TYPE Review
PUBLISHED

22 December 2022

DOI 10.3389/fnsys.2022.1059421

OPEN ACCESS

The role of calcium and CaMKII
in sleep

EDITED BY

Nirinjini Naidoo,
University of Pennsylvania,
United States
REVIEWED BY

Mark Wu,
Johns Hopkins University,
United States
Moitrayee Bhattacharyya,
Yale University, United States

Yuyang Wang1† , Yoichi Minami1† , Koji L. Ode1† andHiroki R. Ueda1,2*
1

Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo,
Tokyo, Japan, 2 Laboratory for Synthetic Biology, RIKEN Center for Biosystems Dynamics Research,
Suita, Japan*CORRESPONDENCE

Hiroki R. Ueda
uedah-tky@umin.ac.jp† These authors have contributed

equally to this work and share firstauthorship
RECEIVED 01 October 2022
ACCEPTED 05 December 2022
PUBLISHED 22 December 2022
CITATION

Wang Y, Minami Y, Ode KL and
Ueda HR (2022) The role of calcium
and CaMKII in sleep.
Front. Syst. Neurosci. 16:1059421.
doi: 10.3389/fnsys.2022.1059421
COPYRIGHT© 2022 Wang, Minami, Ode and Ueda.
This is an open-access article
distributed under the terms of theCreative Commons Attribution License(CC BY). The use, distribution or
reproduction in other forums ispermitted, provided the original
author(s) and the copyright owner(s)are credited and that the original
publication in this journal is cited, in
accordance with accepted academic
practice. No use, distribution or
reproduction is permitted which does
not comply with these terms.

Sleep is an evolutionarily conserved phenotype shared by most of the animals
on the planet. Prolonged wakefulness will result in increased sleep needor sleep pressure. However, its mechanisms remain elusive. Recent findings
indicate that Ca2+ signaling, known to control diverse physiological functions,also regulates sleep. This review intends to summarize research advancesin Ca2+ and Ca2+ /calmodulin-dependent protein kinase II (CaMKII) in sleep
regulation. Significant changes in sleep phenotype have been observed
through calcium-related channels, receptors, and pumps. Mathematicalmodeling for neuronal firing patterns during NREM sleep suggests that thesemolecules compose a Ca2+ -dependent hyperpolarization mechanism. The
intracellular Ca2+ may then trigger sleep induction and maintenance throughthe activation of CaMKII, one of the sleep-promoting kinases. CaMKII and its
multisite phosphorylation status may provide a link between transient calcium
dynamics typically observed in neurons and sleep-wake dynamics observedon the long-time scale.
KEYWORDS

NREM sleep, calcium, CaMKII, phosphorylation, sleep-promoting kinase, Ca2+ dependent hyperpolarization

1 Introduction
Until the mid-20th century, it was not widely accepted that a simple inorganic ion,
calcium (Ca2+ ), could regulate such important and diverse physiological functions,serving as a messenger for regulating specific inter- and intra-cellular signaling. Dr.
Setsuro Ebashi (1922–2006) pioneered the demonstration that Ca2+ is important formuscle contraction, opening the door for research on Ca2+ as a signaling molecule inthe body (Endo, 2011). To date, Ca2+ is well recognized as one of the most commonlyused cellular signaling molecules, acting in signal transduction via membrane proteins[e.g., N-methyl-D-aspartate receptors (NMDARs), voltage-gated calcium channels(VGCCs)] or intra-cellular proteins such as protein kinase PKC and CaMKII. Ca2+
plays an important role in signal transduction pathways, acting as second messenger inneurotransmitter release from neurons (Berridge, 1998), in the contraction of the muscle(Baylor and Hollingworth, 2011), and in fertilization (Kashir et al., 2013).

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10.3389/fnsys.2022.1059421(Nath et al., 2017; Kanaya et al., 2020). The rate that jellyfish
twitch their tentacles decreases during the night, and theanimals’ response to external stimuli slows down during thisperiod (Nath et al., 2017). Zebrafish showed high activity
during the day, and sleep-like behaviors such as inactivity andcaudal fin drooping were observed at night (Yokogawa et al.,2007). These small translucent fish contribute to sleep studies;
they have the advantage of having a central nervous system(CNS) organization similar to that of mammals. C. elegans
also shows sleep-like behavior of reduced responsiveness andreversibility during lethargus period (Raizen et al., 2008).
Drosophila melanogaster also participated in sleep studies withsimilar characteristics as mammals (Huber et al., 2004b). The
required sleep varies between species, ranging from giraffes andhorses that sleep only 3 h to bats that sleep 18 h throughout theday (Siegel, 2005).

Calcium is associated with various temporal dynamics inlife. In neurons, calcium changes neuronal activity on the orderof milliseconds. Calcium waves play an important role in egg
fertilization on the order of seconds (Kashir et al., 2013). In
cardiomyocytes, Ca2+ is involved in the oscillation mechanism
in heartbeats on the order of seconds (Eisner et al., 2017).
Calcium is important in synaptic plasticity and it contributes tolearning and memory formation on the order of hours (MateosAparicio and Rodriguez-Moreno, 2020). Intracellular Ca2+ has
diurnal variations in the suprachiasmatic nucleus (SCN) of theclock center (Enoki et al., 2012), and it is suggested that Ca2+
is important in the formation of circadian rhythms that create adaily activity rhythmicity in the body.
Of note, sleep is an evolutionarily conserved phenotype
shared by most of the animals with nervous systems on theplanet (Joiner, 2016). In humans, recent studies on sleep
dysfunction have linked it to many psychiatric disorders(Anderson et al., 2005; Soehner et al., 2013; Reeve et al.,2015), including schizophrenia (Chan et al., 2017; Manoachand Stickgold, 2019) and bipolar disease (McKenna andEyler, 2012). The molecular mechanisms of mammalian sleep
regulation are still largely unknown. Recent mice genetic
studies have given us more insight into genes and moleculesthat regulate animals’ sleep behavior. For example, a forward
genetic screening in randomly mutagenized mice identified
novel sleep-regulating genes in mammals (Funato et al., 2016).
We also conducted reverse genetics approaches to identify sleepregulating genes including Ca2+ /calmodulin-dependent protein
kinase II (CaMKII) and other genes controlling intracellular
Ca2+ dynamics (Tatsuki et al., 2016). This review describes
the forms of action and physiological functions of calcium and
CaMKII in the different dimensions of sleep control as well as
challenges of current research and future possibilities.

