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The fundamental properties of this clock were then established. For example, SCN neurons contain an internal pacemaker with an endogenous, rhythmic electrical activity. So, when the SCN is completely isolated as a hypothalamic island24, or when individual SCN neurons are cultured in vitro, the cells from this hypothalamic region show high electrical activity during the subjective daytime2527, even after three weeks in culture28. Similarly, other studies have shown high metabolic activity and glucose uptake during subjective daytime in the SCN29,30. It is interesting that the secretion of vasopressin (VP), one of the peptide transmitters of the SCN, has been found to follow the general pattern of electrical and metabolic activity of SCN neurons in both rats and monkeys31,32. So, SCN activity signals inactivity in nocturnal animals such as rats and mice (FIG. 1), and activity in diurnal species such as humans a point that we will revisit later. The definitive evidence showing the role of the SCN as the central clock was provided by transplantation experiments. If the SCN of a mutant hamster, with a rhythm faster than that of wild-type animals, was grafted into the hypothalamus of a wild-type, SCN-lesioned hamster, the recipient animal adopted the faster, mutant rhythm33. Naturally, the question of how individual SCN neurons are capable of maintaining an endogenous circadian activity has challenged many neuroscientists. During the past decade, enormous progress has been made in determining putative molecular clock components. The molecular mechanisms that underlie the function of the clock consist of geneproteingene feedback loops in which proteins can downregulate their own transcription and stimulate the transcription of other clock proteins7,8,34,35. Although progress in this field has been spectacular and has been the topic of several reviews8,34,35, many questions have yet to be answered. For example, how is this molecular loop linked to the mechanism that controls the metabolic or electrical activityinactivity cycle of the neuron (FIG. 1)? Here, we will concentrate on a related issue that has received little attention: the way in which the central clock can synchronize the activity of the peripheral organs and the whole organism.
Targets of the clock

TIMELINE

Hypothalamic integration of central and peripheral clocks

Ruud M. Buijs and Andries Kalsbeek
During sleep, our biological clock prepares us for the forthcoming period of activity by controlling the release of hormones and the activity of the autonomic nervous system. Here, we review the history of the study of circadian rhythms and highlight recent observations indicating that the same mechanisms that govern our central clock might be at work in the cells of peripheral organs. Peripheral clocks are proposed to synchronize the activity of the organ, enhancing the functional message of the central clock. We speculate that peripheral visceral information is then fed back to the same brain areas that are directly controlled by the central clock. Both clock mechanisms are proposed to have a complementary function in the organization of behaviour and hormone secretion.

The rotation of the earth exposes all organisms to a daily change in light intensity. From algae to mammals, nearly all organisms have adapted their lifestyle to cycles of 24 hours. Sunlight penetration has resulted in the development of cellular mechanisms that are sensitive to light and that allow the anticipation of regular changes in the environment; the evolutionary selection pressure has been so strong that this property has been retained in most species from fungi to mammals1. Although most cells of all organisms (including mammals) still have a molecular mechanism able to drive a cellular clock2,3, the coupling of this mechanism to a biochemical system capable of receiving and transducing light has been retained in only a few tissues46. So, until recently, a common idea was that, from lower invertebrates to mammals, the crucial circadian clock elements had moved to the central nervous system (CNS) and had become concentrated close to or within the light-transducing parts of the CNS79. In mammals, for example, the light signal can only reach the CNS through glutamate secretion from retinal terminals10. However, recent findings have refocused our attention on the circadian rhythms of peripheral organs organs that were thought to have lost their rhythmic activity.

