An (n-3) polyunsaturated fatty acid-deficient diet disturbs daily locomotor activity, melatonin rhythm, and striatal dopamine in Syrian hamsters.

Several studies suggest that (n-3) PUFA may play a role in the regulation of cognitive functions, locomotor and exploratory activity, and affective disorders. Additionally, (n-3) PUFA affect pineal function, which is implicated in the sleep-wake rhythm. However, no studies to our knowledge have explored the role of PUFA on the circadian system. We investigated the effect of an (n-3) PUFA-deficient diet on locomotor and pineal melatonin rhythms in Syrian hamsters used as model species in circadian rhythm research. To assess the possible relationship between voluntary wheel running activity and dopaminergic neurotransmission, we also measured endogenous monoamine concentrations in the striatum. Two-month-old male hamsters, fed either an (n-3) PUFA-deficient or an (n-3) PUFA-adequate diet, were housed individually in cages equipped with run wheels. At 3 mo, cerebral structures were extracted for biochemical and cellular analysis. In (n-3) PUFA-deficient hamsters, the induced changes in the pineal PUFA membrane phospholipid composition were associated with a reduction in the nocturnal peak level of melatonin that was 52% lower than in control hamsters (P < 0.001). The (n-3) PUFA-deficient hamsters also had higher diurnal (P < 0.01) and nocturnal (P = 0.001) locomotor activity than the control hamsters, in parallel with activation of striatal dopaminergic function (P < 0.05). The (n-3) PUFA-deficient hamsters exhibited several symptoms: chronic locomotor hyperactivity, disturbance in melatonin rhythm, and striatal hyperdopaminergia. We suggest that an (n-3) PUFA-deficient diet lessens the melatonin rhythm, weakens endogenous functioning of the circadian clock, and plays a role in nocturnal sleep disturbances as described in attention deficit/hyperactivity disorder.

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.

Melatonin induces Cry1 expression in the pars tuberalis of the rat.

In mammals, interacting transcriptional/post-translational feedback loops involving 'clock genes' and their protein products control circadian organisation. These genes are not only expressed in the master circadian clock of the suprachiasmatic nuclei (SCN) but also in many peripheral tissues where they exhibit similar but not identical dynamic to that seen in the SCN. Among these peripheral tissues, the pars tuberalis (PT) of the pituitary expresses clock genes. We show here that the PT of the rat, like that of other rodents, rhythmically expresses Per1. We also report rhythmic expression of another clock gene, Cry1. The peak of Cry1 mRNA expression occurs during the night concomitantly with rising blood plasma melatonin concentrations. Using an acute injection paradigm, we demonstrate that Cry1 expression is directly induced by melatonin in the PT. Melatonin injection at the end of the subjective day also affects Per1 expression, leading to diminished mRNA levels. These data support the existence of a time-measurement model in the PT based on direct opposite actions of melatonin on Per1 and Cry1 expression.

Testosterone-dependent and -independent mechanisms involved in the photoperiodic control of neuropeptide levels in the brain of the jerboa (Jaculus orientalis).

Vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) content in the suprachiasmatic nucleus have been shown to exhibit seasonal changes with an increase in late summer, the period of sexual quiescence in the jerboa (Jaculus orientalis). In this study, VIP content in the SCN and NPY and enkephaline (ENK) content in the geniculo-suprachiasmatic system have been assayed in wild-caught male jerboas (Jaculus orientalis) in order to determine whether these neuropeptides are controlled directly by photoperiod changes or indirectly by short photoperiod induced changes in circulating sex hormones levels. In agreement with previous studies seasonal variations occur in the VIP and NPY content in the SCN. Variations also occur in NPY content in the IGL with an increase in the period of sexual quiescence. In contrast, no seasonal changes are observed in Enk content in the IGL or the SCN. In short photoperiod conditions increases are observed in both VIP and NPY content in the SCN as well as NPY content in the IGL. Castration during the period of sexual activity (spring) or under long photoperiod which drastically reduces testosterone, also induced an increase in the levels of these neuropeptides. Testosterone implants which reproduce the sex hormonal status of the sexual activity period failed to prevent the short photoperiod-induced increase of VIP and NPY in the SCN and of NPY in the IGL. These results clearly show that the photoperiod modulates VIP and NPY in the geniculo-suprachiasmatic system both by testosterone-linked and testosterone-independent mechanisms.

