Comparative analysis of rhythmic parameters of the body temperature in humans measured with thermistors and digital thermometers

In this paper we compare rhythmic parameters of human body temperature simultaneously measured with digital thermometers and thermistors with memory (Thermochron iButtons®). Thirteen healthy male and female volunteers (mean age of 25) measured oral and axillary temperatures with digital thermometers every hour during wakefulness for two days and every three hours for three days, totalling five consecutive days. Concomitantly, they wore badges with thermistors in the thorax and the wrist. Temporal series circadian rhythmicity was evaluated with the Cosinor technique. Acrophases average were 17:32 h±2:02 h for axillary temperature, 10:12 h±7:26 h for thoracic temperature, 17:18 h±00:50 h for oral temperature and 4:15 h±1:55 h for wrist temperature. The rhythmic parameters of the wrist are more robust than those of the thorax. The collection of data in humans, generally performed with digital thermometers, is restricted to the waking phase and depends on the discipline of the volunteers. We suggest that the use of the thermistor with memory in the wrist can be adopted as alternative methodology to studies of human body temperature rhythmicity, especially recommended for long temporal series.     

Chronopharmacological Study of an Antimicrobial Agent,Norfloxacin, in the Rat

Circadian rhythms in physiological processes may affect pharmacological actions of drugs. The purpose of this study was to determine whether pharmacokinetics or acute lethality (LD 50) of norfloxacin, exhibited circadian rhythmicity. Female Sprague- Dawley prepuberal rats (weight 115.8 ± 10.2 g) synchronized with a 12-h-light/ 12-h-dark cycle (lights on 7:00h) were used throughout the study. Norfloxacin pharmacokinetics after intraperitoneal administration at 4:00, 10:00, 16:00 and 22:00h was characterized. Intraperitoneal norfloxacin LD 50 was administered at 2:00, 6:00, 10:00, 14:00, 18:00 and 22:00 h. Pharmacokinetic parameters and lethality percentages were analyzed by the cosinor method for the presence of circadian rhythmicity. The results showed evidence of circadian rhythmicity for norfloxacin k abs, t ½abs, t max, MRT abs, Cl t /f and AUC. Absorption was higher when the drug was administered during the rest (16:00 h) period, meanwhile elimination was higher when administered during the activity (22:00 h) period. No rhythmicity was determined for norfloxacin lethality. It is concluded that, in this study, time of administration modifies the pharmacokinetics of norfloxacin.     

Case study: differences in human Per2 gene expression, body temperature, cortisol, and melatonin parameters in remission and hypersomnia in a patient with recurrent hypersomnia

Recurrent hypersomnia is characterized by recurring episodes of hypersomnia of 18 h or more per day lasting from several days to several weeks. We report the case of a 17-year-old male subject with recurrent hypersomnia who displayed change in the 24 h expression of the hPer2 gene in whole red and white blood cells as well as markers [deep body temperature (DBT) and cortisol] of the circadian time structure during an episode of hypersomnia compared to remission. The patient was studied for the temporal characteristics of hPer2 gene, DBT, cortisol, and melatonin expression during a single 24 h span during an episode of hypersomnia and again during a single 24 h span in the following remission. The approximation of a 24 h cosine curve to the time series data revealed circadian rhythmicity (P < 0.05) only in DBT in the two stages of the disease with differences in amplitude and acrophase. Cortisol circadian rhythmicity was detected during remission, but not during hypersomnia. Statistically significant differences were detected by ANOVA between the remission and active disease stages in the 24 h mean level of hPer2 gene expression (P < 0.05), cortisol (P < 0.05), and DBT (P < 0.05). The findings of this case study suggest the expression of hPer2 gene and alterations in circadian time structure might play an important role in the pathogenesis of recurrent hypersomnia, although additional study is required.     

