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.

Molecular cloning, localization and circadian expression of chicken melanopsin (Opn4): differential regulation of expression in pineal and retinal cell types.

The avian retina and pineal gland contain autonomous circadian oscillators and photo-entrainment pathways, but the photopigment(s) that mediate entrainment have not been definitively identified. Melanopsin (Opn4) is a novel opsin involved in entrainment of circadian rhythms in mammals. Here, we report the cDNA cloning of chicken melanopsin and show its expression in retina, brain and pineal gland. Like the melanopsins identified in amphibians and mammals, chicken melanopsin is more similar to the invertebrate retinaldehyde-based photopigments than the retinaldehyde-based photopigments typically found in vertebrates. In retina, melanopsin mRNA is expressed in cells of all retinal layers. In pineal gland, expression was strong throughout the parenchyma of the gland. In brain, expression was observed in a few discrete nuclei, including the lateral septal area and medial preoptic nucleus. The retina and pineal gland showed distinct diurnal expression patterns. In pineal gland, melanopsin mRNA levels were highest at night at Zeitgeber time (ZT) 16. In contrast, transcript levels in the whole retina reached their highest levels in the early morning (ZT 0-4). Further analysis of melanopsin mRNA expression in retinal layers isolated by laser capture microdissection revealed different patterns in different layers. There was diurnal expression in all retinal layers except the ganglion cell layer, where heavy expression was localized to a small number of cells. Expression of melanopsin mRNA peaked during the daytime in the retinal pigment epithelium and inner nuclear layer but, like in the pineal, at night in the photoreceptors. Localization and regulation of melanopsin mRNA in the retina and pineal gland is consistent with the hypothesis that this novel photopigment plays a role in photic regulation of circadian function in these tissues.

The photoreceptive capacity of the developing pineal gland and eye of the golden hamster (Mesocricetus auratus).

Anatomical and physiological studies have suggested that the pineal gland of neonatal mammals has a photoreceptive capacity. Using the golden hamster (Mesocricetus auratus) as our model, we applied biochemical approaches to look for a functional photopigment within the pineal during early development. Immunocytochemistry and enzyme-linked immunosorbent assay (ELISA) were used to localize and quantify opsin, and high-performance liquid chromatography (HPLC) to identify photopigment chromophore (11-cis and all-trans retinaldehyde) in the developing eye and pineal. For HPLC analysis, retinaldehydes were converted to their corresponding retinoid oximes. Eluted retinoids were identified by comparison with standard vitamin A1 retinoid oxime isomers on the basis of relative elution sequence and characteristic absorbance spectra. Both immunocytochemistry and ELISA suggested an increase in the opsin content of the pineal during the first week of life. In the eye, 11-cis retinaldehyde was first detected between days 3 and 5 after birth. In three separate extractions, and using a considerable excess of pineal tissue, we failed to identify chromophore within the pineal during the first week of postnatal development. The appearance of 11-cis retinaldehyde within the eye between postnatal days 3-5 is consistent with the hypothesis that retinol isomerase activity is coordinated with outer segment development. The failure to identify chromophore within the neonatal pineal suggests that this gland lacks a functional opsin-based photopigment. These data contradict physiological evidence suggesting that the neonatal pineal of mammals contains photoreceptors.

Murine aspartoacylase: cloning, expression and comparison with the human enzyme.

Canavan disease is caused by mutations in aspartoacylase, the enzyme that degrades N-acetylaspartate (NAA) into acetate and aspartate. Murine aspartoacylase (mASPA) was cloned using sequence information from mouse expressed sequence tags homologous to the human cDNA. The open reading frame was cloned into a thioredoxin fusion vector, overexpressed in bacteria, and the protein was purified using affinity chromatography to near homogeneity. Recombinant human ASPA (hASPA) was prepared by a similar method. Both recombinant enzymes were highly specific to NAA, with about 10% of the NAA activity toward N-acetylasparagine. More interestingly, the product of N-acetylasparagine was aspartate but not asparagine, indicating that ASPA catalyzed deacetylation as well as hydrolysis of the beta acid amide. Our success in preparing the recombinant ASPA in high purity should permit multiple lines of investigations to understand the pathogenic mechanisms of Canavan disease and the functional roles of NAA.

A novel human opsin in the inner retina.