2.2 Sleep at the brain level
In mammals, the sleep-wake state can be referred toas peculiar brainwave patterns (Zielinski et al., 2016). Brainrhythms, which refer to the massed neuronal activity that canbe monitored from the surface of the scalp typically by using
electroencephalogram (EEG) (Figure 1B). During wakefulness,
cortical neurons are hardly synchronized, presenting mainly
high frequency and low amplitude beta waves (15–30 Hz) andgamma waves (30–90 Hz). Mammalian sleep can be divided into
rapid eye movement (REM) state and non-rapid eye movement(NREM) state. NREM sleep is characterized by low frequency,
large amplitude EEG rhythms presumably originated from thesynchronous firing of cortical neurons. In humans, NREM sleep
can be further classified according to the brain waves, including
theta waves (3–7 Hz) in stage 1 followed by K-complex (<1 Hz)
in stage 2. Sleep spindles (8–14 Hz) are also observed in stage
2, occasionally grouped with K-complex. Stage 3 in NREM sleep
are clearly defined delta waves (1–4 Hz) and slow waves (<1 Hz)(Patel et al., 2022). On the other hand, EEG of REM sleep is more
like wakefulness, with high frequency and low amplitude EEG
patterns. Note that the presence of NREM and/or REM sleep is
not limited to mammals but it is widely conserved across theanimal kingdom (Tobler and Borbély, 1988; Yokogawa et al.,2007; Shein-Idelson et al., 2016).
Electroencephalogram slow wave activity (0.5–4 Hz, SWA)
during NREM correlates with the expected levels of sleep needthat is well explained by using two process model (Borbély et al.,1981). Process C or the circadian process, controls the timingof sleep and wakefulness during the day-night cycle, whereas
Process S, or the sleep-wake homeostasis, represents the sleep
need, which increases in proportion to the quantity and qualityof the preceding awake state and decreases in the subsequent
sleep state. Although the two-process model supposes Process C
and Process S to act independently, the circadian clock and sleep

2 Sleep in various biological layers
2.1 Sleep at the behavioral level
Sleep seems to be a phenomenon with ancient evolutionary
roots. Sleep is a conserved phenotype within animal species(Joiner, 2016). All mammals with nervous systems, birds, andreptiles seem to sleep. Of all, except humans, mice remain
the most commonly used model for sleep studies (Figure 1A).
Experiments corresponding to sleep studies in mice are wellestablished, e.g., knockout (KO) mice models for specific sleep
genes. Sleep could be characterized by reduced movement,
altered consciousness, and decreased responsiveness to external
stimuli (Zielinski et al., 2016). Since this is a behavioral
definition, it can be applied to animals other than mammals–
fish and invertebrates also show sleep-like states. The jellyfish
Cassiopea and Hydra Vulgaris are some of the most primitive
organisms for which sleep-like behavior has been observed

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FIGURE 1

Sleep in various biological layers. (A) Mice and human sleep patterns. Mice are nocturnal animals and show polyphasic sleep, whereas humans
are diurnal animals and show monophasic sleep. (B) Typical EEG/EMG recordings of wake, REM sleep, and NREM sleep in the adult human.
(C) Schematic diagram of a microelectrode array (MEA) that can measure neuronal firing in vitro. The “wake-like (desynchronized)” and“sleep-like (synchronized)” patterns were observed depending on the culture conditions.

needs may have close interactions at the cellular and molecularlevels (Krueger, 2020). About the circadian clock systems, seeprevious reviews (Takahashi, 2017; Partch, 2020).

have also found that the animals appear to be awake with eyes
open, and their EEG is wake-like, with only a subset of corticalneurons temporarily going offline and firing like slow wave sleep(SWS) (Huber et al., 2006; Vyazovskiy et al., 2011). In contrast,
all the cortical neurons have large and slow rhythms with typicalsleep EEG patterns, and the animals are relatively unresponsive
in real sleep. They also discovered that prolonged wakefulness
would increase these temporary off periods and was found to becorrelated with worsened task performance in rats (Vyazovskiy
et al., 2011). Interestingly, dolphins, whales, and some birds
were found to sleep only one single cerebral hemisphere at atime so that they can stay alert in the demanding environment(Mukhametov et al., 1985; Lyamin et al., 2004, 2017). Someanimals, including humans, also show microsleep (MS), which is

2.3 Local sleep in the cortical areas
Sleep and wakefulness may not be absolutely segregated but
may be controlled heterogeneously among the cortical area. Partof the brain forced to awake for prolonged amounts of time
may take a “local sleep” in a use-dependent manner (Krueger
et al., 2008). Indeed, the level of SWA in the cortex can beheterogeneous depending on the level of neural activity during
the preceding awake period (Huber et al., 2006). Researchers

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bursting and downstate without bursting repeatedly appears(Steriade et al., 1993; Steriade et al., 2001). The frequency
of up-down state switching is well matched with the slower(<1 Hz) component of SWA, while the 1–4 Hz component may
be originated from thalamic and cortical oscillations (Amzica
and Steriade, 1998). Such up-down state oscillation associatedwith sleep need is also observed in Drosophila melanogaster,
implying that the oscillation is a conserved sleep feature acrossphyla (Raccuglia et al., 2019). The synchronous up-down stateoscillation appears in the cortical cell population without inputs
from the other brain area (Hinard et al., 2012).
SNAP25 (synaptosomal-associated protein 25kDa) is at-SNARE protein responsible for synaptic vesicle fusion.
Selective ablation of SNAP25 in neocortical layer V disrupts
calcium-related neurotransmitter release and silences theneurons, which significantly increases wakefulness and reduces
SWA rebound after sleep deprivation (Krone et al., 2021).
A subset of cortical GABAergic interneurons expressing
neuronal nitric oxide synthase (nNOS) might be involved in the
propagation of slow waves through the cortex, along with several
neuropeptides and neurochemicals participating (Schwartz andKilduff, 2015). More specifically, cortical neurons that coexpress nNOS and neurokinin-1 receptor (NK1R) have beenfound to play a critical role in sleep homeostasis, including
NREM sleep time and NREM bout duration (Morairty et al.,2013).
Studies have further found that synchronous neural firing,similar to what is observed in an animal’s intact brain during
sleep, occurs in isolated cortical neurons (Hinard et al., 2012;
Jewett et al., 2015; Colombi et al., 2016; Saberi-Moghadam et al.,2018; Nguyen et al., 2019). Electrical or chemical stimulationof cultured neurons leads to an asynchronous, wake-like firing
pattern, followed by a highly synchronous firing state that can beassociated with rebound sleep response after sleep deprivationin vivo. These studies suggest that the sleep homeostaticmechanism works even in isolated neurons (Figure 1C).

