Alena Sumová

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.

Spontaneous rhythm in c-Fos immunoreactivity in the dorsomedial part of the rat suprachiasmatic nucleus

In rats maintained for 2 days in constant darkness, the suprachiasmatic nucleus exhibited a circadian rhythm in c-Fos immunoreactivity, with the maximum in the morning and trough during the subjective night. In contrast to the night-time photic c-Fos induction occurring in the ventrolateral part of the nucleus, the spontaneous rhythmic c-Fos induction in darkness occurred in the dorsomedial part and might indicate an elevated dorsomedial neuronal activity in the early subjective day.

Hepatic, Duodenal, and Colonic Circadian Clocks Differ in their Persistence under Conditions of Constant Light and in their Entrainment by Restricted Feeding

Physiological functions of the gastrointestinal tract (GIT) are temporally controlled such that they exhibit circadian rhythms. The circadian rhythms are synchronized with the environmental light-dark cycle via signaling from the central circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus, and by food intake. The aim of the study was to determine the extent to which disturbance in the SCN signaling via prolonged exposure to constant light affects circadian rhythms in the liver, duodenum, and colon, as well as to determine whether and to what extent food intake can restore rhythmicity in individual parts of the GIT. Adult male rats were maintained in constant light (LL) for 30 days and fed ad libitum throughout the entire interval or exposed to a restricted feeding (RF) regime for the last 14 days in LL. Locomotor and feeding behaviors were recorded throughout the experiment. On the 30th day, daily expression profiles of clock genes (Per1, Per2, Rev-erbα, and Bmal1) and of clock-controlled genes (Wee1 and Dbp) were measured by real-time reverse transcriptase–polymerase chain reaction (RT-PCR) in the duodenum, colon, and liver. By the end of the LL exposure, rats fed ad libitum had completely lost their circadian rhythms in activity and food intake. Daily expression profiles of clock genes and clock-controlled genes in the GIT were impaired to an extent depending on the tissue and gene studied, but not completely abolished. In the liver and colon, exposure to LL abolished circadian rhythms in expression of Per1, Per2, Bmal1, and Wee1, whereas it impaired, but preserved, rhythms in expression of Rev-erbα and Dbp. In the duodenum, all but Wee1 expression rhythms were preserved. Restricted feeding restored the rhythms to a degree that varied with the tissue and gene studied. Whereas in the liver and duodenum the profiles of all clock genes and clock-controlled genes became rhythmic, in the colon only Per1, Bmal1, and Rev-erbα—but not Per2, Wee1, and Dbp—were expressed rhythmically. The data demonstrate a greater persistence of the rhythmicity of the circadian clocks in the duodenum compared with that in the liver and colon under conditions when signaling from the SCN is disrupted. Moreover, disrupted rhythmicity may be restored more effectively by a feeding regime in the duodenum and liver compared to the colon. (Author correspondence: sumova@biomed.cas.cz)

Photic Entrainment of the Mammalian Rhythm in Melatonin Production

This review summarizes studies on the photic entrainment of the circadian rhythm in the rat pineal melatonin production, namely of the rhythm in N-acetyltransferase (NAT) activity, and compares the NAT rhythm resetting with preliminary results on the resetting of an intrinsic rhythmicity in the suprachiasmatic nucleus (SCN) of the hypothalamus, namely with the entrainment of the rhythm in the light-induced c-fos gene expression. Phase delaying of the NAT rhythm after various light stimuli proceeds within 1 day with almost no transients, whereas during phase advancing of the rhythm only the morning NAT decline is phase advanced within 1 day and the evening rise phase shifts through transients. A light stimulus encompassing the middle of the night may phase delay the evening NAT rise, phase advance the morning decline, compress the rhythm waveform, and eventually lower its amplitude. Similarly, a long photoperiod compresses the NAT rhythm waveform. The magnitude of phase shifts of the NAT rhythm, as well as their direction, depends on a previous photoperiod. Phase shifts of the evening rise in c-fos gene photoinduction in the SCN and of the morning decline are similar to those of the pineal NAT rhythm after all light stimuli studied so far. The data indicate that the resetting of the rhythm in melatonin production in the rat pineal gland reflects changes in the SCN functional state and suggest that the underlying SCN pacemaking system is complex.

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.