Here we report the identification of a novel human opsin, melanopsin, that is expressed in cells of the mammalian inner retina. The human melanopsin gene consists of 10 exons and is mapped to chromosome 10q22. This chromosomal localization and gene structure differs significantly from that of other human opsins that typically have four to seven exons. A survey of 26 anatomical sites indicates that, in humans, melanopsin is expressed only in the eye. In situ hybridization histochemistry shows that melanopsin expression is restricted to cells within the ganglion and amacrine cell layers of the primate and murine retinas. Notably, expression is not observed in retinal photoreceptor cells, the opsin-containing cells of the outer retina that initiate vision. The unique inner retinal localization of melanopsin suggests that it is not involved in image formation but rather may mediate nonvisual photoreceptive tasks, such as the regulation of circadian rhythms and the acute suppression of pineal melatonin. The anatomical distribution of melanopsin-positive retinal cells is similar to the pattern of cells known to project from the retina to the suprachiasmatic nuclei of the hypothalamus, a primary circadian pacemaker.

Retinal projections in mice with inherited retinal degeneration: implications for circadian photoentrainment.

The availability of naturally occurring and transgenic retinal mutants has made the mouse an attractive experimental model to address questions regarding photoentrainment of circadian rhythms. However, very little is known about the retinal cells and the retinal projections to the nuclei of the murine circadian timing system. Furthermore, the effect of inherited retinal degeneration on these projections is not understood. In this report, we have used pseudorabies virus as a neuroanatomical tract tracer in mice to address a series of questions: Which retinal cells mediate circadian responses to light? What is the nature of the retinohypothalamic projection? What is the impact of the inherited retinal disorder, retinal degenerate (rd/rd), on the structures of the photoentrainment pathway? Our results show that a class ofretinal ganglion cell, morphologically similar to the type III ganglion cells of the rat, appears to project to central circadian structures of the mouse. They are few in number and sparsely distributed throughout the retina. The low number and broad distribution of these specialized retinal ganglion cells may be an adaptive mechanism to integrate environmental irradiance without compromising the spatial resolution required for vision. In addition, viral infection of conelike and rodlike photoreceptors and amacrinelike cells suggest that these cells may mediate or contribute to circadian responses to light. Inherited retinal degeneration has no obvious effect on the anatomy of the retinal cells or their projections to the circadian axis. These anatomical findings are consistent with our previous findings showing that aged rd/rd mice are capable of regulating their circadian rhythms by light with unattenuated sensitivity.

Melanopsin: An opsin in melanophores, brain, and eye.

We have identified an opsin, melanopsin, in photosensitive dermal melanophores of Xenopus laevis. Its deduced amino acid sequence shares greatest homology with cephalopod opsins. The predicted secondary structure of melanopsin indicates the presence of a long cytoplasmic tail with multiple putative phosphorylation sites, suggesting that this opsin's function may be finely regulated. Melanopsin mRNA is expressed in hypothalamic sites thought to contain deep brain photoreceptors and in the iris, a structure known to be directly photosensitive in amphibians. Melanopsin message is also localized in retinal cells residing in the outermost lamina of the inner nuclear layer where horizontal cells are typically found. Its expression in retinal and nonretinal tissues suggests a role in vision and nonvisual photoreceptive tasks, such as photic control of skin pigmentation, pupillary aperture, and circadian and photoperiodic physiology.

Circadian rhythms in mice can be regulated by photoreceptors with cone-like characteristics.

In this report we have characterized the photopigments mediating circadian phase shifts in retinal degenerate (rd) mice. In aged rd/rd mice, which lack detectable opsin, high performance liquid chromatography (HPLC) was used to quantify the photopigment chromophore 11-cis-retinaldehyde. This chromophore was photoisomerized in whole eyes, suggesting the presence of a functional opsin-based photopigment system. We also analyzed the spectral sensitivity of phase shifting circadian locomotor rhythms. Our data implicate a photopigment that is consistent with the involvement of the middle wavelength-sensitive cone photoreceptors (M-cones; lambda(max) = 511 nm) found in the mouse retina. In addition, discrete near-ultraviolet (UV-A) pulses were capable of eliciting large phase shifts in circadian locomotor activity rhythms. This result is consistent with the involvement of the short wavelength-sensitive cone photoreceptors (UV-cones; lambda(max) = 359 nm) in photoentrainment. Collectively, these data suggest that both cone classes of the mouse may mediate the photic regulation of circadian rhythms. If this is the case, circadian sensitivity can be maintained by very few degenerate cones. Alternatively, an unknown class of ocular photoreceptor may fulfill this function.

