Circadian Amplitude Research Articles

Inter-Individual Differences In Habitual Sleep Timing and Entrained Phase of Endogenous Circadian Rhythms of BMAL1, PER2 and PER3 mRNA in Human Leukocytes

Individual sleep timing differs and is governed partly by circadian oscillators, which may be assessed by hormonal markers, or by clock gene expression. Clock gene expression oscillates in peripheral tissues, including leukocytes. The study objective was to determine whether the endogenous phase of these rhythms, assessed in the absence of the sleep-wake and light-dark cycle, correlates with habitual sleep-wake timing. Observational, cross-sectional. Home environment and Clinical Research Center. 24 healthy subjects aged 25.0 +/- 3.5 (SD) years. Actigraphy and sleep diaries were used to characterize sleep timing. Circadian rhythm phase and amplitude of plasma melatonin, cortisol, and BMAL1, PER2, and PER3 expression were assessed during a constant routine. Circadian oscillations were more robust for PER3 than for BMAL1 or PER2. Average peak timings were 6:05 for PER3, 8:06 for PER2, 15:06 for BMAL1, 4:20 for melatonin, and 10:49 for cortisol. Individual sleep-wake timing correlated with the phases of melatonin and cortisol. Individual PER3 rhythms correlated significantly with sleep-wake timing and the timing of melatonin and cortisol, but those of PER2 and BMAL1 did not reach significance. The correlation between sleep timing and PER3 expression was stronger in individuals homozygous for the variant of the PER3 polymorphism that is associated with morningness. Individual phase differences in PER3 expression during a constant routine correlate with sleep timing during entrainment. PER3 expression in leukocytes represents a useful molecular marker of the circadian processes governing sleep-wake timing.

Estimating Circadian Sleep/Wake Cycle From Rest/Activity Behaviors In Inbred Strains Of Rats

To establish methods to screen inbred rat strains, we studied EEG/EMG and movement in Brown Norway (BNMCWi), Fawn Hooded (FHMCWi), and Dahl Salt Sensitive (SSMCWi) inbred rat strains in environmental chambers during 3 successive 12L:12D circadian light:dark cycles. The 3rd light:dark cycle was analyzed for the circadian relationship of the onset, offset and duration of sleep state and movement. Although circadian amplitude and phase onset of sleep was different* between strains, onset, offset and duration of sleep and movement were significantly* correlated within strains. Analyzing probability of sleep onset and sleep offset from duration of inactivity we established a 95% probability* of sleep after 35 sec and 0.75 sec, respectively, that was NOT different* within and between strains. We developed an algorithm to determine sleep/wake periods from movement in previously unstudied FHMCWi and BNMCWi rats and found we correctly* estimated sleep onset (90%) and sleep offset (96%) in all observations. We have revised the algorithm to estimate 4 periods of sleep/wake (Active Wake, Quiet Wake, Sleep and Sustained Sleep) to further characterize sleep/wake. In conclusion we have established methods to screen sleep/wake patterns in consomic, congenic and ENU mutated inbred rat strains and to investigate physiological dysfunction and sleep in these models. This study was funded by NHLBI (U01 HL66579) (*P < 0.05)

Assessment of circadian function in fibroblasts of patients with bipolar disorder

Previous studies have implicated the circadian system in the pathophysiology of bipolar disorder, but conclusive evidence for altered circadian clocks is lacking. Cultured fibroblasts harbor circadian clocks representative of those in the master clock resident in the suprachiasmatic nuclei, providing a new avenue to investigate the core clock machinery in patients with bipolar illness. We examined the rhythmic expression patterns of core clock genes (BMAL1, PER1, PER2, REV-ERBalpha, DEC2, DBP) in fibroblasts from 12 bipolar patients and 12 healthy controls. Although we did not detect differences in the circadian period between bipolar patients and controls, the amplitude of rhythmic expression for BMAL1, REV-ERBalpha and DBP, as well as the overall mRNA expression level for DEC2 and DBP was reduced in fibroblasts from bipolar patients. Bonferroni's correction for multiple comparisons still resulted in significantly reduced DBP expression level, and trends toward reduced overall expression level of DEC2 and circadian amplitude of BMAL1, in fibroblasts from bipolar patients. We next examined an expanded cohort of 18 bipolar patients and 35 healthy controls for mRNA expression levels of four kinases (CKIdelta, CKIepsilon, GSK3alpha and GSK3beta) and the protein and phosphorylation levels of two of them (GSK3alpha and GSK3beta). We did not detect differences in steady-state mRNA levels or protein levels of these kinases between bipolar patients and controls, but the level of GSK3beta phosphorylation was significantly reduced in bipolar patients within an Old Order Amish bipolar kindred. Our results suggest that the reduced amplitudes and overall expression levels of circadian genes, and the decreased phosphorylation level of GSK3beta may lead to dysregulation of downstream genes, which could explain some pathological features of bipolar disorder.

