Sato Honma

Melatonin induces γ-glutamylcysteine synthetase mediated by activator protein-1 in human vascular endothelial cells

Abstract In the present study, we show that melatonin induces the expression of γ-glutamylcysteine synthetase (γ-GCS), the rate-limiting enzyme of glutathione (GSH) synthesis, in ECV304 human vascular endothelial cells. One micromolar melatonin induced the expression of γ-GCS mRNA followed by an increase in the concentration of GSH with a peak at 24 h. An electrophoretic mobility shift assay showed that melatonin stimulates the DNA-binding activity of activator protein-1 (AP-1) as well as retinoid Z receptor/retinoid receptor-related orphan receptor α (RZR/RORα). ECV304 cells transiently transfected with a plasmid containing the γ-GCS promoter-luciferase construct showed increased luciferase activity when treated with melatonin. The melatonin-dependent luciferase activity was found in the γ-GCS promoter containing AP-1 site. The luciferase activity mediated by AP-1 was repressed in the promoter containing RZR/RORα site. In addition, cell cycle analysis showed that melatonin increases the number of cells in the G0/G1 phase; however, treatment of the cells with buthionine sulfoximine, a specific inhibitor of γ-GCS, abolished the effect of melatonin on the cell cycle, suggesting induction of cell arrest by melatonin requires GSH. As conclusion, induction of GSH synthesis by melatonin protects cells against oxidative stress and regulates cell proliferation.

Separate oscillating cell groups in mouse suprachiasmatic nucleus couple photoperiodically to the onset and end of daily activity

The pattern of circadian behavioral rhythms is photoperiod-dependent, highlighted by the conservation of a phase relation between the behavioral rhythm and photoperiod. A model of two separate, but mutually coupled, circadian oscillators has been proposed to explain photoperiodic responses of behavioral rhythm in nocturnal rodents: an evening oscillator, which drives the activity onset and entrains to dusk, and a morning oscillator, which drives the end of activity and entrains to dawn. Continuous measurement of circadian rhythms in clock gene Per1 expression by a bioluminescence reporter enabled us to identify the separate oscillating cell groups in the mouse suprachiasmatic nucleus (SCN), which composed circadian oscillations of different phases and responded to photoperiods differentially. The circadian oscillation in the posterior SCN was phase-locked to the end of activity under three photoperiods examined. On the other hand, the oscillation in the anterior SCN was phase-locked to the onset of activity but showed a bimodal pattern under a long photoperiod [light–dark cycle (LD)18:6]. The bimodality in the anterior SCN reflected two circadian oscillatory cell groups of early and late phases. The anterior oscillation was unimodal under intermediate (LD12:12) and short (LD6:18) photoperiods, which was always phase-lagged behind the posterior oscillation when the late phase in LD18:6 was taken. The phase difference was largest in LD18:6 and smallest in LD6:18. These findings indicate that three oscillating cell groups in the SCN constitute regionally specific circadian oscillations, and at least two of them are involved in photoperiodic response of behavioral rhythm.

Circadian periods of single suprachiasmatic neurons in rats

Neuronal activity of a single neuron was monitored continuously for more than 5 days by means of a multi-electrode dish in dispersed cell culture of the rat suprachiasmatic nucleus (SCN). Sixty-seven out of 88 neurons showed a robust circadian rhythm in firing rate. The mean circadian period was 24.2 h, which was almost identical to that of the locomotor activity rhythm in 114 weanling rats blinded on the day of birth. However, the circadian period in individual SCN neurons was scattered from 20.0 to 28.3 h (SD, 1.4 h), while the period of activity rhythm clustered from 24.0 to 24.8 h (SD, 0.2 h). It is concluded that a large number of SCN neurons contain the circadian oscillator, the period of which is more variable than the circadian period of the SCN as a whole. It is suggested that the circadian rhythms in individual SCN neurons are capable of synchronizing to each other and are integrated to constitute a multiple oscillator system(s) within the SCN.

Clock genes outside the suprachiasmatic nucleus involved in manifestation of locomotor activity rhythm in rats

Chronic treatment of methamphetamine (MAP) in rats desynchronized the locomotor activity rhythm from the light–dark cycle. When the activity rhythm was completely phase‐reversed with respect to a light dark‐cycle, 24 h profiles were examined for the clock gene (rPer1, rPer2, rBMAL1, rClock) expressions in several brain structures by in situ hybridization, and for the pineal as well as plasma melatonin levels. In the MAP‐treated rats, the rPer1 expression in the suprachiasmatic nucleus (SCN) showed a robust circadian rhythm which was essentially identical to that in the control rats. Circadian rhythms in pineal as well as plasma melatonin were not changed significantly in the MAP‐treated rats. However, robust circadian rhythms in the rPer1, rPer2 and rBMAL1 expressions detected in the caudate‐putamen (CPU) and parietal cortex were completely phase‐reversed in the MAP‐treated rats, compared with those in the control rats, indicating desynchronization from the SCN rhythm. Such desynchronization was not observed in the circadian rhythms of clock gene expression in the nucleus accumbens and cingulate cortex. The circadian rClock expression rhythm in the MAP‐treated rats was not phase‐reversed in the CPU and parietal cortex. These findings indicate that the locomotor activity rhythm in rats is directly driven by the pacemaker outside the SCN, in which rPer1, rPer2 and rBMAL1 in the CPU and parietal cortex are involved.

