Circadian Gating Research Articles

Neue Erkenntnisse zur Funktion unserer inneren Uhr - Melanopsinhaltige Rezeptoren in der Retina modulieren den Schlaf

Tsai JW, Hannibal J, Hagiwara G et al. Melanopsin as a sleep modulator: circadian gating of the direct effects of light on sleep and altered sleep homeostasis in Opn4-/-mice. PLoS Biol 2009; 7: e1000125

Melanopsin as a Sleep Modulator: Circadian Gating of the Direct Effects of Light on Sleep and Altered Sleep Homeostasis in Opn4−/− Mice

Author SummaryLight affects sleep in two ways: indirectly through the phase adjustment of circadian rhythms and directly through nonvisual mechanisms that are independent of the circadian system. The direct effects of light include the promotion of sleep in night-active animals and of alertness in diurnal species. We analyzed sleep and the electroencephalogram (EEG) under various light–dark regimens in mice lacking melanopsin (Opn4 −/−), a retinal photopigment crucial for conveying light-level information to the brain, to determine the role of melanopsin, as opposed to rod and cones, in mediating these direct effects of light. We show that melanopsin mediates the direct effects of light during the subjective dark period, whereas rods and cones contribute to these effects in the light period. Our finding that “sleep-active” (i.e., galanin-positive) neurons of the anterior hypothalamus are not activated by light in Opn4 −/− mice suggests that these neurons are part of the circuitry whereby light promotes sleep. Also, the alerting effects of transitions into darkness were less pronounced in Opn4 −/− mice judged on the reduced increase in EEG theta and gamma activity. Finally, and unexpectedly, the rate at which the need for sleep, quantified as EEG delta power, accumulated during wakefulness was found to be reduced in Opn4 −/− mice both during baseline and sleep deprivation conditions, implicating a photopigment in the homeostatic regulation of sleep. We conclude that melanopsin contributes to the direct effects of light and darkness, and in interaction with circadian and homeostatic drive, determines the occurrence and quality of both sleep and waking. If confirmed in humans, our observations will have applications for the clinical use of light as well as for societal lighting conditions.

Minimum Criteria for DNA Damage-Induced Phase Advances in Circadian Rhythms

Robust oscillatory behaviors are common features of circadian and cell cycle rhythms. These cyclic processes, however, behave distinctively in terms of their periods and phases in response to external influences such as light, temperature, nutrients, etc. Nevertheless, several links have been found between these two oscillators. Cell division cycles gated by the circadian clock have been observed since the late 1950s. On the other hand, ionizing radiation (IR) treatments cause cells to undergo a DNA damage response, which leads to phase shifts (mostly advances) in circadian rhythms. Circadian gating of the cell cycle can be attributed to the cell cycle inhibitor kinase Wee1 (which is regulated by the heterodimeric circadian clock transcription factor, BMAL1/CLK), and possibly in conjunction with other cell cycle components that are known to be regulated by the circadian clock (i.e., c-Myc and cyclin D1). It has also been shown that DNA damage-induced activation of the cell cycle regulator, Chk2, leads to phosphorylation and destruction of a circadian clock component (i.e., PER1 in Mus or FRQ in Neurospora crassa). However, the molecular mechanism underlying how DNA damage causes predominantly phase advances in the circadian clock remains unknown. In order to address this question, we employ mathematical modeling to simulate different phase response curves (PRCs) from either dexamethasone (Dex) or IR treatment experiments. Dex is known to synchronize circadian rhythms in cell culture and may generate both phase advances and delays. We observe unique phase responses with minimum delays of the circadian clock upon DNA damage when two criteria are met: (1) existence of an autocatalytic positive feedback mechanism in addition to the time-delayed negative feedback loop in the clock system and (2) Chk2-dependent phosphorylation and degradation of PERs that are not bound to BMAL1/CLK.

Circadian Timing of REM Sleep Is Coupled to an Oscillator within the Dorsomedial Suprachiasmatic Nucleus

Sleep is consistently concentrated to a specific time of the day. Its timing and consolidation depend on the interplay between a homeostatic and a circadian process of sleep regulation [1-3]. Sleep propensity rises as a homeostatic response to increasing wake time, whereas a circadian clock determines the specific time when sleep will probably occur. This two-process regulation of sleep also determines which specific sleep stage will be manifested, and the circadian process governs tightly the manifestation of rapid eye movement sleep (REMS) [1, 4]. The role of the hypothalamic suprachiasmatic nucleus (SCN) in the circadian gating of sleep and wakefulness has been unequivocally established by lesion studies [5], but its role in the timing of specific sleep stages has remained unknown. Using a forced desynchrony paradigm that induces the stable dissociation of the ventrolateral (vl) and dorsomedial (dm) SCN, and a jetlag paradigm that induces desynchronization between these SCN subregions, we show that the SCN can time the occurrence of specific sleep stages. Specifically, the circadian regulation of REMS is associated with clock gene expression within the dmSCN. We provide the first neurophysiological model for the disruption of sleep architecture that may result from temporal challenges such as rotational-shift work and jetlag.

