Ontogenetic development of the mammalian circadian system

This review summarizes the current knowledge about the ontogenetic development of the circadian system in mammals. The developmental changes of overt rhythms are discussed, although the main focus of the review is the underlying neuronal and molecular mechanisms. In addition, the review describes ontogenetic development, not only as a process of morpho-functional maturation. The need of repeated adaptations and readaptations due to changing developmental stage and environmental conditions is also considered. The review analyzes mainly rodent data, obtained from the literature and from the author's own studies. Results from other species, including humans, are presented to demonstrate common features and species-dependent differences. The review first describes the development of the suprachiasmatic nuclei as the central pacemaker system and shows that intrinsic circadian rhythms are already generated in the mammalian fetus. As in adult organisms, the period length is different from 24 h and needs continuous correction by environmental periodicities, or zeitgebers. The investigation of the ontogenetic development of the mechanisms of entrainment reveals that, at prenatal and early postnatal stages, non-photic cues deriving from the mother are effective. Light-dark entrainment develops later. At a certain age, both photic and non-photic zeitgebers may act in parallel, even though the respective time information is 12 h out of phase. That leads to a temporary internal desynchronization. Because rhythmic information needs to be transferred to effector organs, the corresponding neural and humoral signalling pathways are also briefly described. Finally, to be able to transform a rhythmic signal into an overt rhythm, the corresponding effector organs must be functionally mature. As many of these organs are able to generate their own intrinsic rhythms, another aspect of the review is dedicated to the development of peripheral oscillators and mechanisms of their entrainment. The latter includes control by the central pacemaker as well as by distinct environmental signals. Ecological aspects of the described developmental changes in the circadian system and some practical consequences are also briefly discussed.     

Social influences on mammalian circadian rhythms: animal and human studies

While light is considered the dominant stimulus for entraining (synchronizing) mammalian circadian rhythms to local environmental time, social stimuli are also widely cited as 'zeitgebers' (time-cues). This review critically assesses the evidence for social influences on mammalian circadian rhythms, and possible mechanisms of action. Social stimuli may affect circadian behavioural programmes by regulating the phase and period of circadian clocks (i.e. a zeitgeber action, either direct or by conditioning to photic zeitgebers), by influencing daily patterns of light exposure or modulating light input to the clock, or by associative learning processes that utilize circadian time as a discriminative or conditioned stimulus. There is good evidence that social stimuli can act as zeitgebers. In several species maternal signals are the primary zeitgeber in utero and prior to weaning. Adults of some species can also be phase shifted or entrained by single or periodic social interactions, but these effects are often weak, and appear to be mediated by social stimulation of arousal. There is no strong evidence yet for sensory-specific nonphotic inputs to the clock. The circadian phase-dependence of clock resetting to social stimuli or arousal (the 'nonphotic' phase response curve, PRC), where known, is distinct from that to light and similar in diurnal and nocturnal animals. There is some evidence that induction of arousal can modulate light input to the clock, but no studies yet of whether social stimuli can shift the clock by conditioning to photic cues, or be incorporated into the circadian programme by associative learning. In humans, social zeitgebers appear weak by comparison with light. In temporal isolation or under weak light-dark cycles, humans may ignore social cues and free-run independently, although cases of mutual synchrony among two or more group-housed individuals have been reported. Social cues may affect circadian timing by controlling sleep-wake states, but the phase of entrainment observed to fixed sleep-wake schedules in dim light is consistent with photic mediation (scheduled variations in behavioural state necessarily create daily light-dark cycles unless subjects are housed in constant dark or have no eyes). By contrast, discrete exercise sessions can induce phase shifts consistent with the nonphotic PRC observed in animal studies. The best evidence for social entrainment in humans is from a few totally blind subjects who synchronize to the 24 h day, or to near-24 h sleep-wake schedules under laboratory conditions. However, the critical entraining stimuli have not yet been identified, and there are no reported cases yet of social entrainment in bilaterally enucleated blind subjects. The role of social zeitgebers in mammalian behavioural ecology, their mechanisms of action, and their utility for manipulating circadian rhythms in humans, remains to be more fully elaborated.     