Arterial stiffness and interdialytic weight gain influence ambulatory blood pressure patterns in hemodialysis patients

Besides an overall increase in blood pressure, hemodialysis patients have marked disturbance in interdialytic ambulatory blood pressure pattern that is characterized by blunted circadian amplitude and a steady rise in blood pressure between dialysis treatments. The pathophysiology of this abnormal blood pressure profile is poorly understood. We hypothesized that the circadian amplitude, the interdialytic increase (linear trend), and the average level of blood pressure (the intercept) are related to the extent of arterial stiffening and the degree of accumulation of salt and water between dialysis treatments. Using a generalized cosinor model, we simultaneously compared the impact of interdialytic weight gain and echo-Doppler-measured aortic pulse wave velocity on the mean level of blood pressure, linear changes over the interdialytic interval, and oscillatory changes in blood pressure. In a cross-sectional analysis of 11,833 blood pressure measurements from 125 long-term hemodialysis patients, we found that aortic pulse wave velocity and interdialytic weight gain had a substantial impact on interdialytic ambulatory blood pressure level, trends, and rhythms. Arterial stiffness was associated with an overall increase in the level (intercept) of systolic, diastolic, and pulse pressure. Interdialytic weight gain, on the other hand, was associated with interdialytic increase (linear trend) in blood pressure. The circadian amplitude was blunted by increments in either arterial stiffness or interdialytic weight gain. Since patterns of ambulatory arterial blood pressure are related to cardiovascular risk factors such as interdialytic weight gain and increased arterial stiffness, the pattern of ambulatory blood pressure recordings may also be of prognostic significance in hemodialysis patients.

Effects of exogenous melatonin and circadian synchronization on tumor progression in melanoma‐bearing C57BL6 mice

Circadian rhythmicity impairment reportedly becomes significant as a tumor progresses, while the incidence of cancer can be affected by disruption of the circadian system. Melatonin has oncostatic effects on several types of cancer (breast, prostate, and colorectal cancers), while it can be self-defeating in others, such as lymphoma. Melanoma is one of the most aggressive cancers in humans; however, it seems to respond positively to melatonin in vitro. The present work tested whether body temperature (BT) rhythms are impaired by tumor progression, and whether exogenous melatonin restricts tumor growth and restores circadian rhythmicity; therefore, enhancing survival. To this end, C57 mice were intraperitoneal implanted with a temperature data logger and subcutaneously inoculated with melanoma cells. Animals were then submitted to light-dark (LD) 12:12 cycles or continuous light (LL), with or without melatonin administration. Under LD light conditions, the BT rhythm exhibited a marked reduction in the first circadian harmonic amplitude, and increased phase instability (Rayleigh vector) as the tumor progressed. Melatonin administration (2 mg/kg BW/day), on the other hand, increased the BT rhythm amplitude and phase stability, reduced tumor weight and prevented intraperitoneal dissemination. Exposure to LL induced a free-running rhythm (1500 min), significantly increasing tumor malignity, and therefore reducing survival. Surprisingly, the highest tumor weights and morbidity by metastasis were seen in the LL group treated with melatonin probably because this indoleamine was being administered at different subjective hours to free-running animals. Circadian rhythmicity can thus be used as a marker rhythm for tumor progression, as rhythm impairment increases along with tumor malignancy. While melatonin administration improves rhythmicity and enhances survival under LD conditions, the results are self-defeating when they coexist with circadian disruption as it occurs under LL. This emphasizes the importance of taking into account endogenous rhythmicity and limiting melatonin administration to the subjective night in order to restrict melanoma progression.

