Pineal function and oviposition in Japanese quail: superior cervical ganglionectomy and photoperiod.

Bilateral ablation of the superior cervical ganglia appears to deprive the pineal body of sympathetic innervation. Although this procedure presumably interrupts the neural circuit for transmission of optic information to the pineal, oviposition rates of ganglionectomized females exposed to stimulatory (15-hour) or to nonstimulatory (4-hour) daily photoperiods do not differ from those of the controls.

Daily rhythm in human urinary melatonin.

The melatonin in urine samples from six healthy adult volunteers was concentrated on Amberlite XAD-2 resin, eluted with organic solvents, and quantitated by use of a bioassay technique (the dermal melanaphore response of larval anurans to melatonin in their bathing medium). The melatonin content of samples collected between 11 p.m. and 7 a.m. was, in each case, several times higher than that of samples collected between 7 a.m. and 3 p.m. or between 3 p.m. and 11 p.m.

Presence of melatonin in plasma and urine or pinealectomized rats.

Melatonin was shown to occur in rat plasma and urine after pinealectomy by bioassay, radioimmunoassay, and thin-layer chromatography. Total daily excretion of melatonin in pinealectomized rats was about 20% of control. Light-dark rhythms of plasma melatonin levels and urinary excretion rates disappeared after pinealectomy.

Melatonin in rat pineal, plasma, and urine: 24-hour rhythmicity and effect of chlorpromazine.

The concentrations of melatonin in rat pineals and in samples of rat plasma and urine were measured by bioassay or radioimmunoassay. Melatonin excretion during the daily dark period (0.98 +/- 0.07 ng/12 h, by bioassay; 1.40 +/- 0.08 ng/12 h, by radioimmunoassay) was much greater than during the light period (0.18 +/- 0.04 ng/12 h, by bioassay; 0.40 +/- 0.04 ng/12 h by radioimmunoassay). (The radioimmunoassay is not completely specific when applied to materials extracted from utine.) Pineal glands and plasmas obtained from animals killed during the daily dark period also contained much more melatonin than samples from animals killed during the light period. Pretreatment with chlorpromazine slowed the disappearance of exogenous melatonin from rat plasma and markedly raised the concentrations of endogenous melatonin in both the pineal gland and the plasma.

Melatonin in human preovulatory follicular fluid.

Melatonin, the major hormone of the pineal gland, has antigonadotrophic activity in many mammals and may also be involved in human reproduction. Melatonin suppresses steroidogenesis by ovarian granulosa and luteal cells in vitro. To determine if melatonin is present in the human ovary, preovulatory follicular fluids (n = 32) from 15 women were assayed for melatonin by RIA after solvent extraction. The fluids were obtained by laparoscopy or sonographically controlled follicular puncture from infertile women undergoing in vitro fertilization and embryo transfer. All patients had received clomiphene citrate, human menopausal gonadotropin, and hCH to stimulate follicle formation. Blood samples were obtained by venipuncture 30 min or less after follicular aspiration. All of the follicular fluids contained melatonin, in concentrations [36.5 +/- 4.8 (+/- SEM) pg/mL] substantially higher than those in the corresponding serum (10.0 +/- 1.4 pg/mL). A positive correlation was found between follicular fluid and serum melatonin levels in each woman (r = 0.770; P less than 0.001). These observations indicate that preovulatory follicles contain substantial amounts of melatonin that may affect ovarian steroidogenesis.

The circadian rhythm of plasma melatonin during the normal menstrual cycle and in amenorrheic women.

Plasma melatonin, PRL, and LH levels were measured in samples collected every 2 h for 24 h from 14 normally cycling women during the early follicular, periovulatory, and luteal phases of their menstrual cycles. Plasma melatonin levels also were measured in samples collected at the same interval from 7 patients with hypothalamic amenorrhea. A distinct daily rhythm in plasma melatonin was evident in all subjects, with peaks occurring around 0300 h. Each woman's rhythm was remarkably consistent throughout the menstrual cycle (in terms of the phase, amplitude, and total melatonin secreted). Plasma PRL levels also exhibited daily rhythms which did not change during the menstrual cycle; the nocturnal peak plasma PRL level tended to occur 1-2 h after that for melatonin. Among the amenorrheic women, both daytime and nighttime melatonin levels were significantly higher (P less than 0.005) than in the normal women. Their plasma PRL levels were similar to those in the normal women. We conclude that, as for PRL, the circadian rhythm of melatonin secretion does not change significantly during the normal menstrual cycle. The elevated plasma melatonin levels in women with hypothalamic amenorrhea suggest that the hormone may be involved in the neuroendocrine pathology underlying this disorder.

Sleep-inducing effects of low doses of melatonin ingested in the evening.

