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In seasonal species, the photoperiod (i.e. day length) tightly regulates reproduction to ensure that birth occurs at the most favourable time of year. In mammals, a distinct photoneuroendocrine circuit controls this process via the pineal hormone melatonin. This hormone is responsible for the seasonal timing of reproduction, but the anatomical substrates and the cellular mechanisms through which melatonin modulates seasonal functions remain imprecise. Recently, several genes have been identified as being regulated by the photoperiod in the brain of seasonal mammals. These genes are thought to play active roles in the regulation of seasonal biology, notably for the adjustment of reproduction and body weight. Here, we briefly review findings associated with the control of seasonal breeding and describe recent data ascribing photoperiodic roles to type 2 and type 3 deiodinases, to the Kiss1/GPR54 system and to the RFamide-related peptides.Interestingly, these systems involve different hypothalamic nuclei, suggesting that several brain loci may be crucial for melatonin to regulate reproduction, and thus represent key starting points to identify the long-sought-after mode and site(s) of action of melatonin. Such findings raise great hopes for the future and could herald a new era of research in the field of seasonal biology., (2009 S. Karger AG, Basel.)

The kisspeptin precursor is the protein transcribed from the Kiss-1 gene and the kisspeptins are the peptides that are posttranslationally processed from the precursor. The kisspeptins activate the G-protein coupled receptor GPR54 and are strongly implicated in puberty onset and in regulation of the hypothalamo-pituitary gonadal axis in mammals. Physiological studies have indicated that these effects occur via a direct activation of the GnRH neurons, and at an unknown site in the median eminence or directly on the gonadotropes. Paradoxically, while the function of kisspeptin is relatively well understood, little data are available about the localization of kisspeptin neurons in the brain, and in particular the projection patterns of kisspeptin containing axons implicated in regulation of the hypothalamo-pituitary gonadal axis. This review covers the current information about the localization of kisspeptin neurons in the mammalian brain and discusses the facts and artifacts of the methods of their detection. The available data suggest that kisspeptins are synthesized in neurons in the anteroventral periventricular nucleus and the arcuate nucleus. Both populations are considered to be involved in control of gonadotropes. In addition, kisspeptin nerve terminals and receptors are found in other hypothalamic area suggesting that kisspeptins are involved in regulation of other yet unknown homeostatic or neuroendocrine functions.

Arylalkylamine N-acetyltransferase (AA-NAT) is the rhythm-generating enzyme for the synthesis of pineal melatonin. Molecular investigations have revealed two biological models for the activation of AA-NAT. In rodent species, Aa-nat gene transcription is turned off during the daytime and markedly activated at night. In primates, sheep, and cows, the Aa-nat gene is constitutively transcripted with no visible daily variations. This inter-species difference in Aa-nat gene regulation leads to different daily profiles in melatonin synthesis and release. Thus, the nighttime onset of the rise in circulating melatonin is delayed and slow in rodents, whereas it is fast and sharp in sheep. In the camel (Camelus dromedarius), we have observed that circulating melatonin rises immediately after sunset, suggesting AA-NAT activity is regulated at the post-transcriptional level. In agreement with this hypothesis, we report herein the amount of Aa-nat mRNA in the camel pineal gland is high, during both the day and night with no daily variations, while melatonin concentration in the same pineal tissue is five times higher during the night than daytime.

In seasonal species, various physiological processes including reproduction are organized by photoperiod via melatonin, but the mechanisms of melatonin action are still unknown. In birds, the peptide gonadotropin-inhibiting hormone (GnIH) has been shown to have inhibitory effects on reproductive activity and displays seasonal changes of expression. Here we present evidence in mammals that the gene orthologous to GnIH, the RFamide-related peptide (RFRP) gene, expressed in the mediobasal hypothalamus, is strongly regulated by the length of the photoperiod, via melatonin. The level of RFRP mRNA and the number of RFRP-immunoreactive cell bodies were reduced in sexually quiescent Syrian and Siberian hamsters acclimated to short-day photoperiod (SD) compared with sexually active animals maintained under long-day photoperiod (LD). This was contrasted in the laboratory Wistar rat, a non-photoperiodic breeder, in which no evidence for RFRP photoperiodic modulation was seen. In Syrian hamsters, the reduction of RFRP expression in SD was independent from secondary changes in gonadal steroids. By contrast, the photoperiodic variation of RFRP expression was abolished in pinealectomized hamsters, and injections of LD hamsters with melatonin for 60 d provoked inhibition of RFRP expression down to SD levels, indicating that the regulation is dependent on melatonin. Altogether, these results demonstrate that in these hamster species, the RFRP neurons are photoperiodically modulated via a melatonin-dependent process. These observations raise questions on the role of RFRP as a general inhibitor of reproduction and evoke new perspectives for understanding how melatonin controls seasonal processes via hypothalamic targets.

Calcium is an important second messenger in the rat pineal gland, as well as cAMP. They both contribute to melatonin synthesis mediated by the three main enzymes of the melatonin synthesis pathway: tryptophan hydroxylase, arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase. The cytosolic calcium is elevated in pinealocytes following alpha(1)-adrenergic stimulation, through IP(3)-and membrane calcium channels activation. Nifedipine, an L-type calcium channel blocker, reduces melatonin synthesis in rat pineal glands in vitro. With the purpose of investigating the mechanisms involved in melatonin synthesis regulation by the L-type calcium channel, we studied the effects of nifedipine on noradrenergic stimulated cultured rat pineal glands. Tryptophan hydroxylase, arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase activities were quantified by radiometric assays and 5-hydroxytryptophan, serotonin, N-acetylserotonin and melatonin contents were quantified by HPLC with electrochemical detection. The data showed that calcium influx blockaded by nifedipine caused a decrease in tryptophan hydroxylase activity, but did not change either arylalkylamine N-acetyltransferase or hydroxyindole-O-methyltransferase activities. Moreover, there was a reduction of 5-hydroxytryptophan, serotonin, N-acetylserotonin and melatonin intracellular content, as well as a reduction of serotonin and melatonin secretion. Thus, it seems that the calcium influx through L-type high voltage-activated calcium channels is essential for the full activation of tryptophan hydroxylase leading to melatonin synthesis in the pineal gland.

