Postnatal Constant Light Compensates Cryptochrome1 and 2 Double Deficiency for Disruption of Circadian Behavioral Rhythms in Mice under Constant Dark

Clock genes Cryptochrome (Cry1) and Cry2 are essential for expression of circadian rhythms in mice under constant darkness (DD). However, circadian rhythms in clock gene Per1 expression or clock protein PER2 are detected in the cultured suprachiasmatic nucleus (SCN) of neonatal Cry1 and Cry2 double deficient (Cry1 ^-/-/Cry2 ^-/-) mice. A lack of circadian rhythms in adult Cry1 ^-/-/Cry2 ^-/- mice is most likely due to developmentally disorganized cellular coupling of oscillating neurons in the SCN. On the other hand, neonatal rats exposed to constant light (LL) developed a tenable circadian system under prolonged LL which was known to fragment circadian behavioral rhythms. In the present study, Cry1 ^-/-/Cry2 ^-/- mice were raised under LL from postnatal day 1 for 7 weeks and subsequently exposed to DD for 3 weeks. Spontaneous movement was monitored continuously after weaning and PER2::LUC was measured in the cultured SCN obtained from mice under prolonged DD. Surprisingly, Chi square periodogram analysis revealed significant circadian rhythms of spontaneous movement in the LL-raised Cry1 ^-/-/Cry2 ^-/- mice, but failed to detect the rhythms in Cry1 ^-/-/Cry2 ^-/- mice raised under light-dark cycles (LD). By contrast, prolonged LL in adulthood did not rescue the circadian behavioral rhythms in the LD raised Cry1 ^-/-/Cry2 ^-/- mice. Visual inspection disclosed two distinct activity components with different periods in behavioral rhythms of the LL-raised Cry1^-/-/Cry2^-/- mice under DD: one was shorter and the other was longer than 24 hours. The two components repeatedly merged and separated. The patterns resembled the split behavioral rhythms of wild type mice under prolonged LL. In addition, circadian rhythms in PER2::LUC were detected in some of the LL-raised Cry1^-/-/Cry2^-/- mice under DD. These results indicate that neonatal exposure to LL compensates the CRY double deficiency for the disruption of circadian behavioral rhythms under DD in adulthood.     

Cell Type-Specific Functions of Period Genes Revealed by Novel Adipocyte and Hepatocyte Circadian Clock Models

In animals, circadian rhythms in physiology and behavior result from coherent rhythmic interactions between clocks in the brain and those throughout the body. Despite the many tissue specific clocks, most understanding of the molecular core clock mechanism comes from studies of the suprachiasmatic nuclei (SCN) of the hypothalamus and a few other cell types. Here we report establishment and genetic characterization of three cell-autonomous mouse clock models: 3T3 fibroblasts, 3T3-L1 adipocytes, and MMH-D3 hepatocytes. Each model is genetically tractable and has an integrated luciferase reporter that allows for longitudinal luminescence recording of rhythmic clock gene expression using an inexpensive off-the-shelf microplate reader. To test these cellular models, we generated a library of short hairpin RNAs (shRNAs) against a panel of known clock genes and evaluated their impact on circadian rhythms. Knockdown of Bmal1, Clock, Cry1, and Cry2 each resulted in similar phenotypes in all three models, consistent with previous studies. However, we observed cell type-specific knockdown phenotypes for the Period and Rev-Erb families of clock genes. In particular, Per1 and Per2, which have strong behavioral effects in knockout mice, appear to play different roles in regulating period length and amplitude in these peripheral systems. Per3, which has relatively modest behavioral effects in knockout mice, substantially affects period length in the three cellular models and in dissociated SCN neurons. In summary, this study establishes new cell-autonomous clock models that are of particular relevance to metabolism and suitable for screening for clock modifiers, and reveals previously under-appreciated cell type-specific functions of clock genes.     

Circadian Regulation of Food-Anticipatory Activity in Molecular Clock–Deficient Mice

