Fast and slow Ca2+-dependent hyperpolarization ...sys-pharm.m.u-tokyo.ac.jp/pdf/Ode_2017b.pdfFast and slow Ca2+-dependent hyperpolarization mechanisms connect membrane potential and

Fast and slow Ca2+-dependent hyperpolarizationmechanisms connect membrane potential and sleephomeostasisKoji L Ode1,2, Takahiro Katsumata1, Daisuke Tone1 andHiroki R Ueda1,2

Available online at www.sciencedirect.com

ScienceDirect

Several lines of evidence indicate that the sleep-wake state of

cortical neurons is regulated not only through neuronal

projections from the lower brain, but also through the cortical

neurons’ intrinsic ability to initiate a slow firing pattern related to

the slow-wave oscillation observed in electroencephalography

of the sleeping brain. Theoretical modeling and experiments

with 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 kinase

cascades, and the activated kinases modify ion channels or

pumps to regulate the cortical sleep/wake firing mode.

Addresses1Department of Systems Pharmacology, Graduate School of Medicine,

The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033,

Japan2 Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center,

1-3 Yamadaoka, Suita, Osaka 565-0871, Japan

Corresponding author: Ueda, Hiroki R ([email protected])

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.

IntroductionSleep 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 conservedfrom flies to humans [3]. This functional conservation


suggests that at least some of the mechanisms that regu-late sleep are closely related to the fundamental proper-ties of neurons in addition to the neural-circuit architec-ture specific to each animal species.

Sleep-wake behavior is apparently controlled by systemsoperating in a variety of time scales [4,5]. In the behav-ioral response, the transition from wakefulness to sleepoccurs 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 ofseconds. Once animals fall asleep, the sleep state con-tinues for hours in great apes (e.g., humans) or for a fewminutes in most animals, including rodents (e.g., mice).Thus, a system designed to measure the length of a sleepepisode 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 increasessleep pressure over the next few days.

What kind of mechanism can control processes with sucha wide range of time scales to control sleep, and beevolutionarily conserved in so many species? In thisreview, we discuss the role of calcium-ion (Ca2+) signalingin cortical neurons as a sleep regulator. Ca2+ is a crucialfactor in regulating neuronal properties across differenttime scales, from milliseconds to hours, in different typesof neurons [6]. On a millisecond time scale, an ion flux ofCa2+ directly depolarizes the membrane potential. Aninflux of Ca2+ also triggers neurotransmitter release atthe synapse [7]. Intracellular Ca2+ further induces severalmolecular cascades that modulate neuronal propertiesover a longer time scale (nearly an hour), such as theformation of synapses [8]. Thus, the evolutionarily con-served and temporally diverse roles of Ca2+ signaling mayhave essential functions in sleep regulation. In addition,we introduce the concept of Ca2+-dependent hyperpolar-ization of the neuronal membrane potential during sleep,which provides further mechanistic and quantitativeinsights connecting sleep and Ca2+ signaling.

Ca2+-dependent hyperpolarization of cortical-membrane potential during non-rapid eyemovement (NREM) sleepTransitions between wake and sleep states are accompa-nied by qualitative alterations in global neuronal firingpatterns in the cortex, which appear as changes in

www.sciencedirect.com

mailto:[email protected]://dx.doi.org/10.1016/j.conb.2017.05.007http://crossmark.crossref.org/dialog/?doi=10.1016/j.conb.2017.05.007&domain=pdfhttp://www.sciencedirect.com/science/journal/09594388

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 anEEG pattern with a high amplitude and a low frequency,typically 0.5–4 Hz. The firing pattern becomes non-syn-chronous when animals go into rapid eye movement(REM) sleep or wake up; these states are characterizedby an EEG pattern with a low amplitude and a highfrequency, and the macroscopic firing pattern loses syn-chrony as individual cortical neurons process variousenvironmental 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 duringNREM sleep is composed of various types of cortical-neuron activities: thalamocortical neurons generateoscillations at 1–4 Hz, while cortical neurons generateslow (

