Randomized trial of polychromatic blue-enriched light for circadian phase shifting, melatonin suppression, and alerting responses.

Wavelength comparisons have indicated that circadian phase-shifting and enhancement of subjective and EEG-correlates of alertness have a higher sensitivity to short wavelength visible light. The aim of the current study was to test whether polychromatic light enriched in the blue portion of the spectrum (17,000 K) has increased efficacy for melatonin suppression, circadian phase-shifting, and alertness as compared to an equal photon density exposure to a standard white polychromatic light (4000 K). Twenty healthy participants were studied in a time-free environment for 7 days. The protocol included two baseline days followed by a 26-h constant routine (CR1) to assess initial circadian phase. Following CR1, participants were exposed to a full-field fluorescent light (1 × 1014 photons/cm2/s, 4000 K or 17,000 K, n = 10/condition) for 6.5 h during the biological night. Following an 8 h recovery sleep, a second 30-h CR was performed. Melatonin suppression was assessed from the difference during the light exposure and the corresponding clock time 24 h earlier during CR1. Phase-shifts were calculated from the clock time difference in dim light melatonin onset time (DLMO) between CR1 and CR2. Blue-enriched light caused significantly greater suppression of melatonin than standard light ((mean ±â€¯SD) 70.9 ±â€¯19.6% and 42.8 ±â€¯29.1%, respectively, p < 0.05). There was no significant difference in the magnitude of phase delay shifts. Blue-enriched light significantly improved subjective alertness (p < 0.05) but no differences were found for objective alertness. These data contribute to the optimization of the short wavelength-enriched spectra and intensities needed for circadian, neuroendocrine and neurobehavioral regulation.

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.

Action spectrum for melatonin regulation in humans: evidence for a novel circadian photoreceptor.

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 light-induced 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 +/- 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 = 627) were completed with wavelengths from 420 to 600 nm. The data were fit to eight univariant, sigmoidal fluence-response curves (R(2) = 0.81-0.95). The action spectrum constructed from these data fit an opsin template (R(2) = 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.

Role of a pineal cAMP-operated arylalkylamine N-acetyltransferase/14-3-3-binding switch in melatonin synthesis.

The daily rhythm in melatonin levels is controlled by cAMP through actions on the penultimate enzyme in melatonin synthesis, arylalkylamine N-acetyltransferase (AANAT; serotonin N-acetyltransferase, EC ). Results presented here describe a regulatory/binding sequence in AANAT that encodes a cAMP-operated binding switch through which cAMP-regulated protein kinase-catalyzed phosphorylation [RRHTLPAN --> RRHpTLPAN] promotes formation of a complex with 14-3-3 proteins. Formation of this AANAT/14-3-3 complex enhances melatonin production by shielding AANAT from dephosphorylation and/or proteolysis and by decreasing the K(m) for 5-hydroxytryptamine (serotonin). Similar switches could play a role in cAMP signal transduction in other biological systems.

cAmp regulation of arylalkylamine N-acetyltransferase (AANAT, EC 2.3.1.87): a new cell line (1E7) provides evidence of intracellular AANAT activation.

Arylalkylamine N-acetyltransferase (serotonin N-acetyltransferase, AANAT, EC ) is the penultimate enzyme in melatonin synthesis. As described here, a cell line (1E7) expressing human AANAT (hAANAT) has been developed to study the human enzyme. 1E7 hAANAT is detectable in immunoblots as a 23-kDa band and is immunocytochemically visualized in the cytoplasm. The specific concentration of hAANAT in homogenates is comparable to that of the night rat pineal gland. Kinetics of AANAT extracted from 1E7 cells are the same as those of bacterially expressed hAANAT; both preparations of hAANAT are equally sensitive to the inhibitor CoA-S-N-acetyltryptamine. Studies of cAMP regulation indicate that treatment with forskolin, dibutyryl cAMP, isobutylmethylxanthine, or isoproterenol activate cellular hAANAT within intact 1E7 cells approximately 8-fold without markedly increasing the abundance of AANAT protein or the activity of AANAT in broken cell preparations; and, that forskolin, isobutylmethylxanthine and isoproterenol elevate cyclic AMP production. These observations extend our understanding of cAMP regulation of AANAT activity, because it is currently thought that this only involves changes in the steady-state levels of AANAT protein. This previously unrecognized switching mechanism could function physiologically to control melatonin production without changing AANAT protein levels.

Human melatonin regulation is not mediated by the three cone photopic visual system.