The relationship between melatonin and dopamine rhythms in the duck retina.

In the retina of duck, levels of dopamine (DA) and its main metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), fluctuate throughout the day, with high values during the light phase. The rhythmic changes in DA content and metabolism are out of phase with the daily oscillations in melatonin (MEL) and serotonin N-acetyltransferase (AA-NAT; the penultimate and key regulatory enzyme in MEL biosynthesis) activity. Acute exposure of ducks to light at night potently increased levels of DA and DOPAC, and decreased AA-NAT activity and MEL content in the retina. Intraocular administration of MEL to light-adapted ducks produced a significant decline in retinal DA and DOPAC concentrations. On the other hand, quinpirole, a D(2)/D(4)-DA receptor agonist, administered intraocularly, markedly suppressed the night-time retinal AA-NAT activity and MEL. These findings provide, for the first time, evidence for an inverse relationship between the DA system and MEL in the duck retina.

Cyclic melatonin synchronizes the circadian rhythm of feeding activity in Japanese quail, Coturnix c. japonica.

In Japanese quail, we can observe the circadian rhythm of feeding activity in constant conditions, especially in birds from selected lines. In order to try to test the importance of melatonin as hormonal output for the circadian system, we gave a 24-h period cycle of exogenous melatonin to some of these birds when they were free running. We used castrated males firstly in order to cancel the known effect of steroids on circadian organisation. Secondly, as castrated birds generally expressed a very short periodicity, it allowed us to check induced synchronisation more easily. We maintained ten castrated males in constant dim light. We divided the experiment into five successive phases. The birds received a 24-h period cycle of melatonin (M phase) or of control solution with only the alcoholic solvent (C phase) as a drink. Before and after each one of these two phases, we gave water continually to drink (W1, W2 and W3 phases). Thus, the successive phases were W1-M-W2-C-W3. We measured intake of liquids and plasma melatonin concentrations to check melatonin ingestion. We automatically recorded individual feeding activity by infrared detectors, and analysed this by spectral analysis. At the beginning of the experiment, eight birds showed a rhythmic feeding activity, with a mean period of 22.9 +/- 0.2 h, and the two others an arrhythmic circadian activity. During the 24-h period cycle of exogenous melatonin, for the rhythmic birds, the circadian period became approximately 24 h (23.9 +/- 0.2 h), the inactive phase corresponding to the period of melatonin availability. During the W2 and C phases, the circadian period was similar to that expressed during the W1 phase. Moreover, when birds only drink water, we found a significant positive relationship between the clarity of the circadian rhythm and the ratio, between the melatonin level of the inactive phase and that of the active phase. These facts support the hypothesis of the role of this hormone in the regulation of the circadian system, at least for feeding activity, in quail.

Radioimmunoassay of N-acetyl-N-formyl-5-methoxykynuramine (AFMK): a melatonin oxidative metabolite.

N-acetyl-N-formyl-5-methoxykynuramine (AFMK) is a melatonin metabolite identified in rat brain by Hirata et al. (The Journal of Biological Chemistry 249 (1974) 1311). Since no assay has been described for its routine measurement, we have developed and validated such a radioimmunoassay. We synthesized AFMK and N-acetyl-5-methoxykynuramine (AMK), in order to produce anti-AFMK antibodies and to standardize the assay. The tracer [3H]-AFMK was obtained from [3H]-melatonin. The assay was preceded by a chromatographic step on Celite microcolumn in order to increase its specificity. The assay was suitable for the measurement of AFMK levels ranging from 59 to 1894 pmol/L. The detection limit of the assay was routinely set at 65 pmol/L. The intra- and inter-assay coefficients of variation were 3.5% and 11% respectively. Investigation of the 24 h plasma pattern in healthy volunteers did not reveal any AFMK levels in plasma samples. In rats, plasma AFMK showed a peak after melatonin injection, which confirmed the in vivo AFMK production as a melatonin metabolite. This AFMK assay is suitable for studies on melatonin metabolism.

