σ Receptor Modulation of Noradrenergic-Stimulated Pineal Melatonin Biosynthesis in Rats

Abstract: Because σ receptors are richly concentrated in the rat pineal gland, the present study was performed to investigate their possible role in the modulation of melatonin production. To this purpose, we assessed in vivo the effects of the σ-receptor ligands 1,3-di(2-tolyl)guanidine and (+)- N -allylnormetazocine on the rat pineal gland activity during either the daytime or the nighttime. Compared with vehicle, 1,3-di(2-tolyl)guanidine and (+)- N -allylnormetazocine potentiated the enhancement of N -acetyltransferase activity and pineal melatonin content induced by isoproterenol administration during the daytime, whereas they did not affect the diurnal basal biosynthetic activity of the gland. Conversely, at night, 1,3-di(2-tolyl)guanidine and (+)- N -allylnormetazocine enhanced significantly the physiological increases in both pineal N -acetyltransferase activity and melatonin levels. This enhancement was prevented by pretreatment with rimcazole, a specific σ-receptor antagonist. These findings suggest that, in rats, the activation of pineal σ-receptor sites does not affect the biosynthetic activity of the pineal gland during daytime, whereas it pontentiates the production of melatonin when the gland is noradrenergically stimulated either by isoproterenol administration or by the endogenously released norepinephrine at nighttime.     

Decreases in pineal melatonin content during the hibernation bout in the golden-mantled group squirrel, Spermophilus lateralis

The role of the pineal gland in modulating the rhythmic bouts of hibernation in the golden-mantled ground squirrel (S. lateralis) was explored by comparing pineal melatonin content in hibernating animals with that of euthermic animals at the same time of year. Significant decreases in pineal melatonin content were found in hibernating versus euthermic animals. In addition, significantly lower values for pineal melatonin were observed in hibernating animals that were sacrificed in the late bout period, just prior to expected spontaneous arousal, as compared to hibernating animals that were sacrificed on the first day of their respective bouts. A strong correlation was evident between pineal melatonin content and the duration of the individual hibernation bout. These data suggest that pineal melatonin may be important in determining the duration of individual bouts of hibernation in this species.     

The profile of melatonin production in tumour-bearing rats

The pineal gland is involved in the regulation of tumour growth through the anticancer activity of melatonin, which presents immunomodulatory, anti-proliferative and anti-oxidant effects. In this study we measured melatonin content directly in the pineal gland, in an attempt to clarify the modulation of pineal melatonin secretory activity during tumour growth. Different groups of Walker 256 carcinosarcoma bearing rats were sacrificed at 12 different time points during 24h (12h:12h light/dark cycle) on different days during the tumour development (on the first, seventh and fourteenth day after tumour inoculation). Melatonin content in the pineal gland was determined by high-performance liquid chromatography with electrochemical detection. During tumour development the amount of melatonin secreted increased from 310.9 ng/mg of protein per day from control animals, to 918.1 ng/mg of protein per day 14 days after tumour implantation, and there were changes in the pineal production profile of melatonin. Cultured pineal glands obtained from tumour-bearing rats turned out to be less responsive to noradrenaline, suggesting the existence, in vivo, of putative factor(s) modulating pineal melatonin production. The results demonstrated that during tumour development there is a modification of pineal melatonin production daily profile, possibly contributing to cachexia, associated to changes in pineal gland response to noradrenaline stimulation.     

Effects of diazepam and its metabolites on nocturnal melatonin secretion in the rat pineal and Harderian glands. A comparative in vivo and in vitro study

We investigated the effects of diazepam (DZP) and its three metabolites: nordiazepam (NZP), oxazepam (OZP), and temazepam (TZP) on pineal gland nocturnal melatonin secretion. We looked at the effects of benzodiazepines on pineal gland melatonin secretion both in vitro (using organ perifusion) and in vivo in male Wistar rats sacrificed in the middle of the dark phase. We also examined the effects of these benzodiazepines on in vivo melatonin secretion in the Harderian glands. Neither DZP (10^-5-10^-6 M) nor its metabolites (10^-4-10^-5 M) affected melatonin secretion by perifused rat pineal glands in vitro. In contrast, a 10^-4 M suprapharmacological concentration of DZP increased melatonin secretion of perifused pineal glands by 70%. In vivo, a single acute subcutaneous administration of DZP (3 mg/kg body weight) significantly affected pineal melatonin synthesis and plasma melatonin levels, while administration of the metabolites under the same conditions did not. DZP reduced pineal melatonin content (-40%), N-acetyltransferase activity (-70%), and plasma melatonin levels (-40%), but had no affects on pineal hydroxyindole-O-methyltransferase activity. Neither DZP nor its metabolites affected Harderian gland melatonin content. Our results indicate that the in vivo inhibitory effect of DZP on melatonin synthesis is not due to the metabolism of DZP. The results also show that the control of melatonin production in the Harderian glands differs from that observed in the pineal gland.     