As a result of this shift in our thinking, molecular clock mechanisms have been uncovered in several peripheral organs2,1113. Together with the older literature on the rhythmic functions of such organs1416, these data indicate a preservation of circadian functions outside the CNS. Here, we review some of the earlier literature on central and peripheral circadian rhythms, and try to provide a tentative functional link between central and peripheral clocks. First, we describe some of the experiments that led to the identification of the suprachiasmatic nucleus (SCN) as the central clock. We then consider the anatomical and physiological evidence for the control that the SCN exerts on peripheral-organ clocks and on the autonomic nervous system. We conclude by proposing a mechanism by which peripheral clocks can feed back their circadian message to the brain, and point to some possible future directions in this field.
The central clock

After the first experimental demonstration that plants have an endogenous rhythm17, nearly 200 years elapsed before a similar endogenous rhythm in body temperature was shown in monkeys18 (TIMELINE). The realization that animals are able to maintain an activity cycle of about 24 hours without any environmental clue led to a search for the location of the master clock. Observations that circadian rhythms also existed in isolated peripheral organs and endocrine glands1416 supported the idea that the rhythm of these organs was responsible for driving the rhythm of the organism. However, the removal of individual organs (including the endocrine glands) or lesions of large parts of the CNS did not abolish the overall rhythm of the organism19,20. The breakthrough in the search for the master clock was the finding that, in addition to the lateral geniculate nucleus, a small part of the hypothalamus the SCN was also the target of retinal fibres21. Selective destruction of the SCN resulted in a complete disappearance of all circadian rhythms22,23 the central clock had been found.

SCN transplantation restores normal patterns of locomotor activity in SCN-lesioned animals33, but an outgrowth of fibres is not observed in the case of most SCN transplants. It was proposed that the diffusion of molecules released by the graft might be sufficient

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Timeline | Endogenous clockworks

Simpson and Galbraith report the existence of an endogenous rhythm in body temperature in monkeys18. Beling90 and von Frisch provide evidence for an endogenous circadian timekeeping system in bees. Bnning detects circadian rhythms in isolated sections of intestine maintained in vitro14. Andrews and Folk record circadian rhythms in adrenal glands maintained in vitro15. The first circadian clock mutants in Drosophila melanogaster are reported by Konopka and Benzer93. Langner and Rensing find circadian rhythms in liver cells maintained in vitro16. Krieger et al. report the maintenance of food anticipatory rhythms in SCNlesioned animals69.

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An endogenous rhythm generator in plants is discovered by de Mairan17.

The endogenous nature of circadian locomotor activity rhythms in rats is shown by Richter89.

The first indication of circadian rhythmicity in adrenal function is shown by Pincus91.

The endogenous nature of the human circadian system is reported by Aschoff and Wever92.

Richter provides evidence pointing to the anterior hypothalamus as the location of the biological clock19,20.

The localization of the mammalian endogenous circadian clock, the suprachiasmatic nucleus (SCN), is reported by Moore and Eichler22, and by Stephan and Zucker23.

Inouye and Kawamura show that circadian rhythms can be maintained in a hypothalamic island24.

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Figure 1 | Lightdark cycles and the clock. Wild-type, cryptochrome (Cry1 and Cry2 )-knockout (Cry-KO) or suprachiasmatic-nucleus-lesioned (SCN-X) animals respond differently to lightdark (L/D) schedules. In plots shown on the left, shaded areas correspond to periods of darkness and red vertical lines represent periods of activity. Intact animals are synchronized to the L/D cycle and, by virtue of their circadian clock, maintain their rhythm under D/D or altered L/D conditions. Cry-KO animals also respond to the lightdark cycle, because their SCN neurons (which lack a clock mechanism) immediately respond to L/D changes by becoming active or inactive. The SCN-X animal does not change its fragmented activity pattern by light exposure. Consequently, SCN neurons with or without a molecular clock are needed for signalling lightinduced inactivity in rodents. The right side of the figure provides a schematic illustration of how an individual SCN neuron is able to mimic the day (L) signal with its molecular clock, resulting in membrane depolarization. Although many molecular components of the clock have been identified, their exact mechanism of action remains elusive, particularly for molecules involved in signalling from and to the membrane. Light exposure will result in the immediate release of glutamate from retinal ganglion cells and membrane depolarization of the targeted SCN neuron. This membrane depolarization results in the propagation of the light signal to target neurons of the SCN and to a phase shift of the molecular clock: phase delay (red arrow) or phase advance (green arrow). Without a molecular clock, the light signal will still be transmitted to SCN targets, explaining the behaviour of the cryptochrome mutants. However, without the SCN, no signal will activate SCN target structures, explaining the unresponsiveness of SCN-X animals to changes in the L/D cycle. Many SCN neurons are not directly connected to retinal terminals, but the observed inhibition of activity by light in intact and Cry-KO animals indicates the involvement of most SCN neurons in transmitting this light signal. Further experiments are needed to clarify what transmitters and what membrane mechanisms are involved.