Hibernation is a fascinating, yet enigmatic, physiological phenomenon during which body temperature and metabolism are reduced to save energy. During the harsh season, this strategy allows substantial energy saving by reducing body temperature and metabolism. Accordingly, biological processes are considerably slowed down and reduced to a minimum. However, the persistence of a temperature-compensated, functional biological clock in hibernating mammals has long been debated. Here, we show that the master circadian clock no longer displays 24-h molecular oscillations in hibernating European hamsters. The clock genes Per1, Per2, and Bmal1 and the clock-controlled gene arginine vasopressin were constantly expressed in the suprachiasmatic nucleus during deep torpor, as assessed by radioactive in situ hybridization. Finally, the melatonin rhythm-generating enzyme, arylalkylamine N-acetyltransferase, whose rhythmic expression in the pineal gland is controlled by the master circadian clock, no longer exhibits day/night changes of expression but constantly elevated mRNA levels over 24 h. Overall, these data provide strong evidence that in the European hamster the molecular circadian clock is arrested during hibernation and stops delivering rhythmic output signals.

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.

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.

Species differences have been reported for the nighttime regulation of arylalkylamine N-acetyltransferase (AA-NAT), the melatonin rhythm-generating enzyme. In particular, de novo synthesis of stimulatory transcription factors is required for Aa-nat transcription in the Syrian hamster but not in the rat pineal gland. The present work investigated the contribution of phosphorylated cAMP-responsive element-binding protein, c-FOS, c-JUN, and JUN-B in the regulation of Aa-nat transcription in Syrian hamsters compared with rats. The nighttime pattern of cAMP-responsive element-binding protein phosphorylation and regulation by norepinephrine observed in the Syrian hamster was similar to those reported in the rat. On the contrary, strong divergences in c-FOS, c-JUN, and JUN-B expression were observed between both species. In Syrian hamster, predominant expression of c-FOS and c-JUN was observed at the beginning of night, whereas a predominant expression of c-JUN and JUN-B was observed in the late night in rat. The early peak of c-FOS and c-JUN, known to form a stimulatory transcription dimer, suggests that they are involved in the nighttime stimulation of Aa-nat transcription. Indeed, early-night administration of a protein synthesis inhibitor (cycloheximide) markedly decreased AA-NAT mRNA levels in Syrian hamster. In the rat, high levels of JUN-B and c-JUN, constituting an inhibitory transcription dimer, are probably involved in the late-night inhibition of Aa-nat transcription. Early-night administration of cycloheximide actually increased AA-NAT mRNA levels toward the late night. Therefore, composition and timing of the pineal activator protein-1 complexes differ between rat and Syrian hamster and may be an activator (Syrian hamster) or an inhibitor (rat) of Aa-nat transcription.

In seasonal species, photoperiod organizes various physiological processes, including reproduction. Recent data indicate that the expression of type 2 iodothyronine deiodinase (Dio2) is modulated by photoperiod in the mediobasal hypothalamus of some seasonal species. Dio2 is believed to control the local synthesis of bioactive T(3) to regulate gonadal response. Here we used in situ hybridization to study Dio2 expression in the hypothalamus of a photoperiodic rodent, the Syrian hamster. Dio2 was highly expressed in reproductively active hamsters in long day, whereas it was dramatically reduced in sexually inhibited hamsters maintained in short day. This contrasted with the laboratory rat, a nonphotoperiodic species, in which no evidence for Dio2 photoperiodic modulation was seen. We also demonstrate that photoperiodic variations of Dio2 expression in hamsters are independent from secondary changes in gonadal steroids. Studies in pinealectomized hamsters showed that the photoperiodic variation of Dio2 expression is melatonin dependent, and injections of long day hamsters with melatonin for only 7 d were sufficient to inhibit Dio2 expression to that of short day levels. Finally, because in some seasonal species thyroid hormones are involved in photorefractoriness, we examined Dio2 expression in short day-refractory hamsters and found that Dio2 mRNA levels remained low despite full reproductive recrudescence. Altogether, these results demonstrate that in the Syrian hamster Dio2 is photoperiodically modulated via a melatonin-dependent process. Furthermore, refractoriness to photoperiod in hamsters appears to occur independently of Dio2. These results raise new perspectives for understanding how thyroid hormones are involved in the control of photoperiodic neuroendocrine processes.

The KiSS-1 gene encodes kisspeptin, the endogenous ligand of the G-protein-coupled receptor GPR54. Recent data indicate that the KiSS-1/GPR54 system is critical for the regulation of reproduction and is required for puberty onset. In seasonal breeders, reproduction is tightly controlled by photoperiod (i.e., day length). The Syrian hamster is a seasonal model in which reproductive activity is promoted by long summer days (LD) and inhibited by short winter days (SD). Using in situ hybridization and immunohistochemistry, we show that KiSS-1 is expressed in the arcuate nucleus of LD hamsters. Importantly, the KiSS-1 mRNA level was lower in SD animals but not in SD-refractory animals, which spontaneously reactivated their sexual activity after several months in SD. These changes of expression are not secondary to the photoperiodic variations of gonadal steroids. In contrast, melatonin appears to be necessary for these seasonal changes because pineal-gland ablation prevented the SD-induced downregulation of KiSS-1 expression. Remarkably, a chronic administration of kisspeptin-10 restored the testicular activity of 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 photoperiod, via melatonin, modulates KiSS-1 signaling 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.

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.

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.

Pineal melatonin synthesis is stimulated at night following an increase in arylalkylamine-N-acetyltransferase (AA-NAT) activity. Depending on the species, two mechanisms of enzyme activation have been described: a cAMP/phospho-cAMP response element-binding protein-dependent stimulation of Aa-nat gene transcription in the rat, presumed to occur in all rodents, or a posttranslational regulation of AA-NAT protein in ongulates. The present data obtained in the Syrian hamster indicate another route of AA-NAT regulation. Elevated nocturnal levels of Aa-nat mRNA were strongly suppressed following light exposure or adrenergic antagonist administration, demonstrating the involvement of norepinephrine in the stimulation of melatonin synthesis. However, administration of adrenergic agonists during the day did not increase Aa-nat mRNA unless a protein synthesis inhibitor was given during the previous night. This indicates that an inhibitory protein, synthesized at night, prevents melatonin synthesis during the day. By contrast, a protein synthesis inhibitor given at the beginning of the night markedly reduced Aa-nat mRNA, suggesting that a stimulatory protein (transcription factor?) is necessary for Aa-nat gene transcription at night. Noteworthy, hamsters raised in long photoperiod were responsive to adrenergic agonist injection only in the first hour after light onset, a response that may be important in this photoperiodic species in which the melatonin peak extends into the morning hours in a short photoperiod.

