Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed^1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere^1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms^3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous^5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects^5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms^5,8-13.Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals^14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection^13,16,17 or stable transduction^5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells²⁰. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.     

The Mouse Microbiome Is Required for Sex-Specific Diurnal Rhythms of Gene Expression and Metabolism

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Fibroblast Circadian Rhythms of PER2 Expression Depend on Membrane Potential and Intracellular Calcium

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 Ca²⁺. 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 Ca²⁺ concentrations, rhythms were advanced or had shorter periods. Buffering intracellular Ca²⁺ 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 (IP₃)-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 Ca²⁺. Changes in intracellular Ca²⁺ may mediate the effects of membrane potential observed in this study. (Author correspondence: welshdk@ucsd.edu)     

Light Pollution Modifies the Expression of Daily Rhythms and Behavior Patterns in a Nocturnal Primate

Among anthropogenic pressures, light pollution altering light/dark cycles and changing the nocturnal component of the environment constitutes a threat for biodiversity. Light pollution is widely spread across the world and continuously growing. However, despite the efforts realized to describe and understand the effects of artificial lighting on fauna, few studies have documented its consequences on biological rhythms, behavioral and physiological functions in nocturnal mammals. To determine the impacts of light pollution on nocturnal mammals an experimental study was conducted on a nocturnal primate, the grey mouse lemur Microcebus murinus. Male mouse lemurs (N = 8) were exposed 14 nights to moonlight treatment and then exposed 14 nights to light pollution treatment. For both treatments, chronobiological parameters related to locomotor activity and core temperature were recorded using telemetric transmitters. In addition, at the end of each treatment, the 14^th night, nocturnal and feeding behaviors were explored using an infrared camera. Finally, throughout the study, body mass and daily caloric food intake were recorded. For the first time in a nocturnal primate, light pollution was demonstrated to modify daily rhythms of locomotor activity and core temperature especially through phase delays and increases in core temperature. Moreover, nocturnal activity and feeding behaviors patterns were modified negatively. This study suggests that light pollution induces daily desynchronization of biological rhythms and could lead to seasonal desynchronization with potential deleterious consequences for animals in terms of adaptation and anticipation of environmental changes.     

Relationships Between Behavioral Rhythms,Plasma Corticosterone and Hypothalamic Circadian Rhythms

Circadian rhythms in physiological processes and behaviors were compared with hypothalamic circadian rhythms in norepinephrine (NE) metabolites, adrenergic transmitter receptors, cAMP, cGMP and suprachiasmatic nucleus (SCN) arginine vasopressin (AVP) in a single population of rats under D: D conditions. Eating, drinking and locomotor activity were high during the subjective night (the time when lights were out in L: D) and low during the subjective day (the time when lights were on in L: D). Plasma corticosterone concentration rose at subjective dusk and remained high until subjective dawn. Binding to hypothalamic α₁- and β-adrenergic receptors also peaked during the subjective night. Cyclic cGMP concentration was elevated throughout the 24-hr period except for a trough at dusk, whereas DHPG concentration peaked at dawn. Arginine vasopressin levels in the suprachiasmatic nucleus peaked in the middle of the day. No rhythm was found either in binding to the α₂-adrenergic receptor, or in MHPG or cAMP concentration. Behavioral and corticosterone rhythms, therefore, are parallel to rhythms in hypothalamic α₁-and β-receptor binding and NE-release. Cyclic GMP falls only at dusk, suggesting the possibility that cGMP inhibits activity much of the day and that at dusk the inhibition of nocturnal activity is removed. SCN AVP, on the other hand, peaking at 1400 hr, may play a role in the pacemaking function of the SCN that drives these other rhythms.     

Role of Sleep Restriction in Daily Rhythms of Expression of Hypothalamic Core Clock Genes in Mice

Lack of sleep time is a menace to modern people, and it leads to chronic diseases and mental illnesses. Circadian processes control sleep, but little is known about how sleep affects the circadian system. Therefore, we performed a 28-day sleep restriction (SR) treatment in mice. Sleep restriction disrupted the clock genes’ circadian rhythm. The circadian rhythms of the Cry1 and Per1/2/3 genes disappeared. The acrophase of the clock genes (Bmal1, Clock, Rev-erbα, and Rorβ) that still had a circadian rhythm was advanced, while the acrophase of negative clock gene Cry2 was delayed. Clock genes’ upstream signals ERK and EIFs also had circadian rhythm disorders. Accompanied by changes in the central oscillator, the plasma output signal (melatonin, corticosterone, IL-6, and TNF-α) had an advanced acrophase. While the melatonin mesor was decreased, the corticosterone, IL-6, and TNF-α mesor was increased. Our results indicated that chronic sleep loss could disrupt the circadian rhythm of the central clock through ERK and EIFs and affect the output signal downstream of the core biological clock.     