Electrical and pharmacological properties of the suprachiasmatic nuclei

The suprachiasmatic nuclei (SCN) of the mammalian hypothalamus are in important circadian pacemaker. The electrical activity of these nuclei exhibits an intrinsic circadian rhythm. The rhythmicity of the SCN is also reflected in cyclic glucose consumption and serotonin metabolism. These rhythms are entrained to the light-dark cycle via the retinohypothalamic projection. This pathway, possibly together with a visual projection via the ventral lateral geniculate nuclei, innervates light-responsive SCN cells, which exhibit the functional properties of luminance detectors. The SCN contain various peptides, acetylcholine, and serotonin either intrinsically or in terminals of afferent projections. For acetylcholine it has been demonstrated that the SCN mediate the process of photic entrainment and light suppression of pineal synthetic activity. In the case of serotonin and vasopressin it seems certain that the SCN do not depend on their presence for generating circadian rhythms or for entrainment. Both substances may modulate the intrinsic pacemaker frequency through mechanisms that remain to be established.     

Synchronization of the mammalian circadian timing system: Light can control peripheral clocks independently of the SCN clock

A vast network of cellular circadian clocks regulates 24‐hour rhythms of behavior and physiology in mammals. Complex environments are characterized by multiple, and often conflicting time signals demanding flexible mechanisms of adaptation of endogenous rhythms to external time. Traditionally this process of circadian entrainment has been conceptualized in a hierarchical scheme with a light‐reset master pacemaker residing in the hypothalamus that subsequently aligns subordinate peripheral clocks with each other and with external time. Here we review new experiments using conditional mouse genetics suggesting that resetting of the circadian system occurs in a more “federated” and tissue‐specific fashion, which allows for increased noise resistance and plasticity of circadian timekeeping under natural conditions.     

Photic phase response curve in Octodon degus: assessment as a function of activity phase preference

Light exposure during the early and late subjective night generally phase delays and advances circadian rhythms, respectively. However, this generality was recently questioned in a photic entrainment study in Octodon degus. Because degus can invert their activity phase preference from diurnal to nocturnal as a function of activity level, assessment of phase preference is critical for computations of phase reference [circadian time (CT) 0] toward the development of a photic phase response curve. After determining activity phase preference in a 24-h light-dark cycle (LD 12:12), degus were released in constant darkness. In this study, diurnal (n = 5) and nocturnal (n = 7) degus were randomly subjected to 1-h light pulses (30-35 lx) at many circadian phases (CT 1-6: n = 7; CT 7-12: n = 8; CT 13-18: n = 8; and CT 19-24: n = 7). The circadian phase of body temperature (Tb) onset was defined as CT 12 in nocturnal animals. In diurnal animals, CT 0 was determined as Tb onset + 1 h. Light phase delayed and advanced circadian rhythms when delivered during the early (CT 13-16) and late (CT 20-23) subjective night, respectively. No significant phase shifts were observed during the middle of the subjective day (CT 3-10). Thus, regardless of activity phase preference, photic entrainment of the circadian pacemaker in Octodon degus is similar to most other diurnal and nocturnal species, suggesting that entrainment mechanisms do not determine overt diurnal and nocturnal behavior.     

A phase dynamics model of human circadian rhythms

Nonphotic entrainment of an overt sleep-wake rhythm and a circadian pacemaker-driving temperature/melatonin rhythm suggests existence of feedback mechanisms in the human circadian system. In this study, the authors constructed a phase dynamics model that consisted of two oscillators driving temperature/melatonin and sleep-wake rhythms, and an additional oscillator generating an overt sleep-wake rhythm. The feedback mechanism was implemented by modifying couplings between the constituent oscillators according to the history of correlations between them. The model successfully simulated the behavior of human circadian rhythms in response to forced rest-activity schedules under free-run situations: the sleep-wake rhythm is reentrained with the circadian pacemaker after release from the schedule, there is a critical period for the schedule to fully entrain the sleep-wake rhythm, and the forced rest-activity schedule can entrain the circadian pacemaker with the aid of exercise. The behavior of human circadian rhythms was reproduced with variations in only a few model parameters. Because conventional models are unable to reproduce the experimental results concerned here, it was suggested that the feedback mechanisms included in this model underlie nonphotic entrainment of human circadian rhythms.     

