Available online at www.sciencedirect.com

ScienceDirect
Fast and slow Ca2+-dependent hyperpolarization
mechanisms connect membrane potential and sleep
homeostasis
Koji L Ode1,2, Takahiro Katsumata1, Daisuke Tone1 andHiroki R Ueda1,2
Several lines of evidence indicate that the sleep-wake state ofcortical neurons is regulated not only through neuronal
projections from the lower brain, but also through the corticalneurons’ intrinsic ability to initiate a slow firing pattern related tothe slow-wave oscillation observed in electroencephalographyof the sleeping brain. Theoretical modeling and experimentswith genetic and pharmacological perturbation suggest that ion
channels and kinases acting downstream of calcium signaling
regulate the cortical-membrane potential and sleep duration. In
this review, we introduce possible Ca2+-dependent
hyperpolarization mechanisms in cortical neurons, in which Ca2+
signaling associated with neuronal excitation evokes kinasecascades, and the activated kinases modify ion channels or
pumps to regulate the cortical sleep/wake firing mode.
Addresses
1
Department of Systems Pharmacology, Graduate School of Medicine,
The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033,
Japan
2
Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center,
1-3 Yamadaoka, Suita, Osaka 565-0871, Japan
Corresponding author: Ueda, Hiroki R (uedah-tky@umin.ac.jp)

Current Opinion in Neurobiology 2017, 44:212–221
This review comes from a themed issue on Neurobiology of sleep
Edited by Yang Dan and Thomas Kilduff

http://dx.doi.org/10.1016/j.conb.2017.05.007
0959-4388/ã 2017 Elsevier Ltd. All rights reserved.

Introduction
Sleep is a fundamental brain state that is observed across awide range of animal species [1,2]. Despite differences incentral nervous system architecture, essential propertiesof sleep appear to be broadly conserved across species.
Sleep is a reversible and homeostatically regulated stateof reduced responsivity to environmental stimuli.
Although the physiological functions of sleep have notbeen fully explored, memory consolidation and otherpotential roles of sleep appear to be highly conserved
from flies to humans [3]. This functional conservation
Current Opinion in Neurobiology 2017, 44:212–221

suggests that at least some of the mechanisms that regulate sleep are closely related to the fundamental properties of neurons in addition to the neural-circuit architecture specific to each animal species.
Sleep-wake behavior is apparently controlled by systems
operating in a variety of time scales [4,5]. In the behavioral response, the transition from wakefulness to sleep
occurs within a minute. In this sense, the system thatswitches the entire brain (or at least most of the corticalregions) to a sleep state may operate in a time window of
seconds. Once animals fall asleep, the sleep state continues for hours in great apes (e.g., humans) or for a few
minutes in most animals, including rodents (e.g., mice).
Thus, a system designed to measure the length of a sleep
episode would operate on a time scale of minutes to hours.
Furthermore, sleep is homeostatically regulated on a timescale of a few days: sleep deprivation on one day increases
sleep pressure over the next few days.
What kind of mechanism can control processes with sucha wide range of time scales to control sleep, and be
evolutionarily conserved in so many species? In thisreview, we discuss the role of calcium-ion (Ca2+) signaling
in cortical neurons as a sleep regulator. Ca2+ is a crucial
factor in regulating neuronal properties across different
time scales, from milliseconds to hours, in different typesof neurons [6]. On a millisecond time scale, an ion flux of
Ca2+ directly depolarizes the membrane potential. An
influx of Ca2+ also triggers neurotransmitter release at
the synapse [7]. Intracellular Ca2+ further induces several
molecular cascades that modulate neuronal properties
over a longer time scale (nearly an hour), such as theformation of synapses [8]. Thus, the evolutionarily conserved and temporally diverse roles of Ca2+ signaling may
have essential functions in sleep regulation. In addition,
we introduce the concept of Ca2+-dependent hyperpolarization of the neuronal membrane potential during sleep,
which provides further mechanistic and quantitative
insights connecting sleep and Ca2+ signaling.

