A network of (autonomic) clock outputs

The circadian clock in the suprachiasmatic nuclei (SCN) is composed of thousands of oscillator neurons, each of which is dependent on the cell-autonomous action of a defined set of circadian clock genes. A major question is still how these individual oscillators are organized into a biological clock producing a coherent output that is able to time all the different daily changes in behavior and physiology. We investigated which anatomical connections and neurotransmitters are used by the biological clock to control the daily release pattern of a number of hormones. The picture that emerged shows projections contacting target neurons in the medial hypothalamus surrounding the SCN. The activity of these pre-autonomic and neuro-endocrine target neurons is controlled by differentially timed waves of, among others, vasopressin, GABA, and glutamate release from SCN terminals. Together our data indicate that, with regard to the timing of their main release period within the light-dark (LD) cycle, at least 4 subpopulations of SCN neurons should be discerned. The different subgroups do not necessarily follow the phenotypic differences among SCN neurons. Thus, different subgroups can be found within neuron populations containing the same neurotransmitter. Remarkably, a similar distinction of 4 differentially timed subpopulations of SCN neurons was recently also discovered in experiments determining the temporal patterns of rhythmicity in individual SCN neurons by way of the electrophysiology or clock gene expression. Moreover, the specialization of the SCN may go as far as a single body structure; i.e., the SCN seems to contain neurons that specifically target the liver, pineal, and adrenal.     

Circadian disruption and SCN control of energy metabolism

In this review we first present the anatomical pathways used by the suprachiasmatic nuclei to enforce its rhythmicity onto the body, especially its energy homeostatic system. The experimental data show that by activating the orexin system at the start of the active phase, the biological clock not only ensures that we wake up on time, but also that our glucose metabolism and cardiovascular system are prepared for increased activity. The drawback of such a highly integrated system, however, becomes visible when our daily lives are not fully synchronized with the environment. Thus, in addition to increased physical activity and decreased intake of high-energy food, also a well-lighted and fully resonating biological clock may help to withstand the increasing "diabetogenic" pressure of today's 24/7 society.     

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.

     

A network of (autonomic) clock outputs

The circadian clock in the suprachiasmatic nuclei (SCN) is composed of thousands of oscillator neurons, each dependent on the cell-autonomous action of a defined set of circadian clock genes. A major question is still how these individual oscillators are organized into a biological clock that produces a coherent output capable of timing all the different daily changes in behavior and physiology. We investigated which anatomical connections and neurotransmitters are used by the biological clock to control the daily release pattern of a number of hormones. The picture that emerged shows projections contacting target neurons in the medial hypothalamus surrounding the SCN. The activity of these pre-autonomic and neuro-endocrine target neurons is controlled by differentially timed waves of vasopressin, GABA, and glutamate release from SCN terminals, among other factors. Together our data indicate that, with regard to the timing of their main release period within the LD cycle, at least four subpopulations of SCN neurons should be discernible. The different subgroups do not necessarily follow the phenotypic differences among SCN neurons. Thus, different subgroups can be found within neuron populations containing the same neurotransmitter. Remarkably, a similar distinction of four differentially timed subpopulations of SCN neurons was recently also discovered in experiments determining the temporal patterns of rhythmicity in individual SCN neurons by way of the electrophysiology or clock gene expression. Moreover, the specialization of the SCN may go as far as a single body structure, i.e., the SCN seems to contain neurons that specifically target the liver, pineal gland, and adrenal gland.     

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.     

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.     

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.     