Studies on the development of growth hormone and prolactin cells in the rat pituitary gland by in situ hybridization

Summary An antiserum raised against N-amino-3-propyl melatonin bound to a protein carrier was used to visualize melatonin by immunohistochemistry and to measure melatonin concentration by radioimmunoassay in the pineal gland of intact mink females killed throughout the 24 h cycle and females killed after a bilateral ablation of the cervical superior ganglion. Melatonin immunoreactivity revealed by immunofluorescence or by the peroxidase-antiperoxidase complex was observed in the cytoplasm of presumed pinealocytes of all the females. Circadian changes in pineal melatonin content were not visualized by immunohistochemistry; furthermore, immunoreactivity was also present in the pineal gland of the ganglionectomized females. However, the melatonin content measured by radioimmunoassay was significantly higher in the pineal gland from intact females killed during the night compared with that of intact females killed during the day or of ganglionectomized females. The discrepancy between the results obtained using the two methods may arise because immunohistochemistry can detect very small amounts of melatonin.     

Determination of pineal melatonin by precolumn derivatization reversed-phase high-performance liquid chromatography and its application to the study of circadian rhythm in rats and mice

Determination of minute amounts of endogenous melatonin in rat and mouse pineal gland was performed using an RP-HPLC system. Melatonin was separated following precolumn derivatization and determined with a fluorescence detector at the emission wavelength of 380 nm with the excitation at 245 nm. The calibration curve of melatonin constructed by adding known amounts of melatonin to the homogenates of mouse pineal gland was linear over the range of 1-500 fmol (injection amount/20 microl). The detection limit of added melatonin was 1 fmol (S/N = 5). Repeatability and day-to-day precision for the melatonin spiked sample of mouse pineal gland was 4.0 and 3.8% (RSD), respectively. Using the present method, circadian changes of melatonin content in rat (Wistar) and mouse (C3H) pineal gland were determined. In addition, a minute amount of melatonin in ddY mouse pineal gland was determined, because pineal melatonin of many inbred mouse strains has been reported to be lower than the detection limit.     

Neuroendocrine effects of light

The light/dark cycle to which animals, and possibly humans, are exposed has a major impact on their physiology. The mechanisms whereby specific tissues respond to the light/dark cycle involve the pineal hormone melatonin. The pineal gland, an end organ of the visual system in mammals, produces the hormone melatonin only at night, at which time it is released into the blood. The duration of elevated nightly melatonin provides every tissue with information about the time of day and time of year (in animals that are kept under naturally changing photoperiods). Besides its release in a circadian mode, melatonin is also discharged in a pulsatile manner; the physiological significance, if any, of pulsatile melatonin release remains unknown. The exposure of animals including man to light at night rapidly depresses pineal melatonin synthesis and, therefore, blood melatonin levels drop precipitously. The brightness of light at night required to depress melatonin production is highly species specific. In general, the pineal gland of nocturnally active mammals, which possess rod-dominated retinas, is more sensitive to inhibition by light than is the pineal gland of diurnally active animals (with cone-dominated retinas). Because of the ability of the light/dark cycle to determine melatonin production, the photoperiod is capable of influencing the function of a variety of endocrine and non-endocrine organs. Indeed, melatonin is a ubiquitously acting pineal hormone with its effects on the neuroendocrine system having been most thoroughly investigated. Thus, in nonhuman photoperiodic mammals melatonin regulates seasonal reproduction; in humans also, the indole has been implicated in the control of reproductive physiology.Summary of a Plenary Lecture presented by the author in Vienna, August, 1990     

Adrenal-mediated depression of N-acetyltransferase activity and melatonin levels in the rat pineal gland

