Melatonin affects nuclear orphan receptors mRNA in the rat suprachiasmatic nuclei.

The pineal hormone melatonin nocturnal synthesis feeds back on the suprachiasmatic nuclei (SCN), the central circadian clock. Indeed, daily melatonin injections in free-running rats resynchronize their locomotor activity to 24 h. However, the molecular mechanisms underlying this chronobiotic effect of the hormone are poorly understood. The endogenous circadian machinery involves positive and negative transcriptional feedback loops implicating different genes (particularly period (Per) 1-3, Clock, Bmal1, cryptochrome (Cry) 1-2). While CLOCK:BMAL1 heterodimer activates the rhythmic transcription of per and cry genes, the PER and CRY proteins inhibit the CLOCK:BMAL1 complex. In previous studies, we observed that the immediate resetting effect of a melatonin injection at the end of the subjective day on the SCN circadian activity did not directly involve the above-mentioned clock genes. Recently, nuclear orphan receptors (NORs) have been presented as functional links between the regulatory loops of the molecular clock. These NORs bind to a retinoic acid receptor-related orphan receptor response element (RORE) domain and activate (RORalpha) or repress (REV-ERBalpha) bmal1 expression. In this study, we investigated whether melatonin exerts its chronobiotic effects through transcriptional regulation of these transcription factors. We monitored roralpha, rorbeta and rev-erbalpha messenger RNA (mRNA) expression levels by quantitative in situ hybridization, up to 36 h following a melatonin injection at circadian time (CT) 11.5. Results clearly showed that, while roralpha was not affected by melatonin, the hormone partially prevented the decrease of the rorbeta mRNA expression observed in control animals during the first hours following the injection. The major result is that the rev-erbalpha mRNA expression rhythm was 1.3+/-0.8-h phase-advanced in melatonin-treated animals during the first subjective night following the melatonin administration. Moreover, the bmal1 mRNA expression was 1.9+/-0.9-h phase-shifted in the second subjective night following the melatonin injection. These results clearly suggest that the NOR genes could be the link between the chronobiotic action of melatonin and the core of the molecular circadian clock.

Melatonin in the multi-oscillatory mammalian circadian world.

In mammals, the complex interaction of neural, hormonal, and behavioral outputs from the suprachiasmatic nucleus (SCN) drives circadian expression of events, either directly or through coordination of the timing of peripheral oscillators. Melatonin, one of the endocrine output signals of the clock, provides the organism with circadian information and can be considered as an endogenous synchronizer, able to stabilize and reinforce circadian rhythms and to maintain their mutual phase-relationship at the different levels of the circadian network. Moreover, exogenous melatonin, through an action on the circadian clock, affects all levels of the circadian network. The molecular mechanisms underlying this chronobiotic effect have also been investigated in rats. REV-ERB alpha seems to be the initial molecular target.

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.

Seasonal changes in melatonin binding sites in the pars tuberalis of male European hamsters and the effect of testosterone manipulation.

Seasonal changes in specific 2-[125I]iodo-melatonin binding were assessed in the pars tuberalis of male European hamsters (Cricetus cricetus) kept under natural environmental conditions. Saturation studies were performed on pars tuberalis membrane preparations every 5-7 weeks for 13 months. The maximum number of melatonin binding sites occurred in spring and summer (Bmax, 4.0 +/- 0.3 fmol/mg protein, n = 9) with significantly reduced binding in winter (Bmax, 1.2 +/- 0.2 fmol/mg protein, n = 2). As an assay control, simultaneous saturation studies were performed on rat pars tuberalis membranes. No summer-winter difference in binding site density was observed in the rat studies. In both hamsters and rats no seasonal changes in binding affinity were found. Although an apparent correlation between Bmax, values and paired testes weight of the hamsters was noted, in early spring the increase in the number of melatonin binding sites preceded the elevation in testicular weight. Castration during the period of sexual activity or sexual inactivity also failed to reduce the elevated number of binding sites observed in the spring and summer. In addition, testosterone implanted sc 5 weeks before had no effect on the low 2-[125I]iodo-melatonin binding site density found in winter (November). These results suggest that the observed seasonal variation in the number of melatonin binding sites is independent of seasonal changes in circulating testosterone concentrations.

