Differential response patterns of kisspeptin and RFamide-related peptide to photoperiod and sex steroid feedback in the Djungarian hamster (Phodopus sungorus).

Many animals synchronise their reproductive activity with the seasons to optimise the survival of their offspring. This synchronisation involves switching on and off their gonadotrophic axis. Ever since their discovery as key regulators of gonadotrophin-releasing hormone (GnRH) neurones, the hypothalamic RF-amide peptides kisspeptin and RFamide-related peptide (RFRP) have been a major focus of research on the seasonal regulation of the gonadotrophic axis. In the present study, we investigated the regulation of both neuropeptides in the Djungarian hamster, a major animal model for the study of seasonal reproduction. During the long-day breeding period, kisspeptin neurones in the anteroventral periventricular area are solely controlled by a positive sex steroid feedback and, in the arcuate nucleus, they are subject to a very strong negative sex steroid feedback associated with a minor photoperiodic effect. During short-day sexual quiescence, the disappearance of this hormonal feedback leads to high levels of kisspeptin in arcuate neurones. Notably, chronic central administration of kisspeptin is able to over-ride the photoperiodic inhibition of the gonadotrophic axis and reactivate the reproductive function. Therefore, our data suggest that kisspeptin secretion by arcuate neurones during sexual quiescence is inhibited by mechanisms upstream of kisspeptin neurones. RFRP expression is solely controlled by photoperiod, being strongly reduced in short days independently of the sex steroid feedback. Thus, kisspeptin and RFRP display contrasting patterns of expression and regulation. Upstream mechanisms controlling these neurones should be the focus of further studies on the roles of these RFamide neuropeptides in the seasonal control of reproduction.

Photoperiodic co-regulation of kisseptin, neurokinin B and dynorphin in the hypothalamus of a seasonal rodent.

In many species, sexual activity varies on a seasonal basis. Kisspeptin (Kp), a hypothalamic neuropeptide acting as a strong activator of gonadotrophin-releasing hormone neurones, plays a critical role in this adaptive process. Recent studies report that two other neuropeptides, namely neurokinin B (NKB) and dynorphin (DYN), are co-expressed with Kp (and therefore termed KNDy neurones) in the arcuate nucleus and that these peptides are also considered to influence GnRH secretion. The present study aimed to establish whether hypothalamic NKB and DYN expression is photoperiod-dependent in a seasonal rodent, the Syrian hamster, which exhibits robust seasonal rhythms in reproductive activity. The majority of Kp neurones in the arcuate nucleus co-express NKB and DYN and the expression of all three peptides is decreased under a short (compared to long) photoperiod, leading to a 60% decrease in the number of KNDy neurones under photo-inhibitory conditions. In seasonal rodents, RFamide-related peptide (RFRP) neurones of the dorsomedial hypothalamus are also critical for seasonal reproduction. Interestingly, NKB and DYN are also expressed in the dorsomedial hypothalamus but do not co-localise with RFRP-immunoreactive neurones, and the expression of both NKB and DYN is higher under a short photoperiod, which is opposite to the short-day inhibition of RFRP expression. In conclusion, the present study shows that NKB and DYN display different photoperiodic variations in the Syrian hamster hypothalamus. In the arcuate nucleus, NKB and DYN, together with Kp, are down-regulated under a short photoperiod, whereas, in the dorsomedial hypothalamus, NKB and DYN are up-regulated under a short photoperiod.

Distribution and seasonal variation in hypothalamic RF-amide peptides in a semi-desert rodent, the jerboa.