Ca2+-dependent hyperpolarization of corticalmembrane potential during non-rapid eye
movement (NREM) sleep
Transitions between wake and sleep states are accompanied by qualitative alterations in global neuronal firing
patterns in the cortex, which appear as changes inwww.sciencedirect.com

Fast and slow Ca2+-dependent hyperpolarization mechanisms connect membrane potential and sleep homeostasis Ode et al. 213

electroencephalogram (EEG) patterns. In the sleep state,
specifically, in non-rapid eye movement (NREM) sleep,
the synchronous firing of cortical neurons produces an
EEG pattern with a high amplitude and a low frequency,
typically 0.5–4 Hz. The firing pattern becomes non-synchronous when animals go into rapid eye movement(REM) sleep or wake up; these states are characterized
by an EEG pattern with a low amplitude and a highfrequency, and the macroscopic firing pattern loses synchrony as individual cortical neurons process various
environmental inputs. The synchronous firing of corticalneurons during NREM sleep has been observed acrossvertebrates from humans to lizards [9].
Intracellular and extracellular electrophysiologicalrecordings have revealed that the EEG pattern during
NREM sleep is composed of various types of corticalneuron activities: thalamocortical neurons generate
oscillations at 1–4 Hz, while cortical neurons generate
slow (<1 Hz) oscillations [10]. Note that ascending
arousal projections, rather than the thalamus, may be
important for the 1–4 Hz EEG oscillations, given thatlesions in the basal forebrain or parabrachial nucleus
cause a loss of EEG power greater than 1 Hz, whereasa thalamic lesion only marginally affects the EEG pattern [11]. Slow-wave oscillations (less than 1 Hz) arelikely to be generated by cortical-neuron activity.
NREM sleep (also called slow-wave sleep) is associatedwith a characteristic firing pattern that appears as slowwave oscillations. Slow-wave oscillations have twoelectrophysiological phases: the depolarized bursting
phase, and the hyperpolarized silent phase [12,13](Figure 1a). Importantly, the bursting phase is fragmented by insertions of the silent phase with a frequency
that is well matched to the slow-wave oscillation EEG
pattern (less than 1 Hz) during NREM sleep. This
electrophysiological finding was recently confirmed bycell-type-specified in vivo membrane-potential imaging[14]. Neurons are hyperpolarized during the silent phaseby hyperpolarization of the membrane potential, whichis over 10 mV lower than a pyramidal neuron’s typicalresting potential. Several models have been proposed to
explain this type of phasic firing, which has also been
observed in non-cortical cells [15]. In sleep studies,
researchers have used computational models of thousands of cortical neurons, called neuron-network models,
to investigate the mechanism of bursting and silent
phases. These models indicate that hyperpolarization
occurs by cell-intrinsic regulation rather than by theintermittent input of synaptic repression [16–18]: bursting-phase firing triggers the silent phase via an activitydependent potassium ion (K+) current mediated bysodium ion (Na+)-dependent K+ channels or Ca2+-dependent K+ channels, depending on the assumptionof the model. However, predictions provided by models
with large numbers of neurons and parameters have
limited generality: the huge parameter space prevents
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a comprehensive survey of parameter combinations, andhence of shared features that could give rise to slowwave oscillation.
A recent study aiming to construct a generalized model ofcortical firing, called an averaged-neuron model, supports
the importance of cell-intrinsic Ca2+-dependent K+ currents [19]. The averaged-neuron model applies a meanfield approximation to the neural-network model to generate a simple computational model. The averaged-neuron model assumes that all excitatory and inhibitory
neurons are connected to each other, and the model isformed as though one neuron were connecting to itself
with both excitatory and inhibitory input. Thus, thismodel has synaptic connections, but its output does
not depend on the specific pattern of the neuron-network
circuit. The averaged-neuron model produced a patternof slow-wave oscillation with alternating bursting andsilent phases in 1000 sets of randomly generated parameters describing the conductance of ion channels/pumps.
The hallmark of the slow-wave activity observed by EEG
is not only the unique firing pattern, but also the synchrony of the firing pattern among cortical neurons.
Because this model implicates the averaged-neuron
behavior, slow-wave oscillations appearing in the model
can be interpreted as synchronous bursting and resting
repeats within a population of neurons connected toexcitatory and inhibitory inputs. A systematic investigation of all of the obtained parameter sets for a slow-wave
oscillation pattern indicated that Ca2+ influx upon excitation (through NMDA receptors and voltage-gated Ca2+
channels) and the activation of Ca2+-dependent K+ channels are the most critical parameters for inducing a
hyperpolarized membrane potential during a silent phase(Figure 1b). Consistent with this view, the activation of
Ca2+ pumps or exchangers might terminate the silent
phase in this model. These theoretical models suggestthat a primitive source of slow-wave oscillations in corticalneurons during NREM sleep may be the cell-intrinsic
control of membrane potential, particularity though firing-induced K+ outward currents such as the Ca2+-dependent hyperpolarization mechanism.

