In mammals many behaviours (e.g. sleep–wake, feeding) as well as physiological (e.g. body temperature, blood pressure) and endocrine (e.g. plasma corticosterone concentration) events display a 24 h rhythmicity. These 24 h rhythms are induced by a timing system that is composed of central and peripheral clocks. The highly co-ordinated output of the hypothalamic biological clock not only controls the daily rhythm in sleep–wake (or feeding–fasting) behaviour, but also exerts a direct control over many aspects of hormone release and energy metabolism. First, we present the anatomical connections used by the mammalian biological clock to enforce its endogenous rhythmicity on the rest of the body, especially the neuro-endocrine and energy homoeostatic systems. Subsequently, we review a number of physiological experiments investigating the functional significance of this neuro-anatomical substrate. Together, this overview of experimental data reveals a highly specialized organization of connections between the hypothalamic pacemaker and neuro-endocrine system as well as the pre-sympathetic and pre-parasympathetic branches of the autonomic nervous system.
The intrinsic period of the master brain oscillator in the hypothalamic SCN (suprachiasmatic nuclei) displays a rhythm of approx. 24 h (i.e. circadian) that is generated and maintained at the molecular level by transcriptional/translational feedback loops of clock genes . As the intrinsic period of the master brain oscillator in the hypothalamic SCN is approximately, but not exactly, 24 h, this oscillator has to be reset on a regular basis in order for the organism and its internal homoeostasis not to drift out of phase with the exact 24 h rhythm of the environment. The circadian rhythm generated by the SCN is entrained with or synchronized to the external day–night cycle mainly by the action of environmental light relayed from the retina through the retinohypothalamic tract. The current model for the light-induced entrainment of the master clock proposes that glutamate and PACAP (pituitary adenylate cyclase-activating peptide) are co-released in the SCN upon photic stimulation . Next to this light-induced entrainment, the SCN is certainly also sensitive to other, i.e. non-photic, stimuli, such as feeding, social interactions, sleep deprivation and exercise . Subsequently the entrained rhythm of the central oscillator in the SCN is relayed to the peripheral oscillators in the brain and periphery. However, whereas the environmental LD (light–dark) cycle is the most important entraining signal for the SCN oscillator, the peripheral oscillators seem to be sensitive to many more stimuli, such as hormones, temperature and metabolic cues [4,5], next to the light-entrained synchronizing signals from the SCN .
In the present chapter, we will provide evidence that the master clock in the SCN imposes a temporal structure on the brain and subsequently the peripheral organs through daily rhythms in the release of its (peptidergic) neurotransmitters in its target areas.
Neural versus humoral
In order for an organism to benefit from its biological clock, the timing signal should be communicated to the rest of the body. Therefore the products (i.e. proteins) of the clock genes in the SCN neurons are not only involved in the maintenance of the 24 h transcriptional/translational feedback loops, but they also drive the day–night rhythm in neuronal firing of the SCN neurons  as well as the expression of CCGs [clock-controlled (output) genes] such as vasopressin and VIP (vasoactive intestinal polypeptide), two well-known peptidergic neurotransmitters of the SCN (Figure 1). Indeed, daily rhythms in the mRNA and peptide expression in SCN neurons, as well as release of these peptidergic neurotransmitters (i.e. neuropeptides) have been described several times. In principle, the SCN has two ways to convey its rhythmic message