Photoperiod-dependent changes in melatonin synthesis in the turkey pineal gland and retina.

The effect of photoperiod on melatonin content and the activity of the melatonin-synthesizing enzymes, namely, serotonin N-acetyltransferase (AANAT) and hydroxyindole-O-methyltransferase, were investigated in the pineal gland and retina of turkeys. The birds were adapted to 3 different lighting conditions: 16L:8D (long photoperiod), 12L:12D (regular photoperiod), and 8L:16D (short photoperiod). Pineal, retinal, and plasma melatonin concentrations oscillated with a robust diurnal rhythm, with high values during darkness. The duration of elevated nocturnal melatonin levels in the turkey pineal gland, retina, and plasma changed markedly in response to the length of the dark phase, being longest during the short photoperiod with 16 h of darkness. These photoperiodic variations in melatonin synthesis appear to be driven by AANAT, because changes in the activity of this enzyme were closely correlated with changes in melatonin. By contrast, pineal and retinal hydroxyindole-O-methyltransferase activities failed to exhibit any significant 24-h variation in the different photoperiods. A marked effect of photoperiod on the level of melatonin production was also observed. Peak values of melatonin and AANAT activity in the pineal gland (but not in the retina) were highest during the long photoperiod. During the light phase, mean melatonin concentrations in the pineal gland and retina of turkeys kept under the long photoperiod were significantly higher compared with those from birds maintained under the regular and short photoperiods. In addition, mean circulating melatonin levels were lowest in the short photoperiod. Finally, the magnitude of the light-evoked suppression of nighttime pineal AANAT activity was also influenced by photoperiod, with suppression being smallest under the long photoperiod. These findings show that in the turkey, photoperiod plays an important role in regulating the melatonin signal.

Phase-advanced daily rhythms of melatonin, body temperature, and locomotor activity in food-restricted rats fed during daytime.

This study was performed to investigate possible effects of a timed caloric restriction on the light-dark (LD) synchronization of four biological rhythms pair-studied in the same animals. In Experiment 1, food-restricted rats kept under a photoperiod of 12 h light:12 h dark received 50% of previous ad libitum food 2 h after the onset of light. Their daily rhythm of pineal melatonin and rhythms of plasma melatonin and corticosterone were examined and compared to those of ad libitum control rats after 1 or 2 months of food restriction. A significant phase advance (about 2 h) was found for the pineal melatonin rhythm and for the daily onset of plasma melatonin. Timing of nocturnal peak of circulating corticosterone was unchanged, and a diurnal peak anticipated food presentation by about 2 h. In Experiment 2, effects of a timed caloric restriction under 12L:12D were studied on the expression of daily rhythms of body temperature and locomotor activity. To discriminate between the effects of timed meal feeding and those of the added caloric restriction, these rhythms were analyzed in food-restricted rats, as in Experiment 1, and were compared to those in sham-restricted rats, concomitantly fed twice more than food-restricted rats (i.e., a timed meal feeding without caloric restriction). Acrophase of the nocturnal peak of body temperature rhythm reached the greatest phase advance (7 h) in food-restricted rats, in which it was close to LD transition. The nocturnal component of locomotor activity rhythm also was markedly phase advanced (6 h) by caloric restriction, as indicated by wheel-running and general activity occurring form early afternoon to midnight. A smaller 4-h phase advance of the nocturnal peak of body temperature also was observed in sham-restricted rats, although the onset of locomotor activity rhythm apparently was unaffected by meal feeding and the end of activity rhythm was phase advanced by 2 h. These results indicate that timed caloric restriction is a potent phase-shifting agent that interacts with the LD cycle zeitgeber. This nonphotic stimulus phase advances melatonin, corticosterone, body temperature, and activity rhythms to different extents and thus suggests a change in the internal synchronization of the circadian system.

Daily variations in pineal melatonin concentrations in inbred and outbred mice.

Melatonin was measured using a specific radioimmunoassay in 1 strain of outbred mice (OF1 Swiss) and 4 strains of inbred mice, 2 of them being known to synthesize melatonin (CBA and C3H) and the 2 others being controversial (BALB/c and C57BL/6). In this study, the 5 mouse strains were able to synthesize melatonin, but the basal levels as well as the diurnal variations were very different from one strain to another. CBA and C3H strains showed a clear-cut day-night rhythm of pineal melatonin concentration, with peak levels of 276 +/- 22 pg/pineal in CBA and 135 +/- 12 pg/pineal in C3H. In BALB/c, the authors confirmed the presence of a very short melatonin peak (15 min) in the middle of the dark period. In C57BL/6 and OF1 Swiss, a very small but significant peak was observed in the middle of the darkness. In the former, another small peak was also observed at light onset. Whether these very small peaks, which may be related to the deficience of N-acetyl transferase activity reported by others, have a physiological meaning remains to be determined.

