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.

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, 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 (∼24h) 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 (3-4weeks old) and adult (9-30weeks 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.

Circadian neural circuits generate near 24-hr physiological rhythms that can be entrained by light to coordinate animal physiology with daily solar cycles. To examine how a circadian circuit reorganizes its activity in response to light, we imaged period (per) clock gene cycling for up to 6 days at single-neuron resolution in whole-brain explant cultures prepared from per-luciferase transgenic flies. We compared cultures subjected to a phase-advancing light pulse (LP) to cultures maintained in darkness (DD). In DD, individual neuronal oscillators in all circadian subgroups are initially well synchronized but then show monotonic decrease in oscillator rhythm amplitude and synchrony with time. The small ventral lateral neurons (s-LNvs) and dorsal lateral neurons (LNds) exhibit this decrease at a slower relative rate. In contrast, the LP evokes a rapid loss of oscillator synchrony between and within most circadian neuronal subgroups, followed by gradual phase retuning of whole-circuit oscillator synchrony. The LNds maintain high rhythmic amplitude and synchrony following the LP along with the most rapid coherent phase advance. Immunocytochemical analysis of PER shows that these dynamics in DD and LP are recapitulated in vivo. Anatomically distinct circadian neuronal subgroups vary in their response to the LP, showing differences in the degree and kinetics of their loss, recovery and/or strengthening of synchrony, and rhythmicity. Transient desynchrony appears to be an integral feature of light response of the Drosophila multicellular circadian clock. Individual oscillators in different neuronal subgroups of the circadian circuit show distinct kinetic signatures of light response and phase retuning.

Shift-work and jet-lag-related disorders are caused by the limited flexibility of the suprachiasmatic nucleus (SCN), a master circadian clock in the hypothalamus, to adjust to new light-dark (LD) cycles. Recent findings confirmed here establish that behavioral jet lag after simulated time-zone travel is virtually eliminated following bifurcated circadian entrainment under a novel and atypical 24-h light:dark:light:dark (LDLD) cycle. To investigate the mechanisms of this fast resetting, we examined the oscillatory stability of the SCN and peripheral tissues in LDLD-bifurcated mice employing the dissection procedure as a perturbing resetting stimulus. SCN, lung, liver, and adrenal tissue were extracted at times throughout the day from female and male PER2::Luciferase knock-in mice entrained to either LDLD or a normal LD cycle. Except for adrenals, the phase of the cultured explants was more strongly set by dissection under LDLD than under normal LD. Acute bioluminescence levels of SCN explants indicate that the rhythm amplitude of PER2 is reduced and phase is altered in LDLD. Real-time quantitative PCR suggests that amplitude and rhythmicity of canonical clock genes in the lung, liver, and kidney are also significantly reduced in LDLD in vivo. Furthermore, spatiotemporal patterns of PER2 peak time in cultured SCN were altered in LDLD. These results suggest that altered gene expression patterns in the SCN caused by bifurcation likely result in fast resetting of behavior and cultured explants, consistent with previously reported mathematical models. Thus, non-invasive, simple light manipulations can make circadian rhythms more adaptable to abrupt shifts in the environmental LD cycle.

Circadian rhythms of mammalian physiology and behavior are coordinated by the suprachiasmatic nucleus (SCN) in the hypothalamus. Within SCN neurons, various aspects of cell physiology exhibit circadian oscillations, including circadian clock gene expression, levels of intracellular Ca2+ ([Ca2+]i), and neuronal firing rate. [Ca2+]i oscillates in SCN neurons even in the absence of neuronal firing. To determine the causal relationship between circadian clock gene expression and [Ca2+]i rhythms in the SCN, as well as the SCN neuronal network dependence of [Ca2+]i rhythms, we introduced GCaMP3, a genetically encoded fluorescent Ca2+ indicator, into SCN neurons from PER2::LUC knock-in reporter mice. Then, PER2 and [Ca2+]i were imaged in SCN dispersed and organotypic slice cultures. In dispersed cells, PER2 and [Ca2+]i both exhibited cell autonomous circadian rhythms, but [Ca2+]i rhythms were typically weaker than PER2 rhythms. This result matches the predictions of a detailed mathematical model in which clock gene rhythms drive [Ca2+]i rhythms. As predicted by the model, PER2 and [Ca2+]i rhythms were both stronger in SCN slices than in dispersed cells and were weakened by blocking neuronal firing in slices but not in dispersed cells. The phase relationship between [Ca2+]i and PER2 rhythms was more variable in cells within slices than in dispersed cells. Both PER2 and [Ca2+]i rhythms were abolished in SCN cells deficient in the essential clock gene Bmal1. These results suggest that the circadian rhythm of [Ca2+]i in SCN neurons is cell autonomous and dependent on clock gene rhythms, but reinforced and modulated by a synchronized SCN neuronal network.