defined as temporary sleep or drowsiness that lasts for seconds.
Different from local sleep, individuals are unconscious and failto respond to the external stimuli in MS, showing behaviors
like nodding and droopy eyelids, etc. (Poudel et al., 2014). MS
often results from sleep deprivation or monotonous tasks and is
extremely dangerous if the job requires alertness, for example,
driving (Innes et al., 2010; Poudel et al., 2014).

2.4 Sleep in cortical neurons
The neural circuits that produce sleep are not yet fully
understood (Weber and Dan, 2016). Previously, people believedthat the ascending reticular activating system (ARAS) promotes
wakefulness while the ventrolateral pre-optic nucleus VLPO
promotes sleep (Saper et al., 2005). With the research efforts
done within these 15 years, however, it is still unknown whether
these circuits connecting brain nuclei play a central role incontrolling sleep need, as animals with disrupted ARAS stillrequire a similar amount of sleep per day (Denoyer et al., 1991;
Gerashchenko et al., 2004; Blanco-Centurion et al., 2007). In
recent years, there has been an increasing number of discoveriesof sleep-promoting and wake-promoting neurons (Weber andDan, 2016; Liu and Dan, 2019; Jacobson et al., 2022). The REM
and NREM sleep neurons constitute highly distributed networks
spanning the forebrain, midbrain, and hindbrain. Liu and Dan
proposed an arousal-action circuit for sleep-wake control inwhich wakefulness is supported by separate arousal and action
neurons, while REM and NREM sleep neurons are part of thecentral somatic and autonomic motor circuits (Liu and Dan,2019). Orexins (hypocretins) are peptide hormones produced
exclusively in the lateral hypothalamus. Orexin receptors arewidely expressed across the brain and participate in various
neurophysiological functions, including sleep and wake, reward,
fear, anxiety, and cognition, etc. And they are associated withenhanced wakefulness and activity (Jacobson et al., 2022).
Adamantidis et al. explain that there are three models of
sleep-wake states: the flip-flop “switch” model, the reciprocalinteraction model, and the thalamocortical loop model(Adamantidis et al., 2019). According to the former twomodels, sleep is defined as neural circuits. However, inthe thalamocortical loop model, sleep is initiated locally at
the neuronal levels. In this model, thalamic neurons have
intrinsic electrical properties that regulate sleep and are notjust a mediator that relays signals (Adamantidis et al., 2019).
Therefore, more evidence could possibly be provided with thestudy of cortical neurons.
Local and heterogenous SWA in the cortex implies thatcortical neurons contain essential properties, at least in part,for the homeostatic regulation of sleep. Synchronous firingof cortical neurons is thought to be the source of SWA in
NREM EEG. During NREM sleep, cortical neurons exhibit acharacteristic firing pattern, where the upstate with neuron

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2.5 Sleep on synapse
Homeostatic control or functions of sleep can be embedded
in synapses. Synaptic plasticity is defined as the ability ofneurons to respond to use or disuse by bringing about changes
in the connections between neuronal networks (Hughes, 1958).
Studies have found that molecules that relate to synaptic
plasticity are actively expressed in regions during wakefulness,and the expression levels were significantly decreased during
sleep (Cirelli and Tononi, 2000). Prolonged wakefulness was
accompanied by difficulty in inducing LTP-like plasticity(Kuhn et al., 2016). Motor-learning tasks could induce anincrease of SWA in subsequent sleep along with increasedtask performance (Huber et al., 2004a), while disruption of
SWA in the motor cortex inhibited performance improvement04

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10.3389/fnsys.2022.1059421(Fattinger et al., 2017). The exact relationship between SWA
and LTP is still under discussion. A pioneering idea called
the synaptic homeostasis hypothesis (SHY) of sleep proposesthat during awake, which is optimal for learning, synapses
are potentiated with increased synaptic strength. While asleep,slow-wave activity during NREM sleep induced downscaling
and renormalization to preserve the synaptic strength at aconserved level (Tononi and Cirelli, 2003). Some findings are
consistent with the model. In vivo recording also revealed
cortical evoked responses increase during awake and decreaseduring sleep (Vyazovskiy et al., 2008). The size of synapses alsochanged with altered synaptic strength. During sleep, especially
NREM sleep, is characterized by SWA, the size of synapse andthe number of localized AMPARs decreases (de Vivo et al.,2017; Diering et al., 2017), which resembles LTD (Vyazovskiy
et al., 2008). Meanwhile, there are some conflicting results.
Post-learning sleep was reported to be critical for forming
filopodia and spines in layer 5 pyramidal neurons in mice (Adler
et al., 2021). Also, sleep deprivation decreased dendritic spine
numbers in hippocampal CA1 but not in CA3 in mice (Havekes
et al., 2016; Raven et al., 2019). In short, some papers support the
SHY hypothesis that synaptic plasticity decreases during sleep,
while others are inconsistent with the hypothesis. Sun et al.
mentioned that changes in synaptic strength during sleep mightvary according to brain regions (Sun et al., 2020).

that the transition of the bursting phase and silent phase ismediated by some most common channels and receptors thatmediate the transportation of Ca2+ , Na+ , and K+ (Compte
et al., 2003; Hill and Tononi, 2005; Sanchez-Vives et al., 2010).
However, the exact channels/receptors that participated are notyet clear. Previously mentioned models are dependent on thegiven parameters and require large-scale calculations, which
is not suitable for the comprehensive exploration of possible
parameter sets or pathways that are involved. Since previous
studies have found that ex vivo cortical slices and even isolated
neurons can reproduce slow wave oscillations (Sanchez-Vives
and McCormick, 2000; Sanchez-Vives et al., 2010; Hinard et al.,2012; Colombi et al., 2016), inputs from other brain regions
may not be necessary for SWA (Ode et al., 2017). Also, studies
have found local sleep where some neurons in the cortex are
temporarily shut off (Funk et al., 2016). Therefore, a dedicatedneural network structure may not be required for the generationof cortical up-down state oscillation in SWS.

