Circadian rhythm disorganization produces profound cardiovascular and renal disease in hamsters

Sleep deprivation, shift work, and jet lag all disrupt normal biological rhythms and have major impacts on health; however, circadian disorganization has never been shown as a causal risk factor in organ disease. We now demonstrate devastating effects of rhythm disorganization on cardiovascular and renal integrity and that interventions based on circadian principles prevent disease pathology caused by a short-period mutation (tau) of the circadian system in hamsters. The point mutation in the circadian regulatory gene, casein kinase-1epsilon, produces early onset circadian entrainment with fragmented patterns of behavior in +/tau heterozygotes. Animals die at a younger age with cardiomyopathy, extensive fibrosis, and severely impaired contractility; they also have severe renal disease with proteinuria, tubular dilation, and cellular apoptosis. On light cycles appropriate for their genotype (22 h), cyclic behavioral patterns are normalized, cardiorenal phenotype is reversed, and hearts and kidneys show normal structure and function. Moreover, hypertrophy does not develop in animals whose suprachiasmatic nucleus was ablated as young adults. Circadian organization therefore is critical for normal health and longevity, whereas chronic global asynchrony is implicated in the etiology of cardiac and renal disease.     

Alteration of period and amplitude of circadian rhythms in shift workers. With special reference to temperature, right and left hand grip strength

48 male shift workers in various industries volunteered to document circadian rhythms in sleeping and working, oral temperature, grip strength of both hands, peak expiratory flow and heart rate. All physiological variables were self-measured 4 to 5 times a day for 2 to 4 weeks. Individual time series were analyzed according to several statistical methods (power spectrum, cosinor, chi squares, ANOVA, correlation, etc.) in order to estimate rhythm parameters such as circadian period (tau) and amplitude (A), and to evaluate subgroup differences with regard to tolerance to shift work, age, duration of shift work, speed of rotation and type of industry. The present study confirms for oral temperature and extends to other variables (grip strength of both hands, heart rate) that intolerance to shift work is frequently associated with both internal desynchronization and small circadian amplitude. The internal desynchronization among several circadian rhythms supports the hypothesis that these latter are driven by several oscillators. Many differences were observed between circadian rhythms in right and left hand grip strength: circadian tau in oral temperature was correlated with that in the grip strength of the dominant hand but not with that of the other hand; changes in tau s of the non-dominant hand were age-related but did not correlate with temperature tau; only the circadian A of the non-dominant hand was associated with a desynchronization. Thus, circadian rhythms in oral temperature and dominant hand grip strength may be driven by the same oscillator while that of the non-dominant hand may be governed by a different one. Internal desynchronization between both hand grip rhythms as well as desynchronization of performance rhythms reported by others provide indirect evidence that circadian oscillator(s) may be located in the human cerebral cortex.     

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)     

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)     

Circadian phase-shifting effects of nocturnal exercise in older compared with young adults

Exercise can phase shift the circadian rhythms of young adults if performed at the right time of day. Similar research has not been done in older adults. This study examined the circadian phase-delaying effects of a single 3-h bout of low-intensity nocturnal exercise in older (n = 8; 55-73 yr old) vs. young (n = 8; 20-32 yr old) adults. The exercise occurred at the beginning of each subject's habitual sleep time, and subjects sat in a chair in dim light during the corresponding time in the control condition. The dim-light melatonin onset (DLMO) was used as the circadian phase marker. The DLMO phase delayed more after the exercise than after the control condition. On average, the difference in phase shift between the exercise and control conditions was similar for older and young subjects, demonstrating that the phase-shifting effects of exercise on the circadian system are preserved in older adults. Therefore, exercise may potentially be a useful treatment to help adjust circadian rhythms in older and young adults.     

Testicular hormones modulate circadian rhythms of the diurnal rodent, Octodon degus