Imaging Multidimensional Therapeutically Relevant Circadian Relationships

Circadian clocks gate cellular proliferation and, thereby, therapeutically target availability within proliferative pathways. This temporal coordination occurs within both cancerous and noncancerous proliferating tissues. The timing within the circadian cycle of the administration of drugs targeting proliferative pathways necessarily impacts the amount of damage done to proliferating tissues and cancers. Concurrently measuring target levels and associated key pathway components in normal and malignant tissues around the circadian clock provides a path toward a fuller understanding of the temporal relationships among the physiologic processes governing the therapeutic index of antiproliferative anticancer therapies. The temporal ordering among these relationships, paramount to determining causation, is less well understood using two- or three-dimensional representations. We have created multidimensional multimedia depictions of the temporal unfolding of putatively causative and the resultant therapeutic effects of a drug that specifically targets these ordered processes at specific times of the day. The systems and methods used to create these depictions are provided, as well as three example supplementary movies.

Circadian modulation of short-term memory in Drosophila

Endogenous biological clocks are widespread regulators of behavior and physiology, allowing for a more efficient allocation of efforts and resources over the course of a day. The extent that different processes are regulated by circadian oscillators, however, is not fully understood. We investigated the role of the circadian clock on short-term associative memory formation using a negatively reinforced olfactory-learning paradigm in Drosophila melanogaster. We found that memory formation was regulated in a circadian manner. The peak performance in short-term memory (STM) occurred during the early subjective night with a twofold performance amplitude after a single pairing of conditioned and unconditioned stimuli. This rhythm in memory is eliminated in both timeless and period mutants and is absent during constant light conditions. Circadian gating of sensory perception does not appear to underlie the rhythm in short-term memory as evidenced by the nonrhythmic shock avoidance and olfactory avoidance behaviors. Moreover, central brain oscillators appear to be responsible for the modulation as cryptochrome mutants, in which the antennal circadian oscillators are nonfunctional, demonstrate robust circadian rhythms in short-term memory. Together these data suggest that central, rather than peripheral, circadian oscillators modulate the formation of short-term associative memory and not the perception of the stimuli.

Interactions between circadian and hormonal signalling in plants

Growth and development of plants is controlled by external and internal signals. Key internal signals are those generated by hormones and the circadian clock. We highlight interactions between the circadian clock and hormonal signalling networks in regulating the physiology and growth of plants. Microarray analysis has shown that a significant proportion of transcripts involved in hormonal metabolism, catabolism, perception and signalling are also regulated by the circadian clock. In particular, there are interactions between the clock and abscisic acid, auxin, cytokinin and ethylene signalling. We discuss the role of circadian modulation ('gating') of hormonal signals in preventing temporally inappropriate responses. A consideration of the daily changes in physiology provides evidence that circadian gating of hormonal signalling couples the rhythmic regulation of carbon and water utilisation to rhythmic patterns of growth.

Modeling the Effects of Cell Cycle M-phase Transcriptional Inhibition on Circadian Oscillation

Circadian clocks are endogenous time-keeping systems that temporally organize biological processes. Gating of cell cycle events by a circadian clock is a universal observation that is currently considered a mechanism serving to protect DNA from diurnal exposure to ultraviolet radiation or other mutagens. In this study, we put forward another possibility: that such gating helps to insulate the circadian clock from perturbations induced by transcriptional inhibition during the M phase of the cell cycle. We introduced a periodic pulse of transcriptional inhibition into a previously published mammalian circadian model and simulated the behavior of the modified model under both constant darkness and light–dark cycle conditions. The simulation results under constant darkness indicated that periodic transcriptional inhibition could entrain/lock the circadian clock just as a light–dark cycle does. At equilibrium states, a transcriptional inhibition pulse of certain periods was always locked close to certain circadian phases where inhibition on Per and Bmal1 mRNA synthesis was most balanced. In a light–dark cycle condition, inhibitions imposed at different parts of a circadian period induced different degrees of perturbation to the circadian clock. When imposed at the middle- or late-night phase, the transcriptional inhibition cycle induced the least perturbations to the circadian clock. The late-night time window of least perturbation overlapped with the experimentally observed time window, where mitosis is most frequent. This supports our hypothesis that the circadian clock gates the cell cycle M phase to certain circadian phases to minimize perturbations induced by the latter. This study reveals the hidden effects of the cell division cycle on the circadian clock and, together with the current picture of genome stability maintenance by circadian gating of cell cycle, provides a more comprehensive understanding of the phenomenon of circading gating of cell cycle.

