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.     

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.     

Scheduled Feeding Alters the Timing of the Suprachiasmatic Nucleus Circadian Clock in Dexras1-Deficient Mice

Restricted feeding (RF) schedules are potent zeitgebers capable of entraining metabolic and hormonal rhythms in peripheral oscillators in anticipation of food. Behaviorally, this manifests in the form of food anticipatory activity (FAA) in the hours preceding food availability. Circadian rhythms of FAA are thought to be controlled by a food-entrainable oscillator (FEO) outside of the suprachiasmatic nucleus (SCN), the central circadian pacemaker in mammals. Although evidence suggests that the FEO and the SCN are capable of interacting functionally under RF conditions, the genetic basis of these interactions remains to be defined. In this study, using dexras1-deficient (dexras1^?/?) mice, the authors examined whether Dexras1, a modulator of multiple inputs to the SCN, plays a role in regulating the effects of RF on activity rhythms and gene expression in the SCN. Daytime RF under 12L:12D or constant darkness (DD) resulted in potentiated (but less stable) FAA expression in dexras1^?/? mice compared with wild-type (WT) controls. Under these conditions, the magnitude and phase of the SCN-driven activity component were greatly perturbed in the mutants. Restoration to ad libitum (AL) feeding revealed a stable phase displacement of the SCN-driven activity component of dexras1^?/? mice by ～2?h in advance of the expected time. RF in the late night/early morning induced a long-lasting increase in the period of the SCN-driven activity component in the mutants but not the WT. At the molecular level, daytime RF advanced the rhythm of PER1, PER2, and pERK expression in the mutant SCN without having any effect in the WT. Collectively, these results indicate that the absence of Dexras1 sensitizes the SCN to perturbations resulting from restricted feeding. (Author correspondence: haiying.cheng@utoronto.ca)     

Circadian rhythms in murine pups develop in absence of a functional maternal circadian clock

A genetic approach was used to investigate whether the emergence of circadian rhythms in murine pups is dependent on a functional maternal clock. Arrhythmic females bearing either the mPer1Brdm1/Per2Brdm1 or mPer2Brdm1/Cry1-/- double-mutant genotype were crossed with wild-type males under constant darkness. The heterozygous offspring have the genetic constitution for a functional circadian clock. Individual pups born to arrhythmic mPer1Brdm1/Per2Brdm1 and mPer2Brdm1/Cry1-/- mothers in constant darkness without external zeitgeber developed normal circadian rhythms, but their clocks were less synchronized to each other compared to wild-type animals. These findings indicate that development of circadian rhythms does not depend on a functional circadian clock in maternal tissue, extending previous findings obtained from pups born to SCN-lesioned mothers.     

Light and feeding entrainment of the molecular circadian clock in a marine teleost (Sparus aurata)

Daily light and feeding cycles act as powerful synchronizers of circadian rhythmicity. Ultimately, these external cues entrain the expression of clock genes, which generate daily rhythmic behavioral and physiological responses in vertebrates. In the present study, we investigated clock genes in a marine teleost (gilthead sea bream). Partial cDNA sequences of key elements from both positive (Bmal1, Clock) and negative (Per2, Cry1) regulatory loops were cloned before studying how feeding time affects the daily rhythms of locomotor activity and clock gene expression in the central (brain) and peripheral (liver) oscillators. To this end, all fish were kept under a light-dark (LD) cycle and were divided into three experimental groups, depending on the time of their daily meal: mid-light (ML), mid-darkness (MD), or at random (RD) times. Finally, the existence of circadian control on gene expression was investigated in the absence of external cues (DD?+?RD). The behavioral results showed that seabream fed at ML or RD displayed a diurnal activity pattern (>91% of activity during the day), whereas fish fed at MD were nocturnal (89% of activity during the night). Moreover, seabream subjected to regular feeding cycles (ML and MD groups) showed food-anticipatory activity (FAA). Regardless of the mealtime, the daily rhythm of clock gene expression in the brain peaked close to the light-dark transition in the case of Bmal1 and Clock, and at the beginning of the light phase in the case of Per2 and Cry1, showing the existence of phase delay between the positive and negative elements of the molecular clock. In the liver, however, the acrophases of the daily rhythms differed depending on the feeding regime: the maximum expression of Bmal1 and Clock in the ML and RD groups was in antiphase to the expression pattern observed in the fish fed at MD. Under constant conditions (DD?+?RD), Per2 and Cry1 showed circadian rhythmicity in the brain, whereas Bmal1, Clock, and Per2 did in the liver. Our results indicate that the seabream clock gene expression is endogenously controlled and in liver it is strongly entrained by food signals, rather than by the LD cycle, and that scheduled feeding can shift the phase of the daily rhythm of clock gene expression in a peripheral organ (liver) without changing the phase of these rhythms in a central oscillator (brain), suggesting uncoupling of the light-entrainable oscillator (LEO) from the food-entrainable oscillator (FEO). These findings provide the basis and new tools for improving our knowledge of the circadian system and entraining pathways of this fish species, which is of great interest for the Mediterranean aquaculture. (Author correspondence: javisan@um.es).     

