Effect of photoperiod on the thyroid-stimulating hormone neuroendocrine system in the European hamster (Cricetus cricetus).

Recent studies have characterised a retrograde mechanism whereby the pineal hormone melatonin acts in the pars tuberalis (PT) of the pituitary gland to control thyroid hormone action in the hypothalamus, leading to changes in seasonal reproductive function. This involves the release of thyroid-stimulating hormone (TSH) from PT that activates type II deiodinase (DIO2) gene expression in hypothalamic ependymal cells, locally generating biologically active T3, and thus triggering a neuroendocrine cascade. In the present study, we investigated whether a similar regulatory mechanism operates in the European hamster. This species utilises both melatonin signalling and a circannual timer to time the seasonal reproductive cycle. We found that expression of betaTSH RNA in the PT was markedly increased under long compared to short photoperiod, whereas TSH receptor expression was localised in the ependymal cells lining the third ventricle, and in the PT, where its expression varied with time and photoperiod. In the ependymal cells at the base of the third ventricle, DIO2 and type III deiodinase (DIO3) expression was reciprocally regulated, with DIO2 activated under long and repressed under short photoperiod, and the reverse case for DIO3. These data are consistent with recent observations in sheep, and suggest that the PT TSH third ventricle-ependymal cell relay plays a conserved role in initiating the photoperiodic response in both long- and short-day breeding mammals.

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.

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.

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.

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.

The circadian clock, light/dark cycle and melatonin are differentially involved in the expression of daily and photoperiodic variations in mt(1) melatonin receptors in the Siberian and Syrian hamsters.

Mechanisms underlying the daily and photoperiodic variations in mt(1) melatonin receptors were investigated in the pars tuberalis (PT) and suprachiasmatic nuclei (SCN) of Siberian and Syrian hamsters. Whatever its daily profile, melatonin receptor density was strongly increased in both structures and species after constant light exposure or pinealectomy, and decreased after a single melatonin injection, indicating melatonin involvement in the daily regulation of the receptor protein. This was confirmed by a strong inverse correlation between melatonin binding capacity and plasma melatonin concentration. In contrast, regulation of mt(1) mRNA appeared more complex. The circadian clock, the light/dark cycle and melatonin are all implicated in mt(1) gene daily fluctuations, but the extent of their involvement depends upon the structure and the species studied. The photoperiodic decrease in melatonin receptor density observed in short photoperiod (PT of the two hamster species and Syrian hamster SCN) seems to be the consequence of a long-term mt(1) gene repression induced by the lengthening of the melatonin peak. Altogether, these results show that during daily variations, mt(1) melatonin receptor mRNA and protein are differentially regulated, while at the photoperiodic level, the mt(1) protein status depends on mRNA transcription.

Photic regulation of mt(1) melatonin receptors and 2-iodomelatonin binding in the rat and Siberian hamster.

We have investigated the photic regulation of melatonin receptors both at the level of binding capacity and mt(1) mRNA expression in the suprachiasmatic nucleus (SCN) and the pars tuberalis (PT) of the pituitary of two species: a highly photoperiodic one, the Siberian hamster, and a nonphotoperiodic one, the Wistar rat. This study has been performed by looking at the effect of a light pulse applied during the night on the two receptor parameters. The results show that the photic regulations of mt(1) mRNA expression and receptor density are distinct from each other in both the SCN and PT of the two species studied. They also show that, depending on the species and the structure, this regulation may implicate either the circadian clock or melatonin.

Melatonin regulates the mRNA expression of the mt(1) melatonin receptor in the rat Pars tuberalis.

