The mammalian circadian system: a hierarchical multi-oscillator structure for generating circadian rhythm
The circadian nature of physiology and behavior is regulated by a circadian clock that generates intrinsic rhythms with a periodicity of approximately 24 h. The mammalian circadian system is composed of a hierarchical multi-oscillator structure, with the central clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus regulating the peripheral clocks found throughout the body. In the past two decades, key clock genes have been discovered in mammals and shown to be interlocked in transcriptional and translational feedback loops. At the cellular level, each cell is governed by its own independent clock; and yet, these cellular circadian clocks in the SCN form regional oscillators that are further coupled to one another to generate a single rhythm for the tissue. The oscillatory coupling within and between the regional oscillators appears to be critical for the extraordinary stability and the wide range of adaptability of the circadian clock, the mechanism of which is now being elucidated with newly advanced molecular tools.
KeywordsCircadian clock Suprachiasmatic nucleus Oscillatory coupling Clock gene Luciferase reporter
Robust circadian rhythms in physiological functions and behaviors are conserved across all organisms, from cyanobacteria to humans. These rhythms are driven by an intrinsic machinery called the circadian clock, which generates self-sustaining rhythms with a periodicity of about (circa) 1 day (dies). In the last two decades, we have come to appreciate the underlying mechanisms of the mammalian circadian clock at the molecular, cellular, network, and organismal levels. Indeed, the 2017 Nobel Prize for Physiology and Medicine was awarded to three chronobiologists who first cloned the Drosophila clock gene, Period (Per) in 1984 [1, 2]. The first mammalian clock gene, clock, was successfully cloned in 1997  and, owing to the remarkable similarities of the circadian pacemaker system between mammals and Drosophila, our understanding of the clock mechanism has advanced rapidly.
Characteristics of circadian rhythms
Although circadian rhythms are intrinsic, living organisms are also under the influence of cyclic changes of environments, such as day–night cycles and seasonal changes in photoperiods and temperature. The alignment of the circadian rhythm to such environmental cues or “Zeitgebers” to maintain periodicity is referred to as entrainment. For all organisms, light is the strongest Zeitgeber. The circadian clock entrains to a 24-h light–dark (LD) cycle resulting in diurnal, nocturnal, or crepuscular behavior.
The circadian clock can be phase-shifted by any of these time cues, and the extent and direction (advance or delay) of the shifts depend on the phase of the circadian clock at which the cues are introduced. The phase–response curve (PRC) summarizes the relationship between the time when the cue, such as light (Fig. 1b) and dexamethasone (Fig. 1d), is introduced and the induced phase shifts . Using the PRC, we can predict how the clock will respond to the environment, and we can calculate the time course of re-entrainment after changes in the LD cycle, as well as the limit of entrainment . For example, a single light pulse at the subjective early night induces a phase-delay shift, while a pulse during the subjective late night to morning results in a phase-advance shift. No shift is observed when a pulse is given during the middle of subjective day. Interestingly, the shape of the PRC has been shown to be remarkably similar across all living organisms recorded thus far , which strongly suggests that the circadian clock developed very early during evolution. Experiments using cyanobacteria mutants with different circadian periodicities have demonstrated the evolutionary advantage of having a circadian clock . Without competitors, cyanobacteria mutants with a short or prolonged periodicity proliferated at the same speed as wild type cyanobacteria. However, when populations with different periodicities were mixed, the colony with a circadian period most similar to the environmental periodicity eventually outpaced their unsynchronized counterparts, highlighting the importance of matching environmental cues to life itself.
Finally, a circadian rhythm requires the ability to compensate for changes in temperature to maintain periodicity. Typically, a circadian oscillation would have a Q10 coefficient (a measure of the rate of change in a biological system dependent upon temperature) of approximately 1.0, meaning that the periodicity is not affected by changes in increasing temperatures that can affect the kinetics of cellular processes . In homothermic animals like mammals and birds, the significance of temperature compensation was initially overlooked as the central circadian clock is located within the central nervous system where the temperature is held constant. However, the discovery of peripheral clocks has reignited the field to research the importance of temperature compensation. While the mechanism of how the circadian clock compensates for the change in temperature is not completely understood, it has been shown that the structure of a clock protein KaiC in cyanobacteria is critical for temperature compensation by slowing down the speed of oscillation and reducing energy expenditure . Furthermore, casein kinase 1 ε was demonstrated to play an important role in temperature compensation in mammals .