Fast dynamics: the Ca2+-dependent
hyperpolarization mechanism acts throughion channels and pumps during NREM sleep
An important question is whether the mechanism ofmembrane-potential regulation, which is a fast, dynamic
process characterized by neuron properties, plays anactive role in the sleep phenotype, or whether the firing
state of cortical neurons is subordinate to other regions of
the brain in sleep control. There are several lines ofevidence suggesting that the cortical-neuron assembly
itself has an intrinsic ability to initiate the bursting andsilent phases during NREM sleep (Figure 2). First,
bursting and silent phases that alternate at 0.5–4 Hz
can be reproduced in ex vivo cortical slices [18,20],
Current Opinion in Neurobiology 2017, 44:212–221

214 Neurobiology of sleep

Figure 1(a)
EEG amplitude
Membranepotential

Membranepotential

EEG recording

Cortical neuronal activity

NREM sleep

EEG amplitude

Wake

Membranepotential(b)

Depolarized up state

Hyperpolarized down state
K+

-

-

-

-

-

+

+

+

+

+

NMDA
receptors

-

-

-

+

+

+

Voltage-gated
Ca2+ channels

Ca2+

+

+

+

+

+

-

-

-

-

-

Ca2+-activated
K+ channels

+

+

+

-

-

-

Voltage-gated
K+ channels

Calmodulin
Current Opinion in Neurobiology

Regulation of cortical membrane potential during NREM sleep. (a) Sleep/wake states are characterized by different behaviors of EEG signals
originating from the collective activity of the cortical neurons. Slow-wave EEG-signal oscillations during NREM sleep, particularly those <1 Hz, arethought to be produced by the intermittent firing of cortical neurons. (b) The intermittent-firing pattern is composed of alternating depolarized
bursting and hyperpolarized silent phases. The silent-phase hyperpolarization may be maintained by Ca2+-dependent K+ channels activated by a
Ca2+ influx accumulated during the preceding bursting phase.

indicating that inputs from other brain regions are notnecessary for this slow-wave oscillation pattern. Second,
the slow-wave oscillation pattern on EEG during NREM
sleep is also observed in the local part of the cortex
during REM sleep and in periods of wakefulness, andthis is called local sleep [21,22]. Local sleep suggests that
Current Opinion in Neurobiology 2017, 44:212–221

the state of the entire cortex during sleep is not fully
defined downstream of other core sleep-regulating circuits, but that a part of the cortex can independently
initiate synchronous slow-wave oscillations. The cortexautonomous ability to alter sleep-related states may be
involved in the ability of fur seals and birds to switch towww.sciencedirect.com

Fast and slow Ca2+-dependent hyperpolarization mechanisms connect membrane potential and sleep homeostasis Ode et al. 215

Figure 2
State of cortical neuron-glia assembly
Sleep

Stable

Awake

LC
TMN
Raphe

VLPO
eVLPO
LC
TMN
Raphe

VLPO
eVLPO

Current Opinion in Neurobiology

Intrinsic ability of cortical cells to transition between awake-firing and sleep-firing states. The cortical neuron–glia assembly is thought to have twostable states corresponding to an awake state and an NREM-sleep state with a characteristic slow-wave oscillation pattern. Wake-promoting andsleep-promoting centers of the brain may stabilize the state of cortical neurons, either in an awake or sleep state. VLPO: ventrolateral preoptic
nucleus; eVLPO: extended ventrolateral preoptic nucleus; LC: locus coeruleus; TMN: tuberomammillary nucleus.