N-acetyltransferase (NAT) is believed to be the rate-limiting enzyme in the synthesis of melatonin from serotonin in the pineal gland. Norepinephrine released from sympathetic nerve endings within the pineal gland stimulates NAT activity and, therefore, melatonin synthesis. When an animal is subjected to a stressful stimulus, it would be expected that the increase in plasma stimulus, it would be expected that the increase in plasma catecholamines originating from the adrenal medulla and/or the sympathetic nervous system would result in a stimulation of pineal NAT activity. Adult male rats were given a 1.5cc injection of physiological saline subcutaneously into the back leg. Compared to non-injected controls, animals stressed in this manner were shown to have significantly lower pineal melatonin content 10 min after the saline injection late in the light phase of the light/dark cycle (at 18.30 h-lights on at 07.00 h). To test this more thoroughly, a time course study was conducted during the dark phase (at 02.00 h-5 hours after lights out) when pineal NAT activity and melatonin levels are either increasing or elevated. NAT activity and melatonin levels in the pineal were significantly depressed in stressed animals as compared to controls by 10 min after the saline injection, and remained so until 60 min after injection. By 90 min they had returned to control values. In the next study the nighttime response of the pineal to stress was compared in intact and adrenalectomized rats. Adrenalectomy prevented the changes in NAT activity and melatonin content associated with the saline injection. Some factor, such as a catecholamine or corticosterone from the adrenal, seems to be eliciting the response in the pineal to the saline injection. It is not known if the factor is acting centrally or directly on the pineal gland.     

Pinealectomy-induced elevation of collagen content in the intact skin is suppressed by melatonin application

The pineal gland is involved in wound repair and collagen deposition in sponge-induced granulomas. The aim of this investigation was to discover whether the pineal gland was able to regulate collagen accumulation in the intact skin. Wistar rats were divided into five groups: control, sham-operated with vehicle application, sham-operated with melatonin injections (30 micrograms/100 g body wt), pinealectomized with vehicle, and pinealectomized with melatonin supplementation. After 8 weeks, the collagen content was estimated as hydroxyproline concentration in the dry tissue of the skin. The results showed that melatonin markedly (p < 0.001) reduced collagen accumulation in the skin. Pinealectomy enhanced collagen deposition in the skin (p < 0.02) and melatonin application reduced the pinealectomy-induced elevation of collagen content (p < 0.001). Results clearly indicate that collagen accumulation in the intact skin is under the control of the pineal gland, and that melatonin, the pineal hormone, is responsible for this control.     

Daily oscillation in melatonin synthesis in the Turkey pineal gland and retina: diurnal and circadian rhythms

The aim of the present study was to examine arylalkylamine N-acetyltransferase (AANAT) activity and melatonin content in the pineal gland and retina as well as the melatonin concentration in plasma of the turkey (Meleagris gallopavo), an avian species in which several physiological processes, including reproduction, are controlled by day length. In order to investigate whether the analyzed parameters display diurnal or circadian rhythmicity, we measured these variables in tissues isolated at regular time intervals from birds kept either under a regular light-dark (LD) cycle or under constant darkness (DD). The pineal gland and retina of the turkey rhythmically produced melatonin. In birds kept under a daily LD cycle, melatonin levels in the pineal gland and retina were high during the dark phase and low during the light phase. Rhythmic oscillations in melatonin, with high night-time concentrations, were also found in the plasma. The pineal and retinal melatonin rhythms mirrored oscillations in the activity of AANAT, the penultimate enzyme in the melatonin biosynthetic pathway. Rhythmic oscillations in AANAT activity in the turkey pineal gland and retina were circadian in nature, as they persisted under conditions of constant darkness (DD). Transferring birds from LD into DD, however, resulted in a potent decline in the amplitude of the AANAT rhythm from the first day of DD. On the sixth day of DD, pineal AANAT activity was still markedly higher during the subjective dark than during the subjective light phase; whereas, AANAT activity in the retina did not exhibit significant oscillations. The results indicate that melatonin rhythmicity in the turkey pineal gland and retina is regulated both by light and the endogenous circadian clock. The findings suggest that environmental light may be of primary importance in the maintenance of the high-amplitude melatonin rhythms in the turkey.     

