High-Resolution Measurement of Circadian Periodicities in Acetabularia

Well-expressed endogenous circadian rhythms in Acetabularia acetabulum were spectrally analyzed and recorded in time-period distributions. The stability of the circadian periods under constant conditions and their changes could be monitored continually in step sizes close to the circadian period length. The resolution of period estimates of the circadian component was increased by a factor of 4-10 by adapting analyzed interval lengths to full period sizes of the corresponding main component. Methodological aspects of the applied algorithms are discussed by means of examples that measure the temperature dependency of the circadian period.     

Regression Models for the Estimation of Orcadian Rhythms in the Presence of Effects Due to Masking

The estimation of human circadian rhythms from experimental data is complicated by the presence of “masking” effects associated with the sleep-wake cycle. The observed rhythm may include a component due to masking, as well as the endogenous component linked to a circadian pacemaker. In situations where the relationship between the sleep-wake cycle and the circadian rhythm is not constant, it may be possible to obtain individual estimates of these two components, but methods commonly used for the estimation of circadian rhythms, such as the cosinor analysis, spectral analysis, average waveforms and complex demodulation, have not generally been adapted to identify the modulations that arise from masking. The estimates relate to the observed rhythms, and the amplitudes and acrophases do not necessarily refer to the endogenous rhythm.

In this paper methods are discussed for the separation of circadian and masking effects using regression models that incorporate a sinusoidal circadian variation together with functions of time since sleep and time during sleep. The basic model can be extended to include a time-varying circadian rhythm and estimates are available for the amplitude and phase at a given time, together with their joint confidence intervals and tests for changes in amplitude and acrophase between any two selected times. Modifications of these procedures are discussed to allow for non-sinusoidal circadian rhythms, non-additivity of the circadian and time-since-sleep effects and the breakdown of the usual assumptions concerning the residual errors.

This approach enables systematic masking effects associated with the sleep-wake cycle to be separated from the circadian rhythm, and it has applications to the analysis of data from experiments where the sleep-wake cycle is not synchronized with the circadian rhythm, for example after time-zone transitions or during irregular schedules of work and rest.     

Characterizing the amplitude dynamics of the human core-temperature circadian rhythm using a stochastic-dynamic model

Two measures, amplitude and phase, have been used to describe the characteristics of the endogenous human circadian pacemaker, a biological clock located in the hypothalamus. Although many studies of change in circadian phase with respect to different stimuli have been conducted, the physiologic implications of the amplitude changes (dynamics) of the pacemaker are unknown. It is known that phase changes of the human circadian pacemaker have a significant impact on sleep timing and content, hormone secretion, subjective alertness and neurobehavioral performance. However, the changes in circadian amplitude with respect to different stimuli are less well documented. Although amplitude dynamics of the human circadian pacemaker are observed in physiological rhythms such as plasma cortisol, plasma melatonin and core temperature data, currently methods are not available to accurately characterize the amplitude dynamics from these rhythms. Of the three rhythms core temperature is the only reliable variable that can be monitored continuously in real time with a high sampling rate. To characterize the amplitude dynamics of the circadian pacemaker we propose a stochastic-dynamic model of core temperature data that contains both stochastic and dynamic characteristics. In this model the circadian component that has a dynamic characteristic is represented as a perturbation solution of the van der Pol equation and the thermoregulatory response in the data that has a stochastic characteristic is represented as a first-order autoregressive process. The model parameters are estimated using data with a maximum likelihood procedure and the goodness-of-fit measures along with the associated standard error of the estimated parameters provided inference about the amplitude dynamics of the pacemaker. Using this model we analysed core temperature data from an experiment designed to exhibit amplitude dynamics. We found that the circadian pacemaker recovers slowly to an equilibrium level following amplitude suppression. In humans this reaction to perturbation from equilibrium value has potential physiological implications.     

Protein kinase C modulates light responses in Neurospora by regulating the blue light photoreceptor WC-1

The Neurospora protein kinase C (NPKC) is a regulator of light responsive genes. We have studied the function of NPKC in light response by investigating its biochemical and functional interaction with the blue light photoreceptor white-collar 1 (WC-1), showing that activation of NPKC leads to a significant decrease in WC-1 protein levels. Furthermore, we show that WC-1 and NPKC interact in a light-regulated manner in vivo, and that protein kinase C (PKC) phosphorylates WC-1 in vitro. We designed dominant negative and constitutively active forms of PKC which are able to induce either a large increase of WC-1 protein level or a strong reduction respectively. Moreover, these changes in PKC activity result in an altered light response. As WC-1 is a key component of Neurospora circadian clock and regulates the clock oscillator component FRQ we investigated the effect of NPKC-mutated forms on FRQ levels. We show that changes in PKC activity affect FRQ levels and the robustness of the circadian clock. Together these data identify NPKC as a novel component of the Neurospora light signal transduction pathway that modulates the circadian clock.     