The pars tuberalis (PT) of the pituitary is a major neuroendocrine target site for melatonin as it contains a large number of high-affinity melatonin receptors. We have previously shown that melatonin autoregulates the density of its own receptors in the PT. However, whether melatonin regulation includes mRNA expression in vivo is unclear. In the present study we have used quantitative in situ hybridization to (1) follow the daily profile of mt(1) mRNA expression in the rat PT and (2) investigate whether mt(1) mRNA expression could be regulated in vivo by melatonin. We found clear diurnal variations of mt(1) mRNA expression that persist in constant darkness. We also showed, on pinealectomized animals, that the rhythmic pineal melatonin secretion is necessary for the expression of these daily variations. In a second step, we studied the effect of an acute suppression of endogenous melatonin synthesis on mt(1) melatonin receptors by applying a 1-hour light pulse during the night. We found that light induced a dramatic increase in mt(1) mRNA which was totally prevented by a melatonin injection showing that the acute effect of melatonin on the receptor mRNA is strongly inhibitory. A light pulse applied to animals with a chronic absence of melatonin was ineffective showing that light only affects melatonin receptors via the light-induced plasma melatonin suppression. Altogether our results show that melatonin regulates mt(1) melatonin receptor mRNA expression. However, this regulation seems to be complex: acute changes in plasma melatonin concentration regulate negatively the gene transcription, even if the daily endogenous nocturnal melatonin peak seems a prerequisite for variations in its receptor expression.

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.

Interstrain differences in activity pattern, pineal function, and SCN melatonin receptor density of rats.

We investigated the possibility that strain-dependent differences in the diurnal pattern of wheel running activity rhythms are also reflected in the melatonin profiles. The inbred rat strains ACI/Ztm, BH/Ztm, and LEW/Ztm. LEW were examined for diurnal [12:12-h light-dark (LD)] wheel running activity, urinary 6-sulphatoxymelatonin (aMT6s) excretion, melatonin concentrations of plasma and pineal glands, and melatonin receptor density in the suprachiasmatic nuclei (SCN). ACI rats displayed unimodal activity patterns with a high level of activity, whereas BH and LEW rats showed multimodal activity patterns with ultradian components and reduced activity levels. In contrast, the individual daily profiles of aMT6s excretion and mean melatonin synthesis followed a unimodal time pattern in all three strains, suggesting that different output pathways of the SCN are responsible for the temporal organization of locomotor activity and pineal melatonin synthesis. In addition, melatonin synthesis at night and SCN melatonin receptor density at day were significantly higher in BH and LEW rats than in ACI rats. These results support the hypothesis of a long-term stimulating effect of melatonin on its own receptor density in the SCN.

Circannual reproductive rhythm in the European hamster (Cricetus cricetus): demonstration of the existence of an annual phase of sensitivity to short photoperiod.

In the European hamster (Cricetus cricetus) short photoperiod (SP) is responsible for the transition between the breeding and the resting season and data obtained previously suggest that a circannual "clock" drives the annual rhythm of reproduction. This hypothesis implies the existence of a SP-sensitive phase of the circannual system that occurs independently of the photoperiodic regime perceived by the animals after their arousal from hibernation at the end of March. In control animals kept outside, testicular atrophy occurs in August. When the animals were transferred from outdoors to controlled SP conditions (LD 10:14 and ambient temperature Ta = 18+/-2 degrees C), immediately (Group II) or 2, 4, 6 wk after capture (Groups IV, V, VI, respectively), sexual arrest occurs at the same time between mid-June and mid-July. In the other groups, transfer from outdoors to SP either after 6, 8, 10, 12 or 14 wk (Groups VI, VII, IX, X, XI, respectively) after capture, is followed directly within 4 wk by the gonadal atrophy. When SP was applied from the beginning of August (Group XII) gonadal atrophy was observed after only 2 wk. In this last group, however, the rapid involution is the consequence of the already initiated decline in sexual activity induced by the short daylengths from July. When comparing the effect of SP in two different ambient temperatures (Ta: 18+/-2 degrees C vs 7+/-2 degrees C), immediately (Groups II vs III), 8 (Groups VII vs VIII) or 16 (Groups XII vs XIII) wk after capture, it appears that low temperature does not affect the physiological process described above. In the European hamster, after the gonadal regrowth at the end of hibernation, the animals do not need to experience increasing long days to react against SP. Gonadal inhibition is induced when, following our hypothesis, SP coincides with an endogenous period of sensitivity that extends from mid-May to at least July-August. The present findings complement and extend earlier evidence to support the existence of an endogenous circannual control of seasonal reproduction in the European hamster.

Vasoconstrictor effects of various melatonin analogs on the rat tail artery in the presence of phenylephrine.

