The use of Constant Routines in Unmasking the Endogenous Component of Human Circadian Rhythms

A major problem in the study of the internal clock(s) that drives human circadian rhythms is that due to the effect produced by rhythmicity of habits and external influences ('masking'). A particularly potent factor in this respect is the sleep-wake cycle. It is anomalous that, even though this masking influence is widely accepted, most studies of circadian rhythmicity have been performed in the presence of such interferences.

A protocol is described, the constant routine, by which these exogenous influences can be minimized, thereby enabling a closer scrutiny of the internal clock(s) to be made. An account is given of the different circumstances in which the constant routines have been used together with the results derived from such studies. Briefly, they indicate that nychthemeral studies can give misleading information about the rate of adjustment of the internal clock to various manipulations, e.g. time-zone transition, shift work.

In addition, future studies making use of constant routines are described, in particular those which might enable the presence of more than one internal clock to be established.     

Investigating the Endogenous Component of Human Circadian Rhythms: A Review of Some Simple Alternatives to Constant Routines

Several types of constant routine are accepted as an important means by which the endogenous component of circadian rhythms can be studied. Nevertheless, they are impracticable to perform and unsuitable for routine use in many individuals. We describe a group of simple methods with which rhythms measured in normal circumstances can be dissociated into the components due to masking and the internal clock. Each method is best suited to a particular type of experimental condition. Results from a variety of protocols are analysed by these and conventional methods to assess the validity of the new methods.     

Circadian Rhythms and Masking: An Overview

Masking, as is well known, enables an organism to act immediately and in an appropriate way to changes of the environment, integrating with internally produced rhythmicity. It now appears that masking can be used to cover a far wider range of problems than was originally intended. To separate masking effects from the effects due to an internal oscillator, several techniques have been used. Such protocols, however, like the constant routine protocol, often replace one form of masking by another. The situation becomes even more complex when one realizes that the output of an internal oscillator modifies the input. The question might be asked whether it is possible to study the properties of the internal oscillator in vivo at all. This article attempts to produce a framework for future discussions.     

Circadian Rhythms and Masking: An Overview

Masking, as is well known, enables an organism to act immediately and in an appropriate way to changes of the environment, integrating with internally produced rhythmicity. It now appears that masking can be used to cover a far wider range of problems than was originally intended. To separate masking effects from the effects due to an internal oscillator, several techniques have been used. Such protocols, however, like the constant routine protocol, often replace one form of masking by another. The situation becomes even more complex when one realizes that the output of an internal oscillator modifies the input. The question might be asked whether it is possible to study the properties of the internal oscillator in vivo at all. This article attempts to produce a framework for future discussions.     

Masking in Humans: The Problem and Some Attempts to Solve IT

Different types of masking are discussed together with an account of the masking effect that the sleep-wake cycle exerts upon the circadian rhythms of body temperature and urinary excretion. The relative importance to masking of the several components of differences between sleeping and wakefulness are then assessed.

Means to deal with the problem of masking fall into two major categories. These attempt to minimise masking effects by protocols such as constant routines or control days, and mathematical models which separate results obtained in the presence of masking influences into endogenous and exogenous components. (The problem of the extent to which masking influences can render the endogenous component of a rhythm an impure reflection of the internal oscillator is considered also.) These different techniques are compared with respect to their usefulness and assumptions.

Finally, a brief speculation is given of the usefulness of masking.     

Circadian Rhythms in Human Body Composition

The study investigates how the human body composition (BC) changes as a function of the day-night cycle. The BC was investigated using bioelec-trical impedance analysis (BIA) of 10 clinically healthy subjects (CHS), mon-itored in supine position (readings at 2-h intervals), avoiding mealtimes, di-etary abuses, and bladder and intestinal retention. Time series data were analyzed for their temporal characteristics and circadian rhythm (CR). Ail the variables of BC (lean body mass, fat body mass, body cell mass, total body water, intracellular and extracellular body water, sodium and potassium exchangeable pool) showed a within-day variability with nighttime crests. Such an oscillatory synchronism corroborates the hypothesis that the rest time plays a fundamental role, via its anabolic effects, in conferring the noc-turnal phase to the CR of the human BC.     

Sleep and Circadian Rhythms of Temperature and Urinary Excretion on a 22.8 hr “Day”

Two groups of subjects (total N = 6) were studied in an isolation chamber for a period of 3 weeks whilst living on a 22.8 hr “day”. Regular samples of urine were taken when the subjects were awake, deep body temperature was recorded continuously and polygraphic EEG recordings were made of alternate sleeps. The excretion in the urine of potassium, sodium, phosphate, calcium and a metabolite of melatonin were estimated.

