Different mechanisms of adjustment to a change of the photoperiod in the suprachiasmatic and liver circadian clocks.

Changes in photoperiod modulate the circadian system, affecting the function of the central clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. The aim of the present study was to elucidate the dynamics of adjustment to a change of a long photoperiod with 18 h of light to a short photoperiod with 6 h of light of clock gene expression rhythms in the mouse SCN and in the peripheral clock in the liver, as well as of the locomotor activity rhythm. Three, five, and thirteen days after the photoperiod change, daily profiles of Per1, Per2, and Rev-erbalpha expression in the rostral, middle, and caudal parts of the SCN and of Per2 and Rev-erbalpha in the liver were determined by in situ hybridization and real-time RT-PCR, respectively. The clock gene expression rhythms in the different SCN regions, desynchronized under the long photoperiod, attained synchrony gradually following the transition from long to short days, mostly via advancing the expression decline. The photoperiodic modulation of the SCN was due not only to the degree of synchrony among the SCN regions but also to different waveforms of the rhythms in the individual SCN parts. The locomotor activity rhythm adjusted gradually to short days by advancing the activity onset, and the liver rhythms adjusted by advancing the Rev-erbalpha expression rise and Per2 decline. These data indicate different mechanisms of adjustment to a change of the photoperiod in the central SCN clock and the peripheral liver clock.

Circadian rhythm in salivary melatonin in narcoleptic patients.

Narcolepsy is characterized, beside other features, by excessive daytime sleepiness and disturbed sleep at night. The pineal hormone melatonin may affect the sleep characteristics. The aim of the study was to compare the circadian rhythm in salivary melatonin in narcoleptic patients with that in control healthy subjects; 18 patients and 21 age- and gender-matched controls were involved. Narcoleptic patients exhibited a nocturnal increase in salivary melatonin similar to the one in control subjects. The morning melatonin decline in the narcoleptic group, as opposed to the control group was, however, not significant, as 8 out of 18 patients exhibited elevated melatonin levels also during the day. In these patients, the mean daytime value of the multiple sleep latency test (MSLT) was decreased when compared with that in patients with undetectable daytime melatonin levels. The results suggest that in some narcoleptic patients the circadian rhythm might be disturbed.

Dynamics of the adjustment of clock gene expression in the rat suprachiasmatic nucleus to an asymmetrical change from a long to a short photoperiod.

The molecular clockwork of the rat suprachiasmatic nucleus, the site of the circadian clock, is affected by the photoperiod (Sumová et al., 2003). The aim of the present study was to partly elucidate the dynamics of the adjustment of the clockwork to a change from a long to a short photoperiod accomplished by an asymmetrical prolongation of the dark period into the morning hours. Rats maintained under a regime with 16 h of light and 8 h of darkness per day (LD 16:8) were transferred to LD 8:16, and after 2, 3, and 13 days, daily profiles of Per1, Per2, Bmal1, and Cry1 mRNA were assessed by in situ hybridization. The rhythms of Per1, Per2, and Bmal1 expression adjusted to the change from a long to a short photoperiod with larger phase delays of the morning Per mRNA rise and Bmal1 mRNA decline than of the evening and nighttime Per mRNA decline and Bmal1 mRNA rise. The rhythm of Cry1 expression adjusted to the change by parallel delays of the Cry1 mRNA rise and decline. Adjustment of the Cry1 mRNA rhythm to short days was almost accomplished within 13 days, whereas adjustment of the Per1 and Bmal1 mRNA rhythms took longer. Different dynamics of the adjustment of rhythms in clock gene expression to a change from a long to a short photoperiod suggests complex resetting effects of the photoperiod change.

The rat circadian clockwork and its photoperiodic entrainment during development.

The mammalian circadian pacemaker is located in the suprachiasmatic nucleus (SCN), which is composed of dorsomedial (dm) and ventrolateral (vl) regions. The molecular clockwork responsible for the SCN rhythmicity consists of clock genes and their transcriptional-translational feedback loops. The rat SCN rhythmicity and clockwork are affected by the photoperiod. The aim of this study was to elucidate development of the rat SCN rhythmicity, namely of the rhythmicity of the dm- and vl-SCN and of expression of clock genes and to ascertain when the photoperiod starts to affect the SCN rhythmicity. Rhythmicity of the dm-SCN, measured as the rhythm in spontaneous c-FOS production, developed earlier than that of the vl-SCN, which was measured as the rhythm in c-FOS photoinduction. However, photoperiodic affection of the rhythmicity occurred earlier in the vl-SCN than in the dm-SCN. From the 4 clock genes (Per1, Per2, Cry1 and Bmal1) studied, the expression of Bmal1 and Per1 was rhythmic already in 1-day-old rats; at this age, the Per2 mRNA rhythm just started to form and no rhythm in Cry1 expression was detected. After the second postnatal day, all 4 genes were expressed in a rhythmic manner. Thereafter, the rhythms matured gradually via increasing amplitude. Per1 and Per2 mRNA rhythms started to be affected by the photoperiod at the 10th postnatal day. The data suggest that the rhythms in clock genes expression in the rat SCN develop mostly postnatally. The molecular clockwork may start to be photoperiod-dependent around the 10th postnatal day.