The mammalian circadian system is composed of a central clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus that regulates additional circadian rhythms found in non-SCN brain regions and in peripheral organs throughout the body [12, 13, 14]. The SCN is the only clock that is reset by the light from the environment; the photic signals are transferred to the SCN via the retinohypothalamic tract (RHT). Once the SCN pacemaker is light-entrained, it synchronizes the peripheral clocks to the same 24-h cycle through mechanisms that remain largely unknown.
The circadian clock allows us to prepare and adapt our physiology to cyclic changes in our environment. Notably, ablation of the circadian clock does not directly terminate the life of organisms, but without a fully functioning clock, the organisms appear to become vulnerable when placed in a naturally or socially competitive environment. The circadian clock thus plays a critical role in the survival of individuals and of species as a whole.
Molecular machinery that generates circadian rhythm at a cellular level
Clock genes and molecular feedback
Due to the similarities between the mammalian and Drosophila clock [1, 2], key members of transcriptional and translational feedbacks were quickly identified in mammals. And in the case of Clock and Bmal1 (called Cycle in Drosophila), Drosophila orthologs were cloned after the cloning of mammalian genes [22, 23]. However, it is important to note that there are also a few differences between mammals and Drosophila. For example, mammals have three Per homologs, Per1, Per2, and Per3, each of them playing a specific role . Furthermore, in Drosophila, TIMELESS protein exhibits photosensitivity and plays a role in regulating the circadian rhythm. In contrast, the mammalian TIMELESS has been shown to regulate cell cycle and carcinogenesis , and its role in the circadian rhythm has remained elusive.
In addition to the core molecular feedback loop consisting of PER1, PER2, CRY1, CRY2, CLOCK, and BMAL1, interlocking feedback loops have also been identified to regulate circadian rhythms. One example is the Bmal1 loop, which interlocks with the core loop  and exhibits an antiphasic gene expression pattern . Bmal1 is regulated by a retinoic acid receptor-related orphan receptor (ROR) enhancer site located upstream of the Bmal1 gene; ROR binding activates gene expression, while REB-ERBα binding inhibits transcription . REB-ERBα itself is regulated by an E-box enhancer located upstream of its transcription start site, resulting in an expression pattern that subsequently puts Bmal1 completely out of phase with Per1 and Per2 . Dec1 and Dec2 are two other elements of an interlocking loop found to negatively affect circadian rhythms . Both Dec1 and Dec2 have E-box enhancers and are positively regulated by CLOCK and BMAL1, and negatively by PER1, PER2, CRY1, and CRY2. Furthermore, the transcription of Dec1 and Dec2 is self-regulated by their encoded proteins. DECs also form a mutually interlocked molecular loop with the Per loop. These interlocked multiple molecular loops are thought to be advantageous by providing stability and fine tuning to the circadian periodicity .
Molecular mechanisms of light entrainment
The circadian clock in the SCN is reset by photic signals that are received by melanopsin-expressing, intrinsically photosensitive retinal ganglion cells (ipRGCs) in the retina [31, 32, 33] and are transmitted via the RHT . Among the five subtypes of ipRGCs (M1–M5), the M1 ipRGCs predominantly (and, to a lesser extent, the M2 ipRGCs) project photic signals to the SCN to entrain the circadian clock . ipRGCs are also involved in light-induced pupil constriction, masking behavior in response to light, and even certain kinds of image-forming visual pathways [36, 37, 38, 39].