Chaos and Ultradian Rhythms

Systems in a chaotic state have apparently random outputs despite a simple underlying kinetic mechanism. For instance, the interaction of two coupled oscillators (the mitotic oscillator and the ultradian clock) can produce chaotic behaviour over a limited range of parameter values. Mathematical modelling shows that physiologically realistic characteristics are thereby exhibited. Cell division cycles of lower eukaryotes (protozoa and yeasts) show both deterministic and stochastic properties. Both dispersion of cell cycle times and quantized values can be generated, as a deterministic chaotic consequence of oscillator interaction rather than from noisy limit cycles. Advantages may stem from chaotic operation; a controlled chaotic attractor could provide multifrequency outputs that determine rhythmic behaviour on different time scales (e.g. ultradian and circadian) with the facility for rapid state changes from one periodicity to another.     

Ultradian Rhythms in Desmodium

The leaves of Desmodium gyrans (L.F.) DC show circadian movements in the terminal and ultradian movements of the lateral leaflets. The movements are due to swelling and shrinking of motor cells in special organs. The anatomy of these pulvini is described for the lateral leaflets. Data from electrophysiological recordings using microelectrodes inserted into the lateral pulvini, together with treatments that affect the proton pumps and ion channels, have been used to develop a physiological model of the ultradian leaflet movement. It explains the oscillations in the motor cells as being due to a change between a pump state and depolarization. During the pump state, ions are taken up, causing water influx and swelling of the motor cells. Depolarization causes loss of ions and water efflux (the motor cells shrink). The roles of calcium and the phosphatidyl inositol signal chain are discussed on the basis of experiments using chemical agents that affect these processes. Since calcium oscillations are known to occur in organisms in both time and space, an attempt has been made to simulate the situation in Desmodium pulvini by a model of specially coupled oscillators. Effects of different other treatments of the lateral pulvini are discussed. Oscillations in the minute range seem to be more common and some might be related to turgor regulation and ion uptake comparable to the situation in Desmodium. The ultradian control of the lateral pulvini and the circadian control of the terminal pulvini are apparently based on different mechanisms.     

Melatonin and Human Rhythms

Melatonin signals time of day and time of year in mammals by virtue of its pattern of secretion, which defines ‘biological night.’ It is supremely important for research on the physiology and pathology of the human biological clock. Light suppresses melatonin secretion at night using pathways involved in circadian photoreception. The melatonin rhythm (as evidenced by its profile in plasma, saliva, or its major metabolite, 6‐sulphatoxymelatonin [aMT6s] in urine) is the best peripheral index of the timing of the human circadian pacemaker. Light suppression and phase‐shifting of the melatonin 24 h profile enables the characterization of human circadian photoreception, and circulating concentrations of the hormone are used to investigate the general properties of the human circadian system in health and disease. Suppression of melatonin by light at night has been invoked as a possible influence on major disease risk as there is increasing evidence for its oncostatic effects. Exogenous melatonin acts as a ‘chronobiotic.’ Acutely, it increases sleep propensity during ‘biological day.’ These properties have led to successful treatments for serveal circadian rhythm disorders. Endogenous melatonin acts to reinforce the functioning of the human circadian system, probably in many ways. The future holds much promise for melatonin as a research tool and as a therapy for various conditions.     

Chaos and Ultradian Rhythms

Systems in a chaotic state have apparently random outputs despite a simple underlying kinetic mechanism. For instance, the interaction of two coupled oscillators (the mitotic oscillator and the ultradian clock) can produce chaotic behaviour over a limited range of parameter values. Mathematical modelling shows that physiologically realistic characteristics are thereby exhibited. Cell division cycles of lower eukaryotes (protozoa and yeasts) show both deterministic and stochastic properties. Both dispersion of cell cycle times and quantized values can be generated, as a deterministic chaotic consequence of oscillator interaction rather than from noisy limit cycles. Advantages may stem from chaotic operation; a controlled chaotic attractor could provide multifrequency outputs that determine rhythmic behaviour on different time scales ( e.g. ultradian and circadian) with the facility for rapid state changes from one periodicity to another.     

Melatonin and Human Rhythms

Melatonin signals time of day and time of year in mammals by virtue of its pattern of secretion, which defines 'biological night.' It is supremely important for research on the physiology and pathology of the human biological clock. Light suppresses melatonin secretion at night using pathways involved in circadian photoreception. The melatonin rhythm (as evidenced by its profile in plasma, saliva, or its major metabolite, 6-sulphatoxymelatonin [aMT6s] in urine) is the best peripheral index of the timing of the human circadian pacemaker. Light suppression and phase-shifting of the melatonin 24 h profile enables the characterization of human circadian photoreception, and circulating concentrations of the hormone are used to investigate the general properties of the human circadian system in health and disease. Suppression of melatonin by light at night has been invoked as a possible influence on major disease risk as there is increasing evidence for its oncostatic effects. Exogenous melatonin acts as a 'chronobiotic.' Acutely, it increases sleep propensity during 'biological day.' These properties have led to successful treatments for serveal circadian rhythm disorders. Endogenous melatonin acts to reinforce the functioning of the human circadian system, probably in many ways. The future holds much promise for melatonin as a research tool and as a therapy for various conditions.