to restore rhythmicity, without the need for synaptic contacts. The first experiments designed to test this idea showed that the restoration of circadian activity could be achieved by placing an SCN transplant encapsulated in a semipermeable membrane into the ventricles36. These data indicated that the SCN message could indeed be conveyed by means of diffusible substances; however, other experiments had shown that the neurotransmitters GABA (-aminobutyric acid) and glutamate were also crucial for transmission of the SCN output37. It was therefore questionable whether all SCN functions could be restored adequately by transplantation without a complete regrowth of connections. In fact, it was subsequently shown that SCN transplants that were capable of restoring circadian locomotor activity in lesioned animals failed to restore the circadian rhythms of cortisol or melatonin secretion38,39. This indicated that the formation of precise connections between SCN grafts and target neurons might be essential for the full transmission of the clock signal. These observations have made it necessary to analyse in detail the targets of SCN projections. Anatomical studies have shown that the SCN projects to at least four different neuronal targets: endocrine neurons, autonomic neurons of the paraventricular nucleus of the hypothalamus (PVN), other hypothalamic structures that transmit the circadian signal to other brain regions, and areas outside the hypothalamus (FIG. 2). Interestingly, although the connection from the SCN to the ventrolateral preoptic nucleus is important for the induction of sleep40,41, the role of the SCN in synchronizing behaviours such as food intake and locomotor activity is not clear at present.

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Groos and Hendriks26, Shibata et al.27 and Green and Gillette25 show that circadian rhythms of neuronal activity in the SCN can be maintained in vitro.

A circadian mutation in the Syrian hamster is discovered by Ralph and Menaker96.

Vitaterna et al. identify the first circadian mutation, Clock, in the mouse97.

An independent circadian clock is found in the mammalian retina by Tosini and Menaker99.

Sun et al.100 and Tei et al.101 identify the mammalian orthologue of the period gene of Drosophila.

The expression of clock genes in mammalian peripheral tissues is shown by Zylka et al.13 and Balsalobre et al.2

Lowrey et al. localize the tau gene103.

The functional unification of peripheral and central clocks.

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Bargiello et al.94 and Reddy et al.95 localize the period (per) gene in Drosophila.

Ralph et al. show that the transplanted SCN determines circadian period33.

Autonomous single-cell circadian oscillators are reported by Welsh et al.98

Giebultowicz and Hege describe an independent circadian clock in the peripheral tissues of Drosophila102.

Van der Horst et al. show that the lightcapturing proteins of plants have an essential role in the mammalian clock6.

The discovery of proteins that link the molecular clock to metabolic functions within the cell.