Oxytocinergic innervation of the brain of the garden dormouse (Eliomys quercinus L.).

The oxytocinergic innervation of the brain of the garden dormouse (Eliomys quercinus L.) was studied by means of immunocytochemistry. In contrast to the sparse oxytocin innervation of the rat forebrain, dense fibre networks in various cortical and limbic brain areas were demonstrated in this animal. These include, e.g., the prefrontal cortex, the claustrum, the septum, and the hippocampus. A very dense innervation was also seen in the caudal regions of the garden dormouse brain; these regions are already known to have a relatively dense oxytocin fibre network in the rat. A dense innervation of oxytocin fibres is seen in several brain regions which, in the rat, have oxytocin binding sites but no visible oxytocin innervation. This discrepancy suggests that the differences in the oxytocinergic innervation of these two rodent brains may be due to an oxytocin system in the rat brain that is more difficult to detect immunocytochemically.

Seasonal changes in vasopressin in the brain of the garden dormouse (Eliomys quercinus L.).

The distribution of vasopressin in the brain of the garden dormouse (Eliomys quercinus L.) was examined by immunocytochemistry at different times of the year. In spring-summer, sexual dimorphism in the density of vasopressin labeling was observed in several areas of the brain. In these regions, such as the lateral septum and the nucleus of the vertical limb of the diagonal band of Broca, male garden dormice showed more vasopressin immunoreactivity than their female counterparts. In autumn-winter, at the time of hibernation, vasopressin was undetectable in the sexually dimorphically innervated brain regions in males as well as females. In early spring, there was large variation in vasopressin staining in the male brain, whereas the female brain was consistently lacking in vasopressin labeling. In brain regions that exhibited no sexually dimorphic innervation in the summer, vasopressin labeling remained the same throughout the year. The difference in the amount of vasopressin immunoreactivity in males in summer and winter correlated significantly with differences in testes weight. In early spring, because of interindividual variations in the start of sexual activation, testes size does not correlate with the density of vasopressin immunoreactivity. Similar seasonal variations in density of vasopressin labeling in the brain were also observed in the European hamster (Cricetus cricetus). In this animal, central vasopressin infusion during the winter prevents hibernation. The presence of a similar pattern of changing vasopressin content in another hibernator, the garden dormouse, suggests an important role for this neuropeptide in seasonal functions.(ABSTRACT TRUNCATED AT 250 WORDS)

Contrary to other non-photic cues, acute melatonin injection does not induce immediate changes of clock gene mRNA expression in the rat suprachiasmatic nuclei.

The suprachiasmatic nuclei (SCN) contain the main clock of the mammalian circadian system. The endogenous oscillation machinery involves interactive positive and negative transcriptional and posttranslational feedback loops involving the clock genes Per1, Per2, Per3, Clock, Bmal1, Cry1 and Cry2. The SCN endogenous oscillation is entrained to 24 h by the light/dark cycle. Light induced regulation of Per1 and Per2 mRNA expression have been suggested to take part in the clock resetting. However, other factors have chronobiotic and synchronizing effects on SCN activity. Especially, the nocturnal pineal gland hormone, melatonin, which is involved in the regulation of both circadian and seasonal rhythms, is known to feedback on the SCN. Melatonin applied on SCN slices immediately phase-shifts their neuronal electrical activity, while daily injections of melatonin to free running rodents resynchronize their locomotor activity to 24 h. To determine whether melatonin feedback control on SCN activity implicates transcriptional regulation of the clock genes, we monitored the expression pattern of Per 1, 2, 3, Bmal1, Cry1 and AVP mRNAs after a single melatonin injection at the end of the subjective day. Results showed that melatonin injection affected none of the mRNA expression pattern during the first circadian night. Per1, Per3, Bmal1 and AVP expression patterns were, however, significantly but differentially affected, during the second subjective night after the melatonin injection. The present results strongly suggest that the immediate phase shifting effect of melatonin on the SCN molecular loop implicates rather post-translational than transcriptional mechanisms.

Photoperiod differentially regulates clock genes' expression in the suprachiasmatic nucleus of Syrian hamster.