The Circadian Clock Component BMAL1 Is a Critical Regulator of p21WAF1/CIP1 Expression and Hepatocyte Proliferation

Most living organisms show circadian (approximately 24 h) rhythms in physiology and behavior. These oscillations are generated by endogenous circadian clocks, present in virtually all cells where they control key biological processes. Although circadian gating of mitosis has been reported for many years in some peripheral tissues, the underlying molecular mechanisms have remained poorly understood. Here we show that the cell cycle inhibitor p21WAF1/CIP1 is rhythmically expressed in mouse peripheral organs. This rhythmic pattern of mRNA and protein expression was recapitulated in vitro in serum-shocked differentiated skeletal muscle cells. p21WAF1/CIP1 circadian expression is dramatically increased and no longer rhythmic in clock-deficient Bmal1-/- knock-out mice. Biochemical and genetic data show that oscillation of p21WAF1/CIP1 gene transcription is regulated by the antagonistic activities of the orphan nuclear receptors REV-ERBalpha/beta and RORalpha4/gamma, which are core clock regulators. Importantly, p21WAF1/CIP1 overexpressing Bmal1-/- primary hepatocytes exhibit a decreased proliferation rate. This phenotype could be reversed using small interfering RNA-mediated knockdown of p21WAF1/CIP1. These data establish a novel molecular link between clock and cell cycle genes and suggest that the G1 progression phase is a target of the circadian clock during liver cell proliferation.

Circadian Gating of Photoinduction of Commitment to Cell-cycle Transitions in Relation to Photoperiodic Control of Cell Reproduction in Euglena¶

Circadian Gating of Photoinduction of Commitment to Cell-cycle Transitions in Relation to Photoperiodic Control of Cell Reproduction in Euglena¶

ELF4Is Required for Oscillatory Properties of the Circadian Clock

Circadian clocks are required to coordinate metabolism and physiology with daily changes in the environment. Such clocks have several distinctive features, including a free-running rhythm of approximately 24 h and the ability to entrain to both light or temperature cycles (zeitgebers). We have previously characterized the EARLY FLOWERING4 (ELF4) locus of Arabidopsis (Arabidopsis thaliana) as being important for robust rhythms. Here, it is shown that ELF4 is necessary for at least two core clock functions: entrainment to an environmental cycle and rhythm sustainability under constant conditions. We show that elf4 demonstrates clock input defects in light responsiveness and in circadian gating. Rhythmicity in elf4 could be driven by an environmental cycle, but an increased sensitivity to light means the circadian system of elf4 plants does not entrain normally. Expression of putative core clock genes and outputs were characterized in various ELF4 backgrounds to establish the molecular network of action. ELF4 was found to be intimately associated with the CIRCADIAN CLOCK-ASSOCIATED1 (CCA1)/LONG ELONGATED HYPOCOTYL (LHY)-TIMING OF CAB EXPRESSION1 (TOC1) feedback loop because, under free run, ELF4 is required to regulate the expression of CCA1 and TOC1 and, further, elf4 is locked in the evening phase of this feedback loop. ELF4, therefore, can be considered a component of the central CCA1/LHY-TOC1 feedback loop in the plant circadian clock.

Reaching out of the shade

Competition for light determines the success of individual plants in dense vegetation. Much depends on the capacity of plants to detect neighbours quickly and on their ability to respond to these signals. Recent findings indicate that although red:far-red ratios, and thus phytochromes, are of major importance in shade-avoidance responses, they do not act alone. Differences in light intensity also provoke shade-avoidance phenotypes, with blue light playing an important role in dense stands. Moreover, links between shade-avoidance signalling and auxins, gibberellins and ethylene have emerged. Additional breakthroughs are based on transcriptome studies that have unveiled new components in the response to shading. Amongst these, the phytochrome interacting factor 3-like proteins PIL1 and PIL2 underline the importance of circadian gating in shade avoidance.