Measurements of the quantity and quality of sleep were made together with assessments of the temperature profiles associated with sleep. In addition, cosinor analysis of circadian rhythmicity in urinary variables and temperature was performed.

The 22.8 hr “days” affected variables and subjects differently. These differences were interpreted as indicating that the endogenous component of half the subjects adjusted to the 22.8 hr “days” but that, for the other three, adjustment did not occur. When the behaviour of different variables was considered then some (including urinary potassium and melatonin, sleep length and REM sleep) appeared to possess a larger endogenous component than others (for example, urinary sodium, phosphate and calcium), with rectal temperature behaving in an intermediate manner. In addition, a comparison between different rhythms in any subject enabled inferences to be drawn regarding any links (or lack of them) that might exist between the rhythms. In this respect also, there was a considerable range in the results and no links between any of the rhythms appeared to exist in the group of subjects as a whole.

Two further groups (total N=8) were treated similarly except that the chamber clock ran at the correct rate. In these subjects, circadian rhythms of urinary excretion and deep body temperature (sleep stages and urinary melatonin were not measured) gave no evidence for deterioration. We conclude, therefore, that the results on the 22.8 hr “day” were directly due to the abnormal “day” length rather than to a prolonged stay in the isolation chamber.     

The Variability in Circadian Phase and Amplitude Estimates Derived from Sequential Constant Routines

Both the constant routine (CR) and the dim light melatonin onset have been suggested as reliable methods to determine circadian phase from a single circadian cycle. However, both techniques lack published studies quantifying the intercycle variability in their phase resolution. To address this question eight healthy male subjects participated in two CRs, 7 days apart. Circadian phase was determined using 3-min samples of core body temperature and two hourly urinary sulphatoxy melatonin excretion rates. Phase and amplitude were estimated using simple (24 h) and complex (24 + 12 h) cosinor models of temperature data and the onset, offset, and a distance-weighted-least-squares (DWLS) fitted acrophase for the melatonin metabolite. The variability in phase estimates was measured using the mean absolute difference between successive CRs. Using the simple 24 h model of temperature data, the mean absolute phase difference was 51 min (SD = 35 min). Using the complex model, the mean absolute phase difference was 62 min (SD = 35 min). Using the DWLS fitted acrophase for the melatonin metabolite, the mean absolute phase difference between CR1 and CR2 was 40 min (SD = 26 min). The results indicate that for CRs a week apart, the mean absolute difference in an individual's phase estimate can vary by 40-60 min depending on the choice of dependent measure and analytic technique. In contrast to the intraindi-vidual variability, the group results showed considerably less variability. The mean algebraic difference between CRs, using temperature- or melatonin-derived estimates, was less than 5 min, and well within the range of normal measurement error.     

Circadian Variations in the Number of Actively Secreting Sebaceous Follicles and Androgen Circadian Rhythms

Sebum excretion has been shown to demonstrate a circadian rhythm using a gravimetric method (cigarette paper). With the newly introduced method of Sebutape, we confirmed this periodicity and showed that the elevation in sebum excretion is correlated with an increase in the number of secreting follicles. We found, furthermore, that the number of secreting follicles on the forehead showed a distinct and statistically significant circadian rhythmicity, in contrast to those of the chest, which remained almost constant. The quantification in plasma levels of cortisol, melatonin, δ-4-androstenedione, dehydroepiandrosterone sulphate, and free testosterone showed no correlation with sebum excretion at either site. These observations suggest that local factors are involved in control of sebum secretion.     

Circadian Variations in the Number of Actively Secreting Sebaceous Follicles and Androgen Circadian Rhythms

Sebum excretion has been shown to demonstrate a circadian rhythm using a gravimetric method (cigarette paper). With the newly introduced method of Sebutape, we confirmed this periodicity and showed that the elevation in sebum excretion is correlated with an increase in the number of secreting follicles. We found, furthermore, that the number of secreting follicles on the forehead showed a distinct and statistically significant circadian rhythmicity, in contrast to those of the chest, which remained almost constant. The quantification in plasma levels of cortisol, melatonin, δ-4-androstenedione, dehydroepiandrosterone sulphate, and free testosterone showed no correlation with sebum excretion at either site. These observations suggest that local factors are involved in control of sebum secretion.     

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.     