The molecular mechanisms of light entrainment have been intensively studied. Photic signals are projected to the SCN core where glutamate, the neurotransmitter of the RHT, induces phosphorylation of Ca2+-cAMP response element-binding (CREB) protein, resulting in the transcription of some key clock genes (Fig. 2). Photic induction of Per1 and Per2 [40, 41], but not Per3 , is detected only during the photosensitive zone of PRC (Fig. 1). Dec1, but not Dec2, expression is also induced by phase-resetting light stimuli . Photic signals administered during the dead-zone of PRC do not induce Per or Dec expression and do not lead to shifts in behavioral rhythms. Interestingly, light induction of these gene expressions is observed at the core of the SCN where the circadian clock gene expression level is low, whereas circadian gene expression is high in the shell region of the SCN . Among these genes, Per1 and Dec1 expressions are induced by lights with the kinetics comparable to those of an immediately early gene. Furthermore, Per1 antisense injection into the SCN blocked light-induced phase shifts, suggesting a critical role of Per1 in mediating light entrainment. Per and Dec genes exhibit robust circadian expression rhythms in the SCN with a peak located during the subjective day, i.e., the dead-zone of PRC (Fig. 2). When light is given in subjective evening when Per expression is decreasing, light-induced gene expression may elongate this part of the cycle, resulting in a phase-delay of the molecular clock . In contrast, when light is given in the subjective morning hours when Per expression is increasing, light-induced gene expression makes an early start of the rising phase of the cycle, resulting in the advancement of the molecular clock. Since the gene expression would already be high during the subjective day at the dead-zone of PRC, we would not expect a light-induced shift of the molecular clock.
Although this mechanism explains light-induced phase shifts and light entrainment, Per1, Per2, or Dec1 knockout mice show LD-entrained behavioral rhythms similar to those of wild type mice, suggesting redundancy in gene functions or alternative mechanisms. Among the light-inducible clock genes, Per1 and Dec1 are expressed immediately following light exposure, and their mRNAs have a relatively short half-life [29, 40]. Subsequently, Per2 expression is observed, and its high mRNA level is maintained for a longer period than for Per1 [41, 42]. Thus, it appears that Per1 and Per2 have non-redundant roles in light entrainment.
Post-transcriptional and post-translational regulation of molecular oscillation
Recent studies have demonstrated that 70–80% of mRNA demonstrating circadian rhythms in their content did not exhibit circadian rhythm in their de novo transcription , indicating a critical role of post-transcriptional regulation in the molecular circadian clock. For example, an RNA editing enzyme, Adarb1, has been shown to have a circadian expression pattern. As a result, its downstream targets also exhibited post-transcriptionally regulated oscillations .
Post-translational modifications also affect the circadian rhythm, especially the period length. Tau was the first mammalian clock gene identified in a mutant hamster that displayed a short circadian period . The cloning of tau was delayed compared to that of Clock  and Per  for many years because it was found in hamsters in which genome information was limited. Tau mutation is a missense mutation in the phosphorylation site of casein kinase 1 ε (CK1ε) , a kinase which phosphorylates many clock proteins, including PER and CRY. Soon after the finding of the Tau mutation in hamsters, patients with advanced sleep-phase syndrome (ASPS) with short circadian periods, were shown to possess a missense mutation in the Per2 gene at the CK1ε phosphorylation site . Interestingly, further studies revealed the Tau mutation to be a gain-of-function mutation, which instead induced hyperphosphorylation in other regions of the PER2 protein, resulting in shortening of the molecular oscillation period . Other kinases were also found to affect the periodicity, such as glycogen synthase kinase 3 β .
In addition to phosphorylation, other post-translational modifications such as ubiquitination [50, 51], acetylation , and SUMOylation  have been shown to play important roles in the fine-tuning of the circadian period. For example, FBXL3, an F-box-type E3 ligase, ubiquitinates CRY1 and CRY2 in the nucleus, which mediates their degradation and shortens the circadian period (Fig. 2). In contrast, FBXL21 ubiquitinates CRYs in the cytosol and stabilizes them. Thus, FBXL21 in the cytosol counteracts the period-shortening effects of FBXL3 in the nucleus [54, 55]. In addition to the familiar ASPS due to Per2 mutation , various mutations have already been identified in different types of sleep–wake rhythm disorders in humans [56, 57, 58]. Clock gene functions have also become research targets for mood [59, 60, 61] and neurodevelopmental [62, 63, 64] disorders that result in disrupted circadian rhythms.