Inducible-cAMP early repressor (ICER) is a potent inhibitor of CRE (cAMP-related element)-driven gene transcription. In the rat pineal gland, it has been proposed to be part of the mechanisms involved in the shutting down of the transcription of the gene coding for arylalkylamine N-acetyltransferase (AA-NAT, the melatonin rhythm-generating enzyme). In this study, we report that ICER is expressed in the pineal gland of the photoperiodic rodent Syrian hamster although with some difference compared to the rat. In the Syrian hamster pineal, Icer mRNA levels, low at daytime, displayed a 20-fold increase during the night. Nighttime administration of a beta-adrenergic antagonist, propranolol, significantly reduced Icer mRNA levels although daytime administration of a beta-adrenergic agonist, isoproterenol, was unable to raise the low amount of Icer mRNA. These observations indicate that Icer mRNA expression is induced by the clock-driven norepinephrine release and further suggest that this stimulation is restricted to nighttime, as already observed for Aa-nat gene transcription. Furthermore, we found that the daily profile of Icer mRNA displayed photoperiodic variation with a lengthening of the nocturnal peak in short versus long photoperiod. These data indicate that ICER may be involved in both daily and seasonal regulation of melatonin synthesis in the Syrian hamster.

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.

The suprachiasmatic nucleus (SCN) controls the circadian rhythm of melatonin synthesis in the mammalian pineal gland by a multisynaptic pathway including, successively, preautonomic neurons of the paraventricular nucleus (PVN), sympathetic preganglionic neurons in the spinal cord and noradrenergic neurons of the superior cervical ganglion (SCG). In order to clarify the role of each of these structures in the generation of the melatonin synthesis rhythm, we first investigated the day- and night-time capacity of the rat pineal gland to produce melatonin after bilateral SCN lesions, PVN lesions or SCG removal, by measurements of arylalkylamine N-acetyltransferase (AA-NAT) gene expression and pineal melatonin content. In addition, we followed the endogenous 48 h-pattern of melatonin secretion in SCN-lesioned vs. intact rats, by microdialysis in the pineal gland. Corticosterone content was measured in the same dialysates to assess the SCN lesions effectiveness. All treatments completely eliminated the day/night difference in melatonin synthesis. In PVN-lesioned and ganglionectomised rats, AA-NAT levels and pineal melatonin content were low (i.e. 12% of night-time control levels) for both day- and night-time periods. In SCN-lesioned rats, AA-NAT levels were intermediate (i.e. 30% of night-time control levels) and the 48-h secretion of melatonin presented constant levels not exceeding 20% of night-time control levels. The present results show that ablation of the SCN not only removes an inhibitory input but also a stimulatory input to the melatonin rhythm generating system. Combination of inhibitory and stimulatory SCN outputs could be of a great interest for the mechanism of adaptation to day-length (i.e. adaptation to seasons).

Melatonin times reproduction with seasons in many photoperiodic mammalian species. Whether sexual hormones reflect on melatonin synthesis is still debated. The aim of this work was to study, using a large panel of technical approaches, whether the daily profile of pineal melatonin synthesis and release varies with the estrous cycle in the female rat. The mRNA levels and enzyme activities of the melatonin synthesizing enzymes, arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase were similar at the four stages of the rat estrous cycle. The endogenous release of melatonin, followed by transpineal microdialysis during six consecutive days in cycling female rats, displayed no significant variation during this interval. Taken together, the present results demonstrate that there is no regular fluctuation in the pineal metabolism leading to melatonin synthesis and release throughout the estrous cycle in female rats.

In the Syrian hamster, the role of noradrenaline in the regulation of melatonin synthesis is less clear than in the rat. During pineal ontogenesis in the rat, noradrenaline is the major transmitter involved in the onset of melatonin synthesis and melatonin rhythm. We analysed the involvement of noradrenaline in the ontogenesis of melatonin synthesis in the Syrian hamster and compared it with that of the rat. We followed the developmental profile of melatonin content in parallel with those of mRNA expression and activity of AA-NAT, the melatonin rhythm-generating enzyme. In addition, we tested the effect of noradrenergic drugs at early steps of pineal ontogenesis. In the Syrian hamster, the night-time Aa-nat mRNA, first detected 3 days after birth, increases progressively up to a maximum reached at 30 days of age and then decreases significantly towards adulthood. The daytime level of Aa-nat mRNA remains always low. A significant day/night rhythm appears 10 days after birth, is maximal (200-fold nocturnal increase) 30 days after birth and decreases slowly towards adulthood. Ontogenesis of the AA-NAT activity rhythm is similar, although with a much lower amplitude of day/night variations (four-fold). The developmental pattern of melatonin content is similar to that of AA-NAT and could be correlated with the appearance of sympathetic innervation in the pineal gland. However, neither alpha- nor beta-adrenergic antagonists inhibit the night-time Aa-nat mRNA transcription in the 9-day-old Syrian hamster, in contrast to what is observed in the adult. For comparison, the beta-adrenergic antagonist propranolol inhibits Aa-nat gene expression in 2-day-old rat. These results show that both species are different in the regulation of the appearance of melatonin synthesis and that Syrian hamster is peculiar from birth in term of noradrenaline involvement in the activation of melatonin synthesis.

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.

Hypocretin-1 (HCRT-1) and hypocretin 2 (HCRT-2), also known as orexin-A and orexin-B, are two neuropeptides derived from the same precursor. Hypocretinergic neurons have been found exclusively in the hypothalamic dorsolateral area. These neurons are implicated in sleep and feeding through activation of specific G-protein-coupled orexin-1 and orexin-2 receptor (OR-R1 and OR-R2). The purpose of this study was to determine the existence of the HCRT peptides in the central input of the rat pineal gland. Further, OR-R1 and OR-R2 expression was determined in the pineal gland and the effect of HCRT-2 on melatonin synthesis and secretion was analysed in dissociated rat pinealocytes. A large contingent of HCRT-positive nerve fibres and terminals were observed in the epithalamus, many of which entered into the pineal parenchyma. A significant number of nerve fibres endowed with positive boutons were identified in the pineal stalk, though the number of positive fibres decreased along the extension of the stalk. So far, no positive fibres have been found in the superficial pineal gland. RT-PCR analysis revealed the expression of OR-R2 mRNA, whereas OR-R1-receptor mRNA was not detected. When tested alone, HCRT-2 had no effect on secretion of melatonin from cultured rat pinealocytes. However, HCRT-2 partially inhibited (by a maximum of 30%) the beta-adrenergic-induced melatonin secretion. The same effect was seen on activation of N-acetyltransferase activity. The distribution and the large number of HCRT-positive fibres together with the effect on noradrenaline-mediated melatonin release through specific receptors suggests that these peptides may be significant central transmitters in pineal function, probably mediating homeostatic signals to the pineal gland.

