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The central circadian clock of the suprachiasmatic nucleus as an ensemble of multiple oscillatory neurons
Journal Pre-proof The central circadian clock of the suprachiasmatic nucleus as an ensemble of multiple oscillatory neurons Michihiro Mieda
PII:
S0168-0102(19)30470-5
DOI:
https://doi.org/10.1016/j.neures.2019.08.003
Reference:
NSR 4304
To appear in:
Neuroscience Research
Received Date:
22 July 2019
Accepted Date:
9 August 2019
Please cite this article as: Mieda M, The central circadian clock of the suprachiasmatic nucleus as an ensemble of multiple oscillatory neurons, Neuroscience Research (2019), doi: https://doi.org/10.1016/j.neures.2019.08.003
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The central circadian clock of the suprachiasmatic nucleus as an ensemble of multiple oscillatory neurons
Michihiro Mieda
Department of Integrative Neurophysiology, Graduate School of Medical Sciences, Kanazawa
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University, 13-1 Takara-machi, Kanazawa, Ishikawa 920-8640, Japan.
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Highlights
The suprachiasmatic nucleus (SCN) functions as the central circadian pacemaker.
The SCN is a network composed of multiple types of GABAergic neurons and glial
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cells.
Intercellular communications are essential for the circadian pacemaking of the SCN.
Multiple types of neurons play differential roles in the SCN network.
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Abstract Circadian rhythms are oscillations with approximately 24-h period that appear in most of physiological events in our body. The suprachiasmatic nucleus (SCN) of the hypothalamus functions as the central circadian pacemaker in mammals and entrains to the environmental light/dark cycle. The SCN is a network structure composed of multiple types of -amino butyric
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acid (GABA)-ergic neurons and glial cells. Although individual SCN neurons have intracellular molecular machinery of circadian clock and the ability to oscillate cell-autonomously, interneuronal communications among these neurons are essential for the circadian pacemaking
of the SCN. However, the mechanisms underlying the SCN network remain largely unknown.
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Here, I briefly review the molecular, cellular, and anatomical structures of the SCN and
introduce recent studies aiming to understand the differential roles of multiple neuropeptides
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Keywords
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and neuropeptide-expressing neurons in the SCN network.
Circadian rhythm, suprachiasmatic nucleus, clock gene, GABA, vasopressin, VIP, neural
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network
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1. The central circadian clock in mammals: the suprachiasmatic nucleus (SCN) Many physiological functions in our body exhibit oscillations with frequencies ranging widely from milliseconds to days, e.g., brain electrical activity, heart rate, breathing, sleep/wakefulness cycle, circadian rhythm, and the estrous cycle. Circadian (the Latin circa, meaning “around,” and diem, meaning “day”) rhythms are oscillations that have an approximately 24-h period, which dominate our lives (Hastings et al., 2003). It is most obvious
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through the regulation of sleep/wakefulness cycle. But in reality, most behaviors and physiological functions of the body are under the control of the circadian rhythms. Those functions include body temperature, secretion of hormones, autonomic functions, metabolisms,
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and so on. Importantly, not only mammals but also most of the living organisms on earth show circadian rhythms in their physiological functions (Dunlap, 1999). These circadian rhythms
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persist stably under the conditions without any external time cues, with periods which are species-specific and slightly different from 24 h (free-running period), providing an evidence
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that the organisms have biological clocks in their bodies and cells. The circadian system has been considered to evolve in order to anticipate and adjust the behaviors and internal
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physiological conditions to the environmental changes associated with day/night cycle. The suprachiasmatic nucleus (SCN) of the hypothalamus functions as the central
circadian clock in mammals to orchestrate multiple circadian biological rhythms in the organism (Figure 1A, B) (Hastings et al., 2018, 2003; Herzog et al., 2017). Because the
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endogenous period of the central clock slightly deviates from 24 h, the environmental light/dark cues adjust its period to just 24 h, conveyed directly from the eye to the SCN via the retinohypothalamic tract (RHT). The critical evidences supporting the role of SCN as the central circadian pacemaker include following findings (Weaver, 1998): Circadian rhythms were abolished by the lesion of SCN. Metabolic and electrophysiological activities in the SCN were rhythmic in vivo. Circadian rhythms of spontaneous firing rate of SCN neurons were sustained
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in vivo even when disconnected from the rest of the brain, as well as in slices. Moreover, circadian behavioral rhythms were restored by the grafting of fetal SCN into the brain of SCN-ablated arrhythmic animals. Importantly, the period of restored circadian rhythm was identical to that of the donor, rather than the host. These observations definitively demonstrated that the SCN is necessary and sufficient to sustain circadian behaviors. Intriguingly, electrical activity of the SCN is higher during the day in both diurnal and nocturnal animals, indicating that the SCN activity encodes solar time and the encoded information is transmitted through the
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efferent pathways differently between diurnal and nocturnal animals.
2. Molecular machinery of the circadian clock: cellular clock
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The SCN is composed of approximately 20,000 neurons (Figure 1). Most of them have
the ability to generate autonomous cellular circadian oscillations, including those of neuronal
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activity and gene expression (Hastings et al., 2018; Herzog et al., 2017). Such individual cellular clocks are driven by the autoregulatory transcriptional/translational feedback loops
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(TTFLs) of clock genes (Figure 1C) (Takahashi, 2017). Making a long story very short, heterodimers of two bHLH-type transcription factors, CLOCK (circadian locomotor output
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cycles protein kaput) and BMAL1 (brain and muscle ARNT-like 1, also known as ARNTL), act as the positive regulators of the loop to drive the transcription of the period (PER1 and PER2) and cryptochrome (CRY1 and CRY2) proteins, the negative regulators, in daytime through binding to E-box regulatory sequences. PER and CRY proteins then make complexes in the
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nucleus and repress their own expression. Subsequent degradation of the existing PER–CRY complexes reinitiates the cycle so that one cycle is approximately 24 h. Therefore, the lack of one of those components, CLOCK (and its homolog NPAS2), BMAL1, PER, or CRY disrupt the cycle, while alteration of the half-lives of PER or CRY proteins changes the cycle speed: stabilities of PER and CRY proteins are regulated in a complicated manner by phosphorylation via kinases including casein kinases, ubiquitination, and other post-translational modifications.
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To stabilize this main loop, there exist additional accessory loops including the nuclear receptors RORα, RORβ, REV-ERBα (also known as NR1D1) and REV-ERBβ (also known as NR1D2) whose expression is also driven by CLOCK/BMAL1, thereby interlinking the main and accessary TTFLs. In addition, oscillations of cytosolic signaling molecules, such as cAMP and Ca2+, further interact with the TTFLs to stabilize and refine the cellular (or molecular) clocks in individual cells (Hastings et al., 2008). TTFLs are likely to modulate the resting membrane potentials of SCN neurons to
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generate daily neuronal firing rhythms by regulating the expression of ion channels and their
modulators. TTX-resistant Na+ leak conductance and L-type and T-type Ca2+ conductances have
been implicated in day-time depolarization, whereas K+ conductance through BK channels seem
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3. Central clock vs. peripheral clocks
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to contribute nocturnal hyperpolarization (Herzog et al., 2017).
Discovery of clock genes uncovered a surprising fact that not only the SCN neurons
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but also most peripheral cells have such intracellular molecular processes of cellular circadian clocks (Balsalobre et al., 1998; Hastings et al., 2003). Thus, our body is full of peripheral
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circadian clocks throughout the peripheral organs as well as the brain. These peripheral clocks are likely to tick to regulate daily rhythms of local functions performed in the individual tissues and organs. Nevertheless, the importance of SCN as the central clock remains solid, ticking and sending the standard time throughout the entire body, according to the environmental day/night
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cycle, via neural, humoral, and other pathways to drive and/or entrain multiple peripheral clocks. Such “internal synchronization” orchestrates individual organs’ physiological functions in a temporally coordinated manner to optimize the performance as the whole-organism level. In that sense, our body includes a multi-oscillator system. Therefore, disturbance of such a temporal coordination, i.e. internal desynchronization, due to jet lag, shift work, irregular lifestyle, and so on, causes various health problems (Bass and Lazar, 2016).