Circadian variations in self-monitoring,a component of executive functions

The objective of this study was to identify circadian rhythms in self-monitoring, a component of executive functions. Participants were 10 undergraduate students, age: 18.5 ± 2.68 years, two male and eight female. They were recorded on a 30-h constant routine protocol; rectal temperature was recorded every minute and performance on a tracking task was assessed every 100 min. Self-monitoring indicators were adjustments of responses to random changes of speed and trajectory of a circle moving on the computer screen. Participants showed better accuracy during the afternoon, with decreases in the morning (06:20 and 08:00 h). These variations showed a phase delay of 2:29 ± 2:19 h with respect to the circadian rhythm of body temperature. In conclusion, there are circadian variations in self-monitoring. The decline in this component of executive functions could cause serious accidents among people working or studying during a morning shift, as well as commuting to and from work or school.     

Different Behavior of the Circadian Rhythms of Activity and Body Temperature During Resynchronization Following an Advance of the LD Cycle

Spontaneous activity and the body temperature of laboratory mice were recorded telemetrically using implantable transmitters. Following ten control days (L : D = 12 : 12; light from 07:00 to 19:00), the LD cycle was phase-advanced by shortening the light time by 8 h. Recordings were continued for a further 3 weeks. The raw temperature data were unmasked or 'purified' — that is, the temperature changes due to locomotor activity were removed, so revealing the endogenous component of the rhythm — using a regression method previously developed by us. The circadian rhythms of activity and measured body temperature resynchronized on average after 8 days. During resynchronization, both rhythms tended to show two components, one adjusting by a phase advance and the other by a phase delay. However, after purification of the body temperature rhythm, only the advancing component remained. These results indicate that the delaying component of the measured temperature rhythm was caused by masking due to activity, and that the endogenous component of this rhythm did not divide into two components during the resynchronization process. Also, the endogenous component of the circadian rhythm of body temperature and one component of the activity rhythm seemed to be controlled by the same oscillator. It remains uncertain how the other component of the activity rhythm is regulated.     

Different Behavior of the Circadian Rhythms of Activity and Body Temperature During Resynchronization Following an Advance of the LD Cycle

Spontaneous activity and the body temperature of laboratory mice were recorded telemetrically using implantable transmitters. Following ten control days (L : D = 12 : 12; light from 07:00 to 19:00), the LD cycle was phase-advanced by shortening the light time by 8 h. Recordings were continued for a further 3 weeks. The raw temperature data were unmasked or ‘purified’ — that is, the temperature changes due to locomotor activity were removed, so revealing the endogenous component of the rhythm — using a regression method previously developed by us. The circadian rhythms of activity and measured body temperature resynchronized on average after 8 days. During resynchronization, both rhythms tended to show two components, one adjusting by a phase advance and the other by a phase delay. However, after purification of the body temperature rhythm, only the advancing component remained. These results indicate that the delaying component of the measured temperature rhythm was caused by masking due to activity, and that the endogenous component of this rhythm did not divide into two components during the resynchronization process. Also, the endogenous component of the circadian rhythm of body temperature and one component of the activity rhythm seemed to be controlled by the same oscillator. It remains uncertain how the other component of the activity rhythm is regulated.     

Ultradian Components of the Sleep-Wake Cycle in Babies

Behavioral states may be analyzed as expressions of underlying cyclic activity involving several physiological systems. The human sleep-wake cycle in the first year of life shows, in addition to the establishment of circadian rhythmic-ity around the second month, the dynamics of its ultradian components, as can be seen in the more or less gradual decline of the polyphasic pattern. To detect these changes, we have analyzed the sleep-wake cycle of five babies of different ages (3, 4, 9, 11, and 13 months) observed for 5 consecutive days (Monday through Friday), 10 h (08:00-18:00 h) per day at a kindergarten by the first author, and during the night (18:00-08:00 h) by the parents. Behavioral observations were designed for minimizing interference with the babies' habits. Sleep/wake data were arranged in 60-min intervals, and the relative amount of time spent asleep per interval constituted the time series submitted for statistical analysis. The five resulting time series were submitted to spectral analysis for detecting the composition of frequencies contributing to the observed sleep/wake cycle. Several frequencies were thus obtained for each baby in the ultradian and circadian domain, ranging from one cycle in 2.0 h to one cycle in 24 h. The circadian component was the strongest rhythmic influence for all individuals except for the youngest (3-month-old) baby, who showed a semicircadian component as the main frequency in the power spectrum. Three individuals showed ultradian frequencies in the domain of 3-4 h. Differences in the spectra derive from three possible, and probably not exclusive. causes: 1) ontogenetic changes, 2) different masking effects, and 3) individual differences.     