We previously observed tht low oral doses of melatonin given at noon increase blood melatonin concentrations to those normally occurring nocturnally and facilitate sleep onset, as assessed using and involuntary muscle relaxation test. In this study we examined the induction of polysomnographically recorded sleep by similar doses given later in the evening, close to the times of endogenous melatonin release and habitual sleep onset. Volunteers received the hormone (oral doses of 0.3 or 1.0 mg) or placebo at 6, 8, or 9 PM. Latencies to sleep onset, to stage 2 sleep, and to rapid eye movement (REM) sleep were measured polysomnographically. Either dose given at any of the three time points decreased sleep onset latency and latency to stage 2 sleep. Melatonin did not suppress REM sleep or delay its onset. Most volunteers could clearly distinguish between the effects of melatonin and those of placebo when the hormone was tested at 6 or 8 PM. Neither melatonin dose induced "hangover" effects, as assessed with mood and performance tests administered on the morning after treatment. These data provide new evidence that nocturnal melatonin secretion may be involved in physiologic sleep onset and that exogenous melatonin may be useful in treating insomnia.

Melatonin: a sleep-promoting hormone.

This review discusses the issue of a dual effect of melatonin on sleep: acute sleep promotion that typically occurs within one hour of administration, and the ability to alter the phase of an underlying circadian pacemaker after a repeated melatonin treatment. The authors suggest that both mechanisms are at work, that they are complementary, and that they may manifest jointly or separately. The review provides some basic information on melatonin, an overview of the literature, and the authors' experience in studying the acute effects of melatonin treatment in humans of different age groups. This review also illustrates the authors' cautious attitude toward melatonin treatment that induces supraphysiologic circulating levels of the hormone.

Effects of low oral doses of melatonin, given 2-4 hours before habitual bedtime, on sleep in normal young humans.

Low oral doses of melatonin raise serum melatonin concentrations to those normally occurring nocturnally and facilitate polysomnographically assessed sleep onset when given at different time points throughout the day, without altering mood or performance on the morning following treatment. In the present study, 12 young healthy volunteers, free of sleep disturbances, received 0.3 or 1.0 mg of melatonin or placebo at 2100 hours, 2-4 hours prior to their habitual bedtime. Polysomnographic recording of overnight sleep began at 2200 hours and continued until 0700 hours the following morning, when subjects were awakened. Sleep onset latency and latency to stage 2 sleep were significantly decreased as a result of melatonin treatment. Neither dose of melatonin significantly altered sleep architecture. Administration of the lower dose of melatonin (0.3 mg) at 2100 hours elevated serum melatonin to levels within the normal nocturnal range (113 +/- 13.5 pg/ml) at the time the sleep test was initiated. Neither melatonin dose caused "hangover effects", as assessed by self-reports or by mood and performance tests administered on the morning following treatment. These observations provide additional evidence that nocturnal melatonin secretion has a sleep-promoting function. They also indicate that an increase in serum melatonin concentrations, within the normal physiologic range, does not significantly alter sleep architecture in subjects with normal sleep who receive the treatment several hours prior to their habitual bedtime.

Effect of pharmacological daytime doses of melatonin on human mood and performance.

Melatonin (10, 20, 40, or 80 mg, PO) or placebo was administered at 1145 hours on five separate occasions to 20 healthy male volunteers and the effects on serum melatonin levels, mood, performance, and oral temperature were monitored. Subjects were studied between 0930 and 1700 hours. A battery of interactive computer tasks designed to assess performance and mood was completed, oral temperature was measured, and blood samples were taken for serum melatonin radioimmunoassay. The areas under the time-melatonin concentration curve (AUC) varied significantly in proportion to the various melatonin doses. Compared with placebo treatment, all melatonin doses significantly decreased oral temperature, number of correct responses in auditory vigilance, response latency in reaction time, and self-reported vigor. Melatonin also increased self-reported fatigue, confusion, and sleepiness.

Possible behavioral consequences of light-induced changes in melatonin availability.

Melatonin is a hormone secreted at night, in the dark, by the human pineal organ. This nocturnal release of melatonin, in humans and other species, is rapidly suppressed by exposure to sufficiently bright light. In humans, the function, if any, of this circadian pattern of melatonin release has not been determined. In fact, no function has been definitively attributed to the hormone melatonin in humans. In one study, conducted in our laboratory, pharmacologic doses of oral melatonin (240 mg over two hours) were administered to volunteers, and various behavioral parameters were assessed. Melatonin had substantial, but brief, sedative-like effects on mood and performance. Thus it appears that a mechanism exists, whereby light, of sufficient intensity to affect melatonin release in humans, can affect behavior. It can be hypothesized that sufficiently bright light, acting by way of the suppression of melatonin release, can acutely increase alertness or act as a zeitgeber (synchronizer of circadian cycles). The light intensity necessary to suppress melatonin secretion in humans is well above typical indoor lighting conditions, but well below normal outdoor daytime levels of illumination. Therefore, the hypothesis that light may affect behavior or circadian patterns of sleep and waking, if found to be true, could have considerable impact on the design of interior lighting.