The aim of this study was to test if the three cone photopic visual system is the primary ocular photoreceptor input for human circadian regulation by determining the effects of different wavelengths on light-induced melatonin suppression. Healthy subjects with stable sleeping patterns (wake-up time 7:30 AM +/- 12 min) and normal color vision were exposed at night to full-field 505 nm or 555 nm monochromatic stimuli or darkness for 90 min. Plasma collected before and after exposures was quantified for melatonin. Subjects exposed to 10 irradiances at 505 nm showed no significant differences across mean pre-exposure melatonin values (F=0.505). A sigmoidal fluence-response curve fitted to the melatonin suppression data (R(2)=0.97) indicated that 9.34 x 10(12) photons/cm(2)/sec induced a half-saturation response (ED(50)) while 6.84 x 10(13) photons/cm(2)/sec induced a saturation melatonin suppression response. Further, a dose of 4.19 x 10(13) photon/cm(2)/sec at 505 nm was significantly stronger (P < 0.01) than an equal photon dose at 555 nm for melatonin suppression. These data demonstrate that the cone system that mediates human photopic vision is not the primary photoreceptor system to tranduce light stimuli for melatonin regulation.

The effect of polarized versus nonpolarized light on melatonin regulation in humans.

The aim of this study was to compare the effects of polarized light versus nonpolarized light on melatonin secretion in healthy, humans (mean age, 25 years; N = 6). On separate evenings, each subject was exposed to four different light intensities (20, 40, 80 and 3200 lx) of both polarized and nonpolarized light, as well as to a control, dark exposure. Each evening experiment consisted of a 120 min dark exposure (0000-0200 h) followed by a 90 min light exposure (0200-0330 h). Subjects' pupils were dilated prior to exposures. Blood samples were drawn at the start and end of each light-exposure period and later assayed for melatonin by radioimmunoassay. When compared to control exposures, both polarized and nonpolarized light elicited significant suppression of plasma melatonin at each illuminance (P < 0.03 to P < 0.0001), There were no significant differences between the effects of polarized light and nonpolarized light at any illuminance. The two light stimuli modalities demonstrated very similar fluence-response relationships between illuminance and melatonin suppression. Thus, the human pineal gland is responsive to ocular exposure with polarized light in a dose-dependent manner similar to that of nonpolarized light, although no significant differences were detected between polarized and nonpolarized light on melatonin regulation.

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.

Light-emitting diodes and cool white fluorescent light similarly suppress pineal gland melatonin and maintain retinal function and morphology in the rat.

BACKGROUND AND PURPOSE: A novel light-emitting diode (LED) light source for use in animal-habitat lighting was evaluated. METHODS: The LED was evaluated by comparing its effectiveness with that of cool white fluorescent light (CWF) in suppressing pineal gland melatonin content and maintaining normal retinal physiology, as evaluated by use of electroretinography (ERG), and morphology. RESULTS: Pineal melatonin concentration was equally suppressed by LED and CWF light at five light illuminances (100, 40, 10, 1, and 0.1 lux). There were no significant differences in melatonin suppression between LED and CWF light, compared with values for unexposed controls. There were no differences in ERG a-wave implicit times and amplitudes or b-wave implicit times and amplitudes between 100-lux LED-exposed rats and 100-lux CWF-exposed rats. Results of retinal histologic examination indicated no differences in retinal thickness, rod outer segment length, and number of rod nuclei between rats exposed to 100-lux LED and 100-lux CWF for 14 days. Furthermore, in all eyes, the retinal pigmented epithelium was intact and not vacuolated, whereas rod outer segments were of normal thickness. CONCLUSION: LED light does not cause retinal damage and can suppress pineal melatonin content at intensities similar to CWF light intensities.

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.

Photic regulation of melatonin in humans: ocular and neural signal transduction.

Light is a potent stimulus for regulating the pineal gland's production of melatonin and the broader circadian system in humans. It initially was thought that only very bright photic stimuli (> or = 2500 lux) could suppress nocturnal melatonin secretion and induce other circadian responses. It is now known that markedly lower illuminances (< or = 200 lux) can acutely suppress melatonin or entrain and phase shift melatonin rhythms when exposure conditions are optimized. The elements for physical/biological stimulus processing that regulate photic influences on melatonin secretion include the physics of the light source, gaze behavior relative to the light source, and the transduction of light energy through the pupil and ocular media. Elements for sensory/neural signal processing become involved as photons are absorbed by retinal photopigments and neural signals are generated in the retinohypothalamic tract. Aspects of this physiology include the ability of the circadian system to integrate photic stimuli spatially and temporally as well as the wavelength sensitivity of the operative photoreceptors. Acute, light-induced suppression of melatonin is proving to be a powerful tool for clarifying how these elements of ocular and neural physiology influence the interaction between light and the secretion of melatonin from the human pineal gland.

Pharmacokinetics of extracellular melatonin in Siberian hamster forebrain.

