The suprachiasmatic nucleus (SCN), the master circadian clock of mammals, is composed of multiple circadian oscillator neurons. Most of them exhibit significant circadian rhythms in their clock gene expression and spontaneous firing when cultured in dispersed cells, as well as in an organotypic slice. The distribution of periods depends on the SCN tissue organization, suggesting that cell-to-cell interaction is important for synchronization of the constituent oscillator cells. This cell-to-cell interaction involves both synaptic interactions and humoral mediators. Cellular oscillators form at least three separate but mutually coupled regional pacemakers, and two of them are involved in the photoperiodic regulation of behavioral rhythms in mice. Coupling of cellular oscillators in the SCN tissue compensates for the dysfunction due to clock gene mutations, on the one hand, and desynchronization within and between the regional pacemakers that suppresses the coherent rhythm expression from the SCN, on the other hand. The multioscillator pacemaker structure of the SCN is advantageous for responding to a wide range of environmental challenges without losing coherent rhythm outputs.
A biological timing system necessarily consists of an intrinsic clock mechanism that measures time, an input mechanism that allows the clock to become synchronized or reset by changes in the environment, and output pathways that lead to generation of overt rhythms such as daily changes in locomotor activity, sleep, and hormone levels. The mammalian circadian timing system is considerably more complicated than this simple linear scheme, because it is composed of a hierarchy of circadian oscillators (Fig. 39.3). At the pinnacle of this oscillatory system is a small brain area, the suprachiasmatic nuclei (SCN) of the anterior hypothalamus. The SCN are often called the master circadian clock, because these nuclei (one on each side of the brain) play a key role in coordinating oscillations in other tissues and in regulating behavior. Many other cells and tissues also have the capacity to display an approximately 24-hour rhythmicity. The molecular mechanism underlying these cell-autonomous circadian oscillations is a transcriptional feedback loop.
Figure 39.3. The mammalian circadian timing system consists of a hierarchy of oscillators. Oscillatory neurons in the SCN interact with each other to produce a set of coherent outputs. These outputs, which include behavioral and physiological rhythms, synchronize cell-autonomous oscillations in other brain regions and in peripheral tissues.
From Reppert & Weaver, 2002, with permission. Copyright 2002, Nature.
The primary input pathway to the SCN circadian clock is through retinal detection of light. Remarkably, the retinal photoreceptors that lead to visual image formation are not needed for circadian photoreception. Instead, a specialized population of retinal ganglion cells directly detect light, project to the SCN, and are necessary for photic entrainment of the SCN clock.
The main output of the SCN is encoded in neuronal firing rates. In addition, rhythmic neuropeptide secretion into the cerebrospinal fluid may be an important mechanism for regulation of downstream targets. Anatomically, the outputs of the SCN are focused within the hypothalamus but have widespread influence, consistent with the pervasive influence of the SCN on physiological functions ranging from the modulation of cognitive function to neuroendocrine responses. Output rhythms dependent on the SCN synchronize autonomous oscillators in other tissues. Local circadian oscillators regulate gene expression in a tissue-specific manner. Microarray studies conducted on brain, liver, heart, and kidney tissue in mammals reveal that 5 to 10% of the genome is rhythmically transcribed, and many of the genes that are rhythmically expressed represent key, tissue-specific metabolic control points. Thus, the circadian timing system has a pervasive impact on physiological processes via its output pathways.
The Suprachiasmatic Nuclei are the Site of the Primary Circadian Pacemaker in Mammals
The SCN have been recognized as the master circadian clock in rodents since the early 1970s (see Weaver, 1998). At that time, “new” techniques for anterograde tract-tracing were used to assess retinal projections, and they revealed a direct retina-to-SCN projection, the retinohypothalamic tract. To determine if the SCN were an important node on the anatomical pathway for detection of light for regulating rhythms, Moore and Eichler (1972) destroyed the rat SCN and observed loss of rhythmic serum corticosterone in a population of rats. Using a similar approach, Stephan and Zucker (1972) discovered that the rhythm of drinking in individual rats was lost following destruction of the SCN. Together, these two studies demonstrated the necessity of the SCN for circadian rhythmicity. Hundreds of subsequent studies have confirmed the necessity of the SCN for rhythmicity in physiology and behavior (Weaver, 1998; Welsh, Takahashi, & Kay 2010).