Endogenous melatonin levels and the fate of exogenous melatonin: age effects.

This study examines the range of serum melatonin concentrations that occur among young and older adults, and tests the effects of orally administered melatonin on the serum and saliva concentrations of the hormone. Healthy volunteers (20-36 per study), aged 20-73 years, were divided into two groups on the basis of age (29.2 +/- 6.5 and 60 +/- 8.8 years). For study 1: Serum melatonin levels were measured at 15 to 60 min intervals over a 25 h period using a radioimmunoassay. For study 2: serum and saliva melatonin levels were measured before and at intervals after the administration of a 0.3 mg dose of melatonin at 11.00 h. The younger subjects had significantly higher peak endogenous melatonin concentrations (+/- SD) and greater inter-individual variability (100.9 +/- 48.6 pg/ml) than the older subjects (34.5 +/- 15.4 pg/ml). Mean melatonin levels following treatment with the hormone tended to be higher and were significantly more variable among the group of older volunteers (254.5 +/- 145.7) than among the younger group (170.2 +/- 22.0 pg/ml). We conclude that although the peak endogenous serum melatonin levels are lower in elderly adults, the increment in serum melatonin levels induced by a low oral dose of the hormone is greater and more variable among people over 48 years old.

Effect of cyclic changes in environmental lighting and ambient temperature on the daily rhythm in melatonin excretion by rats.

Melatonin excretion was measured by radioimmunoassay in 6 h or 12 h urine specimens from individual control rats and from animals previously blinded by bilateral orbital enucleation. Among sighted rats, the rate of melatonin excretion was greatest during the daily 12 h of darkness (1.44 +/- 0.06 ng/12 h dark period vs. 0.53 +/- 0.07 ng/12 h light period; P less than 0.001). Moreover, greater quantities of melatonin were excreted in the latter half of the dark period than in the first half (e.g. 0.55 +/- 0.08 ng/first 6 h vs 0.97 +/- 0.05 ng/second 6 h; P less than 0.001). When the onset of the daily light period was shifted forward by 12 h, 5--7 days were needed for the daily rhythm in melatonin excretion to become re-entrained to the new light--dark cycle. Among blinded rats, the rate of melatonin excretion also varied rhythmically; however, the rhythm was neither synchronized with the light-dark cycle nor influenced by alterations in the lighting schedule. Similarly, artificial cycles in environmental temperature were not effective in entraining the daily rhythm in melatonin excretion among blinded rats.

Melatonin and its precursors in Y79 human retinoblastoma cells: effect of sodium butyrate.

We studied the release of melatonin and the production of its precursors, 5-hydroxytryptophan and serotonin, in cultured Y79 human retinoblastoma cells. This biosynthetic capability was found to be dependent on cell differentiation, which was initiated by culturing Y79 cells for 7 days in dishes coated with poly-D-lysine to promote cell adhesion to the surface of the culture dishes. Differentiation was further induced by exposing the cell monolayer to sodium butyrate (3 mM) for 3 days. This protocol dramatically increased the release of melatonin and the syntheses of 5-hydroxytryptophan and serotonin in response to forskolin stimulation. Exposure to dopamine (10 microM) or L-DOPA (100 microM) markedly diminished the forskolin-stimulated release of melatonin, as well as the production of 5-hydroxytryptophan and serotonin. These observations indicate that Y79 cells represent a primitive cell line which, following appropriate differentiation (e.g. treatment with sodium butyrate) can display biochemical characteristics similar to those of the human retina. Moreover, serotonin synthesis and melatonin release appear to be coupled in Y79 cells. The inhibition of melatonin release by dopamine supports the hypothesis that in these cells, melatonin and dopamine are components of a retinal feedback loop.

Effects of melatonin on human mood and performance.

The function of melatonin, a hormone secreted by the pineal gland primarily at night, has not been definitively established in humans. To determine if pharmacologic doses of melatonin had any behavioral effects it was administered acutely to 14 healthy men. Their mood, performance, memory and visual sensitivity were assessed. Plasma melatonin concentration was assayed as well. Melatonin significantly decreased self-reported alertness and increased sleepiness as measured by the Profile of Mood States and the Stanford Sleepiness Scale self-report mood questionnaires. The effects were brief. Melatonin also affected performance, slowing choice-reaction time but concurrently decreasing errors of commission. Sustained fine motor performance was not impaired after melatonin administration nor were the tests of memory and visual sensitivity that were administered. It is concluded that melatonin, administered orally in pharmacological quantities, has significant but short acting sedative-like properties.