3.2 Calcium importance in sleep
highlighted by “average neuron” model
In 2016, Tatsuki et al. put this idea into practice (Tatsuki
et al., 2016). They first try to establish an “average neuron” using
mean-field approximation, where the neuron could interact
with itself directly or indirectly through excitatory or inhibitory
synaptic currents (extrinsic currents). Aside from the extrinsic
currents, intrinsic (non-synaptic) currents are also implemented
based on previous models (Timofeev et al., 2000; Bazhenov et al.,
2002; Hill and Tononi, 2005; Sanchez-Vives et al., 2010; Chenet al., 2012).
More than 10,000,000 random parameter sets weregenerated, and 1,113 were selected that show SWS-like firing
patterns. Bifurcation analysis was performed to understandwhich channels or pumps are of vital importance to theoscillation. Altogether, four candidates are proved to beimportant to generate SWS-like firing, which are NMDA
receptors, voltage-gated Ca2+ channels (Cav ), Ca2+ -dependent
potassium channels (KCa ), and Ca2+ pumps. These four classes
work in a calcium-dependent manner. In the up-state bursting
phase, Ca2+ goes into the cell through NMDAR. Calcium influx
depolarizes the membrane potential and further activates the
Cav . Accumulated Ca2+ activates the KCa , and K+ effluxes themembrane to decrease the membrane potential to suppress
the firing to go into the silent phase. Accumulated Ca2+ isexported through the Ca2+ pump. The suppression of calcium
entry disappeared; thus, another firing could be observed. This
Ca2+ -dependent hyperpolarization pathway predicts that the
impairment of the ion channels that allow calcium influx would
decrease sleep duration, while impairment of the Ca2+ pumpwill increase sleep duration by facilitating hyperpolarization

3 Calcium in sleep
3.1 Theoretical analysis of neuralactivity during NREM sleep
Computational and theoretical biology has been a powerfultool to understand the molecular mechanisms underlying thecortical up-down state oscillation observed in SWS. Several
models have been established to reconstruct SWS firing patterns(Timofeev et al., 2000; Hill and Tononi, 2005; Sanchez-Vives
et al., 2010; Chen et al., 2012). In these studies, scientists
attempt to reconstruct neuronal networks that include hundreds
to thousands of neurons that reproduce the behavior of thecerebral cortex and thalamus (Compte et al., 2003; Hill andTononi, 2005). The network model used by Compete et al.
consists of 1,024 pyramidal neurons and 256 interneurons
connected to each other by biologically rational synaptic
dynamics, which also achieved the reproduction of slow
wave oscillations (Compte et al., 2003). Hill and Tononi
constructed a large-scale computer model that included partsof two visual areas, as well as the associated thalamic andreticular thalamic nuclei (Hill and Tononi, 2005). Their model
assumed 28,880 model neurons in the primary visual area
(Vp) layer and designed various connections between themin a computer, which successfully reproduced the slow-wave
oscillations. Their studies share some interesting conclusions

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10.3389/fnsys.2022.1059421(Tatsuki et al., 2016). In this model, calcium accumulation iscorrelated with sleep duration (Figures 2A,B).
To explore the role of each gene involved in the Ca2+ dependent hyperpolarization pathway by generating geneknockout mice, we developed a CRISPR/Cas9-based highthroughput gene modification system called the triple-CRISPR
technique (Sunagawa et al., 2016). The technique allows

scientists to generate gene KO mice within 4 months.
Sleep phenotypes were monitored using the respiration-based
automated sleep phenotyping system Snappy Sleep Stager (SSS),
which allows sleep phenotyping on a large scale (Sunagawa
et al., 2016). Using these systems, we successfully disrupted
all 29 genes that were involved in the AN model (Sunagawa
et al., 2016; Tatsuki et al., 2016). KO mice with impaired

FIGURE 2
Ca2+ -dependent hyperpolarization pathway neuronal firing during NREM sleep. (A) Neurotransmitter receptors, ion channels, and Ca2+

pumps/exchangers, which are involved in the “averaged neuron” (AN) model (Tatsuki et al., 2016). This model simplifies the neuronal network to
average neurons by applying a rationale similar to the mean-field approximation. VGCC, voltage-gated calcium channels; VGSC, voltage-gatedsodium channels; VGPC, voltage-gated potassium channels; IRK, inwardly rectifying K+ channels. (B) Ca2+ influx evokes SWS-like oscillations
in silico. Ca2+ influx dominantly through Cav and NMDA receptors generates the bursting up-state in SWS-like oscillations, while K+ dominantly
efflux through the KCa generates the down-state in SWS-like oscillations (right). Inhibition of Ca2+ influx or acceleration of Ca2+ efflux leads toawake-like firing without a down state (left). (C) Gene knockout studies demonstrated that ion channels/pumps suggested by the AN model
play an important role in sleep regulations. Studies using mice also demonstrated that CaMKII α and β are important in the regulation of sleep
duration.