The multiple interacting molecular loops, post-transcriptional, and post-translational modifications of the clock genes increase the stability as well as adaptability of the system. Further study is needed to fully understand the mechanisms that regulate the clock at the molecular and cellular levels.
Technical advances in long-term monitoring of physiological functions
Canonical clock genes such as Per1, Per2, and Bmal1 exhibit robust circadian rhythms in their transcriptional activity and protein levels [15, 17, 41, 65]. To monitor their circadian rhythms in living cells for prolonged periods of time, bioluminescent and fluorescent reporters have been introduced. Among them, luciferase reporters have been widely used to analyze clock gene expression and circadian rhythms because detection of luminescence produced by the luciferin–luciferase reaction requires no excitation. This low phototoxicity is advantageous for long-term, continuous recording of the reporters. To detect these signals, a sensitive photon counting device called a photomultiplier tube (PMT) can be used; however, PMTs lack spatial resolution [14, 66]. To overcome this, a charge coupled device (CCD) camera can be used to image bioluminescence to monitor circadian rhythms at the cellular level. To detect very dim luminescence, CCD and electron multiplying CCD cameras can be cooled to − 60 to −80 ℃ to reduce background noise [67, 68].
The central circadian pacemaker, SCN
Architecture of the SCN
Regional oscillators and networks
Dispersed SCN neurons exhibit circadian rhythms in their clock gene expression and spontaneous firing [81, 82, 83]. Importantly, SCN neurons in wild type animals exhibit independent circadian rhythms, with cell-specific periods with a Gaussian distribution covering a wide range of 20–30 h. In contrast, the circadian periods of SCN neurons in a slice culture are synchronized to one another, and the slice-to-slice variability is much smaller than that observed in dissociated cell culture . Free-running periods of behavior were distributed in an even narrower range than in slices . These findings suggest that in an intact SCN, individual neurons communicate to achieve synchronized periodicity representative of the majority of constitutional cellular oscillators. The coupling of the oscillator network in the SCN is critical for generating a coherent output from the SCN and, subsequently, for the regulation of the peripheral clocks.
Bioluminescence imaging of coronal SCN slices of either transgenic mice expressing a Per1-promoter-driven luciferase reporter (Per1-luc) or knock-in mice carrying a PER2::LUCIFERASE fusion reporter (PER2::LUC) demonstrated robust cellular circadian rhythms with synchronized periodicity. However, the phase distribution within the SCN was specific to topology and changed dynamically in response to a number of perturbing signals, in particular, photic signals [86, 87]. Abrupt phase shifts in an LD cycle induced phase shifts in behavioral rhythm with a transient period of approximately 1 week. This transient period is regarded as the lag between the immediate phase shift of the central clock and the gradual shifts of the peripheral clocks involved in overt rhythm expression. Interestingly, the rate of change in clock gene expression rhythms appears to be region-dependent; a shift was immediately observed in the SCN core following the LD perturbation, while a more gradual change was recorded over several days in the shell . This finding supports the presence of regional oscillators in the SCN, which are synchronized under steady-state conditions, but become distinct in the presence of external stimuli. Previously, we reported the separation of the core and shell rhythms by measuring AVP and VIP releases from an organotypic SCN slice culture . We found that the circadian rhythms of the release of the two peptides were initially synchronized; however, if they were treated with antimitotic drugs at the beginning of the culture, the two systems became gradually desynchronized over time. This demonstrated that the synchrony was kept among the identical peptidergic cells, but AVP and VIP neurons could be separated, suggesting the presence of two types of cellular networks within the SCN.