214 Neurobiology of sleep

Figure 1

(a)

(b)

EEG recording

Wake NREM sleep

Cortical neuronal activity

EE

G a

mpl

itude

EE

G a

mpl

itude

Mem

bran

epo

tent

ial

Mem

bran

epo

tent

ial

NMDAreceptors

Voltage-gatedCa2+ channels

+ + +

- - -

+ +

- -

+ + +

- - -

Ca2+-activatedK+ channels

+ + +

- - -

+ +

- -

+ + +

- - -

Voltage-gatedK+ channels

Ca2+

K+

Depolarized up state Hyperpolarized down state

Mem

bran

epo

tent

ial

CalmodulinCurrent 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


Figure 2

State of cortical neuron-glia assembly

Sta

ble

Awake SleepSleepAwake

VLPOeVLPO

LCTMN

RapheVLPO

eVLPO

LCTMN

Raphe

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 two

stable states corresponding to an awake state and an NREM-sleep state with a characteristic slow-wave oscillation pattern. Wake-promoting and

sleep-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 reconstructsleep in cultured neurons and glial cells in vitro [25,26].Notably, Jewett et al. recapitulated the homeostaticregulation of a sleep-like state in vitro: electrical stimu-lation to induce a wake-like firing state in culturedneurons was followed by an increase in the sleep-likefiring state on the next day of stimulation, suggestingthat the cultured neuron itself retains a homeostaticproperty that allows it to maintain constant excitabilityover a day. Taken together, the cortex-intrinsic changein 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 itselfcan autonomically switch between states of sleep andwakefulness (Figure 2).

If slow-wave oscillations in the cortex are producedautonomously in the cortex, the next question is whethersuch an electrophysiological pattern itself actively regu-lates sleep duration in the entire organism. Massiminiet al. showed that using 5-Hz transcranial magnetic stim-ulation of the local cortex to mimic the EEG pattern ofNREM sleep deepened slow-wave EEG activity in asleeping human subject [27]. Thus, the state of the localcortical 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


produced mice with longer or shorter sleep durationsby manipulating the Ca2+-dependent hyperpolarizationpathway, which is important for inducing a hyperpolar-ized silent phase. The pharmacological inactivation orCRISPR/Cas9-mediated knockout of ion channels toblock Ca2+ entry (i.e., NMDA receptors and voltage-gatedCa2+ channels), along with the downstream Ca2+-depen-dent K

+

channels, shortens the daily sleep duration inmice [19��,28�]. Given the critical roles of Ca2+ in generalneural function, impairing Ca2+-dependent hyperpolari-zation pathways might produce a non-specific phenotype(e.g., non-specific neural damage). However, knocking outplasma-membrane Ca2+ ATPase (PMCA) genes to inhibitCa2+ efflux lengthens sleep duration. Therefore, theopposing sleep-duration phenotypes induced by inhibit-ing the Ca2+-dependent hyperpolarization pathway or theCa2+-efflux pathway strongly suggest that these effectsare not explained by a simple disruption of the physio-logical controls.

The importance of cortical ion flux was furtherhighlighted by a study that focused on the extracellularion environment. Ding et al. found that natural sleep isassociated with a higher extracellular Ca2+ and magne-sium ion (Mg2+) concentration and a lower extracellularK+ concentration [29��]. This concentration bias wouldsupport an influx of Ca2+ and efflux of K+. Consideringthat astrocytes regulate the homeostasis of the extracel-lular ion environment, the importance of extracellular ionconditions implies the active involvement of not onlyneurons, but also glial cells for inducing the sleep state.