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levels, NMDA receptor expression, and structural markers ofsynaptic strength (Liu et al., 2016).
T-Type Ca2+ channels are one of the low-voltage activated.
They respond to small amplitudes and are inactivated while
the cell membrane is hyperpolarized and then activated during
depolarization. Ca2+ influx through the T-Type Ca2+ channels
induces low-threshold spikes, which triggers the burst firing
activity in corticothalamic neurons. T-Type Ca2+ channels
have been implicated in sleep-related brain rhythms and thequantity and quality of sleep. CaV 3.1 KO mice showed asignificant decrease in total sleep duration, resulting from adecrease in NREM sleep duration during the lighted period,
and also accompanied by fragmented NREM sleep by brief
wakefulness (Lee et al., 2004). Thalamic-specific CaV 3.1 KO
also displays this NREM sleep fragmentation, which indicatesthe essential role of the thalamic T-type Ca2+ channel inblocking the transmission of arousal signals and stabilizing
sleep (Anderson et al., 2005). An antagonist of the T-type
Ca2+ channel was reported to enhance sleep (Yang et al.,2010). The same group reported another T-type Ca2+ channel
antagonist, TTA-A2, in 2010, which suppresses active wakeand promotes SWS in wild-type mice but not in mice lacking
both Cav 3.1 and Cav 3.3 (Kraus et al., 2010). The T-Type Ca2+
channels are also linked to the brain waves associated withsleep. Cav 3.1 KO mice showed impaired delta oscillations,altered spindle oscillations, and intact slow oscillations (<1 Hz)(Lee et al., 2004). Meanwhile, Cav 3.3 mutant mice showedsignificantly decreased power density of spindle waves (Astori
et al., 2011). The results suggest that Cav 3.1 plays an essential
role in generating thalamocortical oscillations, and Cav 3.3 isresponsible for the spindle oscillations.
Another type of Ca2+ channel, Cacna1c encoding for
L-type voltage-dependent calcium channel, may be involved
in sleep regulation. Cacna1c is a risk gene for bipolar andschizophrenia, characterized by sleep disturbance (Ament et al.,2015; O’Connell et al., 2019). Genetic knockout of the geneis lethal (Tatsuki et al., 2016), and heterozygous knockout of
Cacna1c in mice induce reduced REM sleep recovery comparedwith their wild-type littermate (Kumar et al., 2015). A recent
study indicates that it also plays a role in sleep regulation. Several
Cacna1c variants are found to be associated with sleep latency(Kantojarvi et al., 2017).

KCa showed a decreased total sleep duration and also NREM
sleep (SWS) duration, with a lower transition probability froman awake state to sleep (Pws) . KCa KO mice also have sleep
homeostasis dysfunction after sleep deprivation (SD), withno significant SWS increase after SD. A study using local
cortical depletion of KCa channels showed that KCa channels areimportant for the induction of SWA in the local cortex (Muheim
et al., 2019). Then α1 subunits of Cav , which determine
the channel’s responsiveness to membrane depolarization, arealso knocked out, and KO of Cav 3.1 and Cav 3.2 showedsignificantly decreased sleep duration, which is consistent withthe previous findings (Lee et al., 2004; Anderson et al., 2005).
The NMDAR subfamilies include the GluN1 (Nr1) subunit, four
distinct GluN2 (Nr2) subunits, and two GluN3 (Nr3) subunits.
Nr3 KO mice exhibit a significant short-sleeper phenotype(Sunagawa et al., 2016). KO mice of Nr1 or Nr2b exhibit lethal
phenotypes (Forrest et al., 1994; Li et al., 1994) and were
blocked pharmacologically by NMDAR antagonist MK-801 or
phencyclidine (PCP). NMDAR perturbed mice showed a dosedependent decrease in total sleep duration and also NREM sleep(SWS) duration compared to the controls. Blockade of NMDAR
using MK-801 also increased neuronal excitability as measured
by whole-brain imaging (Tatsuki et al., 2016). On the other
hand, mice with impaired Ca2+ pumps decrease calcium effluxand increase sleep duration significantly. These results support
that the Ca2+ -dependent hyperpolarization pathway controls
the sleep duration in mice (Figure 2C).

3.3 Evidence of calcium importance insleep
Sleep researches other than mice have also highlighted
calcium’s importance in sleep. Drosophila melanogaster, or fruit
fly, has been widely used in biological research in the lastcentury owing to its simple genetics and fast reproduction
speed. Over the past decades, sleep studies on flies have
led to gradual findings on genes and neuronal circuits that
regulate sleep (Hendricks et al., 2000; Shaw et al., 2000;
Lee et al., 2004). In line with our study, knockdown of
the NMDA receptor gene (Nmdar1) or application of the
NMDAR antagonist, MK-801, reduced sleep in flies (Tomita
et al., 2015) which is consistent with the result in mice.
Ca2+ influx through the NMDAR activates Calcineurin,a heterodimeric Ca2+ /calmodulin-dependent serine/threonine
protein phosphatase. Knockdown of calcineurin catalytic
subunit A (CanA1), calcineurin regulatory subunit B (CanB),and calcineurin regulator sarah (sra) also results in a significant
reduction in sleep (Nakai et al., 2011; Tomita et al., 2015). These
results suggest that NMDAR-calcineurin signaling may play arole in sleep regulation. In 2016, Liu et al. found that elevatedsleep pressure triggered reversible increases in cytosolic Ca2+

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3.4 Calcium in glial transmission
Astrocytes, a subtype of glial cells that tile the entire brain,
may also play a key role in regulating sleep (Halassa et al., 2009;
Pelluru et al., 2016; Clasadonte et al., 2017). Glial transmission isa phenomenon that describes the regulation of neuronal activity
by astrocytes through the release of chemical transmitters.
Inhibition of glial transmission reduced the accumulation of
sleep stress and prevented the cognitive deficits associated with07