We performed a pharmacologic analysis of the increase in perfusion pressure induced by melatonin and related analogues in the perfused rat tail artery precontracted by 1 microM phenylephrine. Melatonin, 2-iodomelatonin, 6-chloromelatonin, and S20098 (N-[2-(7-methoxy-1-naphthyl)ethyl]acetamide) produced a concentration-dependent enhancement of the vasoconstrictor response evoked by 1 microM phenylephrine with a rank order of potency compatible with the pharmacologic profile defined for high-affinity melatonin receptors. Melatonin had no effect on electrically induced [3H]noradrenaline release, but the neurogenic vasoconstriction was increased at melatonin concentrations of 100 and 300 nM. Increasing concentrations of the naphthalenic-based antagonist S20928 (N-[2-(1-naphthyl)ethyl]cyclobutanecarboxamide) caused a parallel rightward shift in the melatonin concentration-response curve without depressing the maximal response. The pA2 value of S20928 was 7.01 +/- 0.08. Luzindole, 1 microM, an antagonist of Mel1b melatonin receptors, was without effect on melatonin-induced responses. By using reverse transcription-polymerase chain reaction (RT-PCR), we found that messenger RNA (mRNA) encoding for Mel1a is transcribed in the rat tail artery. In conclusion, the results show that melatonin produced an enhancement of the contractile response elicited by phenylephrine in the perfused rat tail artery. This vasoconstrictor response appears to be mediated through activation of Mel1a receptors located on smooth-muscle cells. No evidence for an action of melatonin on either the endothelium or sympathetic nerve endings was found.

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.

Effects of two melatonin analogues, S-20098 and S-20928, on melatonin receptors in the pars tuberalis of the rat.

By using quantitative autoradiography, we studied the effects of two drugs related to melatonin on the 2-(125)I-melatonin binding in the pars tuberalis (PT) of rats. The drugs tested were two naphthalenic analogues of melatonin, S-20098 (N-[2-(7-methoxy-1-naphthyl) ethyl] acetamide), an agonist, and S-20928 (N-[2-(1-naphthyl) ethyl] cyclobutyl carboxamide), a putative antagonist. Melatonin (s.c. and i.p.), S-20098 (s.c.), and S-20928 (i.p.) were injected 4 hr before sacrifice. Acute administration of both melatonin and S-20098 decreased melatonin receptor density. In contrast, the putative antagonist S-20928, at a low dose (1 mg/kg), was ineffective on melatonin receptors. It neither affected the 2-(125)I-melatonin specific binding observed in the control group nor did it prevent the decrease in binding induced by melatonin when injected 5 min before the hormone. At a high dose (10 mg/kg), S-20928 totally blocked the effect of melatonin on melatonin receptor density and induced a decrease in binding capacity as melatonin did when injected alone. These results indicate that in the rat pars tuberalis, the melatonin agonist, S-20098, is able to down-regulate melatonin receptors, whereas S-20928 seems to behave as a partial agonist.

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.

Annual variations of the NPYergic innervation of the pineal gland in the European hamster (Cricetus cricetus): a quantitative immunohistochemical study.

A prominent innervation of the pineal gland of the European hamster with nerve fibres containing neuropeptide Y (NPY) and tyrosine hydroxylase (TH) was demonstrated by means of immunohistochemistry. Nearly all the TH- and NPY-immunoreactive nerve fibres in the superficial pineal gland disappeared after bilateral superior cervical ganglionectomy, showing that the majority of NPY- and TH-immunoreactive nerve fibres belonged to the sympathetic nervous system. Since, in the European hamster, preliminary studies of the NPY-fibre density in the pineal gland had indicated seasonal changes, the density of NPY-immunoreactive nerve fibre profiles was ascertained in the superficial pineal gland in a series of animals between the first part of November and late April. The highest density of NPY-immunoreactive nerve fibre profiles was observed during midwinter. On the other hand, during the same period of the year, the number of sympathetic TH-immunoreactive sympathetic nerve fibre profiles did not exhibit seasonal variation, nor did substitution of testosterone, during the sexually inactive period, affect the density of NPY-containing nerve fibres in the gland. Our results show the presence of a testosterone-independent annual variation in the content of NPY in the sympathetic nerve fibres innervating the pineal gland of the European hamster. This variation can be correlated with the changes in the daily pattern of melatonin production observed by others in the same species at this period of the year.