The photopigment in the human eye that transduces light for circadian and neuroendocrine regulation, is unknown. The aim of this study was to establish an action spectrum for lightinduced melatonin suppression that could help elucidate the ocular photoreceptor system for regulating the human pineal gland. Subjects (37 females, 35 males, mean age of 24.5 Ϯ 0.3 years) were healthy and had normal color vision. Full-field, monochromatic light exposures took place between 2:00 and 3:30 A.M. while subjects' pupils were dilated. Blood samples collected before and after light exposures were quantified for melatonin. Each subject was tested with at least seven different irradiances of one wavelength with a minimum of 1 week between each nighttime exposure. Nighttime melatonin suppression tests (n ϭ 627) were completed with wavelengths from 420 to 600 nm. The data were fit to eight univariant, sigmoidal fluence-response curves (R 2 ϭ 0.81-0.95). The action spectrum constructed from these data fit an opsin template (R 2 ϭ 0.91), which identifies 446-477 nm as the most potent wavelength region providing circadian input for regulating melatonin secretion. The results suggest that, in humans, a single photopigment may be primarily responsible for melatonin suppression, and its peak absorbance appears to be distinct from that of rod and cone cell photopigments for vision. The data also suggest that this new photopigment is retinaldehyde based. These findings suggest that there is a novel opsin photopigment in the human eye that mediates circadian photoreception.

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 melanopsinpositive 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.

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.Photopigments consist of an integral membrane apoprotein, opsin, that is covalently linked to a retinaldehyde chromophore. Upon illumination, the chromophore is photoisomerized, forcing a conformational change in the surrounding opsin and subsequently initiating an intracellular signaling cascade. The conversion of light into neural signals within photoreceptor cells of the retina is the best studied example of opsin-mediated signaling.Dermal melanophores of Xenopus laevis, like retinal photoreceptors, are photosensitive (1). When maintained in vitro, the melanosomes in dermal melanophores migrate to the cellular periphery in response to illumination (2) or the activation of a wide variety of G protein-coupled receptors (3). The melanophore response to these agents has led to the development of innovative systems for studying functional interactions between ligands and their receptors (4, 5). Melanophore photosensitivity is reversibly activated by retinaldehydes (6) and has an action spectrum characteristic of opsinlike photopigments (2). In an effort to identify an opsin that may regulate melanosome migration, we have analyzed protein extracts of cultured melanophores for opsin immunoreactivity and screened a melanophore cDNA library for opsin-like nucleotide sequences. MATERIALS AND METHODSWestern Blot. Approximately 10 5 cultured Xenopus laevis dermal melanophores were washed twice with 1ϫ calcium-and magnesium-free Dulbecco's phosphate buffer and lysed with lysis buffer [1% IGEPAL CA-630 (Sigma)͞0.2 M NaBH 3 CN͞1 mM phenylmethylsulfonyl fluoride͞aprotinin (0.1 trypsin inhibitor unit͞ml)͞5 mM EDTA͞0.086 M NaC 2 H 3 O 2 , pH 5.0, 4°C]. A homogenate of one early postmetamorphic adult eye was also extracted as above. Lysates were centrifuged and the supernatants subjected to SDS͞PAGE analysis and subsequent electroblotting onto a poly(vinylidene difluoride) membrane. The blot was probed with a 1:2,000 dilution of antisera (CERN 886) raised against bovine rhodopsin and detected by enhanced chemiluminescence.cDNA Library Screen. A X. laevis dermal melanophore oligo(dT) cDNA library was...

The master circadian oscillator in the hypothalamic suprachiasmatic nucleus is entrained to the day/night cycle by retinal photoreceptors. Melanopsin (Opn4), an opsin-based photopigment, is a primary candidate for photoreceptor-mediated entrainment. To investigate the functional role of melanopsin in light resetting of the oscillator, we generated melanopsin-null mice (Opn4-/-). These mice entrain to a light/dark cycle and do not exhibit any overt defect in circadian activity rhythms under constant darkness. However, they display severely attenuated phase resetting in response to brief pulses of monochromatic light, highlighting the critical role of melanopsin in circadian photoentrainment in mammals.