The jerboa is a semi-desert rodent, in which reproductive activity depends on the seasons, being sexually active in the spring-summer. The present study aimed to determine whether the expression of two RF-amide peptides recently described to regulate gonadotrophin-releasing hormone neurone activity, kisspeptin (Kp) and RF-amide-related peptide (RFRP)-3, displays seasonal variation in jerboa. Kp and/or RFRP-3 immunoreactivity was investigated in the hypothalamus of jerboas captured in the field of the Middle Atlas mountain (Morocco), either in the spring or autumn. As in other rodents, the Kp-immunoreactive (-IR) neurones were found in the anteroventro-periventricular and arcuate nuclei. RFRP-3 neurones were noted within the dorso/ventromedial hypothalamus. A marked sexual dimorphism in the expression of Kp (but not RFRP-3) was observed. The number of Kp-IR neurones was nine-fold higher, and the density of Kp-IR fibres and terminal-like elements in the median eminence was two-fold higher in females than in males. Furthermore, a significant seasonal variation in peptide expression was obtained with an increase in both Kp- and RFRP-3-IR cell bodies in sexually active male jerboas captured in the spring compared to sexually inactive autumn animals. In the arcuate nucleus, the level of Kp-IR cells and fibres was significant higher during the sexually active period in the spring than during the autumnal sexual quiescence. Similarly, the number of RFRP-3-IR neurones in the ventro/dorsomedial hypothalamus was approximately three-fold higher in sexually active jerboa captured in the spring compared to sexually inactive autumn animals. Altogether, the present study reports the distribution of Kp and RFRP-3 neurones in the hypothalamus of a desert species and reveals a seasonal difference in their expression that correlates with sexual activity. These findings suggest that these two RF-amide peptides may act in concert to synchronise the gonadotrophic activity of jerboas with the seasons.

Circannual variation in thyroid hormone deiodinases in a short-day breeder.

At temperate latitudes, many mammals and birds show internally timed, long-term changes in seasonal physiology, synchronised to the seasons by changing day length (photoperiod). Photoperiodic control of thyroid hormone levels in the hypothalamus dictates the timing. This is effected through reciprocal regulation of thyroid hormone deiodinase gene expression. The local synthesis of type 2 deiodinase (Dio2) promotes triiodothyronine (T3) production and summer biology, whereas type 3 deiodinase (Dio3) promotes T3 degradation and winter biology. In the present study, we investigated the extent to which the hypothalamic expression of Dio2 and Dio3 is circannually regulated in the Soay sheep, a short-day breeding mammal. Male sheep were exposed to a long photoperiod (LP; 16 : 24 h light/dark cycle) or a short photoperiod (SP; 8 : 24 h light/dark cycle), for up to 28 weeks to establish four different endocrine states: (i) LP animals in a spring/summer-like state of reproductive arrest; (ii) LP refractory (LPR) animals showing spontaneous reproductive reactivation; (iii) SP animals showing autumn/winter-like reproductive activation; and (iv) SP refractory (SPR) animals showing spontaneous reproductive arrest. A complex pattern of hypothalamic Dio2 and Dio3 expression was observed, revealing distinctive photoperiod-driven and internally timed effects for both genes. The patterns of expression differed both spatially and temporally, with phases of peak Dio2 expression in the median eminence and tuberoinfundibular sulcus, as well as in the paraventricular zone (PVZ) (maximal under LP), whereas Dio3 expression was always confined to the PVZ (maximal under SP). These effects likely reflect the distinct roles of these enzymes in the localised control of hypothalamic T3 levels. The spontaneous decline in Dio2 and spontaneous increase in Dio3 in LPR animals occurred with a corresponding decline in thyroid-stimulating hormone ß expression in the neighbouring pars tuberalis (PT), although this relationship did not hold for the corresponding Dio2 increase/Dio3 decrease seen in SPR animals. We conclude that internally timed and spatially regulated changes in Dio2 and Dio3 expression may drive the cycling between breeding and nonbreeding states in long-lived seasonal species, and may be either PT-dependent or PT-independent at different phases of the circannual cycle.

Plasma corticosterone elevation inhibits the activation of nuclear factor kappa B (NFKB) in the Syrian hamster pineal gland.