Growth and development of children with a special focus on sleep

The first two decades of life are characterised complex biological processes involving growth and maturation as well as differentiation. The Central Nervous System (CNS) where among others internal and external stimuli are integrated and responses of the body are prepared starts to evolve quite early during ontogenesis. One of the complex behaviours, which are regulated by the brain, is the sleep-wake cycle.The discussion of age-related changes in sleep comprises changes at the physiological level (e.g. changes in the frequency and amplitude of EEG signal, as well as development and distribution of sleep stages), changes in the corresponding behaviour (e.g. changes in the absolute amount of sleep and its distribution in 24 h perspective), and finally the subjective perception of sleep and sleep as a measure of well-being.Studies on the impact of a specific factor on sleep during childhood and adolescence have to consider chronological and biological age as well as sex as relevant biological parameters. Even when these factors are controlled for large interindividual differences persist, that is why prospective instead of cross-sectional approaches should be used whenever possible. Furthermore, it has to be distinguished between sleep assessed at the level of brain functioning (i.e. by polysomnography), which gives information on effects at the physiological level and at the level of self-assessment, which focuses on behaviour. Both, sleep at the subjective as well as at the objective level, can to a considerable degree be affected by life style factors, which hence have to be considered as potential confounders.     

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.     

Temporal phasing of locomotor activity, heart rate rhythmicity, and core body temperature is disrupted in VIP receptor 2-deficient mice

Neurons of the brain's biological clock located in the hypothalamic suprachiasmatic nucleus (SCN) generate circadian rhythms of physiology (core body temperature, hormone secretion, locomotor activity, sleep/wake, and heart rate) with distinct temporal phasing when entrained by the light/dark (LD) cycle. The neuropeptide vasoactive intestinal polypetide (VIP) and its receptor (VPAC2) are highly expressed in the SCN. Recent studies indicate that VIPergic signaling plays an essential role in the maintenance of ongoing circadian rhythmicity by synchronizing SCN cells and by maintaining rhythmicity within individual neurons. To further increase the understanding of the role of VPAC2 signaling in circadian regulation, we implanted telemetric devices and simultaneously measured core body temperature, spontaneous activity, and heart rate in a strain of VPAC2-deficient mice and compared these observations with observations made from mice examined by wheel-running activity. The study demonstrates that VPAC2 signaling is necessary for a functional circadian clock driving locomotor activity, core body temperature, and heart rate rhythmicity, since VPAC2-deficient mice lose the rhythms in all three parameters when placed under constant conditions (of either light or darkness). Furthermore, although 24-h rhythms for three parameters are retained in VPAC2-deficient mice during the LD cycle, the temperature rhythm displays markedly altered time course and profile, rising earlier and peaking ～4-6 h prior to that of wild-type mice. The use of telemetric devices to measure circadian locomotor activity, temperature, and heart rate, together with the classical determination of circadian rhythms of wheel-running activity, raises questions about how representative wheel-running activity may be of other behavioral parameters, especially when animals have altered circadian phenotype.     

Temporal orientation: circadian clocks in animals and humans

By evolutionary adaptation to the regular day-night changes in environmental conditions, most organisms have acquired a temporal programme which matches the 24-h day. It rests on periodic processes which have the characteristics of self-sustaining oscillations, and which, in constant conditions, free-run with periods slightly deviating from 24 h. When entrained to 24 h, these circadian rhythms can be used by the organism as a clock for temporal orientation, e.g. in the occupation of one of the temporal niches provided by the environment or in the coordination of activities among individuals and species. The circadian clock also provides the basis for using the sun as a compass in spatial orientation, and for the recognition of the time of day as exemplified by the time sense in honey bees. Within the human organism, almost every function is modulated in a circadian fashion. Usually, all rhythms keep a distinct phase-relationship t to each other, providing a high degree of temporal order within the organism. When living in an isolation unit without time cues, most subjects develop free-running rhythms with periods close to 25 h in all functions. Sometimes, however, the sleep-wake cycle is lengthened to 30 h and more, or shortened to less than 22 h. In those instances, other rhythms such as that of body temperature become uncoupled from the sleep-wake cycle and continue to free-run with a period of about 25 h. During such states of ‘internal desynchronization’, subjects can be awake continuously for about 32 h, and sleep without interruption for 16 h. Nevertheless, they experience their ‘days’ as equal to 24 h. This is due to a profound change in time estimation: the intervals of 1-h estimates made by the subject are positively correlated with the duration of wakefulness. In contrast, short-time estimates (in the range of seconds) remain unaffected by desynchronization, indicating that short- and long-time estimates are based on different mechanisms.     