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.     

Daily restricted feeding resets the circadian clock in the suprachiasmatic nucleus of CS mice

Circadian rhythms in clock gene expressions in the suprachiasmatic nucleus (SCN) of CS mice and C57BL/6J mice were measured under a daily restricted feeding (RF) schedule in continuous darkness (DD), and entrainment of the SCN circadian pacemaker to RF was examined. After 2-3 wk under a light-dark cycle with free access to food, animals were released into DD and fed for 3 h at a fixed time of day for 3-4 wk. Subsequently, they returned to having free access to food for 2-3 wk. In CS mice, wheel-running rhythms entrained to RF with a stable phase relationship between the activity onset and feeding time, and the rhythms started to free run from the feeding time after the termination of RF. mPer1, mPer2, and mBMAL1 mRNA rhythms in the SCN showed a fixed phase relationship with feeding time, indicating that the circadian pacemaker in the SCN entrained to RF. On the other hand, in C57BL/6J mice, wheel-running rhythms free ran under RF, and clock gene expression rhythms in the SCN showed a stable phase relation not to feeding time but to the behavioral rhythms, indicating that the circadian pacemaker in the SCN did not entrain. These results indicate that the SCN circadian pacemaker of CS mice is entrainable to RF under DD and suggest that CS mice have a circadian clock system that can be reset by a signal associated with feeding time.     

The cell adhesion molecule EphA4 is involved in circadian clock functions

Circadian (～24 h) rhythms of cellular network plasticity in the central circadian clock, the suprachiasmatic nucleus (SCN), have been described. The neuronal network in the SCN regulates photic resetting of the circadian clock as well as stability of the circadian system during both entrained and constant conditions. EphA4, a cell adhesion molecule regulating synaptic plasticity by controlling connections of neurons and astrocytes, is expressed in the SCN. To address whether EphA4 plays a role in circadian photoreception and influences the neuronal network of the SCN, we have analyzed circadian wheel‐running behavior of EphA4 knockout (EphA4^?/?) mice under different light conditions and upon photic resetting, as well as their light‐induced protein response in the SCN. EphA4^?/? mice exhibited reduced wheel‐running activity, longer endogenous periods under constant darkness and shorter periods under constant light conditions, suggesting an effect of EphA4 on SCN function. Moreover, EphA4^?/? mice exhibited suppressed phase delays of their wheel‐running activity following a light pulse during the beginning of the subjective night (CT15). Accordingly, light‐induced c‐FOS (FBJ murine osteosarcoma viral oncogene homolog) expression was diminished. Our results suggest a circadian role for EphA4 in the SCN neuronal network, affecting the circadian system and contributing to the circadian response to light.     

Sex-related difference in food-anticipatory activity of mice

The expression of food-anticipatory activity (FAA) is induced by restricted feeding (RF), and its entrainment requires food-entrainable oscillators, the neuroanatomical basis of which is currently unclear. Although RF impacts various hormones, sex-related differences in FAA are unclear. 'Here, we report significantly more food-anticipatory wheel-running activity in male than in female mice during RF. In parallel with the sex-related difference in FAA, male and female mice display different food intake and body weight in response to RF. Since gonadal hormones could be involved in the sex-specific difference in FAA, we compared sham and gonadectomized male and female wild-type mice. In gonadectomized mice, the sex difference in FAA was abolished, indicating a role for gonadal hormones in FAA. Further, plasma concentrations of the hormone ghrelin were higher in female than in male mice during ad libitum (AL) feeding, and RF induced a temporal advance in its peak in both sexes. RF also shifted the expression peak of the circadian gene mPer1 in the hippocampus and liver, although no sex difference was found in either the level or the cyclic phase of its expression. Per1^Brdm1 mutant mice were still sexually dimorphic for FAA, but diminished FAA was noted in both male and female Per2^Brdm1 mutant mice. In summary, our results imply that gonadal hormones contribute to the sex difference in FAA, possibly through modulating ghrelin activity.     

The Clock Genes Period 2 and Cryptochrome 2 Differentially Balance Bone Formation

Background

Clock genes and their protein products regulate circadian rhythms in mammals but have also been implicated in various physiological processes, including bone formation. Osteoblasts build new mineralized bone whereas osteoclasts degrade it thereby balancing bone formation. To evaluate the contribution of clock components in this process, we investigated mice mutant in clock genes for a bone volume phenotype.