The rodent pineal gland is the end point of several peripheral and central fibers innervating the superficial and deep parts of the gland. Up to now, only the sympathetic transmitter norepinephrine is thought to regulate melatonin synthesis, although numerous biochemical experiments have reported in vitro effects of various transmitters on melatonin synthesis. To find out whether there is non-noradrenergic regulation of in vivo pineal metabolism, the mRNA encoding the enzyme arylalkylamine N-acetyltransferase was studied using the highly sensitive technique of in situ hybridization. The existence of a marked nocturnal increase of arylalkylamine N-acetyltransferase mRNA in the superficial pineal gland was confirmed. Interestingly and for the first time, a similar daily variation was observed in the deep pineal. After removal of superior cervical ganglia, the daily rhythm in arylalkylamine N-acetyltransferase mRNA was abolished in both the superficial and deep pineal indicating that the rhythm is driven by sympathetic input in the entire pineal complex. Interestingly, the remaining arylalkylamine N-acetyltransferase mRNA level in the pineal of day- and night-time ganglionectomized rats was significantly higher than in the pineal of day-time intact animals. These data reveal a sympathetic-dependent day-time inhibition of arylalkylamine N-acetyltransferase gene expression. In addition, the day-time pineal arylalkylamine N-acetyltransferase mRNA expression in ganglionectomized rats persisted after adrenal gland removal but was reduced by 50% after propranolol injection. These results indicate that arylalkylamine N-acetyltransferase mRNA in ganglionectomized rats is not induced by circulating catecholamines and may be caused by both a centrally originated norepinephrine, as already suggested, and other non-adrenergic transmitter(s). In conclusion, this work shows that norepinephrine drives the nocturnal increase of arylalkylamine N-acetyltransferase gene expression both in the superficial and deep pineal and strongly suggests that other neurotransmitters are involved in day-time inhibition and night-time stimulation of pineal metabolism.

Despite a pronounced inhibitory effect of light on pineal melatonin synthesis, usually the daily melatonin rhythm is not a passive response to the surrounding world. In mammals, and almost every other vertebrate species studied so far, the melatonin rhythm is coupled to an endogenous pacemaker, i.e. a circadian clock. In mammals the principal circadian pacemaker is located in the suprachiasmatic nuclei (SCN), a bilateral cluster of neurons in the anterior hypothalamus. In the present paper we show in the rat that bilateral abolition of gamma-aminobutyric acid (GABA), but not vasopressin, neurotransmission in an SCN target area, i.e. the paraventricular nucleus of the hypothalamus, during (subjective) daytime results in increased pineal melatonin levels. The fact that complete removal of the SCN results in a pronounced increase of daytime pineal mRNA levels for arylalkylamine N-acetyltransferase (AA-NAT), i.e. the rate-limiting enzyme of melatonin synthesis, further substantiates the existence of a major inhibitory SCN output controlling the circadian melatonin rhythm.

The effects of long-term daily melatonin infusions on the melatonin synthetic pathway in the Harderian glands and eyes of male Siberian hamsters were studied. Hamsters were pinealectomized (PX) and infused daily for 8 hr with either melatonin (6 microg/hr) or vehicle for 7 days in short photoperiod (SP, 10L:14D), followed by 14 wk in either SP (SP group) or in constant darkness (DD group). After the infusion period (15 wk), the infusion was stopped and animals were transferred into SP for 3 wk. The hamsters were then killed at midday or midnight. Exogenous melatonin infusion caused an increase in the Harderian gland weight, which was still evident 3 wk after the end of the treatment. In addition, exogenous melatonin increased endogenous melatonin concentrations (4-fold) and hydroxyindole-O-methyltransferase (HIOMT) activity (2-fold). N-acetyltransferase (NAT) activity, however, was not increased, and no day/night difference in melatonin content and HIOMT activity was observed in the Harderian glands. In the eye, melatonin infusions significantly increased day and night-time melatonin levels (up to 3-fold) and both NAT and HIOMT activities (up to 3.5-fold). This effect of melatonin treatment was observed in both SP and DD groups. These observations demonstrate that exogenously-infused melatonin at relatively high doses activates the synthesis of endogenous melatonin in the Harderian gland and eye of the Siberian hamster. Circulating levels of melatonin were also markedly increased, indicating that in these conditions melatonin may be released from extra-pineal sites.

In the pineal, melatonin (Mel) is synthesized from serotonin by arylalkylamine-N-acetyltransferase (AA-NAT) and hydroxyindole-O-methyltransferase (HIOMT). Although it is clear that AA-NAT drives the daily rhythm in Mel synthesis, the mechanisms involved in the photoperiodic changes of the amplitude of the Mel peak, as observed in the Siberian hamster, remain to be determined. We investigated the characteristics of AA-NAT and HIOMT in Siberian hamsters kept either under a short (SP) or a long photoperiod (LP). The amplitude of the nocturnal peak of Mel was about two times higher under SP than under LP, whereas AA-NAT activity was about two times smaller under SP. In contrast, a twofold increase of HIOMT activity was observed under SP compared with LP. No change in the affinity of the enzymes for their substrates was observed between the two photoperiods. Our data strongly suggest that the photoperiodic variations in the amplitude of the nocturnal peak of Mel are driven by HIOMT, thereby promoting an important physiological role for this enzyme in the seasonal regulation of Mel production.

The aims of the current investigation were (1) to establish an efficient procedure for the isolation of rodent harderian gland cells and to define conditions for maintenance of viable differentiated cells; (2) to compare the in vitro growth pattern of cultured epithelial cells; and (3) to characterise the cultured epithelial cells from 3 rodent species: Wistar rats, Syrian hamsters and Djungarian hamsters. We have established primary culture conditions that permit the maintenance of viable and differentiated secretory cells from adult rodent harderian gland. This study demonstrates that the cell growth pattern is faster in hamsters than in rats and despite morphological changes, epithelial cells reestablish their distinctive (biochemical/metabolic) phenotype as indicated by lipid-containing vacuoles, porphyrin pigment and serotonin and tryptophan hydroxylase labelling.