Neurotransmitters of the central clock

Individual SCN neurons contain different neuropeptides such as VP, vasoactive intestinal peptide, gastrin-releasing peptide and somatostatin, reflecting their possible functional specialization. Moreover, ultrastructural studies have shown that about 30% of SCN axons contain both GABA and a peptide transmitter42,43. In addition, electrophysiological studies have shown that glutamate is also an SCN transmitter37, which conveys the circadian signal to hypothalamic target structures44. The presence of all of these transmitters in different combinations endows SCN neurons with a rich diversity of substances to transmit its signals. So, for example, the circadian peak of blood corticosterone is controlled by the SCN indirectly through the PVN, where neurons containing corticotropin-releasing hormone (CRH) regulate the secretion of adrenocorticotropic hormone (ACTH) from the pituitary44. Similarly, melatonin secretion from the pineal is regulated by autonomic PVN neurons that project to the sympathetic preganglionic neurons of the spinal cord45. It has therefore been proposed that a circadian pattern of SCN activity might impose the same rhythm on its target structures. Interestingly, it has recently been shown that SCN neurons have no unified wave of activity, but might have different peaks, depending on their location in the SCN46. In other words, different populations of SCN neurons might have different rhythms, in which case it would be important to establish the specific patterns of connectivity of different SCN neuronal populations and their relationship to the peptide/transmitter content of these neurons. So, what is the role of the different transmitters of the SCN? Previous experiments had shown that the circadian curve of corticosterone secretion (FIG. 2) is organized by the

SCN in at least two ways: a stimulatory component of undetermined nature and an inhibitory component that consists of the daily secretion of VP from SCN terminals44,4750. This rhythm drives the daily corticosterone peak in mammals and it is also involved in the regulation of, for example, stress-induced corticosterone release, which might vary in intensity at different points of the lightdark cycle51.

Several anatomical and physiological studies indicate that the influence of the SCN on the release of other hormones might be arranged in a similar fashion. One clear example is the regulation of the reproductive cycle. SCN fibres contact oestrogen-containing neurons in the medial preoptic area (MPOA)52, and also have limited direct contacts with neurons that contain gonadotropin-releasing hormone

PVT LGN sPVN

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Figure 2 | Targets of the SCN. The hypothalamic structures through which the suprachiasmatic nucleus (SCN) signal is translated into a hormonal pattern are shown. We propose that the SCN uses four important means to organize hormonal secretion: first, by direct contact with neuroendocrine neurons, for example, those containing gonadotropin-releasing hormone (GnRH) or corticotropin-releasing hormone (CRH); second, by contact with neuroendocrine neurons via intermediate neurons, for example, those of the medial preoptic nucleus (MPN), the dorsomedial hypothalamic nucleus (DMH), or the sub-paraventricular nucleus (sPVN); third, by projections to the autonomic PVN (aPVN) to influence the autonomic nervous system, preparing the endocrine organs for the arrival of hormones; and fourth, by influencing its own feedback. The roles of the SCN projections to the paraventricular nucleus of the thalamus (PVT) or to the lateral geniculate nucleus (LGN) are not well understood at present. The bottom panel shows the circadian rhythm in blood levels of corticosterone, luteinizing hormone (controlled by GnRH) and melatonin.

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the final levels of corticosterone secretion from the adrenal gland50. So, the SCN uses a dual mechanism to provide an optimum secretion of corticosterone: direct control of the hypothalamic neuroendocrine neurons and of neurons of the autonomic nervous system. We propose that this is a general principle that might hold, not only for endocrine glands50,60, but for other organs as well (FIG. 3). For example, recent evidence indicates that, just before the onset of activity, the SCN increases insulin sensitivity, resulting in a physiologically relevant increase in glucose uptake in muscle, and simultaneously causes increased hepatic glucose production61,62.
The human SCN

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Figure 3 | Interaction between peripheral and central clocks. An illustration of the main pathways by which peripheral and central clocks might communicate with the central nervous system. The hypothalamus is the chief target for both. Information from the suprachiasmatic nucleus (SCN) is translated mainly by the paraventricular nucleus of the hypothalamus (PVN) into a hormonal and autonomic signal. These hormonal, parasympathetic and sympathetic signals will reach peripheral organs such as the adrenal gland, liver, fat tissue and gonads. From these organs, both visceral sensory and hormonal information will reach the hypothalamus. Sympathetic sensory information enters the brain at the level of layers I and V in the dorsal horn and will reach the hypothalamus via the nucleus of the solitary tract (NTS) and the parabrachial nucleus (PBN). Vagal sensory information enters the brain at the level of the NTS and reaches the hypothalamus directly or indirectly via the PBN. These connections provide the hypothalamus with information that allows the organism to adjust and balance peripheral lightdark information with metabolic information from the peripheral organs. DMV, dorsal motor nucleus of the vagus; IML, intermediolateral columns.