to the rest of the brain and subsequently the rest of the organism: a humoral or a neural pathway. Transplantation experiments provided support for the humoral mechanism; transplants of fetal donor SCN tissue to animals bearing SCN lesions (i.e. arrhythmic animals) resulted in the restoration of behavioural rhythms matching the circadian phenotype of the donor . These experiments indicate that the SCN-derived output that drives circadian rhythms of (e.g. locomotor) behaviour must include the rhythmic secretion of paracrine factors in its immediate surroundings and in the third ventricle, because encapsulated transplants could also restore rhythms to SCN-lesioned hosts. To date, a number of peptides have been proposed to serve as a humoral SCN output, the most notable examples being vasopressin, TGF-α (transforming growth factor-α), prokineticin-2 and cardiotrophin-like cytokine [8–11]. The same transplantation experiments, however, also provided evidence for the existence of a neural transmission pathway due to the absence of hormonal rhythms in the animals in which (encapsulated) grafts had re-instated behavioural rhythms [12,13]. But the clearest evidence for the existence of hardwired neural connections was provided by elegant experiments by de la Iglesia et al. . First, these authors demonstrated, in hamsters showing ‘splitting' of their daily behavioural and endocrine rhythms (i.e. one bout of activity and sleep or one daily surge of cortisol every 12 h instead of every 24 h), that the daily activity rhythms of the left and right side of their bilaterally paired SCN are exactly 12 h out of phase. Next, they demonstrated that, in female hamsters showing a splitting of the daily surge of LH (luteinizing hormone), each 12 h surge of LH was coupled to the preferential activation of GnRH (gonadotropin-releasing hormone) neurons on either the left side or the right side of the brain, in concert with the activity of the SCN. Clearly, the alternating left- and right-sided activation of GnRH neurons is more likely to be explained by point-to-point axonal projections from the SCN than by diffusible factors.
SCN output rhythms
Given the pivotal role of the hypothalamus in homoeostatic regulation, the discovery in 1972 that the master circadian clock resides in this region was not surprising . After this discovery, numerous neuro-anatomical tracing studies have shown that the projection fibres from the SCN are surprisingly limited and, by and large, restricted to a few hypothalamic nuclei, prime targets being the PVN (paraventricular nuclei of the hypothalamus), the MPOA (medial pre-optic area) and the DMH (dorsomedial nuclei of the hypothalamus). Therefore propagation of the timing signal from the SCN mainly proceeds through its contacts with the neuro-endocrine and pre-autonomic motorneurons of the hypothalamus . In the following paragraphs, we will show examples of how the SCN enforces its circadian rhythmicity on to the endocrine rhythms via its neuronal projections to the neuro-endocrine neurons within the hypothalamus, as well as how it controls metabolic rhythms via its projections to the sympathetic and parasympathetic pre-autonomic neurons within the hypothalamus. It is most likely that the SCN control of behavioural rhythms, such as the daily sleep–wake rhythm, is effected via its projections to intermediate neurons in integrative hypothalamic nuclei, such as the DMH and subPVN (subparaventricular zone; also called the SPZ) . Indeed, we will show an example of how the SCN control of glucose metabolism and sleep–wake behaviour can be integrated via its projections to the orexin (also known as hypocretin) neurons in the lateral DMH. Finally, we will provide evidence that light-induced phase-shifts of the SCN can be transmitted instantaneously from the SCN to the molecular clocks of the peripheral organs through the ANS (autonomic nervous system).