ANALYSIS OF INTESTINAL CELL PROLIFERATION AFTER GUANETHIDINE-INDUCED SYMPATHECTOMY

Rats were treated with guanethidine-sulphate every 48 hr from birth until 15 days and then maintained until young adulthood. Sympathectomy was verified by dissection and light microscopic preparation of the superior cervical and coeliac ganglia which showed at least a 78% reduction in the number of perikarya. The effect of the chemical sympathectomy was a decrease in the amplitude of the circadian mitotic rhythm from 44·7 to 27·1%, 67·0 to 25·3% and 54·9 to 24·7%, in the duodenum, jejunum, and ileum, respectively. The shape of the mitotic index curve was altered and the mean mitotic index was significantly decreased (P < 0·01) in all three segments of the small intestine. The mean mitotic index of control intestinal epithelium was 3·2 ± 0·1%, 3·6 ± 0·1% and 4·0 ± 0·1% in the duodenum, jejunum, and ileum, respectively, and 2·3 ± 0·1%, 2·4 ± 0·1%, and 2·5 ± 0·1% in guanethidine-treated rats. Stathmokinetic estimates of cycle time were obtained by use of the metaphase arrest agent, colchicine. The longest cell generation cycle time (T_c) and lowest mitotic index occurred between 12.00 and 16.00 hours and the shortest T_c and highest mitotic index occurred between 00.00 and 04.00 hours, in all three segments of the small intestine. Guanethidine-treatment lengthens T_c throughout the small intestinal epithelium and reduces the range of variation in T_c over a 24-hr period. It is suggested that norepinephrine depletion induced by guanethidine may be the cause of the inhibition in circadian periodicity and that norepinephrine and the sympathetic nervous system may be essential for the maintenance of circadian rhythms in mitotic activity.     

In-vitro circadian rhythm of murine bone marrow progenitor production

Hematopoietic processes display 24h rhythms both in rodents and in human beings. We hypothesized these rhythms to be in part generated by a circadian oscillator within the bone marrow. The ability of murine bone marrow granulo-monocytic (GM) precursors to form colonies following colony-stimulating factor (rm GM-CSF) exposure was investigated in liquid culture samples obtained every 3 h for a span of up to 198 h. The CFU-GM count varied rhythmically over the first 4 d of culture, with a reproducible maximum in the early morning hours, similar to that observed in vivo. These experiments provide the first evidence that bone marrow progenitors sustain in vitro circadian rhythmicity, and they demonstrate the presence of a circadian time-keeping system within these cells. The results support the potential usefulness of bone marrow cultures for investigating chronopharmacologic effects of anticancer drugs and cytokines on this target system.     

The Effect of Activity on the Waking Temperature Rhythm in Humans

Nine healthy female subjects were studied when exposed to the natural light-dark cycle, but living for 17 “days” on a 27h day (9h sleep, 18h wake). Since the circadian endogenous oscillator cannot entrain to this imposed period, forced desynchronization between the sleep/activity cycle and the endogenous circadian temperature rhythm took place. This enabled the effects of activity on core temperature to be assessed at different endogenous circadian phases and at different stages of the sleep/activity cycle. Rectal temperature was measured at 6-minute intervals, and the activity of the nondominant wrist was summed at 1-minute intervals. Each waking span was divided into overlapping 3h sections, and each section was submitted to linear regression analysis between the rectal temperatures and the total activity in the previous 30 minutes. From this analysis were obtained the gradient (of the change in rectal temperature produced by a unit change in activity) and the intercept (the rectal temperature predicted when activity was zero). The gradients were subjected to a two-factor analysis of variance (ANOVA) (circadian phase/ time awake). There was no significant effect of time awake, but circadian phase was highly significant statistically. Post hoc tests (Newman-Keuls) indicated that gradients around the temperature peak were significantly less than those around its trough. The intercepts formed a sinusoid that, for the group, showed a mesor (±SE) of 36.97 (±0.12) and amplitude (95% confidence interval) of 0.22°C (0.12°C, 0.32°C). We conclude that this is a further method for removing masking effects from circadian temperature rhythm data in order to assess its endogenous component, a method that can be used when subjects are able to live normally. We suggest also that the decreased effect of activity on temperature when the endogenous circadian rhythm and activity are at their peak will reduce the possibility of hyperthermia.     