P.4.a.001 Sleep disorders and obsessive-compulsive disorder

Disturbances in sleep and circadian rest-activity rhythms are key features of depression. Actigraphy, a non-invasive method for monitoring motor activity, can be used to objectively assess circadian rest-activity rhythms and sleep patterns. While recent studies have measured sleep and daytime activity of depressed patients using wrist-worn actigraphy, the actigraphic 24-h rest-activity rhythm in depression has not been well documented. We aimed to examine actigraphically measured sleep and circadian rest-activity rhythms in depressed outpatients. Twenty patients with DSM-IV major depressive episode and 20 age- and sex-matched healthy controls participated in this study. Participants completed 7 consecutive days of all-day actigraphic activity monitoring while engaging in usual activities. For sleep parameters, total sleep time, wake after sleep onset, and sleep fragmentation index were determined. Circadian rhythms were estimated by fitting individual actigraphy data to a cosine curve of a 24-h activity rhythm using the cosinor method, which generated three circadian activity rhythm parameters, i.e., MESOR (rhythm-adjusted mean), amplitude, and acrophase. Subjective sleep was also assessed using a sleep diary and the Pittsburgh Sleep Quality Index. Patients showed significantly lower MESOR and more dampened amplitude along with significant sleep disturbances. Logistic regression analysis revealed that lower MESOR and more fragmented sleep emerged as the significant predictors of depression. Correlations between subjectively and actigraphically measured parameters demonstrated the validity of actigraphic measurements. These results indicate marked disturbances in sleep and circadian rest-activity rhythms of depression. By simultaneously measuring sleep and rest-activity rhythm parameters, actigraphy might serve as an objective diagnostic aid for depression.

Does shift work cause cancer?

Does shift work cause cancer?

Optimization of Biomathematical Model Predictions for Cognitive Performance Impairment in Individuals: Accounting for Unknown Traits and Uncertain States in Homeostatic and Circadian Processes

Current biomathematical models of fatigue and performance do not accurately predict cognitive performance for individuals with a priori unknown degrees of trait vulnerability to sleep loss, do not predict performance reliably when initial conditions are uncertain, and do not yield statistically valid estimates of prediction accuracy. These limitations diminish their usefulness for predicting the performance of individuals in operational environments. To overcome these 3 limitations, a novel modeling approach was developed, based on the expansion of a statistical technique called Bayesian forecasting. The expanded Bayesian forecasting procedure was implemented in the two-process model of sleep regulation, which has been used to predict performance on the basis of the combination of a sleep homeostatic process and a circadian process. Employing the two-process model with the Bayesian forecasting procedure to predict performance for individual subjects in the face of unknown traits and uncertain states entailed subject-specific optimization of 3 trait parameters (homeostatic build-up rate, circadian amplitude, and basal performance level) and 2 initial state parameters (initial homeostatic state and circadian phase angle). Prior information about the distribution of the trait parameters in the population at large was extracted from psychomotor vigilance test (PVT) performance measurements in 10 subjects who had participated in a laboratory experiment with 88 h of total sleep deprivation. The PVT performance data of 3 additional subjects in this experiment were set aside beforehand for use in prospective computer simulations. The simulations involved updating the subject-specific model parameters every time the next performance measurement became available, and then predicting performance 24 h ahead. Comparison of the predictions to the subjects' actual data revealed that as more data became available for the individuals at hand, the performance predictions became increasingly more accurate and had progressively smaller 95% confidence intervals, as the model parameters converged efficiently to those that best characterized each individual. Even when more challenging simulations were run (mimicking a change in the initial homeostatic state; simulating the data to be sparse), the predictions were still considerably more accurate than would have been achieved by the two-process model alone. Although the work described here is still limited to periods of consolidated wakefulness with stable circadian rhythms, the results obtained thus far indicate that the Bayesian forecasting procedure can successfully overcome some of the major outstanding challenges for biomathematical prediction of cognitive performance in operational settings.

A physiologically based mathematical model of melatonin including ocular light suppression and interactions with the circadian pacemaker

The rhythm of plasma melatonin concentration is currently the most accurate marker of the endogenous human circadian pacemaker. A number of methods exist to estimate circadian phase and amplitude from the observed melatonin rhythm. However, almost all these methods are limited because they depend on the shape and amplitude of the melatonin pulse, which vary among individuals and can be affected by environmental influences, especially light. Furthermore, these methods are not based on the underlying known physiology of melatonin secretion and clearance, and therefore cannot accurately quantify changes in secretion and clearance observed under different experimental conditions. A published physiologically-based mathematical model of plasma melatonin can estimate synthesis onset and offset of melatonin under dim light conditions. We amended this model to include the known effect of melatonin suppression by ocular light exposure and to include a new compartment to model salivary melatonin concentration, which is widely used in clinical settings to determine circadian phase. This updated model has been incorporated into an existing mathematical model of the human circadian pacemaker and can be used to simulate experimental protocols under a number of conditions.

