Valérie Simonneaux

Melatonin synthesis in rodents is tightly regulated at the transcriptional level by stimulatory and inhibitory transcription factors. Among them, phosphorylated cAMP‐related element binding protein (pCREB) and inducible cAMP early repressor (ICER), a strong inhibitor of cAMP‐related element‐driven genes, have an antagonistic action in activating/inhibiting the transcription of theAa‐natgene, which is an important enzyme in melatonin synthesis. In the Syrian hamster, a rodent displaying a seasonal control of reproduction, melatonin synthesis is strongly gated to the second part of the night. Indeed, exogenous adrenergic stimulation is unable to stimulateAa‐natgene transcription and melatonin synthesis during daytime. In the present study, we investigated whether ICER may be the cause of this daytime repression by comparing the dynamic of ICER and the adrenergic regulation of two genes whose expression is rapidly activated by cAMP‐dependant mechanisms, c‐fosandIcer. Adrenergic induction of c‐fosandIcerexpression was not possible during daytime, except at early day. ICER immunoreactivity was elevated throughout the daily cycle but reached the highest levels at early day, when gene expression can be induced by adrenergic agonists. Additionally, CREB phosphorylation was subjected to the same daily gating with an adrenergic induction occurring in the early but not in the late day. Taken together, our results indicate that the diurnal gating of pineal activity in the Syrian hamster is not caused by the repressor ICER and that it may occur at the level of noradrenergic receptor signalling.

Pineal melatonin is synthesized with daily and seasonal rhythms following the hypothalamic clock‐driven release of norepinephrine (NE). The pineal gland of rats and mice, like the biological clock, expresses a number of clock genes. However, the role of pineal clock elements in pineal physiology is still unknown. We examined the expression and regulation of several clock genes (Per1, Cry2, Bmal1 and Rev‐erbα) under different lighting conditions or following adrenergic treatments in the Syrian hamster, a seasonal rodent. We found that Per1 and Cry2 genes were similarly regulated by the nocturnal release of NE: levels of Per1 and Cry2 mRNA displayed a nocturnal increase that was maintained after 2 days in constant darkness (DD) but abolished after 2 days under constant light (LL), a condition that suppresses endogenous NE release, or after an early night administration of the adrenergic antagonist propranolol. In contrast, Bmal1 and Rev‐erbα exhibited a different pattern of expression and regulation. mRNA levels of both clock genes displayed a marked daily variation, maintained in DD, with higher values at midday for Bmal1 and at day/night transition for Rev‐erbα. Remarkably, the daily variation of both Bmal1 and Rev‐erbα mRNA was maintained in LL conditions and was not affected by propranolol. This study confirms the daily regulation of Per1 and Cry2 gene expression by NE in the pineal gland of rodents and shows for the first time that a second set of clock genes, Bmal1 and Rev‐erbα are expressed with a circadian rhythm independent of the hypothalamic clock‐driven noradrenergic signal.

In seasonal species, photoperiod (i.e. daylength) tightly regulates reproduction to ensure that birth occurs at the most favorable time of year. In mammals, a distinct photoneuroendocrine circuit controls this process via the pineal hormone melatonin. This hormone is responsible for the seasonal regulation of reproduction, but the anatomical substrate and the cellular mechanism through which melatonin modulates sexual activity is far from understood. The Syrian hamster is widely used to explore the photoneuroendocrine system, because it is a seasonal model in which sexual activity is promoted by long summer days (LD) and inhibited by short winter days (SD). Recent evidences indicate that the products of the KiSS-1 gene, kisspeptins, and their specific receptor GPR54, represent potent stimulators of the sexual axis. We have shown that melatonin impacts on KiSS-1 expression to control reproduction in the Syrian hamster. In this species, KiSS-1 is expressed in the antero-ventral-periventricular and arcuate nuclei of the hypothalamus at significantly higher levels in hamsters kept in LD as compared to SD. In the arcuate nucleus, the downregulation of KiSS-1 expression in SD appears to be mediated by melatonin and not by secondary changes in gonadal hormones. Remarkably, a chronic administration of kisspeptin restores testicular activity in SD hamsters, despite persisting photoinhibitory conditions. Overall, these findings are consistent with a role of KiSS-1/GPR54 in the seasonal control of reproduction. We propose that the photoperiod, via melatonin, modulates KiSS-1 neurons to drive the reproductive axis.