Circadian and Ultradian Periodicities of Grooming Behavior in Family Groups of Common Marmosets (Callithrix jacchus) in Captivity

Three families' groups of common marmosets were observed to describe the characteristics of their grooming rhythmicity, as the duration of the episodes of self- and social grooming made and received by the animals, in captivity under natural environmental conditions. Data were collected by focal animal sampling, at 20 min hourly intervals during 16 non-consecutive days, from January to March in 1994 (sunrise: 05:24 h ± 2 min; sunset: 17:34 h ± 1 min). Time series were obtained for each individual and for each family through hourly duration of grooming. Family time serie was obtained through the mean of its individuals time series. Spectral analysis revealed statistically significant circadian rhythms for all families and individuals. Ultradian components were detected in 50% of the families and in 46.7% of the animals. Acrophases of self- and social grooming calculated by Single Cosinor took place between 9:21 h and 10:39 h, for the families. For individuals self- and social grooming made and received, acrophases occurred from 8:35 h to 12:43 h. The confidence limits of acrophases did not show differences between the families and the individuals within the families, irrespective of their sex, age and reproductive condition, suggesting that this behavior has a stronger temporal marking. Grooming has circadian and ultradian components of rhythmicity in captive families' groups of common marmosets, under natural environmental conditions. Since only some animals showed the ultradian component, it may be consequence of social or environmental masking, or yet phenotypic plasticity of temporal genotype. Further studies are needed to test these possibilities.     

Early development of circadian rhythmicity in the suprachiamatic nuclei and pineal gland of teleost,flounder (Paralichthys olivaeus), embryos

Circadian rhythms enable organisms to coordinate multiple physiological processes and behaviors with the earth's rotation. In mammals, the suprachiasmatic nuclei (SCN), the sole master circadian pacemaker, has entrainment mechanisms that set the circadian rhythm to a 24‐h cycle with photic signals from retina. In contrast, the zebrafish SCN is not a circadian pacemaker, instead the pineal gland (PG) houses the major circadian oscillator. The SCN of flounder larvae, unlike that of zebrafish, however, expresses per2 with a rhythmicity of daytime/ON and nighttime/OFF. Here, we examined whether the rhythm of per2 expression in the flounder SCN represents the molecular clock. We also examined early development of the circadian rhythmicity in the SCN and PG. Our three major findings were as follows. First, rhythmic per2 expression in the SCN was maintained under 24 h dark (DD) conditions, indicating that a molecular clock exists in the flounder SCN. Second, onset of circadian rhythmicity in the SCN preceded that in the PG. Third, both 24 h light (LL) and DD conditions deeply affected the development of circadian rhythmicity in the SCN and PG. This is the first report dealing with the early development of circadian rhythmicity in the SCN in fish.     

Twenty-four-hour variation of vestibular function in young and elderly adults

ABSTRACT

Animal and human studies demonstrate anatomical and functional links between the vestibular nuclei and the circadian timing system. This promotes the hypothesis of a circadian rhythm of vestibular function. The objective of this study was to evaluate the vestibular function through the vestibulo-ocular reflex using a rotatory chair at different times of the day to assess circadian rhythmicity of vestibular function. Two identical studies evaluating temporal variation of the vestibulo-ocular reflex (VOR) were performed, the first in young adults (age: 22.4 ± 1.5 y), and the second in older adults (70.7 ± 4.7 y). The slow phase velocity and time constant of the VOR were evaluated in six separate test sessions, i.e., 02:00, 06:00, 10:00, 14:00, 18:00, and 22:00 h. In both studies, markers of circadian rhythmicity (temperature, fatigue, and sleepiness) displayed expected usual temporal variation. In young adults, the time constant of the VOR showed variation throughout the day (p < .005), being maximum 12:25 h (06:00 h test session) before the acrophase of temperature circadian rhythm. In older adults, the slow phase velocity and time constant also displayed temporal variation (p < .05). Maximum values were recorded at 10:35 h (06:00 h test session) before the acrophase of temperature circadian rhythm. The present study demonstrates that vestibular function is not constant throughout the day. The implication of the temporal variation in vestibular system in equilibrium potentially exposes the elderly, in particular, to differential risk during the 24 h of losing balance and falling.     

