Melatonin: daily cycle in plasma and cerebrospinal fluid of calves.

Melatonin was measured by radioimmunoassay in jugular vein plasma and lateral ventricle cerebrospinal fluid collected from calves at 12 times of the day and night. Melatonin in cerebrospinal fluid increased 17-fold from an average (+/- standard error) of 38 +/- 8 picograms per milliliter during the day to an average of 637 +/- 133 picograms per milliliter during the night (P less than .001). Plasma concentrations of melatonin increased sixfold from an average, per milliliter, of 19 +/- 4 picograms during the day to 121 +/- 24 picograms during the night (P less than .001).

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.

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.

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.

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.

Radioimmunoassay of serum concentrations of melatonin in sheep exposed to different lighting regimens.

A specific and sensitive double-antibody radioimmunoassay for melatonin (N-acetyl-5-methoxytryptamine) has been developed utilizing rabbit antisera to a bovine serum albumin conjugate of N-succinyl-5-methoxytryptamine and utilizing N-3-(4-hydroxyphenl)-propionyl-5-methoxytryptamine for radioiodination. The least detectable concentration of melatonin standard was 10 pmolar (2.3 pg/tube) with 50% inhibition resultinhibition curves obtained with increasing quantities of melatonin or increasing quantities of chloroform extracts of ovine sera were parallel. The immunoreactivity found in ovine sera c-migrated with [3H]melatonin on silica gel G when developed with chloroform:methanol (9:1). N-Acetylserotonin, 5-methoxytryptamine, serotonin, tryptophan, 6-hydroxymelatonin, 6-methoxytetrahydroharmalan, and several other indole and beta-carboline compounds do not influence the estimation of melatonin in the radioimmunoassay. Concentrations of melatonin could be accurately determined when 31 to 1000 pg were added to 1 ml ovine serum. Serum samples with melatonin concentrations of 1000 pg/ml, 500 pg/ml and 75 pg/ml had intra-assay coefficients of variation of 9.1%, 8.6%, and 17.4%, respectively. The respective inter-assay coefficients of variation were 22.7%, 18.1%, and 37.1%. Ewes exposed to a 12 h light: 12 h dark lighting regimen demonstrated a circadian rhythm in serum concentrations of melatonin. Concentrations ranged from 10-30 pg/ml during periods of light to 100-300 pg/ml during periods of dark. During exposure to continuous light, the circadian rhythm was abolished and concentrations of melatonin were maintained at 10-50 pg/ml. When exposed to conditions of continuous dark the circadian rhythm persisted. A precipitous drop in serum concentrations of melatonin resulted when ewes experiencing peak melatonin concentrations were exposed to light. Concentrations returned to peak levels when the lights were turned off 3.5 h later.

The suppression of nocturnal pineal melatonin in the Syrian hamster: dose-response curves at 500 and 360 nm.

It has recently been shown that wavelengths in the near-UV range (UV-A, 320-400 nm) are capable of influencing pineal melatonin content in the hamster. The purpose of this study was to compare the capacities of monochromatic visible and UV radiation for suppressing nocturnal pineal melatonin. Groups of male Syrian hamsters adapted to a 14-h light, 10-h dark cycle (lights on, 1700-0700 h) were exposed to irradiances of 500 or 360 nm light for 5 min during their dark phase. Both wavelengths suppressed pineal melatonin in a dose-related manner. The resultant fluence-response curves were similar in shape, although their corresponding threshold irradiances were markedly different. The calculated ED50 values for 500 and 360 nm light were 0.022 microW/cm2 (1.66 X 10(13) photons/cm2) and 0.306 microW/cm2 (1.66 X 10(14) photons/cm2), respectively. These data show that the induction of a 50% depression of pineal melatonin requires 10 times the number of 360-nm photons compared to 500-nm photons at the level of the cornea. Despite this difference in sensitivity to wavelength, environmental irradiances of UV-A are well above the threshold for melatonin suppression in the hamster. These results thus demonstrate the importance of considering UV-A, in addition to the visible wavelengths, in the regulation of hamster pineal physiology.

Pineal melatonin concentrations in the Syrian hamster.

Pineal melatonin concentrations exhibited a marked diurnal rhythmicity in gold hamsters maintained in a light-dark cycle of 15 h of light and 10 h of darkness. When tissue was collected at 3-h intervals throughout a 24-h period, daytime levels of 95--232 pg/pineal gland rose to concentrations of 760--1335 pg/pineal gland (P greater than 0.001 vs. all other values) at 0400 h, 8 h after the onset of darkness. When tissue was collected sequentially during the dark phase, pineal melatonin concentrations remained significantly elevated from 0200--0500 h (P less than 0.01 and P less than 0.001 vs. daytime values, respectively). Superior cervical ganglionectomy abolished the rhythm of pineal melatonin concentrations, and the concentrations were maintained at 60--105 pg/pineal gland throughout a 24-hr period.