The suprachiasmatic nuclei (SCN) contain the master circadian pacemaker in mammals. Generation and maintenance of circadian oscillations involve clock genes which interact to form transcriptional/translational loops and constitute the molecular basis of the clock. There is some evidence that the SCN clock can integrate variations in day length, i.e. photoperiod. However, the effects of photoperiod on clock-gene expression remain largely unknown. We here report the expression pattern of Period (Per) 1, Per2, Per3, Cryptochrome (Cry) 1, Cry2, Bmal1 and Clock genes in the SCN of Syrian hamsters when kept under long (LP) and short (SP) photoperiods. Our data show that photoperiod differentially affects the expression of all clock genes studied. Among the components of the negative limb of the feedback loop, Per1, Per2, Per3, Cry2 but not Cry1 genes show a shortened duration of their peak expression under SP compared with LP. Moreover, mRNA expression of Per1, Per3 and Cry1 are phase advanced in SP compared with LP. Per3 shows an mRNA peak of higher amplitude under SP conditions whereas Per1 and Per2 peak amplitudes are unaffected by photoperiod changes. Bmal1 expression is phase advanced without a change of duration in SP compared with LP. Furthermore, the expression of Clock is rhythmic under SP whereas no rhythm is observed under LP. These results, which provide further evidence that the core clock mechanisms of the SCN integrate photoperiod, are discussed in the context of the existing molecular model.

Failure of N-(2,4-dinitrophenyl)-5-methoxytryptamine (a putative melatonin antagonist) and of N-(3,5-dinitrophenyl)-5-methoxytryptamine to prevent the effects of injections of melatonin in the late afternoon on testicular activity of the golden hamster.

Daily injections of 10 micrograms melatonin in the late afternoon into male golden hamsters kept under a long photoperiod (14 h light: 10 h darkness) and at low ambient temperature 6 +/- 1 degrees C) induced a complete gonadal atrophy after 4 weeks. When administered under the same conditions at doses of 25 micrograms, neither N-(3,5-dinitrophenyl)-5-methoxytryptamine or N-(2,4-dinitrophenyl)-5-methoxytryptamine, a putative melatonin antagonist termed ML-23 in the literature, showed any effect on testicular activity. Moreover, these two drugs were also unable to prevent melatonin-induced gonadal atrophy when injected 30 min before melatonin. The results demonstrate that in the golden hamster and in the present experimental conditions, these drugs do not have the melatonin-antagonistic properties as described 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.

Cloning experiments and developmental expression of both melatonin receptor Mel1A mRNA and melatonin binding sites in the Syrian hamster suprachiasmatic nuclei.

The suprachiasmatic nuclei (SCN) are implicated in the control of circadian biological rhythms, and especially the melatonin nocturnal synthesis. In numerous rodents, melatonin has been shown to feed back on the SCN activity through high affinity receptors. In contrast, Syrian hamster SCN activity is unresponsive to melatonin injections. As this lack of effect could be linked to a developmental loss of SCN melatonin receptors, the goals of the present study were 1) to report in Syrian hamster SCN, and pars tuberalis (PT) as a control, a complete pattern of the postnatal (PN) development of the melatonin receptor density and 2) to investigate whether the regulation of the Mel1a mRNA expression could be implicated in the post natal variations of the melatonin binding capacities. We first subcloned by PCR a partial cDNA encoding the Mel1a receptor from Syrian hamster SCN. Subsequent quantification of Mel1a mRNA expression and melatonin receptor density revealed that in the PT and SCN, both Mel1a mRNA expression and melatonin binding capacities declined abruptly between PN 0 and PN 8. Afterwards, in the PT, both parameters went up until they got stabilized in adulthood. Therefore, in the PT, post natal melatonin receptor density variations were highly correlated with post natal variations of the Mel1a mRNA expression. In the SCN, after PN 8, the melatonin receptor density followed its drop and then declined by more than 92% between post natal day 0 (PN 0) and PN 60 (12.11+/-0. 27 vs. 0.94+/-0.08 fmol/mg protein at PN 0 and PN 60 respectively). In contrast, Mel1a mRNA expression only slightly went down after PN 8 and got stabilized in adult age at 42% of the birth day expression level. These results show that Syrian hamster SCN undergo a dramatic post natal loss of their melatonin receptors that could explain the lack of effect of melatonin injections on SCN circadian activity. Furthermore, this SCN binding capacities decline could not be attributed to an inhibition of the mRNA expression, but rather to a post transcriptional blockade of the Mel1a receptor expression.