Daily Oscillation in Melatonin Synthesis in The Turkey Pineal Gland and Retina: Diurnal and Circadian Rhythms

The aim of the present study was to examine arylalkylamine N‐acetyltransferase (AANAT) activity and melatonin content in the pineal gland and retina as well as the melatonin concentration in plasma of the turkey (Meleagris gallopavo), an avian species in which several physiological processes, including reproduction, are controlled by day length. In order to investigate whether the analyzed parameters display diurnal or circadian rhythmicity, we measured these variables in tissues isolated at regular time intervals from birds kept either under a regular light‐dark (LD) cycle or under constant darkness (DD). The pineal gland and retina of the turkey rhythmically produced melatonin. In birds kept under a daily LD cycle, melatonin levels in the pineal gland and retina were high during the dark phase and low during the light phase. Rhythmic oscillations in melatonin, with high night‐time concentrations, were also found in the plasma. The pineal and retinal melatonin rhythms mirrored oscillations in the activity of AANAT, the penultimate enzyme in the melatonin biosynthetic pathway. Rhythmic oscillations in AANAT activity in the turkey pineal gland and retina were circadian in nature, as they persisted under conditions of constant darkness (DD). Transferring birds from LD into DD, however, resulted in a potent decline in the amplitude of the AANAT rhythm from the first day of DD. On the sixth day of DD, pineal AANAT activity was still markedly higher during the subjective dark than during the subjective light phase; whereas, AANAT activity in the retina did not exhibit significant oscillations. The results indicate that melatonin rhythmicity in the turkey pineal gland and retina is regulated both by light and the endogenous circadian clock. The findings suggest that environmental light may be of primary importance in the maintenance of the high‐amplitude melatonin rhythms in the turkey.     

Pineal sensitivity to nighttime swimming stress changes during the active season in Richardson's ground squirrels (Spermophilus richardsonii)

Melatonin synthesis in the pineal gland, which is primarily regulated by the environmental lighting regime, can also be influenced by other factors that elicit modifications in sympathetic tone. The objectives of this study were to determine if forced swimming alters the normal pattern of melatonin production in the pineal gland of the Richardson's ground squirrel (Spermophilus richardsonii). In early June, the squirrels were forced to swim for 10 min during the photophase or during the scotophase. In mid-July squirrels swam only during the scotophase. Animals were sacrificed 15, 30, or 60 min after the onset of swimming. Activities of pineal N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) were assessed by radioenzyme assay, and pineal melatonin content was measured by radioimmunoassay. Daytime swimming elicited no major changes in enzyme activity or pineal melatonin. In June, swimming at night prevented the normal rises in NAT activity and pineal melatonin seen in nonswimming controls. In contrast, the pineals of squirrels that were tested 6 weeks later in mid-July did not appear to be as sensitive to nighttime swimming, as there were only minor differences in both NAT activity and melatonin content compared to controls. These results demonstrate that forced nighttime swimming, unlike several other aversive stimuli, can evoke changes in the normal pattern of pineal melatonin production in this species. Furthermore, the pineal's response to such stimuli may not be stable over the course of the active season.     

Swimming-induced suppression of rat pineal melatonin is prevented by pretreatment with calcium channel blockers

Young adult male rats were treated with isoproterenol during the day to induce high levels of pineal N-acetyltransferase (NAT) activity and melatonin. Roughly 2 hr later when pineal NAT activity and melatonin levels were elevated, animals were given either an injection of a calcium channel blocker, i.e., either nifedipine or verapamil, or diluent. The rats were then forced to swim for 10 min in room temperature (22 degrees C) water. Fifteen minutes after swimming onset, pineal glands were collected for measurement of NAT activity and melatonin. Swimming caused a dramatic reduction in pineal melatonin content without influencing NAT activity. Nifedipine substantially and verapamil completely blocked the drop in pineal melatonin levels due to swimming without influencing NAT activity. The results suggest that calcium may be somehow directly or indirectly involved in melatonin release from the rat pineal gland.     

Factors other than Light Affecting the Pineal Gland: Hypophysectomy, Testosterone, Dihydrotestosterone, Estradiol, Cryptorchidism, and Stress

This article reviews those factors other than light that affectthe activity of the pineal gland. Both testosterone and dihydrotestosteronewere shown to have tissue specific inhibitory effects on pinealMAO activity concomitant with an increased activity of the gland.Estradiol stimulated pineal MAO activity and decreased the activityof this gland. This effect also was tissue-specific. Bilateraland unilateral experimental cryptorchidism also decreased pinealMAO activity 3 to 4 weeks after surgery. Acute stresses appearto increase adrenal catecholamine output (epinephrine and norepinephrine)as well as to stimulate local adrenergic pathways, while chronicstress, such as starvation, appears to act through the adrenalcorticosteroids by decreasing pineal MAO activity thereby indirectlyincreasing melatonin synthesis. Thusly, both components of theadrenal gland appear to act in concert to increase effectivelymelatonin synthesis by the pineal gland. Irradiation and histaminehave also been reported to affect pineal function—thelatter specifically inhibits HIOMT activity. These observationsindicate that many factors other than light affect pineal morphologyand melatonin synthesis. The pineal appears to be a true neuroendocrineorgan that is affected by hypophysectomy and is responsive tofeedback and control from other organs within the mammalianorganism.     