Serotonergic antagonists impair arousal-induced phase shifts of the circadian system of the Syrian hamster

Single episodes of arousal of Syrian hamsters 2 h before projected activity onset (i.e., CT 10) phase-advanced their free-running circadian rhythm of wheel-running. Serial arousal once every 23 h or once every 23.5 h for 7 days caused large composite phase-advances to the wheel-running rhythm, the latter period being more effective in supporting an interval of stable entrainment. Pre-treatment of hamsters at CT 6 with the serotonergic antagonist ritanserin (1-5 mg/kg, which acts at both 5-HT2 and the putative 5-HT7 receptor, impaired the phase-advancing response to arousal at CT 10 but the drug was without effect on phase advances induced by exposure to light. Pre-treatment with a second serotonergic antagonist, ketanserin (1-5 mg/kg), which is without effect at 5-HT7 but has high affinity for 5-HT2 receptors, was also effective in attenuating the phase advancing effect of arousal at CT 10. However, neither agent was able to achieve complete blockade of the phase advances. These results are discussed in relation to in vitro and in vivo studies in the rat which have identified a role for 5-HT7 receptors in serotonin-mediated circadian entrainment.

Daily profiles of arginine vasopressin mRNA in the suprachiasmatic, supraoptic and paraventricular nuclei of the rat hypothalamus under various photoperiods

Daily rhythm of arginine vasopressin (AVP) mRNA levels in the suprachiasmatic nucleus (SCN) of rats maintained under a short, LD 8:16 photoperiod differed from that of rats maintained under a long, LD 16:8 photoperiod: under the short photoperiod the morning AVP rise occurred significantly later than under the long one. Daily profiles of AVP mRNA in the supraoptic and paraventricular nuclei were not rhythmic and AVP mRNA levels under LD 8:16 did not differ from those under LD 16:8. The data indicate that photoperiod affects selectively the clock driven AVP gene expression in the SCN.

Salivary Melatonin Rhythm as a Marker of the Circadian System in Healthy Children and Those With Attention-Deficit/Hyperactivity Disorder

Attention-deficit/hyperactivity disorder (ADHD) is the most common neurobehavioral disorder of childhood. Problems with sleep structure, efficiency, and timing have been reported in some, but not all, studies on ADHD children. As the sleep-wake cycle belongs to circadian rhythms, the timekeeping circadian system might be involved in ADHD. To assess whether the circadian system of ADHD children differs from that of controls, the rhythm of the pineal hormone melatonin was used as a reliable marker of the system. Saliva from 34 ADHD and 43 control 6- to 12-yr-old children was sampled at 2-h intervals throughout the entire 24-h cycle, and the melatonin profiles of the ADHD and control children were compared. The nocturnal melatonin peaks of the ADHD and control group did not differ significantly. The high nocturnal interindividual variability of the peaks seen in adulthood was present already in the studied children. The 24-h melatonin profiles of all the ADHD subjects did not differ significantly from those of the control subjects. Categorization of subjects according to age, into groups of 6- to 7-yr-old (9 ADHD, 5 control), 8- to 9-yr-old (16 ADHD, 26 control), and 10- to 12-yr-old (9 ADHD, 12 control) children, revealed significant differences between the ADHD and control group in the melatonin rhythm waveform, but not in nocturnal melatonin peaks; the peaks were about the same in both groups and did not change significantly with increasing age. In the oldest, but not in the younger, children, the melatonin signal duration in the ADHD group was shorter than in the control group. The difference might be due to the fact that whereas in the control group both the evening melatonin onset and the morning offset phase delayed in the oldest children relative to those in the youngest children, in the ADHD group only the onset, but not the offset, phase delayed with increasing age. The data may indicate subtle differences between the circadian system of ADHD and control children during development. (Author correspondence: sumova@biomed.cas.cz)

Memory on long but not on short days is stored in the rat suprachiasmatic nucleus

In order to follow adjustment of the rat circadian pacemaking system to a change of the photoperiod, the rhythm in the light-induced c-fos expression was used as a marker of the intrinsic rhythmicity of the suprachiasmatic nucleus (SCN). After a change from a long photoperiod with 16 h of light per day to a short one with 8 h of light, the interval enabling high c-fos photoinduction decompressed gradually and the full extension by 5-6 h was achieved only within 2 weeks. After a change from a short to a long photoperiod, the interval was compressed by 5-6 h within 3 days. The data indicate a rapid adjustment of the photoperiod dependent-state of the SCN pacemaker to long days but only a slow one to short days.

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.