Characterization of melatonin binding sites in the pars tuberalis of the European hamster.

Melatonin binding sites in the pars tuberalis of the European hamster (Cricetus cricetus) have been characterized using the radioligand 2-[(125) I]iodomelatonin. Specific 2-[(125) I]iodomelatonin binding was assessed using radioreceptor studies of pars tuberalis membrane preparations. Saturation studies revealed a single, high affinity site (K(d) 39.8 (± 7.6 SEM) pM and B(max) 4.1 (± 0.5 SEM) fmol/mg protein, n=4). Kinetic experiments showed the 2-[(125) I]iodomelatonin binding to be rapid, saturable and reversible. The K(d) calculated from the dissociation and association rate constants was 19.4 pM. The order of potency of different indoles for inhibition of 2-[(125) I]iodomelatonin binding was 6-chloromelatonin > melatonin > 6-hydroxymelatonin > N-acetylserotonin > 5-methoxytryptophol > serotonin > 5-methoxytryptamine. GTP caused a dose-dependent inhibition of the 2-[(125) I]iodomelatonin binding. A saturation study showed that GTP reduced the number of binding sites by a third without altering their affinity. These results imply the presence of a G-protein-coupled melatonin receptor in the pars tuberalis of sexually active European hamsters.

Effect of constant light, pinealectomy and guanosine triphosphate gamma-s on the density of melatonin receptors in the rat suprachiasmatic nucleus: a possible implication of melatonin action.

We report here the effects of pinealectomy and exposition to constant light on the density of melatonin receptors in the suprachiasmatic nuclei of the rat using quantitative autoradiography. The B(max) values were significantly increased when the animals were maintained in constant light for 3 days (8.22 ± 0.95 fmol/mg protein versus 4.55±0.14 fmol/mg protein in control group, 12 h light/12 h dark cycle (12L/12D), n = 6). A similar increase was also observed in rats pinealectomized 3 days before sacrifice and then maintained either under 12L/12D (B(max) 7.56±0.80 fmol/mg protein) or in constant light (B(max) 7.85±1.02 fmol/mg protein), while K(d) values failed to show any variations after constant light and/or pinealectomy. The effect of GTPγS on the density of rnelatonin binding sites was also investigated in control animals and after 3 days of constant light. In 12L/12D animals, the B(max) shifted from 5.94 ± 0.14 fmol/mg protein in the absence of GTPγS to 3.97±0.22 fmol/ mg protein in the presence of 50µiM GTPγS. In animals maintained for 3 days in constant light, a similar decrease in the B(max) value was observed (8.95 ± 0.25 fmol/mg protein in absence and 5.95 ± 0.22 fmol/mg protein in presence of 50 µ GTPγS). In both cases, K(d) values were not affected by GTPγS. Pinealectomy and constant light exposition are known to induce a suppression of the nocturnal peak of plasma rnelatonin and to keep plasma rnelatonin concentrations at a very low level. These results could suggest a regulatory effect of rnelatonin on the density of its own receptors which are shown here to be also coupled with a G-protein.

Regulation of melatonin receptors in the pars tuberalis of Syrian hamsters transferred from long to short photoperiod: implication of melatonin and testosterone.

The exposure of long day seasonal breeders to a short photoperiod (SP) induces both sexual quiescence and a decrease in pars tuberalis (PT) melatonin receptor density. Therefore, we studied the respective roles of melatonin and testosterone on the regulation of PT melatonin receptors in Syrian hamsters transferred from long photoperiod (LP) to SP. Compared with intact sexually active animals in LP, the density of melatonin receptors was not affected by the absence of melatonin after removal of the pineal gland from animals kept in either SP or LP. In contrast, the presence of a long melatonin peak in the blood which induces gonadal atrophy induced a significant decrease in binding capacity. The SP-induced decrease in PT melatonin receptor density was also observed in castrated animals showing that it was directly regulated by melatonin, independently of circulating testosterone concentrations. However, the absence of testosterone induced an increased binding in LP, while increasing blood testosterone concentration after implantation of one testosterone-filled silastic tube resulted in a decrease in binding both in LP-and in SP-animals. These results indicate that testosterone induces a photoperiod-independent decrease in PT melatonin receptor density. In summary, these results show that both melatonin and testosterone have negative regulatory effects on the density of PT melatonin receptors.

