Synchronized ATP oscillations have a critical role in prechondrogenic condensation during chondrogenesis.

The skeletal elements of embryonic limb are prefigured by prechondrogenic condensation in which secreted molecules such as adhesion molecules and extracellular matrix have crucial roles. However, how the secreted molecules are controlled to organize the condensation remains unclear. In this study, we examined metabolic regulation of secretion in prechondrogenic condensation, using bioluminescent monitoring systems. We here report on ATP oscillations in the early step of chondrogenesis. The ATP oscillations depended on both glycolysis and mitochondrial respiration, and their synchronization among cells were achieved via gap junctions. In addition, the ATP oscillations were driven by Ca(2+) oscillations and led to oscillatory secretion in chondrogenesis. Blockade of the ATP oscillations prevented cellular condensation. Furthermore, the degree of cellular condensation increased with the frequency of ATP oscillations. We conclude that ATP oscillations have a critical role in prechondrogenic condensation by inducing oscillatory secretion.

Phase-advance shifts of human circadian pacemaker are accelerated by daytime physical exercise.

Effects of forced sleep-wake schedules with and without physical exercise were examined on the human circadian pacemaker under dim light conditions. Subjects spent 15 days in an isolation facility separately without knowing the time of day and followed a forced sleep-wake schedule of a 23 h 40-min period for 12 cycles, and physical exercise was imposed twice per waking period for 2 h each with bicycle- or rowing-type ergometers. As a result, plasma melatonin rhythm was significantly phase advanced with physical exercise, whereas it was not changed without exercise. The difference in phase was already significant 6 days after the start of exercise. The amplitude of melatonin rhythm was not affected. A single pulse of physical exercise in the afternoon or at midnight significantly phase delayed the melatonin rhythms when compared with the prepulse phase, but the amount of phase shift was not different from that observed in the sedentary controls. These findings indicate that physical exercise accelerates phase-advance shifts of the human circadian pacemaker associated with the forced sleep-wake schedule.

Effects of light and sleep stages on heart rate variability in humans.

Effects of light intensity and sleep stages on heart rate variability (HRV) were investigated in young healthy subjects. The low-frequency (LF)/high-frequency (HF) ratio was significantly increased by exposing either to bright lights of 10 000 lx or to extreme darkness (< 0.01 lx), while HF and LF components of HRV were not changed, when compared with those under dim light (100 lx). However, LF was significantly increased at REM sleep, when compared with that at the pre-sleep wake. In contrast, HF was increased at all stages of sleep, and the LF/HF ratio was decreased at slow wave sleep during the baseline night.

Continuous measurement of targeted promoter activity by a secreted bioluminescence reporter, Vargula hilgendorfii luciferase.

The promoter activity of growth hormone (GH) was continuously monitored in rat pituitary adenoma cells (GH3) by a secreted bioluminescence reporter, Vargula hilgendorfii (Vh) luciferase. The sensitivity of the reporter was approximately 60-times higher than that of the firefly luciferase. GH3 cells were transfected with a plasmid containing a DNA sequence of the GH promoter (1.8 kb) and a full length of the Vh luciferase cDNA. Using the stable transformants, the Vh luciferase activity was monitored in the plate culture through the bioluminescence of Vh luciferase secreted into the culture medium. The reporter activity was well correlated with GH mRNA as well as GH when the GH promoter activity was activated by thyroid hormone. To develop a continuous monitoring system of the promoter, the reporter activity was sequentially measured in the perfusion system. When the promoter activity of the stable transformants was suppressed by a transcription inhibitor, the reporter activity and GH in the perfusate were simultaneously decreased. The Vh luciferase reporter is a sensitive and convenient tool for continuous and prolonged measurement of promoter activity in living cell culture systems.

Synchronization of circadian firing rhythms in cultured rat suprachiasmatic neurons.

The circadian clock in mammals is located in the suprachiasmatic nucleus (SCN) which consists of multiple oscillating neurons. Integration of the cellular oscillations is essential for the generation of a single circadian period in the SCN. By using a multielectrode dish (MED), we measured circadian firing rhythms in individual SCN neurons for more than 2 weeks continuously, and examined the involvement of synaptic communication in the synchronization of circadian rhythms. Cross-correlation analysis of spontaneous action potentials revealed that a neuron pair was functionally connected by synapses when their circadian rhythms were synchronized. No correlation was found between the paired neurons whose circadian rhythms were not synchronized. Calcium (Ca2+)-dependent synaptic transmission in the cellular communication was indicated by dose-dependent lengthening of an intercellular spike interval and loss of spike correlation with a Ca2+ channel blocker. Approximately 60% of the SCN neurons in culture were immunoreactive to antibodies against gamma-aminobutyric acid (GABA) or glutamic acid decarboxylase (GAD). Spontaneous firing of all the neurons tested was either increased or decreased by bicuculline, the GABAA receptor antagonist. These findings indicate that synaptic communication plays a critical role in the synchronization of circadian rhythms in individual SCN neurons and the GABAergic transmission is involved in the synchronization mechanism.

cDNA cloning of a novel bHLH-PAS transcription factor superfamily gene, BMAL2: its mRNA expression, subcellular distribution, and chromosomal localization.

