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In mammals, the master circadian clock resides in the suprachiasmatic nucleus (SCN). The SCN is characterized by robust circadian oscillations of clock gene expression and neuronal firing. The synchronization of circadian oscillations among individual cells in the SCN is attributed to intercellular coupling. Previous studies have shown that gap junctions, specifically those composed of connexin-36 (Cx36) subunits, are required for coupling of electrical firing among SCN neurons at a time scale of milliseconds. However, it remains unknown whether Cx36 gap junctions also contribute to coupling of circadian (~24 h) rhythms of clock gene expression. Here, we investigated circadian expression patterns of the clock gene Period 2 (Per2) in the SCN of Cx36-deficient mice using luminometry and single-cell bioluminescence imaging. Surprisingly, we found that synchronization of circadian PER2 expression rhythms is maintained in SCN explants from Cx36-deficient mice. Since Cx36 expression levels change with age, we also tested circadian running-wheel behavior of juvenile (3–4 weeks old) and adult (9–30 weeks old) Cx36-deficient mice. We found that impact of connexin-36 expression on circadian behavior changes greatly during postnatal development. However, consistent with the intact synchrony among SCN cells in cultured explants, Cx36-deficient mice had intact locomotor circadian rhythms, although adults displayed a lengthened period in constant darkness. Our data indicate that even though Cx36 may be required for electrical coupling of SCN cells, it does not affect coupling of molecular clock gene rhythms. Thus, electrical coupling of neurons and coupling of circadian clock gene oscillations can be regulated independently in the SCN.

Human exposure to lead (Pb) due to uncontrolled Pb-acid battery recycling has been an environmental health issue in newly developed industrial regions. We conducted a human monitoring survey in Dong Mai, a battery recycling village in Vietnam, to assess exposure status to Pb. Lead level was measured in hair, blood and urine samples of residents in Dong Mai and two reference sites during 4 years spanning 2007–2011. In Dong Mai, Pb levels in three matrixes were significantly higher than those in reference sites. Blood Pb levels of all adults and children exceeded 10 μg/dL, the Centers for Disease Control and Prevention definition of an elevated blood Pb level. Clear increase of urinary δ-aminolevulinic acid (ALA) level with increasing blood Pb level indicated disruption of heme synthesis. One adult exceeded 100 μg/dL of blood Pb, where encephalopathy is of concern. The blood Pb levels achieved various toxic effect threshold values, and elevated blood Pb was not limited to recycling workers, but was also in children and women of reproductive age. Serious pollution status of Dong Mai village suggests an importance of further monitoring surveys in various developing Asian countries.

Like neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker in the brain, single fibroblasts can function as independent oscillators. In the SCN, synaptic and paracrine signaling among cells creates a robust, synchronized circadian oscillation, whereas there is no evidence for such integration in fibroblast cultures. However, interactions among single-cell fibroblast oscillators cannot be completely excluded, because fibroblasts were not isolated in previous work. In this study, we tested the autonomy of fibroblasts as single-cell circadian oscillators in high- and low-density culture, by single-cell imaging of cells from PER2::LUC circadian reporter mice. We found greatly reduced PER2::LUC rhythmicity in low-density cultures, which could result from lack of either constitutive or rhythmic paracrine signals from neighboring fibroblasts. To discriminate between these 2 possibilities, we mixed PER2::LUC wild-type (WT) cells with nonluminescent, nonrhythmic Bmal1–/– cells, so that density of rhythmic cells was low but overall cell density remained high. In this condition, WT cells showed clear rhythmicity similar to high-density cultures. We also mixed PER2::LUC WT cells with nonluminescent, long period Cry2–/– cells. In this condition, WT cells showed a period no different from cells cultured with rhythmic WT cells or nonrhythmic Bmal1–/– cells. In previous work, we found that low K+ suppresses fibroblast rhythmicity, and we and others have found that either low K+ or low Ca2+ suppresses SCN rhythmicity. Therefore, we attempted to rescue rhythmicity of low-density fibroblasts with high K+ (21 mM), high Ca2+ (3.6 mM), or conditioned medium. Conditioned medium from high-density fibroblast cultures rescued rhythmicity of low-density cultures, whereas high K+ or Ca2+ medium did not consistently rescue rhythmicity. These data suggest that fibroblasts require paracrine signals from adjacent cells for normal expression of rhythmicity, but that these signals do not have to be rhythmic, and that rhythmic signals from other cells do not affect the intrinsic periods of fibroblasts.