Indeed, other studies have investigated how adenosinesignaling through gliotransmission affects cortical slow-wave oscillations [30] and promotes rebound sleep aftersleep deprivation [31]. Moreover, optogenetically stimu-lating astrocytes induces synchronized slow-wave oscilla-tion in cortical neurons in vivo [32�]. Thus, neuron–gliainteractions would act cooperatively to modulate slow-wave oscillation patterns.

The cell-intrinsic properties that elicit slow-wave oscilla-tion might be synchronized and organized through theneural-network architecture. Studies using optogeneticsand designer receptors exclusively activated by designer-drug (DREADD) perturbation revealed a causal relation-ship between the sleep-wake transition and the activity ofa subset of neurons. The core neuron subsets for sleepregulation 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 ofthe brain such that the state of the entire organismswitches 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 ofsleep/wake cycles through Ca2+-dependentphosphorylation cascadesThe close relationship between membrane-potential con-trol and sleep-wake dynamics presents a mystery as to themechanism that coordinates the differences in time scalesbetween ion-flux regulation, which occurs in the subse-cond 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 ion-flux dynamics. A common biological architecture to pro-vide homeostatic control over longer time scales is the useof signaling cascades. Interestingly, the Ca2+-dependenthyperpolarization hypothesis of sleep control proposesthat higher hyperpolarization activity (sleep state) canbe triggered via Ca2+ influx by activating a Ca2+-depen-dent pathway such as calcium/calmodulin-dependentkinase II (CaMKII) a/b [19

��], which is important in

synaptic learning processes [39]. Knocking outCaMKIIa/b shortens sleep duration, suggesting thatCaMKIIa/b play a role in inducing sleep. The Ca2+-dependent hyperpolarization hypothesis proposes thatactive neuron firing during an awake state causes an influxof Ca2+, and a subsequent influx of CaMKIIa/b activatesa Ca2+-dependent hyperpolarization mechanism toinduce 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 comple-mented by a kinase cascade downstream of CaMKIIa/b.


Several studies on long-term potentiation (LTP) indicatethat CaMKII further triggers the downstream mitogen-activated protein kinases (MAPKs) ERK1/2, the activityof which is sustained for over an hour [41,42]. Interest-ingly, a recent study demonstrated that knocking outERK2 specifically in cortical neurons reduces theNREM-sleep duration, and that the level of phosphory-lated (active) MAPK differs between NREM and wakestates [43], although other studies indicate that MAPKactivation is associated with REM rather than NREM-sleep [44,45]. Ca2+ also potentiates PKC activity, whichmay increase with sleep deprivation [46]. The activities ofMAPK and PKC are altered by sleep-modulating sub-stances (D2 agonist, melatonin, and pentobarbital) [47].These results suggest that the sleep-wake cycle affectsthe activity of kinases downstream of Ca2+ signaling.

Another candidate kinase cascade downstream ofCaMKIIa/b was recently identified by a forward geneticscreening of sleep-controlling genes in mice [48��].NREM duration was significantly longer in mice with asleepy mutation, which involves exon skipping across theSIK3 kinase gene, with the loss of several amino acidsincluding a PKA-phosphorylation site. This phosphory-lation inhibits SIK3 activity, suggesting that the sleepySIK3 mutant is a gain-of-function mutant. Sleep depriva-tion did not appear to affect the level of PKA-responsiblephosphorylation in SIK3. On the other hand, sleep dep-rivation increased the SIK3-activating phosphorylationlevel; thus, kinases other than PKA may be involved inthe sleep deprivation-dependent phosphorylation ofSIK3.

The role of PKA in regulating sleep/wake cycles appearsto be the opposite of that of CaMKIIa/b, SIK3, andERK1/2. For example, sleep deprivation reducescAMP-PKA signaling in the hippocampus, therebyimpairing memory consolidation [49–51]. Partially sup-pressing PKA activity increased fragmentation and EEGdelta power during NREM sleep [52]. Since the down-stream pathway of PKA in sleep regulation is stillunknown, it will be interesting to investigate the molec-ular interactions between sleep-promoting kinases (e.g.,CaMKIIa/b, SIK3, and ERK1/2) and the awake-promot-ing kinase PKA in the context of sleep/wake cycles.