The arylalkylamine-N-acetyltransferase (AA-NAT) expressed in the vertebrate pineal gland catalyzes the N-acetylation of the serotonin into N-acetylserotonin and is considered to be the rate limiting enzyme of the pineal melatonin synthesis. Indeed, dramatic changes in its activity throughout the 24-h period drive the large day/night variations in plasma melatonin concentrations. Recently, AA-NAT was cloned in the rat pineal. In this species, AA-NAT mRNA variations were demonstrated to be responsible of the well known AA-NAT activity and plasma melatonin circadian fluctuations. In the Syrian hamster, the pineal melatonin secretion pattern is characterized by a late-night short-duration peak of melatonin synthesis. We investigated whether this typical pattern could be due to a late-night delayed pineal AA-NAT mRNA expression. The first part of our study was dedicated to the molecular cloning of a Syrian hamster AA-NAT cDNA. A PCR-generated clone of 1045 bp encoding the AA-NAT has been isolated and sequenced. In situ hybridization using an AA-NAT cRNA probe revealed that the AA-NAT mRNA expression undergoes strong daily fluctuations in the Syrian hamster pineal, with undetectable level in the second half of the light period and a dramatic increase at night. After lights off, the AA-NAT mRNA expression requires 6-7 h to reach its maximum expression. This result thus suggests that the transcription of the AA-NAT mRNA in the Syrian pineal gland determines the lag period in pineal responsiveness and melatonin synthesis to darkness., (Copyright 1999 Elsevier Science B.V.)

The Harderian gland is considered as being an extrapineal source of melatonin. In most rodents, the Harderian gland contains two epithelial cell types (I and II). The aim of this study has been to define which cell type is involved in indoleamine synthesis. The presence and localization of serotonin (melatonin precursor) and tryptophan hydroxylase (the rate-limiting enzyme for serotonin synthesis) have been investigated by immunohistochemistry in male Wistar rats, Syrian hamsters and Djungarian hamsters. The results of the present study show that immunoreactivity for tryptophan hydroxylase and serotonin is confined to the type I cell, suggesting that this cell type is involved in indoleamine synthesis in the rodent Harderian gland.

Photoperiodic changes of pineal melatonin (MEL) profile are accompanied by parallel changes of arylalkylamine-N-acetyltransferase (AA-NAT) activity. In the present study, the authors investigated, for the first time, whether two other important variables of pineal metabolism, AA-NAT and hydroxyindole-O-methyltransferase (HIOMT) gene expression, also may be affected by the photoperiod. Evening rises in AA-NAT and HIOMT mRNA and in circulating MEL occurred concomitantly with an increased delay from dark onset as scotophase shortened. On the opposite, the morning declines of all three variables occurred with different kinetics but were locked to light onset. These observations demonstrate that the daily rhythms in AA-NAT and HIOMT gene expression are modulated by the photoperiod and bring further evidence in favor of nor adrenaline as the possible link between the endogenous clock and MEL. Interestingly, the duration of the nocturnal peak in HIOMT mRNA was positively correlated with HIOMT activity. In conclusion, this study adds two important links to the chain of mechanisms involved in the photoperiodic control of pineal metabolism. First, photoperiodic modulation of the MEL rhythm primarily results from changes in the AA-NAT gene expression. Second, the photoperiodic regulation of HIOMT activity occurs at the transcriptional level.

In the pineal gland, synthesis of melatonin requires O-methylation catalyzed by hydroxyindole-O-methyltransferase (HIOMT; EC 2.1.1.4). We investigated in vivo the molecular mechanisms involved in the regulation of rat pineal HIOMT messenger RNA (mRNA) expression and activity using in situ hybridization and radioenzymatic assay. HIOMT mRNA levels and activity are both detectable during the daytime and display nocturnal increases of 100% and 30%, respectively. These variations are controlled by the endogenous clock, as they persist in constant darkness. The nocturnal increase in HIOMT mRNA mainly results from a beta-adrenergic stimulation of HIOMT gene expression without requiring de novo synthesis of a transcription factor. In contrast, the nocturnal increase in HIOMT activity appears independent of beta1/alpha1-adrenergic stimulation. A light pulse at night abolishes the nighttime increase in HIOMT mRNA, but not HIOMT activity. Constant light application for up to 11 days does not depress HIOMT mRNA levels lower than the daytime levels, but decreases enzyme activity down to 50% of the daytime level. This finding indicates that the nocturnal stimulation of HIOMT gene expression is required for sustaining a basal level of activity over a few days. Our data suggest 1) that HIOMT gene expression is partly regulated by beta1-stimulation; and 2) that HIOMT activity is regulated over the short term by a nonnoradrenergic stimulus and over the long term by noradrenergic stimulation.

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 beta1-adrenergic-induced increase in cyclic adenosine monophosphate (cAMP) concentration potentiated by an alpha1-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+.

Postnatal development of hydroxyindole-O-methyltransferase (HIOMT) mRNA expression, HIOMT activity and melatonin content was investigated in the rat pineal gland from birth to adulthood (62-day old). For each age, animals were sacrificed at two different time-points: midday and midnight. HIOMT mRNA was first detectable one day after birth and maximal diurnal levels were reached at the end of the first week. A 2-fold nocturnal increase appeared significantly 11 days after birth. HIOMT activity was detectable from 5 days of life and significant day/night variations did not appear before 21 days after birth. Appearance of melatonin synthesis and rhythm followed that of HIOMT mRNA. The 10-day delay observed between the appearance of the nocturnal increase in HIOMT activity and expression is discussed in term of differential regulation of both HIOMT mRNA expression and HIOMT activity., (Copyright 1998 Elsevier Science B.V.)

Melatonin secretion from the mammalian pineal gland is strongly stimulated by noradrenaline and also by vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP). Three types of receptors for VIP and PACAP have been characterized so far: VIP1/PACAP receptors and VIP2/PACAP receptors, which possess similar high affinities for VIP and PACAP, and PACAP1 receptors which exhibit a 100-1000-fold higher affinity for PACAP. The aim of the present study was to characterize the receptor subtype(s) mediating the stimulatory effects of VIP and PACAP on melatonin synthesis in the rat pineal gland. Autoradiographic studies showed that PACAP and VIP were equally potent in displacing binding of radioiodinated PACAP27 from pineal sections. Amplification of pineal complementary DNAs by polymerase chain reaction using specific primers for the different receptor subtypes revealed that all three receptor messenger RNAs are expressed and that VIP1/PACAP receptor messenger RNA was predominant over VIP2/PACAP receptor messenger RNA. In vitro, VIP and PACAP stimulated melatonin synthesis with similar high potency and the effect of the two peptides were not additive. The selective VIP1/PACAP receptor agonists [R16]chicken secretin (1-25) and [K15, R16, L27]VIP(1-7)/growth hormone releasing factor(8-27) were significantly more potent than the selective VIP2/PACAP receptor agonist RO 25-1553 in stimulating melatonin secretion. The stimulatory effects of VIP and PACAP were similarly inhibited by the VIP1/PACAP antagonist [acetyl-His1, D-Phe2, K15, R16, L27]VIP(3-7)/growth hormone releasing factor(8-27). These data strongly suggest that VIP and PACAP exert a stimulatory effect on melatonin synthesis mainly through activation of a pineal VIP1/PACAP receptor subtype.