Clock Gene Expression in the Murine Gastrointestinal Tract: Endogenous Rhythmicity and Effects of a Feeding Regimen

Based on observations that the gastrointestinal tract is subject to various 24-hour rhythmic processes, it is conceivable that some of these rhythms are under circadian clock gene control. We hypothesized that clock genes are present in the gastrointestinal tract and that they are part of a functional molecular clock that coordinates rhythmic physiologic functions. The effects of timed feeding and vagotomy on temporal clock gene expression (clock, bmal1, per1-3, cry1-2) in the gastrointestinal tract and suprachiasmatic nucleus (bmal, per2) of C57BL/6J mice were examined using real-time polymerase chain reaction and Western blotting (BMAL, PER2). Colonic clock gene localization was examined using immunohistochemistry (BMAL, PER1-2). Clock immunoreactivity was observed in the myenteric plexus and epithelial crypt cells. Clock genes were expressed rhythmically throughout the gastrointestinal tract. Timed feeding shifted clock gene expression at the RNA and protein level but did not shift clock gene expression in the central clock. Vagotomy did not alter gastric clock gene expression compared with sham-treated controls. The murine gastrointestinal tract contains functional clock genes, which are molecular core components of the circadian clock. Daytime feeding in nocturnal rodents is a strong synchronizer of gastrointestinal clock genes. This synchronization occurs independently of the central clock. Gastric clock gene expression is not mediated through the vagal nerve. The presence of clock genes in the myenteric plexus and epithelial cells suggests a role for clock genes in circadian coordination of gastrointestinal functions such as motility, cell proliferation, and migration.

Assessment of circadian rhythms throughout the menstrual cycle of female rhesus monkeys

Reproductive cyclicity has a significant influence on the regulation of circadian rhythms in rodents. Studies have suggested that there are changes in body temperature rhythms between the follicular and luteal phases in human females. This study examined the effects of menstrual cyclicity on physiological and behavioral circadian rhythms in female rhesus monkeys (Macaca mulatta), an acknowledged biomedical model. Seven unrestrained subjects were implanted with a biotelemetry transmitter to measure body temperature and heart rate and an accelerometer was used to measure physical activity. Water was available ad libitum and drinking was measured via an electronic circuit attached to a water lixit. A video-based task system, the Psychomotor Test System, provided environmental enrichment and delivered a pelletized diet. Mean, phase, and amplitude of each rhythm were calculated. Estrogen and progesterone conjugates were assayed and quantified from daily urine samples to identify follicular and luteal phases of the menstrual cycle. Average circadian variables were then compared between these phases. Heart rate was significantly (P< or =0.05) delayed in the luteal phase. Albeit non-significant, analysis showed a trend toward decreased circadian amplitude of body temperature in the luteal phase.

Circadian Physiology Is a Toxicity Target of the Anticancer Drug Gemcitabine in Mice

The circadian timing system determines the optimal timing and waveform of drug tolerability, yet treatment itself can alter this system. Gemcitabine is an antimetabolite agent that is active against lung and pancreatic cancers. Tolerability for this drug is best following dosing at ZT 11 in mice. The authors investigated the effects of gemcitabine on the circadian rhythms in body temperature and rest activity as physiological markers of the circadian timing system. Healthy unrestrained B6D2F(1) mice implanted with radiotelemetry transmitters were kept in LD 12:12 prior to receiving a single intravenous dose of gemcitabine (200, 400, or 600 mg/kg) at ZT 11 or 23. Gemcitabine (400 mg/kg) transiently suppressed the body temperature rhythm in 50% of the mice dosed at ZT 23, as compared to none of the mice treated at ZT 11 within the 2 days following drug dosing (Fisher 's exact test p = 0.04). The rest-activity circadian rhythm was suppressed in 40% (ZT 11) and 50% (ZT 23) of the mice, respectively. In the mice with persistent circadian rhythms, gemcitabine delivery at ZT 23 resulted in more prominent decreases and slower recovery of circadian mesor and amplitude of both rhythms as compared to mice treated at ZT 11. Gemcitabine also induced a transient internal desynchronization between temperature and activity rhythms following dosing at ZT 23 but not at ZT 11. The delivery of a single therapeutic dose of gemcitabine near its time of least toxicity produced least alterations in circadian physiological outputs, a finding that suggests that the extent of circadian disruption contributes to toxicokinetic processes.