Another type of regional oscillator was reported in hamsters exhibiting split rhythms (two activity components that were 180 degrees out of phase) under constant light (LL). When rhythm splitting was observed in their behavior, gene expression rhythms of clock genes such as Per1, Per2, and Bmal1 were desynchronized and exhibited anti-phasic rhythms between the right and left SCN , even though the phase relation among these clock genes was intact unilaterally. Additionally, each SCN independently regulated their output functions, such as the release of gonadotrophin-releasing hormone . This result demonstrates that under certain circumstances, the circadian clocks of the right and left SCN can also be separated.
Oscillators in the SCN that regulate seasonal changes in behavior
Another set of regional oscillations were found to change their phase relations when animals were exposed to different photoperiods. Nocturnal rodents are known to entrain to a wide range of photoperiods, from an LD cycle of light 0.5 h and dark 23.5 h (LD 0.5:23.5) to an LD 20:4. In order to explain this wide range of adaptability, the two-oscillator model was proposed in 1976 . In this model, the evening (E) oscillator is reset by a light-off signal and regulates the onset of activity, whereas the morning (M) oscillator is reset by a light-on signal and regulates the end of activity. In order to study the localization and the molecular mechanisms of the E and M oscillators, we exposed the Per1-luc mice to either LD18:6 (long day) or LD 6:18 (short day) for 3 weeks. On the last day of the experiment, we collected two consecutive coronal SCN slices and found that the Per1-luc rhythm of the posterior SCN phase-led the anterior counterpart without exception . Notably, the phase difference between the two SCN slices increased with the length of the light phase, and in mice exposed to LD18:6, the Per1-luc signal peaked twice a day in the anterior SCN slice, in the morning and in the evening. Furthermore, we found that the Per1-luc rhythm in the anterior SCN was phase-locked to the onset of behavior activity, while that of the posterior SCN coincided with the end of behavior activity, indicating the anterior and posterior SCN as sites of the E and M oscillators, respectively. We also observed a third oscillator, which exhibited a peak in the early morning in the anterior SCN only under LD18:6; the function of this was unknown.
To identify the precise location of the three oscillators in the SCN, we made horizontal SCN slices that contained both the anterior and posterior SCNs in a single slice. A geometrical transformation technique was applied to set the shape of the cultured SCNs to the same shape as the arbitrary-selected template SCN so that the bioluminescence image data could be statistically evaluated . We found that the oscillator cells located in the posterior tip corresponded to the M oscillator; cells of the open-ring shape covering the anterior SCN corresponded to the E oscillator; and cells in the center appeared to respond to light. The light-responsive area may transfer the lights-on and -off signals to the M and E oscillators, respectively.
We also analyzed the rhythms of PER2::LUC mice to compare the roles of Per1 and Per2 in photoperiodic responses. Intriguingly, the peak phases of Per1-luc rhythms changed almost proportionally to the changes in photoperiods (Fig. 4c–f), whereas those of PER2:LUC exhibited minimal changes in the circadian phase within a slice. The distinct localization of the E and M oscillator cells were not detected in the PER2::LUC mice. Per1 and Per2 were shown to have non-redundant roles in the photoperiodic response.
Clock gene mutation revealed clock mechanisms at cellular and network levels
Gene mutations of clock and clock-related genes can induce rhythm disturbances at multiple levels in the organism. In most mutant mice that exhibited altered circadian periods in behavior, similar deviations were also observed in their dispersed SCN cells [94, 95]. However, behavior rhythms are also affected by the mutation affecting rhythm coupling and output. Despite no known role of VIP in the generation of circadian rhythm in the SCN, mice lacking its receptor, VIP Receptor 2 (VIPR2), often exhibit behavioral arrhythmicity in DD . They also display a variety of abnormalities such as splitting and relative coordination . VIPR2 is widely expressed in neurons of both the SCN core and shell. In cultured SCN slices, individual SCN cells exhibited significant circadian rhythms, but their circadian periods were distributed over a wider range of time compared to that of wild type mice . Collectively, these results demonstrate the importance of coupling in vivo, and that the mechanism that translates circadian rhythm in the SCN to behavior is far more complicated than we had previously expected.