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So, what is different between the central and peripheral circadian clocks? There are many studies showing that, in contrast to peripheral clocks, the circadian oscillations generated by the SCN central clock is highly stable even when isolated in the culture and strikingly resistant to genetic and environmental perturbations (Hastings et al., 2018; Herzog et al., 2017). It is the intercellular communications within the SCN neural network that differentiate the SCN from peripheral tissues to generate highly robust and coherent oscillations at the population level. Although individual dispersed neurons of the SCN have the ability to maintain
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cell-autonomous circadian oscillations of their firing rate, intracellular calcium concentration
([Ca2+]i) and clock gene expression, which are driven by their cellular clocks, these
cell-autonomous rhythms are fairly sloppy with a large variation of the period length. These are
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features shared with peripheral cells. By contrast, individual cells in SCN slice cultures, in
which its neural circuit connectivity is largely preserved, can maintain robust, stable, and
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precise circadian oscillations. Indeed, blocking interneuronal communications via action potentials by tetrodotoxin (TTX) application causes damping and gradual desynchronization of
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clock gene expression and [Ca2+]i rhythms of individual SCN neurons in neonatal SCN slice cultures (Enoki et al., 2012; Noguchi et al., 2017; Yamaguchi et al., 2003). Moreover,
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PER2::LUC (a bioluminescence reporter of clock gene Per2 expression (Yoo et al., 2004)) rhythms in adult SCN slices, which are intrinsically resistance to temperature entrainment, become sensitive to temperature changes in the presence of TTX, as peripheral tissues are (Buhr et al., 2010). Intriguingly, the individual cells’ oscillations are differentially phased so that
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activation of firing, clock gene expression and [Ca2+]i demonstrates stereotypically organized waves that move from dorsomedial to ventrolateral in the coronal SCN slices (Enoki et al., 2012; Welsh et al., 1995; Yamaguchi et al., 2003). Collectively, the central circadian clock of SCN is also a network of multiple neuronal oscillators.
4. Structure of the SCN
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The SCN is composed of approximately 20,000 -amino butyric acid (GABA)-ergic neurons (Figure 1A, B) (Antle and Silver, 2005; Moore et al., 2002). They include several types of neurons characterized by co-expression of neuropeptides, such as arginine vasopressin (AVP), vasoactive intestinal peptide (VIP), and gastrin releasing peptide (GRP). Anatomically, the SCN is divided into dorsal (shell) and ventral (core) subdivisions. The shell is represented by AVP-producing GABAergic neurons, whereas the core contains VIP-producing GABAergic
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neurons and GRP-producing GABAergic neurons. The SCN core neurons receives projections from the retina (i.e., RHT), respond immediately to the environmental light stimuli, and convey
the light information to the SCN shell (Shigeyoshi et al., 1997; Silver et al., 1996). On the other
hand, cells in the shell show rhythmic Period (Per) expression in constant darkness (DD) most
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prominently in the SCN (Hamada et al., 2004). The SCN core is innervated also by the median raphe and intergeniculate leaflet, while afferents from the hypothalamus and limbic areas
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terminate mainly in the SCN shell (Moga and Moore, 1997). Efferent fibers of SCN neurons terminate principally within the diencephalon, such as dorsomedial hypothalamic nucleus, hypothalamic
nucleus,
lateral
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paraventricular
hypothalamus,
and
especially
in
the
subparaventricular zone, the area just dorsal to the SCN. The efferent projections originates
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mostly in the shell, but the core also send projections in a manner different from the shell neurons (Leak and Moore, 2001). Within the SCN, core neurons send dense projections to the shell, whereas fibers of shell neurons in the core are sparse (Leak et al., 1999).
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5. Factors implicated in the intercellular communications in the SCN Here, I just introduce three factors that have been relatively well-studied. However, many neuropeptides are expressed in small populations of the SCN neurons and may have some contribution to the SCN network functions. The contribution of glutamate released from astrocyte will be described later.
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5.1 GABA Despite the fact that all SCN neurons contains GABA, its role in the central circadian clock remains controversial (Moore and Speh, 1993; Ono et al., 2018). GABA was initially reported to inhibit firing, phase-shift and synchronize the firing rhythms of dispersed SCN neurons through GABAA receptors (Liu and Reppert, 2000). By contrast, experiments using PER2::LUC reporter in the SCN slices suggested that GABAA receptor signaling instead opposes the synchrony of cellular clocks of individual SCN neurons (Aton et al., 2006; Freeman et al.,
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2013). Another study reported that GABAA signaling re-synchronizes the firing rhythms of the SCN shell and core after a transient phase-dissociation between these two regions caused by a
sudden 6-h phase delay in the light/dark cycle (Vansteensel et al., 2005). GABAA signaling also
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enhances the re-synchronization of the shell and core when PER2::LUC luminescence in the
two subdivisions are oscillating in anti-phasic configurations after mice are housed in an
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extremely long photoperiod, while it opposes the synchrony when these two oscillations are in phase with each other under the standard lighting condition (Evans et al., 2013). Because the
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SCN network is composed of multiple types of neurons, pharmacological approaches using GABAA receptor agonists and antagonists in the above-mentioned reports might complicate the
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interpretation of their results. In addition, it should be also taken into account that both inhibitory and excitatory effects of GABA on SCN neurons have been reported, which may differ according to the time of the day, photoperiod, or the region within the SCN (Albers et al.,
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2017; Farajnia et al., 2014; Ono et al., 2018; Vansteensel et al., 2005; Wagner et al., 1997).
5.2 VIP
VIP has been considered as a factor critical for the maintenance and synchronization of cellular clocks of the individual SCN neurons (Herzog et al., 2017). The absence of VIP signaling in mice, as in Vip- or VIP receptor Vipr2-deficient mice, substantially attenuated behavioral rhythms, often with multiple period components (Aton et al., 2005; Colwell et al., 2003; Harmar
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AJ et al., 2002). In the SCN slices of those mice, the number of neurons showing circadian oscillations of electrical firing and clock gene expression was reduced, and synchrony between still rhythmic neurons was drastically attenuated (Aton et al., 2005; Brown et al., 2007; Maywood et al., 2006). Daily application of VIP receptor agonist, as well as coculture with wildtype SCN grafts, restored the circadian rhythmicity to VIP-deficient SCN neurons (Aton et al., 2005; Maywood et al., 2011). Optogenetic stimulation of VIP neurons in the SCN phase-shifted cellular clocks
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(circadian PER2::LUC oscillations) ex vivo in a VIP-dependent manner and entrained the behavior rhythm in vivo (Jones et al., 2015; Mazuski et al., 2018), futher confirming the role of
VIP as the principle synchronizer of SCN neurons. Conversely, the light-induced phase-shift of
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circadian behavior rhythm was reduced by chemogenetic inhibition of SCN VIP neurons (Jones et al., 2018). In explants, prolonged chemogenetic stimulation of these neurons further
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reprograms the global spatiotemporal dynamics of the SCN cellular clocks (Brancaccio et al.,
5.3 AVP
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2013).
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The AVP level in the cerebrospinal fluid (CSF) has been long known to show circadian rhythm with a peak in the morning (Kalsbeek et al., 2010; Stark and Daniel, 1989). Such a daily fluctuation originates from the AVP content in the SCN (Södersten et al., 1985). Consistent with these observations, the transcription of Avp gene in the SCN is under the control of
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CLOCK/BMAL1, the positive regulators of TTFLs of cellular clocks (Jin et al., 1999; Mieda et al., 2015). Because AVP-deficient Brattleboro rats show reduced amplitude of circadian rhythms but little abnormality in circadian pacemaking, AVP has been considered to serve as an SCN output (Brown and Nunez, 1989; Groblewski et al., 1981; Kalsbeek et al., 2010). A recent optogenetic study directly demonstrated clock-driven AVP neurotransmission that mediates anticipatory thirst prior to sleep (Gizowski et al., 2016).
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Nevertheless, AVP may also play some role in the intercellular communications of SCN neurons. Mice lacking V1a receptor, the principal receptor for AVP in the SCN, demonstrated the lengthening in the activity time (the length of activity period) of circadian behavior rhythm, a sign of attenuated coupling of the SCN neurons (Li et al., 2009). By contrast, mice lacking both V1a and V1b, AVP receptors in the brain, showed normal circadian behavior rhythm, while they immediately reentrained to phase-shifted LD cycles (i.e., no jet lag!), suggesting that AVP-mediated intercellular communications confer a resistance to changes of
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the environmental light condition on the central clock of SCN (Yamaguchi et al., 2013). In
coculture experiments of SCN explants, the contribution of AVP signaling to the
synchronization of SCN neurons was uncovered when VIP signaling was eliminated (Maywood
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et al., 2011; Ono et al., 2016).