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sleep loss (Halassa et al., 2009). Two-photon calcium imaging of
neocortical astrocytes showed that an increase in calcium level inastrocytes precedes the spontaneous circuit shift to a slow-wave
oscillatory state (Poskanzer and Yuste, 2016). Direct stimulationof astrocytes using optogenetic tools induced sleep (Pelluru
et al., 2016) and switched the local neuronal circuit to this slowoscillation state by altering extracellular glutamate (Poskanzer
and Yuste, 2016). Recent studies have shown that astrocytic
channel IP3 -receptors (IP3 Rs), which predominantly control theCa2+ release from the endoplasmic reticulum (ER), are strongly
implicated in sleep regulation. Transgenic overexpression of
IP3 -5-phosphatase mice in astrocytes attenuated IP3-mediated
Ca2+ signaling in astrocytes and increased REM sleep andtheta power (Foley et al., 2017). These findings suggest that
IP3 /Ca2+ signaling via IP3 R is involved in the regulation of
sleep. Although previous studies have focused on IP3 R2, its
KO in mice does not affect sleep (Cao et al., 2013). The activerole of IP3-mediated Ca2+ signaling in astrocytes may also be
supported by the observation that a general anesthetic at a lower
concentration than inhibits neuronal responses is known tosuppress IP3-mediated astrocyte Ca2+ responses (Thrane et al.,2012). In Drosophila, scientists detected Ca2 + levels within
astrocytes vary with prolonged wake/sleep need using in vivo
and ex vivo imaging (Blum et al., 2021). And sleep need is furthertransmitted to sleep drive circuit by releasing an interleukin-1
analog to act on the Toll receptor and R5 neurons (Blum et al.,2021).
Different neuronal and glial states in the sleep-wake cycle
alter the extracellular ion environment, including Ca2+ (Dinget al., 2016). Extracellular K+ concentration is higher during thewake state, while extracellular Ca2+ and Mg2+ concentrations
are higher during sleep. Such ion composition changes are
not significantly affected by the inhibition of α-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA)dependent synaptic inputs, but anesthesia reproduces the sleeplike extracellular ion conditions. Furthermore, it has been
demonstrated that sleep and wakefulness can be induced byintroducing artificial cerebrospinal fluid (ACSF) into the brain,
which mimics the ionic environment during the sleep or wakephase, suggesting the causal role of extracellular ion conditions
for the induction of sleep and awake neuronal firing patterns.
Different extracellular ion conditions have been incorporated
into mathematical models based on the AN model (Rasmussen
et al., 2017). Mathematical analysis predicted that changes in
the K+ conductance of KCa channels in response to changes
in the extracellular ionic environment are important for theformation of slow-wave firing patterns. In addition, a largescale computer model that reconstructs the circuit structure ofcortical nerves predicts that an increase in extracellular Ca2+
concentration triggers synchronous firing of cortical nerves(Markram et al., 2015).
The extracellular ion concentrations are also altered between
UP and DOWN states. The extracellular Ca2+ concentration

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increases toward the end of the DOWN state and decreases in
the UP state (Massimini and Amzica, 2001). On the other hand,the extracellular K+ concentration increases in the UP state anddecreases in the DOWN state (Amzica and Massimini, 2002;
Amzica et al., 2002).

4 CaMKII in sleep
4.1 Phosphorylation hypothesis ofsleep
The role of the Ca2+ -dependent hyperpolarization pathway
in sleep implies that Ca2+ is also important for sleep serving as asignaling molecule inside the neurons, leading us to the findingof Ca2+ /calmodulin-dependent protein kinase II (CaMKII)
α and β as sleep-promoting kinases (Tatsuki et al., 2016).
Physiological post-translational regulation of sleep-controlling
protein appears to be associated with multiple kinase activities.
Genetic and biochemical studies further revealed other potent
sleep-promoting kinases, including salt-inducible kinase (SIK)
1/2/3 (Funato et al., 2016; Honda et al., 2018; Park et al., 2020),
extracellular signal-regulated kinase (ERK) (Mikhail et al., 2017)(Figure 3A).
Funato et al. performed a large-scale forward genetic
screening of more than 8,000 randomly mutagenized mice andidentified a Sleepy mutant (Funato et al., 2016). Sleepy mice
exhibited prolonged NREM sleep and increased NREM SWA.
This particular phenotype was shown to result from a gainof-function exon skipping of the salt-inducible kinase (SIK)
3. SIK3 is a member of the AMP-activated protein kinase(AMPK) family that is widely expressed in the brain. Of note,the exon skipping involves a phosphorylation site of SIK3, S551,
targeted by protein kinase A (PKA) (Funato et al., 2016). S551A
point mutation reproduced the NREM sleep promotion (Honda
et al., 2018; Xu et al., 2022). Although the S551D mutant may
not work for the phosphomimicking mutant and produced asimilar sleep promotion effect, these results at least indicatethat proper control of phosphorylation at S551 is importantto the SIK3-mediated sleep regulation. The role of SIK3 insleep promotion was conserved in Drosophila and C. elegans,indicating the conservation of sleep regulation in invertebrates(Funato et al., 2016).
Extracellular signal-regulated kinase (ERK) is another sleeppromoting kinase. The sleep-promoting activity of ERK was
suggested in a study with Drosophila, where researchers foundthat sleep deprivation increases ERK phosphorylation in wildtype flies (Vanderheyden et al., 2013). Pan-neuronal expressionof active ERK increased sleep, while ERK inhibitor reduced sleep(Vanderheyden et al., 2013). The role of ERK1/2 in the natural
sleep-wake cycle was then confirmed in mice by embryonic andconditional knockout experiments (Mikhail et al., 2017). Also,in vitro studies showed that wake-like stimulation of primary08

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FIGURE 3

Sleep promoting kinases. (A) Genetic studies revealed sleep-promoting kinases, including CaMKIIα/β, SIK3, and ERK1/2. (B) Phosphorylation
functions in many physiological phenomena. Environmental information and sleep-associated function could be integrated by sleep-promoting
kinases. (C) CaMKII is a dodecameric protein complex with a ring-like structure. When the kinase is inhibited, CaMKII has a compacted structure.
The compact inhibited state is under equilibrium with the open or extended active state. The active form is stabilized by the binding of theCa2+ /calmodulin (CaM) complex that allows for further autophosphorylation events. The residue number is based on CaMKIIβ. Details areshown in the text.

cortical cultures could induce similar changes in gene expression
as in sleep-deprived animals, including the overexpression ofseveral serine-threonine protein kinases that inhibit MAPKs in
ERK pathways (Hinard et al., 2012).
These and other kinases associated with sleep control
may markedly affect the global phosphorylation status inneurons. A phosphoproteomics study revealed that among
the quantified 4,500 proteins and ∼10,000 phosphopeptides
in the post-synaptic density (PSD), about 20% of PSD
proteins and phosphorylation sites showed changes in theexpression level and/or phosphorylation level between day
and night (Diering et al., 2017). Another phosphoproteomics
study quantitatively compares the phosphorylation profile
between mice with sleep deprivation and the Sleepy mutant
mice, both of which should have a higher sleep need andelevated SWA (Wang et al., 2018). The study revealed thesimilarity of the phosphoproteomics profile between the two
conditions against the untreated control mice. Furthermore,
the study annotates 80 phosphoproteins whose phosphorylationlevels are correlated with the expected amount of sleep

Frontiers in Systems Neuroscienceneed. The dominant role of the sleep-wake cycle for thephosphorylation level of synaptic proteins is further supportedby the observation that sleep-wake cycles induce daily rhythms
of many synaptic phosphoproteins, whereas sleep deprivation
abolishes almost all phosphorylation cycles (Bruning et al.,2019). These findings lead us to propose the idea of thephosphorylation hypothesis of sleep, which assumes that sleep
is induced by various kinases and such sleep-promoting kinases
connect various signals to sleep-related physiological functions(Figure 3B).