Sex differences have been identified in a variety of circadian rhythms, including free-running rhythms, light-induced phase shifts, sleep patterns, hormonal fluctuations, and rates of reentrainment. In the precocial, diurnal rodent Octodon degus, sex differences have been found in length of free-running rhythm (tau), phase response curves, rates of reentrainment, and in the use of social cues to facilitate reentrainment. Although gonadal hormones primarily organize circadian rhythms during early development, adult gonadal hormones have activational properties on various aspects of circadian rhythms in a number of species examined. Gonadectomy of adult female O. degus did not influence tau, phase angle of entrainment, or activity patterns in previous experiments. The present experiment examined the role of gonadal hormones in adult male degus' circadian wheel-running rhythms. We predicted that male gonadal hormones would have an activational effect on some aspects of circadian rhythms, particularly those in which we see sex differences. Phase angles of entrainment, tau, length of the active period (alpha), maximum and mean activity levels, and activity amplitude were examined for intact and castrated males housed in LD 12:12. Responses to light pulses while housed in constant darkness (DD) were also compared. Castration had no significant effect on tau or light-induced phase shifts. However, castration significantly increased phase angle of entrainment and decreased activity levels. The data indicate that adult gonadal steroids are not responsible for the sex differences in endogenous circadian mechanisms of O. degus (tau, PRC), although they influence activity level and phase angle of entrainment. This is most likely due to masking properties of testosterone, similar to the activity-increasing effects of estrogen during estrus in O. degus females.     

How to trick mother nature into letting you fly around or stay up all night

Night shift work and rapid transmeridian travel result in a misalignment between circadian rhythms and the new times for sleep, wake, and work, which has health and safety implications for both the individual involved and the general public. Entrainment to the new sleep/wake schedule requires circadian rhythms to be phase-shifted, but this is often slow or impeded. The authors show superimposed light and melatonin PRCs to explain how to appropriately time these zeitgebers to promote circadian adaptation. They review studies in which bright light and melatonin were administered to try to counteract jet lag or to produce circadian adaptation to night work. They demonstrate how jet lag could be prevented entirely if rhythms are shifted before the flight using their preflight plan and discuss the combination of interventions that they now recommend for night shift workers.     

Period and phase control in a multioscillatory circadian system (Iguana iguana)

The circadian system of the lizard Iguana iguana is composed of several independent pacemakers that work in concert: the pineal gland, retinae of the lateral eyes, and a fourth oscillator presumed to be located in the hypothalamus. These pacemakers govern the circadian expression of multiple behaviors and physiological processes, including rhythms in locomotor activity, endogenous body temperature, electroretinogram, and melatonin synthesis. The numerous, easily measurable rhythmic outputs make the iguana an ideal organism for examining the contributions of individual oscillators and their interactions in governing the expression of overt circadian rhythms. The authors have examined the effects of pinealectomy and enucleation on the endogenous body temperature rhythm (BTR) and locomotor activity rhythm (LAR) of juvenile iguanas at constant temperature both in LD cycles and in constant darkness (DD). They measured the periods (tau) of the circadian rhythms of LAR and BTR, the phase relationships between them in DD (psiAT), and the phase relationship between each rhythm and the light cycle (psiRL). Pinealectomy lengthened tau of locomotor activity in all animals tested and abolished the BTR in two-thirds of the animals. In those animals in which the BTR did persist following pinealectomy, tau lengthened to the same extent as that of locomotor activity. Pinealectomy also delayed the onset of activity with respect to its normal phase relationship with body temperature in DD. Enucleation alone had no significant effect on tau of LAR or BTR; however, after enucleation, BTR became 180 degrees out of phase from LAR in DD. After both pinealectomy and enucleation, 4 of 16 animals became arrhythmic in both activity and body temperature. Their data suggest that rhythmicity, period, and phase of overt circadian behaviors are regulated through the combined output of multiple endogenous circadian oscillators.     

The intergeniculate leaflet partially mediates effects of light on circadian rhythms

Photic signals affect circadian activity rhythms by both phasic and tonic mechanisms that modulate pacemaker phase and period. In mammals, the effects of light on circadian activity are mediated by the retina, which communicates with the suprahiasmatic nucleus (SCN) by two different anatomical routes: the retino-hypothalamic tract (RHT), originating in the retina, and the geniculo-hypothalamic tract (GHT), arising from a retino-recipient nucleus, the intergeniculate leaflet (IGL). We assessed the roles of these two afferent systems in mediating phasic and tonic effects of light on circadian activity in IGL-lesioned animals. Destruction of the IGL significantly affected phase shifts produced by brief light pulses (phasic effect) and modified the change in period (tau) of the free-running activity rhythm produced by changing the level of constant light (LL) (tonic effect). Phase advances produced by brief light pulses were decreased in amplitude while phase delays were increased in IGL-lesioned animals as compared to controls. The free-running period in constant dark (tau DD) of IGL-lesioned animals was greater than tau DD of controls, and the lengthening of tau normally produced by LL was not observed or was greatly reduced in IGL-lesioned animals. Entrainment to light-dark cycles was unaffected by the lesions, as were other aspects of the circadian activity rhythm that normally change in response to LL (e.g., activity-rest ratio, total activity, splitting). Our data support the interpretation that the IGL plays a significant role in relaying information regarding illumination intensity to the SCN.     