Sleep and Circadian Rhythms in Spousally Bereaved Seniors

A laboratory study of sleep and circadian rhythms was undertaken in 28 spousally bereaved seniors (≥60 yrs) at least four months after the loss event. Measures taken included two nights of polysomnography (second night used), ～36 h of continuous core body temperature monitoring, and four assessments of mood and alertness throughout a day. Preceding the laboratory study, two‐week diaries were completed, allowing the assessment of lifestyle regularity using the 17‐item Social Rhythm Metric (SRM) and the timing of sleep using the Pittsburgh Sleep Diary (PghSD). Also completed were questionnaires assessing level of grief (Texas Revised Inventory of Grief [TRIG] and Index of Complicated Grief [ICG]), subjective sleep quality (Pittsburgh Sleep Quality Index [PSQI]), morningness‐eveningness (Composite Scale of Morningness [CSM]), and clinical interview yielding a Hamilton Depression Rating Scale (HDRS) score. Grief was still present, as indicated by an average TRIG score of about 60. On average, the bereaved seniors habitually slept between ～23:00 and ～06:40 h, achieving ～6 h of sleep with a sleep efficiency of ～80%. They took about 30 min to fall asleep, and had their first REM episode after 75 min. About 20% of their sleep was in Stage REM, and about 3% in Stages 3 or 4 (slow wave sleep). Their mean PSQI score was 6.4. Their circadian temperature rhythms showed the usual classic shape with a trough at ～01:00 h, a fairly steep rise through the morning hours, and a more gradual rise to mid‐evening, with an amplitude of ～0.8°C. In terms of lifestyle regularity, the mean regularity (SRM) score was 3.65 (slightly lower than that usually seen in seniors). Mood and alertness showed time‐of‐day variation with peak alertness in the late morning and peak mood in the afternoon. Correlations between outcome sleep/circadian variables and level of grief (TRIG score) were calculated; there was a slight trend for higher grief to be associated with less time spent asleep (p=0.07) and reduced alertness at 20:00 h (p=0.05). Depression score was not correlated with TRIG score (p>0.20). When subjects were divided into groups by the nature of their late spouse's death (expected/after a long‐term chronic illness [n=18] versus unexpected [n=10]), no differences emerged in any of the variables. In conclusion, when studied at least four months after the loss event, there appears to be some sleep disruption in spousally bereaved seniors. However, this disruption does not appear to be due to bereavement‐related disruptions in the circadian system.     

Circadian Rhythms of Locomotor Activity in the Nile Tilapia Oreochromis niloticus

Behavioral rhythms of the Nile tilapia were investigated to better characterize its circadian system. To do so, the locomotor activity patterns of both male and female tilapia reared under a 12:12 h light-dark (LD) cycle were studied, as well as in males the existence of endogenous rhythmicity under free-running conditions (DD and 45 min LD pulses). When exposed to an LD cycle, the daily pattern of activity differed between individuals: some fish were diurnal, some nocturnal, and a few displayed an arrhythmic pattern. This variability would be typical of the plastic circadian system of fish. Moreover, reproductive events clearly affected the behavioral rhythms of female tilapia, a mouth-brooder teleost species. Under DD, 50% (6 of 12) of male fish showed circadian rhythms with an average period (τ) of 24.1±0.2 h, whereas under the 45 min LD pulses, 58% (7 of 12) of the fish exhibited free-running activity rhythms with an average τ of 23.9±0.5 h. However, interestingly in this case, activity was always confined to the dark phase. Furthermore, when the LD cycle was reversed, a third of the fish showed gradual resynchronization to the new phase, taking 7–10 days to be completely re-entrained. Taken together, these results suggest the existence of an endogenous circadian oscillator that controls the expression of locomotor activity rhythms in the Nile tilapia, although its anatomical localization remains unknown.     

A Plastic Clock: How Circadian Rhythms Respond to Environmental Cues in Drosophila

Circadian clocks synchronize the physiology and behavior of most animals with the day to night cycle. A fundamental property of the molecular pacemakers generating circadian rhythms is their self-sustained nature: they keep oscillating even under constant conditions, with a period close to, but not exactly, 24 h. However, circadian pacemakers have to be sensitive to environmental cues to be beneficial. They need to be reset every day to keep a proper phase relationship with the day to night cycle, and they have to be able to adjust to seasonal changes in day length and temperature. Here, we review our current knowledge of the molecular and neural mechanisms contributing to the plasticity of Drosophila circadian rhythms, which are proving to be remarkably sophisticated and complex.     