In the mammalian brain, the suprachiasmatic nucleus (SCN) of the anterior hypothalamus is considered to be the principal circadian pacemaker, keeping the rhythm of most physiological and behavioral processes on the basis of light/dark cycles. Because restriction of food availability to a certain time of day elicits anticipatory behavior even after ablation of the SCN, such behavior has been assumed to be under the control of another circadian oscillator. According to recent studies, however, mutant mice lacking circadian clock function exhibit normal food-anticipatory activity (FAA), a daily increase in locomotor activity preceding periodic feeding, suggesting that FAA is independent of the known circadian oscillator. To investigate the molecular basis of FAA, we examined oscillatory properties in mice lacking molecular clock components. Mice with SCN lesions or with mutant circadian periods were exposed to restricted feeding schedules at periods within and outside circadian range. Periodic feeding led to the entrainment of FAA rhythms only within a limited circadian range. Cry1^−/− mice, which are known to be a “short-period mutant,” entrained to a shorter period of feeding cycles than did Cry2^−/− mice. This result indicated that the intrinsic periods of FAA rhythms are also affected by Cry deficiency. Bmal1 ^−/− mice, deficient in another essential element of the molecular clock machinery, exhibited a pre-feeding increase of activity far from circadian range, indicating a deficit in circadian oscillation. We propose that mice possess a food-entrainable pacemaker outside the SCN in which canonical clock genes such as Cry1, Cry2 and Bmal1 play essential roles in regulating FAA in a circadian oscillatory manner.     

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.     

Signalling entrains the peripheral circadian clock

In mammals, 24-h rhythms of behaviour and physiology are regulated by the circadian clock. The circadian clock is controlled by a central clock in the brain's suprachiasmatic nucleus (SCN) that synchronizes peripheral clocks in peripheral tissues. Clock genes in the SCN are primarily entrained by light. Increasing evidence has shown that peripheral clocks are also regulated by light and hormones independent of the SCN. How the peripheral clocks deal with internal signals is dependent on the relevance of a specific cue to a specific tissue. In different tissues, most genes that are under circadian control are not overlapping, revealing the tissue-specific control of peripheral clocks. We will discuss how different signals control the peripheral clocks in different peripheral tissues, such as the liver, gastrointestinal tract, and pancreas, and discuss the organ-to-organ communication between the peripheral clocks at the molecular level.     

Chronic Ethanol Consumption Disrupts the Core Molecular Clock and Diurnal Rhythms of Metabolic Genes in the Liver without Affecting the Suprachiasmatic Nucleus

Chronic ethanol consumption disrupts several metabolic pathways including β-oxidation and lipid biosynthesis, facilitating the development of alcoholic fatty liver disease. Many of these same metabolic pathways are directly regulated by cell autonomous circadian clocks, and recent studies suggest that disruption of daily rhythms in metabolism contributes to multiple common cardiometabolic diseases (including non-alcoholic fatty liver disease). However, it is not known whether ethanol disrupts the core molecular clock in the liver, nor whether this, in turn, alters rhythms in lipid metabolism. Herein, we tested the hypothesis that chronic ethanol consumption disrupts the molecular circadian clock in the liver and potentially changes the diurnal expression patterns of lipid metabolism genes. Consistent with previous studies, male C57BL/6J mice fed an ethanol-containing diet exhibited higher levels of liver triglycerides compared to control mice, indicating hepatic steatosis. Further, the diurnal oscillations of core clock genes (Bmal1, Clock, Cry1, Cry2, Per1, and Per2) and clock-controlled genes (Dbp, Hlf, Nocturnin, Npas2, Rev-erbα, and Tef) were altered in livers from ethanol-fed mice. In contrast, ethanol had only minor effects on the expression of core clock genes in the suprachiasmatic nucleus (SCN). These results were confirmed in Per2^Luciferase knock-in mice, in which ethanol induced a phase advance in PER2::LUC bioluminescence oscillations in liver, but not SCN. Further, there was greater variability in the phase of PER2::LUC oscillations in livers from ethanol-fed mice. Ethanol consumption also affected the diurnal oscillations of metabolic genes, including Adh1, Cpt1a, Cyp2e1, Pck1, Pdk4, Ppargc1a, Ppargc1b and Srebp1c, in the livers of C57BL/6J mice. In summary, chronic ethanol consumption alters the function of the circadian clock in liver. Importantly, these results suggest that chronic ethanol consumption, at levels sufficient to cause steatosis, disrupts the core hepatic clock as well as the diurnal rhythms of key lipid metabolism genes.     

Circadian oscillators in the mouse brain: molecular clock components in the neocortex and cerebellar cortex