We isolated a human cDNA encoding a novel member of the bHLH-PAS transcription factor superfamily, BMAL2, which is highly similar to, but distinct from, BMAL1. The composite cDNA covered a 1720-bp sequence consisting of a putative 1653-bp open reading frame encoding a polypeptide of 551 amino acids. The deduced BMAL2 product contains a bHLH-PAS domain in its N-terminal region and a variable C-terminus. The overall identity of BMAL2 polypeptide to that of human BMAL1 is 49%. RNA analysis revealed that expression of BMAL2 transcripts was restricted to the fetal brain and to the adult liver in human, while human BMAL1 mRNA was expressed in the brain and skeletal muscle. The chromosomal localization of the human BMAL2 gene was determined by fluorescent in situ hybridization to be localized on chromosome 12 at region p12.2-p11.2. These results suggest that BMAL2 may play different roles from BMAL1 in the embryonic brain and in adult mammals.

After-effect of entrainment on the period of human circadian system.

The purpose of the present study is to examine changes of the circadian period in humans during temporal isolation, where the circadian rhythm is free-running. Twelve young males each spent 22 d alone in a temporal isolation room. Rectal temperature was continuously recorded and plasma melatonin was measured on day 3 (D3) and from days 10 to 13 (D10-D13). The light intensity in the room was less than 100 lx during the waking period. The free-run period of temperature rhythm from D3 to D10 (24.75 h) was not significantly different from those of the plasma melatonin rhythm (24.63 h) and sleep-wake cycle (24.69 h). The free-run period of temperature rhythm was 24.58 h in S-1 (D2-D6), which was gradually and significantly lengthened to 24.84 h in S-2 (D6-D10), 25.16 h in S-3 (D12-D16), and 25.18 h in S-4 (D18-D22). The free-run period of rectal temperature rhythm was steadily lengthened throughout the isolation period, which was probably due to the after-effect of previous entrainment to 24-h time cues.

Circadian modulation in photic induction of Fos-like immunoreactivity in the suprachiasmatic nucleus cells of diurnal chipmunk, Eutamias asiaticus.

Photic induction of immediate early genes including c-fos in the suprachiasmatic nucleus (SCN) has been well demonstrated in the nocturnal rodents. On the other hand, in diurnal rodents, no data is available whether the light can induce c-fos or Fos in the SCN. We therefore examined whether 60 min light exposure induces Fos-like immunoreactivity (Fos-lir) in the SCN cells of diurnal chipmunks and whether the induction is phase dependent, comparing with the results in nocturnal hamsters. We also examined an effect of light on the locomotor activity rhythm under continuous darkness. Fos-lir was induced in the chipmunk SCN. The induction was clearly phase dependent. The light during the subjective night induced strong expression of Fos-lir. This phase dependency is similar to that in hamsters. However, unlike in hamsters, the Fos-lir was induced in some SCN cells of chipmunks exposed to light during the subjective day. In the locomotor rhythm, on the other hand, the light pulse failed to induce the phase shift at phases at which the Fos-lir was induced. These results suggest that the photic induction of Fos-lir in the diurnal chipmunks is gated by a circadian oscillator as well as in the nocturnal hamsters. However, the functional role of Fos protein may be different in the diurnal rodents from in the nocturnal rodents.

Maternal phase setting of fetal circadian oscillation underlying the plasma corticosterone rhythm in rats.

We examined the entraining effect of the maternal circadian system on the fetal circadian oscillation during pregnancy. The circadian rhythm of locomotor activity and plasma corticosterone of pregnant rats was abolished by bilateral ablation of the suprachiasmatic nuclei at day 10 of gestation. At term, pups were removed by Cesarean section and were blinded immediately. To avoid possible rhythmic influences of a nursing mother on the pups' circadian rhythms, alternating nursing was imposed on blinded infants. Thus, pups were exchanged every 12 h between two foster mothers, one entrained to a light-dark cycle and the other to dark-light, so that one group of pups were always nursed in the light period and the other in the dark. Both groups of pups showed free-running circadian hormone rhythms with similar phase angles. However, the circadian rhythm of these pups was always phase delayed by about 8 h to that of blinded control pups which were born to unoperated mothers. Furthermore, blinded pups born to and nursed by a suprachiasmatic nuclei lesioned mother developed a circadian hormone rhythm which was phase delayed by 4 h to that of the control. It is concluded that the circadian oscillation underlying the rhythm of corticosterone release in rats has entrained to the maternal circadian system during fetal life. It is further suggested that the entrainment starts before day 10 of gestation.