Circadian and Circannual Rhythms of Hormonal Variables in Elderly Men and Women

A group of fourteen men (73 ± 5 yr of age), and eighteen women (77 ± 7 yr of age) institutionalized at the Berceni Clinical Hospital, Bucharest, Romania, were studied over a 24-hr span once during each season (winter, spring, summer and fall). All subjects followed a diurnal activity pattern with rest at night and ate three meals per day with breakfast at about 0830, lunch at about 1300 and dinner at about 1830. The meals were similar, although not identical for all subjects during all seasons. On each day of sampling blood was collected at 4-hr intervals over a 24-hr span. Seventeen hormonal variables were determined by radioimmunoassay. Statistically significant circadian rhythms were detected and quantitated by population mean cosinor analysis in pooled data from all four seasons in both sexes for ACTH, aldosterone, Cortisol, C-peptide, dehydroepiandrosterone-sulfate (DHEA-S), immunoreactive insulin, prolactin, 17-OH progesterone, testosterone, total T₄ and TSH. In women, estradiol and progesterone also were determined and showed a circadian rhythm during all seasons. Total T, and FSH showed circadian rhythm detection by cosinor analysis in the men only; LH showed no consistent circadian rhythm as group phenomenon in men or women.

A circannual rhythm was detected using the circadian means of each subject at each season as input for the population mean cosinor in the women for ACTH, C-peptide, DHEA-S, FSH, LH, progesterone, 17-OH progesterone and TSH. In the men, a circannual rhythm was detected for ACTH, FSH, insulin, LH, testosterone and T₃. There were phase differences between men and women in ACTH, FSH and LH. In those functions in which both the circadian and circannual rhythms were statistically significant, a comparison of the amplitudes showed in the women a higher circannual rather than circadian amplitude for DHEA-S. In 17-OH progesterone, TSH and C-peptide, the circadian amplitude in women was larger. In men, the circannual amplitude of T₃ was larger than the circadian amplitude and in insulin the circadian amplitude was larger than the circannual amplitude. There was no statistically significant difference between the circadian and circannual amplitudes in the women in ACTH and progesterone and in the men in ACTH and testosterone.     

Circadian Rhythms in 6-Sulphatoxymelatonin and Nocturnal Sleep in Blind Children

This article describes the relationship between melatonin secretion and sleep quality and subjective complaints about sleep in totally blind children. Eleven boarding-school children (mean age 15.2 years) participated. The major urinary melatonin metabolite 6-sulphatoxymelatonin (aMT6s) was measured five times a day for 48 h. Sleep-wake cycles were recorded by continuous actigraphic recordings during the same time period. Results showed that delayed secretory peaks in aMT6s were significantly associated with disturbed nocturnal sleep and with complaints about morning fatigue.     

Circadian Rhythms in 6-Sulphatoxymelatonin and Nocturnal Sleep in Blind Children

This article describes the relationship between melatonin secretion and sleep quality and subjective complaints about sleep in totally blind children. Eleven boarding-school children (mean age 15.2 years) participated. The major urinary melatonin metabolite 6-sulphatoxymelatonin (aMT6s) was measured five times a day for 48 h. Sleep-wake cycles were recorded by continuous actigraphic recordings during the same time period. Results showed that delayed secretory peaks in aMT6s were significantly associated with disturbed nocturnal sleep and with complaints about morning fatigue.     

Effects of Pregnancy and Parturition on Free-Running Circadian Activity Rhythms in the Rat

Alterations in circadian rhythms have previously been associated with estrous and seasonal changes in reproductive state. In the present study we explored the effects of the reproductive events of pregnancy and parturition on free-running circadian activity rhythms in the rat. Free-running rhythms were monitored before mating, during pregnancy, and following parturition and removal of pups. Systematic and long-lasting alterations of the period of the free-running activity rhythm were seen following parturition. The effects of estrous, seasonal, and gestational reproductive states on circadian rhythms may be mediated by the endocrine events which accompany these states.     

Effects of Pregnancy and Parturition on Free-Running Circadian Activity Rhythms in the Rat

Alterations in circadian rhythms have previously been associated with estrous and seasonal changes in reproductive state. In the present study we explored the effects of the reproductive events of pregnancy and parturition on free-running circadian activity rhythms in the rat. Free-running rhythms were monitored before mating, during pregnancy, and following parturition and removal of pups. Systematic and long-lasting alterations of the period of the free-running activity rhythm were seen following parturition. The effects of estrous, seasonal, and gestational reproductive states on circadian rhythms may be mediated by the endocrine events which accompany these states.