Melatonin content and release from Harderian glands (HGs) has been measured by an in vitro perifusion technique in three rodent species: Wistar rat, Syrian hamster, and Siberian hamster. Melatonin immunoreactive concentrations in HGs of animals killed at 10.00 hr were 0.31 +/- 0.031 pg/mg gland in male Wistar rat, 0.54 +/- 0.026 pg/mg gland in male Siberian hamster, 0.17 +/- 0.070 and 0.20 +/- 0.059 pg/mg gland in male and female Syrian hamster, respectively. In all species examined, isolated HGs perifused for 9-15 hr released melatonin but did not stabilize their melatonin release rate. No sex-related difference could be noted in the HG melatonin release rate. The total amount of melatonin released over a 15 hr long perifusion was about 0.075 +/- 0.004 ng/15 h/mg gland and 0.063 +/- 0.010 ng/15 hr/mg gland in male and female Wistar rat, respectively; 0.155 +/- 0.019 ng/15 hr/mg gland and 0.141 +/- 0.006 ng/15 hr/mg gland in male and female Siberian hamster, respectively; 0.035 +/- 0.003 ng/15 hr/mg gland and 0.045 +/- 0.004 ng/15 hr/mg gland in male and female Syrian hamster, respectively. This amount, which is higher than the tissue levels, demonstrates the de novo melatonin synthesis. This is confirmed by the fact that infusion of the indoleamine precursor, tryptophan (TRP), stimulated melatonin secretion from HGs. The melatonin release is increased by 2.5-fold in male and female Wistar rat, 1.5-fold in male and female Siberian hamster, and 2.0- and 3.0-fold in male and female Syrian hamster, respectively. Treatment with a TRP hydroxylase inhibitor, para-chlorophenylalanine, reduced basal melatonin release and inhibited the TRP-induced melatonin stimulation. Kinetics and amounts of melatonin released were not affected by pinealectomy, ruling out a possible plasmatic origin of the HG melatonin. Isoproterenol, a beta-adrenergic agonist, and dibutyryl cyclic AMP, a cyclic AMP analogue, failed to stimulate HG melatonin secretion. In conclusion, these results confirm the presence of melatonin in the HGs and demonstrate that melatonin is synthesized in and released from isolated rodent HGs.

Hydroxyindole-O-methyltransferase (HIOMT) is the enzyme involved in the last step of the melatonin synthesis pathway. Recently, a cDNA encoding HIOMT has been isolated from a rat pineal gland library. Using this cDNA, we developed a highly sensitive in situ hybridisation protocol to investigate the distribution of HIOMT mRNA in both the rat brain and dissociated pinealocytes maintained in primary cell culture. In the rat brain, HIOMT mRNA was only detected in the three parts of the pineal complex: the superficial pineal, the stalk and the deep pineal. No extra-pineal hybridisation labelling was observed. These results strongly suggest that melatonin synthesis also occurs in the deep part and the stalk of the pineal gland. HIOMT mRNA was markedly expressed in cultured pinealocytes. No particular subcellular area was observed to express HIOMT mRNA specifically, as the labelling was homogeneously distributed in the cytosol and in the axon-like processes. In conclusion, the use of in situ and in vitro hybridisation with a pineal riboprobe has detected notable HIOMT expression restricted to pinealocytes.

This study investigated whether the activation of pinealocyte beta-adrenergic receptors is involved in the regulation of serotonin (5-HT) synthesis and release, as it is for melatonin production. In addition, the role of the intra- and extra-cellular 5-HT in modulating the synthesis of melatonin induced by the beta-adrenergic agonist isoproterenol (ISO) was also studied. The incubation of dissociated pinealocytes with 0.1-10 microM ISO resulted in a concentration-dependent increase of melatonin synthesis. 5-HT release and intracellular 5-HT content were increased by 0.1 and 1 microM ISO but they were reduced after ISO 10 microM. Moreover, when incubated with the tryptophan hydroxylase inhibitor p-chlorophenylalanine (PCPA), the secretion of 5-HT as well as the intracellular 5-HT levels were markedly reduced in both ISO-stimulated and unstimulated conditions. Melatonin release was also inhibited by PCPA, although it responded in the expected manner to increasing concentrations of ISO. These data indicate that the release of 5-HT from pinealocytes depends on the availability of cytoplasmic 5-HT, which in turn is highly dependent on the tryptophan hydroxylase activity. In cells stimulated with moderate ISO concentrations, 5-HT release may be an important regulatory process of pineal 5-HT. After a large stimulation of N-acetyltransferase (NAT) activity by ISO, the synthesis of melatonin prevails on 5-HT release, whose decrease is associated to a deficit of intracellular 5-HT. On the other hand, the present study shows that the incubation of pineal cells with high concentrations of 5-HT or with a selective 5-HT2 receptor agonist, alpha-methyl-5-hydroxytryptamine, reverses partially the inhibitory effect of PCPA on the ISO-stimulated melatonin synthesis. In contrast the 5-HT2 antagonist, ketanserin, results in an inhibiton of the release of melatonin following ISO stimulation. These results suggest that released 5-HT may have a role in the full expression of the beta-adrenergically induced NAT activity and, thus, may contribute to the optimal melatonin synthesis at night.