Using triple knockout mice lacking VIPR2, CRY1, and CRY2 (VIPR2−/−/Cry1−/−/Cry2−/−), we demonstrated that the CRY-independent coupling found during the neonatal period was dependent on VIP signaling, and that AVP and VIP had redundant roles in the coupling of cellular rhythms . AVP expression was attenuated in the Cry1−/−/Cry2−/− SCN and VIP signaling naturally lost its ability to regulate rhythm coupling over time, thus resulting in the loss of synchronous cellular rhythms in adult Cry1−/−/Cry2−/− SCN slices. Recovery of AVP signaling from the neonatal wild type SCN rescued circadian rhythms in both the adult VIPR2−/−/Cry1−/−/Cry2−/− (Fig. 5b) and Cry1−/−/Cry2−/− SCNs. It appears that peptidergic signaling is critical for generating coordinated rhythm expression from the SCN, and may be advantageous over the fast classical neurotransmitter signaling, which exerts its effect in the order of milliseconds.
Circadian rhythms in calcium levels and membrane potential
Intracellular Ca2+ mediates input signals to the molecular clock of the cell (e.g., phase-resetting stimuli to the SCN) and output signals from the molecular clock (e.g., neurotransmitter release). Taking advantage of the improvements in long-term and large-scale Ca2+ imaging, we were able to monitor circadian Ca2+ rhythms across all neurons of cultured SCN slices [102, 103]. In every section of the SCN neuron examined so far — including the regions with low clock gene expression — a robust circadian Ca2+ rhythm with a topologically specific phase distribution was observed . Circadian Ca2+ rhythms of the dorsal SCN neurons phase-led those of the ventral neurons, similarly to the phase-distribution pattern observed for Per1 and Per2 . The gap junction blocker carbenoxolone had minimal effects on the Ca2+ rhythm at the single cell as well as at the network levels, while the Na+ channel blocker TTX uncoupled the Ca2+ rhythms between the dorsal and ventral SCNs. Under TTX treatment, Ca2+ rhythms continued although the amplitude was reduced by approximately 30% . These findings suggest that neural firing may be necessary to establish the hierarchy among the multiple regional oscillators in the SCN, even though the synchronization within the regional oscillator are independent of the cellular transmission via Na+ channels or gap junctions. The 30% reduction of amplitude under TTX treatment is thought to reflect the Ca2+ increase due to input signals in the overall Ca2+ rhythms.
The SCN outputs circadian signals to multiple brain areas and peripheral clocks to orchestrate the circadian rhythms throughout the body. In addition to the multielectrode array dish (MED) to record spontaneous firing from multiple neurons of the SCN, we found that the genetically encoded voltage sensor ArchLightD is a powerful tool to monitor circadian rhythm of the membrane potential from entire SCN slices. Unexpectedly, the circadian voltage rhythms were synchronous throughout the coronal SCN slice [105, 106]. Circadian rhythms of spontaneous firing, simultaneously monitored by the MED system, were also synchronous between the dorsal and ventral SCN, despite the topologically specific phase distribution of Ca2+ rhythms within the SCN. The similarity in phase distribution between the Per1-luc/PER2::LUC and the Ca2+ rhythms indicates that the synchronous rhythm of the neural outputs at the network level is independent of the molecular clock of individual neurons. The differences in regulation of the firing and clock gene expression rhythms [86, 87] may allow for the precise phase regulation of rhythm outputs.
The circadian clock regulates neuronal, metabolic, and hormonal functions, and many clock gene mutants exhibit disturbances in sleep–wake rhythms and metabolisms [112, 113]. Further research is needed to reveal how the SCN orchestrates all of the peripheral clocks, how these clocks communicate their circadian rhythm, and how the hierarchical multi-oscillator circadian system is established. Finally, the structure and functions of the extra-SCN oscillators would be fascinating research areas to target.
Compliance with ethical standards
Conflict of interest
The author declares no conflicts of interest.
Research involving human participants and/or animals
All applicable international, national, and institutional guidelines for the care and use of animals were followed. This article did not contain any studies involving human participants.
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