(GnRH)53. In the MPOA of female rats, VP secretion from the SCN serves to shape the GnRH peak54 and, consequently, the concentration of luteinizing hormone (FIG. 2). It is noteworthy that the stimulatory role of VP on the GnRH axis coincides with an inhibitory role of SCN-derived VP on corticosterone secretion. So, by the secretion of VP, the SCN simultaneously reduces the stress response while stimulating sexual receptivity51,54. As mentioned above, a significant fraction of SCN neurons use GABA as transmitter. What is the role of this molecule? We have some important leads on this question. It has been found that the SCN regulates melatonin secretion, mainly by the release of GABA during the daytime, and that this effect is mediated by the inhibition of autonomic PVN neurons45,55,56. At night, when this inhibition is removed, the resulting activation of PVN neurons induces the melatonin peak that is commonly associated with darkness (FIG. 2). These experiments show that both GABA and VP are essential for transmitting the daytime message of the SCN. It is interesting to note that VP is excitatory to most neurons, whereas GABA is inhibitory. As GABA is present in 30% of the VP projections, it would be of great interest to identify the precise targets of VP terminals that do and do not con-

tain GABA. The GABA-mediated inhibition of melatonin secretion by day indicates that activation of PVN sympathetic neurons might be suppressed by projections from the SCN in nocturnal animals.

How can the central clock synchronize and control the activity of the peripheral organs and the whole organism?
SCN and the autonomic nervous system

The SCN-mediated control of the melatonin surge indicates that autonomic control is at least one important aspect of SCN function. Early data showed a pronounced circadian change in the sensitivity of the adrenal cortex to ACTH (REFS 5759). Transneuronal tracing and physiological experiments provided proof that, in addition to the classical neuroendocrine control of the adrenal cortex by the release of CRH from the PVN, and the subsequent release of ACTH, an important neuronal SCNPVNsympathetic nervous systemadrenal cortex axis also determines

As most data on SCN anatomy and function have been obtained in rodents, it is important to examine whether similar observations can be made in the human brain. Indeed, recent immunocytochemical post-mortem neuronal tracing experiments have shown that hypothalamic projections of the human SCN are identical to those observed in the rat brain63,64. However, there might be important differences in their functional properties when compared with the observations in rodents that we have just reviewed. For example, increased electrical activity of the SCN during the daytime occurs in both nocturnal and diurnal mammals30,65. In humans, however, secretion of corticosterone and the regulation of activity, heart rate and temperature take place at a time in the lightdark cycle opposite to that observed in the rat. So, the signal of the SCN to the human PVN and other hypothalamic targets will be interpreted in a different way. It will be of great importance to clarify how the activity of the SCN is translated into a signal that induces activity instead of inactivity in diurnal animals. Interestingly, this observation leads to the prediction that only the sympathetic output to the pineal, which causes melatonin secretion, should be active at night in humans, whereas the sympathetic output to other organs is suppressed during the rest period. Retrograde-tracing studies in rats have revealed that SCN neurons project to both sympathetic and parasympathetic systems66,67, indicating that the SCN might indeed support both the activity and the rest period of the circadian cycle. A shift in the presence of glutamate and GABA in SCN terminals from nocturnal animals to diurnal animals might be one solution to such adaptation. In fact, such a shift is supported by the 12-hour phase reversal of multi-unit activity in brain areas that directly surround the SCN when comparing diurnal and nocturnal species68.