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6. Dissecting the network mechanism underlying the central circadian clock of the SCN Thus, SCN contains tens of thousands of cellular clocks, each of which has the
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molecular mechanisms to determine the amplitude, period, and the phase of its circadian oscillation driven by the TTFLs within individual cells. However, these cell-autonomous
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rhythms are fairly sloppy with a large variation of the period length (Herzog et al., 2017). Being connected via intercellular communications, how are the amplitude, period, and phase of the circadian rhythm determined at the SCN network level? Are those multiple cellular clocks all cell-autonomous and functionally equivalent? Or do different types of SCN neurons contribute
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differentially in the generation of circadian rhythms by the whole SCN? Is there a particular class of neurons that functions as the pacemaker to entrain the entire SCN?
6.1 Do all SCN neurons contribute equally to the circadian oscillation of the SCN network? Webb et al. used fully isolated SCN neurons in culture and found no evidence for a
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specialized or anatomically localized class of cell-autonomous pacemakers, concluding the SCN neurons are weak, inducible, and probabilistic oscillators (Webb et al., 2009). Low-Zeddies and Takahashi utilized a genetic approach to address the network mechanism of the circadian period determination of the SCN by generating and analyzing chimera mice of wildtype and long-period Clock19/19 mutant cells (Low-Zeddies and Takahashi, 2001). In the SCN of these mice, random subsets of wildtype SCN cells were
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replaced with Clock19/19 cells. The ratio of Clock19/19 to wildtype cells in the SCN largely determined circadian behavior in chimeric individuals. This result suggested that the ensemble
period of the SCN network is determined by simple averaging of periods of equally contributing individual SCN neurons. Nevertheless, some of balanced chimeras showed parameters of
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circadian rhythm diverged from the overall chimerism of the SCN, indicating the involvement
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contributions among SCN cells.
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of nonlinear mechanism to the determination of SCN ensemble period and therefore unequal
6.2 Neuron type-specific functions in the SCN network Although such a random chimera analysis was an elegant strategy, neuron
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type-specific genetic manipulations of cellular clocks might provide a different perspective (Table 1). Initially, two studies utilizing such an approach were reported for Neuromedin S (NMS, a neuropeptide) -producing neurons and AVP neurons, respectively (Lee et al., 2015; Mieda et al., 2015). NMS neurons constitute approximately 40% of SCN neurons and include
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VIP neurons, AVP neurons, and some other types of neurons. Lee et al. showed that lengthening period of NMS-cellular clocks by specific overexpression of Clock19 lengthened period of circadian behavioral rhythm and PER2::LUC rhythm of SCN explant as well (Table 1) (Lee et al., 2015). In addition, disruption of cellular clocks specifically in NMS neurons by deleting Bmal1 or overexpressing Per2 abolished the behavior rhythm. Furthermore, blocking synaptic transmission from NMS neurons also abolished the behavior rhythm. Based on these
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observations, the authors concluded that the NMS neurons define a subpopulation of pacemakers that control SCN network synchrony and in vivo circadian rhythms through intercellular synaptic transmission. Intriguingly, NMS peptide is dispensable for the pacemaker function of NMS neurons, implicating that other transmitters mediate NMS cellular function. By contrast, similar genetic manipulation to lengthen cellular period specifically in VIP neurons did not alter the behavior free-running period (Lee et al., 2015). Therefore, VIP neurons may not contribute significantly to the pacemaking of the SCN network, although VIP is a critical
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synchronizer for SCN neurons as described previously.
Similarly, Mieda et al. found that AVP neuron-specific disruption of cellular clocks by
Bmal1 deletion drastically attenuated circadian behavior rhythm, resulting in the unstable,
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lengthened free-running period and activity time (the length of activity period) (Figure 2, Table 1) (Mieda et al., 2015). A small number of those mice were completely arrhythmic, but most of
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the mice retained certain circadian rhythmicity. Therefore, the extent of circadian impairment was weaker than that of NMS neuron-specific manipulations. Additional behavioral and clock
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gene expression analyses suggested that cellular clocks of AVP neurons may modulate the coupling of the SCN network by regulating expression of multiple factors implicated in
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intercellular communications. Because the lack of AVP signaling in the SCN has not been reported to cause large defects in circadian rhythms, those function of AVP neurons may be mediated by transmitters expressed in these neurons but other than AVP, such as GABA and prokineticin 2 (Mieda et al., 2015). Furthermore, lengthening cellular circadian period
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specifically in AVP neurons by deleting casein kinase 1 (CK1) was sufficient to lengthen behavior circadian period whereas shortening cellular period in these cells by overexpressing CK11shortened behavior period as well, suggesting AVP neurons function as at least a part of pacemaker cells in the SCN network (Figure 2, Table 1) (Mieda et al., 2016). Because the extent of period lengthening by CK1 deletion specific to AVP neurons (~50 min) was comparable to that of pan-SCN CK1 deletion (Table 1) (van der Vinne et al., 2018), AVP neurons may be the
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principal determinant of circadian period generated by the SCN network in vivo. Smyllie et al. manipulated dopamine 1a receptor-expressing (Drd1a) cells in CK1εTau/Tau mice, which has short free-running period (~20 h) due to the floxed CK1εTau alleles that encode a mutant CK1ε that destabilizes PER proteins and thereby accelerates cellular clocks (Table 1) (Smyllie et al., 2016). Cre expressed in Drd1a cells excised floxed CK1εTau alleles and thereby made these cells CK1ε-/- that has an intrinsic cellular period of ~24 h, while non-Drd1a cells remained to be CK1εTau/Tau with a period of ~20 h. Intriguingly, 60% of such
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chimeric mice showed 24 h periods of behavior and SCN PER2::LUC rhythms like CK1ε-/- mice (i.e., revertants), whereas 33% showed 20 h periods as in CK1εTau/Tau mice (i.e., non-revertants).
Approximately 63% of all SCN cells were Drd1a-positive, and 62% of AVP neurons and 81% of
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VIP neurons were also Drd1a-positive (Smyllie et al., 2016). The fact that the behavioral period
did not always follow the cellular period of 80% of VIP neurons is in accordance with the
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earlier finding by Lee et al. that lengthening the period of cellular clocks in VIP neurons caused little effect on the behavioral free-running period (Lee et al., 2015). Considering data indicating
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AVP neurons as pacemaker cells reported by Mieda et al., the variation in the ratio of 24 h AVP neurons to 20 h AVP neurons within Drd1a cells might result in a bimodal distribution of
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behavior periods in Smyllie et al. Regardless of period length, the chimeric SCN sustained a wave of PER2::LUC bioluminescence comparable to that of nonchimeric SCN (Smyllie et al., 2016). This made a clear contrast to AVP neuron-specific lengthening of cellular period in which the shell-core phase relationship in the SCN was altered, although such an alteration did
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not impair the generation per se of circadian behavior rhythm (Mieda et al., 2016). Therefore, although AVP neurons may be sufficient to set the free-running behavior period, they may need to work in concert with other types of Drd1a-positive neurons to completely reorganize the entire SCN network. Taken together, studies of neuron type-specific genetic manipulations described above all argued against the equal contributions of individual cellular clocks in the SCN and
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implicated some functional differences between neuron types.
6.3 Important contribution of astrocytic clocks Recently, unexpected contributions of astrocytes in the SCN circadian pacemaking were reported (Table 1) (Barca-Mayo et al., 2017; Brancaccio et al., 2017; Tso et al., 2017). [Ca2+]i imaging in SCN organotypic slice cultures by Brancaccio et al. demonstrated that astrocytes and neurons are likely to act as two arms of the central circadian pacemaker network,
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which shows oscillations anti-phasic to each other (Brancaccio et al., 2017). These neuronal and
astrocytic clocks are likely to be coupled via glutamate released from astrocytes that increases presynaptic GABA release and subsequently suppresses activity of postsynaptic neurons
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assumedly during night. Deleting floxed CK1εTau alleles specifically in SCN astrocytes was
sufficient to reverse behavior free-running period of CK1εTau/Tau mice from 20 h to 24 h,
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suggesting that SCN astrocytes are able to control the period of circadian behavior rhythms. On the other hand, neuron-specific deletion of CK1εTau alleles in the SCN also corrected
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free-running period of CK1εTau/Tau mice to 24 h. Therefore, both SCN astrocytes and neurons are equally able to impart timekeeping information to the rest of the body (Brancaccio et al., 2017).