Glia-pinealocyte network: the paracrine modulation of melatonin synthesis by tumor necrosis factor (TNF)

The pineal gland, a circumventricular organ, plays an integrative role in defense responses. The injury-induced suppression of the pineal gland hormone, melatonin, which is triggered by darkness, allows the mounting of innate immune responses. We have previously shown that cultured pineal glands, which express toll-like receptor 4 (TLR4) and tumor necrosis factor receptor 1 (TNFR1), produce TNF when challenged with lipopolysaccharide (LPS). Here our aim was to evaluate which cells present in the pineal gland, astrocytes, microglia or pinealocytes produced TNF, in order to understand the interaction between pineal activity, melatonin production and immune function. Cultured pineal glands or pinealocytes were stimulated with LPS. TNF content was measured using an enzyme-linked immunosorbent assay. TLR4 and TNFR1 expression were analyzed by confocal microscopy. Microglial morphology was analyzed by immunohistochemistry. In the present study, we show that although the main cell types of the pineal gland (pinealocytes, astrocytes and microglia) express TLR4, the production of TNF induced by LPS is mediated by microglia. This effect is due to activation of the nuclear factor kappa B (NF-kB) pathway. In addition, we observed that LPS activates microglia and modulates the expression of TNFR1 in pinealocytes. As TNF has been shown to amplify and prolong inflammatory responses, its production by pineal microglia suggests a glia-pinealocyte network that regulates melatonin output. The current study demonstrates the molecular and cellular basis for understanding how melatonin synthesis is regulated during an innate immune response, thus our results reinforce the role of the pineal gland as sensor of immune status.     

Pineal melatonin synthesis in Syrian hamsters: A summary

During the past decade there has been ample documentation of the proposition that the pineal gland mediates photoperiodic influences upon reproductive behavior of hamsters. It is commonly hypothesized that the pineal gland expresses its activity by transformation of photoperiodic information into an hormonal output, that hormone being melatonin. If this hypothesis is correct, there must be some essential diffrence in melatonin's output when hamsters are exposed to different photoperiodic environments. The experiments summarized in this communication analyze pineal melatonin contents in Syrian hamsters maintained in a variety of photoperiodic conditions during different physiological states. The results demonstrate that adult hamsters have a daily surge in pineal melatonin content throughout their lifetime when exposed to simulated annual photoperiodic cycles. There is some fluctuation in the amount of pineal melatonin produced during different physiological states and photoperiodic environments, but these fluctuations seem small when compared to those normally found for other regulatory hormones. When hamsters are exposed to different photoperiodic regimens, the daily melatonin surge maintains a relatively constant phase relationship with respect to the onset of daily activity. There is a concomitant change in its phase relationship with respect to light-dark transitions.Presented at the Ninth International Congress of Biometeorology, 23 September–1 October 1981, Osnabrück and Stuttgart-Hohenheim, Federal Republic of Germany.     

Hormonal modulation of pineal melatonin synthesis in rats and Syrian hamsters: effects of streptozotocin-induced diabetes and insulin injections

Pineal levels of tryptophan, 5-hydroxytryptophan, serotonin, N-acetylserotonin, melatonin, 5-hydroxyindoleacetic acid and the enzyme activities of N-acetyltransferase and hydroxyindole-O-methyltransferase were determined in male albino rats and Syrian hamsters that were injected with insulin twice daily for three days, or injected with streptozotocin to induce diabetes. Neither insulin injections nor streptozotocin diabetes had any effect on pineal melatonin production in rats. In hamsters, diabetes reduced the nocturnal peak of pineal melatonin content by approximately one half, while insulin injections had no effect on pineal melatonin levels; however, insulin injections did cause a slight increase in pineal N-acetyltransferase activity. These findings indicate that the pineal gland of the hamster may be more sensitive to alterations in plasma insulin levels than the same organ in rats.     

