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.     

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 of Plasma Renin Activity and Aldosterone: Changes Related to Age, Sex, Recumbency and Sodium Restriction. Chronobiologic Specification for Reference Values

Plasma renin activity (PRA) and aldosterone (PA) levels are characterized by a circadian rhythmicity (CR). The present study revealed that this rhythmicity is influenced by several factors including posture, sodium intake and age. Time-qualified PRA and PA reference intervals can reduce the incidence of false positives and false negatives in a diagnostic work-up. The circadian rhythmicity of PRA and PA have been quantified in relation to posture, sodium intake and age. The cosinor procedure has been applied to quantify the properties of the circadian rhythmicity under these conditions.

Chronograms and circadian parameters can be used to optimize the use of PRA and PA measurements in clinical practice. The chronobiological specification of reference values for PRA and PA is of valuable importance since the assessment of PRA and PA circadian rhythmicity has a diagnostic interest for a certain type of clinical disorder. It should be noted that several studies have described circannual variations for renin and aldosterone. The next step in the optimation of laboratory time-qualified reference values is the assessment of changes induced by the deterministic factors on a circannual domain.     

The circadian clock as a molecular calendar

There are two dominant environmental oscillators shaping the living conditions of our world: the day-night cycle and the succession of the seasons. Organisms have adapted to these by evolving internal clocks to anticipate these variations. An orchestra of finely tuned peripheral clocks slaved to the master pacemaker of the suprachiasmatic nuclei (SCN) synchronizes the body to the daily 24h cycle. However, this circadian clockwork closely interacts with the seasonal time-teller.

Recent experiments indeed show that photoperiod—the dominant Zeitgeber of the circannual clock—might be deciphered by the organism using the tools of the circadian clock itself. From the SCN, the photoperiodic signal is transferred to the pineal where it is decoded as a varying secretion of melatonin.

Different models have been proposed to explain the mechanism by which the circadian clock measures day-length. Recent work using mutant mice suggests a set of two molecular oscillators tracking dusk and dawn, respectively, thereby translating day-length to the body. However, not every aspect of photoperiodism is covered by this theory and major adjustments will need to be made to establish a widely acceptable uniform model of circadian/circannual timekeeping.     

Getting through to circadian oscillators: why use constant routines?

Overt 24-h rhythmicity is composed of both exogenous and endogenous components, reflecting the product of multiple (periodic) feedback loops with a core pacemaker at their center. Researchers attempting to reveal the endogenous circadian (near 24-h) component of rhythms commonly conduct their experiments under constant environmental conditions. However, even under constant environmental conditions, rhythmic changes in behavior, such as food intake or the sleep-wake cycle, can contribute to observed rhythmicity in many physiological and endocrine variables. Assessment of characteristics of the core circadian pacemaker and its direct contribution to rhythmicity in different variables, including rhythmicity in gene expression, may be more reliable when such periodic behaviors are eliminated or kept constant across all circadian phases. This is relevant for the assessment of the status of the circadian pacemaker in situations in which the sleep-wake cycle or food intake regimes are altered because of external conditions, such as in shift work or jet lag. It is also relevant for situations in which differences in overt rhythmicity could be due to changes in either sleep oscillatory processes or circadian rhythmicity, such as advanced or delayed sleep phase syndromes, in aging, or in particular clinical conditions. Researchers studying human circadian rhythms have developed constant routine protocols to assess the status of the circadian pacemaker in constant behavioral and environmental conditions, whereas this technique is often thought to be unnecessary in the study of animal rhythms. In this short review, the authors summarize constant routine methodology and what has been learned from constant routines and argue that animal and human circadian rhythm researchers should (continue to) use constant routines as a step on the road to getting through to central and peripheral circadian oscillators in the intact organism.     