Connecting fast and slow dynamics: Ca2+-dependent phosphorylation cascadeshomeostatically regulate the Ca2+-dependenthyperpolarization mechanismGenetic studies have identified genes that regulate sleepduration, 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



in animals with an impaired or enhanced activity of geneproducts: to understand the molecular network of sleepregulation, we need to illustrate a path from neuronalactivity 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 inthe cortex appears to respond to the intensity of synaptictransmissions processed during the wake state in the samelocal area [53–55], indicating that some part of the mech-anism of sleep homeostasis is completed within the localcortical area or even within the individual neurons.

Slow-wave oscillation is typically observed in NREMsleep, which suggests that the ion channels involved inthis firing pattern act differently during the NREM-sleepand wake states. Otherwise, an influx of Ca2+ uponneuronal firing during the awake state would suddenlyinduce Ca2+-dependent hyperpolarization, and the neu-ron would not able to maintain an active awake state.Thus, it may be reasonable to assume that during NREMsleep, (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+ pumpsare 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 involvedare regulated. The mechanism that switches corticalneurons between the awake and sleep states may involvethe 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 hasyet to be identified. One simple hypothesis is thatsleep-regulating kinases directly phosphorylate sleep-regulating ion channels or pumps. To homeostaticallyinduce sleep, kinases that are activated downstream ofincreased excitability (e.g., accumulated Ca2+ signals) mayphosphorylate ion channels or pumps to accelerate Ca2+-dependent hyperpolarization (Figure 3). Thus, the Ca2+-dependent phosphorylation cascade may form a slowCa2+-dependent hyperpolarization mechanism. Forexample, the voltage-gated T-type Ca2+ channel CAC-NA1H (Cav3.2) is activated when phosphorylated byCaMKII [57]. Considering that both molecules promotesleep, based on the phenotypes of knockout mice, andthat CaMKII is activated by synaptic input that is gener-ally higher during the awake state, CaMKII might acti-vate CACNA1H to promote wake-driven sleep. Identify-ing novel phosphorylation sites will provide potentiallinks between sleep-regulating kinases and substrates.Advances in mass spectrometry allow us to identify phos-phorylation and other modifications comprehensivelyeven in complex membrane proteins. Blesneac et al. tookthis approach with CACNA1H [58] and identified tens of


phosphorylation sites in vivo, some of which shifted thewindow current of this channel to make it more sensitiveto 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-dependentprotein regulation is its reversibility. Thus, the impor-tance of kinases in sleep regulation implies that theirreverse enzymes, phosphatases, are also critical forregulation. Currently, no potent sleep-related phospha-tases have been discovered in mammals; however, nota-bly, 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 unphosphorylatedproteins with an effect similar to the dephosphorylationof phosphorylated proteins. Indeed, the transcriptionfactor CREB, which is regulated by PKA, is involvedin regulating sleep duration. Reducing CREB expres-sion sharply increases the sleep time in mice [59], andthe CREB, PKA, and MAPK activities change duringthe sleep-wake cycle [44]. In flies, PKA–CREB activityis important for sleep particularly in the mushroombody, a part of the brain that is critical for regulatingsleep-wake behavior in the fly. As in mammals, blockingthe CREB activity increases sleep in flies, [60] whilePKA overexpression inhibits sleep [61]. These resultssuggest that elevated cAMP signaling modulates CREBactivity 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 proteinregulating the Cullin-3 ubiquitin ligase complex [62].We also note that ion-channel regulation at the tran-scription or translation level has been implicated as anunderlying mechanism for periodic changes in mem-brane excitability during day/night cycles in mice andflies [63–65], and that some parts of the sleep/wakecycle may involve periodic regulation at the transcrip-tion or translation level.