The circadian timekeeper of the mammalian brain resides in the suprachiasmatic nucleus of the hypothalamus (SCN), and is characterized by rhythmic expression of a set of clock genes with specific 24-h daily profiles. An increasing amount of data suggests that additional circadian oscillators residing outside the SCN have the capacity to generate peripheral circadian rhythms. We have recently shown the presence of SCN-controlled oscillators in the neocortex and cerebellum of the rat. The function of these peripheral brain clocks is unknown, and elucidating this could involve mice with conditional cell-specific clock gene deletions. This prompted us to analyze the molecular clockwork of the mouse neocortex and cerebellum in detail. Here, by use of in situ hybridization and quantitative RT-PCR, we show that clock genes are expressed in all six layers of the neocortex and the Purkinje and granular cell layers of the cerebellar cortex of the mouse brain. Among these, Per1, Per2, Cry1, Arntl, and Nr1d1 exhibit circadian rhythms suggesting that local running circadian oscillators reside within neurons of the mouse neocortex and cerebellar cortex. The temporal expression profiles of clock genes are similar in the neocortex and cerebellum, but they are delayed by 5 h as compared to the SCN, suggestively reflecting a master–slave relationship between the SCN and extra-hypothalamic oscillators. Furthermore, ARNTL protein products are detectable in neurons of the mouse neocortex and cerebellum, as revealed by immunohistochemistry. These findings give reason to further pursue the physiological significance of circadian oscillators in the mouse neocortex and cerebellum.     

Establishment of cell lines derived from the rat suprachiasmatic nucleus

Physiological and behavioral circadian rhythms in mammals are orchestrated by a central circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Photic input entrains the phase of the central clock, and many peripheral clocks are regulated by neural or hormonal output from the SCN. We established cell lines derived from the rat embryonic SCN to examine the molecular network of the central clock. An established cell line exhibited the stable circadian expression of clock genes. The circadian oscillation was abruptly phase-shifted by forskolin, and abolished by siBmal1. These results are compatible with in vivo studies of the SCN.     

Postnatal ontogenesis of the circadian clock within the rat liver

In mammals, the circadian oscillator within the suprachiasmatic nuclei (SCN) entrains circadian clocks in numerous peripheral tissues. Central and peripheral clocks share a molecular core clock mechanism governing daily time measurement. In the rat SCN, the molecular clockwork develops gradually during postnatal ontogenesis. The aim of the present work was to elucidate when during ontogenesis the expression of clock genes in the rat liver starts to be rhythmic. Daily profiles of mRNA expression of clock genes Per1, Per2, Cry1, Clock, Rev-Erbalpha, and Bmal1 were analyzed in the liver of fetuses at embryonic day 20 (E20) or pups at postnatal age 2 (P2), P10, P20, P30, and in adults by real-time RT-PCR. At E20, only a high-amplitude rhythm in Rev-Erbalpha and a low-amplitude variation in Cry1 but no clear circadian rhythms in expression of other clock genes were detectable. At P2, a high-amplitude rhythm in Rev-Erbalpha and a low-amplitude variation in Bmal1 but no rhythms in expression of other genes were detected. At P10, significant rhythms only in Per1 and Rev-Erbalpha expression were present. At P20, clear circadian rhythms in the expression of Per1, Per2, Rev-Erbalpha, and Bmal1, but not yet of Cry1 and Clock, were detected. At P30, all clock genes were expressed rhythmically. The phase of the rhythms shifted between all studied developmental periods until the adult stage was achieved. The data indicate that the development of the molecular clockwork in the rat liver proceeds gradually and is roughly completed by 30 days after birth.     

In vivo monitoring of peripheral circadian clocks in the mouse

The mammalian circadian system is comprised of a central clock in the suprachiasmatic nucleus (SCN) and a network of peripheral oscillators located in all of the major organ systems. The SCN is traditionally thought to be positioned at the top of the hierarchy, with SCN lesions resulting in an arrhythmic organism. However, recent work has demonstrated that the SCN and peripheral tissues generate independent circadian oscillations in Per1 clock gene expression in vitro. In the present study, we sought to clarify the role of the SCN in the intact system by recording rhythms in clock gene expression in vivo. A practical imaging protocol was developed that enables us to measure circadian rhythms easily, noninvasively, and longitudinally in individual mice. Circadian oscillations were detected in the kidney, liver, and submandibular gland studied in about half of the SCN-lesioned, behaviorally arrhythmic mice. However, their amplitude was decreased in these organs. Free-running periods of peripheral clocks were identical to those of activity rhythms recorded before the SCN lesion. Thus, we can report for the first time that many of the fundamental properties of circadian oscillations in peripheral clocks in vivo are maintained in the absence of SCN control.     