Seasonal variations of clock gene expression in the suprachiasmatic nuclei and pars tuberalis of the European hamster (Cricetus cricetus).

In mammals, day length (photoperiod) is read and encoded in the main circadian clock, the suprachiasmatic nuclei (SCN). In turn, the SCN control the seasonal rhythmicity of various physiological processes, in particular the secretion pattern of the pineal hormone melatonin. This hormone then operates as an essential mediator for the control of seasonal physiological functions on some tissues, especially the pars tuberalis (PT). In the European hamster, both hormonal (melatonin) and behavioral (locomotor activity) rhythms are strongly affected by season, making this species an interesting model to investigate the impact of the seasonal variations of the environment. The direct (on SCN) and indirect (via melatonin on PT) effect of natural short and long photoperiod was investigated on the daily expression of clock genes, these being expressed in both tissues. In the SCN, photoperiod altered the expression of all clock genes studied. In short photoperiod, whereas Clock mRNA levels were reduced, Bmal1 expression became arrhythmic, probably resulting in the observed dramatic reduction in the rhythm of Avp expression. In the PT, Per1 and Rev-erbalpha expressions were anchored to dawn in both photoperiods. The daily profiles of Cry1 mRNA were not concordant with the daily variations in plasma melatonin although we confirmed that Cry1 expression is regulated by an acute melatonin injection in the hamster PT. The putative role of such seasonal-dependent changes in clock gene expression on the control of seasonal functions is discussed.

Diurnal and circadian rhythms in melatonin synthesis in the turkey pineal gland and retina.

The pineal gland and retina of the turkey rhythmically produce melatonin. In birds kept under a daily light-dark (LD) illumination cycle melatonin concentrations in the pineal gland and retina were low during the light phase and high during the dark phase. A similar melatonin rhythm with high night-time values was also observed in the plasma. The pineal and retinal melatonin rhythms mirror oscillations in the activity of serotonin N-acetyltransferase (AANAT; the penultimate enzyme in the melatonin biosynthetic pathway). In contrast, in both the pineal gland and retina the activity of the enzyme hydroxyindole-O-methyltransferase (HIOMT) did not exhibit significant changes throughout the 24-h period. Acute exposure of turkeys to light at night dramatically decreased melatonin levels in the pineal gland, retina and plasma. The rhythms in AANAT activity and melatonin concentrations in the turkey pineal gland and retina were circadian in nature as they persisted under conditions of constant darkness (DD). Under DD, however, the amplitudes of AANAT and melatonin rhythms were significantly lower (by 50-80%) than those found under the LD cycle. The findings indicate that melatonin rhythmicity in the turkey pineal gland and retina is regulated both by light and the endogenous circadian clock. The rapid dampening of the rhythms under DD suggests that of these two regulatory factors, environmental light may be the primary stimulus in the maintenance of the high amplitude melatonin rhythms in the turkey.

Environmental control and adrenergic regulation of pineal activity in the diurnal tropical rodent, Arvicanthis ansorgei.

Like nocturnal rodents, the diurnal tropical rodent Arvicanthis ansorgei shows a daily rhythm in pineal melatonin content. Seasonal and photoperiodic variations in the biosynthetic activity of the pineal gland: arylalkylamine-N-acetyltransferase (AA-NAT), hydroxyindole-O-methyltransferase (HIOMT) activities and melatonin content were measured in male and female A. ansorgei captured near Samaya, Mali, and kept either under artificial laboratory photoperiods [light-dark (LD) cycles: LD 14:10, LD 12:12 or LD 10:14 or caught in the field in Mali and killed at four different times of the year (January, April, June and November). Under artificial photoperiod, the duration of the nocturnal peak of AA-NAT activity and melatonin content increased with the duration of the dark period while the amplitude did not significantly change. In the field, annual variations in the amplitude of the nocturnal melatonin peak were observed with a maximum in April (highest temperature, low humidity and no grass availability, only seeds) and a minimum in November (high humidity, maximum green grass availability). The variations in the amplitude of the melatonin peak were not correlated with changes in AA-NAT HIOMT activities, suggesting that seasonal variations in the amplitude of the melatonin peak are not driven by these enzymes. Daytime injections of the beta-adrenergic agonist, isoproterenol, stimulated melatonin synthesis in January, April and June, but not in November. The annual differences in the amplitude of the melatonin peak as well as the seasonal differences in the response to an adrenergic stimulation suggest that environmental factors other than photoperiod, such as temperature, humidity and consequent food availability, could be important in the regulation of the annual variations in the pineal biosynthetic activity in this species.