Expression of Clock Genes in Human Peripheral Blood Mononuclear Cells throughout the Sleep/Wake and Circadian Cycles

The rhythmic expression of circadian clock genes in the neurons of the suprachiasmatic nucleus (SCN) underlies the manifestation of endogenous circadian rhythmicity in behavior and physiology. Recent evidence demonstrating rhythmic clock gene expression in non‐SCN tissues suggests that functional clocks exist outside the central circadian pacemaker of the brain. In this investigation, the nature of an oscillator in peripheral blood mononuclear cells (PBMCs) is evaluated by assessing clock gene expression throughout both a typical sleep/wake cycle (LD) and during a constant routine (CR). Six healthy men and women aged (mean±SEM) 23.7±1.6 yrs participated in this five‐day investigation in temporal isolation. Core body temperature and plasma melatonin concentration were measured as markers of the central circadian pacemaker. The expression of HPER1, HPER2, and HBMAL1 was quantified in PBMCs sampled throughout an uninterrupted 72 h period. The core body temperature minimum and the midpoint of melatonin concentration measured during the CR occurred 2:17±0:20 and 3:24 ±0:09 h before habitual awakening, respectively, and were well aligned to the sleep/wake cycle. HPER1 and HPER2 expression in PBMCs demonstrated significant circadian rhythmicity that peaked early after wake‐time and was comparable under LD and CR conditions. HBMAL1 expression was more variable, and peaked in the middle of the wake period under LD conditions and during the habitual sleep period under CR conditions. For the first time, bi‐hourly sampling over three consecutive days is used to compare clock gene expression in a human peripheral oscillator under different sleep/wake conditions.     

Circadian rhythm in plasma aldosterone concentration and its seasonal modulation in the camel (Camelus dromedarius) living in the Algerian Sahara desert

Five male camels dwelling in the Algerian Sahara were studied for circadian rhythmicity in plasma aldosterone concentration and its seasonal modulation. Blood was sampled at a frequency of 1 h or less for a span of 27 h during each season of the year. The mean plasma aldosterone concentration exhibited a significant circadian rhythmicity in every season of the year. Plasma aldosterone concentration was lowest in the morning, increased in the afternoon, and generally highest in the late evening. The peak of the circadian rhythm exhibited seasonal variation; it occurred at 20:04h in October, 16:41h in December, 20:40h in March, and 24:16h in June. The rhythm's 24h mean also exhibited seasonal variability, being significantly higher in March and June compared to October.     

Biological clock function is linked to proactive and reactive personality types

Background

Many physiological processes in our body are controlled by the biological clock and show circadian rhythmicity. It is generally accepted that a robust rhythm is a prerequisite for optimal functioning and that a lack of rhythmicity can contribute to the pathogenesis of various diseases. Here, we tested in a heterogeneous laboratory zebrafish population whether and how variation in the rhythmicity of the biological clock is associated with the coping styles of individual animals, as assessed in a behavioural assay to reliably measure this along a continuum between proactive and reactive extremes.

Results

Using RNA sequencing on brain samples, we demonstrated a prominent difference in the expression level of genes involved in the biological clock between proactive and reactive individuals. Subsequently, we tested whether this correlation between gene expression and coping style was due to a consistent change in the level of clock gene expression or to a phase shift or to altered amplitude of the circadian rhythm of gene expression. Our data show a remarkable individual variation in amplitude of the clock gene expression rhythms, which was also reflected in the fluctuating concentrations of melatonin and cortisol, and locomotor activity. This variation in rhythmicity showed a strong correlation with the coping style of the individual, ranging from robust rhythms with large amplitudes in proactive fish to a complete absence of rhythmicity in reactive fish. The rhythmicity of the proactive fish decreased when challenged with constant light conditions whereas the rhythmicity of reactive individuals was not altered.