Daily and photoperiodic melatonin binding changes in the suprachiasmatic nuclei, paraventricular thalamic nuclei, and pars tuberalis of the female Siberian hamster (Phodopus sungorus).

Using quantitative autoradiography, 2-(125)I-melatonin binding was investigated throughout the light:dark cycle in the suprachiasmatic nuclei (SCN), paraventricular nuclei (PVT), and pars tuberalis (PT) of adult female Siberian hamsters kept for 10 weeks in either long or short photoperiods (LP or SP, respectively). Plasma melatonin concentrations were measured by radioimmunoassay, and the sexual status of the animals was established by visual inspection of vaginal smears and by weighing uteri after sacrifice. The SCN displayed neither daily nor photoperiod-dependent variations in specific binding. Melatonin receptors in these nuclei would be regulated neither by plasma melatonin nor by the light:dark cycle or sexual steroids. By contrast, melatonin receptor density in the PT displayed both strong daily (maximal values during the first half of the light period and minimal values during the night) and photoperiod-dependent (maximal values in LP) variations. These variations dependent on changes in the maximal binding (Bmax) without differences in the dissociation constant (Kd). Daily melatonin receptor densities in the PT of LP- and SP-exposed animals might be regulated by the dark:light transition but not by melatonin. Daily profiles of 2-(125)I-melatonin-specific binding in the PT were independent of photoperiod. Factors underlying the photoperiod-related variations presently are unknown. Concerning the PVT, weak variations in specific binding were detected in SP only when time points were grouped according to the light or dark periods. It is not yet possible to conclude whether they have any physiological relevance. These results show clearly that the regulation of melatonin receptors varies among structures (SCN, PVT, and PT) in the Siberian hamster and is also totally different from that found in the rat.

Effect of different photoperiods on concentrations of 5-methoxytryptophol and melatonin in the pineal gland of the Syrian hamster.

Specific, sensitive and direct radioimmunoassays have been used to determine the daily patterns of 5-methoxytryptophol (ML) and melatonin in the pineal glands of Syrian hamsters kept in different photoperiods: 8 h light: 16 h darkness (8L:16D), 14L:10D and 16L:8D. A rhythm in pineal ML was evident in animals in all the photoperiods, with high daytime levels (641 +/- 35 (S.E.M.) fmol/gland; n = 162) which dropped to 119 +/- 16 fmol/gland (n = 44) 7.1-7.5 h after lights out. The duration of low night-time ML levels was proportional to the length of the dark phase (1.2 h in 16L:8D, 5.4 h in 14L:10D and 8.4 h in 8L:16D). A marked daily rhythm in melatonin was also present in hamsters in the different photoperiods, with daytime levels of 323 +/- 34 fmol/gland (n = 129) and night-time peak concentrations of 3676 +/- 336 fmol/gland (n = 22). The duration of high nocturnal melatonin levels was dependent upon the length of the dark phase (4.1 h in 16L:8D, 4.5 h in 14L:10D and 12.5 h in 8L:16D). Linear regression analysis revealed a statistically significant inverse relationship between pineal ML and melatonin levels in 8L:16D (P less than 0.001), 14L:10D normal (P less than 0.05) and 14L:10D shifted (P less than 0.001) photoperiods. After advancing the lighting schedule by 10 h (14L:10D, lights off at 04.00 h), pineal ML and melatonin rhythms became entrained to the new lighting regimen. The daily rhythms in pineal ML and melatonin in the Syrian hamster thus depend on the prevailing photoperiod, a reciprocal relationship existing between pineal ML and melatonin concentrations.

The effect of 5-methoxytryptamine on golden hamster gonads is not a consequence of its acetylation into melatonin.

Radioimmunoassay and high performance liquid chromatography were used to determine if the gonadal atrophy induced by late afternoon injections of 5-methoxytryptamine (5-MT) in golden hamsters kept under long photoperiod could be due to the acetylation of this compound into melatonin. An increase in plasma concentrations of melatonin (10-13 nmol/l) was detected 15 min after injection of 130 nmol 5-MT. An injection of 4.3 nmol melatonin generated a similar plasma concentration of melatonin. 5-MT (130 nmol) and melatonin (4.3 nmol) were then injected daily in the late afternoon to golden hamsters kept under long photoperiod. After 8 weeks, 5-MT induced total testicular regression, while melatonin induced partial atrophy only. Thus under these experimental conditions, 5-MT had a physiological activity independent of that of melatonin.