This paper describes the effects of beta-adrenergic and peptidergic inputs on serotonin (5-HT) synthesis, outflow and metabolism into melatonin in cultured dissociated rat pinealocytes. The spontaneous outflow of 5-HT from pinealocytes was high as demonstrated by the elevated levels of extracellular 5-HT accumulated in the medium (about 5 ng/h/70,000 pineal cells). The beta-adrenergic agonist isoproterenol (ISO) used at concentrations up to 10(-6) M induced a moderate (+20-40%) increase in intra- and extracellular 5-HT levels together with a large release of melatonin. At a higher ISO stimulation (10(-5) M), the intra- and extracellular levels of 5-HT were significantly (-25-30%) reduced whereas melatonin secretion was dramatically increased. This is interpreted as a large 5-HT mobilization for melatonin synthesis and release, consequently reducing both the intracellular pool and outflow of 5-HT. The peptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) up to 10(-7) M induced always a moderate (+20-30%) increase in intra- and extracellular levels of 5-HT. However, the use of nM concentrations of VIP or PACAP together with 10(-6) M ISO induced a decrease in 5-HT outflow (-25-30%) and a dramatic increase in melatonin secretion as did 10(-5) M ISO alone. Neuropeptide Y (NPY) is another pineal peptide which induced a stimulation of 5-HT outflow (+30-40%) although its effect on melatonin release was marginal. The above results are discussed in term of the multineuronal regulation of the synthetic and secretory activities of the rat pineal gland.

The pineal gland of mammals is richly innervated by nerve fibres containing various peptides of central or sympathetic origin. In this study, we have investigated the presence, regulation and effect of secretoneurin (SN), a 33-amino-acid peptide discovered recently in the central and sympathetic nervous systems. The rat pineal gland contains about 34 fmol SN/pineal, although only 20% of the secretoneurin immunoreactivity (SN-IR) is the free peptide. SN-IR is localised in only a few nerve fibres found in the parenchyma of the gland and along the pineal stalk. In the rat pineal, SN-IR shows no midday/midnight differences but is significantly decreased (55%-65%) after superior cervical ganglionectomy or exposure to constant light. These observations suggest that, in the rat pineal, SN-IR originates partly from central areas and partly from the superior cervical ganglia. The pineal of the Syrian hamster contains much more SN-IR: 120 fmol SN-IR/pineal (about 20% being the free peptide). SN-IR is found both in nerve fibres and pineal cells. Some of the fibres run along the pineal stalk, indicating that they are of central origin, although a sympathetic origin cannot be ruled out. Pineal SN-IR is below detectable levels in the Siberian hamster. In cultured rat pinealocytes, SN significantly inhibits serotonin release and, to a lower extent, melatonin release.

We recently determined that melatonin stimulated serotonin (5-HT) secretion from rat pineal glands by increasing 5-HT release from the pinealocytes (microM melatonin concentrations) and by inhibiting 5-HT uptake in the pineal sympathetic nerve endings (mM melatonin concentrations). The present study investigated whether a single melatonin injection could alter the content of indoleamines in the rat pineal gland, as well as its possible dependence on the daytime of administration. Melatonin (150 micrograms/kg) was i.p. injected at 8 time points (11.00 h, 14.00 h, 17.00 h, 20.00 h, 23.00 h, 02.00 h, 05.00 h and 08.00 h) to rats kept in 12:12 h light:dark cycle (lights on at 07.00 h). Melatonin injections in the afternoon (17:00 h) and late in the nighttime (02.00 h and 05.00 h) decreased pineal 5-HT content 90 min later. The levels of 5-hydroxyindoleacetic acid (5-HIAA) were also decreased 90 min after the melatonin treatment at 14.00 h, 17.00 h and 02.00 h. The effect of melatonin on 5-HT content was a long-fasting effect (still evident after 180 min) only when injected at 02.00 h, whereas 5-HIAA levels were found to be decreased 180 min after melatonin treatment at 14.00 h and 23.00 h. No changes in these compounds were detected 240 min after melatonin treatment. Moreover, melatonin did not change 5-hydroxytryptophan levels at any of the daytime points studied. By contrast, 90 min after the injection of melatonin at 20.00 h, an increased content of pineal N-acetylserotonin was observed. This effect of melatonin could be mediated through a phase alteration of the pineal N-acetyltransferase activity rhythm by acting on the suprachiasmatic clock, although a direct melatonin effect on the pineal rhythmic function cannot be excluded. The effects of the hormone on 5-HT and 5-HIAA contents agree with previous findings on the inhibitory effect of pharmacological doses of melatonin on pineal 5-HT uptake, which presumably would result in a decreased intraneuronal content of 5-HT and its acid metabolite. These data point to an acute regulatory action of exogenous melatonin on the pineal melatonin synthesis pathway which seems to be limited to two daytime phases; the afternoon-early evening period and the second half of the night.

The expression of mRNAs coding for tryptophan hydroxylase (TPOH), the first enzyme involved in melatonin synthesis, has been studied in the rat adult pineal gland at four different circadian time points by in-situ hybridization using an oligonucleotide probe. TPOH mRNAs were detected at all chosen time-points. The lowest level was seen at 13:00 h. Quantification of the hybridization signals demonstrated a significant increase in expression (+16.5%) between 13:00 h and 19:30 h, with the values at 23:30 h and 09:00 h being similar to that of 19:30 h. These results coincide with the nycthemeral rhythm of TPOH activity. Day/night rhythms in the rat pineal gland are controlled by noradrenaline release at lights-off, which results in an increase in cAMP levels and in gene activation. This study suggests that the TPOH gene might be one such gene whose activation is stimulated in this way.

The pineal gland releases the "time-keeping' hormone melatonin following a rhythmic sympathetic input which translates light information. The aim of this work was to study the role and mechanism of action of the central vasopressinergic input on pineal cAMP-dependent melatonin synthesis in the rat. The pineal was found to display vasopressin receptors of the V1a subtype, as the V1a antagonist [125I]HO-LVA bound in a saturable manner to pineal membranes with a high affinity (kd = 10 pM) and a maximal binding capacity (B(max)) of 13 fmol/mg protein. Vasopressin was able to displace [125I]HO-LVA binding in a dose-dependent manner (k(i) = 1.9 nM). Vasopressin had no effect on the basal cAMP level and melatonin secretion in cultured rat pinealocytes. However, it clearly and dose-dependently (EC50 = 7 nM) potentiated by 2-3 times cAMP accumulation and by 1.5-2.5 times melatonin secretion induced by moderate noradrenergic stimulation. On strongly stimulated pinealocytes, however, vasopressin could potentiate cAMP accumulation, but not melatonin secretion. The potentiatory effect of vasopressin was inhibited in the presence of the V1a antagonist. These results indicate that vasopressin is a potent modulator of rat pineal synthetic activity.