Tryptophan hydroxylase is modulated by L-type calcium channels in the rat pineal gland

Calcium is an important second messenger in the rat pineal gland, as well as cAMP. They both contribute to melatonin synthesis mediated by the three main enzymes of the melatonin synthesis pathway: tryptophan hydroxylase, arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase. The cytosolic calcium is elevated in pinealocytes following alpha(1)-adrenergic stimulation, through IP(3)-and membrane calcium channels activation. Nifedipine, an L-type calcium channel blocker, reduces melatonin synthesis in rat pineal glands in vitro. With the purpose of investigating the mechanisms involved in melatonin synthesis regulation by the L-type calcium channel, we studied the effects of nifedipine on noradrenergic stimulated cultured rat pineal glands. Tryptophan hydroxylase, arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase activities were quantified by radiometric assays and 5-hydroxytryptophan, serotonin, N-acetylserotonin and melatonin contents were quantified by HPLC with electrochemical detection. The data showed that calcium influx blockaded by nifedipine caused a decrease in tryptophan hydroxylase activity, but did not change either arylalkylamine N-acetyltransferase or hydroxyindole-O-methyltransferase activities. Moreover, there was a reduction of 5-hydroxytryptophan, serotonin, N-acetylserotonin and melatonin intracellular content, as well as a reduction of serotonin and melatonin secretion. Thus, it seems that the calcium influx through L-type high voltage-activated calcium channels is essential for the full activation of tryptophan hydroxylase leading to melatonin synthesis in the pineal gland.     

Ex vivo studies on the acute and chronic effects of DHEA and DHEA-sulfate on melatonin synthesis in young- and old-rat pineal glands

This study investigates the effects of acute and chronic injections of the neurosteroid dehydroepiandrosterone (DHEA) and its sulfate DHEA-S on pineal gland melatonin synthesis. Pineal melatonin production and plasma melatonin levels were investigated in young (9-week-old) and old (27-month-old) male Wistar rats. DHEA or DHEA-S have been administered acutely in a single intraperitoneal injection at a dosage of 50, 250, or 500 microg per animal, or on a long-term basis, i.e., for 8 days at a dosage of 100 microg per animal, 1 h before the onset of darkness. DHEA, at a dose of 50, 250, or 500 microg per animal, administered acutely to rats had no significant effects on pineal melatonin production whatever the age of the animals. In contrast, 500 microg DHEA-S induced a significant increase in the pineal melatonin content (15% in young animals and 35% in old animals) and the activity of N-acetyltransferase, the rate-limiting enzyme for melatonin synthesis in the pineal gland, (40% in young animals and 20% in old animals), without altering the activity of hydroxyindole-O-methyltransferase whatever the age of the animals. At lower concentrations (50 or 250 microg) DHEA-S had no effect on pineal melatonin production regardless of the age of the rats. Chronic injection of DHEA or DHEA-S at a dose of 100 microg had no effect on pineal melatonin or NAT and HIOMT activities in the two age groups. This work shows that DHEA-S (and not DHEA) is able, at pharmacological concentrations, to stimulate melatonin production by rat pineal glands regardless of the age of the animals.     

Vitamin A deficiency reduces the responsiveness of pineal gland to light in Japanese quail (Coturnix japonica)

Synthesis of melatonin in pineal gland is under the control of light environment. The recent finding of the presence of rhodopsin-like photopigment (pinopsin) and retinal in the avian pinealocytes has led to a hypothesis that vitamin A is involved in photoresponses of the pineal gland. We have thus analyzed the effect of vitamin A deficiency on the regulatory system of melatonin synthesis in the pineal gland of Japanese quail. Depletion of vitamin A from Japanese quails was attained by feeding them with a vitamin A-free diet supplemented with retinoic acid. In the vitamin A-deficient birds, diurnal rhythm in melatonin production persisted such that the phase of the wave was similar to that seen in the control birds. However, the amplitude of the nighttime surge of pineal melatonin was damped by vitamin A deficiency. When the control birds were briefly exposed to light at night, pineal melatonin dropped to the daytime level. In contrast, only slight decrease was observed in the vitamin A-deficient quails. The light responsiveness was restored after feeding the vitamin A-deficient quails with the control diet for 1 week. These results indicate that vitamin A plays essential roles in maintaining sufficient responsiveness of the avian pineal gland to photic input.