Brain and pituitary melatonin receptors in male rat during post-natal and pubertal development and the effect of pinealectomy and testosterone manipulation.

Using quantitative autoradiography, melatonin receptors have been studied during post-natal and pubertal development of the rat in 2 brain and 2 pituitary structures. In the pars distalis of anterior pituitary, melatonin receptors decrease gradually in density after birth and disappear in 30 day-old animals. In contrast melatonin binding is only expressed in the paraventricular nuclei of the thalamus at the age of 21-23 days and is always present in adult animals. In the suprachiasmatic nuclei and in the pars tuberalis of the pituitary, melatonin receptor density decreases after birth, remains stable for approximately 1 month and increases again at puberty to reach the birth values in the adult. This increase was absent in pinealectomized and in castrated animals but present in castrated animals receiving testosterone suggesting that it depends upon circulating testosterone and melatonin levels. These results show that melatonin receptors are differentially regulated during post-natal development in each of the 4 structures studied, and that melatonin and testosterone are 2 factors which could be involved in the regulation of melatonin receptor density in the suprachiasmatic nuclei and pars tuberalis.

Serotonergic modulation of photically induced increase in melatonin receptor density and Fos immunoreactivity in the suprachiasmatic nuclei of the rat.

The mammalian suprachiasmatic nuclei (SCN) contain a circadian clock which is regulated by neuronal photic and non-photic afferences. Among these, the serotonergic input originating from the dorsal raphe nucleus (DRN) is extremely important. In rats, a light pulse administered during the dark period is known to induce the expression of the immediate early gene c-fos and to increase melatonin receptor density in the SCN. The aim of this study was to assess whether, in rats, these two phenomena were regulated by serotonin, acting via 5-HT1A receptors. Three days after pinealectomy, 4 groups of rats were injected i.p. 90 min before sacrifice with respectively: (1) vehicle, (2) the 5-HT1A-agonist 8-OH-DPAT (5 mg/kg), (3) the 5-HT1A-antagonist NAN-190 (10 mg/kg) or (4) NAN-190 and the 8-OH-DPAT. Half of the animals from each group were exposed to light for 60 min before sacrifice and the other half remained in darkness. Sacrifice took place 5 to 6 h after lights off. Our results show that the antagonist NAN-190: (1) completely blocked the photically-induced increase of melatonin receptor density in the SCN, with an IC50 = 0.352 +/- 0.103 mg/kg, and (2) partially blocked (30%) the photic induction of Fos (the protein product of c-fos) in the ventrolateral subdivision of the SCN. The agonist 8-OH-DPAT enhanced the photically-induced increase of melatonin receptors by 10% and decreased the photically-induced increase in Fos by 18%. Both drugs were devoid of any effect in non-light exposed animals. From these results we may suggest that, in rats, there is a serotonergic control of the neuronal path driving photic information to the SCN. This regulation seems to occur through 5-HT1A or 5-HT1a-like receptors.

MT1 melatonin receptor mRNA expressing cells in the pars tuberalis of the European hamster: effect of photoperiod.

Melatonin, secreted only during the night by the pineal gland, transduces the photoperiodic message to the organism. One important target for the hormone is the pars tuberalis (PT) of the adenohypophysis which displays a very high number of melatonin binding sites in mammals and is implicated in the seasonal regulation of prolactin secretion. To gain insight into the mechanism by which the melatonin signal is decoded in the PT, we studied the effect of photoperiod on the PT cells expressing the MT1 melatonin receptor in a highly photoperiodic species, the European hamster. Recently, we showed that, in the rat, the MT1 receptor mRNA is expressed in PT-specific cells characterized by their expression of beta-thyroid stimulating hormone (beta-TSH) along with the alpha-glycoprotein subunit (alpha-GSU). As the cellular composition of the PT shows variability among species, we first identified the cell type expressing the MT1 receptor in the European hamster by combining immunocytochemistry and nonradioactive in situ hybridization for the MT1 receptor mRNA. Our results show that, in the European hamster, as in the rat, the MT1 receptor is only expressed by the PT-specific-cells, beta-TSH and alpha-GSU positive. In a second step, we analysed the effects of photoperiod on the MT1 mRNA, and on beta-TSH and alpha-GSU both at the mRNA and protein levels. Our data show that, compared to long photoperiod, short photoperiod induces a dramatic decrease of MT1, beta-TSH and alpha-GSU expression. Protein levels of beta-TSH and alpha-GSU were also dramatically reduced in short photoperiod. Together, our data suggest that melatonin exerts its seasonal effects in the PT by signalling to PT specific-cells through the MT1 receptor subtype.