The biological clock: the bodyguard of temporal homeostasis

In order for any organism to function properly, it is crucial that it be table to control the timing of its biological functions. An internal biological clock, located, in mammals, in the suprachiasmatic nucleus of the hypothalamus (SCN), therefore carefully guards this temporal homeostasis by delivering its message of time throughout the body. In view of the large variety of body functions (behavioral, physiological, and endocrine) as well as the large variety in their preferred time of main activity along the light:dark cycle, it seems logical to envision different means of time distribution by the SCN. In the present review, we propose that even though it presents a unimodal circadian rhythm of general electrical and metabolic activity, the SCN seems to use several sorts of output connections that are active at different times along the light: dark cycle to control the rhythmic expression of different body functions. Although the SCN is suggested to use diffusion of synchronizing factors in the rhythmic control of behavioral functions, it also needs neuronal connections for the control of endocrine functions. The distribution of the time-of-day message to neuroendocrine systems is either directly onto endocrine neurons or via intermediate neurons located in specific SCN targets. In addition, the SCN uses its connections with the autonomic nervous system for spreading its time-of-day message, either by setting the sensitivity of endocrine glands (i.e., thyroid, adrenal, ovary) or by directly controlling an endocrine output (i.e., melatonin synthesis). Moreover, the SCN seems to use different neurotransmitters released at different times along the light: dark cycle for each of the different connection types presented. Clearly, the temporal homeostasis of endocrine functions results from a diverse set of biological clock outputs.     

Nutrition,metabolism, and epigenetics: pathways of circadian reprogramming

Food intake profoundly affects systemic physiology. A large body of evidence has indicated a link between food intake and circadian rhythms, and ~24‐h cycles are deemed essential for adapting internal homeostasis to the external environment. Circadian rhythms are controlled by the biological clock, a molecular system remarkably conserved throughout evolution. The circadian clock controls the cyclic expression of numerous genes, a regulatory program common to all mammalian cells, which may lead to various metabolic and physiological disturbances if hindered. Although the circadian clock regulates multiple metabolic pathways, metabolic states also provide feedback on the molecular clock. Therefore, a remarkable feature is reprogramming by nutritional challenges, such as a high‐fat diet, fasting, ketogenic diet, and caloric restriction. In addition, various factors such as energy balance, histone modifications, and nuclear receptor activity are involved in the remodeling of the clock. Herein, we review the interaction of dietary components with the circadian system and illustrate the relationships linking the molecular clock to metabolism and critical roles in the remodeling process.     

Modeling the circadian clock: from molecular mechanism to physiological disorders

Based on genetic and biochemical advances on the molecular mechanism of circadian rhythms, a computational model for the mammalian circadian clock is used to examine the dynamical bases of circadian-clock-related physiological disorders in humans. Entrainment by the light-dark cycle with a phase advance or a phase delay is associated with the Familial advanced sleep phase syndrome (FASPS) or the Delayed sleep phase syndrome (DSPS), respectively. Lack of entrainment corresponding to the occurrence of quasiperiodic oscillations with or without phase jump can be associated with the non-24 h sleep-wake syndrome. In the close vicinity of the entrainment domain, the model uncovers the possibility of infradian oscillations of very long period. Perturbation in the form of chronic jet lag, as used in mice, prevents entrainment of the circadian clock and results in chaotic or quasiperiodic oscillations. It is important to clarify the conditions for entrainment and for its failure because dysfunctions of the circadian clock may lead to physiological disorders, which pertain not only to the sleep-wake cycle but also to mood and cancer.     