bilateral-hemispheric sleep on land and unihemisphericsleep when flying or swimming [23,24]. Based on thisview of sleep, researchers have attempted to reconstruct
sleep in cultured neurons and glial cells in vitro [25,26].
Notably, Jewett et al. recapitulated the homeostatic
regulation of a sleep-like state in vitro: electrical stimulation to induce a wake-like firing state in cultured
neurons was followed by an increase in the sleep-likefiring state on the next day of stimulation, suggestingthat the cultured neuron itself retains a homeostatic
property that allows it to maintain constant excitability
over a day. Taken together, the cortex-intrinsic change
in state may provide a modified version of a flip-flopmodel: regions of the brain, including the ventrolateralpreoptic nucleus (VLPO) and other sleep/wake centers,
induce a bias in the firing state of the cortex, which itself
can autonomically switch between states of sleep andwakefulness (Figure 2).
If slow-wave oscillations in the cortex are produced
autonomously in the cortex, the next question is whether
such an electrophysiological pattern itself actively regulates sleep duration in the entire organism. Massimini
et al. showed that using 5-Hz transcranial magnetic stimulation of the local cortex to mimic the EEG pattern of
NREM sleep deepened slow-wave EEG activity in asleeping human subject [27]. Thus, the state of the local
cortical neurons during sleep may actively control theanimals’ sleep. Several genetic studies support an activerole for the cortical firing state; consistent with thecomputational prediction, researchers successfully
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produced mice with longer or shorter sleep durations
by manipulating the Ca2+-dependent hyperpolarization
pathway, which is important for inducing a hyperpolarized silent phase. The pharmacological inactivation or
CRISPR/Cas9-mediated knockout of ion channels toblock Ca2+ entry (i.e., NMDA receptors and voltage-gatedalong with the downstream Ca2+-depenCa2+ channels),+dent K channels, shortens the daily sleep duration inmice [19,28]. Given the critical roles of Ca2+ in general
neural function, impairing Ca2+-dependent hyperpolarization pathways might produce a non-specific phenotype(e.g., non-specific neural damage). However, knocking out
plasma-membrane Ca2+ ATPase (PMCA) genes to inhibit
Ca2+ efflux lengthens sleep duration. Therefore, theopposing sleep-duration phenotypes induced by inhibiting the Ca2+-dependent hyperpolarization pathway or the
Ca2+-efflux pathway strongly suggest that these effects
are not explained by a simple disruption of the physiological controls.
The importance of cortical ion flux was further
highlighted by a study that focused on the extracellular
ion environment. Ding et al. found that natural sleep is
associated with a higher extracellular Ca2+ and magnesium ion (Mg2+) concentration and a lower extracellular
K+ concentration [29]. This concentration bias would
support an influx of Ca2+ and efflux of K+. Consideringthat astrocytes regulate the homeostasis of the extracellular ion environment, the importance of extracellular ion
conditions implies the active involvement of not only
neurons, but also glial cells for inducing the sleep state.
Current Opinion in Neurobiology 2017, 44:212–221

216 Neurobiology of sleep

Indeed, other studies have investigated how adenosine
signaling through gliotransmission affects cortical slowwave oscillations [30] and promotes rebound sleep after
sleep deprivation [31]. Moreover, optogenetically stimulating astrocytes induces synchronized slow-wave oscillation in cortical neurons in vivo [32]. Thus, neuron–glia
interactions would act cooperatively to modulate slowwave oscillation patterns.
The cell-intrinsic properties that elicit slow-wave oscillation might be synchronized and organized through theneural-network architecture. Studies using optogenetics
and designer receptors exclusively activated by designerdrug (DREADD) perturbation revealed a causal relationship between the sleep-wake transition and the activity ofa subset of neurons. The core neuron subsets for sleep
regulation are in the basal forebrain for NREM control[33] and in the pons/midbrain for REM control [34,35].
These dedicated neural circuits coordinate the state of
the brain such that the state of the entire organism
switches between sleep and wake states [36], and lossof this coordination at the whole-brain level may lead tothe abnormal EEG patterns associated with psychiatricdiseases [37,38].

Slow dynamics: the homeostatic regulation of
sleep/wake cycles through Ca2+-dependent
phosphorylation cascades
The close relationship between membrane-potential control and sleep-wake dynamics presents a mystery as to themechanism that coordinates the differences in time scales
between ion-flux regulation, which occurs in the subsecond order, and the regulation of sleep homeostasis,
which occurs over a period of hours. It is difficult toaccount for the slow sleep dynamics based only on ionflux dynamics. A common biological architecture to provide homeostatic control over longer time scales is the useof signaling cascades. Interestingly, the Ca2+-dependent
hyperpolarization hypothesis of sleep control proposesthat higher hyperpolarization activity (sleep state) canbe triggered via Ca2+ influx by activating a Ca2+-dependent pathway such as calcium/calmodulin-dependentkinase II (CaMKII) a/b [19 ], which is important insynaptic learning processes [39]. Knocking out
CaMKIIa/b shortens sleep duration, suggesting that
CaMKIIa/b play a role in inducing sleep. The Ca2+-dependent hyperpolarization hypothesis proposes that
active neuron firing during an awake state causes an influxof Ca2+, and a subsequent influx of CaMKIIa/b activatesa Ca2+-dependent hyperpolarization mechanism to
induce NREM sleep [19].
Compared with the duration of a single episode of wake orsleep, the dynamics of CaMKIIa/b activation itself uponneuronal excitation are relatively fast—within a minute,at least at synapses [40]. This time gap may be complemented by a kinase cascade downstream of CaMKIIa/b.
Current Opinion in Neurobiology 2017, 44:212–221