Circadian G2 Arrest as Related to Circadian Gating of Cell Population Growth in Euglena

Cell population growth is gated to occur in particular circadian phases, which has been known for over four decades in various organisms including cyanobacteria and human. However, little is known as to which cell cycle phases from G1 to M are primarily regulated by the circadian rhythm or when in a circadian cycle this primary regulation takes place. We report here that in the flagellate alga Euglena gracilis grown photoautotrophically, the circadian rhythm primarily prevented developmentally matured G2 cells from progressing to mitosis, such that cell population growth occurred only during subjective night. In addition, we found that the circadian rhythm also arrests G1-to-S and S-to-G2 transitions at particular circadian phases.

Pineal circadian clocks gate arylalkylamine N‐acetyltransferase gene expression in the mouse pineal gland

In mammals it has been thought that the circadian clock localizes only in the suprachiasmatic nucleus of the hypothalamus. Recent studies have revealed that certain brain regions and peripheral tissues may also have intrinsic circadian clocks. However, the roles played by 'peripheral circadian clocks' have not been fully elucidated. In this study, we investigated their function using mouse pineal glands, and found that expression of the arylalkylamine N-acetyltransferase (Aa-Nat, EC 2.3.1.87, the rate-limiting enzyme of melatonin synthesis) gene after adrenergic receptor stimulation depended on the time of day even in vitro (gating). Phase-dependent Aa-Nat responses were observed in both melatonin-proficient and melatonin-deficient mouse pineal glands. Phosphodiesterases are unlikely to suppress Aa-Nat induction because a phosphodiesterase inhibitor itself had no effect on the mRNA levels. Puromycin was ineffective in inducing Aa-Nat mRNA levels in either the presence or absence of isoproterenol, suggesting that newly synthesized proteins may not be necessary to gate the Aa-Nat responses. We also discovered circadian dependence of the expression of Period1-luminescence in Period1-luciferase transgenic mouse pineal glands: circadian clocks may be functional in culture. Aa-Nat mRNA levels showed no significant circadian rhythms in the absence of isoproterenol, thus suggesting that Aa-Nat mRNA levels are induced by adrenergic mechanisms, not by a pineal circadian clock. Our results suggest that the pineal circadian clock may determine timing when Aa-Nat gene expression can respond to inputs from the master circadian clock in the suprachiasmatic nucleus, e.g. adrenergic stimulation.

Feeding Cues Alter Clock Gene Oscillations and Photic Responses in the Suprachiasmatic Nuclei of Mice Exposed to a Light/Dark Cycle

The suprachiasmatic nuclei (SCN) of the hypothalamus contain the master mammalian circadian clock, which is mainly reset by light. Temporal restricted feeding, a potent synchronizer of peripheral oscillators, has only weak influence on light-entrained rhythms via the SCN, unless restricted feeding is coupled with calorie restriction, thereby altering phase angle of photic synchronization. Effects of daytime restricted feeding were investigated on the mouse circadian system. Normocaloric feeding at midday led to a predominantly diurnal (60%) food intake and decreased blood glucose in the afternoon, but it did not affect the phase of locomotor activity rhythm or vasopressin expression in the SCN. In contrast, hypocaloric feeding at midday led to 2-4 h phase advances of three circadian outputs, locomotor activity rhythm, pineal melatonin, and vasopressin mRNA cycle in the SCN, and it decreased daily levels of blood glucose. Furthermore, Per1 and Cry2 oscillations in the SCN were phase advanced by 1 and 3 h, respectively, in hypocalorie- but not in normocalorie-fed mice. The phase of Per2 and Bmal1 expression remained unchanged regardless of feeding condition. Moreover, the shape of behavioral phase-response curve to light and light-induced expression of Per1 in the SCN were markedly modified in hypocalorie-fed mice compared with animals fed ad libitum. The present study shows that diurnal hypocaloric feeding affects not only the temporal organization of the SCN clockwork and circadian outputs in mice under light/dark cycle but also photic responses of the circadian system, thus indicating that energy metabolism modulates circadian rhythmicity and gating of photic inputs in mammals.

The TIME FOR COFFEE gene maintains the amplitude and timing of Arabidopsis circadian clocks.

Plants synchronize developmental and metabolic processes with the earth's 24-h rotation through the integration of circadian rhythms and responses to light. We characterize the time for coffee (tic) mutant that disrupts circadian gating, photoperiodism, and multiple circadian rhythms, with differential effects among rhythms. TIC is distinct in physiological functions and genetic map position from other rhythm mutants and their homologous loci. Detailed rhythm analysis shows that the chlorophyll a/b-binding protein gene expression rhythm requires TIC function in the mid to late subjective night, when human activity may require coffee, in contrast to the function of EARLY-FLOWERING3 (ELF3) in the late day to early night. tic mutants misexpress genes that are thought to be critical for circadian timing, consistent with our functional analysis. Thus, we identify TIC as a regulator of the clock gene circuit. In contrast to tic and elf3 single mutants, tic elf3 double mutants are completely arrhythmic. Even the robust circadian clock of plants cannot function with defects at two different phases.