Whole body and muscle energy metabolism in preruminant calves: effects of nutrient synchrony and physical activity

The effects of asynchronous availability of amino acids and glucose on muscle composition and enzyme activities in skeletal muscle were studied in preruminant calves. It was hypothesized that decreased oxidative enzyme activities in muscle would explain a decreased whole body heat production with decreasing nutrient synchrony. Preruminant calves were assigned to one of six degrees of nutrient synchrony, step-wise separating the intake of protein and lactose over the two daily meals. Calves at the most synchronous treatment received two identical meals daily. At the most asynchronous treatment, 85% of the daily protein and 20% of the daily lactose supply were fed in one meal and the remainder in the other meal. Daily intakes of all dietary ingredients were identical for all treatments. Oxidative enzyme activities and fat content increased with decreasing nutrient synchrony in M. Rectus Abdominis (RA), but not in M. Semitendinosus. Cytochrome-c-oxidase activity was positively correlated with fat content in RA (r 0.49; P < 0.01). Oxidative enzyme activities in both muscles were not correlated with average daily heat production, but citrate synthase activity in RA was positively correlated (P < 0.01) with the circadian amplitude (r 0.53) and maximum (r 0.61) of heat production associated with physical activity. In conclusion, this study indicates that muscle energy stores are regulated by nutrient synchrony. The lack of correlation between muscle oxidative enzyme activities and average daily heat production was in contrast with findings in human subjects. Therefore, oxidative enzyme activity in muscle should not be used as an indicator for whole body heat production in growing calves.

Long Term Effects of Sleep Deprivation on The Mammalian Circadian Pacemaker

In mammals, sleep is controlled by a homeostatic process, which regulates depth of sleep, and by the circadian clock of the suprachiasmatic nucleus (SCN), which regulates 24-h rhythms in timing of sleep. Sleep deprivation is known to cause molecular and physiological changes and results in an alteration in the timing of sleep. It is generally assumed that following sleep deprivation, homeostatic mechanisms overrule the circadian clock, allowing animals to sleep during their active phase. However, recent evidence indicates that sleep states have direct access to the circadian pacemaker of the SCN. We questioned therefore whether sleep deprivation may have long-term effects on the circadian pacemaker, which may explain altered sleep patterns following sleep deprivation. To test this hypothesis, we combined SCN recordings of electrical impulse frequency through stationary implanted electrodes in freely moving rats with electroencephalogram recordings in the same animal before, during, and after a mild 6-h sleep deprivation. Following sleep deprivation, SCN neuronal activity was significantly reduced to about 60% of baseline levels. The decrements in SCN activity were most obvious during NREM sleep and REM sleep and lasted for 6-7 hours. The data show that sleep deprivation influences not only sleep homeostatic mechanisms, but also SCN electrical activity, resulting in a strong reduction in circadian amplitude in the major output signal from the SCN.

Central and Peripheral Signals Set the Circadian Liver Clock

Central and Peripheral Signals Set the Circadian Liver Clock

VIP Activates and Couples Clock Cells. Focus on “Disrupted Neuronal Activity Rhythms in the Suprachiasmatic Nucleus of Vasoactive Intestinal Polypeptide-Deficient Mice”

Neurons of the suprachiasmatic nucleus (SCN), located in the ventromedial hypothalamus, comprise the central mammalian circadian pacemaker ([Reppert and Weaver 2002][1]). In dispersed culture, these neurons can generate autonomous circadian (ca. 24 h) oscillations in spontaneous firing rate ([Welsh

Diurnal mood variation in depression: A signal of disturbed circadian function?

Background Diurnal variation in mood is a prominent symptom of depression, and is typically experienced as positive mood variation (PMV — mood being worse upon waking and better in the evening). The present study sought to advance understanding of PMV by measuring daily mood variation in non-clinical individuals with varying levels of depressed mood. Based on research into normative variation in mood and evidence that circadian amplitudes may be decreased in depression, it was hypothesised that compared to those with low levels of depression, individuals meeting Centre for Epidemiological Studies-Depression Scale (CES-D) cut-off for probable depression would exhibit an attenuated circadian component in diurnal variation of Positive Affect (PA). Method Ninety-nine young healthy women (mean age = 21.5, SD = 3.0) living on a normal sleep–wake schedule provided mood reports every two hours between 0800 h and 2200 h for 7 days. Results The high depression group (CES-D ≥ 23, n = 22) exhibited a pattern of diurnal variation consistent with PMV (increased PA in the evening relative to the morning). As predicted, evidence was also found that the high depression group was characterized by a decreased circadian component to diurnal variation in PA relative to the low depression group (CES-D < 23, n = 77). Conclusions It is provisionally concluded that diurnal mood variation in depression can usefully be understood from the perspective of weakened circadian function. Findings are discussed in terms of limitations of the study's naturalistic design and future research avenues identified.