Feeding-associated corticosterone peak in rats under various feeding cycles.

Features of the anticipatory (prefeeding) corticosterone peak under restricted feeding were examined in rats by forcing them to various feeding schedules. Meal feeding appears specific in inducing the anticipatory corticosterone peak, since other regular manipulations such as daily cleaning of the rat cage and sampling of vaginal smear were without effect. Under meal intervals of 22,24, and 26 h, the phase relation of prefeeding hormone peak to meal was 30 min, 2.5 h, and 4.5 h, respectively. When the interval was extended to 30 h, a hormone peak appeared not immediately but several hours before meal, and when it was shortened to 18 h, hormone peak was not observed before meal but appeared when regular meal was omitted; the interval between a preceding meal and the hormone peak was similarly 18-20 h in both cases. On the other hand, a single meal feeding had a bidirectional immediate effect on plasma hormone levels, namely, a postprandial elevation of the basal hormone level and a postprandial reduction of the already elevated prefeeding hormone level under restricted feeding. Both effects were observed alternately when rats were fed twice a day for 2 wk. It is concluded that the 24-h pattern of plasma corticosterone level in rats under restricted feeding is determined primarily by the interaction of three different factors: a light- entrainable circadian rhythm, a separate oscillation associated with meal feeding, and a bidirectional effect of a single meal.

Effects of elimination of maternal circadian rhythms during pregnancy on the postnatal development of circadian corticosterone rhythm in blinded infantile rats.

The influence of maternal circadian rhythms on fetal circadian oscillations during pregnancy was examined. Circadian rhythms of spontaneous locomotor activity and plasma corticosterone level in pregnant rats were eliminated by bilateral lesions of the suprachiasmatic nuclei (SCN) at different stages of gestation and the postnatal manifestation of the circadian corticosterone rhythm was examined in individual pups. Effective lesions of SCN at day 3 of gestation resulted in an abortion in all rats examined. Rats whose SCN were lesioned at day 10 or 17 of gestation maintained their pregnancies. At term, pups were removed by Cesarean operation and immediately blinded by bilateral ocular enucleation; they were reared by unoperated foster mother afterwards. All pups from SCN lesioned mothers showed a clear free running circadian rhythm of plasma corticosterone levels after the 4th week of postnatal life. Furthermore, the circadian rhythm that developed in pups from SCN-lesioned mother at day 10 of gestation (G10 pups) was always phase-delayed about 4 h as compared with that in pups from mothers whose SCN were lesioned at day 17 of gestation (G17 pups) and in pups from sham-operated mothers (S pups). It is concluded that the circadian hormone rhythmicity develops in normal fashion postnatally when the maternal SCN are effectively lesioned after day 10 of gestation. The phase-angle difference in the circadian rhythm between G10 pups and G17 or S pups suggests that fetal circadian oscillation is entrainable at least after day 10 of gestation.

Critical role of food amount for prefeeding corticosterone peak in rats.

The effects of food on plasma corticosterone levels were examined in rats under restricted daily feeding or prolonged food deprivation. High hormone levels before feeding were observed when the daily meal was restricted to 2 h at a fixed time of day, but it was not detected when food availability was extended to 6 h. The amount of food intake under the latter condition was comparable to that in 24 h of ad libitum feeding. After the termination of restricted feeding, the prefeeding hormone peak was maintained in rats fasted subsequently but disappeared when rats were returned to ad libitum feeding. Food deprivation for 10 days increased plasma corticosterone levels in the light period, resulting in abolition of the circadian rhythm. A subsequent meal decreased the hormone level such that the 24-h mean hormone level after food ingestion was inversely related to the amount of food intake. When rats were allowed to feed for 6 h after prolonged food deprivation, the prefeeding hormone peak observed at the second meal disappeared at the fourth meal. The amount of food consumption in these rats increased and reached a level comparable to that with ad libitum feeding at the third meal. It is concluded that the amount of food intake is critical for the development and maintenance of the prefeeding hormone peak under restricted feeding; prolonged fasting.

Effects of parachlorophenylalanine and 5, 6-dihydroxytryptamine on the free-running rhythms of locomotor activity and plasma corticosterone in the rat exposed to continuous light.

