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).

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

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

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

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

Circadian profile and photic regulation of clock genes in the suprachiasmatic nucleus of a diurnal mammal Arvicanthis ansorgei.

The molecular mechanisms of the mammalian circadian clock located in the suprachiasmatic nucleus have been essentially studied in nocturnal species. Currently, it is not clear if the clockwork and the synchronizing mechanisms are similar between diurnal and nocturnal species. Here we investigated in a day-active rodent Arvicanthis ansorgei, some of the molecular mechanisms that participate in the generation of circadian rhythmicity and processing of photic signals. In situ hybridization was used to characterize circadian profiles of expression of Per1, Per2, Cry2 and Bmal1 in the suprachiasmatic nucleus of A. ansorgei housed in constant dim red light. All the clock genes studied showed a circadian expression. Per1 and Per2 mRNA increased during the subjective day and decreased during the subjective night. Also, Bmal1 exhibited a circadian expression, but in anti-phase to that of Per1. The expression of Cry2 displayed a circadian pattern, increasing during the late subjective day and decreasing during the late subjective night. We also obtained the phase responses to light for wheel-running rhythm and clock gene expression. At a behavioral level, light was able to induce phase shifts only during the subjective night, like in other diurnal and nocturnal species. At a molecular level, light pulse exposure during the night led to an up-regulation of Per1 and Per2 concomitant with a down-regulation of Cry2 in the suprachiasmatic nucleus of A. ansorgei. In contrast, Bmal1 expression was not affected by light pulses at the circadian times investigated. This study demonstrates that light exposure during the subjective night has opposite effects on the expression of the clock genes Per1 and Per2 compared with that of Cry2. These differential effects can participate in photic resetting of the circadian clock. Our data also indicate that the molecular mechanisms underlying circadian rhythmicity and photic synchronization share clear similarities between diurnal and nocturnal mammals.

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.

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

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