SCN: Oscillator and Pacemaker
Lesion studies leading to loss of rhythmicity do not necessarily show that the SCN function as the primary circadian pacemaker. An alternative possibility is that the SCN could be a necessary element on a key output pathway leading to physiological rhythmicity. The presence of rhythms in SCN metabolic activity, electrical activity, and gene expression profiles in vivo do not distinguish between these possibilities. Studies demonstrating rhythms in neuropeptide secretion, metabolic activity, and electrical firing rate in SCN tissue maintained in vitro do show that the SCN contain a functional oscillator. The SCN do not simply oscillate, however: through its outputs, the SCN regulate rhythms in physiology and behavior, and thus serve as a circadian pacemaker. This unique pacemaker role of the SCN is revealed most clearly by studies showing that rodents made arrhythmic by SCN lesion can have rhythmicity restored by transplantation of fetal hypothalamic tissue containing the SCN into the third ventricle of the lesioned adult. Furthermore, transplants between hamsters with different circadian cycle lengths revealed that the period of restored rhythmicity is dictated by the genotype of the SCN tissue donor (Ralph, Foster, Davis, & Menaker 1990). Thus, the SCN serve as a pacemaker that communicates rhythmicity to tissues regulating behavioral rhythms.
Summary
The SCN function as the master circadian pacemaker in the mammalian brain. The SCN oscillate in vivo and also when placed in vitro. More importantly, however, the SCN generate output signals that lead to physiological and behavioral rhythms. The SCN is positioned at the interface between the outside world (detected by retinal photoreceptors) and light-insensitive effector tissues.
The suprachiasmatic nucleus (SCN) transmits its rhythm by precise anatomical connections that release peptides and amino acid neurotransmitters. The SCN uses at least four different types of neuronal targets in the hypothalamus to pass on its circadian signal: (1) endocrine neurons; (2) pre-autonomic neurons; (3) structures that dissipate the circadian signal within and outside the hypothalamus; and (4) areas outside the hypothalamus. The SCN has a major role in organizing metabolism and diminishing the effect of stress. The relationship between the different hypothalamic functions and the SCN is discussed. Hereby special attention will be given to how a disturbance of our daily balance in activity, sleep, and food intake may lead to obesity and diabetes.
The SCN has the capacity to maintain a 24-h rhythm in electrical activity, which results in a rhythmic release of its neurotransmitters from the nerve terminals.7 The rhythm in neuronal activity is maintained in vitro and is proposed to be dependent on a molecular machinery of clock genes that, by mean of positive and negative feedback loops, give a possibility for the SCN to have this 24-h rhythm in electrical activity. Although it is assumed that individual neurons may have the capacity to generate an endogenous rhythm,8 it has become likely that only when SCN neurons form a network, these neurons have the possibility to be rhythmic for long-time periods.9 Consequently, it is not only the molecular machinery that gives the SCN neurons their capacity to have an endogenous rhythm of 24 h, but in addition, these neurons need to function within a network. That a neuronal network is essential for the SCN rhythmic properties can also be concluded from studies in which knocking out a vasoactive intestinal polypeptide (VIP) receptor, which is one of the most abundant neuropeptides within the SCN, results in the incapacity to maintain rhythmic behavioral patterns.10 As these VIP receptors are highly expressed in the SCN and the electrical activity in the SCN of the knockout animals is strongly disturbed,11 it can be concluded that intra-SCN neuronal communication is essential to sustain the SCN rhythm. On this basis, we propose that similar network connections are also important in the functioning of the whole circadian system, that is, the SCN needs signals back from the body and the brain in order to function optimally.
Without a doubt, the rhythmic message of the SCN to mainly hypothalamic structures drives hormonal and autonomic output in a 24-h pattern, resulting in a rhythm in behavior that is synchronized with, for example, the adequate hormone or glucose levels together with adequate temperature and cardiovascular parameters to ensure an optimal physiology.12–14 In addition, these rhythmic patterns are not fixed but are highly adaptive and take into account the homeostatic situation of the animal. For example, the response of corticosterone to psychological stress is the highest during the beginning of the sleep period,14–16 while the corticosterone response to hypoglycemia is the highest in the beginning of the active period.17 Such interactions lead to a rhythmic organization of body functions, which is dependent on the integrity of the SCN and its output pathways.