Differential seasonal regulation of melatonin receptor density in the pars tuberalis and the suprachiasmatic nuclei: a study in the hedgehog (Erinaceus europaeus, L.).

Using quantitative autoradiography, we have studied the seasonal changes of high affinity melatonin receptor density in both the SCN and PT of the hedgehog, a seasonal breeder and an hibernator. Animals in 3 different physiological states were studied: sexually active animals, and sexually inactive animals during the hibernation period, being then either euthermic or hypothermic. In sexually active animals, Bmax were 75.8 +/- 7.1 fmol/mg protein in PT and 9.1 +/- 0.5 fmol/mg protein in SCN; and Kd values were: 94 +/- 22 pM in the PT and 101 +/- 15 pM in the SCN. This specific binding was strongly decreased in the PT of sexually inactive animals. Moreover, this decrease was significantly stronger in hypothermic than in euthermic hedgehogs. Saturation studies and Scatchard analysis revealed that the observed decrease in the PT resulted from change in the Bmax but not in the Kd, Bmax values being respectively 56.4 +/- 5.9 and 29.5 +/- 1.9 fmol/mg protein in euthermic and hypothermic sexually at rest animals. In none of the different physiological states, did the density of melatonin receptors of the SCN show any changes, Bmax values being respectively 9.8 +/- 0.5 and 9.8 +/- 0.4 fmol/mg protein in euthermic and hypothermic sexually at rest animals. This shows for the first time a tissue-specific regulation of melatonin receptor density occurring in the PT but not in the SCN. Furthermore, this decrease of binding in the PT is correlated with both sexual inactivity and hibernation period. This strongly suggests that the mediation of the photoperiodic effect on seasonal functions like seasonal hypothermia and reproduction involves an effect of melatonin on the PT rather than on the SCN.

Photic regulation of mt1 melatonin receptors in the Siberian hamster pars tuberalis and suprachiasmatic nuclei: involvement of the circadian clock and intergeniculate leaflet.

In the Siberian hamster suprachiasmatic nuclei and pars tuberalis of the pituitary, high affinity mt1 melatonin receptors are present. We have previously shown that night applied light pulse induced an increase in mt1 mRNA expression in the suprachiasmatic nuclei of this species, independently of the endogenous melatonin. Here, we report the photic regulation of melatonin receptor density and mRNA expression in the suprachiasmatic nuclei and pars tuberalis of pinealectomized Siberian hamsters and the implication in this control of either the circadian clock or the intergeniculate leaflet. The results show that: (1) A 1-h light pulse, delivered during the night, induces a transitory increase in mt1 mRNA expression in the suprachiasmatic nuclei and pars tuberalis. After 3 h this increase has totally disappeared (suprachiasmatic nuclei) or is greatly reduced (pars tuberalis). (2) The melatonin receptor density, in the suprachiasmatic nuclei, is not affected by 1 or 3 h of light, while it is strongly increased in the pars tuberalis. (3) In hamsters kept in constant darkness, the mt1 mRNA rise is gated to the subjective night in the suprachiasmatic nuclei and pars tuberalis. In contrast, the light-induced increase in melatonin binding is also observed in the subjective day in the pars tuberalis. (4) intergeniculate leaflet lesion totally inhibits the mt1 mRNA expression rise in the suprachiasmatic nuclei, while it has no effect on the light-induced increase in mt1 mRNA in the pars tuberalis. However, the light-induced increase in melatonin receptor density is totally prevented by the intergeniculate leaflet lesion in the pars tuberalis. These results show that: (1) the photic regulations of mt1 mRNA expression and receptor density are independent of each other in both the suprachiasmatic nuclei and pars tuberalis; and (2) the circadian clock and the intergeniculate leaflet are implicated in the photic regulation of melatonin receptors but their level of action differs totally between the suprachiasmatic nuclei and pars tuberalis.