Effects of prolonged exposure to darkness on circadian photic responsiveness in the mouse

Circadian rhythms in mammals are generated by an endogenous pacemaker but are modulated by environmental cycles, principally the alternation of light and darkness. Although much is known about nonparametric effects of light on the circadian system, little is known about other effects of photic stimulation. In the present study, which consists of a series of five experiments in mice, various manipulations of photic stimulation were used to dissect the mechanisms responsible for a variation in the magnitude of light-induced phase-shifts that results from prolonged exposure to darkness. The results confirmed previous observations that prolonged exposure to darkness causes an increase in the magnitude of phase shifts (both phase advances and phase delays) evoked by discrete light pulses. The results also indicated that the increase in responsiveness results from the lack of exposure to light per se and not from collateral effects of exposure to constant darkness such as the lack of previous entrainment. The lack of exposure to light causes the circadian system to undergo a process of dark adaptation similar to dark adaptation in the visual system but with a much slower temporal course. The results suggest that circadian dark adaptation may take place at the retinal level, but it is not clear whether it involves a change in the sensitivity or maximal responsiveness of the system.     

Effects of Prolonged Exposure to Darkness on Circadian Photic Responsiveness in the Mouse

Circadian rhythms in mammals are generated by an endogenous pacemaker but are modulated by environmental cycles, principally the alternation of light and darkness. Although much is known about nonparametric effects of light on the circadian system, little is known about other effects of photic stimulation. In the present study, which consists of a series of five experiments in mice, various manipulations of photic stimulation were used to dissect the mechanisms responsible for a variation in the magnitude of light-induced phase-shifts that results from prolonged exposure to darkness. The results confirmed previous observations that prolonged exposure to darkness causes an increase in the magnitude of phase shifts (both phase advances and phase delays) evoked by discrete light pulses. The results also indicated that the increase in responsiveness results from the lack of exposure to light per se and not from collateral effects of exposure to constant darkness such as the lack of previous entrainment. The lack of exposure to light causes the circadian system to undergo a process of dark adaptation similar to dark adaptation in the visual system but with a much slower temporal course. The results suggest that circadian dark adaptation may take place at the retinal level, but it is not clear whether it involves a change in the sensitivity or maximal responsiveness of the system.     

Period and phase adjustments of human circadian rhythms in the real world

Entrainment of the circadian rhythm has 2 aspects, period and phase adjustments, which are established simultaneously in most nonhuman circadian systems. The human circadian system is unique in its functional structure in which 2 different subsystems are involved; one is the circadian pacemaker analogous to that located in the suprachiasmatic nucleus, and the other is the oscillatory system of unknown nature that drives the rest-activity cycle. The human circadian system shows the endogenous period very close to 24 h under entrainment and less sensitive to photic stimuli than under free running, which may explain stable entrainment in the real word where natural sun lights are unpredictable in terms of the intensity and time of appearance. On the other hand, nonphotic entrainment seems to play a significant role in phase adjustment of the human circadian system. Nonphotic zeitgebers initially directed to the rest-activity cycle may affect the circadian pacemaker through feedback and/or associated LD cycles.     

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.     

Photic and nonphotic inputs to the diurnal circadian clock

Diurnal animals occupy a different temporal niche from nocturnal animals and are consequently exposed to different amounts of light as well as different dangers. Accordingly, some variation exists in the way that diurnal animals synchronize their internal circadian clock to match the external 24-hour daily cycle. First, though the brain mechanisms underlying photic entrainment are very similar among species with different daily activity patterns, there is evidence that diurnal animals are less sensitive to photic stimuli compared to nocturnal animals. Second, stimuli other than light that synchronize rhythms (i.e. nonphotic stimuli) can also entrain and phase shift daily rhythms. Some of the rules that govern nonphotic entrainment in nocturnal animals as well as the brain mechanisms that control nonphotic influences on rhythms do not appear to apply to diurnal animals, however. Some evidence supports the idea that arousal or activity plays an important role in entraining rhythms in diurnal animals, either during the light (active) or dark (inactive) phases, though no consistent pattern is seen. GABAergic stimulation induces phase shifts during the subjective day in both diurnal and nocturnal animals. In diurnal Arvicanthis niloticus (Nile grass rats), SCN GABA_A receptor activation at this time results in phase delays while in nocturnal animals phase advances are induced. It appears that the effect of GABA at this circadian phase results from the inhibition of period gene expression in both diurnal and nocturnal animals. Nonetheless, the resulting phase shifts are in opposite directions. It is not known what stimuli or behaviours ultimately induce changes in GABA activity in the SCN that result in alterations of circadian phase in diurnal grass rats. Taken together, studies such as these suggest that it may be problematic to apply the principles governing nocturnal nonphotic entrainment and its underlying mechanisms to diurnal species including humans.     