Photopigments and circadian systems of vertebrates.

In the retinal degeneration (rd) mouse the absence of rod cells and the progressive loss of cones does not result in a decrease in circadian phase shifting responses to light. By contrast, rd/rd mice are unable to perform simple visual tasks. In addition, rodless transgenic mice, and mice homozygous for the retinal degeneration slow (rds) mutation, show unattenuated circadian responses to light. Collectively these data suggest that cone cells lacking outer segments are sufficient to maintain normal circadian responses to light, or some unidentified photoreceptor within the retina. An action spectrum for circadian responses to light in rd/rd mice, and molecular analysis of retinally degenerate mice and blind mole rat eyes, suggests the involvement of a mid-to-long wavelength sensitive cone opsin in photoentrainment. Extraocular photoreceptors of non-mammalian vertebrates are currently being analyzed in order to identify functional and evolutionary similarities between visual and non-visual photoreceptor systems.

Visual and circadian responses to light in aged retinally degenerate mice.

The progression of photoreceptor degeneration in retinally degenerate (rd) mice commences early in postnatal development resulting in the complete loss of rods by 60-70 days of age followed by the more protracted loss of cones. We have previously shown that rd mice 80 days of age are capable of phase shifting their circadian locomotor rhythms in response to brief pulses of light and these animals show the same sensitivity as wild-type (+/+) controls. If surviving cones mediate these circadian responses, then one would expect the sensitivity of the circadian system in rd mice to decline with age and parallel the loss of cones. We demonstrate that aging rd mice (80-767 days of age) remain capable of photically regulating circadian locomotor rhythms in a manner indistinguishable from +/+ mice. Circadian responses to light do not parallel cone cell degeneration in rd mice. In contrast to the circadian responses to light, old (> 210 days of age) rd mice show no visually-evoked behavioral or electroretinogram (ERG) responses.

Identification of vertebrate deep brain photoreceptors.

Since the beginning of this century evidence has accumulated which demonstrates that nonmammalian vertebrates possess photoreceptors situated deep within the brain. These photoreceptors have been implicated in several different areas of physiology, but in all species examined, they play a critical role in the regulation of circadian and reproductive responses to light. Many attempts have been made to localize these sensory cells over the past 50 years, but until recently all attempts have failed. As a result, this important sensory system remains largely unexplored. Recent attempts to localize these photoreceptors, in a range of vertebrates, using combined antibody and biochemical approaches has met with some success. However, inconsistencies have emerged. Published and preliminary data raise the possibility of several types of encephalic photoreceptor photopigment (cone-like, rod-like or different from both), and depending on species at least two types of photoreceptor cell: CSF-contacting neurons (larval lamprey, reptiles and birds) and classical neurosecretory neurons within the nucleus magnocellularis preopticus (NMPO)(fish and amphibians).

Vitamin A2-based photopigments within the pineal gland of a fully terrestrial vertebrate.

Fully terrestrial vertebrates were previously thought to exclusively employ vitamin A1 to generate visual pigments. However, recent studies on the visual system of the lizard Anolis carolinensis have shown that its visual pigments are vitamin A2-based. This unexpected result prompted an investigation of the pineal photopigments in this species [13]. HPLC analysis has shown that this extraretinal photoreceptor also exclusively utilizes a vitamin A2-derived chromophore. The adaptive significance of this chromophore within the pineal is unclear. The extended long wavelength sensitivity characteristic of vitamin A2-based visual pigment systems may enhance important visual tasks such as prey detection or mate selection [13]. A similar argument cannot be made for the pineal, whose role is not image formation, but rather detection of the irradiance changes associated with dawn and dusk. We suggest that the pineal may passively utilize whatever retinoids have been adaptively selected by the visual system.

Photoreceptors regulating circadian behavior: a mouse model.