In mammals, the SCN is a paired nucleus of small neurons lying above the optic chiasm on each side of the third ventricle (Fig. 8-3). Four lines of evidence support the conclusion that the SCN is an important circadian pacemaker:
1
The SCN is the site of termination of an entraining pathway—the RHT.
2
SCN ablation abolishes many circadian rhythms, but SCN lesions typically alter only the temporal organization of a function; the function itself is unchanged.
3
Isolation of the SCN, either in vivo or in vitro, does not alter the expression of circadian rhythms in the SCN, but most circadian rhythms in other brain and peripheral areas are lost.
4
Transplantation of a fetal SCN into the third ventricle of arrhythmic hosts with SCN lesions restores the circadian rest-activity rhythm with a period that reflects donor, not host, rhythmicity.
Functional Divisions of the SCN
The SCN is made up of two distinct subdivisions, which differ in neuronal morphology, peptide phenotype, and connections. The central region of the SCN lying immediately above the optic chiasm is designated the “core,” and it is surrounded by the second subdivision, the “shell.” In Golgi-stained material, neurons in the shell are quite small and have sparse dendritic arbors, whereas neurons in the core are larger with more extensive dendritic arbors, often extending beyond the apparent boundary of the SCN. The majority of shell neurons contain arginine vasopressin (AVP) co-localized with the inhibitory transmitter γ-aminobutyric acid (GABA). Afferents to the SCN shell arise predominantly from the brain stem, hypothalamus, basal forebrain, and limbic cortex. Core neurons, however, typically contain vasoactive intestinal polypeptide (VIP) or gastrin-releasing peptide (GRP) co-localized with GABA. Visual afferents, the primary retinal input from the RHT, and some secondary visual projections from other visual nuclei that receive retinal afferents terminate in the core. Another important input to the core is from the serotonin neurons of the midbrain raphe nuclei. These SCN subdivisions are found in all mammals, and there is evidence that they can function as independent pacemakers.10–16
SCN Neurons Are Circadian Oscillators
Both physiologic and molecular studies indicate that SCN neurons are circadian oscillators that are coupled by neural connections to form a pacemaker. Individual SCN neurons maintained in cell culture each have a rhythmic firing rate that approximates 24 hours. The free-running period of SCN neurons in culture approximates 24 hours, as would be expected, but the variance in period among individual neurons is greater than that of free-running rhythms in intact animals.17,18 The coupling of individual oscillators is critical to the function of the SCN as a pacemaker. Recent work indicates that GABA is important to the process of synchronization of SCN neurons. Evidence also suggests that gap junctions and neural cell adhesion molecules participate in the coupling of SCN neurons that underlies pacemaker function, and that these factors all interact to provide the neuronal coupling that produces an SCN pacemaker that has a reliable and uniform output that can be entrained to the solar cycle.
In Fetal Life the SCN Pacemaker Is Entrained to Maternal Rhythms
Overt circadian rhythms are typically expressed in mammals after birth. The SCN in the rat is formed in late gestation, between embryonic days 14 and 17 (E14–E17; gestation in the rat is 21 days). Circadian function in the SCN is first expressed at E19 as an intrinsic rhythm in glucose utilization, entrained to maternal rhythms. Maternal rhythmicity is not necessary, however, for the development of fetal SCN function. When the SCN of pregnant females is ablated early in gestation, before the formation of SCN neurons in the fetus, development of the fetal SCN rhythmicity progresses normally. In this situation, however, individual pups develop rhythms independent of one another and of their environment. The signal for entrainment to maternal rhythms is not known with certainty, but melatonin appears to play an important role. Limited data on development of neural mechanisms of circadian function in humans indicate that the SCN establishes circadian rhythmicity prenatally but behavioral and physiologic rhythms do not appear until after birth.