The suprachiasmatic nucleus (SCN) of the hypothalamus synchronizes circadian rhythms of cells and tissues throughout the body. In SCN neurons, rhythms of clock gene expression are suppressed by manipulations that hyperpolarize the plasma membrane or lower intracellular Ca(2+). However, whether clocks in other cells also depend on membrane potential and calcium is unknown. In this study, the authors investigate the effects of membrane potential and intracellular calcium on circadian rhythms in mouse primary fibroblasts. Rhythms of clock gene expression were monitored using a PER2::LUC knockin reporter. Rhythms were lost or delayed at lower (hyperpolarizing) K(+) concentrations. Bioluminescence imaging revealed that this loss of rhythmicity in cultures was due to loss of rhythmicity of single cells rather than loss of synchrony among cells. In lower Ca(2+) concentrations, rhythms were advanced or had shorter periods. Buffering intracellular Ca(2+) by the calcium chelator 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis acetoxymethyl ester (BAPTA-AM) or manipulation of inositol triphosphate (IP(3))-sensitive intracellular calcium stores by thapsigargin delayed rhythms. These results suggest that the circadian clock in fibroblasts, as in SCN neurons, is regulated by membrane potential and Ca(2+). Changes in intracellular Ca(2+) may mediate the effects of membrane potential observed in this study.

In mammals, circadian rhythms are driven by a pacemaker located in the suprachiasmatic nucleus (SCN), which is composed of multiple, single-cell oscillators. Isolated SCN tissue shows clear circadian oscillation in release of arginine vasopressin (AVP) in organotypic slice cultures. Previously, we reported that the oscillators in the dorsal SCN have shorter periods than those in the ventral part. Here, we examined whether a correlation between the period and the rostral-caudal co-ordination could exist. The rostral, central and caudal SCN were cultured separately and the periods of circadian rhythms of AVP release were measured. The rostral and caudal parts of the SCN showed shorter periods than the central SCN. Together with previous findings, it is suggested that the shorter period region originates from AVP containing areas, while the longer period region corresponds with vasoactive intestinal polypeptide (VIP) containing cells. In our VIP-immunoreactive slices, the application of VIP antagonists shortened the periods of the AVP-releasing rhythm. These data indicate that the oscillators in AVP cells have short periods and are entrained by VIP cells to form a single integrated rhythm.

Circadian clocks impact vital cardiac parameters such as blood pressure and heart rate, and adverse cardiac events such as myocardial infarction and sudden cardiac death. In mammals, the central circadian pacemaker, located in the suprachiasmatic nucleus of the hypothalamus, synchronizes cellular circadian clocks in the heart and many other tissues throughout the body. Cardiac ventricle explants maintain autonomous contractions and robust circadian oscillations of clock gene expression in culture. In the present study, we examined the relationship between intrinsic myocardial function and circadian rhythms in cultures from mouse heart. We cultured ventricular explants or dispersed cardiomyocytes from neonatal mice expressing a PER2::LUC bioluminescent reporter of circadian clock gene expression. We found that isoproterenol, a β-adrenoceptor agonist known to increase heart rate and contractility, also amplifies PER2 circadian rhythms in ventricular explants. We found robust, cell-autonomous PER2 circadian rhythms in dispersed cardiomyocytes. Single-cell rhythms were initially synchronized in ventricular explants but desynchronized in dispersed cells. In addition, we developed a method for long-term, simultaneous monitoring of clock gene expression, contraction rate, and basal intracellular Ca2+ level in cardiomyocytes using PER2::LUC in combination with GCaMP3, a genetically encoded fluorescent Ca2+ reporter. In contrast to robust PER2 circadian rhythms in cardiomyocytes, we detected no rhythms in contraction rate and only weak rhythms in basal Ca2+ level. In summary, we found that PER2 circadian rhythms of cardiomyocytes are cell-autonomous, amplified by adrenergic signaling, and synchronized by intercellular communication in ventricle explants, but we detected no robust circadian rhythms in contraction rate or basal Ca2+.