Several studies on long-term potentiation (LTP) indicatethat CaMKII further triggers the downstream mitogenactivated protein kinases (MAPKs) ERK1/2, the activityof which is sustained for over an hour [41,42]. Interestingly, a recent study demonstrated that knocking out
ERK2 specifically in cortical neurons reduces the
NREM-sleep duration, and that the level of phosphorylated (active) MAPK differs between NREM and wakestates [43], although other studies indicate that MAPK
activation is associated with REM rather than NREMsleep [44,45]. Ca2+ also potentiates PKC activity, which
may increase with sleep deprivation [46]. The activities of
MAPK and PKC are altered by sleep-modulating substances (D2 agonist, melatonin, and pentobarbital) [47].
These results suggest that the sleep-wake cycle affects
the activity of kinases downstream of Ca2+ signaling.
Another candidate kinase cascade downstream of
CaMKIIa/b was recently identified by a forward genetic
screening of sleep-controlling genes in mice [48].
NREM duration was significantly longer in mice with asleepy mutation, which involves exon skipping across the
SIK3 kinase gene, with the loss of several amino acids
including a PKA-phosphorylation site. This phosphorylation inhibits SIK3 activity, suggesting that the sleepy
SIK3 mutant is a gain-of-function mutant. Sleep deprivation did not appear to affect the level of PKA-responsible
phosphorylation in SIK3. On the other hand, sleep deprivation increased the SIK3-activating phosphorylationlevel; thus, kinases other than PKA may be involved in
the sleep deprivation-dependent phosphorylation of
SIK3.
The role of PKA in regulating sleep/wake cycles appears
to be the opposite of that of CaMKIIa/b, SIK3, andERK1/2. For example, sleep deprivation reducescAMP-PKA signaling in the hippocampus, thereby
impairing memory consolidation [49–51]. Partially suppressing PKA activity increased fragmentation and EEG
delta power during NREM sleep [52]. Since the downstream pathway of PKA in sleep regulation is still
unknown, it will be interesting to investigate the molecular interactions between sleep-promoting kinases (e.g.,
CaMKIIa/b, SIK3, and ERK1/2) and the awake-promoting kinase PKA in the context of sleep/wake cycles.

Connecting fast and slow dynamics: Ca2-dependent phosphorylation cascades
homeostatically regulate the Ca2+-dependent
hyperpolarization mechanism+

Genetic studies have identified genes that regulate sleep
duration, but the molecular pathways that connect thesegenes are unknown. Sleep and sleep deprivation canaffect numerous molecules such as ion channels, pumps,
and kinases both directly and indirectly. It is not enoughto measure protein activity upon sleep deprivation (or thenatural sleep-wake cycle) or to quantify sleep phenotypes
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Fast and slow Ca2+-dependent hyperpolarization mechanisms connect membrane potential and sleep homeostasis Ode et al. 217