Circadian Oscillators Occur Widely in Neural and Non-neural Tissues
It was established quite early that circadian pacemakers other than the SCN are present in avian species. The evidence for non-SCN pacemakers in mammals, however, was quite limited until a circadian rhythm in melatonin production was shown in cultured hamster neural retina.19 This study provided definitive evidence that the mammalian eye contains a circadian pacemaker, probably functioning to maintaining a circadian rhythm of visual sensitivity. With the development of understanding of the molecular basis of clock function, additional non-SCN oscillators have been described in many tissues and organs, including brain areas outside the SCN. Although these oscillators maintain circadian rhythmicity in the absence of SCN input, the timing of individual cells begins to differ and the cellular oscillators go out of phase, indicating that the SCN functions to coordinate the timing of functions throughout the body20–23 (Fig. 8-4).
Summary
The SCN is the dominant mammalian pacemaker. It is composed of two subdivisions made up of neurons that are born as individual circadian oscillators coupled to form a pacemaker. One subdivision, the shell, contains AVP/GABA neurons and receives nonvisual input. The shell surrounds the core, which contains VIP/GABA and GRP/GABA neurons that receive visual input from the retina and from the intergeniculate leaflet (IGL) of the lateral geniculate.
Decreased VP, vasoactive intestinal polypeptide, and neurotensin activity in hypertensive suprachiasmatic nucleus
The suprachiasmatic nucleus of hypothalamus is a core structure of circadian clock networks that regulate the physiological and behavioral rhythms in mammals, helps to adapt the body to the day/night cycle, as well as to seasonal changes in the environment. In the SCh, both neuronal and astroglial cells contain individual oscillators (cellular clocks), which are driven by autoregulatory transcriptional/translational feedback loops of clock genes in cooperation with cytosolic signaling molecules, such as cAMP and Ca2 +. Being synchronized with each other and entrained to solar time by both direct retinal innervation via the retinohypothalamic tract and indirectly through retinally innervated cells of the intergeniculate leaflet, the SCh cells generate very stable and coherent oscillations, providing a consistent output for entrainment of countless peripheral cellular clocks across the body (Welsh et al., 2010; Takahashi, 2017; Hastings et al., 2018).
In the SCh of hypertensive patients, we found a noticeable decrease in the number of VP-, VIP-, and NT-immunostained neurons, compared with the SCh of healthy people from the control group (Fig. 24.5). It should be noted that, despite the decrease in the density of VP neurons in SCh, the density of VP neurons in both Pa (Fig. 24.6) and supraoptic nucleus (SO) (Goncharuk et al., 2001) of hypertensive patients did not differ from the density of VP neurons in the Pa and SO of healthy individuals.
Fig. 24.5. Represents total number of vasopressin (VP), vasoactive intestinal peptide (VIP) or neurotensin (NT) cell bodies in the unilateral suprachiasmatic nucleus (SCh) of controls (Ctr) and hypertensives (Hyp). Only neurons with a visible nucleus were counted as a cell body; neurons that were only partially sectioned or of which only a cap was stained were excluded. As soon as the staining intensity of a neuronal profile became detectable above background value, this was counted as a positive cell body. This difference in staining intensity is, however, not taken into account for these quantitative data. The Mann–Whitney U test indicated that the difference in vasopressin (VP), vasoactive intestinal peptide (VIP), and neurotensin (NT) cell number between the hypertensives and controls was highly significant (P < 0.001; P < 0.05; and P < 0.001), the bar indicates the mean.
From Goncharuk VD, van Heerikhuize J, Dai JP et al. (2001). Neuropeptide changes in the suprachiasmatic nucleus in primary hypertension indicate functional impairment of the biological clock. J Comp Neurol 431: 320–330, with permission.
Fig. 24.6. Coronal sections through the middle level of suprachiasmatic (SCh) nucleus from the hypothalamus of normotensive individual (case #20) (A) and hypertensive patient (case #5) (B) immunohistochemically stained for vasopressin (VP). Boxed areas within the hypothalamic paraventricular (Pa) nucleus in (A) and (B) are presented under higher magnification in (C) and (D), respectively. Boxed areas within the SCh in (A) and (B) are shown under higher magnification in (E) and (F), respectively. Note that VP-positive neuronal profiles and fibers in the control (C) and hypertensive (D) Pa have comparable density. At the same time, the number of VP neurons in hypertensive SCh (F) is markedly decreased compared to control (E). The differences look even more dramatic if to take into account that the only upper part of the control SCh from (A) is presented in (E), whereas almost the whole hypertensive SCh from (B) is shown in (F). Bar = 100 mkm in (A, B) and 40 mkm in (C–F).