Expression of clock and clock-driven genes in the rat suprachiasmatic nucleus during late fetal and early postnatal development.

The SCN as a site of the circadian clock itself exhibits rhythmicity. A molecular clockwork responsible for the rhythmicity consists of clock genes and their negative and positive transcriptional-translational feedback loops. The authors' previous work showed that rhythms in clock gene expression in the rat SCN were not yet detectable at embryonic day (E) 19 but were already present at postnatal day (P) 3. The aim of the present study was to elucidate when during the interval E19-P3 the rhythms start to develop in clock gene expression and in clock-controlled, namely in arginine-vasopressin (AVP), gene expression. Daily profiles of Per1, Per2, Cry1, Bmal1, and Clock mRNA and of AVP heteronuclear (hn) RNA as an indicator of AVP gene transcription were assessed in the SCN of fetuses at E20 and of newborn rats at P1 and P2 by the in situ hybridization method. At E20, formation of a rhythm in Per1 expression was indicated, but no rhythms in expression of other clock genes or of the AVP gene were detected. At P1, rhythms in Per1, Bmal1, and AVP and a forming rhythm in Per2 but no rhythm in Cry1 expression were present in the SCN. The Per1 mRNA rhythm was, however, only slightly pronounced. The Bmal1 mRNA rhythm, although pronounced, exhibited still an atypical shape. Only the AVP hnRNA rhythm resembled that of adult rats. At P2, marked rhythms of Per1, Per2, and Bmal1 and a forming rhythm of Cry1, but not of Clock, expression were present. The data suggest that rhythms in clock gene expression for the most part develop postnatally and that other mechanisms besides the core clockwork might be involved in the generation of the rhythmic AVP gene expression in the rat SCN during early ontogenesis.

Setting the biological time in central and peripheral clocks during ontogenesis.

In mammals, the principal circadian clock within the suprachiasmatic nucleus (SCN) entrains the phase of clocks in numerous peripheral tissues and controls the rhythmicity in various body functions. During ontogenesis, the molecular mechanism responsible for generating circadian rhythmicity develops gradually from the prenatal to the postnatal period. In the beginning, the maternal signals set the phase of the newly developing fetal and early postnatal clocks, whereas the external light-dark cycle starts to entrain the clocks only later. This minireview discusses the complexity of signaling pathways from mothers and the outside world to the fetal and newborn animals' circadian clocks.

Effect of photic stimuli disturbing overt circadian rhythms on the dorsomedial and ventrolateral SCN rhythmicity.

To ascertain how photic stimuli disturbing overt circadian rhythms affect the endogenous rhythmicity of the suprachiasmatic nucleus (SCN), rats were subjected to constant light (LL) or to a 9-h light pulse encompassing midnight, and rhythms of abundance of the c-Fos-immunoreactive (c-Fos-ir) and the PER1-immunoreactive (PER1-ir) cells were studied during the first 1-2 cycles following release into LL or darkness (DD) within the whole SCN as well as in its ventrolateral (vl) and the dorsomedial (dm) part. LL seemingly abolished the c-Fos rhythm in the whole SCN, while the rhythm persisted in the vl- and dm-SCN. In the dm-SCN, the rhythm of c-Fos-ir was phase-delayed by about 4 h in LL, whereas the rhythm of PER1-ir was affected just slightly. In the vl-SCN, the rhythm of c-Fos photo-induction might be delayed by 5-6 h as compared with the reported rhythm [A. Sumova and H. Illnerova, Am. J. Physiol. 274 (1998) R857-R863], whereas the PER1 profile appeared to be out of phase with that in DD. After a 9-h light pulse encompassing midnight, the rhythm of PER1-ir in the dm-SCN changed just slightly, whereas the PER1 rhythm in the vl-SCN was abolished and there was just an indication of extension of elevated PER1-ir. Altogether, the data indicate that photic stimuli disturbing circadian rhythms affect more dramatically the vl- than the dm-SCN rhythmicity within the first cycles and that in the dm-SCN shifting of the c-Fos rhythm proceeds more rapidly than that of the Per1 rhythm.

Insight into molecular core clock mechanism of embryonic and early postnatal rat suprachiasmatic nucleus.