4.2 CaMKII
Ca2+ /calmodulin-dependent protein kinase II is widely
expressed in the brain and is also a major downstream kinaseof calcium signaling. It is known to interact with some of thecandidates in the Ca2+ -dependent hyperpolarization pathway.
For example, CaMKII can directly bind to the NMDA receptor
subunits NR1 and NR2B in response to Ca2+ influx and09

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participate in LTP and memory (Leonard et al., 1999; Rose et al.,2009; Tao et al., 2021).

The knockout and brain-wide inhibition of CaMKII
activity demonstrate that CaMKII is a sleep-promoting kinase.
However, we note that CaMKII has brain area-specific roles in
the control of sleep. In brainstem pedunculopontine tegmentum(PPT) neurons, the phosphorylation level of CaMKIIα T286 iscorrelated with increased wakefulness (Stack et al., 2010), but
the injection of CaMKII inhibitor KN-93 to the PPT decreased
wakefulness, suggesting that in pedunculopontine tegmentum(PPT or PPN) neurons CaMKII is associated with promoting
wakefulness (Datta et al., 2011; Urbano et al., 2014). Conversely,in the dorsal raphe nucleus (DRN), KN-93 microinjection
suppressed wakefulness and enhanced NREM sleep and REM
sleep (Cui et al., 2016). In the cortex and hippocampus in rats,
the phosphorylation status of Thr286 in CaMKII but not the
total protein level was also found to be higher in the waking
group compared to the sleeping group (Vyazovskiy et al., 2008).
Phosphorylation states of CaMKIIα and CaMKIIβ are
also affected by the sleep-wake cycle. Phosphoproteomics of
PSD protein and western blotting of synaptosome showedthat activated CaMKIIα with autophosphorylation at T286 ishigher in the dark (active) phase. Interestingly, CaMKIIα withphosphorylated T310 is lower in the dark phase (Vyazovskiy
et al., 2008; Diering et al., 2017), suggesting that CaMKIIαundergoes different phosphorylation states depending on thetime of day and/or sleep-wake states. Western blot results also
showed that CaMKIIα pT286 increases during wakefulness anddecreases during sleep (Bruning et al., 2019). The changes in
the phosphorylation of CaMKIIα T286 and CaMKIIβ T287
appeared to be correlated to the level of sleep need because
these phosphorylation levels were increased by sleep deprivation(Wang et al., 2018; Tone et al., 2022).
The role of the phosphorylation status of CaMKII in
sleep promotion is recently demonstrated by using AAVmediated expression of CaMKIIβ and its phosphomimetic
mutants (Tone et al., 2022). Through the comprehensive
sleep phenotyping of mice expressing CaMKIIβ mutants,
each of which had a phosphomimetic mutation at eitherof all serine or threonine residues, researchers found that
phosphomimetic mutation at T287 (T287D) induced theincrease in NREM sleep and SWA. This phenotype is opposite
to what is observed in Camk2b knockout mice, further
supporting the importance of CaMKII as a sleep-promoting
kinase. CaMKIIβ having multiple phosphomimetic mutationsat T306/T307 as well as T287 (T287D:T306D:T307D) also
increases NREM sleep. Interestingly, however, the mode ofsleep induction is qualitatively different between T287Dexpression and T287D:T306D:T307D-expression. CaMKIIβT287D expression increases the transition probability fromawake to sleep, namely, the mutant promotes the sleep induction
step. On the other hand, T287D:T306D:T307D-expression leads
to a decrease in the transition probability from sleep to awake.
Thus, this mutant promotes sleep maintenance (Tone et al.,2022). It has been suggested that phosphorylation of T305

4.2.1 Structural and functional properties of
CaMKII
Ca2+ /calmodulin-dependent protein kinase II forms adodecameric complex with a ring-like structure and has aunique activity regulation through its autophosphorylation
activity (Figure 3C). The kinase is made up of 12 subunits
which together form the central organizing hub. Each subunit
is composed of a catalytic kinase domain, regulatory segment,
and central hub (Hoelz et al., 2003). When the kinase is autoinhibited, CaMKII has a very compact structure. The regulatory
segment blocks the substrate-binding site (S-site) and isolates
the T286 (CaMKIIα) and T287 (CaMKIIβ) phosphorylationsites, which avoids calmodulin binding (Rosenberg et al.,2005). When Ca2+ /calmodulin complex binds to CaMKII, theregulatory segment is stripped off and allows substrate binding
and phosphorylation. The activated CaMKII phosphorylates
T286/T287 through autophosphorylation, which allows thekinase to maintain an “autonomous” open active structure
without Ca2+ /calmodulin. However, CaMKII is not fully
activated yet but could be further stimulated by Ca2+ /CaM
(Coultrap et al., 2010). CaMKII further autophosphorylates
other CaMKII residues, including T305/T306 (CaMKIIα) andT306/T307 (CaMKIIβ), of which phosphorylation inhibits
Ca2+ /calmodulin complex binding abilities and vice versa(Colbran and Soderling, 1990; Hudmon and Schulman, 2002).
The downstream substrate of CaMKII includes several neuronalproteins (e.g., GluN2B, GluA1, TIAM1) (Yasuda et al., 2022).
These unique features of kinase modification offer the possibility
that CaMKII maintains its kinase activity over a period of time.
In Drosophila, the auto-regulatory activity of CaMKII acts as atimer within a time frame of a few minutes (Thornquist et al.,2020).