in animals with an impaired or enhanced activity of geneproducts: to understand the molecular network of sleep
regulation, we need to illustrate a path from neuronal
activity during the wake state to sleep-regulating factors.
As a perspective for future studies, we here discuss apossible signaling cascade among sleep-regulating factors.
Notably, the local power of the slow-wave oscillation in
the cortex appears to respond to the intensity of synaptictransmissions processed during the wake state in the same
local area [53–55], indicating that some part of the mechanism of sleep homeostasis is completed within the local
cortical area or even within the individual neurons.
Slow-wave oscillation is typically observed in NREM
sleep, which suggests that the ion channels involved inthis firing pattern act differently during the NREM-sleep
and wake states. Otherwise, an influx of Ca2+ uponneuronal firing during the awake state would suddenly
induce Ca2+-dependent hyperpolarization, and the neuron would not able to maintain an active awake state.
Thus, it may be reasonable to assume that during NREM
sleep, (1) Ca2+ channels are more permeable to Ca2+, (2)Ca2+-dependent K+ channels become more sensitive andactive in response to Ca2+ influx, and/or (3) Ca2+ pumps
are less permeable to Ca2+. If the state of ion channels andpumps is modified by highly excitable neuronal firing,then this activity-dependent modification would close thehomeostatic loop: prolonged wakefulness promotes sleep.
The question, then, is how the channels/pumps involved
are regulated. The mechanism that switches corticalneurons between the awake and sleep states may involve
the phosphorylation of ion channels or pumps. Althoughseveral studies have investigated phospho-proteomicchanges in the sleep and awake states [56], a criticalkinase substrate for homeostatic sleep regulation has
yet to be identified. One simple hypothesis is thatsleep-regulating kinases directly phosphorylate sleepregulating ion channels or pumps. To homeostaticallyinduce sleep, kinases that are activated downstream ofincreased excitability (e.g., accumulated Ca2+ signals) may
phosphorylate ion channels or pumps to accelerate Ca2+-dependent hyperpolarization (Figure 3). Thus, the Ca2+-dependent phosphorylation cascade may form a slow
Ca2+-dependent hyperpolarization mechanism. For
example, the voltage-gated T-type Ca2+ channel CACNA1H (Cav3.2) is activated when phosphorylated by
CaMKII [57]. Considering that both molecules promote
sleep, based on the phenotypes of knockout mice, andthat CaMKII is activated by synaptic input that is generally higher during the awake state, CaMKII might activate CACNA1H to promote wake-driven sleep. Identifying novel phosphorylation sites will provide potential
links between sleep-regulating kinases and substrates.
Advances in mass spectrometry allow us to identify phosphorylation and other modifications comprehensively
even in complex membrane proteins. Blesneac et al. took
this approach with CACNA1H [58] and identified tens ofwww.sciencedirect.com

phosphorylation sites in vivo, some of which shifted thewindow current of this channel to make it more sensitive
to membrane-potential depolarization. As the amount ofmolecular information increases, the next challenge is toevaluate which molecular pathway is responsible forregulating sleep duration in vivo.
The important feature of phosphorylation-dependent
protein regulation is its reversibility. Thus, the importance of kinases in sleep regulation implies that theirreverse enzymes, phosphatases, are also critical for
regulation. Currently, no potent sleep-related phosphatases have been discovered in mammals; however, notably, phosphorylation can also be reversibly regulated byproteolysis-synthesis protein turnover. In other words,
the degradation of old phosphorylated proteins and thesynthesis of new proteins can supply unphosphorylated
proteins with an effect similar to the dephosphorylationof phosphorylated proteins. Indeed, the transcription
factor CREB, which is regulated by PKA, is involved
in regulating sleep duration. Reducing CREB expression sharply increases the sleep time in mice [59], andthe CREB, PKA, and MAPK activities change during
the sleep-wake cycle [44]. In flies, PKA–CREB activity
is important for sleep particularly in the mushroom
body, a part of the brain that is critical for regulating
sleep-wake behavior in the fly. As in mammals, blocking
the CREB activity increases sleep in flies, [60] while
PKA overexpression inhibits sleep [61]. These results
suggest that elevated cAMP signaling modulates CREB
activity to reduce the amount of sleep. Alternatively,
the protein degradation pathway is also important forcontrolling sleep: a genetic study in flies identified theshort-sleep insomniac gene, which encodes a protein
regulating the Cullin-3 ubiquitin ligase complex [62].
We also note that ion-channel regulation at the transcription or translation level has been implicated as anunderlying mechanism for periodic changes in membrane excitability during day/night cycles in mice andflies [63–65], and that some parts of the sleep/wakecycle may involve periodic regulation at the transcription or translation level.
Sleep duration and structure depends markedly on thedevelopmental stage [66], and the Ca2+-dependent
hyperpolarization pathway may be involved in sleep
regulation over the animal’s life span. The NMDA receptor member Nr3a is known for its age-dependent expression in rodents, which peaks during a critical period ofexperience-dependent synaptic plasticity [67,68]. The
Nr3a expression is low in adult rodents and humans.
Notably, knocking out the Nr3a gene shortens sleep,
but knocking out the other non-lethal NMDA receptor
genes does not (Nr2a, Nr2c, Nr2d, and Nr3a) [28]. Nr3a’s
unique sleep-promoting role may be part of an underlying
mechanism to ensure a higher amount of sleep
during childhood and a lower amount after adolescence.
Current Opinion in Neurobiology 2017, 44:212–221

