Seiichi Takekida

Light-Induced Resetting of a Mammalian Circadian Clock Is Associated with Rapid Induction of the mPer1 Transcript

To understand how light might entrain a mammalian circadian clock, we examined the effects of light on mPer1, a sequence homolog of Drosophila per, that exhibits robust rhythmic expression in the SCN. mPer1 is rapidly induced by short duration exposure to light at levels sufficient to reset the clock, and dose-response curves reveal that mPer1 induction shows both reciprocity and a strong correlation with phase shifting of the overt rhythm. Thus, in both the phasing of dark expression and the response to light mPer1 is most similar to the Neurospora clock gene frq. Within the SCN there appears to be localization of the induction phenomenon, consistent with the localization of both light-sensitive and light-insensitive oscillators in this circadian center.

Per1 and Per2 gene expression in the rat suprachiasmatic nucleus: circadian profile and the compartment-specific response to light.

Expression profiles of rPer1 and rPer2 messenger RNAs, rat homologues of the Drosophila clock gene period, were examined in the rat suprachiasmatic nucleus, a main locus of circadian oscillation, with special reference to the topographical compartmentation of the suprachiasmatic nucleus. Quantitative in situ hybridization of rPer1 and rPer2 messenger RNAs showed a robust circadian rhythm in the suprachiasmatic nucleus, with a characteristic peak/trough profile in each gene: the peak of rPer1 messenger RNA was in the daytime and that of rPer2 messenger RNA was at the transition time of day to night in both light-dark and constant dark conditions. Light exposure at circadian time 16 increased both rPer1 and rPer2 messenger RNAs in the suprachiasmatic nucleus. In a detailed histological analysis, we found that light exposure at circadian time 16 induced the expression of rPer1 and rPer2 genes in neurons limited to the ventrolateral part of the suprachiasmatic nucleus, although the usual circadian rPer1 and rPer2 messenger RNA oscillation in light-dark and constant dark conditions occurred strongly in neurons in the dorsomedial part but weakly in neurons in the ventrolateral part of the suprachiasmatic nucleus. These rPer expression profiles indicate that the two major subpopulations of neurons in the suprachiasmatic nucleus play different roles in the generation of circadian rhythm: a strong autonomous expression ability with no light response in dorsomedial neurons and a strong light responsiveness with a weak autonomous expression in ventrolateral neurons.

A light-independent oscillatory gene mPer3 in mouse SCN and OVLT.

A new member of the mammalian period gene family, mPer3, was isolated and its expression pattern characterized in the mouse brain. Like mPer1, mPer2 and Drosophila period, mPer3 has a dimerization PAS domain and a cytoplasmic localization domain. mPer3 transcripts showed a clear circadian rhythm in the suprachiasmatic nucleus (SCN). Expression of mPer3 was not induced by exposure to light at any phase of the clock, distinguishing this gene from mPer1 and mPer2. Cycling expression of mPer3 was also found outside the SCN in the organum vasculosum lamina terminalis (OVLT), a potentially key region regulating rhythmic gonadotropin production and pyrogen‐induced febrile phenomena. Thus, mPer3 may contribute to pacemaker functions both inside and outside the SCN.

Phase-dependent responses of Per1 and Per2 genes to a light-stimulus in the suprachiasmatic nucleus of the rat.

Single brief and discrete light treatments are sufficient to reset the overt mammalian rhythms of nocturnal rodents. In the present study, we examined the phase-dependent response of the mammalian clock genes, Per1 and Per2, to a brief strong light-stimulus (1000 lux) in the circadian oscillator center, the suprachiasmatic nucleus (SCN) of rats. Light-induced elevation of Per1 mRNA was observed through the subjective night (CT16, CT20 and CT0 (=CT24)) with a marked peak at the subjective dawn (CT0). However, the light influence was very limited for the induction of Per2; only weak elevation of Per2 mRNA was detected at CT16. The effect of light-stimulus on the Per1 gene was transient, and the effect was restricted to ventrolateral SCN neurons in both CT0 and CT16 after light exposure. Since it is known that these rats show a light-induced behavioral phase-shift throughout the subjective night with being strongest at subjective dawn, the present results suggest that the transient induction of Per1 in ventrolateral SCN neurons is a critical step in the resetting of the biological clock to environmental light-dark schedule.

Differential adrenergic regulation of the circadian expression of the clock genes Period1 and Period2 in the rat pineal gland

Precise temporal regulation of transcription is pivotal to the role of the mammalian pineal gland as a transducer of circadian and seasonal information. The circadian clock genes Per1 and Per2 encode factors implicated in temporally gated transcriptional programmes in brain and pituitary. Here we show that the nocturnal circadian expression of Per1 and Per2 in the rat pineal gland parallels that of serotonin N‐acetyltransferase (NAT) mRNA, which encodes the rate‐limiting enzyme of melatonin biosynthesis. This rhythm is dependent upon an intact sympathetic innervation. Increases in rPer1 (r indicates rat) and rPer2, as well as rNAT, expression during subjective night were blocked completely by superior cervical ganglionectomy (SCGX). In SCGX rats, the β‐adrenergic receptor agonist isoproterenol rapidly induced the rPer1 mRNA with dynamics very similar to its effect on rNAT mRNA. In contrast, isoproterenol was without effect on expression of rPer2 mRNA. These findings demonstrate that circadian pineal expression of both rPer1 and rPer2 is controlled by sympathetic afferent innervation, but whereas β‐adrenergic signalling regulates rPer1 and rNAT, an alternative route mediates sympathetic regulation over rPer2 expression.

A mammalian ortholog of Drosophila timeless, highly expressed in SCN and retina, forms a complex with mPER1.

It is now becoming clear that the circadian rhythm of behaviours and hormones arises from a rhythm at the level of gene expression, and that mammals and Drosophila essentially use homologous genes as molecular gears in the control of circadian oscillation. In Drosophila, the period and timeless genes form a functional unit of the clock and its autoregulatory feedback loop for circadian rhythm. However, in mammals, the counterpart of timeless has not been found.

Circadian and light induced expression of Per1 and Per2 in the rat suprachiasmatic nucleus

The hypothalamic suprachiasmatic nucleus (SCN) contains a master circadian oscillator that controls daily rhythm of behaviors and hormonal secretion in mammals. Per1 and Per2 are mammalian homologue of the Drosophila clock gene period, and it is known that these transcripts show the circadian rhythm in the mouse SCN. In the present study, we examined the expression of these genes in the rat SCN, since the intranuclear functional subdivision of SCN of this species are well established. In the present study, we investigated the circadian profiles, and the light-inducibility of Per2 and Per2 mRNA, particularly focusing its expression in side the SCN. Using in situ hybridization of 33P-labeled or digoxigeninlabeled cRNA probe, we found that Per1 and Per2 showed a robust sircadian rhythm in the SCN. The amount of rat Per1 and Per2 mRNA showed a peak in the day (subjective day), and trough in the night (subjective night), in both LD (12hours light : 12 hours dark) and DD (constant darkness) conditions. Per1 and Per2 were induced in the SCN by short duration of light exposure at CT16, when the amount of Perl and Per2 mRNA levels are very low. In the SCN, these gene transcripts were expressed in cells located in the ventrolateral part. It is interesting to note that the induction of per genes occurs in the same subdivision of the SCN where VIPergic and GRPergic neurons localize, which peptide-expression was controled by environmental light-dark cycle.