In vivo brain microdialysis was used to characterize the pharmacokinetics of subcutaneously injected melatonin in the anterior hypothalamic-preoptic area (AH-POA) of the male Siberian hamster. Animals with a microdialysis probe implanted in the AH-POA were treated with a subcutaneous melatonin injection at 0900 h (3 h after lights-on) or 2000 h (2 h prior to lights-off). Treatment with 2.5 or 0.25 mg/kg dosages of melatonin in saline vehicle induced peak concentrations of melatonin in AH-POA microdialysates within 20 min of injection (165 +/- 34 and 18 +/- 8 pg/20 min, respectively). For the 2.5 and 0.25 mg/kg dosages, the half-life of the absorbed melatonin (t 1/2 elimination) was less than 20 min, and the concentrations fell to baseline within 60 min after injection. There were no significant time of day effects on the kinetic profile of extracellular melatonin associated with either of these dosages. These results confirm the rapid accumulation and clearance of extracellular melatonin in the vicinity of its putative target tissues.

Melatonin regulation in humans with color vision deficiencies.

Light can induce an acute suppression and/or circadian phase shift of plasma melatonin levels in subjects with normal color vision. It is not known whether this photic suppression requires an integrated response from all photoreceptors or from a specialized subset of photoreceptors. To determine whether normal cone photoreceptor systems are necessary for light-induced melatonin suppression, we tested whether color vision-dificient human subjects experience light-induced melatonin suppression. In 1 study, 14 red-green color vision-deficient subjects and 7 normal controls were exposed to a 90-min, 200-lux, white light stimulus from 0200-0330 h. Melatonin suppression was observed in the controls (t = -7.04; P < 0.001), all color vision-deficient subjects (t = -4.76; P < 0.001), protanopic observers (t = -6.23; P < 0.005), and deuteranopic observers (t = -3.48; P < 0.05), with no significant difference in the magnitude of suppression between groups. In a second study, 6 red/green color vision-deficient males and 6 controls were exposed to a broad band green light stimulus (120 nm with lambda max 507 nm; mean +/- SEM, 305 +/- 10 lux) or darkness from 0030-0100 h. Hourly melatonin profiles (2000-1000 h) were not significantly different in onset, offset, or duration between the two groups. Melatonin suppression was also observed after exposure to the green light source at 0100 h (color vision deficient: t = -2.3; df = 5; P < 0.05; controls: t = -3.61; df = 5; P < 0.01) and 0115 h (color vision deficient: t = -2.74; df = 5; P < 0.05; controls: t = -3.57; df = 5; P < 0.01). These findings suggest that a normal trichromatic visual system is not necessary for light-mediated neuroendocrine regulation.

Effects of photoperiod on pineal melatonin in the marsh rice rat (Oryzomys palustris).

Pineal melatonin content was examined under four different photoperiods (10L:14D, 12L:12D, 14L:10D, and 16L:8D) in adult female rice rats (Experiment 1). Pineal melatonin was basal during the light and increased beginning 1 hr after lights off. Within 2 hr after lights off, melatonin increased to levels that were maintained throughout the dark period. In all but one photoperiod (10L:14D), melatonin remained elevated prior to light onset and decreased markedly within one hour after lights on. In addition, the duration of pineal melatonin was inversely related to the length of the photoperiod. In Experiment 2, the time course of pineal melatonin content on 16L:8D was examined every 20 min during the first hour after lights off and the first hour after lights on. Melatonin content increased gradually during the first hour and decreased markedly within 20 min after lights on. These data show that pineal melatonin in female rice rats is regulated by photoperiod.

Ultraviolet regulation of neuroendocrine and circadian physiology in rodents.

UV wavelengths can regulate neuroendocrine and circadian responses in some rodent species. Appropriately timed UV exposures can block the short photoperiod-induced collapse of the reproductive system, cause a rapid suppression of nocturnal melatonin synthesis, regulate melatonin rhythms and phase shift wheel running rhythms. These biological effects of UV are not dependent on the Harderian gland or melanin in the eye, but appear to be related to the degree of transmission through the ocular lens. Such results are consistent with the hypothesis that elements in the retina can transduce UV stimuli for circadian and neuroendocrine regulation.

Effect of MR imaging on the normal human pineal body: measurement of plasma melatonin levels.

Production of melatonin, a hormone synthesized and secreted by the pineal body, has been suppressed by electromagnetic fields in some but not all animal studies. Magnetic resonance (MR) imaging at 1.5 T was evaluated for its ability to modulate the level of melatonin in eight male volunteers. Subjects were exposed to three conditions, respectively, between 1:00 and 2:00 AM on different nights: (a) a series of routine MR pulse sequences for brain imaging in dark conditions, (b) dark control conditions, and (c) bright-light control conditions. Plasma was analyzed for melatonin and cortisol levels. Hormonal changes were analyzed by one-factor repeated measures within-subject analysis of variance. These conditions were associated with significant differences in melatonin levels: F(2, 6) = 7.95, and P = .021. Subjects exposed to darkness showed a typical increase in melatonin concentration. Subjects exposed to bright light showed a characteristic suppression of melatonin concentration. Those exposed to the MR imaging fields showed an increase in melatonin level similar to that seen in the dark control condition. Light and MR imaging had no significant effects on cortisol levels. Thus, MR imaging at field strengths known to modulate melatonin levels in rats did not suppress melatonin production in human subjects.