Circadian gating of photoinduction of commitment to cell-cycle transitions in relation to photoperiodic control of cell reproduction in Euglena.

A novel type of circadian and photoperiodic control of the cell division cycle was found in photoautotrophic Euglena gracilis. When algae entrained to 24 h light-dark (LD) cycles (14 h L) were transferred to continuous darkness (DD) at the eighth hour of the final LD photoperiod, cell-cycle transition was arrested in phase G1, S or G2. The subsequent exposure of these dark-arrested cells to a 6 h light-break allowed the dark-arrested cells to undergo cell-cycle progression in DD, in a manner dependent on the circadian phase; maximum photoinduction occurred around dusk. Inhibitor experiments suggested that the photoinduced commitment of G2 cells to cell division required light for a signal originating in noncyclic photosynthetic electron transport (PET), particularly cytochrome b6-f but not for the metabolic energy required by the process. The fact that the circadian rhythm of photoinduction ran out-of-phase from that of noncyclic PET signaling suggests that the site of regulation by the former rhythm is downstream of noncyclic PET. The occurrence of maximum photoinduction around dusk suggests that the 'external coincidence' model of photoperiodic induction describes the activation of the photoinductive phase. Further evidence supporting this hypothesis is the relationship between cell reproduction and day length; the resulting sigmoidal curve indicates a combined effect of photosynthesizing period and circadian stimulation around dusk. Circadian control is shown to be an integral part of the mechanism for 24 h LD cycle-induced synchronous cell division.

Role of feedback loops in the scorpion circadian system

Scorpions are nocturnal animals whose circadian activity rhythm is entrained via light/dark programs to the median and lateral eyes. Timing of light signals conveyed by the median eyes is modified by feedback from the circadian pacemaker. Using a simulation program, NeuroDynamix, we demonstrated that these median-eye feedback loops cause a prolonged dead zone in the phase response curve. Also, entrainment by median eyes during simulated T cycles resulted primarily from brief photoreceptor twilight signals. We propose that feedback loops reduce entrainment stability, but serve a necessary function by limiting light-generated inputs to the pacemaker to a narrow circadian gate.

The PAS Protein VIVID Defines a Clock-Associated Feedback Loop that Represses Light Input, Modulates Gating, and Regulates Clock Resetting

vvd, a gene regulating light responses in Neurospora, encodes a novel member of the PAS/LOV protein superfamily. VVD defines a circadian clock–associated autoregulatory feedback loop that influences light resetting, modulates circadian gating of input by connecting output and input, and regulates light adaptation. Rapidly light induced, vvd is an early repressor of light-regulated processes. Further, vvd is clock controlled; the clock gates light induction of vvd and the clock gene frq so identical signals yield greater induction in the morning. Mutation of vvd severely dampens gating, especially of frq, consistent with VVD modulating gating and phasing light-resetting responses. vvd null strains display distinct alterations in the phase–response curve to light. Thus VVD, although not part of the clock, contributes significantly to regulation within the Neurospora circadian system.

The ELF3 zeitnehmer regulates light signalling to the circadian clock.

The circadian system regulates 24-hour biological rhythms and seasonal rhythms, such as flowering. Long-day flowering plants like Arabidopsis thaliana, measure day length with a rhythm that is not reset at lights-off, whereas short-day plants measure night length on the basis of circadian rhythm of light sensitivity that is set from dusk, early flowering 3 (elf3) mutants of Arabidopsis are aphotoperiodic and exhibit light-conditional arrhythmias. Here we show that the elf3-7 mutant retains oscillator function in the light but blunts circadian gating of CAB gene activation, indicating that deregulated phototransduction may mask rhythmicity. Furthermore, elf3 mutations confer the resetting pattern of short-day photoperiodism, indicating that gating of phototransduction may control resetting. Temperature entrainment can bypass the requirement for normal ELF3 function for the oscillator and partially restore rhythmic CAB expression. Therefore, ELF3 specifically affects light input to the oscillator, similar to its function in gating CAB activation, allowing oscillator progression past a light-sensitive phase in the subjective evening. ELF3 provides experimental demonstration of the zeitnehmer ('time-taker') concept.