Conclusion

These results shed new light on the role of the biological clock by demonstrating that large variation in circadian rhythmicity of individuals may occur within populations. The observed correlation between coping style and circadian rhythmicity suggests that the level of rhythmicity forms an integral part of proactive or reactive coping styles.

     

Female conspecifics restore rhythmic singing behaviour in arrhythmic male zebra finches

The present study investigated whether pairing with a conspecific female would restore rhythmicity in the singing behaviour of arrhythmic male songbirds. We recorded the singing and, as the circadian response indicator, monitored the activity–rest pattern in male zebra finches (Taeniopygia guttata) housed without or with a conspecific female under 12 h light: 12 h darkness (12L:12D) or constant bright light (LL_bright). Both unpaired and paired birds exhibited a significant daily rhythm in the singing and activity behaviour, but paired birds, under 12L:12D, showed a ~2 h extension in the evening. Exposure to LL_bright decayed rhythmicity, but the female presence restored rhythmic patterns without affecting the 24 h song output. In the acoustic features, we found a significant difference in the motif duration between unpaired and paired male songs. Overall, these results demonstrated for the first time the role of the female in restoring the circadian phenotype of singing behaviour in male songbirds with disrupted circadian functions, although how interaction between sexes affects the circadian timing of male singing is not understood yet. It is suggested that social cues rendered by a conspecific female could improve the circadian performance by restoring rhythmicity in the biological functions of the cohabiting arrhythmic male partner.     

Circadian and Ultradian Rhythmicities in Very Premature Neonates Maintained in Incubators

Six very premature babies (born at 26-28 weeks gestational age) have been studied in hospital for 11-17 weeks, while in intensive care and in an incubator. Apart from suffering occasionally from the neonatal disorders of haemolytic jaundice and 'respiratory distress of the newborn', the babies were healthy and developed normally. Initially, the babies were continuously fed intravenously, and the lighting in the ward was on continuously. Routine care was given round the clock. When their medical condition permitted it, the babies were moved in their incubator to an adjacent ward, where they took frequent (2-4 hourly) small meals by mouth, the lighting was dimmed at night, and routine care tended to be given more in the daytime. Hourly recordings of insulated skin temperature were taken throughout the study, and it is the detection of rhythmicity in these measurements that has been the subject of the present study. The methods used were Phase-weighted Stacks, Phasor Walkout and Power Spectral Analysis. These methods have previously been used mainly in geophysical studies, and their value is that they can detect weak signals in noisy data and do not assume a particular waveform of any signal. Circadian rhythmicity was found in all babies for much of the time that were in the constant environment provided by the incubator. Ultradian rhythms were sometimes present also, but they were shorter-lived, and showed a wide range of changing periods, generally in excess of 8 h. When the babies were being treated for jaundice or respiratory distress, there was a tendency for the circadian rhythms to become weaker and for a broader spectrum of ultradian periods to appear. Placing babies in the 12 h : 12 h light : dark environment provided by the ward, and instituting feeding by mouth, had, in most cases, only modest effects upon either circadian or ultradian rhythms. Thus, circadian rhythms continued (but generally with a period not exactly equal to 24 h), and ultradian rhythms, when present, often did not show periods that could be related easily to feeding or care-giving. These results are discussed in terms of evidence for endogenous and exogenous origins of the observed rhythms, and of theories that have postulated the relationship between circadian and ultradian rhythms. It is concluded that the results from the present analyses are difficult to reconcile with the view that circadian rhythms develop from interactions between ultradian oscillators. We suggest that they indicate a matu-ration of the circadian system as a consequence of increasing associations between the circadian elements that are present in the suprachiasmatic nuclei and in other oscillators of the circadian system. The new analytical methods used here also indicate that the results obtained from time-frequency analysis depend to some extent upon the method used.     