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Note that astrocyte-specific CK1εTau deletion made 24 h astrocytic clocks and left 20 h neuronal clocks whereas neuron-specific CK1εTau deletion made 24 h neuronal clocks and left 20 h astrocytic clocks, but curiously these reversed temporal misalignments of the SCN caused a same outcome, i.e. behavior free-running period of 24 h. One explanation for such apparently
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contradicting observations may be that cellular clocks and the SCN network are optimized to work at 24 h and therefore would be advantaged in the chimeras over the 20 h cells, regardless of which cell type has been targeted (Brancaccio et al., 2017). Therefore, it would be very interesting to examine whether artificial deviation of the astrocytic cellular period from 24 h by Clock19 overexpression, CK1 deletion, or CK11 overexpression alters the free-running period of behavior rhythm as much as neuronal manipulations do.
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Brancaccio et al. further demonstrated that rhythmic behavior can be rescued in behaviorally arrhythmic Cry1-/-;Cry2-/- mice by restoring Cry1 expression specifically in astrocytes in the SCN (Brancaccio et al., 2019). Restoring clocks in SCN neurons can also restore the circadian rhythms in these mice. These observations suggested that cell-autonomous clocks of astrocytes, as well as of neurons, drives circadian behavior in mammals, although it should be noted that some weak and unstable pacemaker function may be still present even in
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the SCN of Cry1-/-;Cry2-/- mice (Ono et al., 2013).
7. Concluding remarks
Understanding of intracellular molecular machinery of circadian clocks has been greatly
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advanced, as the Nobel prize in physiology or medicine was awarded in 2017 to three pioneer
researchers in this field. However, understanding of the network mechanisms of the SCN, the
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mammalian central circadian clock, have only just begun. The intracellular TTFLs in themselves are undoubtedly insufficient to explain the unique nature of the generation of
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circadian rhythms by the SCN, which is robust and stable, but can be efficiently entrained by external light conveyed from the eye in a time-of-day-dependent manner. Although many
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studies of anatomical, molecular, and electrophysiological architectures have been reported since the identification of the SCN as the central circadian clock in 1972 (Moore and Eichler, 1972; Stephan and Zucker, 1972), they still remain far from the complete descriptions. With the aid of more detailed information about gene expression profiling, connection patterns, and
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electrophysiological characteristics of individual neuron types of the SCN, combination of neuron type-specific genetic manipulations, in vivo recordings of neuronal activity and [Ca2+]i, and artificial manipulations of neural activity in vivo via optogenetics and chemogenetics would help further understand the SCN network mechanism.
Acknowledgement
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This work was supported by MEXT/JSPS KAKENHI Grant Numbers JP18H04941,
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JP18H04972, 19H03399.
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Figure Legends Figure 1. The central circadian clock of the SCN and molecular machinery of the circadian clock in the individual cells. (A) Schematic diagram of the SCN. Almost all SCN neurons are GABAergic. Some populations of these neurons coexpress neuropeptides. Light input is conveyed from the eye first to the core via retinohypothalamic tract (RHT). 3v, third ventricle; oc, optic chiasm. (B) A coronal section of mouse SCN, showing the dorsal shell region delineated by the expression of tdTomato fluorescent protein in AVP neurons (red) and the
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ventral core region containing VIP neurons labeled immunofluorescently (green). (C) A simplified schematic of cellular clock, the molecular machinery of circadian clock composed of TTFLs of clock genes. For the detailed mechanism, please see other reviews, such as Takahashi,
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2017. Transcription factors CLOCK and BMAL1 bind to E-box sequences to drive expression
of PER and CRY proteins, which in turn suppress CLOCK-BMAL1 activity, closing a negative
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feedback loop. CLOCK/BMAL1 also drive expression of ROR and REV-ERB nuclear receptors, which in turn regulate expression of BMAL1 via REV response elements (RRE), forming
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additional feedback loop. Phosphorylation of PER proteins by CK1 enhances those proteins’
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degradation and consequently accelerates the speed of cellular clocks.
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Figure 2. Representative actograms of circadian behavior rhythms in mice with AVP
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neuron-specific genetic manipulations. The free-running period is lengthened in both Avp-Bmal1-/- and Avp-CK1-/- mice, while the activity time was lengthened only in Avp-Bmal1-/-
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mice. Modified from Mieda et al. (2015, 2016).
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Table
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Table 1. Effects of neuron type-specific manipulations of cellular circadian clocks on the free-running circadian behavior rhythms.
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NMS neurons
NMS neurons
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Behavi or Genetic periods manipulatio in n control s
~60 min longer period
Arrhythm ic
Arrhythm ic
Control mice
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NMS neurons
AVP neurons, VIP neurons, other neurons AVP neurons, VIP neurons, other neurons AVP neurons, VIP neurons, other neurons
Changes in circadia n behavior rhythm
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Manipulat ed cells
Compositi on of manipulat ed cells
Crossed mice/ Injected AAV vectors
Names in original papers
Reference s
~24 h
Clock19 overexpressi on
Rosa26-LSL-tT A ;TRE-Clock19 *
Nms-iCre mice
Nms-Clock19
Lee et al., 2015
~24 h
Bmal1 deletion
Bmal1flox/flox
Nms-iCre mice
Nms-Bmal1fl/fl
Lee et al., 2015
~24 h
Per2 overexpressi on
Rosa26-LSL-tT A ;TRE-Per2 *
Nms-iCre mice
Nms-Per2
Lee et al., 2015
28
~50 min longer period ~5 h longer activity time ~50 min longer period ~40 min longer period
AVP neurons
AVP neurons
AVP neurons
GABA neurons
Most SCN neurons
Drd1a neurons
62% of AVP neurons, 81% of VIP neurons, other neurons
~4 h longer period in 60%, no change in 33%
SCN neurons
~4 h longer period
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SCN neurons
Bmal1 deletion
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~24 h
Rosa26-LSL-tT A ;TRE-Clock19 *
Vip-ires-Cre mice
Vip-Clock19
Lee et al., 2015
Bmal1flox/flox
Avp-iCre mice
Avp-Bmal1-/-
Mieda et al, 2015
CK1 deletion
CK1flox/flox
Avp-iCre mice
Avp-CK1-/-
Mieda et al, 2016
~24 h
CK1 deletion
CK1flox/flox
Vgat-ires-Cre mice
Vgat-Cre+ CK1fl/fl
van der Vinne et al., 2018
~20 h
CK1Tau deletion
CK1flox-Tau/floxTau #
Drd1a-Cre mice
DCR+/CK1Tau/Tau
Smyllie et al., 2016
~20 h
CK1Tau deletion
CK1flox-Tau/flox-
Syn-mCherry::Cr e AAV
CK1Tau/Tau + Syn-mCherry::Cr e
Brancacci o et al., 2017
~24 h
al P
AVP neurons
~24 h
Clock19 overexpressi on
ro
no change
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VIP neurons
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VIP neurons
Tau
29
of
~20 h
CK1Tau deletion
CK1flox-Tau/flox-
~24 h
Bmal1 deletion
Bmal1flox/flox
~24 h
Bmal1 deletion
~22 h
~22 h
GFAP-mCherry:: Cre AAV
-p
ro
Tau
Rosa26-LSL-C as9 *
CK1Tau deletion
CK1wildtype/flox-
CK1Tau deletion
CK1wildtype/flox-
al P
SCN astrocytes
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~4 h longer period ~10 min Astrocyte Astrocytes longer period ~30 min SCN SCN longer astrocytes astrocytes period ~70 min Astrocyte Astrocytes longer period ~50 min SCN SCN longer astrocytes astrocytes period * LSL: loxP-STOP-loxP cassette SCN astrocytes
Tau
Tau
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# also contains a Rosa26-LSL-EYFP allele
30
CK1Tau/Tau + GFAP-mCherry:: Cre
Brancacci o et al., 2017 Barca-Ma yo et al, 2017
Glast-CreERT2 mice
Bmal1 cKO
Aldh1L1-Cre mice & sgBmal1 AAV
Aldh1L1-Bmal1-/-
Tso et al, 2017
Aldh1L1-Cre mice
Aldh1L1-CK1tau/
Tso et al, 2017
GFAP-Cre::GFP AAV
CK1tau/+ + AAV8-GFAP-Cre
+
Tso et al, 2017
PII:
S0168-0102(19)30470-5
DOI:
https://doi.org/10.1016/j.neures.2019.08.003
Reference:
NSR 4304
To appear in:
Neuroscience Research
Received Date:
22 July 2019
Accepted Date:
9 August 2019
Please cite this article as: Mieda M, The central circadian clock of the suprachiasmatic nucleus as an ensemble of multiple oscillatory neurons, Neuroscience Research (2019), doi: https://doi.org/10.1016/j.neures.2019.08.003
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The central circadian clock of the suprachiasmatic nucleus as an ensemble of multiple oscillatory neurons
Michihiro Mieda
Department of Integrative Neurophysiology, Graduate School of Medical Sciences, Kanazawa
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University, 13-1 Takara-machi, Kanazawa, Ishikawa 920-8640, Japan.