The Syrian hamster is a rodent species in which the photoperiodic change in the melatonin peak duration is pivotal for the synchronization of annual functions, like reproduction. In this species, the activity of arylalkylamine N‐acetyltransferase (AANAT), the key enzyme for the rhythmic synthesis of melatonin, is precisely controlled and time‐gated, suggesting regulatory mechanisms different from those in the rat or mouse. At the beginning of the night, norepinephrine (NE) elicits a rapid and sustained phosphorylation of CREB into pCREB and a transient synthesis of the immediate early gene products c‐FOS and c‐JUN that peak 3 h after dark onset. c‐FOS synthesis requires both pCREB and the pERK1/2 pathways. Interestingly, injection of the protein synthesis inhibitor cycloheximide before, but not after, the c‐FOS/c‐JUN peak markedly reduces Aanat mRNA levels. This finding suggests that the c‐FOS/c‐JUN dimer is required for transcriptional activation of the Aanat gene. During daylight, exogenous noradrenergic stimulation cannot stimulate Aanat expression and, therefore, melatonin synthesis. The inhibitory transcription factor ICER is present in the pineal gland but with highest values when AANAT may be activated, suggesting the blockade takes place upstream of Aanat expression. Preliminary experiments indicate that the diurnal inhibition of AANAT occurs at the level of the adrenergic receptor signalling pathway, but it is not known whether this is sufficient to explain the pineal resistance to NE during the daytime. Together, these findings demonstrate that AANAT regulation in the Syrian hamster requires a complex intracellular signalling cascade, different from that described in laboratory rodents like mice and rats.

In seasonal species, photoperiod exerts tight regulation of reproduction to ensure that birth occurs at the most favorable time of yr. A distinct photoneuroendocrine circuit composed of the retina, suprachiasmatic nucleus (SCN) of the hypothalamus, and pineal gland transduces daylength into a rhythmic secretion of melatonin. The duration of the night‐time rise of this hormone conveys daylength information to the organism. Melatonin is known to mediate the control of seasonal reproduction, but how it modulates sexual activity is far from understood. Recent data indicate that the product of the KiSS‐1 gene is a potent stimulator of the hypothalamic‐pituitary‐gonadal axis and may play, together with its receptor GPR54, a central role in the neuroendocrine regulation of gonadotropin secretion. This article briefly reviews these findings and presents arguments that KiSS‐1 could take part in the seasonal control of reproduction.

Melatonin, the major hormone produced by the pineal gland, displays characteristic daily and seasonal patterns of secretion. These robust and predictable rhythms in circulating melatonin are strong synchronizers for the expression of numerous physiological processes in photoperiodic species. In mammals, the nighttime production of melatonin is mainly driven by the circadian clock, situated in the suprachiasmatic nucleus of the hypothalamus, which controls the release of norepinephrine from the dense pineal sympathetic afferents. The pivotal role of norepinephrine in the nocturnal stimulation of melatonin synthesis has been extensively dissected at the cellular and molecular levels. Besides the noradrenergic input, the presence of numerous other transmitters originating from various sources has been reported in the pineal gland. Many of these are neuropeptides and appear to contribute to the regulation of melatonin synthesis by modulating the effects of norepinephrine on pineal biochemistry. The aim of this review is firstly to update our knowledge of the cellular and molecular events underlying the noradrenergic control of melatonin synthesis; and secondly to gather together early and recent data on the effects of the nonadrenergic transmitters on modulation of melatonin synthesis. This information reveals the variety of inputs that can be integrated by the pineal gland; what elements are crucial to deliver the very precise timing information to the organism. This also clarifies the role of these various inputs in the seasonal variation of melatonin synthesis and their subsequent physiological function.

The time giver hormone melatonin is secreted with both daily and seasonal rhythms in the mammalian pineal gland. We report here that the two main melatoninsynthesizing enzymes N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) are differently involved in the regulation of the daily and photoperiodic rhythms of melatonin synthesis. The activity of the unstable NAT enzyme is stimulated acutely at night by norepinephrine via cAMP-dependent transcriptional mechanisms, indicating that it triggers the nocturnal increase in melatonin synthesis and drives its duration according to the night length. In contrast, the activity of the stable HIOMT enzyme is regulated chronically by a norepinephrine-induced, 2-fold increase in HIOMT mRNA that occurs every night. Such regulation leads to a photoperiodic variation in HIOMT activity which is positively related to duration of the night. Whether in vivo or in vitro, at high NAT activity, HIOMT activity appears to limit the amount of melatonin release. This suggests that HIOMT activity may tune the amplitude of the nocturnal increase in pineal melatonin secretion with photoperiodic variations.

: The pineal gland is mainly innervated by sympathetic fibres containing noradrenaline (NA) and neuropeptide Y (NPY). NA released at night stimulates melatonin synthesis via a β₁‐adrenergic‐induced increase in cyclic adenosine monophosphate (cAMP) concentration potentiated by an α₁‐adrenergic‐induced increase in Ca²⁺ concentration. We previously showed that NPY acted on presynaptic Y2 receptors inhibiting NA release and on postsynaptic Y1 receptors stimulating melatonin synthesis. Here we used Fura‐2 imaging to assess the effect of NPY on the intracellular Ca²⁺ concentration, [Ca²⁺]_i, in cultured rat pineal cells. In 84% of cells, on average, 10 nm NPY induced a progressive rise of [Ca²⁺]_i from its basal value of 102 ± 3 nm to a plateau of 180 ± 6 nm (n = 467 cells), which lasted the time of NPY application. This effect of NPY appeared dependent on extracellular Ca²⁺.