We evaluated how the mild stress-induced increase in endogenous corticosterone affected the pineal gland in Syrian hamsters (Mesocricetus auratus). The animals were maintained under constant light for 1 day, instead of a cycle of 14:10-h, to increase the circulating corticosterone levels during the daytime. The nuclear translocation of nuclear factor kappa B (NFKB), which is the pivotal transcription factor for stress and injury, presented a daily rhythm in normal animals. NFKB nuclear content increased linearly from the onset of light [Zeitgeber Time 0 (ZT0)] until ZT11 and decreased after ZT12 when the plasma corticosterone peak was detected in normal animals. However, the 24-h profiles of the two curves were different, and they did not clearly support an exclusive relationship between corticosterone levels and NFKB content. Therefore, we tested the effect of increased endogenous corticosterone through inducing mild stress by maintaining daytime illumination for one night. This stressful condition, which increased daytime corticosterone levels, resulted in a daytime decrease in NFKB nuclear content, and this was inhibited by mifepristone. Overall, this study shows that NFKB has a daily rhythm in Syrian hamster pineal glands and, by increasing endogenous corticosterone with a stressful condition, NFKB activity is regulated. Therefore, this study suggests that the pineal gland in the Syrian hamster is a sensor of stressful conditions.

Peripheral kisspeptin reverses short photoperiod-induced gonadal regression in Syrian hamsters by promoting GNRH release.

In seasonal breeders, reproduction is synchronised by day length via the pineal hormone melatonin. In short winter days (short day, SD), the Syrian hamster displays a complete gonadal atrophy together with a marked reduction in expression of kisspeptins (Kp), a family of potent hypothalamic stimulators of GNRH neurons. Both central and peripheral acute injections of Kp have been reported to activate the gonadotropic axis in mammals. The aim of this study was to determine if and how peripheral administration of Kp54 could restore gonadal function in photo-inhibited hamsters. Testicular activity of hamsters kept in SD was reactivated by two daily i.p. injections of Kp54 but not by chronic subcutaneous delivery of the same peptide via mini-pumps. Acute i.p. injection of Kp54-induced FOS (c-Fos) expression in a large number of GNRH neurons and pituitary gonadotrophs together with a strong increase in circulating testosterone. The activation of pituitary cells by Kp was inhibited by preadministration of the GNRH receptor antagonist acyline. Altogether, our results demonstrate that peripheral Kp54 activates the gonadotropic axis by stimulating GNRH release and indicate that an appropriate protocol of long-term systemic Kp administration can recrudesce a photo-inhibited reproductive axis.

Mapping of kisspeptin fibres in the brain of the pro-oestrous rat.

Kisspeptins are a family of small peptides that play a key role in the neuroendocrine regulation of the reproductive function through neural pathways that have not yet been completely identified. The present study aimed to investigate the distribution of kisspeptin neurone fibres in the female rat brain by comparing precisely the immunoreactive pattern obtained with two antibodies: one specifically directed against kisspeptin-52 (Kp-52), the longest isoform, and the other directed against kisspeptin-10 (Kp-10), whose sequence is common to all putative mature isoforms. With both antibodies, immunoreactive cell bodies were exclusively observed in the arcuate nucleus, and immunoreactive fibres were confined to the septo-preoptico-hypothalamic continuum of the brain. Fibres were observed in the preoptic area, the diagonal band of Broca, the septohypothalamic area, the anteroventral periventricular, suprachiasmatic, supraoptic, paraventricular and periventricular nuclei, the dorsal border of the ventromedian nucleus, the dorsomedial and arcuate nuclei, and the median eminence. In the latter structure, varicose fibres were mainly distributed in the internal layer and were detected to a lesser extent throughout the external layer, including around the deeper part of the infundibular recess. Most regions of immunoreactive cells and fibres matched perfectly for the two antibodies. However, fibres in the dorsolateral septum, anterior fornix, accumbens nucleus and the lateral bed nucleus of the stria terminalis were only recognised by antibody anti-Kp-10, suggesting that anti-Kp-10 may recognise a wider range of kisspeptin isoforms than anti-Kp-52 or cross-react with molecules other than kisspeptin in rat tissue. Overall, these results illustrate the variety of projection sites of kisspeptin neurones in the rat and suggest that these peptides play a role in different functions.