Lithium is widely used as a treatment of bipolar disorder, a neuropsychiatric disorder associated with disrupted circadian rhythms. Lithium is known to lengthen period and increase amplitude of circadian rhythms. One possible pathway for these effects involves inhibition of glycogen synthase kinase-3β (GSK-3β), which regulates degradation of CRY2, a canonical clock protein determining circadian period. CRY1 is also known to play important roles in regulating circadian period and phase, although there is no evidence that it is similarly phosphorylated by GSK-3β. In this paper, we tested the hypothesis that lithium affects circadian rhythms through CRYs. We cultured fibroblasts and slices of the suprachiasmatic nucleus (SCN), the master circadian pacemaker of the brain, from Cry1-/-, Cry2-/-, or wild-type (WT) mice bearing the PER2:LUC circadian reporter. Lithium was applied in the culture medium, and circadian rhythms of PER2 expression were measured. In WT and Cry2-/- fibroblasts, 10mM lithium increased PER2 expression and rhythm amplitude but not period, and 1mM lithium did not affect either period or amplitude. In non-rhythmic Cry1-/- fibroblasts, 10mM lithium increased PER2 expression. In SCN slices, 1mM lithium lengthened period ∼1h in all genotypes, but did not affect amplitude except in Cry2-/- SCN. Thus, the amplitude-enhancing effect of lithium in WT fibroblasts was unaffected by Cry2 knockout and occurred in the absence of period-lengthening, whereas the period-lengthening effect of lithium in WT SCN was unaffected by Cry1 or Cry2 knockout and occurred in the absence of rhythm amplification, suggesting that these two effects of lithium on circadian rhythms are independent of CRYs and of each other.

In mammals, circadian rhythms are driven by a pacemaker located in the suprachiasmatic nucleus (SCN) of the hypothalamus. We measured the rhythm of arginine vasopressin release in rat organotypic SCN slices following application of tetrodotoxin (TTX) or N-methyl-D-aspartate (NMDA) at various times throughout the circadian cycle. TTX resets the clock in a manner similar to dark pulses. A 4-h application of TTX starting in mid subjective day, at around circadian time (CT) 7.0, induced phase advances, while TTX treatment started in early subjective morning, at about CT 2.0, induced phase delays. On the other hand, NMDA resets the clock in a manner similar to a light pulse; that is, NMDA treatment in the early evening induced phase delays while treatment in the late night induced phase advances. The data indicate that deprivation of neuronal firing changes the circadian rhythm.

In mammals, circadian rhythms are driven by a pacemaker located in the suprachiasmatic nuclei (SCN) of the hypothalamus. The pacemaker is composed of an ensemble of multiple, single-cell oscillators in the SCN. We measured arginine-vasopressin (AVP) release in organotypic SCN slices. The SCN slice culture showed circadian oscillation of AVP release with a period length (+/- SEM) of 23.84 +/- 00.03 h. This period is very similar to the one we previously reported in dispersed SCN cultures and is also close to that of behavioural rhythms. When the ventral part was removed by a surgical cut across the slice in the horizontal plane, however, the period became shorter (23.22 +/- 00.08 h). On the other hand, the removal of the dorsal part did not affect period length. These results suggest that the oscillators in ventral and dorsal cells contribute differently to period length and that the dorsal oscillators are entrained by the ventral ones to form a single integrated oscillator.