Sleep duration and structure depends markedly on thedevelopmental stage [66], and the Ca2+-dependenthyperpolarization pathway may be involved in sleepregulation over the animal’s life span. The NMDA recep-tor member Nr3a is known for its age-dependent expres-sion in rodents, which peaks during a critical period ofexperience-dependent synaptic plasticity [67,68]. TheNr3a expression is low in adult rodents and humans.Notably, knocking out the Nr3a gene shortens sleep,but knocking out the other non-lethal NMDA receptorgenes does not (Nr2a, Nr2c, Nr2d, and Nr3a) [28�]. Nr3a’sunique sleep-promoting role may be part of an underlyingmechanism to ensure a higher amount of sleepduring childhood and a lower amount after adolescence.



Figure 3

NMDAreceptors

Voltage-gatedCa2+ channels

Ca2+-activated K+ channels

Voltage-gatedK+ channels

K+

Ca2+

Wake-associated activation of kinase signaling cascade Induction of up-state / down-state oscillation

Ca2+ channelsGPCRs

CaMKIIα/β (sleep promoting kinases)

PKA, PKC etc

SIK3 (a sleep promoting kinase)

Ca2+

ERK1/2 (sleep promoting kinases)

Current Opinion in Neurobiology

Possible relationship between sleep-regulating kinases, ion channels and pumps, and the neuronal firing-dependent control of membrane

potential. Several kinases are activated through Ca2+ influx and other awake-associated signals. Kinases are slow-regulation factors in the sleep-

wake 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 agedhumans [69]. Knocking out Cacna1g shortens sleep dura-tion; thus, decreased Cacna1g expression may decreasenighttime sleep duration in the elderly.


Evolutionary conservation of cell-intrinsicsleep regulationSeveral studies in flies show that ion-channel activities aremodulated during the sleep-wake cycle. One study



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]. Thisstudy also connected the roles of the classical sleep-related mutants found in Fmn [71] and Shaker flies[72], and demonstrated that the Shaker molecule itselfis dynamically regulated by the sleep-wake cycle.Another study identified a set of neurons responsiblefor sleep deprivation and subsequent rebound sleep inflies [73��]; interestingly, sleep deprivation results in anincrease in Ca2+ levels and NMDA receptors in thisneuron set. The study further demonstrated that Ca2+

is released from ER storage through the IP3 receptor inresponse to sleep deprivation. In a broad sense, mousegenetics focusing on cortical excitability overlap with flygenetics in identifying key players: intracellular Ca2+

signal, altered K+ current, and kinases/phosphatasespotentially regulate channel properties [60,61,74–76].Although studies in flies suggest that these ion channelsoperate mostly in a defined set of sleep-regulating neu-rons, we would nonetheless point out that even in flies,the excitability of individual neurons in response tostimuli is reduced depending on the preceding awakeperiod [77�]. This finding is consistent with local sleep inmammals; thus, neuron-intrinsic homeostasis may also bevalid in flies. Another fly study showed that blocking theNMDA receptor decreases sleep, as was also observed inmammals, and that pan-neurons (rather than a specific setof neurons) appear to contribute to this effect [78��]. Inaddition, the Ca2+-dependent phosphatase calcineurinpromotes sleep in the fruit fly [75,76]. The NMDAreceptor and Ca2+-dependent phosphatase might actcooperatively rather than in opposition to induce sleepin fruit flies.

ConclusionThe genetic identification of sleep-regulating genes hasprovided a list of ion channels/pumps and kinases thatmay function pan-neuronally. Connecting the enzyme-substrate networks among these molecules may reveal acellular cascade that is conserved across neuron subtypesand across phyla, in which case the molecular pathwayshould be understood in the context of neural networks.Revisiting the homeostatic action of mammalian sleep,sleep deprivation increases the power of slow-wave activ-ity observed on EEG. This effect might be due tochanges in the intraneuronal molecular properties ofindividual cortical neurons and to functional changes inthe neural circuits that synchronize the cortical neuronstate; it is highly possible that both mechanisms areimportant for brain function. To understand the mecha-nism of sleep control in a complex neural network, webelieve that it is necessary to understand the properties ofeach neuron during the sleep-wake cycle, because the


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

Conflict of interest statementThe authors have no conflict of interest regarding on thismanuscript.