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Highlights
The suprachiasmatic nucleus (SCN) functions as the central circadian pacemaker.
The SCN is a network composed of multiple types of GABAergic neurons and glial
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cells.
Intercellular communications are essential for the circadian pacemaking of the SCN.
Multiple types of neurons play differential roles in the SCN network.
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Abstract Circadian rhythms are oscillations with approximately 24-h period that appear in most of physiological events in our body. The suprachiasmatic nucleus (SCN) of the hypothalamus functions as the central circadian pacemaker in mammals and entrains to the environmental light/dark cycle. The SCN is a network structure composed of multiple types of -amino butyric
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acid (GABA)-ergic neurons and glial cells. Although individual SCN neurons have intracellular molecular machinery of circadian clock and the ability to oscillate cell-autonomously, interneuronal communications among these neurons are essential for the circadian pacemaking
of the SCN. However, the mechanisms underlying the SCN network remain largely unknown.
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Here, I briefly review the molecular, cellular, and anatomical structures of the SCN and
introduce recent studies aiming to understand the differential roles of multiple neuropeptides
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Keywords
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and neuropeptide-expressing neurons in the SCN network.
Circadian rhythm, suprachiasmatic nucleus, clock gene, GABA, vasopressin, VIP, neural
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network
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1. The central circadian clock in mammals: the suprachiasmatic nucleus (SCN) Many physiological functions in our body exhibit oscillations with frequencies ranging widely from milliseconds to days, e.g., brain electrical activity, heart rate, breathing, sleep/wakefulness cycle, circadian rhythm, and the estrous cycle. Circadian (the Latin circa, meaning “around,” and diem, meaning “day”) rhythms are oscillations that have an approximately 24-h period, which dominate our lives (Hastings et al., 2003). It is most obvious
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through the regulation of sleep/wakefulness cycle. But in reality, most behaviors and physiological functions of the body are under the control of the circadian rhythms. Those functions include body temperature, secretion of hormones, autonomic functions, metabolisms,
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and so on. Importantly, not only mammals but also most of the living organisms on earth show circadian rhythms in their physiological functions (Dunlap, 1999). These circadian rhythms
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persist stably under the conditions without any external time cues, with periods which are species-specific and slightly different from 24 h (free-running period), providing an evidence
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that the organisms have biological clocks in their bodies and cells. The circadian system has been considered to evolve in order to anticipate and adjust the behaviors and internal
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physiological conditions to the environmental changes associated with day/night cycle. The suprachiasmatic nucleus (SCN) of the hypothalamus functions as the central
circadian clock in mammals to orchestrate multiple circadian biological rhythms in the organism (Figure 1A, B) (Hastings et al., 2018, 2003; Herzog et al., 2017). Because the
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endogenous period of the central clock slightly deviates from 24 h, the environmental light/dark cues adjust its period to just 24 h, conveyed directly from the eye to the SCN via the retinohypothalamic tract (RHT). The critical evidences supporting the role of SCN as the central circadian pacemaker include following findings (Weaver, 1998): Circadian rhythms were abolished by the lesion of SCN. Metabolic and electrophysiological activities in the SCN were rhythmic in vivo. Circadian rhythms of spontaneous firing rate of SCN neurons were sustained
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in vivo even when disconnected from the rest of the brain, as well as in slices. Moreover, circadian behavioral rhythms were restored by the grafting of fetal SCN into the brain of SCN-ablated arrhythmic animals. Importantly, the period of restored circadian rhythm was identical to that of the donor, rather than the host. These observations definitively demonstrated that the SCN is necessary and sufficient to sustain circadian behaviors. Intriguingly, electrical activity of the SCN is higher during the day in both diurnal and nocturnal animals, indicating that the SCN activity encodes solar time and the encoded information is transmitted through the
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efferent pathways differently between diurnal and nocturnal animals.
2. Molecular machinery of the circadian clock: cellular clock
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The SCN is composed of approximately 20,000 neurons (Figure 1). Most of them have
the ability to generate autonomous cellular circadian oscillations, including those of neuronal
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activity and gene expression (Hastings et al., 2018; Herzog et al., 2017). Such individual cellular clocks are driven by the autoregulatory transcriptional/translational feedback loops
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(TTFLs) of clock genes (Figure 1C) (Takahashi, 2017). Making a long story very short, heterodimers of two bHLH-type transcription factors, CLOCK (circadian locomotor output
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cycles protein kaput) and BMAL1 (brain and muscle ARNT-like 1, also known as ARNTL), act as the positive regulators of the loop to drive the transcription of the period (PER1 and PER2) and cryptochrome (CRY1 and CRY2) proteins, the negative regulators, in daytime through binding to E-box regulatory sequences. PER and CRY proteins then make complexes in the
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nucleus and repress their own expression. Subsequent degradation of the existing PER–CRY complexes reinitiates the cycle so that one cycle is approximately 24 h. Therefore, the lack of one of those components, CLOCK (and its homolog NPAS2), BMAL1, PER, or CRY disrupt the cycle, while alteration of the half-lives of PER or CRY proteins changes the cycle speed: stabilities of PER and CRY proteins are regulated in a complicated manner by phosphorylation via kinases including casein kinases, ubiquitination, and other post-translational modifications.
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To stabilize this main loop, there exist additional accessory loops including the nuclear receptors RORα, RORβ, REV-ERBα (also known as NR1D1) and REV-ERBβ (also known as NR1D2) whose expression is also driven by CLOCK/BMAL1, thereby interlinking the main and accessary TTFLs. In addition, oscillations of cytosolic signaling molecules, such as cAMP and Ca2+, further interact with the TTFLs to stabilize and refine the cellular (or molecular) clocks in individual cells (Hastings et al., 2008). TTFLs are likely to modulate the resting membrane potentials of SCN neurons to
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generate daily neuronal firing rhythms by regulating the expression of ion channels and their
modulators. TTX-resistant Na+ leak conductance and L-type and T-type Ca2+ conductances have
been implicated in day-time depolarization, whereas K+ conductance through BK channels seem
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3. Central clock vs. peripheral clocks
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to contribute nocturnal hyperpolarization (Herzog et al., 2017).
Discovery of clock genes uncovered a surprising fact that not only the SCN neurons
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but also most peripheral cells have such intracellular molecular processes of cellular circadian clocks (Balsalobre et al., 1998; Hastings et al., 2003). Thus, our body is full of peripheral
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circadian clocks throughout the peripheral organs as well as the brain. These peripheral clocks are likely to tick to regulate daily rhythms of local functions performed in the individual tissues and organs. Nevertheless, the importance of SCN as the central clock remains solid, ticking and sending the standard time throughout the entire body, according to the environmental day/night
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cycle, via neural, humoral, and other pathways to drive and/or entrain multiple peripheral clocks. Such “internal synchronization” orchestrates individual organs’ physiological functions in a temporally coordinated manner to optimize the performance as the whole-organism level. In that sense, our body includes a multi-oscillator system. Therefore, disturbance of such a temporal coordination, i.e. internal desynchronization, due to jet lag, shift work, irregular lifestyle, and so on, causes various health problems (Bass and Lazar, 2016).