Three dimensions of individual variation in phase angle between sleep timing and timing of nocturnal rise of the feeling of sleepiness

Studies of melatonin and body temperature rhythms revealed that women, younger adults, and morning-oriented types show a relatively larger phase angle between entrained circadian phase and sleep timing than men, older adults, and evening-oriented types, respectively. However, none of these studies has been designed to compare participants representing all these three dimensions of individual variation. Since daily fluctuations in self-reported level of alertness–sleepiness closely follow the circadian rhythms of melatonin and body temperature, one can predict that a study of circadian phase characteristics of fluctuations of sleepiness shell reveals identical sex-, age-, and diurnal type-related differences in phase angle between circadian phase and sleep timing. Analysis of self-scorings of alertness–sleepiness provided by 130 healthy participants of sleep deprivation experiments confirmed this prediction. It seems that both fundamental research and field studies of sleep-deprived individuals can benefit from the evaluation of circadian phase through self-assessment of nocturnal rise of alertness–sleepiness.     

Familial advanced sleep-phase syndrome: A short-period circadian rhythm variant in humans.

Biological circadian clocks oscillate with an approximately 24-hour period, are ubiquitous, and presumably confer a selective advantage by anticipating the transitions between day and night. The circadian rhythms of sleep, melatonin secretion and body core temperature are thought to be generated by the suprachiasmatic nucleus of the hypothalamus, the anatomic locus of the mammalian circadian clock. Autosomal semi-dominant mutations in rodents with fast or slow biological clocks (that is, short or long endogenous period lengths; tau) are associated with phase-advanced or delayed sleep-wake rhythms, respectively. These models predict the existence of familial human circadian rhythm variants but none of the human circadian rhythm disorders are known to have a familial tendency. Although a slight 'morning lark' tendency is common, individuals with a large and disabling sleep phase-advance are rare. This disorder, advanced sleep-phase syndrome, is characterized by very early sleep onset and offset; only two cases are reported in young adults. Here we describe three kindreds with a profound phase advance of the sleep-wake, melatonin and temperature rhythms associated with a very short tau. The trait segregates as an autosomal dominant with high penetrance. These kindreds represent a well-characterized familial circadian rhythm variant in humans and provide a unique opportunity for genetic analysis of human circadian physiology.     

Absence of apparent circadian rhythms of gonadotropins and free alpha-subunit in postmenopausal women: evidence for distinct regulation relative to other hormonal rhythms

Aging is associated with a decrease in gonadotropin levels in postmenopausal women (PMW) and is also associated with alterations in a number of circadian rhythms. The goals of this study were to determine the presence of circadian rhythms of gonadotropins and glycoprotein free alpha-subunit (FAS) in young and old PMW. Healthy, euthyroid PMW, ages 45 to 55 years (n = 11) and 70 to 80 years (n = 11), were admitted in the morning to start a 24-h constant routine of light, temperature, position, and activity. Subjects remained awake and semirecumbent for the duration of the study and were fed hourly snacks, and activity was monitored continuously. Blood was sampled every 5 min for two 8-h periods corresponding to the estimated acrophase and nadir of the temperature rhythm. Luteinizing hormone (LH) and FAS were measured in all samples and follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and cortisol in 20-min serum pools. Mean LH (p < 0.001), FSH (p < 0.002), and FAS (p < 0.002) were lower in older compared with younger PMW. Day/night differences in cortisol and TSH (p < 0.001) were present in all subjects. However, there were no day/night differences in LH in younger or older PMW or in FSH in younger or older PMW. There were no day/night differences in mean FAS in younger or older PMW or in FAS pulse frequency or amplitude. Thus, in controlled studies in which differences in cortisol and TSH were demonstrated, there were no day/night differences in LH, FSH, or FAS in PMW. These studies suggest that despite evidence of intact circadian rhythms of cortisol and TSH, gonadotropin secretion does not appear to follow a circadian pattern in PMW. Thus, the age-related decline in gonadotropin secretion in PMW is not associated with a dampening of circadian rhythmicity. The absence of day/night differences in FAS suggests that GnRH plays a more prominent role in FAS regulation than does thyrotropin-releasing hormone in PMW.     