4.2.2 CaMKII in sleep in vivo
Ca2+ /calmodulin-dependent protein kinase II KOs arelethal in fruit flies and cause reproductive abnormalities inthe second generation of mice. But scientists successfully
produced homozygous Camk2 KO mice using the triple CRISPR
technique (Tatsuki et al., 2016). Embryonic knockout of Camk2aor Camk2b showed a significant decrease in sleep duration inmice (Tatsuki et al., 2016). Additionally, Camk2a or Camk2b
knockout also reduced the transition probability between sleep
and awake (Tatsuki et al., 2016). Consistent with the phenotypeof these knockout mice, the post-natal expression of CaMKII
kinase inhibitor AIP2 by the adeno-associated virus (AAV)
vector resulted in a decrease in sleep duration (Tone et al.,2022). The reduced sleep duration of the AIP2-expressed mice,and at least for Camk2b knockout mice, was attributed to the
reduced NREM sleep duration. Consistently, these mice showed
an altered NREM delta-power profile (Tone et al., 2022).

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and T306 (CaMKIIα) affects substrate specificity and neuronal
localization of CaMKII and switches CaMKII’s contribution toneuronal plasticity from LTP to LTD (Coultrap et al., 2014).
The multi-site and multi-step phosphorylation of CaMKII
may also control the sleep-wake transition cycle at different
steps. As discussed in this review, changes in neuronal firing
patterns associated with the sleep-wake cycle led to different
Ca2+ dynamics in neurons. On the other hand, multi-site
autophosphorylation of CaMKII has been shown to be efficiently
phosphorylated by Ca2+ signals of different frequencies (DeKoninck and Schulman, 1998; Coultrap et al., 2014). Thus,
neuronal Ca2+ dynamics and the regulation of sleep-waketransitions by CaMKII are probably closely interacting with eachother.
There are four different CaMKII isoforms: α, β, δ,and γ that are different but highly homologous (Zalcman
et al., 2018). The isoforms of CaMKII subunits differ inthe linker sequence that connects the kinase domain to thecentral hub (Bhattacharyya et al., 2020). Notably, CaMKII
can form homo- and hetero-polymeric holoenzymes with no
known preference for isoforms (Bayer et al., 2002; Bayerand Schulman, 2019). Previous studies have revealed thatwhen CaMKIIα and CaMKIIβ are expressed together, thetwo isoforms form heterooligomers. A small portion of
CaMKIIβ is enough to dock the predominant CaMKIIα tothe actin cytoskeleton, which supports the hypothesis that the
primary function of CaMKIIβ is to alter the localization of
CaMKIIα, first forming CaMKIIα/β heterooligomers and thenlocalizing the hybrid complex to the new docking site (Shenet al., 1998). In sleep studies, whether and how CaMKIIα/βheterooligomers participate in sleep induction and maintenanceis future work. Also, the different isoforms of CaMKII
determine the phosphorylation outcomes (Bhattacharyya et al.,2020). CaMKIIα with a shorter linker tends to acquire
activating autophosphorylation on T286, while CaMKIIβ witha longer residue linker is biased to be phosphorylated
at T305/T306, which is the inhibitory autophosphorylation.
Further studies unveil the secrets of CaMKII isoforms in sleep
research.

sleep. Phosphoproteomicsgen analysis revealed that protein
phosphorylation levels, including CaMK2α/β, were dynamicallychanged according to sleep/wake status. Together withfindings of SIK3 and ERK phosphorylation level changes,
these findings support the idea of the phosphorylation
hypothesis in which phosphorylation level changes of proteins
are key features of sleep regulations. Because calcium is
involved in almost all neural behaviors, what kind of calcium
behaviors are recognized by specific sleep-controlling kinases
such as CaMKII to regulate sleep-related functions and atwhich phosphorylation sites require further investigation.
Therefore, future efforts could be devoted to identifying which
phosphorylation sites are important and the corresponding
sleep phenotypes.

Author contributions
HU conceived, supervised the study, and all aspects of
the work. YW, YM, and KO wrote the first draft of themanuscript and designed the figures. All authors contributed tothe manuscript revision and approved the submitted version.

Funding
This work was supported by grants from theBrain/MINDS (JP21dm0207049), Science and Technology
Directorate Platform Program for Advanced Biological
Medicine (JP21am0401011), AMED CREST JP21gm0610006(AMED/MEXT) (HU), Grant-in-Aid for Scientific Research(S) JP25221004 (JSPS KAKENHI) (HU) and Scientific
Research (C) JP20K07282 (JSPS KAKENHI) (YM), HFSP
Research Grant Program RGP0019/2018 (HFSP) (HU), ERATOJPMJER2001 (JST) (HU), MEXT Quantum Leap Flagship
Program (MEXT QLEAP) Grant Number (JPMXS0120330644),
and an intramural Grant-in-Aid from the RIKEN BDR (HU).

Acknowledgments
5 Concluding remarks

We thank all the lab members at the University of Tokyoand RIKEN BDR, especially Fumiya Tatsuki, Daisuke Tone, andZhiqing Wen for their constructive comments. We apologize toresearchers whose work is not cited due to space limitations.

In this review, we have discussed calcium, CaMKII, andsleep. An important discussion is about sleep in different layers.
The sleep-like behavior found in primary cultured neurons
suggests that organismal-level sleep behavior may be assembled
from neuronal assemblies. Therefore, molecular studies may
provide good evidence for the regulatory mechanisms of
sleep homeostasis. The insight from the neuronal firing
activity during NREM sleep highlighted the importance of
calcium in sleep regulation, and mice studies supported theidea. Noteworthy, gene perturbation of Camk2a/b disrupted

Frontiers in Systems Neuroscience

Conflict of interest
HU was a co-inventor on a patent covering Triple
CRISPR [PCT/JP2015/086259 (pending), patent applicant was
RIKEN] and patent applications covering the CUBIC reagents[PCT/JP2014/070618 (pending), patent applicant was RIKEN,

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Publisher’s note

PCT/JP2017/016410 (pending), patent applicant was RIKEN]
and a co-founder of CUBICStars Inc. HU was filed a patentapplication regarding the sleep/wake classification algorithm.
HU was the founder and Chief Technology Officer of
ACCELStars Inc.
The remaining authors declare that the research was
conducted in the absence of any commercial or financial
relationships that could be construed as a potential conflict ofinterest.

All claims expressed in this article are solely those of theauthors and do not necessarily represent those of their affiliatedorganizations, or those of the publisher, the editors and thereviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteedor endorsed by the publisher.

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