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So, what is different between the central and peripheral circadian clocks? There are many studies showing that, in contrast to peripheral clocks, the circadian oscillations generated by the SCN central clock is highly stable even when isolated in the culture and strikingly resistant to genetic and environmental perturbations (Hastings et al., 2018; Herzog et al., 2017). It is the intercellular communications within the SCN neural network that differentiate the SCN from peripheral tissues to generate highly robust and coherent oscillations at the population level. Although individual dispersed neurons of the SCN have the ability to maintain
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cell-autonomous circadian oscillations of their firing rate, intracellular calcium concentration
([Ca2+]i) and clock gene expression, which are driven by their cellular clocks, these
cell-autonomous rhythms are fairly sloppy with a large variation of the period length. These are
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features shared with peripheral cells. By contrast, individual cells in SCN slice cultures, in
which its neural circuit connectivity is largely preserved, can maintain robust, stable, and
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precise circadian oscillations. Indeed, blocking interneuronal communications via action potentials by tetrodotoxin (TTX) application causes damping and gradual desynchronization of
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clock gene expression and [Ca2+]i rhythms of individual SCN neurons in neonatal SCN slice cultures (Enoki et al., 2012; Noguchi et al., 2017; Yamaguchi et al., 2003). Moreover,
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PER2::LUC (a bioluminescence reporter of clock gene Per2 expression (Yoo et al., 2004)) rhythms in adult SCN slices, which are intrinsically resistance to temperature entrainment, become sensitive to temperature changes in the presence of TTX, as peripheral tissues are (Buhr et al., 2010). Intriguingly, the individual cells’ oscillations are differentially phased so that
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activation of firing, clock gene expression and [Ca2+]i demonstrates stereotypically organized waves that move from dorsomedial to ventrolateral in the coronal SCN slices (Enoki et al., 2012; Welsh et al., 1995; Yamaguchi et al., 2003). Collectively, the central circadian clock of SCN is also a network of multiple neuronal oscillators.
4. Structure of the SCN
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The SCN is composed of approximately 20,000 -amino butyric acid (GABA)-ergic neurons (Figure 1A, B) (Antle and Silver, 2005; Moore et al., 2002). They include several types of neurons characterized by co-expression of neuropeptides, such as arginine vasopressin (AVP), vasoactive intestinal peptide (VIP), and gastrin releasing peptide (GRP). Anatomically, the SCN is divided into dorsal (shell) and ventral (core) subdivisions. The shell is represented by AVP-producing GABAergic neurons, whereas the core contains VIP-producing GABAergic
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neurons and GRP-producing GABAergic neurons. The SCN core neurons receives projections from the retina (i.e., RHT), respond immediately to the environmental light stimuli, and convey
the light information to the SCN shell (Shigeyoshi et al., 1997; Silver et al., 1996). On the other
hand, cells in the shell show rhythmic Period (Per) expression in constant darkness (DD) most
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prominently in the SCN (Hamada et al., 2004). The SCN core is innervated also by the median raphe and intergeniculate leaflet, while afferents from the hypothalamus and limbic areas
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terminate mainly in the SCN shell (Moga and Moore, 1997). Efferent fibers of SCN neurons terminate principally within the diencephalon, such as dorsomedial hypothalamic nucleus, hypothalamic
nucleus,
lateral
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paraventricular
hypothalamus,
and
especially
in
the
subparaventricular zone, the area just dorsal to the SCN. The efferent projections originates
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mostly in the shell, but the core also send projections in a manner different from the shell neurons (Leak and Moore, 2001). Within the SCN, core neurons send dense projections to the shell, whereas fibers of shell neurons in the core are sparse (Leak et al., 1999).
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5. Factors implicated in the intercellular communications in the SCN Here, I just introduce three factors that have been relatively well-studied. However, many neuropeptides are expressed in small populations of the SCN neurons and may have some contribution to the SCN network functions. The contribution of glutamate released from astrocyte will be described later.
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5.1 GABA Despite the fact that all SCN neurons contains GABA, its role in the central circadian clock remains controversial (Moore and Speh, 1993; Ono et al., 2018). GABA was initially reported to inhibit firing, phase-shift and synchronize the firing rhythms of dispersed SCN neurons through GABAA receptors (Liu and Reppert, 2000). By contrast, experiments using PER2::LUC reporter in the SCN slices suggested that GABAA receptor signaling instead opposes the synchrony of cellular clocks of individual SCN neurons (Aton et al., 2006; Freeman et al.,
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2013). Another study reported that GABAA signaling re-synchronizes the firing rhythms of the SCN shell and core after a transient phase-dissociation between these two regions caused by a
sudden 6-h phase delay in the light/dark cycle (Vansteensel et al., 2005). GABAA signaling also
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enhances the re-synchronization of the shell and core when PER2::LUC luminescence in the
two subdivisions are oscillating in anti-phasic configurations after mice are housed in an
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extremely long photoperiod, while it opposes the synchrony when these two oscillations are in phase with each other under the standard lighting condition (Evans et al., 2013). Because the
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SCN network is composed of multiple types of neurons, pharmacological approaches using GABAA receptor agonists and antagonists in the above-mentioned reports might complicate the
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interpretation of their results. In addition, it should be also taken into account that both inhibitory and excitatory effects of GABA on SCN neurons have been reported, which may differ according to the time of the day, photoperiod, or the region within the SCN (Albers et al.,
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2017; Farajnia et al., 2014; Ono et al., 2018; Vansteensel et al., 2005; Wagner et al., 1997).
5.2 VIP
VIP has been considered as a factor critical for the maintenance and synchronization of cellular clocks of the individual SCN neurons (Herzog et al., 2017). The absence of VIP signaling in mice, as in Vip- or VIP receptor Vipr2-deficient mice, substantially attenuated behavioral rhythms, often with multiple period components (Aton et al., 2005; Colwell et al., 2003; Harmar
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AJ et al., 2002). In the SCN slices of those mice, the number of neurons showing circadian oscillations of electrical firing and clock gene expression was reduced, and synchrony between still rhythmic neurons was drastically attenuated (Aton et al., 2005; Brown et al., 2007; Maywood et al., 2006). Daily application of VIP receptor agonist, as well as coculture with wildtype SCN grafts, restored the circadian rhythmicity to VIP-deficient SCN neurons (Aton et al., 2005; Maywood et al., 2011). Optogenetic stimulation of VIP neurons in the SCN phase-shifted cellular clocks
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(circadian PER2::LUC oscillations) ex vivo in a VIP-dependent manner and entrained the behavior rhythm in vivo (Jones et al., 2015; Mazuski et al., 2018), futher confirming the role of
VIP as the principle synchronizer of SCN neurons. Conversely, the light-induced phase-shift of
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circadian behavior rhythm was reduced by chemogenetic inhibition of SCN VIP neurons (Jones et al., 2018). In explants, prolonged chemogenetic stimulation of these neurons further
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reprograms the global spatiotemporal dynamics of the SCN cellular clocks (Brancaccio et al.,
5.3 AVP
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2013).
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The AVP level in the cerebrospinal fluid (CSF) has been long known to show circadian rhythm with a peak in the morning (Kalsbeek et al., 2010; Stark and Daniel, 1989). Such a daily fluctuation originates from the AVP content in the SCN (Södersten et al., 1985). Consistent with these observations, the transcription of Avp gene in the SCN is under the control of
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CLOCK/BMAL1, the positive regulators of TTFLs of cellular clocks (Jin et al., 1999; Mieda et al., 2015). Because AVP-deficient Brattleboro rats show reduced amplitude of circadian rhythms but little abnormality in circadian pacemaking, AVP has been considered to serve as an SCN output (Brown and Nunez, 1989; Groblewski et al., 1981; Kalsbeek et al., 2010). A recent optogenetic study directly demonstrated clock-driven AVP neurotransmission that mediates anticipatory thirst prior to sleep (Gizowski et al., 2016).
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Nevertheless, AVP may also play some role in the intercellular communications of SCN neurons. Mice lacking V1a receptor, the principal receptor for AVP in the SCN, demonstrated the lengthening in the activity time (the length of activity period) of circadian behavior rhythm, a sign of attenuated coupling of the SCN neurons (Li et al., 2009). By contrast, mice lacking both V1a and V1b, AVP receptors in the brain, showed normal circadian behavior rhythm, while they immediately reentrained to phase-shifted LD cycles (i.e., no jet lag!), suggesting that AVP-mediated intercellular communications confer a resistance to changes of
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the environmental light condition on the central clock of SCN (Yamaguchi et al., 2013). In
coculture experiments of SCN explants, the contribution of AVP signaling to the
synchronization of SCN neurons was uncovered when VIP signaling was eliminated (Maywood
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et al., 2011; Ono et al., 2016).