The role of VP, VIP, and NT neuronal circuits within the SCh have further been elucidated in animal experiments. Thus diurnal changes of the VP mRNA and heteronuclear (hn) RNA, as indicator for gene transcription, were found in the rat SCh. However, unlike SCh, VP hnRNA or mRNA levels in SO, where VP is synthesized in response to plasma osmolarity and/or volume, did not show any circadian rhythm (Yambe et al., 2002). Moreover, recent experiments on genetically chimeric mice with a functionally deficient cell clock in their VP neurons demonstrated that the SCh VP neuronal network plays a critical role in generating a robust circadian rhythm of behavior by regulating the coupling of SCh neurons, as well as determining the circadian period (Mieda, 2019). Furthermore, a new optogenetic study showed that clock-controlled release of VP from axon terminals in the SCh mediates anticipatory presleep water intake in mice (Gizowski et al., 2016, 2018). In general, these results indicate that activity of the SCh VP network is necessary and sufficient to provide the mechanisms that are used by the central clock to generate circadian rhythms of the body.
The SCh VIP-expressing neurons receive synaptic inputs from the retinal interneurons (Lokshin et al., 2015) and perform two vital circadian functions: they maintain synchronized circadian oscillations across the SCh in the absence of entraining signal and, by photically regulated release of VIP, they integrate light–dark cycle cues with the intrinsic oscillation of the SCh network (Hastings et al., 2018; Hamnett et al., 2019). Both NT (Watts and Swanson, 1987) and NT receptors (Alexander and Leeman, 1998) have been localized in cell bodies of the rat SCh and in vitro experiments have shown that NT alters spontaneous discharge rates of individual SCh neurons (Coogan et al., 2001). Moreover, application of NT during the projected day resulted in a large advance in the timing of the peak in firing rate rhythm, whereas treatments during the projected night had no effect. Thus the observed in vitro pattern of phase-resetting effects of NT was similar to that evoked by nonphotic stimuli in vivo. This provides evidence that NT may play a role in regulating the rat circadian pacemaker (Meyer-Spasche et al., 2002). Based on the experimental data presented, it can be assumed that a significant decrease in the numbers of VP, VIP, and NT neurons we observed in the SCh of hypertensive patients may indicate an insufficient function of the main circadian clock.
Seasonal fluctuations originate in the SCN (Hofman and Swaab, 1992). Since the prevalence for SAD is five times higher for women than for men (Agustini et al., 2019), sex differences in the interaction of sex hormones and the SCN can be expected. Indeed, nuclear AR is higher expressed in the SCN of men than of women while ERα and ERβ are expressed more in the SCN of women (Fernandez-Guasti et al., 2000; Kruijver and Swaab, 2002; Kruijver et al., 2003). In postmortem brains of mood disorder patients, we found a disorder of SCN function, characterized by an increased number of AVP-expressing neurons, in combination with a decreased amount of AVP-mRNA in this nucleus, and diminished circadian fluctuation of AVP-mRNA (Zhou et al., 2001). Recently, Wu et al. (2017) confirmed this finding with the new observation that a significant increase of SCN-AVP neuropeptide levels was present only in female and not in male depression patients.
Isolated SCN neurons all have slightly different periods and phases, and the average of these individual pacemakers makes up the final output. This concept is supported by studies using mouse chimeras in which the SCN is composed of a mixture of wild-type and Clock mutant neurons. The resulting circadian period depends on the proportion of wild-type and mutant cells in the SCN (Low-Zeddies & Takahashi, 2001). Several mechanisms have been proposed that underlie the coupling between individual SCN neurons, such as synaptic transmission, gap junctions, as well as paracrine mechanisms.