Differential regulation of kiss1 expression by melatonin and gonadal hormones in male and female Syrian hamsters.

In seasonal breeders, reproduction is synchronized to seasons by day length via the pineal hormone melatonin. Recently, we have demonstrated that Kiss1, a key activator of the reproductive function, is down-regulated in sexually inactive hamsters maintained in inhibitory short days (SDs). In rodents, Kiss1 is expressed in the anteroventral periventricular nucleus (AVPV) and in the arcuate nucleus (ARC). Because both the duration of the nocturnal peak of melatonin and circulating sex steroid levels vary with photoperiod, the aim of this study was to determine whether melatonin and sex steroids differentially regulate Kiss1 expression in the ARC and the AVPV. Kiss1 expression was examined by in situ hybridization in both male and female hamsters kept in various experimental conditions, and we observed that 1) SD exposure markedly reduced Kiss1 expression in the ARC and AVPV of male and female hamsters as compared to LD animals, 2) sex steroid treatment in SD-adapted male and female hamsters increased the number of Kiss1 neurons in the AVPV but decreased it in the ARC, 3) melatonin administration to LD-adapted hamsters decreased Kiss1 mRNA level in both the AVPV and the ARC in intact animals, whereas in castrated hamsters, melatonin rapidly inhibited Kiss1 expression in the ARC but not in the AVPV, and 4) pinealectomy of male or female SD-adapted hamsters increased the number of Kiss1 neurons in the ARC but not in the AVPV. In conclusion, our data demonstrate that Kiss1 expression in the Syrian hamster hypothalamus is down-regulated in SD via different mechanisms. In the ARC, melatonin inhibits Kiss1 via a direct effect on the hypothalamus, and this effect is probably sex steroid dependent, whereas in the AVPV, the decrease in Kiss1 expression appears to be secondary to the melatonin-driven reduction of sex steroid levels. Taken together, our data support the hypothesis that ARC Kiss1 neurons mediate melatonin effects on the gonadotropic axis of the Syrian hamster.

Daytime gating in the Syrian hamster pineal gland.

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 the Aa-nat gene, 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 stimulate Aa-nat gene 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-fos and Icer. Adrenergic induction of c-fos and Icer expression 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.

Local corticosterone infusion enhances nocturnal pineal melatonin production in vivo.

Melatonin, an important marker of the endogenous rhythmicity in mammals, also plays a role in the body defence against pathogens and injuries. In vitro experiments have shown that either pro- or anti-inflammatory agents, acting directly in the organ, are able to change noradrenaline-induced pineal indoleamine production. Whereas corticosterone potentiates melatonin production, incubation of the gland with tumour necrosis factor-alpha decreases pineal hormonal production. In the present study, we show that nocturnal melatonin production measured by intra-pineal microdialysis is enhanced in pineals perfused with corticosterone at concentrations similar to those measured in inflamed animals. In vitro experiments suggest that this enhancement may be due to an increase in the activity of the two enzymes that convert serotonin to N-acetylserotonin (NAS) and NAS to melatonin. The present results support the hypothesis that the pineal gland is a sensor of inflammation mediators and that it plays a central role in the control of the inflammatory response.

Daily rhythm and regulation of clock gene expression in the rat pineal gland.