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6. Dissecting the network mechanism underlying the central circadian clock of the SCN Thus, SCN contains tens of thousands of cellular clocks, each of which has the
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molecular mechanisms to determine the amplitude, period, and the phase of its circadian oscillation driven by the TTFLs within individual cells. However, these cell-autonomous
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rhythms are fairly sloppy with a large variation of the period length (Herzog et al., 2017). Being connected via intercellular communications, how are the amplitude, period, and phase of the circadian rhythm determined at the SCN network level? Are those multiple cellular clocks all cell-autonomous and functionally equivalent? Or do different types of SCN neurons contribute
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differentially in the generation of circadian rhythms by the whole SCN? Is there a particular class of neurons that functions as the pacemaker to entrain the entire SCN?
6.1 Do all SCN neurons contribute equally to the circadian oscillation of the SCN network? Webb et al. used fully isolated SCN neurons in culture and found no evidence for a
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specialized or anatomically localized class of cell-autonomous pacemakers, concluding the SCN neurons are weak, inducible, and probabilistic oscillators (Webb et al., 2009). Low-Zeddies and Takahashi utilized a genetic approach to address the network mechanism of the circadian period determination of the SCN by generating and analyzing chimera mice of wildtype and long-period Clock19/19 mutant cells (Low-Zeddies and Takahashi, 2001). In the SCN of these mice, random subsets of wildtype SCN cells were
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replaced with Clock19/19 cells. The ratio of Clock19/19 to wildtype cells in the SCN largely determined circadian behavior in chimeric individuals. This result suggested that the ensemble
period of the SCN network is determined by simple averaging of periods of equally contributing individual SCN neurons. Nevertheless, some of balanced chimeras showed parameters of
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circadian rhythm diverged from the overall chimerism of the SCN, indicating the involvement
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contributions among SCN cells.
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of nonlinear mechanism to the determination of SCN ensemble period and therefore unequal
6.2 Neuron type-specific functions in the SCN network Although such a random chimera analysis was an elegant strategy, neuron
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type-specific genetic manipulations of cellular clocks might provide a different perspective (Table 1). Initially, two studies utilizing such an approach were reported for Neuromedin S (NMS, a neuropeptide) -producing neurons and AVP neurons, respectively (Lee et al., 2015; Mieda et al., 2015). NMS neurons constitute approximately 40% of SCN neurons and include
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VIP neurons, AVP neurons, and some other types of neurons. Lee et al. showed that lengthening period of NMS-cellular clocks by specific overexpression of Clock19 lengthened period of circadian behavioral rhythm and PER2::LUC rhythm of SCN explant as well (Table 1) (Lee et al., 2015). In addition, disruption of cellular clocks specifically in NMS neurons by deleting Bmal1 or overexpressing Per2 abolished the behavior rhythm. Furthermore, blocking synaptic transmission from NMS neurons also abolished the behavior rhythm. Based on these
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observations, the authors concluded that the NMS neurons define a subpopulation of pacemakers that control SCN network synchrony and in vivo circadian rhythms through intercellular synaptic transmission. Intriguingly, NMS peptide is dispensable for the pacemaker function of NMS neurons, implicating that other transmitters mediate NMS cellular function. By contrast, similar genetic manipulation to lengthen cellular period specifically in VIP neurons did not alter the behavior free-running period (Lee et al., 2015). Therefore, VIP neurons may not contribute significantly to the pacemaking of the SCN network, although VIP is a critical
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synchronizer for SCN neurons as described previously.
Similarly, Mieda et al. found that AVP neuron-specific disruption of cellular clocks by
Bmal1 deletion drastically attenuated circadian behavior rhythm, resulting in the unstable,
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lengthened free-running period and activity time (the length of activity period) (Figure 2, Table 1) (Mieda et al., 2015). A small number of those mice were completely arrhythmic, but most of
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the mice retained certain circadian rhythmicity. Therefore, the extent of circadian impairment was weaker than that of NMS neuron-specific manipulations. Additional behavioral and clock
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gene expression analyses suggested that cellular clocks of AVP neurons may modulate the coupling of the SCN network by regulating expression of multiple factors implicated in
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intercellular communications. Because the lack of AVP signaling in the SCN has not been reported to cause large defects in circadian rhythms, those function of AVP neurons may be mediated by transmitters expressed in these neurons but other than AVP, such as GABA and prokineticin 2 (Mieda et al., 2015). Furthermore, lengthening cellular circadian period
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specifically in AVP neurons by deleting casein kinase 1 (CK1) was sufficient to lengthen behavior circadian period whereas shortening cellular period in these cells by overexpressing CK11shortened behavior period as well, suggesting AVP neurons function as at least a part of pacemaker cells in the SCN network (Figure 2, Table 1) (Mieda et al., 2016). Because the extent of period lengthening by CK1 deletion specific to AVP neurons (~50 min) was comparable to that of pan-SCN CK1 deletion (Table 1) (van der Vinne et al., 2018), AVP neurons may be the
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principal determinant of circadian period generated by the SCN network in vivo. Smyllie et al. manipulated dopamine 1a receptor-expressing (Drd1a) cells in CK1εTau/Tau mice, which has short free-running period (~20 h) due to the floxed CK1εTau alleles that encode a mutant CK1ε that destabilizes PER proteins and thereby accelerates cellular clocks (Table 1) (Smyllie et al., 2016). Cre expressed in Drd1a cells excised floxed CK1εTau alleles and thereby made these cells CK1ε-/- that has an intrinsic cellular period of ~24 h, while non-Drd1a cells remained to be CK1εTau/Tau with a period of ~20 h. Intriguingly, 60% of such
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chimeric mice showed 24 h periods of behavior and SCN PER2::LUC rhythms like CK1ε-/- mice (i.e., revertants), whereas 33% showed 20 h periods as in CK1εTau/Tau mice (i.e., non-revertants).
Approximately 63% of all SCN cells were Drd1a-positive, and 62% of AVP neurons and 81% of
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VIP neurons were also Drd1a-positive (Smyllie et al., 2016). The fact that the behavioral period
did not always follow the cellular period of 80% of VIP neurons is in accordance with the
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earlier finding by Lee et al. that lengthening the period of cellular clocks in VIP neurons caused little effect on the behavioral free-running period (Lee et al., 2015). Considering data indicating
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AVP neurons as pacemaker cells reported by Mieda et al., the variation in the ratio of 24 h AVP neurons to 20 h AVP neurons within Drd1a cells might result in a bimodal distribution of
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behavior periods in Smyllie et al. Regardless of period length, the chimeric SCN sustained a wave of PER2::LUC bioluminescence comparable to that of nonchimeric SCN (Smyllie et al., 2016). This made a clear contrast to AVP neuron-specific lengthening of cellular period in which the shell-core phase relationship in the SCN was altered, although such an alteration did
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not impair the generation per se of circadian behavior rhythm (Mieda et al., 2016). Therefore, although AVP neurons may be sufficient to set the free-running behavior period, they may need to work in concert with other types of Drd1a-positive neurons to completely reorganize the entire SCN network. Taken together, studies of neuron type-specific genetic manipulations described above all argued against the equal contributions of individual cellular clocks in the SCN and
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implicated some functional differences between neuron types.
6.3 Important contribution of astrocytic clocks Recently, unexpected contributions of astrocytes in the SCN circadian pacemaking were reported (Table 1) (Barca-Mayo et al., 2017; Brancaccio et al., 2017; Tso et al., 2017). [Ca2+]i imaging in SCN organotypic slice cultures by Brancaccio et al. demonstrated that astrocytes and neurons are likely to act as two arms of the central circadian pacemaker network,
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which shows oscillations anti-phasic to each other (Brancaccio et al., 2017). These neuronal and
astrocytic clocks are likely to be coupled via glutamate released from astrocytes that increases presynaptic GABA release and subsequently suppresses activity of postsynaptic neurons
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assumedly during night. Deleting floxed CK1εTau alleles specifically in SCN astrocytes was
sufficient to reverse behavior free-running period of CK1εTau/Tau mice from 20 h to 24 h,
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suggesting that SCN astrocytes are able to control the period of circadian behavior rhythms. On the other hand, neuron-specific deletion of CK1εTau alleles in the SCN also corrected
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free-running period of CK1εTau/Tau mice to 24 h. Therefore, both SCN astrocytes and neurons are equally able to impart timekeeping information to the rest of the body (Brancaccio et al., 2017).