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Peripheral and central clocks meet

Despite the fact that cells of many organs have the ability to retain a rhythmic function for a few days, this property disappears without daily enforcement (that is, without the SCN)3. It is interesting that soon after the SCN was identified as the site of the biological clock, other studies showed the capacity of an animal to respond to a 24-hour rhythm of food availability without an SCN69,70. Although many attempts were made to pinpoint the mechanism of the observed anticipatory behaviour and accompanying changes in hormone secretion, it has remained enigmatic. However, we would like to propose that peripheral clocks of different organs, most notably of the liver, are essential for this anticipatory behaviour. These peripheral clocks, which lose their rhythm without the SCN, can be entrained with food or hormone stimulation11,71,72. They are coupled to the metabolism of the cell and, consequently, their synchronized activity could send an important signal to the brain (FIG. 3). This hypothesis is supported by several observations in relation to the entrainment of behaviour in intact and SCN-lesioned animals. One of the entrainment properties of the SCN by light is that the clock responds within certain limits, that is, a 2228-hour period73,74. If peripheral clocks have a molecular mechanism similar to the central clock, one might expect a similar limit of entrainment as well. Interestingly, Stephan75 showed more than 20 years ago that entrainment to restricted feeding showed similar limits in SCN-lesioned animals, an observation that led this investigator to propose a multi-oscillatory clock model in mammals. However, some differences in the properties of central and peripheral clocks exist. For example, in contrast to the SCN, peripheral oscillators do not seem to have a dead zone for entrainment; phase shifts can be induced at any time of the lightdark cycle72. On the basis of these observations, we would like to suggest a role for peripheral oscillators in synchronizing the metabolic activity of the organ by their signalling to the brain, mainly to the hypothalamus. Visceral information could enter the hypothalamus directly through hormones and/or through axons from the nucleus of the solitary tract (NTS), and indirectly through projections from the parabrachial nucleus (FIG. 3). As one might expect, lesions of the vagus nerve or the parabrachial nucleus lead to impaired entrainment to feeding schedules76,77. In this regard, it is worth noting that the NTS and parabrachial nucleus heavily target the same hypothalamic structures that are innervated by the SCN7881. These observations indicate a common ground for the functioning of clock systems. In our view, periph-

eral clocks, like the central clock, control hypothalamic nuclei to maintain a balance between body and brain.
Conclusions and future prospects

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Although peripheral organs were first shown to have a rhythm about 100 years ago, attention quickly moved to the study of circadian rhythms controlled by the CNS. Now the pendulum swings back as we try to understand the interaction between the central and peripheral clocks. Evolution has equipped our organs with the capacity to anticipate and adapt their activity in relation to the activity pattern of the whole organism. With hindsight, it is easy to see why the function of the period (per) gene in the circadian clock of Drosophila initially confused scientists. As homogenates of the whole body were used to detect circadian changes in per mRNA concentrations, the rhythms of the individual organs were masked by the conspicuous noncycling nature of this transcript in the female ovary82. Similarly, we can now begin to explain why clock rhythms in different mammalian organs have a different momentum, and how they relate to the pace of the central biological clock. Organisms have to anticipate and adapt to a changing external and internal environment. Information from these two environments is picked up by sensory and endogenous systems that evolution has connected to cellular circadian pacemakers. Information between these different systems is constantly exchanged; in mammals, this exchange takes place mainly within the hypothalamus. Here, the physiological state of the whole body is evaluated and the appropriate hormonal and autonomic output is selected. This idea is supported by several reports of circadian rhythm disturbances in humans suffering from chronic diseases disturbances that are manifest even before the onset of other symptoms8385. Furthermore, these observations are bolstered by correlated changes in the anatomical integrity of the human SCN8688. We hope that these insights will help to bridge the gap, not only between body and brain, but also between neurology and internal medicine.
Ruud M. Buijs and Andries Kalsbeek are at the Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ, Amsterdam, The Netherlands. Correspondence to R.M.B. e-mail: r.buijs@nih.knaw.nl

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DATABASE LINKS Vasopressin | Vasoactive intestinal peptide | Gastrin-releasing peptide | Somatostatin | CRH | ACTH | GnRH | Insulin | per | Cry1 | Cry2

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Acknowledgements
We would like to thank all the members of our group at the Netherlands Institute for Brain Research for their contributions, and H. Stoffels for his artwork.

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