Methodology/Principal Findings

We found that Per2^Brdm1 mutant mice as well as mice lacking Cry2^−/− displayed significantly increased bone volume at 12 weeks of age, when bone turnover is high. Per2^Brdm1 mutant mice showed alterations in parameters specific for osteoblasts whereas mice lacking Cry2^−/− displayed changes in osteoclast specific parameters. Interestingly, inactivation of both Per2 and Cry2 genes leads to normal bone volume as observed in wild type animals. Importantly, osteoclast parameters affected due to the lack of Cry2, remained at the level seen in the Cry2^−/− mutants despite the simultaneous inactivation of Per2.

Conclusions/Significance

This indicates that Cry2 and Per2 affect distinct pathways in the regulation of bone volume with Cry2 influencing mostly the osteoclastic cellular component of bone and Per2 acting on osteoblast parameters.     

Effects of bisphenol A and light conditions on the circadian rhythm of the goldfish Carassius auratus

We investigated how exposure to bisphenol A (BPA) under different photoperiodic conditions affected the expression of clock genes in the brain and liver of the goldfish, Carassius auratus. Three photoperiodic conditions were used: control, LD; continuous light, LL; and continuous dark, DD; the fish were exposed to three concentrations of BPA, namely 0, 10, or 100 μg/L. We measured changes in the expression of cryptochrome 1 (Cry1), period 2 (Per2), and melatonin receptor 1 (MT-R1). The levels of Cry1, Per2, and MT-R1 mRNAs decreased with increasing BPA concentration and with increasing exposure time. Expression of Cry1 and Per2 increased more in the LL group than in the LD and DD groups. However, for MT-R1, the DD group showed increased expression compared to the LL and LD groups. Our analysis shows that circadian rhythms in goldfish can be disrupted by exposure to BPA and that the response can be modified by regulating the photoperiod.     

Circadian Oscillations of Molecular Clock Components in the Cerebellar Cortex of the Rat

The central circadian clock of the mammalian brain resides in the suprachiasmatic nucleus (SCN) of the hypothalamus. At the molecular level, the circadian clockwork of the SCN constitutes a self-sustained autoregulatory feedback mechanism reflected by the rhythmic expression of clock genes. However, recent studies have shown the presence of extrahypothalamic oscillators in other areas of the brain including the cerebellum. In the present study, the authors unravel the cerebellar molecular clock by analyzing clock gene expression in the cerebellum of the rat by use of radiochemical in situ hybridization and quantitative real-time polymerase chain reaction. The authors here show that all core clock genes, i.e., Per1, Per2, Per3, Cry1, Cry2, Clock, Arntl, and Nr1d1, as well as the clock-controlled gene Dbp, are expressed in the granular and Purkinje cell layers of the cerebellar cortex. Among these genes, Per1, Per2, Per3, Cry1, Arntl, Nr1d1, and Dbp were found to exhibit circadian rhythms in a sequential temporal manner similar to that of the SCN, but with several hours of delay. The results of lesion studies indicate that the molecular oscillatory profiles of Per1, Per2, and Cry1 in the cerebellum are controlled, though possibly indirectly, by the central clock of the SCN. These data support the presence of a circadian oscillator in the cortex of the rat cerebellum. (Author correspondence: mrath@sund.ku.dk)     

“Feeding time” for the brain: A matter of clocks

Circadian clocks are autonomous time-keeping mechanisms that allow living organisms to predict and adapt to environmental rhythms of light, temperature and food availability. At the molecular level, circadian clocks use clock and clock-controlled genes to generate rhythmicity and distribute temporal signals. In mammals, synchronization of the master circadian clock located in the suprachiasmatic nuclei of the hypothalamus is accomplished mainly by light stimuli. Meal time, that can be experimentally modulated by temporal restricted feeding, is a potent synchronizer for peripheral oscillators with no clear synchronizing influence on the suprachiasmatic clock. Furthermore, food-restricted animals are able to predict meal time, as revealed by anticipatory bouts of locomotor activity, body temperature and plasma corticosterone. These food anticipatory rhythms have long been thought to be under the control of a food-entrainable clock (FEC). Analysis of clock mutant mice has highlighted the relevance of some, but not all of the clock genes for food-entrainable clockwork. Mutations of Clock or Per1 do not impair expression of food anticipatory components, suggesting that these clock genes are not essential for food-entrainable oscillations. By contrast, mice mutant for Npas2 or deficient for Cry1 and Cry2 show more or less altered responses to restricted feeding conditions. Moreover, a lack of food anticipation is specifically associated with a mutation of Per2, demonstrating the critical involvement of this gene in the anticipation of meal time. The actual location of the FEC is not yet clearly defined. Nevertheless, current knowledge of the putative brain regions involved in food-entrainable oscillations is discussed. We also describe several neurochemical pathways, including orexinergic and noradrenergic, likely to participate in conveying inputs to and outputs from the FEC to control anticipatory processes.