Mechanisms regulating the marked seasonal variation in melatonin synthesis in the European hamster pineal gland.

Like many wild species, the European hamster (Cricetus cricetus) adapts to the marked seasonal changes in its environment, namely by hibernation and inhibition of sexual activity in winter. These annual functions are driven by the variation in the environmental factors (light, temperature) that are transmitted to the body through large variations in the duration and amplitude of the nocturnal melatonin rhythm. Here we report that the seasonal variation in melatonin synthesis is mainly driven by arylalkylamine N-acetyltransferase gene transcription and enzyme activation. This, however, does not exclude participation of hydroxyindole-O-methyltransferase, which may relay environmental temperature information. The in vivo experiments show that norepinephrine stimulates melatonin synthesis, this effect being gated at night. The possibility that the variation in pineal metabolism depends on a seasonal change in the suprachiasmatic nuclei clock circadian activity that is transmitted by norepinephrine is discussed.

Pineal arylalkylamine N-acetyltransferase gene expression is highly stimulated at night in the diurnal rodent, Arvicanthis ansorgei.

The different mechanisms underlying the control of diurnal vs. nocturnal activity are still unknown. Regarding the nocturnal synthesis of the pineal hormone, melatonin, experiments performed on diurnal sheep or bovine and on nocturnal rat or hamster revealed important differences in the regulation of the melatonin rate-limiting enzyme, arylalkylamine N-acetyltransferase (AA-NAT). These observations raised the hypothesis that melatonin synthesis may be different in nocturnal vs. diurnal animals. In this study, we cloned the cDNA coding for Aa-nat and analysed the mechanisms of AA-NAT enzyme activation in the pineal gland of the diurnal grass rat, Arvicanthis ansorgei, and compared them to those of the nocturnal Wistar rat, Rattus norvegicus. Aa-nat gene sequences of both species are 86.6% identical. In Arvicanthis, Aa-nat gene expression is markedly increased at the beginning of the night and is followed by a large increase in AA-NAT activity and melatonin content. In contrast, at the end of the night, the decrease in AA-NAT activity and melatonin content precedes that of Aa-nat mRNA. A beta-adrenergic agonist given at daytime reproduces the nocturnal activation of melatonin synthesis, whereas, a beta-adrenergic antagonist given at night-time inhibits AA-NAT activity and melatonin synthesis independently of Aa-nat mRNA. The day-night regulation of melatonin synthesis in the pineal of the diurnal Arvicanthis, involving a transcriptional activation in early night and a post-translational inhibition at late night, is very similar to that of the nocturnal Wistar rat. In conclusion, the fundamental differences underlying melatonin synthesis among species rely upon phylogenetic rather than behavioural differences.

Suppression of melatonin biosynthesis in the chicken pineal gland by retinally perceived light - involvement of D1-dopamine receptors.

In this study the role of retinal dopamine (DA) receptors in the light-induced suppression of melatonin biosynthesis in the chicken pineal gland was examined. Exposure of dark-adapted chickens to low intensity light (4 lux) at night significantly decreased the activity of serotonin N-acetyltransferase (AA-NAT; the penultimate and key regulatory enzyme in melatonin production) and melatonin content in the pineal gland. This suppressive action of light was blocked by intraocular (i.oc.) administration of SCH 23390 (a selective antagonist of D1-DA receptors), but was not affected by sulpiride (a selective antagonist of D2-DA receptors). Injection of DA (i.oc.) to dark-adapted chickens significantly decreased pineal AA-NAT activity and melatonin content in a dose- and time-dependent manner. The action of DA was mimicked by selective agonists of D1-DA receptors, SKF 38393 and SKF 81297, and non-hydrolyzable analogs of cyclic AMP (cAMP), dibutyryl-cAMP and 8-bromo-cAMP. However, i.oc. administration of quinpirole, a selective agonist of D2-DA receptors, did not modify pineal AA-NAT activity. In contrast, quinpirole potently decreased nocturnal AA-NAT activity in the retina. Systemic administration of SCH 23390 to chickens blocked the i.oc. DA-evoked decline in nighttime pineal AA-NAT activity, whereas sulpiride was ineffective. These findings indicate that light activation of retinal dopaminergic neurotransmission, with concomitant stimulation of D1-DA receptors positively coupled to the cAMP generating system, plays an important role in a cascade of events regulating pineal activity.