光对植物生物钟的调节

植物中的许多生理和生化反应都表现出一种内源的近似于24小时的昼夜节律现象,这些昼夜节律现象受生物钟的调节。高等植物的生物钟系统由输入途径、中央振荡器、输出途径以及一个阀门效应器组成。光信号通过光敏色素和隐花色素进入生物钟,使中央振荡器产生振荡,改变生物钟的输出信号,引起各种生理反应。本文综述了光信号对高等植物生物钟的调节作用和转导途径。     

Systemic PPARγ Deletion Impairs Circadian Rhythms of Behavior and Metabolism

Compelling evidence from both human and animal studies suggests a physiological link between the circadian rhythm and metabolism but the underlying mechanism is still incompletely understood. We examined the role of PPARγ, a key regulator of energy metabolism, in the control of physiological and behavioral rhythms by analyzing two strains of whole-body PPARγ null mouse models. Systemic inactivation of PPARγ was generated constitutively by using Mox2-Cre mice (MoxCre/flox) or inducibly by using the tamoxifen system (EsrCre/flox/TM). Circadian variations in oxygen consumption, CO(2) production, food and water intake, locomotor activity, and cardiovascular parameters were all remarkably suppressed in MoxCre/flox mice. A similar phenotype was observed in EsrCre/flox/TM mice, accompanied by impaired rhythmicity of the canonical clock genes in adipose tissues and liver but not skeletal muscles or the kidney. PPARγ inactivation in isolated preadipocytes following exposure to tamoxifen led to a similar blockade of the rhythmicity of the clock gene expression. Together, these results support an essential role of PPARγ in the coordinated control of circadian clocks and metabolic pathways.     

Changing the dosing schedule minimizes the disruptive effects of interferon on clock function

The effectiveness and toxicity of many drugs vary depending on the relationship between the dosing schedule and the 24-hour rhythms of biochemical, physiological and behavioral processes. In addition, several drugs can cause alterations to the 24-hour rhythms leading to illness and altered homeostatic regulation. However, the mechanisms of this drug-based disruption of circadian 'clock' genes remain unclear. Here, we show the disruptive effect of interferon-alpha on the rhythm of locomotor activity, body temperature and clock-gene mRNA expression in the periphery and suprachiasmatic nuclei, a primary circadian pacemaker. The rhythmicity of clock genes and the photic induction of the Per gene in suprachiasmatic nuclei were disturbed by the repetitive administration of interferon-alpha. Moreover, alteration of clock function, a new concept of adverse effects, can be overcome by optimizing the dosing schedule to minimize adverse drug effects.     

Output pathways of the mammalian suprachiasmatic nucleus: coding circadian time by transmitter selection and specific targeting

Every day, we experience profound changes in our mental and physical condition as body and brain alternate between states of high activity during the waking day and rest during night-time sleep. The fundamental evolutionary adaptation to these profound daily changes in our physiological state is an endogenous 24-h clock. This biological clock enables us to prepare ourselves to these daily changes, instead of only being able to show a passive and delayed response. During the past decade, enormous progress has been made in determining possible molecular components of the biological clock. An important question remains, however, regarding how the rhythmic signal from the biological clock is spread throughout the body to control its physiology and behavior. Indeed, ultimately, the only raison d'etre for the biological clock is its output (Green 1998). In the present review, we propose that the main mechanism for the spreading time-of-day information throughout the body consists of different circadian waves of suprachiasmatic nucleus (SCN) transmitter release, directed to a restricted number of specific SCN target areas, and affecting both neuroendocrine mechanisms and the peripheral autonomic nervous system.     

光对植物生物钟的调节

植物中的许多生理和生化反应都表现出一种内源的近似于24小时的昼夜节律现象,这些昼夜节律现象受生物钟的调节.高等植物的生物钟系统由输入途径、中央振荡器、输出途径以及一个阀门效应器组成.光信号通过光敏色素和隐花色素进入生物钟,使中央振荡器产生振荡,改变生物钟的输出信号,引起各种生理反应.本文综述了光信号对高等植物生物钟的调节作用和转导途径.