Circadian clock assessment method in humans

A Circadian Clock Assessment (CCA) method was developed for identifying circadian phase, period (τ), mesor and amplitude using human core temperature (Tc) records showing normal or disrupted circadian Tc rhythms (CTcR). CCA uses an algorithm for identifying daily Tc nadirs and a series of fit polynomial curves for identifying circadian characteristics. CCA fit irregular Tc curve shapes better and produced more accurate estimates of circadian characteristics than Cosinor when applied to artificially generated data. CCA estimates showed less day-to-day variability than Cosinor on normal, entrained CTcR data. When CTcR was obviously disrupted, CCA produced estimates of τ that better agreed with visual estimates. CCA failed to identify τ once, in one data set that included both abnormally long (~30 h) and short (~ 12 h) days. Cosinor failed in this case as well. Cosinor had difficulty fitting abnormally long days, occasionally estimated τ in the hundreds of hours, and frequently produced estimates of τ that disagreed, by several hours, with τ estimated using a series of fit cosine curves. In conclusion, CCA is capable of estimating circadian characteristics when CTcR appears normal, as well as when CTcR is obviously disrupted. Applications include individualization of chronotherapy, therapies aimed at clock resynchronization, and basic research studying day-to-day changes in circadian characteristics.

Multiple phytohormones influence distinct parameters of the plant circadian clock

Circadian systems coordinate endogenous events with external signals. In mammals, hormone-clock feedbacks are a well-known integration system. Here, we investigated phytohormone effects on plant-circadian rhythms via the promoter:luciferase system. We report that many hormones control specific features of the plant-circadian system, and do so in distinct ways. In particular, cytokinins delay circadian phase, auxins regulate circadian amplitude and clock precision, and brassinosteroid and abscisic acid modulate circadian periodicity. We confirmed the pharmacology in hormone synthesis and perception mutants, as rhythmic expression is predictably altered in an array of hormone-related mutants. We genetically dissected one mechanism that integrates hormone signals into the clock, and showed that the hormone-activated ARABIDOPSIS RESPONSE REGULATOR 4 and the photoreceptor phytochrome B are elements in the input of the cytokinin signal to circadian phase. Furthermore, molecular-expression targets of this signal were found. Collectively, we found that plants have multiple input/output feedbacks, implying that many hormones can function on the circadian system to adjust the clock to external signals to properly maintain the clock system.

Regulating a Circadian Clock's Period, Phase and Amplitude by Phosphorylation: Insights from Drosophila

Much progress has been made in understanding the molecular underpinnings governing circadian ( approximately 24 h) rhythms. Despite the increased complexity in metazoans whereby inter-cellular networks form the basis for driving overt rhythms, such as wake-sleep cycles in animals, single isolated cells can exhibit all the formal properties of a circadian pacemaker. How do these cell-autonomous rhythm generators operate? Breakthrough studies in Drosophila melanogaster led to the realization that the molecular logic underlying circadian clocks are highly shared. Most notably, interconnected transcriptional-translational feedback loops produce coordinated rhythms in "clock" RNAs and proteins that are required for the daily progression of clocks, synchronization to local time and transducing temporal signals to downstream effector pathways. More recent findings indicate prominent roles for reversible phosphorylation of clock proteins in the core oscillatory mechanism. In this review we focus on findings in Drosophila to explore the multiple levels that reversible phosphorylation plays in clock function. Specific clock proteins in this system are subjected to different phosphorylation programs, which affect three key properties of a circadian oscillator, its period, amplitude and phase. The role of phosphorylation in clocks is of clear relevance to human health because mutations that affect the PERIOD (PER) phosphorylation program are associated with familial sleep disorders. In addition, the central role of phosphorylation in the assembly of a circadian oscillator was dramatically shown recently by the ability to reconstitute a circadian phosphorylation/dephosphorylation cycle in vitro, suggesting that the dynamics of clock protein phosphorylation are at the "heart" of circadian time-keeping.