Circadian rhythms in the retina of rats with photoreceptor degeneration

Previous studies have demonstrated that the mammalian retina contains a circadian clock system that controls several retinal functions. In mammals the location of the retinal circadian clock is unknown whereas, in non-mammalian vertebrates, earlier work has demonstrated that photoreceptor cells contain the circadian clock. New experimental evidence has suggested that in mammals the retinal circadian clock may be located outside the photoreceptor cells. In this study we report that circadian rhythms in Aa-nat mRNA (in vivo) and melatonin synthesis (in vitro) are still present in the retina of rats lacking photoreceptors. The circadian pacemaker(s) controlling such rhythms is probably located in kainic acid sensitive neurons in the inner retina since kainic acid injections abolished the rhythmicity. These data are the first direct demonstration that circadian rhythmicity in the mammalian retina can be generated independently from the photoreceptors and the suprachiasmatic nuclei of the hypothalamus.     

Variation in Meals and Sleep-Activity Patterns in Aged Subjects; its Relevance to Orcadian Rhythm studies

The study was performed upon a sample of aged and non-institutionalized subjects. Information was obtained by questionnaires and diaries on personal factors during a typical week. A random subset was subjected to a more detailed analysis of the composition of their meals.

Results showed that increasing age was correlated with: a decreased day-by-day variability in an individual's time of retiring, rising and eating meals; earlier sleep times; increased frequency of daytime naps and nocturnal awakenings; and decreased physical activity. These results occurred both in subjects living alone and in those living with company. Day-by-day differences in the composition of meals tended to decrease with age. When differences between individuals were considered then these tended to increase with age.

Some implications of these findings for studies of circadian rhythmicity in aged subjects-in whom the timing of circadian rhythms becomes more erratic and amplitude falls-are discussed.     

The Neurospora circadian clock: simple or complex?

The fungus Neurospora crassa is being used by a number of research groups as a model organism to investigate circadian (daily) rhythmicity. In this review we concentrate on recent work relating to the complexity of the circadian system in this organism. We discuss: the advantages of Neurospora as a model system for clock studies; the frequency (frq), white collar-1 and white collar-2 genes and their roles in rhythmicity; the phenomenon of rhythmicity in null frq mutants and its implications for clock mechanisms; the study of output pathways using clock-controlled genes; other rhythms in fungi; mathematical modelling of the Neurospora circadian system; and the application of new technologies to the study of Neurospora rhythmicity. We conclude that there may be many gene products involved in the clock mechanism, there may be multiple interacting oscillators comprising the clock mechanism, there may be feedback from output pathways onto the oscillator(s) and from the oscillator(s) onto input pathways, and there may be several independent clocks coexisting in one organism. Thus even a relatively simple lower eukaryote can be used to address questions about a complex, networked circadian system.     

The Cellular Mechanism of Orcadian Rhythms-A View on Evidence, Hypotheses and Problems

A stable period length is a characteristic property of circadian oscillations. The question about whether higher frequency oscillators (0.5-8 hr) contribute to or establish the stable circadian periodicity cannot be answered at present. A sequential coupling of quantal subcycles appears possible on the basis of known “ultradian” oscillations. There is, however, no supporting evidence for such a concept. Phase response curves of the circadian clock derived from various perturbing pulses allow qualitative conclusions concerning the perturbed clock process. Deductions from computer simulations also allow conclusions about the phase of this oscillatory process.

The distinction between processes (a) essential to the clock mechanism, (b) maintaining and controlling the clock (inputs) and (c) depending on the clock (outputs) on the basis of “oscillatory” and “change of φ or τ after perturbation” seems to be useful but not stringent. Protein synthesis may be an essential or input process. Oscillatory changes of this process may be due to periodic translational control or RNA-supply. Circadian changes in protein concentration and/or activity may depend on periodic synthesis, proteolysis, covalent modifications or aggregations. Specific essential proteins have not been identified conclusively. The large overlap between the group of agents and treatments that phase shift the clock and the group that induces stress proteins suggest that the latter may play a role in the controlling (input) or essential domain.

The role of membranes in the clock mechanism is not clear: concepts assuming an essential function are based on circumstantial evidence. The membrane potential as well as Ca²⁺ may be involved in either input or essential function. Ca²⁺ -calmodulin may also be important as concluded from inhibitor experiments. It is tempting to assume that a calmodulin-dependent kinase is part of a periodic protein phosphorylation process, yet it is not clear whether the periodic protein phosphorylation that has been observed is essential or is just another output process.     