Parachlorophenylalanine (PCPA) and 5,6-dihydroxytryptamine (5,6-DHT), depletors of brain serotonin, were administered to the rat and circadian rhythms of locomotor activity and plasma corticosterone were determined simultaneously in individual rats in light-dark cycles (LD) and in 200 lux continuous light (LL). Free-running periods and acrophases on the 12th day in LL (LL12) were calculated by a least squares spectrum method. In PCPA-treated rats which showed 70% depletion of brain serotonin, circadian rhythms of locomotor activity in LL and of plasma corticosterone and ACTH in LD disappeared for several days after the drug injection. Circadian rhythms of locomotor activity reappeared after the LL7 day and free-ran with a phase shift. Free-running periods of these rats did not differ significantly from that of control rats. However, the acrophase of PCPA-treated group on the LL11 day was 5 h advanced as compared to that of control. Circadian rhythm of plasma corticosterone in the PCPA-treated rats was detected on the LL12 day but their peak times were distributed around 24:00 h instead 08:00 h observed rats. The 5,6-DHT-treated rats which showed only 40% depletion of brain serotonin exhibited normal free-running rhythms in both locomotor activity and plasma corticosterone in LL and no difference in the acrophases of these functions on the LL12 day as compared to controls. These results suggest that PCPA affects the circadian clock (or clocks) itself in such a way that it blocks the clock to free-run or at least it effectively shortens the free-running periods of locomotor activity and plasma corticosterone in the rat.

Participation of brain catecholaminergic neurons in a self-sustained circadian oscillation of plasma corticosterone in the rat.

Circadian rhythms of locomotor activity and plasma corticosterone were determined simultaneously in individual rats which were injected intraventricularly with 6-hydroxydopamine (6-OHDA), a specific depletor of catecholamines. In light-dark cycles (LD), 6-OHDA-treated rats showed essentially normal circadian rhythms of both functions. In 200 lux continuous light (LL) the locomotor activity in the drug-treated rats ran freely with a period slightly longer than 24 h: free-running parameters (mesor, amplitude and period) of the locomotor activity were the same as those control rats. The circadian rhythm of plasma corticosterone in control rats also ran freely in LL, resulting in a reversal of their phase after 12 days exposure to LL. However, the rhythm disappeared in 6-OHDA-treated rats under LL: instead in these rats 3--4 episodes of secretion were obsered over a 24 h period. These results suggest a difference in the biochemical background between the regulatory mechanism of circadian rhythm of locomotor actitivity and that of plasma corticosterone. It is also apparent that brain catecholaminergic components are indispensable for the circadian rhythm of plasma corticosterone to freerun normally in LL, but are dispensable in LD. A plausible model consisting of two oscillators is proposed to explain these findings.

Internal synchronization among several circadian rhythms in rats under constant light.

Three biological rhythms (locomotor activity, body temperature, and plasma corticosterone) were measured simultaneously in individual rats under light-dark cycles and continuous light. Spontaneous locomotor activity was recorded on an Animex and body temperature was telemetrically monitored throughout the experiments. Blood samples were obtained serially at 2-h intervals on the experimental days. Phase angles of these rhythms were calculated by a least-squares spectrum analysis. Under light-dark cycles, the acrophases of locomotor activity, body temperature, and plasma corticosterone were found at 0029, 0106, and 1940 h, respectively. When rats were exposed to 200 lx continuous light, locomotor activity and body temperature showed free-running rhythms with a period of 25.2 h on the average. Plasma corticosterone levels determined at 12 days after exposure to continuous light exhibited a circadian rhythm with the acrophase shifted to 0720. The acrophases of locomotor activity and body temperature, determined simultaneously on the same day, were found to be located at 1303 and 1358 h, respectively. Phase-angle differences among the three rhythms on the 12th day of continuous light were essentially the same with those under the light-dark cycle. These results suggest that circadian rhythms of locomotor activity, body temperature, and plasma corticosterone are most probably coupled to a common internal oscillator in the rat.

Endogenous ultradian rhythms in rats exposed to prolonged continuous light.

Circadian rhythms of locomotor activity, body temperature, and plasma corticosterone were determined simultaneously in individual rats that were exposed to 200 lx continuous light for over 3 mo. Free-running circadian rhythms of locomotor activity persisted for about 2 mo under continuous light and then the rhythms gradually decomposed. After 3 mo of exposure, circadian rhythms disappeared and activity bursts of 1- to 2-h duration manifested themselves several times during a 24-h period. Body temperature also exhibited several bursts of fluctuation and these bursts were closely correlated in their temporal sequence with those of locomotor activity. A least-squares spectrum analysis revealed that the burst had regular 4- to 6-h periods. Plasma corticosterone, determined by serial sampling at 2-h intervals from individual rats, also exhibited several secretion episodes in a day. These episodic secretions synchronized with bursts of locomotor activity. These results suggest that the ultradian component, manifested under prolonged continuous light, is a fundamental unit of the circadian rhythm and an oscillator for the ultradian rhythm is common to the three functions examined.