Pineal responses to stress.

When laboratory rats are sensitized by appropriate environmental manipulations (e.g., protracted exposure to light, fasting), significant increases in melatonin synthesis and secretion can be induced by the acute imposition of stress (e.g., physical immobilization). In the absence of such priming pre-treatment, however, a stress-induced increment in melatonin levels may not be detectable. The mechanisms responsible may involve concurrent changes in the sensitivity of the pineal to catecholamines, sympathetic neural input to the gland, and circulating levels of catecholamines. The experimental use of stress-induced changes in pineal function may enhance the utility of the laboratory rat's pineal gland as a model for studying changes in the rhythmic secretion of melatonin in humans as a consequence of endogenous processes.

The measurement of melatonin in mammalian tissues and body fluids.

A quantitative bioassay for melatonin and a radioimmunoassay for melatonin are described and evaluated in terms of their specificity, sensitivity, and practicability. A thin-layer chromatographic method is presented whereby the specificity of the radioimmunoassay for melatonin in blood and tissue extract is affirmed and its specificity in the assay of urinary melatonin is described. Preliminary observations on the relationship between the rate of melatonin excretion and blood levels of melatonin are also reported.

Light intensities required to suppress nocturnal melatonin secretion in albino and pigmented rats.

Sprague-Dawley albino rats or Long-Evans pigmented rats were exposed during the dark phase of the daily light:dark cycle to various intensities of a sunlight-stimulating white fluorescent light (0.022, 0.044, 0.110, 0.220, 0.440 or 2.200 microW/cm2) for 30 min; pineal glands and trunk blood samples were then collected and assayed for melatonin by radioimmunoassay. Albino rats exposed to irradiances of 0.110 microW/cm2 or less had pineal melatonin levels that were not significantly different from those of unexposed animals; higher irradiances significantly (P less than 0.001) reduced melatonin levels. In contrast, as little as 0.022 microW/cm2 significantly (P less than 0.02) reduced pineal and serum melatonin levels in the pigmented rats. These results suggest that something other than the simple presence or absence of eye pigmentation is the critical factor in determining the sensitivity of the rat's pineal to retinal-mediated photic suppression of melatonin synthesis.

Effects of illumination on human nocturnal serum melatonin levels and performance.

In humans, exposure to bright light at night suppresses the normal nocturnal elevation in circulating melatonin. Oral administration of pharmacological doses of melatonin during the day, when melatonin levels are normally minimal, induces fatigue. To examine the relationship between illumination, human pineal function, and behavior, we monitored the overnight serum melatonin profiles and behavioral performance of 24 healthy male subjects. On each of three separate occasions subjects participated in 13.5 h (1630-0800 h) testing sessions. Each subject was assigned to an individually illuminated workstation that was maintained throughout the night at an illumination level of approximately 300, 1500, or 3000 lux. Melatonin levels were significantly diminished by light treatment, F(2, 36) = 12.77, p < 0.001, in a dose-dependent manner. Performance on vigilance, reaction time, and other tasks deteriorated throughout the night, consistent with known circadian variations in these parameters, but independent of ambient light intensity and circulating melatonin levels.

Melatonin concentrations in the sudden infant death syndrome.

To examine a possible relationship between pineal function and the sudden infant death syndrome (SIDS), samples of whole blood, ventricular cerebrospinal fluid (CSF) and/or vitreous humor (VH) were obtained at autopsy from 68 infants (45 male, 23 female) whose deaths were attributed to either SIDS (n = 32, 0.5-5.0 months of age; mean +/- S.E.M., 2.6 +/- 0.2 months) or other causes (non-SIDS, n = 36, 0.3-8.0 months of age 4.3 +/- 0.3 months). The melatonin concentrations were measured by radioimmunoassay. A significant correlation was observed for melatonin levels in different body fluids from the same individual. After adjusting for age differences, CSF melatonin levels were significantly lower among the SIDS infants (91 +/- 29 pmol/l; n = 32) than among those dying of other causes (180 +/- 27; n = 35, P less than 0.05). A similar, but non-significant trend was also noted in blood (97 +/- 23, n = 30 vs. 144 +/- 22 pmol/l, n = 33) and vitreous humor (68 +/- 21, n = 10 vs. 81 +/- 17 pmol/l, n = 15). These differences do not appear to be explainable in terms of the interval between death and autopsy, gender, premortem infection or therapeutic measures instituted prior to death. Diminished melatonin production may be characteristic of SIDS and could represent an impairment in the maturation of physiologic circadian organization.