Influence of the quantity and quality of light on photosynthetic periodicity in coral endosymbiotic algae

Symbiotic corals, which are benthic organisms intimately linked with their environment, have evolved many ways to deal with fluctuations in the local marine environment. One possible coping mechanism is the endogenous circadian clock, which is characterized as free running, maintaining a ～24 h periodicity of circuits under constant stimuli or in the absence of external cues. The quantity and quality of light were found to be the most influential factors governing the endogenous clock for plants and algae. Unicellular dinoflagellate algae are among the best examples of organisms that exhibit circadian clocks using light as the dominant signal. This study is the first to examine the effects of light intensity and quality on the rhythmicity of photosynthesis in the symbiotic dinoflagellate Symbiodinium sp., both as a free-living organism and in symbiosis with the coral Stylophora pistillata. Oxygen production measurements in Symbiodinium cultures exhibited rhythmicity with a periodicity of approximately 24 h under constant high light (LL), whereas under medium and low light, the cycle time increased. Exposing Symbiodinium cultures and corals to spectral light revealed different effects of blue and red light on the photosynthetic rhythm, specifically shortening or increasing the cycle time respectively. These findings suggest that the photosynthetic rhythm is entrained by different light cues, which are wired to an endogenous circadian clock. Furthermore, we provide evidence that mRNA expression was higher under blue light for two potential cryptochrome genes and higher under red light for a phytochrome gene isolated from Symbiodinium. These results offer the first evidence of the impact of the intensity and quality of light on the photosynthetic rhythm in algal cells living freely or as part of a symbiotic association. Our results indicate the presence of a circadian oscillator in Symbiodinium governing the photosynthetic apparatus through a light-induced signaling pathway that has yet to be described.     

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.     

On the Role of Energy Metabolism in Neurospora Circadian Clock Function

Neurospora crassa (bdA) mycelia were kept in liquid culture. Without rhythmic conidiation the levels of adenine nucleotides undergo circadian changes in constant darkness. Maxima occur 12-17 hr and 33-35 hr after initiation of the rhythm, i.e., at CT 0-6 hr. Pulses of metabolic inhibitors such as vanadate (Na₃Vo₄), molybdate (Na₂MoO₄: 2 H₂O), N-ethylmaleimide (NEM), azide (NaN₃), cyanide (NaCN) and oligomycin phase shift the circadian conidiation rhythm of Neurospora crassa. Maximal advance phase shifts are observed at about CT 6 with all inhibitors.

Pulses of N,N'dicyclohexylcarbodiimide (DCCD) and light phase shift the conidiation rhythm following a phase response curve different from those of the other agents (maximal advance at about CT 18-24). The phase shifts with DCCD and light are significantly larger in the wild type compared to the mitochrondrial mutant poky. Such differences are not found in PRCs of the protein synthesis inhibitor cycloheximide.

[³¹P] NMR spectra of wild type Neurospora crassa and the clock mutants frq 1 and frq 7 which differ in their circadian period lengths did not reveal differences in the concentrations of adenine nucleotides, pyridine nucleotides or sugar phosphates. Starvation causes drastic changes of the levels of adenine nucleotides, phosphate and mobile polyphosphate without effecting phase or period length of the circadian rhythm.     

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 adaptive value of circadian clocks: an experimental assessment in cyanobacteria

Circadian clocks are thought to enhance the fitness of organisms by improving their ability to adapt to extrinsic influences, specifically daily changes in environmental factors such as light, temperature, and humidity. Some investigators have proposed that circadian clocks provide an additional "intrinsic adaptive value," that is, the circadian clock that regulates the timing of internal events has evolved to be such an integral part of the temporal regulation that it is useful in all conditions, even in constant environments. There have been practically no rigorous tests of either of these propositions. Using cyanobacterial strains with different clock properties growing in competition with each other, we found that strains with a functioning biological clock defeat clock-disrupted strains in rhythmic environments. In contrast to the expectations of the "intrinsic value model," this competitive advantage disappears in constant environments. In addition, competition experiments using strains with different circadian periods showed that cyanobacterial strains compete most effectively in a rhythmic environment when the frequency of their internal biological oscillator and that of the environmental cycle are similar. Together, these studies demonstrate the adaptive value of circadian temporal programming in cyanobacteria but indicate that this adaptive value is only fulfilled in cyclic environments.     

