the present study, we investigated the effects of various cell growth factors on the differentiation of photoreceptor and neural cell types using pineal cell cultures from quail embryos. The results show that IGF-1 promotes differentiation of rhodopsin-immunoreactive cells, but had no effect on neural cell differentiation. Simultaneous administration of EGF and IGF-1 further enhanced differentiation of rhodopsin-immunoreactive cells, although the mechanism of the synergistic effect is unknown. FGF-1 did not stimulate proliferation of neural progenitor cells, but intensively promoted and maintained expression of a neural cell phenotype. FGF-1 appeared to lead to the conversion from an epithelial (endocrinal) to a neuronal type. It also enhanced phenotypic expression of retinal ganglion cell markers but rather suppressed expression of an amacrine cell marker. These results indicate that growth factors are important regulatory cues for pineal cell differentiation and suggest that they play roles in determining the fate of the pineal organ and the eye. It can be speculated that the differences in environmental cues between the retina and pineal may result in the transition of the pineal primordium from a potentially ocular (retinal) organ to a photoendocrine organ.     

Expression of novel opsins and intrinsic light responses in the mammalian retinal ganglion cell line RGC-5. Presence of OPN5 in the rat retina

The vertebrate retina is known to contain three classes of photoreceptor cells: cones and rods responsible for vision, and intrinsically photoresponsive retinal ganglion cells (RGCs) involved in diverse non-visual functions such as photic entrainment of daily rhythms and pupillary light responses. In this paper we investigated the potential intrinsic photoresponsiveness of the rat RGC line, RGC-5, by testing for the presence of visual and non-visual opsins and assessing expression of the immediate-early gene protein c-Fos and changes in intracellular Ca(2+) mobilization in response to brief light pulses. Cultured RGC-5 cells express a number of photopigment mRNAs such as retinal G protein coupled receptor (RGR), encephalopsin/panopsin (Opn3), neuropsin (Opn5) and cone opsin (Opn1mw) but not melanopsin (Opn4) or rhodopsin. Opn5 immunoreactivity was observed in RGC-5 cells and in the inner retina of rat, mainly localized in the ganglion cell layer (GCL). Furthermore, white light pulses of different intensities and durations elicited changes both in intracellular Ca(2+) levels and in the induction of c-Fos protein in RGC-5 cell cultures. The results demonstrate that RGC-5 cells expressing diverse putative functional photopigments display intrinsic photosensitivity which accounts for the photic induction of c-Fos protein and changes in intracellular Ca(2+) mobilization. The presence of Opn5 in the GCL of the rat retina suggests the existence of a novel type of photoreceptor cell.     

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.     

Melanopsin in chicken melanocytes and retina

The vertebrate pigment cell, with the exception of mammals and birds, is able to provide the animal with rapid colour changes, which involve dispersion and aggregation of pigment granules in response to hormonal and neuronal agents, and in some cases as a direct response to light. The search for the mechanisms through which Xenopus leavis melanophores respond to light led to the discovery of a new photopigment, melanopsin, with a different spectral sensitivity to that of rhodopsin. This photopigment was also found in mammalian retinal ganglion cells that project to the suprachiasmatic nucleus and other non-visual retinorecipient areas. Herein we demonstrate (by RT-PCR, cloning and sequencing) for the first time that chick melanocytes express melanopsin, and confirmed the presence of the protein by immunocytochemistry. In the chicken retina, we revealed by immunocytochemistry that ganglion cells express melanopsin, but the highest density of immunopositive cells was found in the inner nuclear layer. Quantitative PCR showed that the retina of animals kept in 6 h light: 18 h dark possessed three-fold higher melanopsin mRNA content than animals kept in longer photoperiod, thus demonstrating that light modulates melanopsin expression in chickens.     

The Avian Pineal Gland

The pineal gland plays a key role in the control of the daily and seasonal rhythms in most vertebrate species. In mammals, rhythmic melatonin (MT) release from the pineal gland is controlled by the suprachiasmatic nucleus via the sympathetic nervous system. In most non‐mammalian species, including birds, the pineal gland contains a self‐sustained circadian oscillator and several input channels to synchronize the clock, including direct light sensitivity. Avian pineal glands maintain rhythmic activity for days under in vitro conditions. Several physical (light, temperature, and magnetic field) and biochemical (Vasoactive intestinal polypeptide (VIP), norepinephrine, PACAP, etc.) input channels, influencing release of melatonin are also functional in vitro, rendering the explanted avian pineal an excellent model to study the circadian biological clock. Using a perifusion system, we here report that the phase of the circadian melatonin rhythm of the explanted chicken pineal gland can be entrained easily to photoperiods whose length approximates 24 h, even if the light period is extremely short, i.e., 3L:21D. When the length of the photoperiod significantly differs from 24 h, the endogenous MT rhythm becomes distorted and does not follow the light‐dark cycle. When explanted chicken pineal fragments were exposed to various drugs targeting specific components of intracellular signal transduction cascades, only those affecting the cAMP‐protein kinase‐A system modified the MT release temporarily without phase‐shifting the rhythm in MT release. The potential role of cGMP remains to be investigated.     

Physiological colour change in the Moorish gecko,Tarentola mauritanica (Squamata: Gekkonidae): effects of background,light, and temperature

Colour has many different functions in animals, such as an involvement in thermoregulation, crypsis, and social interactions. Species capable of physiological colour change may alter their coloration in response to ecological conditions. The Moorish gecko, Tarentola mauritanica, is capable of actively changing its body coloration. In the present study, we investigated colour change in this gecko as a function of background, temperature, and light. Our results demonstrate that the Moorish gecko indeed changes its dorsal colour in response to changes in environmental conditions. By contrast to several other reptilian species, this rapid colour change does not appear to be associated with thermoregulation. Background matching, however, did appear to be a prominent function, although illumination appears to be an essential trigger. Future research should concentrate on individual variation and its effectiveness with respect to antipredatory mechanisms. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ?? , ??–??.     

Differential expression of melanopsin isoforms Opn4L and Opn4S during postnatal development of the mouse retina

Photosensitive retinal ganglion cells (pRGCs) respond to light from birth and represent the earliest known light detection system to develop in the mouse retina. A number of morphologically and functionally distinct subtypes of pRGCs have been described in the adult retina, and have been linked to different physiological roles. We have previously identified two distinct isoforms of mouse melanopsin, Opn4L and Opn4S, which are generated by alternate splicing of the Opn4 locus. These isoforms are differentially expressed in pRGC subtypes of the adult mouse retina, with both Opn4L and Opn4S detected in M1 type pRGCs, and only Opn4L detected in M2 type pRGCs. Here we investigate the developmental expression of Opn4L and Opn4S and show a differential profile of expression during postnatal development. Opn4S mRNA is detected at relatively constant levels throughout postnatal development, with levels of Opn4S protein showing a marked increase between P0 and P3, and then increasing progressively over time until adult levels are reached by P10. By contrast, levels of Opn4L mRNA and protein are low at birth and show a marked increase at P14 and P30 compared to earlier time points. We suggest that these differing profiles of expression are associated with the functional maturation of M1 and M2 subtypes of pRGCs. Based upon our data, Opn4S expressing M1 type pRGCs mature first and are the dominant pRGC subtype in the neonate retina, whereas increased expression of Opn4L and the maturation of M2 type pRGCs occurs later, between P10 and P14, at a similar time to the maturation of rod and cone photoreceptors. We suggest that the distinct functions associated with these cell types will develop at different times during postnatal development.