218 Neurobiology of sleep

Figure 3

Wake-associated activation ofkinase signaling cascade

GPCRs

Ca2+ channels

Induction of up-state / down-state oscillation

NMDA
receptors

Voltage-gated
Ca2+ channels

Ca2+-activated
K+ channels

Voltage-gated
K+ channels
K+

Ca2+

Ca2+

CaMKIIα/β (sleep promoting kinases)
SIK3 (a sleep promoting kinase)
ERK1/2 (sleep promoting kinases)
PKA, PKC etc

Current Opinion in Neurobiology

Possible relationship between sleep-regulating kinases, ion channels and pumps, and the neuronal firing-dependent control of membranepotential. Several kinases are activated through Ca2+ influx and other awake-associated signals. Kinases are slow-regulation factors in the sleepwake cycle. The activated kinases modify ion channels involved in the slow-wave oscillation pattern, and the ion channels act as fast-regulation
factors. This connection between slow and fast regulation factors provides a plausible mechanism for the homeostatic control of wake-induced
sleepiness. GPCRs: G protein-coupled receptors.

Similarly, the expression of Cacna1g decreases in aged
humans [69]. Knocking out Cacna1g shortens sleep duration; thus, decreased Cacna1g expression may decreasenighttime sleep duration in the elderly.
Current Opinion in Neurobiology 2017, 44:212–221

Evolutionary conservation of cell-intrinsic
sleep regulation
Several studies in flies show that ion-channel activities are
modulated during the sleep-wake cycle. One study
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Fast and slow Ca2+-dependent hyperpolarization mechanisms connect membrane potential and sleep homeostasis Ode et al. 219

showed that a reduction of voltage-gated K+ current(through Shaker and Slab) and induction of leak K+ current(through Sandman) form the underlying mechanism forthe dopamine-mediated hyperpolarization of the dFB, asleep-promoting region of the brain in flies [70]. This
study also connected the roles of the classical sleeprelated mutants found in Fmn [71] and Shaker flies[72], and demonstrated that the Shaker molecule itself
is dynamically regulated by the sleep-wake cycle.
Another study identified a set of neurons responsible
for sleep deprivation and subsequent rebound sleep inflies [73]; interestingly, sleep deprivation results in an
increase in Ca2+ levels and NMDA receptors in thisneuron set. The study further demonstrated that Ca2+is released from ER storage through the IP3 receptor in
response to sleep deprivation. In a broad sense, mouse
genetics focusing on cortical excitability overlap with fly
genetics in identifying key players: intracellular Ca2+
signal, altered K+ current, and kinases/phosphatases
potentially regulate channel properties [60,61,74–76].
Although studies in flies suggest that these ion channels
operate mostly in a defined set of sleep-regulating neurons, we would nonetheless point out that even in flies,
the excitability of individual neurons in response tostimuli is reduced depending on the preceding awake
period [77]. This finding is consistent with local sleep inmammals; thus, neuron-intrinsic homeostasis may also bevalid in flies. Another fly study showed that blocking the
NMDA receptor decreases sleep, as was also observed in
mammals, and that pan-neurons (rather than a specific setof neurons) appear to contribute to this effect [78]. In
addition, the Ca2+-dependent phosphatase calcineurin
promotes sleep in the fruit fly [75,76]. The NMDA
receptor and Ca2+-dependent phosphatase might act
cooperatively rather than in opposition to induce sleepin fruit flies.

system behavior emerges from the properties of both thearchitecture and the components.

Conflict of interest statement
The authors have no conflict of interest regarding on thismanuscript.

Acknowledgements
This work was supported by AMED-CREST, CREST, Brain/MINDS, theBasic Science and Platform Technology Program for Innovative Biological
Medicine, JSPS (25221004, 23115006, 25830146, 16K14653), RIKEN, andthe Takeda Science Foundation.

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Conclusion
The genetic identification of sleep-regulating genes has
provided a list of ion channels/pumps and kinases that
may function pan-neuronally. Connecting the enzymesubstrate networks among these molecules may reveal acellular cascade that is conserved across neuron subtypes
and across phyla, in which case the molecular pathway
should be understood in the context of neural networks.
Revisiting the homeostatic action of mammalian sleep,
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