Emergence of circadian and photoperiodic system level properties from interactions among pacemaker cells

Daily patterns of behavior and physiology in animals in temperate zones often differ substantially between summer and winter. In mammals, this may be a direct consequence of seasonal changes of activity of the suprachiasmatic nucleus (SCN). The purpose of this study was to understand such variation on the basis of the interaction between pacemaker neurons. Computer simulation demonstrates that mutual electrical activation between pacemaker cells in the SCN, in combination with cellular electrical activation by light, is sufficient to explain a variety of circadian phenomena including seasonal changes. These phenomena are: self-excitation, that is, spontaneous development of circadian rhythmicity in the absence of a light-dark cycle; persistent rhythmicity in constant darkness, and loss of circadian rhythmicity in pacemaker output in constant light; entrainment to light-dark cycles; aftereffects of zeitgeber cycles with different periods; adjustment of the circadian patterns to day length; generation of realistic phase response curves to light pulses; and relative independence from day-to-day variation in light intensity. In the model, subsets of cells turn out to be active at specific times of day. This is of functional importance for the exploitation of the SCN to tune specific behavior to specific times of day. Thus, a network of on-off oscillators provides a simple and plausible construct that behaves as a clock with readout for time of day and simultaneously as a clock for all seasons.     

The mammalian retina as a clock

Many physiological, cellular, and biochemical parameters in the retina of vertebrates show daily rhythms that, in many cases, also persist under constant conditions. This demonstrates that they are driven by a circadian pacemaker. The presence of an autonomous circadian clock in the retina of vertebrates was first demonstrated in Xenopus laevis and then, several years later, in mammals. In X. laevis and in chicken, the retinal circadian pacemaker has been localized in the photoreceptor layer, whereas in mammals, such information is not yet available. Recent advances in molecular techniques have led to the identification of a group of genes that are believed to constitute the molecular core of the circadian clock. These genes are expressed in the retina, although with a slightly different 24-h profile from that observed in the central circadian pacemaker. This result suggests that some difference (at the molecular level) may exist between the retinal clock and the clock located in the suprachiasmatic nuclei of hypothalamus. The present review will focus on the current knowledge of the retinal rhythmicity and the mechanisms responsible for its control.     

Assay and Characteristics of Circadian Rhythmicity in Liquid Cultures of Neurospora crassa

Previous work on circadian rhythms of Neurospora crassa has been done almost exclusively with cultures expressing rhythmic conidiation and growing on solid agar medium. Such conditions severely restrict the kinds of biochemical experiments that can be carried out. We have now developed systems which allow indirect assay of circadian rhythmicity in liquid culture. Neurospora was grown in glucose and acetate liquid media under conditions which result in a range of growth rates and morphologies. Liquid media were inoculated with conidia and the cultures were grown in constant light for 33 or 48 hours, by which time floating mycelial pads had formed. Experimental pieces of mycelium then were cut and placed in fresh new liquid medium. As controls, other pieces of mycelium were cut and put directly on solid agar medium in race tubes. All cultures were transferred to constant darkness at this time. This light-to-dark transition set the phase of the circadian clock of both the liquid and solid cultures. At various times after the light-to-dark transition, the mycelial pieces in the liquid were transferred in the dark to solid medium in race tubes, where they grew normally and conidiated rhythmically. Comparison of the phase of the rhythm in these race tubes to the controls demonstrated that, under appropriate conditions, the circadian clock of the liquid cultures functions normally for at least two cycles in constant conditions. Using these culture systems, a significantly greater variety of biochemical studies of circadian rhythmicity in Neurospora is now possible.