Rhythms are everywhere
SCN control of neuro-endocrine rhythms
Under baseline conditions, plasma concentrations of the glucocorticoid hormones released from the cortex of the adrenal gland vary predictably across the day–night cycle. Both in nocturnal and diurnal species, plasma corticosterone (cortisol in humans) concentrations are highest around the time of arousal (i.e. morning for humans and evening for rats). Adrenal glucocorticoid hormones have highly integrated effects on both energy metabolism and behaviour . It is thought that the increased levels of corticosterone at awakening act to enable foraging behaviour by increasing the amount of available energy. CRH (corticotrophin-releasing hormone) is the principal neural signal controlling the release of corticosterone from the adrenal gland via its stimulatory action on the ACTH (adrenocorticotrophic hormone)-producing cells in the anterior pituitary. Together, this neuro-endocrine pathway is known as the HPA (hypothalamo–pituitary–adrenal) axis. CRH is synthesized in neuroendocrine neurons in the medial parvocellular part of the PVN. Therefore we set out to test the hypothesis that the circadian rhythms generated in the SCN were incorporated in the daily activity of the HPA axis via the release of SCN neurotransmitters on to the CRH neurons in the medial PVN [8,16]. The combined results of this and several follow-up experiments are shown in Figures 2 and 3. Vasopressin is an important signal from the SCN necessary to shape the daily rhythm in plasma corticosterone. As for now, the hunt for the hypothesized excitatory SCN signal is still open, although VIP seems a likely candidate. Contrary to our initial hypothesis, however, indirect connections via GABA- (γ-aminobutyric acid) ergic interneurons in the subPVN and the DMH, but not the direct connections between SCN fibres and CRH neurons, were most important for this SCN control. The intermediate neurons in the subPVN and DMH explain the sparse contacts between SCN fibres and CRH neurons (Figure 4) as well as the inhibitory effect of an excitatory transmitter, such as vasopressin, on the activity of the HPA axis. More importantly, however, these intermediate neurons provide a degree of flexibility to the system that helps to explain how a 12 h shift can occur in the timing of the corticosterone peak between nocturnal and diurnal species, if the timing of the daily peak in vasopressin release is not changing between diurnal and nocturnal species.
The daily rhythm in vasopressin release is also important for the control of the daily surge in LH in female rats . Again, the SCN projection to intermediate neurons, in this case in the MPOA, is most important for the control of the daily LH surge, despite the existence of direct SCN projections also to the GnRH neurons .
SCN control of the ANS
In the mammalian pineal gland, information on the environmental lighting conditions is converted into a hormonal timing signal by the rhythmic release of melatonin. The rhythmic pattern of activity in the melatonin pathway is a conserved feature of vertebrate biology, with high levels signalling night and the absence of light and low levels signalling day and the presence of light. At night, postganglionic projections from the superior cervical ganglion that innervate the pineal gland are activated and release noradrenaline into the pineal perivascular space, increasing the activity of the rate-limiting enzyme for melatonin >100-fold after its binding to the adrenergic receptors . Using similar experiments to those described above to unravel the function of vasopressin in the circadian control of the HPA and HPG (hypothalamo–pituitary–gonadal) axes, we found that this daily rhythm in plasma melatonin concentrations is generated by a combination of glutamatergic and GABAergic SCN outputs. However, in this case, the prime targets of the SCN projections are not the neuro-endocrine or intermediate neurons, but the pre-autonomic neurons that are at the origin of the sympathetic innervation of the pineal gland. We proposed that the activity of the pre-autonomic PVN neurons that are in charge of the sympathetic input to the pineal gland is controlled by the combination of glutamatergic and GABAergic inputs from the SCN . The circadian and light-induced daytime activity of the GABAergic SCN projections to the PVN ensures low melatonin levels during the light period. The nocturnal arrest of the inhibitory GABAergic inputs, combined with the continuously active glutamatergic inputs, enables the pre-autonomic PVN neurons that control the sympathetic input to the pineal gland to become active again and start a new period of melatonin synthesis and release (Figure 5).
To investigate further the SCN control of the ANS, especially its parasympathetic branch, we next focused our attention on the daily rhythm in plasma glucose concentrations. Maintaining a constant blood glucose level is essential for normal physiology in the body, particularly for the CNS (central nervous system), as the CNS can neither synthesize nor store the amount of glucose required for its normal cellular function. The liver plays a pivotal role in maintaining optimal glucose levels by balancing glucose entry into and removal from the circulation. From a hypothalamic and chronobiological viewpoint, glucose production by the liver is especially interesting because of the clear involvement of both the sympathetic and parasympathetic inputs to the liver in glucose metabolism  and the recently demonstrated strong circadian control of (glucose) metabolism in the liver . Using local intra-hypothalamic administration of GABA and glutamate receptor (ant)agonists, we probed the contribution of changes in ANS activity to the daily control of plasma glucose and plasma insulin concentrations. The daily rhythm in plasma glucose concentrations is controlled predominantly by the activity of sympathetic liver innervation. In fact, the activity of these liver-dedicated sympathetic pre-autonomic neurons in the PVN is controlled according to a mechanism very similar to the mechanism just described for the SCN control of the daily rhythm in melatonin release, i.e., a combination of rhythmic GABAergic inputs and continuous glutamatergic stimulation. The major difference between the liver-dedicated and pineal-dedicated pre-autonomic neurons is the timing of the GABAergic inputs. In case of the pineal-dedicated pre-autonomic neurons, this inhibitory input is present during the major part of the light period with an acrophase around ZT6 (Zeitgeber time 6), whereas for the liver-dedicated pre-autonomic neurons the acrophase of the GABAergic inhibition is somewhere around ZT2 (Figure 6).