How does the indoleamine production of the pineal gland respond to variations of the environment in a non-mammalian vertebrate, Testudo hermanni Gmelin?

The relative effects of environmental factors, photoperiod and temperature on pineal indoleamine production have been investigated in male Tortoises, Testudo hermanni, at different times of their seasonal reproductive cycle. Photoperiod appears to be an important factor in the regulation of the seasonal and diurnal variations of pineal serotonin content, while temperature has a more drastic effect on the diurnal production of melatonin. These results reinforce, at least partly, the concept that the pineal gland responds to several kinds of information about the state of the environment in order to synchronize some cyclical functions such as reproduction.

Effect of NMDA receptor antagonist MK-801 on light-induced Fos expression in the suprachiasmatic nuclei and on melatonin production in the Syrian hamster.

In mammals, circadian rhythms generated by the suprachiasmatic nuclei (SCN) are daily synchronized by a light-dark cycle. Photic information is transmitted to the SCN mainly through the direct retinohypothalamic tract, the neurotransmitters involved being excitatory amino acids. It is also commonly accepted that photoperiodic information coming from the retina via the SCN is transduced by the pineal into a nocturnal signal, i.e. melatonin production. Light exposure at night induces (1) an inhibition of melatonin synthesis and (2) an expression of c-fos in numerous cells of SCN. To determine the role of the NMDA receptor in these effects, we treated Syrian hamsters with ip injections of MK-801, a noncompetitive NMDA receptor antagonist. Several subpopulations of light-sensitive cells in the SCN are affected by MK-801. According to previous studies, MK-801 inhibits light-induced Fos immunoreactivity mainly in the most ventral part of the SCN. However, we observed that numerous other cells are still activated by light. When light is applied in the middle of the night, MK-801 pretreatment does not reduce Fos-ir in the dorsal SCN. At the beginning of the night, labeled cells in this part of the nucleus appear even more numerous after MK-801. We also found that MK-801 fails to reduce the light-induced inhibition of melatonin synthesis. Moreover, in control animals, which received no light stimulation, ip injection of MK-801 induces by itself a dose-dependent inhibition of melatonin production.

The photoperiodic response in Syrian hamster depends upon a melatonin-driven circadian rhythm of sensitivity to melatonin.

The pineal gland, via the daily pattern of melatonin (MEL) secretion, is directly involved in the conduction of photoperiodic information. The duration of MEL secretion is proportional to the duration of the dark period and, whatever the photoperiod is, MEL synthesis occurs 3 or 4 h after the dark onset in Syrian hamsters. In order to determine the relative importance of the duration or the coincidence hypothesis, a daily infusion protocol was used in sexually active pinealectomized hamsters. Long duration of MEL infusion (10 h) completely inhibit testes whereas short duration infusion (5 h) had no effect. When the animals were infused twice within 2 h 30 min separated by 3 h, they presented a complete gonadal atrophy, similar to the one observed with the 10 h infusion. Measurement of plasma MEL during the infusion and separation periods revealed that MEL reached physiological nighttime values during the infusion period and fell to daytime values 1 h after the end of an infusion period. Thus, the results could not be due to a time additive action of the two MEL pulses. An intermediate response was observed when the 2 signals were applied across the light/dark transition. Gonadal regression did not occur when the 2 periods of infusion were separated by 5 h 30 min. The efficiency of this type of infusion was not dependent on the ambiant photoperiod since similar results were obtained in long and short photoperiods. The infusion was also as effective during the day as well as during the night. These results suggest that there is a rhythm of sensitivity to MEL, based on the coincidence hypotheses, that are important for transmission of photoperiodic information. This rhythm of sensitivity to MEL seems to be entrained by MEL itself, since the efficiency of the two pulses of MEL is not dependent of time of application and/or of photoperiod.

Potentiation effect of vasopressin on melatonin secretion as determined by trans-pineal microdialysis in the Rat.