Contributions Made to Chronobiology by Studies of Fiddler Crab Rhythms

One of the classic organisms used in chronobiological research is the fiddler crab (genus Uca), an animal unique in that it displays both circadian and tidal (i.e., circalunidian) rhythms. The pioneering work on this animal helped produce the early evidence for many of the standard properties now recognized for all circadian rhythms: near temperature independence of the period, phase lability and setability, the light and temperature sensitivity rhythms expressed by phase response curves, and the persistence of rhythms in organs isolated from a multicellular animal. Importantly, results arising from studies of this crab–and a few other organisms–resulted in the development of the exogenous timing hypothesis. While philosophically sound, the lack of supporting evidence for this hypothesis has resulted in it being discarded by most chronobiologists; but while still in its prime, it drew great interest, and therefore grant support, to the field in general, stimulated a great deal of research that otherwise might not have been performed, and resulted in the discovery of environmental stimuli previously unsuspected to influence organisms. As could be expected, continuing work with this crab, using modern approaches and statistical techniques, has modified earlier findings and interpretations, has revealed new properties, and has resulted in the creation of new hypotheses. The review and update is a synthesis of 45 years of this work.     

Circadian Phase in Adults of Contrasting Ages

There is evidence that aging may impair phase‐shifting responses to light synchronizers, which could lead to disturbed or malsynchronized circadian rhythms. To explore this hypothesis, 62 elder participants (age, 58 to 84 years) and 25 young adults (age, 19 to 40 years) were studied, first with baseline 1‐wk wrist actigraphy at home and then by 72 h in‐laboratory study using an ultra‐short sleep‐wake cycle. Subjects were awake for 60 minutes in 50 lux followed by 30 minutes of darkness for sleep. Saliva samples were collected for melatonin, and urine samples were collected for aMT6s (a urinary metabolite of melatonin) and free cortisol every 90 minutes. Oral temperatures were also measured every 90 minutes. The timing of the circadian rhythms was not significantly more variable among the elders. The times of lights‐out and wake‐up at home and urinary free cortisol occurred earlier among elders, but the acrophases (cosinor analysis‐derived peak time) of the circadian rhythm of salivary melatonin, urinary aMT6s, and oral temperature were not significantly phase‐advanced among elders. The estimated duration of melatonin secretion was 9.9 h among elders and 8.4 h among young adults (p<0.025), though the estimated half‐life of blood melatonin was shorter among elders (p<0.025), and young adults had higher saliva melatonin and urinary aMT6s levels. In summary, there was no evidence for circadian desynchronization associated with aging, but there was evidence of some rearrangement of the internal phase‐angles among the studied circadian rhythms.     

Rats with minimal hepatic encephalopathy show reduced cGMP-dependent protein kinase activity in hypothalamus correlating with circadian rhythms alterations

Patients with liver cirrhosis show disturbances in sleep and in its circadian rhythms which are an early sign of minimal hepatic encephalopathy (MHE). The mechanisms of these disturbances are poorly understood. Rats with porta-caval shunt (PCS), a model of MHE, show sleep disturbances reproducing those of cirrhotic patients. The aims of this work were to characterize the alterations in circadian rhythms in PCS rats and analyze the underlying mechanisms. To reach these aims, we analyzed in control and PCS rats: (a) daily rhythms of spontaneous and rewarding activity and of temperature, (b) timing of the onset of activity following turning-off the light, (c) synchronization to light after a phase advance and (d) the molecular mechanisms contributing to these alterations in circadian rhythms. PCS rats show altered circadian rhythms of spontaneous and rewarding activities (wheel running). PCS rats show more rest bouts during the active phase, more errors in the onset of motor activity and need less time to re-synchronize after a phase advance than control rats. Circadian rhythm of body temperature is also slightly altered in PCS rats. The internal period length (tau) of circadian rhythm of motor activity is longer in PCS rats. We analyzed some mechanisms by which hypothalamus modulate circadian rhythms. PCS rats show increased content of cGMP in hypothalamus while the activity of cGMP-dependent protein kinase was reduced by 41% compared to control rats. Altered cGMP-PKG pathway in hypothalamus would contribute to altered circadian rhythms and synchronization to light.     

Contributions made to chronobiology by studies of fiddler crab rhythms.