Melatonin has been proposed to exert some regulatory actions within the pineal gland itself. The present study examined the effect of melatonin on the release of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) from rat pineal glands by using an in vitro perifusion system. Melatonin induced a concentration-dependent stimulatory effect on 5-HT secretion from 10(-6) M to 10(-3) M. Maximal effects were obtained with melatonin 10(-3) M and concentrations lower than 10(-6) M were without effect. The secretion of 5-HIAA was inhibited by melatonin 10(-3) and 10(-4) M, but it was increased when pineals were incubated with 10(-5) and 10(-6) M of melatonin. The indoleamine secretion was also studied on peripherally denervated rat pineal glands. Basal output of 5-HT from these glands was increased when compared with those from control rats. In contrast, the secretion of 5-HIAA was strongly reduced after removal of the sympathetic input to the pineal gland. Melatonin 10(-3) M failed to stimulate 5-HT release from denervated pineal glands, although it inhibited 5-HIAA secretion. In contrast, melatonin 10(-5) M enhanced 5-HT release without altering 5-HIAA output. Fluoxetine, a 5-HT uptake inhibitor, produced similar effects than mM concentrations of melatonin on the indoleamine secretion from control pineal glands, but it had no effect on glands taken from peripherally denervated rats. These data suggest that mM concentrations of the pineal hormone are able to stimulate 5-HT release from the pinealocyte, while mM concentrations of melatonin increase extracellular 5-HT by inhibiting its reuptake in the adrenergic nerve endings. These findings are discussed in relation to the possible role of melatonin regulating the intra- and extracellular availability of 5-HT in the pineal gland and its significance as an autocrine factor.

Neuropeptide Y is colocalized with noradrenaline in sympathetic fibers innervating the rat pineal gland. In this article we present a study of the effects and mechanisms of action of neuropeptide Y on the pineal noradrenergic transmission, the main input leading to the rhythmic secretion of melatonin. At the presynaptic level, neuropeptide Y inhibits by 45%, with an EC50 of 50 nM, the potassium-evoked noradrenaline release from pineal nerve endings. This neuropeptide Y inhibition occurs via the activation of pertussis toxin-sensitive G protein-coupled neuropeptide Y-Y2 receptors and is independent from, but additive to, the alpha 2-adrenergic inhibition of noradrenaline release. At the postsynaptic level, neuropeptide Y decreases by a maximum of 35%, with an EC50 of 5 nM, the beta-adrenergic induction of cyclic AMP elevation via the activation of neuropeptide Y-Y1 receptors. This moderate neuropeptide Y-induced inhibition of cyclic AMP accumulation, however, has no effect on the melatonin secretion induced by a beta-adrenergic stimulation. On the contrary, in the presence of 1 mM ascorbic acid, neuropeptide Y potentiates (up to threefold) the melatonin secretion. In conclusion, this study has demonstrated that neuropeptide Y modulates the noradrenergic transmission in the rat pineal gland at both presynaptic and postsynaptic levels, using different receptor subtypes and transduction pathways.

Transcription factor CREM appears to play a key physiological and developmental role within the hypothalamic-pituitary-gonadal axis. This axis is modulated by the pineal hormone melatonin, whose production is in turn driven by the endogenous clock. There is striking circadian fluctuation of a novel CREM isoform, ICER, which is expressed at high levels during the night. ICER is generated from an alternative, intronic promoter and functions as a powerful repressor of cyclic AMP-induced transcription. Rhythmic adrenergic signals originated by the clock direct ICER expression by stimulation of the cAMP signal transduction pathway.

Dopamine and norepinephrine are involved in regulation of melatonin synthesis in the pineal gland. In bovine pineal gland, D1- and D2-dopaminergic and alpha 1-adrenergic receptors have been characterized pharmacologically in several laboratories, while beta 1-adrenergic receptors have been studied using physiological technique. The current study presents a quantitative autoradiographic analysis of these four dopaminergic and noradrenergic receptors in bovine pineal gland. The density order of the receptors is D1 greater than alpha 1 greater than D2 greater than or equal to beta 1. The Bmax of dopamine D1 receptors is about 5 to 6 times higher than the Bmax for alpha 1-adrenergic receptors and about 20 times higher than the Bmax values for beta 1-adrenergic and D2-dopaminergic receptors. Dopamine D1 receptors are significantly denser in the pineal cortex than in the medulla. Both dopamine receptors are more concentrated in the distal area than in the proximal area (close to the habenula), whereas both noradrenergic receptors are homogeneously distributed along the longitudinal axis. Only D1-dopaminergic receptors display a heterogeneous distribution between the superior and the inferior areas, being denser in the inferior area. The observation of a much higher concentration of D1-dopaminergic receptors relative to the other receptors suggests an important role for dopamine in the regulation of bovine pineal physiology.

The rat pineal gland is known to release melatonin in response to noradrenergic stimulation. The effect of vasoactive intestinal peptide (VIP), one of the neuropeptides present in the pineal, was examined on perifused rat pineal glands. VIP stimulated melatonin release with a dose-dependent effect above 10(-7) M. In regard of kinetic characteristics, the pattern of melatonin release after VIP stimulation was similar to that after isoproterenol stimulation. 10(-6) M VIP-stimulated melatonin release was not altered when the pineal glands were treated with 10(-5) M propranolol (a beta-adrenergic antagonist) or 10(-5) M prazosin (an alpha 1-adrenergic antagonist). Thus VIP has a noradrenergic-independent effect on melatonin secretion. Conversely, this VIP effect is greatly inhibited by the specific action of a VIPergic antagonist. This suggests that VIP acts on melatonin synthesis through its own binding sites. This study demonstrates that melatonin secretion from rat pineal glands may be elicited through a VIPergic system which is independent of the well-known noradrenergic system.

In the posterior intestine of the sea-water eel, mucus plays an important role in biocrystallization of calcium ions. By means of transmission and scanning electron microscopy associated with X-ray microanalysis and X-ray diffraction it has been possible to determine the role of mucous fibers as nucleation sites. Biocrystallization occurs in 2 steps: (1) Calcification of mucus. As soon as mucus is excreted in the intestinal lumen, it is loaded with calcium, as shown by lanthanum affinity and X-ray microanalysis on freeze-dried tissues. (2) Genesis of crystals. Needle-shaped crystallites build up in coalescent spherites in the intestinal lumen near the microvilli. Genesis occurs as follows: (a) crystallite mineralization by nucleation in an organic matrix composed of glycoproteinaceous mucous fibers, followed by the appearance of spherites; (b) coalescence in spherites and association of spherites in rhombohedra; (c) extrusion of organic material during the final step of crystallization.