The mammalian pineal gland is known to receive a noradrenergic innervation originating from the superior cervical ganglion which corresponds to the primary regulatory input for melatonin synthesis. However, many peptidergic fibers containing peptides such as vasopressin and oxytocin have also been found in the rat pineal gland. The present study was performed to investigate the possible role of vasopressin and oxytocin on melatonin secretion in vivo. Therefore, both neuropeptides were delivered for 2 h through a trans-pineal microdialysis probe directly into the gland at different times during the nocturnal phase of the light:dark cycle. At the same time pineal dialysates were collected continuously. Melatonin concentrations were measured by radioimmunoassay. Melatonin synthesis potentiation was achieved when vasopressin was infused locally in the pineal, during the onset of nocturnal melatonin secretion. In order to assess the possible role of a physiological increase of endogenous circulating vasopressin on pineal metabolism, melatonin synthesis was recorded in the same animals before and after a prolonged dehydration period. Night time melatonin concentration was increased after the water deprivation vs control conditions. Contrary to that, oxytocin seems not to affect pineal metabolism in the rat since no significant change was observed on melatonin secretion in response to a local oxytocin infusion. These results show that vasopressin can modulate melatonin synthesis in the rat pineal whereas no effect was obtained with oxytocin, at least under the present experimental conditions.

Sexual dimorphism of lipids in Harderian glands of golden hamsters.

The Harderian gland of golden hamsters excretes alkyldiacylglycerol (ADG), the fatty acid and alkyl compositions of which differ between males and females. ADG in males contains mostly straight chain fatty acids, even- and odd-numbered, the major one being 15:0, while ADG in females contains iso- and anteiso-branched chain acids (34.0%). Iso-branching was found in both even- and odd-numbered acids, but anteiso-branching was found mostly in odd-numbered acids. The presence of propionic acid at the 3 position of the glycerol moiety in male ADG, and of isovaleric and 2-methylbutyric acids at the same position in female ADG was demonstrated by NMR spectrometry. Alkyl portions also exhibited sexual dimorphism in these lipids. ADG from males consisted of straight aliphatic chains, but branched chain components occupied almost half (45%) in ADG from females, and the branching resided at the iso- and anteiso-positions. The ADGs in glands from the two sexes were separated by Iatrobeads column chromatography into three and two subfractions, respectively. The fatty acid and alkyl compositions of these subfractions coincided with the above-mentioned results and with the behavior of the ADGs on thin-layer plates. These findings suggest that a sex hormone affects the metabolism of valine, leucine and isoleucine, and sexual dimorphism of ADGs occurs in the Harderian gland.

Sexual diversity of the lipid metabolism in the Harderian gland of the golden hamster.

Fatty acid and alkyl compositions of 1-alkyl-2,3-diacylglycerols (ADG) in the Harderian gland of the golden hamster were determined. Fatty acids of male ADG consisted of straight chain saturated acids ranging from C12 to C22. Both even- and odd-numbered acids were observed, indicating that acetyl- and propionyl-CoAs were equally used as primers in the fatty acid synthesis. In female ADG a large amount of iso- and anteiso-branched fatty acids were detected. Odd-numbered acids (C15, 17, 19, and 21) contained iso- together with anteiso-branchings, and even-numbered acids (C16 and 18) contained iso-branchings. These findings suggested that isobutyryl-, isovaleryl-, and 2-methylbutyryl-CoAs were used as primers in addition to acetyl- and propionyl-CoAs in fatty acid synthesis in the female gland. Such unusual primers are catabolic intermediates of valine, leucine, and isoleucine, respectively. Male ADG contained straight chain saturated alkyl residues ranging from C14 to 21. In the case of female ADG, though the main component was C18:O, most alkyl components had branchings at the iso- and anteiso-positions. NMR data suggested the presence of propionic acid at the C3 position of glycerol in the male ADG and of isovaleric and 2-methylbutyric acids at the same position in the female ADG. These results suggest that the sexual differences of the lipids in the Harderian gland of the golden hamster are determined at the step of fatty acid synthesis depending on the available precursors in the male and female glands. We speculate that sex hormones affect the metabolism of branched chain amino acids and bring about the dramatic diversities of the lipid compositions in the male and female glands.

Dopamine and melatonin interactions in the intact chicken eye. Electrooculographic and biochemical study.

Electrophysiological and biochemical techniques were used to investigate the interactions between dopamine (DA) and melatonin (MEL) in the intact chicken eye. Endogenous DA depletion induced by intraocular administration of alpha-methyl-para-tyrosine (alpha-MPT), a selective tyrosine hydroxylase inhibitor, decreases the transepithelial potential (TEP) of the retinal pigment epithelium and reduces the light peak (LP) recorded by an indirect electro-oculographic (EOG) method. An intraocular injection of MEL also reduces the TEP but does not reduce the LP. Retinal MEL is increased after endogenous DA depletion and a tight inverse correlation between DA and MEL contents was found. The present data, together with other findings support the hypothesis (1) that in the intact chicken eye, DA and MEL play respectively a role of light and dark signals on the TEP, and (2) that a balance between these two neurohormones may be responsible for the regulation of RPE events which are dependent on light-dark conditions.