Activity rhythms in the circadian domain appear in suprachiasmatic nuclei lesioned rats given methamphetamine

Female rats were lesioned in the suprachiasmatic nuclei (SCN) electrolytically and treated with methamphetamine. The SCN lesions abolished the circadian locomotor rhythm completely. When methamphetamine was administered in the drinking water, robust rhythmicities in locomotor activity appeared in the SCN lesioned rats, which did not entrain to the 24 hr light cycle. The period of the activity rhythm was dose-dependent; the lower the concentration of methamphetamine was, the shorter the period of the rhythm became. When rats were treated with 0.005% methamphetamine, the mean period was 26.4 hours. In addition, activity time (alpha) became shorter, rest time (rho) longer and alpha/rho ratio lower, when methamphetamine concentration was decreased. After methamphetamine withdrawal, the rhythmicity disappeared and locomotor activity became aperiodic again. When methamphetamine was administered continuously by means of an osmotic minipump, similar rhythmicities appeared in locomotor activity of the SCN lesioned rats. It is concluded that methamphetamine manifests an activity rhythm whose period is in the circadian range. The rhythmicity is independent of the SCN and is not entrained by the light-dark cycle.

Human blood metabolite timetable indicates internal body time.

A convenient way to estimate internal body time (BT) is essential for chronotherapy and time-restricted feeding, both of which use body-time information to maximize potency and minimize toxicity during drug administration and feeding, respectively. Previously, we proposed a molecular timetable based on circadian-oscillating substances in multiple mouse organs or blood to estimate internal body time from samples taken at only a few time points. Here we applied this molecular-timetable concept to estimate and evaluate internal body time in humans. We constructed a 1.5-d reference timetable of oscillating metabolites in human blood samples with 2-h sampling frequency while simultaneously controlling for the confounding effects of activity level, light, temperature, sleep, and food intake. By using this metabolite timetable as a reference, we accurately determined internal body time within 3 h from just two anti-phase blood samples. Our minimally invasive, molecular-timetable method with human blood enables highly optimized and personalized medicine.

Clock mutation lengthens the circadian period without damping rhythms in individual SCN neurons

Spontaneous discharges of individual neurons in the suprachiasmatic nucleus (SCN) of Clock mutant mice were recorded for over 5 days in organotypic slice cultures and dispersed cell cultures using a multi-electrode dish. Circadian rhythms with periods of about 27 hours were detected in 77% of slice cultures and 15% of dispersed cell cultures derived from Clock/Clock homozygotes. These findings indicate that the Clock mutation lengthens the circadian period but does not abolish the circadian oscillation, and suggest an important role of intercellular communication in the expression of circadian rhythm in the SCN.

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 γ‐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.

Regional pacemakers composed of multiple oscillator neurons in the rat suprachiasmatic nucleus

Regional specificities of the dorsal and ventral regions of the suprachiasmatic nucleus (SCN) were examined to elucidate the structure of multioscillator circadian organization. The circadian rhythms of arginine vasopressin (AVP) and vasoactive intestinal polypeptide (VIP) release, and of electrical activity of individual neurons were measured in an organotypic, static slice culture of the SCN obtained from neonatal rats. Five days after the start of culture, robust circadian rhythms were detected in AVP release with a peak located consistently at the middle of the original light phase, while the 24 h profiles of VIP release were either arrhythmic or rhythmic. In the latter case, a phase delay of 5–7 h was observed in the circadian peak from the AVP rhythm. Multi‐channel, extracellular recording revealed that 51 (76.1%) out of 67 firing neurons, examined in the SCN, showed circadian rhythms in their firing rate. The percentage of rhythmic neurons was significantly larger in the dorsal (86.8%) than in the ventral (62.1%) region of the SCN, where the AVP and VIP containing neurons predominate, respectively. Twenty‐seven percent of the firing rhythms were almost antiphasic from the majority of rhythms. There was no regional specificity in the distribution of the antiphasic rhythm. These findings, that the dorsal and ventral regions of the SCN both contain circadian pacemakers with different properties that regulate the AVP and VIP release separately, is probably due to differences in the number and, hence, the coupling strength of oscillating neurons.

Phase-dependent shift of free-running human circadian rhythms in response to a single bright pulse

Responsiveness of free-running human circadian rhythms to a single pulse of bright light was examined in a temporal isolation unit. Bright light (5000 lx) of either 3 or 6 h duration, applied during the early subjective day, produced phase-advance shifts in both the sleep-wake cycle and the rhythm of rectal temperature; the light pulse had essentially no effect on the phase of the circadian rhythms, when it was introduced during the late subjective day or the early subjective night. The results indicate that bright light can reset the human circadian pacemaker.