Possible linkage between the ability to change the period (tau) of the prolactin and cortisol rhythms in women and breast cancer risk

The 24 h profiles of plasma hormone concentrations are rhythmic. The circadian period (τ) changes in development, with seasons, and in women with different stages of the menstrual cycle. It is known that the rhythms of prolactin and cortisol are sensitive to environmental time cues, such as changes in day length and phase; however, the importance of these changes is not yet understood. This study investigates whether there is a relation between the ability of a subject to respond to external cues that are associated with seasonal changes causing alteration of the rhythm's periods in cortisol and prolactin and the epidemiologically determined susceptibility to breast cancer. It is shown that the rhythmic output pattern of prolactin and cortisol in vivo is generated by more than one oscillator and structured by more than one rhythmic component. Each cohort of American women, classified on an epidemiologic basis as high risk (HR) or low risk (LR) to develop breast cancer, expresses different rhythmic output patterns of both variables, suggesting that the genetic background as defined by the risk state is related to differences in the circadian time structure, including the ability of the subject to change the rhythm's τ. The LR cohort exhibited a statistically significant change between seasons in the rhythm's τ of both the prolactin and cortisol patterns. In contrast, the HR cohort showed no change in the rhythm's τ between seasons for prolactin and cortisol patterns. These results show that in human beings, the presence of a circannual rhythm in the circadian time structure or the ability to adapt the circadian rhythmic pattern of these variables to external cues, such as seasons, is related to the partly genetically determined risk state to develop breast cancer and may be of importance for human health.     

Intracellular and intercellular processes determine robustness of the circadian clock

Circadian clocks are present in most organisms and provide an adaptive mechanism to coordinate physiology and behavior with predictable changes in the environment. Genetic, biochemical, and cellular experiments have identified more than a dozen component genes and a signal transduction pathway that support cell-autonomous, circadian clock function. One of the hallmarks of biological clocks is their ability to reset to relevant stimuli while ignoring most others. We review recent results showing intracellular and intercellular mechanisms that convey this robust timekeeping to a variety of circadian cell types.     

Circadian inputs influence the performance of a spiking, movement-sensitive neuron in the visual system of the blowfly.

Long-term extracellular recordings from a spiking, movement-sensitive giant neuron (H1) in the third optic ganglion of the blowfly Calliphora vicina (L.) revealed periodic endogenous sensitivity fluctuations. The sensitivity changes showed properties typical of an endogenous circadian rhythm. This was true for the responses in reaction to intensity changes of visual patterns as well as for the responses elicited by pattern movement. For these two types of stimuli, the circadian fluctuations were comparable, but the envelope in the case of responses to movement was more robust. A circadian fluctuation in responses to movement is, therefore, present at the level of single elementary movement detectors. The tonic activity of the neuron was also shown to be under circadian control. In constant darkness (DD) the fluctuation was circadian, whereas in constant light it was not. The subjective light-dark (LD) transitions in the tonic activity in DD closely followed the LD transitions in the holding cages initially; that is, there was low activity at night and high activity during the daytime. The sensitivity fluctuations in response to visual stimuli led the tonic spike activity fluctuations by several hours.     

"Demasking" the temperature rhythm after simulated time zone transitions.

Simulated time zone transitions were performed in an isolation unit upon groups of one to four human subjects. In the first series of experiments, the adjustment of the circadian rhythm of body temperature, measured in the presence of sleep and other masking factors, was assessed by cosinor analysis and by cross-correlation methods. These methods modeled the circadian timing system either as a single component or as the sum of two components, those due to exogenous and endogenous influences. The one-component models described a more rapid adjustment of the temperature rhythm to the time zone transition than did the two-component models; we attribute this difference to the masking effects of the exogenous component. In a second series of experiments, we showed that the shift of the endogenous component, as assessed by the two-component models, was not significantly different from that measured during constant routines. The results also showed that, if the zeitgebers were phased in advance of the endogenous component, then advances of the endogenous component were produced only if this mismatch was less than about 10 hr. Mismatches greater than this, and cases where the zeitgebers were delayed with respect to the endogenous component, both produced delays of the endogenous component. We conclude that the two-component cross-correlation methods can be used to estimate shifts of the endogenous component of a circadian rhythm in the presence of masking factors. They are therefore an alternative to constant routines when these latter are impracticable to carry out.     