Rhythmicity of the rat suprachiasmatic nucleus (SCN), a site of the circadian clock, develops prenatally. A molecular clockwork responsible for the rhythmicity consists of clock genes and their negative and positive transcriptional-translational feedback loops. The aim of the present study was to discover the development of the clockwork during ontogenesis. Daily profiles of Per1, Per2, Cry1, Bmal1, and Clock mRNA in the SCN of fetuses at the embryonic day (E)19 and of newborn rats at the postnatal day (P)3 and P10 were assessed by the in situ hybridization method. In addition, daily profiles of PER1, PER2, and CRY1 proteins at E19 were assessed by immunohistochemistry. As early as at E19, all the studied clock genes were already expressed in the SCN. However, no SCN rhythm in their expression was detected; Per1, Cry1, and Clock mRNA levels were low, whereas Bmal1 mRNA levels were high and Per2 mRNA levels were medium. Moreover, no rhythms of PER1, PER2, and CRY1 were detectable, as no immunoreactive cells were present at E19. At P3, rhythms in Per1, Per2, Cry1, and Bmal1, but not in Clock mRNA, were expressed in the SCN. The rhythm matured gradually; at P10, the amplitude of Per1, Per2, and Bmal1 mRNA rhythms was more pronounced than at P3. Altogether, the data show a gradual development of both the positive and negative elements of the molecular clockwork, from no detectable rhythmicity at E19 to highly developed rhythms at P10.

Development of circadian rhythmicity and photoperiodic response in subdivisions of the rat suprachiasmatic nucleus.

To ascertain whether the circadian rhythmicity of the ventrolateral (vl) suprachiasmatic nucleus (SCN) develops concurrently with that of the dorsomedial (dm) SCN and when the rhythmicity starts to respond to day length, i.e., to the photoperiod, rats with their offspring were maintained under either a long photoperiod with 16 h of light and 8 h of darkness per day (LD 16:8) or under a short, LD 8:16 photoperiod. The rhythms of spontaneous c-Fos immunoreactivity in the dm-SCN and of the light-induced c-Fos immunoreactivity in the vl-SCN were studied in the pups. In 3- and 10-day-old rats, the dm-SCN rhythm in spontaneous c-Fos immunoreactivty was already well expressed but a response to a photoperiod similar to that in adult rats has not yet been developed. The vl-SCN gate for insensitivity of c-Fos production to light at certain times was detected in 10-day but not yet in 3-day-old rats: in the latter, light exposure at any daytime induced high c-Fos immunoreactivity. In the 10-day-old pups, similarly as with adult rats, the gate was shorter under LD 8:16 than under LD 16:8, but the difference in the gate duration between the short and the long photoperiod did not yet attain that of adult animals. The data indicate that the circadian rhythmicity may develop sooner in the dm-SCN, than in the vl-SCN, whereas the photoperiodic response may develop sooner in the vl-SCN.

Clock gene daily profiles and their phase relationship in the rat suprachiasmatic nucleus are affected by photoperiod.

Rhythmicity of the rat suprachiasmatic nucleus (SCN), a site of the circadian pacemaker, is affected by daylength; that is, by the photoperiod. Whereas various markers of rhythmicity have been followed, so far there have been no studies on the effect of the photoperiod on the expression of the clock genes in the rat SCN. To fill the gap and to better understand the photoperiodic modulation of the SCN state, rats were maintained either under a long photoperiod with 16 h of light and 8 h of darkness per day (LD16:8) or under a short LD8:16 photoperiod, and daily profiles of Per1, Cry1, Bmal1 and Clock mRNA in darkness were assessed by in situ hybridization method. The photoperiod affected phase, waveform, and amplitude of the rhythmic gene expression as well as phase relationship between their profiles. Under the long period, the interval of elevated Per1 mRNA lasted for a longer and that of elevated Bmal1 mRNA for a shorter time than under the short photoperiod. Under both photoperiods, the morning and the daytime Per1 and Cry1 mRNA rise as well as the morning Bmal1 mRNA decline were closely linked to the morning light onset. Amplitude of Per1, Cry1, and Bmal1 mRNA rhythms was larger under the short than under the long photoperiod. Also, under the short photoperiod, the daily Clock mRNA profile exhibited a significant rhythm. Altogether, the data indicate that the whole complex molecular clockwork in the rat SCN is photoperiod dependent and hence may differ according to the season of the year.

The circadian rhythm of Per1 gene product in the rat suprachiasmatic nucleus and its modulation by seasonal changes in daylength.

The suprachiasmatic nucleus (SCN) of rats maintained under a 12-h light, 12-h dark cycle (LD12:12) as well as of those released into darkness exhibited the rhythm of a clock gene Per1 product, PER1 protein, with the maximum late in the subjective day and early night and minimum in the morning. The rhythm was phase delayed by 6-8 h compared with the reported rhythm of Per1 mRNA in the rat SCN [L. Yan et al. Neuroscience 94 (1999) 141]. Under a long, LD16:8, artificial photoperiod, the interval of elevated PER1-immunoreactivity was at least 4 h longer than that under a short, LD 8:16 photoperiod, due mainly to an earlier PER1 day-time rise under the long photoperiod. Under a natural photoperiod, profiles of the PER1 rhythm in summer and in winter resembled those under corresponding artificial photoperiods; therefore, twilight did not affect the rhythm in a substantial way. Under all photoperiods, when PER1 immunoreactivity was elevated, immunopositive cells were localized in the dorsomedial rather than in the ventrolateral part of the SCN. As the Per1 gene is a part of a molecular clockwork and as the rhythm of its product is modulated by the photoperiod, it appears that the whole molecular clockwork in the rat SCN is photoperiod-dependent and thus shaped by the season of the year.