SCN neurons are not the only cells that express circadian oscillations. In fact, there are circadian oscillators throughout the body. Several genes making up the intracellular clock mechanism (see next) have been found to be rhythmically expressed in other brain areas as well as various peripheral organs (Buijs et al., 2001; Yamazaki et al., 2000). In contrast to SCN neurons, these slave oscillators sustain their 24-hour oscillations for a few days only when kept in culture, deprived of master clock input. It is thought that the collective SCN output synchronizes the timing of peripheral oscillators, which in turn regulate local rhythms in physiology and behavior. This hierarchical system confers precise period and phase control as well as stability of the widespread physiological systems that are regulated.
The SCN has long been known to receive one of the densest serotonergic innervations in the brain. This monoaminergic input to the SCN arises from ascending projections of serotonin (5-hydroxytryptamine; 5-HT) neurons in the midbrain median raphe nucleus (Morin, 1999; Ciarleglio et al., 2011). 5-HT fibers are distributed throughout much of the SCN, but they are most heavily concentrated in the ventral aspect of the nucleus, forming a curved-shaped terminal plexus that is co-extensive to a considerable degree with the RHT terminal field (Fig. 1). In this region of overlap between the two afferent fiber systems, 5-HT fibers preferentially make axo-dendritic and axo-somatic synaptic contacts with VIP neurons that also receive retinal synapses (Bosler and Beaudet, 1985; Fernandez et al., 2016). Ultrastructural analyses have also shown that 5-HT varicosities in the SCN are very often apposed to retinal and GABA terminals (Bosler, 1989); these 5-HT axo–axonic interfaces represent additional important sites for serotonergic modulation of SCN activity.
Figure 1. Representation of the retinohypothalamic tract (RHT) and serotonin (5-hydroxytryptamine; 5-HT) terminal fields in the mouse suprachiasmatic nucleus (SCN). The region of the SCN containing the heaviest concentration of retinal fibers is shown in green and the region of densest 5-HT fibers is depicted in red; the yellow region represents the area where the terminal fields are co-extensive.
Early on it was demonstrated that destruction of the 5-HT input to the SCN was not essential for the expression of circadian behavior (Block and Zucker, 1979). Genetic elimination of 5-HT in the central nervous system has confirmed that 5-HT is not required for the SCN to generate a circadian rhythm (Ciarleglio et al., 2014). However, removal of the serotonergic input to the SCN following ablation of the median raphe nucleus does produce an increased sensitivity to photic input in the SCN and an alteration in the phase angle of entrainment of the circadian activity rhythm to the light/dark cycle (Morin, 1999). These findings are consistent with the theme that 5-HT neurons do not play a role in the initiation of physiological and behavioral processes but rather exert modulatory influences on their targets. It had been generally accepted that the modulatory effects of the serotonergic system were based on slow alterations in tonic activity levels. This long standing idea is being modified as data accumulate indicating that 5-HT neurons can function on multiple timescales not only to modulate but also to dynamically shape ongoing information processing (Ranade and Mainen, 2009; Cohen et al., 2015).
The SCN exerts its controls over its outputs via humoral and neural pathways. The SCN’s direct efferent projections are sparse and mostly confined within the medial hypothalamus, reaching the medial preoptic area (MPO), the paraventricular nucleus of the hypothalamus (PVN), the subparaventricular nucleus (sPVN or SPZ) and the dorsomedial nucleus of the hypothalamus (DMH). Outside the hypothalamus, the SCN projects to the paraventricular nucleus of the thalamus, the IGL and indirectly to the ventral tegmentum through the median preoptic nucleus of the anterior hypothalamus. This latter connection could be responsible for circadian regulation of behavioral processes that include arousal and motivation.115b
At least three different types of neuronal targets of the SCN fibers can be discriminated within the medial hypothalamus: (1) endocrine neurons [such as those containing corticotropin-releasing hormone (CRH), thyrotropin-releasing hormone (TRH) and gonadotropin-releasing hormone (GnRH)], (2) projections to the autonomic PVN to influence autonomic neurons projecting to preganglionic parasympathetic and sympathetic neurons in the spinal cord116–119, and (3) neurons reached by the direct SCN connections in the MPO, SPZ, and DMH, also called intermediate neurons, that likely integrate circadian information with other hypothalamic inputs before the information is passed on to endocrine and possibly autonomic neurons.