Effects of photoperiod on rat motor activity rhythm at the lower limit of entrainment

The experiment described here studied the rat motor activity pattern as a function of the photoperiod of circadian light-dark cycles in the limits of entrainment (22-and 23-h periods). In most cases, the overt rhythm showed 2 circadian components: 1 that followed the external LD cycle and a 2nd rhythm that was free run. The expression of these components was directly dependent on the photoperiod, and there was a gradual transition in the manifestation of 1 or the other. The component with a period equal to that of the external cycle was more manifested under long photoperiods, while the other 1 was more expressed during short photoperiods. Also, the period of the free-running component was longer under T22 than T23. For each period, the free-running component was longer under a longer photoperiod. At first sight, the presence of these 2 components in most of the rats might appear to be due to the fact that in the limits of entrainment, some rats do not entrain and thus show a free-running rhythm plus masking. However, the gradation observed in the different patterns of the overt motor activity rhythm, especially those patterns related to the different balance between the 2 components and the length of the period of the free-running component under LD as a function of the photoperiod, suggests that the circadian system can be functionally dissociated.     

A Nonlinear Interrelationship Between Period Length and the Amount of Activity - Age-Dependent changes

The circadian activity rhythm undergoes changes in the course of postnatal development. Experiments without external time cues were performed to characterize the endogenous component and to investigate any age-dependent changes. Female laboratory mice were used. At the beginning of the experiment they were 3 (juvenile), 23 (adult) or 72 (senile) weeks old. Animals were kept in climatic chambers (constant darkness, food and water ad libitum, temperature: 22±2°C, rel. humidity: 55±5%). Locomotor activity was recorded continuously using infrared detectors. The data were stored and analysed by means of the “Chronobiology Kit” (Stanford University). The mean period lengths were not statistically different between age groups. The stability of the spontaneous activity rhythms was highest in adult mice, however. The mean activity/day decreased from juvenile to senile mice. A nonlinear interrelationship between period length and amount of activity was obtained. At lower activity levels the period length became shorter with increasing activity; at higher levels it became longer again. The general shape of the curve was similar in all age groups. With respect to the nonlinear curve, one could not establish a general age dependency of period length. At similar ranges of activity the period length would be shortest in senile animals. Taking into account, however, the decline with age of the amount of activity the period of old mice could be shorter than, equal to or longer than that of adult mice. The results show that the endogenous component of the circadian activity rhythm, including feedback loops, matures and stabilizes from the juvenile to the adult. An expected loss of stability in senile mice was not demonstrated, probably due to a high variance of the animals' biological age. These age-dependent changes contribute to the changes of circadian activity rhythms obtained under entrained conditions.     

Contribution of circadian physiology and sleep homeostasis to age-related changes in human sleep

The circadian pacemaker and sleep homeostasis play pivotal roles in vigilance state control. It has been hypothesized that age-related changes in the human circadian pacemaker, as well as sleep homeostatic mechanisms, contribute to the hallmarks of age-related changes in sleep, that is, earlier wake time and reduced sleep consolidation. Assessments of circadian parameters in healthy young (∼20-30 years old) and older people (∼65-75 years old)—in the absence of the confounding effects of sleep, changes in posture, and light exposure—have demonstrated that an earlier wake time in older people is accompanied by about a 1h advance of the rhythms of core body temperature and melatonin. In addition, older people wake up at an earlier circadian phase of the body temperature and plasma melatonin rhythm. The amplitude of the endogenous circadian component of the core body temperature rhythm assessed during constant routine and forced desynchrony protocols is reduced by 20-30% in older people. Recent assessments of the intrinsic period of the human circadian pacemaker in the absence of the confounding effects of light revealed no age-related reduction of this parameter in both sighted and blind individuals. Wake maintenance and sleep initiation are not markedly affected by age except that sleep latencies are longer in older people when sleep initiation is attempted in the early morning. In contrast, major age-related reductions in the consolidation and duration of sleep occur at all circadian phases. Sleep of older people is particularly disrupted when scheduled on the rising limb of the temperature rhythm, indicating that the sleep of older people is more susceptible to arousal signals genernpated by the circadian pacemaker. Sleep-homeostatic mechanisms, as assayed by the sleep-deprivation-induced increase of EEG slow-wave activity (SWA), are operative in older people, although during both baseline sleep and recovery sleep SWA in older people remains at lower levels. The internal circadian phase advance of awakening, as well as the age-related reduction in sleep consolidation, appears related to an age-related reduction in the promotion of sleep by the circadian pacemaker during the biological night in combination with a reduced homeostatic pressure for sleep. Early morning light exposure associated with this advance of awakening in older people could reinforce the advanced circadian phase. Quantification of the interaction between sleep homeostasis and circadian rhythmicity contributes to understanding age-related changes in sleep timing and quality. (Chronobiology International, 17(3), 285-311, 2000)