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Note that astrocyte-specific CK1εTau deletion made 24 h astrocytic clocks and left 20 h neuronal clocks whereas neuron-specific CK1εTau deletion made 24 h neuronal clocks and left 20 h astrocytic clocks, but curiously these reversed temporal misalignments of the SCN caused a same outcome, i.e. behavior free-running period of 24 h. One explanation for such apparently
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contradicting observations may be that cellular clocks and the SCN network are optimized to work at 24 h and therefore would be advantaged in the chimeras over the 20 h cells, regardless of which cell type has been targeted (Brancaccio et al., 2017). Therefore, it would be very interesting to examine whether artificial deviation of the astrocytic cellular period from 24 h by Clock19 overexpression, CK1 deletion, or CK11 overexpression alters the free-running period of behavior rhythm as much as neuronal manipulations do.
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Brancaccio et al. further demonstrated that rhythmic behavior can be rescued in behaviorally arrhythmic Cry1-/-;Cry2-/- mice by restoring Cry1 expression specifically in astrocytes in the SCN (Brancaccio et al., 2019). Restoring clocks in SCN neurons can also restore the circadian rhythms in these mice. These observations suggested that cell-autonomous clocks of astrocytes, as well as of neurons, drives circadian behavior in mammals, although it should be noted that some weak and unstable pacemaker function may be still present even in
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the SCN of Cry1-/-;Cry2-/- mice (Ono et al., 2013).
7. Concluding remarks
Understanding of intracellular molecular machinery of circadian clocks has been greatly
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advanced, as the Nobel prize in physiology or medicine was awarded in 2017 to three pioneer
researchers in this field. However, understanding of the network mechanisms of the SCN, the
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mammalian central circadian clock, have only just begun. The intracellular TTFLs in themselves are undoubtedly insufficient to explain the unique nature of the generation of
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circadian rhythms by the SCN, which is robust and stable, but can be efficiently entrained by external light conveyed from the eye in a time-of-day-dependent manner. Although many
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studies of anatomical, molecular, and electrophysiological architectures have been reported since the identification of the SCN as the central circadian clock in 1972 (Moore and Eichler, 1972; Stephan and Zucker, 1972), they still remain far from the complete descriptions. With the aid of more detailed information about gene expression profiling, connection patterns, and
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electrophysiological characteristics of individual neuron types of the SCN, combination of neuron type-specific genetic manipulations, in vivo recordings of neuronal activity and [Ca2+]i, and artificial manipulations of neural activity in vivo via optogenetics and chemogenetics would help further understand the SCN network mechanism.
Acknowledgement
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This work was supported by MEXT/JSPS KAKENHI Grant Numbers JP18H04941,
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JP18H04972, 19H03399.
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Figure Legends Figure 1. The central circadian clock of the SCN and molecular machinery of the circadian clock in the individual cells. (A) Schematic diagram of the SCN. Almost all SCN neurons are GABAergic. Some populations of these neurons coexpress neuropeptides. Light input is conveyed from the eye first to the core via retinohypothalamic tract (RHT). 3v, third ventricle; oc, optic chiasm. (B) A coronal section of mouse SCN, showing the dorsal shell region delineated by the expression of tdTomato fluorescent protein in AVP neurons (red) and the
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ventral core region containing VIP neurons labeled immunofluorescently (green). (C) A simplified schematic of cellular clock, the molecular machinery of circadian clock composed of TTFLs of clock genes. For the detailed mechanism, please see other reviews, such as Takahashi,
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2017. Transcription factors CLOCK and BMAL1 bind to E-box sequences to drive expression
of PER and CRY proteins, which in turn suppress CLOCK-BMAL1 activity, closing a negative
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feedback loop. CLOCK/BMAL1 also drive expression of ROR and REV-ERB nuclear receptors, which in turn regulate expression of BMAL1 via REV response elements (RRE), forming
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additional feedback loop. Phosphorylation of PER proteins by CK1 enhances those proteins’
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degradation and consequently accelerates the speed of cellular clocks.
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Figure 2. Representative actograms of circadian behavior rhythms in mice with AVP
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neuron-specific genetic manipulations. The free-running period is lengthened in both Avp-Bmal1-/- and Avp-CK1-/- mice, while the activity time was lengthened only in Avp-Bmal1-/-
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mice. Modified from Mieda et al. (2015, 2016).
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Table
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Table 1. Effects of neuron type-specific manipulations of cellular circadian clocks on the free-running circadian behavior rhythms.
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NMS neurons
NMS neurons
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Behavi or Genetic periods manipulatio in n control s
~60 min longer period
Arrhythm ic
Arrhythm ic
Control mice
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al P
NMS neurons
AVP neurons, VIP neurons, other neurons AVP neurons, VIP neurons, other neurons AVP neurons, VIP neurons, other neurons
Changes in circadia n behavior rhythm
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Manipulat ed cells
Compositi on of manipulat ed cells
Crossed mice/ Injected AAV vectors
Names in original papers
Reference s
~24 h
Clock19 overexpressi on
Rosa26-LSL-tT A ;TRE-Clock19 *
Nms-iCre mice
Nms-Clock19
Lee et al., 2015
~24 h
Bmal1 deletion
Bmal1flox/flox
Nms-iCre mice
Nms-Bmal1fl/fl
Lee et al., 2015
~24 h
Per2 overexpressi on
Rosa26-LSL-tT A ;TRE-Per2 *
Nms-iCre mice
Nms-Per2
Lee et al., 2015
28
~50 min longer period ~5 h longer activity time ~50 min longer period ~40 min longer period
AVP neurons
AVP neurons
AVP neurons
GABA neurons
Most SCN neurons
Drd1a neurons
62% of AVP neurons, 81% of VIP neurons, other neurons
~4 h longer period in 60%, no change in 33%
SCN neurons
~4 h longer period
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SCN neurons
Bmal1 deletion
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~24 h
Rosa26-LSL-tT A ;TRE-Clock19 *
Vip-ires-Cre mice
Vip-Clock19
Lee et al., 2015
Bmal1flox/flox
Avp-iCre mice
Avp-Bmal1-/-
Mieda et al, 2015
CK1 deletion
CK1flox/flox
Avp-iCre mice
Avp-CK1-/-
Mieda et al, 2016
~24 h
CK1 deletion
CK1flox/flox
Vgat-ires-Cre mice
Vgat-Cre+ CK1fl/fl
van der Vinne et al., 2018
~20 h
CK1Tau deletion
CK1flox-Tau/floxTau #
Drd1a-Cre mice
DCR+/CK1Tau/Tau
Smyllie et al., 2016
~20 h
CK1Tau deletion
CK1flox-Tau/flox-
Syn-mCherry::Cr e AAV
CK1Tau/Tau + Syn-mCherry::Cr e
Brancacci o et al., 2017
~24 h
al P
AVP neurons
~24 h
Clock19 overexpressi on
ro
no change
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VIP neurons
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VIP neurons
Tau
29
of
~20 h
CK1Tau deletion
CK1flox-Tau/flox-
~24 h
Bmal1 deletion
Bmal1flox/flox
~24 h
Bmal1 deletion
~22 h
~22 h
GFAP-mCherry:: Cre AAV
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ro
Tau
Rosa26-LSL-C as9 *
CK1Tau deletion
CK1wildtype/flox-
CK1Tau deletion
CK1wildtype/flox-
al P
SCN astrocytes
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~4 h longer period ~10 min Astrocyte Astrocytes longer period ~30 min SCN SCN longer astrocytes astrocytes period ~70 min Astrocyte Astrocytes longer period ~50 min SCN SCN longer astrocytes astrocytes period * LSL: loxP-STOP-loxP cassette SCN astrocytes
Tau
Tau
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# also contains a Rosa26-LSL-EYFP allele
30
CK1Tau/Tau + GFAP-mCherry:: Cre
Brancacci o et al., 2017 Barca-Ma yo et al, 2017
Glast-CreERT2 mice
Bmal1 cKO
Aldh1L1-Cre mice & sgBmal1 AAV
Aldh1L1-Bmal1-/-
Tso et al, 2017
Aldh1L1-Cre mice
Aldh1L1-CK1tau/
Tso et al, 2017
GFAP-Cre::GFP AAV
CK1tau/+ + AAV8-GFAP-Cre
+
Tso et al, 2017