Near-ultraviolet radiation suppresses pineal melatonin content.

UV radiation (200-400 nm) is generally considered to be outside the range of visible illumination for mammals. The aim of this study was to determine if UV wavelengths can influence pineal physiology. Experiments on hamsters confirm that wavelengths as low as 305 nm are transmitted through the clear ocular media to the retina. Furthermore, low irradiances of broadband (340-405 nm) and monochromatic (360 nm) UV radiation are capable of suppressing high nocturnal levels of pineal melatonin in intact, but not blind, hamsters. These data indicate that the hamster eye and neuroendocrine system are able to detect and respond to near-UV wavelengths.

Pupil size regulation of threshold of light-induced melatonin suppression.

The capacity of pupil dilation to affect light-induced plasma melatonin suppression was tested by exposing human subjects with freely constricting or pharmacologically dilated pupils to either 50 (n = 6), 100 (n = 8), or 200 lux (n = 5) of white light presented over the entire visual field. Pupil dilation significantly enhanced low level white light-induced melatonin suppression over that elicited with freely constricting pupils. Although 100 and 200 lux white light exposures resulted in significant melatonin suppression over control (no light) conditions, the effects of 50 lux were not strong enough to demonstrate statistically significant suppression with six subjects. Linear regression did not reveal a systematic relationship between theoretical retinal illuminance in Trolands and magnitude of melatonin suppression. These results suggest that pupil diameter may be a factor in the effectiveness of light stimuli used to shift circadian rhythms or to treat seasonal depression or sleep disorders.

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.

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.

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.

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.

Suppression of pineal melatonin in Peromyscus leucopus by different monochromatic wavelengths of visible and near-ultraviolet light (UV-A).

The purpose of this study was to examine the effects of monochromatic visible and near-ultraviolet radiation (UV-A) on pineal melatonin suppression in the white-footed mouse, Peromyscus leucopus. To this end, mice were entrained to a daily cycle of 8 h of light and 16 h of darkness. During the night when pineal melatonin contents were high, mice were individually exposed for 5 min to specific wavelengths of monochromatic light (10 nm half-peak bandwidths). Control animals received the same handling conditions but no experimental exposure. Pineal glands were collected from animals 18 min after the 5 min experimental exposure and were later assayed for melatonin content. In groups of animals exposed to equal photon densities (2.64 X 10(15) photons/cm2) of either 320, 340, 360, 500, or 560 nm, mean pineal melatonin content was significantly suppressed as compared to the unexposed control animals. The 640 nm wavelength (red) at the same photon density did not suppress pineal melatonin. These experiments are the first to demonstrate light-induced suppression of pineal melatonin in Peromyscus leucopus. In addition, these data reveal a novel finding: the suppression of pineal melatonin content by ultraviolet wavelengths as low as 320 and 340 nm.

Light-induced plasma melatonin suppression in seasonal affective disorder.

1. Subjects with seasonal affective disorder were exposed to 0, 500 and 1000 lux of white light for one hour beginning at 0300 hours. 2. Plasma samples were taken periodically and analysed for melatonin. 3. Plasma melatonin levels were suppressed by exposure to both 500 and 1000 lux light levels, suggesting that SAD patients show no neuroendocrine insensitivity to light but may show supersensitive responses to light.

Pertussis toxin sensitive photoaggregation of pigment in isolated Xenopus tail-fin melanophores.

Direct illumination of Xenopus laevis tail-fin melanophores results in rapid, reversible translocation of intracellular pigment granules to a perinuclear location, an effect distinct from and opposite to the photodispersion of pigment found in melanophores isolated from Xenopus embryos. In this report we show that both pertussis toxin and dibutyryl-adenosine-3',5'-monophosphate block the ability of light to cause photoaggregation of pigment in cultured tail-fin melanophores, whereas dibutyryl-guanosine-3',5'-monophosphate is without effect.

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.

Melatonin: reproductive effects.

N-acetyl-5-methoxytryptamine, melatonin, is synthesized within and secreted from the pineal gland. Although the concentration of this constituent in the blood is diminished after surgical removal of the pineal gland it does not completely disappear. Other potential contributors to blood titers of melatonin include the retinas, the Harderian glands and the gastro-intestinal tract. Melatonin has a potent antigonadotrophic action in the Syrian hamster ( a highly photosensitive species) provided the indole is given during a restricted portion of the light phase of the light-dark cycle. This so-called sensitive period falls late in the light phase; melatonin acutely administered at other times has virtually no inhibitory influence on the reproductive physiology of hamsters. When melatonin is continuously available (from a subcutaneous deposit) it counteracts the antigonadotrophic influence of the pineal gland in light restricted or blinded hamsters, i.e., it causes a "functional pinealectomy". Furthermore, chronically available melatonin negates the antigonadotrophic capability of acute melatonin injections.