Potent synchronization of peripheral circadian clocks by glucocorticoid injections in PER2::LUC-Clock/Clock mice

In mammals, the central clock (the suprachiasmatic nuclei, SCN) is entrained mainly by the light-dark cycle, whereas peripheral clocks in the peripheral tissues are entrained/synchronized by multiple factors, including feeding patterns and endocrine hormones such as glucocorticoids. Clock-mutant mice (Clock/Clock), which have a mutation in a core clock gene, show potent phase resetting in response to light pulses compared with wild-type (WT) mice, owing to the damped and flexible oscillator in the SCN. However, the phase resetting of the peripheral clocks in Clock/Clock mice has not been elucidated. Here, we characterized the peripheral clock gene synchronization in Clock/Clock mice by daily injections of a synthetic glucocorticoid (dexamethasone, DEX) by monitoring in vivo PER2::LUCIFERASE bioluminescence. Compared with WT mice, the Clock/Clock mice showed significantly decreased bioluminescence and peripheral clock rhythms with decreased amplitudes and delayed phases. In addition, the DEX injections increased the amplitudes and advanced the phases. In order to examine the robustness of the internal oscillator, T-cycle experiments involving DEX stimulations with 24- or 30-h intervals were performed. The Clock/Clock mice synchronized to the 30-h T-cycle stimulation, which suggested that the peripheral clocks in the Clock/Clock mice had increased synchronizing ability upon DEX stimulation, to that of circadian and hour-glass type oscillations, because of weak internal clock oscillators.     

Intercellular coupling confers robustness against mutations in the SCN circadian clock network

Molecular mechanisms of the mammalian circadian clock have been studied primarily by genetic perturbation and behavioral analysis. Here, we used bioluminescence imaging to monitor Per2 gene expression in tissues and cells from clock mutant mice. We discovered that Per1 and Cry1 are required for sustained rhythms in peripheral tissues and cells, and in neurons dissociated from the suprachiasmatic nuclei (SCN). Per2 is also required for sustained rhythms, whereas Cry2 and Per3 deficiencies cause only period length defects. However, oscillator network interactions in the SCN can compensate for Per1 or Cry1 deficiency, preserving sustained rhythmicity in mutant SCN slices and behavior. Thus, behavior does not necessarily reflect cell-autonomous clock phenotypes. Our studies reveal previously unappreciated requirements for Per1, Per2, and Cry1 in sustaining cellular circadian rhythmicity and demonstrate that SCN intercellular coupling is essential not only to synchronize component cellular oscillators but also for robustness against genetic perturbations.     

Photoperiod differentially regulates circadian oscillators in central and peripheral tissues of the Syrian hamster

In many seasonally breeding rodents, reproduction and metabolism are activated by long summer days (LD) and inhibited by short winter days (SD). After several months of SD, animals become refractory to this inhibitory photoperiod and spontaneously revert to LD-like physiology. The suprachiasmatic nuclei (SCN) house the primary circadian oscillator in mammals. Seasonal changes in photic input to this structure control many annual physiological rhythms via SCN-regulated pineal melatonin secretion, which provides an internal endocrine signal representing photoperiod. We compared LD- and SD-housed animals and show that the waveform of SCN expression for three circadian clock genes (Per1, Per2, and Cry2) is modified by photoperiod. In SD-refractory (SD-R) animals, SCN and melatonin rhythms remain locked to SD, reflecting ambient photoperiod, despite LD-like physiology. In peripheral oscillators, Per1 and Dbp rhythms are also modified by photoperiod but, in contrast to the SCN, revert to LD-like, high-amplitude rhythms in SD-R animals. Our data suggest that circadian oscillators in peripheral organs participate in photoperiodic time measurement in seasonal mammals; however, circadian oscillators operate differently in the SCN. The clear dissociation between SCN and peripheral oscillators in refractory animals implicates intermediate factor(s), not directly driven by the SCN or melatonin, in entrainment of peripheral clocks.     

Circadian time-place learning in mice depends on Cry genes

Endogenous biological clocks allow organisms to anticipate daily environmental cycles. The ability to achieve time-place associations is key to the survival and reproductive success of animals. The ability to link the location of a stimulus (usually food) with time of day has been coined time-place learning, but its circadian nature was only shown in honeybees and birds. So far, an unambiguous circadian time-place-learning paradigm for mammals is lacking. We studied whether expression of the clock gene Cryptochrome (Cry), crucial for circadian timing, is a prerequisite for time-place learning. Time-place learning in mice was achieved by developing a novel paradigm in which food reward at specific times of day was counterbalanced by the penalty of receiving a mild footshock. Mice lacking the core clock genes Cry1 and Cry2 (Cry double knockout mice; Cry1(-/-)Cry2(-/-)) learned to avoid unpleasant sensory experiences (mild footshock) and could locate a food reward in a spatial learning task (place preference). These mice failed, however, to learn time-place associations. This specific learning and memory deficit shows that a Cry-gene dependent circadian timing system underlies the utilization of time of day information. These results reveal a new functional role of the mammalian circadian timing system.