Our recent studies have examined circadian photoreception in mice with hereditary retinal disorders (rd/rd and rds/rds). Despite the loss of visual function in these mice, circadian responses to light remain unaffected. Using c-fos expression within the suprachiasmatic nuclei (SCN) as a marker of neural activation of the circadian entrainment pathway, we have found identical levels of Fos in the SCN of rd/rd and +/+ mice in response to retinal illumination. On the basis of action spectrum studies, and measurements of photopigment retinoids using high-pressure liquid chromatography, we believe that the photopigment mediating circadian responses to light is based upon an opsin, and that 11-cis-retinaldehyde is the photopigment chromophore. Preliminary measurements of mouse rod opsin, blue cone, and green-red cone opsin messenger RNA in retinally degenerate mice suggest that none of these opsins is exclusively used to mediate circadian responses to light. Collectively, our data suggest that circadian photoreception can be maintained by a very small number of rod or cone cells without outer segments, or, alternatively, is performed by an unrecognized class of photoreceptive cell within the mammalian retina.

Vitamin A2-based visual pigments in fully terrestrial vertebrates.

As part of a broad study of the ocular and extraocular photoreceptors of reptiles, we have used high performance liquid chromatography (HPLC) to identify the retinoids present in whole eye extracts of the arboreal lizard Anolis carolinensis and the non-arboreal ruin lizard Podarcis sicula. Unexpectedly, only vitamin A2-derived chromophore was detected in Anolis, while a mixture of vitamin A1- and vitamin A2-derived chromophores was detected in Podarcis. These are the first examples of fully terrestrial vertebrates using vitamin A2-derived chromophore for visual pigment generation. Furthermore, microspectrophotometric (MSP) data for Anolis show a class of photoreceptor having a visual pigment with maximum absorbance at about 625 nm, some 40 nm further into the red than has been found in any terrestrial vertebrate examined to date.

Circadian photoreception in the retinally degenerate mouse (rd/rd).

We have examined the effects of light on circadian locomotor rhythms in retinally degenerate mice (C57BL/6J mice homozygous for the rd allele: rd/rd). The sensitivity of circadian photoreception in these mice was determined by varying the irradiance of a 15 min light pulse (515 nm) given at circadian time 16 and measuring the magnitude of the phase shift of the locomotor rhythm. Experiments were performed on animals 80 days of age. Despite the loss of visual photoreceptors in the rd/rd retina, animals showed circadian responses to light that were indistinguishable from mice with normal retinas (rd/+ and +/+). While no photoreceptor outersegments were identified in the retina of rd/rd animals (80-100 days of age), we did identify a small number of perikarya that were immunoreactive for cone opsins, and even fewer cells that contained rod opsin. Using HPLC, we demonstrated the presence and photoisomerization of the rhodopsin chromophore 11-cis retinaldehyde. The rd/rd retinas contained about 2% of 11-cis retinaldehyde found in +/+ retinas. We have yet to determine whether the opsin immunoreactive perikarya or some other unidentified cell type mediate circadian light detection in the rd/rd retina.

Melatonin receptors and signal transduction in photorefractory Siberian hamsters (Phodopus sungorus).

In seasonally breeding mammals, seasonal alterations in day length are perceived via the pineal hormone melatonin. When exposed to short day lengths, hamsters and other long-day breeders undergo gonadal regression. With chronic exposure (greater than 20 weeks) to short days, however, the animals become photorefractory, or insensitive to the inhibitory influence of short-day melatonin patterns, and gonadal recrudescence occurs. In this report, we examined photorefractory Siberian hamsters and long-day housed control hamsters to examine whether this apparent insensitivity to melatonin is due to alterations in melatonin receptors or signal transduction. In vitro autoradiographic assessment of melatonin receptors using 125I-labeled 2-iodomelatonin (I-MEL) revealed that melatonin receptor distribution, affinity, density, and G protein coupling are unaltered in photorefractory animals. In each animal, high-affinity (dissociation constant approximately 40 pM) 2-iodomelatonin binding sites were observed in the hypophysial pars tuberalis, in the suprachiasmatic nucleus of the hypothalamus, and in the thalamus (paraventricular nucleus, reuniens nucleus, and nucleus of the stria medullaris). The nonhydrolyzable GTP analog, GTP gamma S (100 microM) caused a 10-fold reduction in melatonin receptor affinity in the pars tuberalis in both photorefractory and control hamsters, demonstrating receptor-G protein coupling in both groups. Furthermore, melatonin (10 nM) inhibited forskolin-stimulated cAMP accumulation in median eminence/pars tuberalis explants in photorefractory animals, just as previously observed in explants from long-day hamsters. These results suggest that melatonin receptors, receptor-G protein coupling, and inhibition of adenylyl cyclase by melatonin are not altered in photorefractory hamsters.