In the rat, exogenous melatonin increases the amplitude of pineal melatonin secretion by a direct action on the circadian clock.

The effect of exogenous melatonin on pineal melatonin synthesis was studied in the rat in vivo. Daily melatonin profiles were measured by transpineal microdialysis over 4 consecutive days in rats maintained on a 12-h light : 12-h dark schedule (LD 12 : 12). Curve-fitting was used to determine the amplitude of the peak of melatonin production, and the times of its onset (IT50) and offset (DT50). A subcutaneous injection of melatonin (1 mg/kg) at the onset of darkness (ZT12) induced an advance of IT50 on the second day after the treatment, in 50% of the animals kept in LD. When the animals were switched to constant darkness, the treatment caused no detectable advance of IT50, while 70% of individuals showed a significant delay in DT50 2 days after the injection. Locally infusing the drug by reverse microdialysis into the suprachiasmatic nuclei (SCN) failed to enhance the shift in melatonin onset. Following subcutaneous melatonin injection, a significant increase ( approximately 100%) in melatonin peak amplitude was observed. This increase persisted over 2 days and occurred only when the melatonin was applied at ZT12, but not at ZT6, 17 or 22. The effect was also observed when the drug was infused directly into the SCN, but not into the pineal. Thus, the SCN are the target site for the effect of exogenous melatonin on the amplitude of the endogenous melatonin rhythm, with a similar window of sensitivity as its phase-shifting effect on the pacemaker.

Daily variation in the concentration of melatonin and 5-methoxytryptophol in the goose pineal gland, retina, and plasma.

The goose pineal gland rhythmically produces two 5-methoxyindole compounds, namely melatonin and 5-methoxytryptophol. Melatonin concentrations were high at night and low during the day, while in contrast 5-methoxytryptophol levels were markedly higher during the day compared to the night-time values. Rhythmic oscillations in melatonin content, with high night-time values, have also been found in plasma and the retina of goose. The pineal and retinal melatonin rhythm mirrored oscillations in the activity of serotonin N-acetyltransferase (AA-NAT; the penultimate and key regulatory enzyme in the melatonin biosynthetic pathway). Acute exposure of geese to light at night markedly decreased melatonin levels in the pineal, plasma, and retina. In addition, this light exposure resulted in a significant increase in pineal 5-methoxytryptophol content. Our results demonstrate, for the first time, the ability of the goose pineal gland and retina to synthesise melatonin and 5-methoxytryptophol in a rhythmic manner.

Daily variation in the concentration of 5-methoxytryptophol and melatonin in the duck pineal gland and plasma.

The duck pineal gland rhythmically produces two 5-methoxyindole compounds, i.e. 5-methoxytryptophol and melatonin. 5-Methoxytryptophol levels are low at night and high during the day, while melatonin concentrations are high at night and low during the day. The melatonin rhythm reflects oscillations in the activity of serotonin N-acetyltransferase (AA-NAT; a penultimate and key regulatory enzyme in the melatonin biosynthetic pathway). The activity of hydroxyindole-O-methyltransferase (HIOMT; an enzyme involved in the synthesis of both 5-methoxytryptophol and melatonin) does not exhibit any significant rhythmic changes throughout the 24-hr period. Plasma levels of melatonin exhibited daily changes that were parallel to fluctuations in pineal melatonin content. Although plasma concentrations of 5-methoxytryptophol were low in ducks, they showed daily variations. The mean 5-methoxytryptophol concentration between zeitgeber time 9 (ZT9) and ZT15 was 2.4-times higher than the mean value for samples collected between ZT18 and ZT3. These findings indicate that in the duck the pineal production of 5-methoxytryptophol and melatonin may be inversely correlated.