Daily variation in the concentration of melatonin and 5-methoxytryptophol in the human pineal gland: effect of age and Alzheimer's disease.

Melatonin and 5-methoxytryptophol (ML) were measured in human pineals (38 controls, 16 subjects with Alzheimer's disease). Time of death had a major influence on the indole concentrations with significantly higher melatonin levels occurring at night (22.00-10.00 h) and significantly higher ML levels occurring during the day (10.00-22.00 h). This daily variation disappeared in both the older subjects (55-92 years) and in the Alzheimer patients (55-89 years).

Seasonal variation in vasopressin innervation in the brain of the European hamster (Cricetus cricetus).

Using immunocytochemistry, vasopressin innervation was determined in the brain of the European hamster (Cricetus cricetus) during different seasons. It was found that the spring period coincides with a dense vasopressin innervation in many brain regions in the male hamster, and lower vasopressin fibre density in some brain regions in the female hamster. In autumn just before hibernation an almost complete disappearance of vasopressin innervation is noted in those brain regions that are sexually dimorphically innervated in spring. These results suggest that vasopressin activity in certain areas of the brain might be required for some seasonal functions to find expression.

5-Methoxytryptophol injections in the Syrian hamster: plasma and pineal concentrations.

Plasma and pineal 5-methoxytryptophol (ML) levels were determined by radioimmunoassay (RIA) following subcutaneous (s.c.) injection of ML (1, 25 and 100 micrograms) to hamsters, Mesocricetus auratus. A dose-dependent increase in plasma ML occurred, with the duration of the supra-physiological levels being related to the dose. However, only the 100 micrograms dose significantly increased pineal ML levels above control values. Plasma profiles after 1 or 25 micrograms ML fitted a one-compartment model with half-lives of 18.6 and 25.2 min, respectively. Following 100 micrograms ML a biphasic decay curve was evident, the half-lives of the 2 phases being 6.6 min and 2.95 h.

Pineal 5-methoxytryptophol rhythms in the box turtle: effect of photoperiod and environmental temperature.

The effect of different photoperiods and temperatures on pineal 5-methoxytryptophol (ML) content was investigated in male box turtles, Terrapene carolina triunguis. A rhythm in pineal ML was evident in the long photoperiod (18 h light (L)-6 h dark (D] with high daytime levels of 178 +/- 48 pg/gland (means +/- S.E.M.) which dropped to 38 +/- 6 pg/gland during lights off. In the short photoperiod (8L:16D) no clearcut ML rhythm was observed. Diurnal (10.00-12.00 h) ML concentrations rose linearly (P less than 0.05) with increasing ambient temperatures (5, 15, 20 and 27 degrees C). Day/night differences in ML levels, however, were not significant. Pineal ML in the box turtle thus seems to be modified by the photoperiod and, to a lesser extent, by temperature.

Identification of melatonin in the compound eyes of an insect, the locust (Locusta migratoria), by radioimmunoassay and gas chromatography-mass spectrometry.

Melatonin, a well-known pineal substance implicated in conveying photoperiodic information in vertebrates, appears also to be present in the compound eyes of an insect, the locust. The identification of melatonin in the eyes of an invertebrate suggests that it may be an evolutionary conservative molecule, principally involved in the time transduction of photoperiodic information in all living organisms.

Adenosine analogs elevate N-acetylserotonin and melatonin in rat pineal gland.

Adenosine, S-adenosyl-L-homocysteine, adenosine analogs such as 5'-N-ethylcarboxamidoadenosine and mioflazine, a nucleoside transport inhibitor, injected intraperitoneally at 09.00 h during the light period increased melatonin levels in the pineal gland of the rat. The largest increase occurred with 1 mg/kg 5'-N-ethylcarboxamidoadenosine. A representative time-response curve with 5'-N-ethylcarboxamidoadenosine (1 mg/kg) showed a maximal peak of N-acetylserotonin and melatonin 2 and 4 h after injection, respectively. These results are discussed in relation with the possible modulation through A2 receptors of melatonin synthesis in the pineal gland.

[Circadian and circannuel fluctuations of pineal metatonin content in Testudo hermanni G. (Reptilia-Chelonia) under natural conditions of photoperiod and temperature (author's transl)]. / Variations circadiennes et circannuelles de la mélatonine épiphysaire chez Testudo hermani G. (reptile-chelonien) dans des conditions naturelles d'éclairement et de température.

Pineal content of melatonin is measured by RIA. The tortoise, Testudo hermanni, in natural lighting, shows peak night-time melatonin levels during summer (may to september) at the period of greatest activity. During hibernation, melatonin levels are very low and no circadian variation can be detected.