Drosophila melanogaster deficient in protein kinase A manifests behavior-specific arrhythmia but normal clock function.

Drosophila melanogaster bearing mutations in the DCO gene, which encodes the major catalytic subunit of cAMP-dependent protein kinase (PKA), displays arrhythmic locomotor activity strongly suggesting a role for PKA in the circadian timing system. This arrhythmicity might result from a requirement for PKA activity in photic resetting pathways, the timekeeping mechanism itself, or downstream effector pathways controlling overt behavioral rhythms. To address these possibilities, we examined the protein and mRNA products from the clock gene period (per) in PKA-deficient flies. The per protein (PER) and mRNA products undergo daily cycles in the heads and bodies of DCO mutants that are indistinguishable from those observed in control wild-type flies. These results indicate that PKA deficiencies affect the proper functioning of elements downstream of the Drosophila timekeeping mechanism. The requirement for PKA in the manifestation of rhythmic activity was preferentially greater in the absence of environmental cycles. However, PKA does not appear to play a universal role in output functions because the clock-controlled eclosion rhythm is normal in DCO mutants. Our results suggest that PKA plays a critical role in the flow of temporal information from circadian pacemaker cells to selective behaviors.     

Relative coordination to unknown "weak zeitgebers" in free-running blind individuals

Light is the primary synchronizer of the human biological clock. In more than half of those blind individuals who completely lack light perception, the absence of photic input to the hypothalamic circadian pacemaker results in rhythms that free-run (blind free-runners [BFRs]) with a period typically greater than 24 h. The remainder are entrained, although sometimes at an abnormal phase angle. It is presumed that weak as-yet-to-be-identified time cues provide the necessary resetting stimulus in these entrained individuals. These weak zeitgebers might be expected to modulate the observed circadian period in blind people who are not actually entrained by them. The authors report here the results from 5 BFRs (average linear regression period +/-SD of 24.31 +/- 0.06 h) who had high-resolution (many and frequent) phase assessments. All 5 subjects demonstrated a similar and reproducible pattern of changes in observed period (period response curves) indicative of relative coordination. The precise shape of the period response curve to weak zeitgebers has implications for the entrainment of BFRs using exogenous melatonin administration or other nonphotic stimuli. Sighted individuals may also be affected by such weak zeitgebers, which may be obscured by the stronger light/dark cycle.     

Reply To-Healy D. and Waterhouse J.M.: the Circadian System and Affective Disorders: Clocks or Rhythms? Chronobiologic Disorders, Social Cues and the Light-Dark Cycle

In their monograph (1), Healy and Waterhouse quite thoughtfully distinguish between the altered shapes of circadian rhythms and their entrainment (synchronization). Although there is a great deal of evidence that various influences can alter the shape of circadian rhythms (“masking”), the literature on multiple time cues (zeitgebers) entraining different pacemakers is less convincing. In humans, evidence for nonphotic (social and activity-rest cycle) zeitgebers is restricted to human studies of anchor sleep (2), to data from Wever (3) and Czeisler (4) and to animal studies by Mrosovksy (5) and Turek (6). Until proven otherwise, it seems most likely that social cues primarily affect the sleep-wake cycle (activity-rest cycle), which- being loosely coupled to the endogenous circadian pacemaker-can be dissociated from it and the overt rhythms that are driven by it.     

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.     

Phase sensitivity analysis of circadian rhythm entrainment

As a biological clock, circadian rhythms evolve to accomplish a stable (robust) entrainment to environmental cycles, of which light is the most obvious. The mechanism of photic entrainment is not known, but two models of entrainment have been proposed based on whether light has a continuous (parametric) or discrete (nonparametric) effect on the circadian pacemaker. A novel sensitivity analysis is developed to study the circadian entrainment in silico based on a limit cycle approach and applied to a model of Drosophila circadian rhythm. The comparative analyses of complete and skeleton photoperiods suggest a trade-off between the contribution of period modulation (parametric effect) and phase shift (nonparametric effect) in Drosophila circadian entrainment. The results also give suggestions for an experimental study to (in)validate the two models of entrainment.