A pronounced daily rhythm in plasma glucose concentrations has been described in experimental animals as well as humans . Although the peak time of plasma glucose levels shows a 12 h difference between nocturnal and diurnal species , in both species peak plasma glucose concentrations are attained every day shortly before awakening at the start of the main activity period. Plasma glucose concentrations are the result of a glucose influx from the gut and liver, and glucose efflux by its uptake in brain, muscle and adipose tissue. In order to investigate in more detail through which glucoregulatory mechanism the just-described SCN output mechanism contributes to the increased plasma glucose concentrations at awakening, we combined our hypothalamic administration studies with the systemic infusion of a stable glucose isotope. The use of the stable glucose isotope enabled us to distinguish between changes in glucose production and glucose uptake. These experiments showed that the most pronounced increase in hepatic glucose production was caused by the disinhibition of neurons in the perifornical area lateral to the DMH, and that orexin- [but not MCH (melatonin-concentrating hormone)]-containing neurons were strongly activated. Further studies revealed that the hyperglycaemic effect of bicuculline could indeed be blocked by the concomitant intracerebroventricular administration of an orexin antagonist, and that orexin fibres impinge upon sympathetic preganglionic neurons in the IML (intermediolateral nucleus) of the spinal cord that project to the liver . Previously, we demonstrated that the hyperglycaemic effect of a focal blockade of GABAergic transmission was very much time dependent , indicating an SCN control (Figure 7). Indeed, using an approach very much similar to ours, Alam et al.  had previously demonstrated that perifornical orexin neurons are subject to an increased endogenous GABAergic inhibition during sleep. Together, these results indicate that by a disinhibition of the orexin system at the end of the light period the SCN not only promotes arousal, but at the same time may also cause an increase of endogenous glucose production to ensure adequate concentrations of plasma glucose when the animal awakes . A drawback of such a nicely integrated system, however, may be that sleep problems will also disturb glucose metabolism .
As has become evident from the daily variation in meal-induced insulin responses , intestinal glucose uptake  and respiratory functioning , the parasympathetic branch of the ANS is also under control of the circadian timing system. Our intra-hypothalamic infusion studies revealed that the daily changes in the activity of the parasympathetic pre-autonomic neurons also involved a combination of GABAergic and glutamatergic inputs. The daily rhythm in SCN-derived GABAergic inputs is a general principle. However, a major difference between the circadian control of parasympathetic and sympathetic pre-autonomic neurons is the origin of the excitatory glutamatergic inputs (Figure 8). SCN-lesion studies clearly proved that the glutamatergic inputs to the parasympathetic pancreas-dedicated pre-autonomic neurons are not derived from SCN neurons . At present, it is not clear from which extra-SCN source the glutamatergic inputs to the parasympathetic pancreas-dedicated pre-autonomic neurons originate, but likely candidates are the VMH (ventromedial hypothalamus) and ARC (arcuate nucleus).
SCN control of peripheral oscillators?