Circadian and Ultradian Rhythmicities in Very Premature Neonates Maintained in Incubators

Six very premature babies (born at 26–28 weeks gestational age) have been studied in hospital for 11–17 weeks, while in intensive care and in an incubator. Apart from suffering occasionally from the neonatal disorders of haemolytic jaundice and ‘respiratory distress of the newborn’, the babies were healthy and developed normally. Initially, the babies were continuously fed intravenously, and the lighting in the ward was on continuously. Routine care was given round the clock. When their medical condition permitted it, the babies were moved in their incubator to an adjacent ward, where they took frequent (2–4 hourly) small meals by mouth, the lighting was dimmed at night, and routine care tended to be given more in the daytime. Hourly recordings of insulated skin temperature were taken throughout the study, and it is the detection of rhythmicity in these measurements that has been the subject of the present study. The methods used were Phase-weighted Stacks, Phasor Walkout and Power Spectral Analysis. These methods have previously been used mainly in geophysical studies, and their value is that they can detect weak signals in noisy data and do not assume a particular waveform of any signal. Circadian rhythmicity was found in all babies for much of the time that were in the constant environment provided by the incubator. Ultradian rhythms were sometimes present also, but they were shorter-lived, and showed a wide range of changing periods, generally in excess of 8 h. When the babies were being treated for jaundice or respiratory distress, there was a tendency for the circadian rhythms to become weaker and for a broader spectrum of ultradian periods to appear. Placing babies in the 12 h : 12 h light : dark environment provided by the ward, and instituting feeding by mouth, had, in most cases, only modest effects upon either circadian or ultradian rhythms. Thus, circadian rhythms continued (but generally with a period not exactly equal to 24 h), and ultradian rhythms, when present, often did not show periods that could be related easily to feeding or care-giving. These results are discussed in terms of evidence for endogenous and exogenous origins of the observed rhythms, and of theories that have postulated the relationship between circadian and ultradian rhythms. It is concluded that the results from the present analyses are difficult to reconcile with the view that circadian rhythms develop from interactions between ultradian oscillators. We suggest that they indicate a matu-ration of the circadian system as a consequence of increasing associations between the circadian elements that are present in the suprachiasmatic nuclei and in other oscillators of the circadian system. The new analytical methods used here also indicate that the results obtained from time-frequency analysis depend to some extent upon the method used.     

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.     

Predicting the actigraphy-based acrophase using the Morningness–Eveningness Questionnaire (MEQ) in college students of North Italy

Actigraphy is the reference objective method to measure circadian rhythmicity. One simpler subjective approach to assess the circadian typology is the Morningness–Eveningness Questionnaire (MEQ) by Horne and Ostberg. In this study, we compared the MEQ score against the actigraphy-based circadian parameters MESOR, amplitude and acrophase in a sample of 54 students of the University of Milan in Northern Italy. MEQ and the acrophase resulted strongly and inversely associated (r = ?0.84, p < 0.0001), and their relationship exhibited a clear-cut linear trend. We thus used linear regression to develop an equation enabling us to predict the value of the acrophase from the MEQ score. The parameters of the regression model were precisely estimated, with the slope of the regression line being significantly different from 0 (p < 0.0001). The best-fit linear equation was: acrophase (min) = 1238.7–5.49·MEQ, indicating that each additional point in the MEQ score corresponded to a shortening of the acrophase of approximately 5 min. The coefficient of determination, R², was 0.70. The residuals were evenly distributed and did not show any systematic pattern, thus indicating that the linear model yielded a good, balanced prediction of the acrophase throughout the range of the MEQ score. In particular, the model was able to accurately predict the mean values of the acrophase in the three chronotypes (Morning-, Neither-, and Evening-types) in which the study subjects were categorized. Both the confidence and prediction limits associated to the regression line were calculated, thus providing an assessment of the uncertainty associated with the prediction of the model. In particular, the size of the two-sided prediction limits for the acrophase was about ±100 min in the midrange of the MEQ score. Finally, k-fold cross-validation showed that both the model’s predictive ability on new data and the model’s stability to changes in the data set used for parameter estimation were good. In conclusion, the actigraphy-based acrophase can be predicted using the MEQ score in a population of college students of North Italy.