Rhythms in pineal melatonin synthesis are controlled by the biological clock located in the suprachiasmatic nuclei. The endogenous clock oscillations rely upon genetic mechanisms involving clock genes coding for transcription factors working in negative and positive feedback loops. Most of these clock genes are expressed rhythmically in other tissues. Because of the peculiar role of the pineal gland in the photoneuroendocrine axis regulating biological rhythms, we studied whether clock genes are expressed in the rat pineal gland and how their expression is regulated.Per1, Per3, Cry2 and Cry1 clock genes are expressed in the pineal gland and their transcription is increased during the night. Analysis of the regulation of these pineal clock genes indicates that they may be categorized into two groups. Expression of Per1 and Cry2 genes shows the following features: (1) the 24 h rhythm persists, although damped, in constant darkness; (2) the nocturnal increase is abolished following light exposure or injection with a beta-adrenergic antagonist; and (3) the expression during daytime is stimulated by an injection with a beta-adrenergic agonist. In contrast, Per3 and Cry1 day and night mRNA levels are not responsive to adrenergic ligands (as previously reported for Per2) and daily expression of Per3 and Cry1 appears strongly damped or abolished in constant darkness. These data show that the expression of Per1 and Cry2 in the rat pineal gland is regulated by the clock-driven changes in norepinephrine, in a similar manner to the melatonin rhythm-generating enzyme arylalkylamine N-acetyltransferase. The expression of Per3 and Cry1 displays a daily rhythm not regulated by norepinephrine, suggesting the involvement of another day/night regulated transmitter(s).

Analysis of adrenergic regulation of melatonin synthesis in Siberian hamster pineal emphasizes the role of HIOMT.

Seasonal variations of environmental factors are translated into annual fluctuations in synthesis and release of melatonin, which in turn acts as a neuroendocrine messenger for the synchronization of annual functions. So far, most studies performed to understand the regulation of melatonin synthesis have used the non seasonal laboratory rat. It was demonstrated that nocturnal melatonin synthesis depends on alpha- and beta-adrenergic activation of the enzyme arylalkylamine N-acetyltransferase (AA-NAT). In this study, we investigated the mechanisms of melatonin synthesis in the Siberian hamster, a seasonal species with marked photoperiodic variation in melatonin peak duration and amplitude. A beta-adrenergic receptor agonist alone markedly stimulated AA-NAT activity and melatonin synthesis and release. An alpha-adrenergic receptor agonist, while having no effect per se, potentiated the beta-adrenergic stimulation of AA-NAT activity both in vitro and in vivo. Strikingly, the potentiation of AA-NAT activity did not result in a potentiation of melatonin synthesis, suggesting that the rate of melatonin production is limited downstream in the metabolic pathway, most probably at the level of hydroxyindole-O-methyltransferase (HIOMT). HIOMT presented a constitutively high activity that was not acutely (within hours) stimulated by beta-adrenergic agonist, but was rather up-regulated by chronic application of the agonist. This long-term beta-adrenergic regulation may explain the reported large photoperiodic variation of HIOMT activity that drives the photoperiodic variation in melatonin peak.

Syrian hamster and rat display developmental differences in the regulation of pineal arylalkylamine N-acetyltransferase.

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.

Hypocretin (orexin) in the rat pineal gland: a central transmitter with effects on noradrenaline-induced release of melatonin.

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.

In vivo observation of a non-noradrenergic regulation of arylalkylamine N-acetyltransferase gene expression in the rat pineal complex.

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.

Melatonin sees the light: blocking GABA-ergic transmission in the paraventricular nucleus induces daytime secretion of melatonin.

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.

Long-term daily melatonin infusion induces a large increase in N-acetyltransferase activity, hydroxyindole-O-methyltransferase activity, and melatonin content in the Harderian gland and eye of pinealectomized male Siberian hamsters (Phodopus sungorus).

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.

HIOMT drives the photoperiodic changes in the amplitude of the melatonin peak of the Siberian hamster.

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.

Molecular cloning of the arylalkylamine-N-acetyltransferase and daily variations of its mRNA expression in the Syrian hamster pineal gland.

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.