The discovery of clock genes outside the SCN initially caused the status of the SCN as the master oscillator to be scrutinized. However, viable transplants of non-SCN tissue do not restore behavioural rhythms in SCN-lesioned animals , and co-culture models showed that only SCN cells, but not fibroblasts, can confer molecular and metabolic rhythms on co-cultured cells [33a]. According to the current opinion, the central pacemaker in the SCN co-ordinates the activity of local oscillators in peripheral tissues through behavioural, neuroendocrine and autonomic pathways [34,35]. However, a clear understanding of the role of peripheral oscillators in the regulation of the physiological functions of peripheral organs is still lacking. We investigated whether peripheral oscillators in the liver are a necessary link in the transfer of circadian information from the central biological clock to hepatic glucose production. Removal of either the sympathetic or the parasympathetic input to the liver indeed caused an obliteration of the daily rhythm in plasma glucose concentrations [36,37], but to our surprise the transcript levels of all five clock genes studied maintained their rhythmicity in the liver . Liver-specific deletion of the clock-gene Bmal1 (brain and muscle arnt-like 1) caused an exaggerated drop of plasma glucose concentrations during the light period , and demonstrated that >90% of the >300 rhythmically expressed hepatic genes are dependent on a functional clock in the hepatocyte . Consequently, a rhythmic expression of clock genes in the liver is not sufficient to maintain a rhythmic glucose output from the liver, although suppressing the rhythmic expression of >90% of the rhythmically expressed genes in the liver severely disturbs glucose homoeostasis. The ~10% of cyclically expressed liver genes not affected by the liver-specific Bmal1 deletion must be regulated by oscillating systemic signals. Previously, corticosterone, feeding behaviour and body temperature have already been implicated as important systemic signals for liver clocks , and now autonomic innervation can also be added to this list. Using nocturnal light exposure, we found immediate changes in the expression levels of both clock genes and glucoregulatory genes in the liver. Interestingly, a selective denervation of the autonomic liver innervation completely prevented the light-induced changes in both clock genes and glucoregulatory genes , again pinpointing the ANS as an important gateway for the SCN to (immediately) reset peripheral physiology.
From the above review it is clear that, in mammals, the output of the biological clock not only controls daily rhythms in behaviour, such as the sleep–wake or feeding–fasting rhythms, but also exerts direct control over many physiological and endocrine rhythms. Our studies indicate that, by balancing its stimulatory and inhibitory output pathways, the biological clock uses a ‘push-and-pull' mechanism to control the different hormone rhythms and the ANS. In our opinion, one of the most important aspects of the output of the biological clock is its control of the sympathetic–parasympathetic balance. In view of the widespread influence of the ANS on the physiological state of an organism, it is obvious that a ‘yin–yang' balanced regulation of their antagonistic properties is essential for healthy control of our daily lives. However, there is a reverse to every medal: a malfunctioning of the biological clock (or a misalignment of the central and peripheral oscillators) due to either aging, shift-work or a modern Western lifestyle [8,28,41,42] may be an important pre-disposing factor for pathologies characterized by an imbalanced ANS, such as hypertension, Type 2 diabetes and the metabolic syndrome.
• The projection fibres from the SCN (to mediate the outflow of its timing information) are surprisingly limited and, by and large, restricted to a few hypothalamic nuclei.
• The biological clock in the SCN imposes a temporal structure on the brain, and subsequently the peripheral organs, via day–night rhythms in the release of its neurotransmitters into its target areas.
• The biological clock imposes its daily rhythmicity on the different hormone rhythms and the ANS by balancing its stimulatory and inhibitory inputs to the neuroendocrine and pre-autonomic neurons according to a ‘push-and-pull' mechanism.
• The presence of SCN target areas with intermediate neurons has the great advantage of enabling the integration of information from different sources (such as circadian, hormonal, metabolic, stress, etc.) before a definitive signal is sent to the endocrine and pre-autonomic motor neurons.
• Intermediate neurons also provide a degree of flexibility to the system that helps to explain how a 12 h shift can occur in the timing of daily rhythms between nocturnal and diurnal species, when the timing of the daily peak in SCN activity is not changing between diurnal and nocturnal species.
- © The Authors Journal compilation © 2011 Biochemical Society