One of the classic organisms used in chronobiological research is the fiddler crab (genus Uca), an animal unique in that it displays both circadian and tidal (i.e., circalunidian) rhythms. The pioneering work on this animal helped produce the early evidence for many of the standard properties now recognized for all circadian rhythms: near temperature independence of the period, phase lability and setability, the light and temperature sensitivity rhythms expressed by phase response curves, and the persistence of rhythms in organs isolated from a multicellular animal. Importantly, results arising from studies of this crab--and a few other organisms--resulted in the development of the exogenous timing hypothesis. While philosophically sound, the lack of supporting evidence for this hypothesis has resulted in it being discarded by most chronobiologists; but while still in its prime, it drew great interest, and therefore grant support, to the field in general, stimulated a great deal of research that otherwise might not have been performed, and resulted in the discovery of environmental stimuli previously unsuspected to influence organisms. As could be expected, continuing work with this crab, using modern approaches and statistical techniques, has modified earlier findings and interpretations, has revealed new properties, and has resulted in the creation of new hypotheses. The review and update is a synthesis of 45 years of this work.     

Racial Differences in the Human Endogenous Circadian Period

The length of the endogenous period of the human circadian clock (tau) is slightly greater than 24 hours. There are individual differences in tau, which influence the phase angle of entrainment to the light/dark (LD) cycle, and in doing so contribute to morningness-eveningness. We have recently reported that tau measured in subjects living on an ultradian LD cycle averaged 24.2 hours, and is similar to tau measured using different experimental methods. Here we report racial differences in tau. Subjects lived on an ultradian LD cycle (1.5 hours sleep, 2.5 hours wake) for 3 days. Circadian phase assessments were conducted before and after the ultradian days to determine the change in circadian phase, which was attributed to tau. African American subjects had a significantly shorter tau than subjects of other races. We also tested for racial differences in our previous circadian phase advancing and phase delaying studies. In the phase advancing study, subjects underwent 4 days of a gradually advancing sleep schedule combined with a bright light pulse upon awakening each morning. In the phase delaying study, subjects underwent 4 days of a gradually delaying sleep schedule combined with evening light pulses before bedtime. African American subjects had larger phase advances and smaller phase delays, relative to Caucasian subjects. The racial differences in tau and circadian phase shifting have important implications for understanding normal phase differences between individuals, for developing solutions to the problems of jet lag and shift work, and for the diagnosis and treatment of circadian rhythm based sleep disorders such as advanced and delayed sleep phase disorder.     

Circadian and solar clocks interact in seasonal flowering

The plant maintains a 24‐h circadian cycle that controls the sequential activation of many physiological and developmental functions. There is empirical evidence suggesting that two types of circadian rhythms exist. Some plant rhythms appear to be set by the light transition at dawn, and are calibrated to circadian (zeitgeber) time, which is measured from sunrise. Other rhythms are set by both dawn and dusk, and are calibrated to solar time that is measured from mid‐day. Rhythms on circadian timing shift seasonally in tandem with the timing of dawn that occurs earlier in summer and later in winter. On the other hand, rhythms set to solar time are maintained independently of the season, the timing of noon being constant year‐round. Various rhythms that run in‐phase and out‐of‐phase with one another seasonally may provide a means to time and induce seasonal events such as flowering.     

Maternal entrainment of tau mutant hamsters.

Maternal entrainment of the circadian wheel-running activity rhythm was examined in Syrian hamsters heterozygous for a single gene mutation (tau) that affects the free-running period of circadian rhythms. Heterozygous tau pups were born to and raised by wild-type mothers under constant dim light. The pups' wheel-running activity was recorded after weaning on postnatal day 18 or 24. Pups weaned on day 18 had an average free-running period of 21.70 hr, demonstrating that the tau phenotype was fully expressed at this age. Using the activity onset of the postnatal free-running rhythms as a phase reference, we estimated the phase relationships between the pups and their mothers on days 18 and 24. In contrast to results with wild-type pups, the activity rhythms of tau pups were not in phase with the rhythms of their wild-type mothers; that is, activity onsets of mothers and pups did not coincide. The pups did, however, show synchrony among themselves, indicating that they had been exposed to a synchronizing signal sometime during development. It is likely that this synchronizing signal was provided by the mothers, since pups from different litters showed phase relationships similar to those of their mothers. Thus the mothers provided a signal that was sufficient to cause entrainment, despite the 2-hr difference in free-running period between the mothers and pups. Although the pups' activity rhythms appeared to have been entrained by the mothers, they were clearly free-running by postnatal day 18. The mechanism for entrainment is lost during the course of development, despite continued interaction between the mothers and pups.     

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.