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

References and recommended readingPapers of particular interest, published within the period of review,have been highlighted as:

� of special interest�� of outstanding interest

1. Siegel JM: Sleep viewed as a state of adaptive inactivity. Nat.Rev. Neurosci. 2009, 10:747-753.

2. Siegel JM: Do all animals sleep? Trends Neurosci. 2008, 31:208-213.

3. Krueger JM, Frank MG, Wisor JP, Roy S: Sleep function: towardelucidating an enigma. Sleep Med. Rev. 2016, 28:46-54.

4. Olbrich E, Claussen JC, Achermann P: The multiple time scalesof sleep dynamics as a challenge for modelling the sleepingbrain. Philos. Trans. A Math. Phys. Eng. Sci. 2011, 369:3884-3901.

5. Krueger JM, Rector DM, Roy S, Van Dongen HP, Belenky G,Panksepp J: Sleep as a fundamental property of neuronalassemblies. Nat. Rev. Neurosci. 2008, 9:910-919.

6. Berridge MJ: Neuronal calcium signaling. Neuron 1998, 21:13-26.

7. Greengard P: The neurobiology of slow synaptic transmission.Science 2001, 294:1024-1030.

8. Hopf FW, Waters J, Mehta S, Smith SJ: Stability and plasticity ofdeveloping synapses in hippocampal neuronal cultures. J.Neurosci. 2002, 22:775-781.

9.��

Shein-Idelson M, Ondracek JM, Liaw HP, Reiter S, Laurent G:Slow waves, sharp waves, ripples, and REM in sleepingdragons. Science 2016, 352:590-595.

The authors recorded local field potential (LFP) in the dorsal forebrain areaof reptiles, and monitored eye movement and other behavior. They foundthat reptiles showed two alternating types of sleep—with and withoutslow oscillation—in the LFP. Sleep without slow oscillation was accom-panied by eye movement. Thus, the electrophysiological features ofNREM and REM sleep are conserved across homeothermal and poiki-lothermal vertebrates.

10. Amzica F, Steriade M: Electrophysiological correlates of sleepdelta waves. Electroencephalogr. Clin. Neurophysiol. 1998,107:69-83.

11. Fuller PM, Sherman D, Pedersen NP, Saper CB, Lu J:Reassessment of the structural basis of the ascending arousalsystem. J. Comp. Neurol. 2011, 519:933-956.

12. Steriade M, McCormick DA, Sejnowski TJ: Thalamocorticaloscillations in the sleeping and aroused brain. Science 1993,262:679-685.

13. Steriade M, Timofeev I, Grenier F: Natural waking and sleepstates: a view from inside neocortical neurons. J. Neurophysiol.2001, 85:1969-1985.

14. Marshall JD, Li JZ, Zhang Y, Gong Y, St-Pierre F, Lin MZ,Schnitzer MJ: Cell-type-specific optical recording ofmembrane voltage dynamics in freely moving mice. Cell 2016,167:1650-1662 e1615.


http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0005http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0005http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0010http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0010http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0015http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0015http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0020http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0020http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0020http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0025http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0025http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0025http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0030http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0030http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0035http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0035http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0040http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0040http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0040http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0045http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0045http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0045http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0050http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0050http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0050http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0055http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0055http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0055http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0060http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0060http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0060http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0065http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0065http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0065http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0070http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0070http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0070http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0070


15. Izhikevich EM: Dynamical Systems in Neuroscience: TheGeometory of Excitability and Bursting. Cambridge,Massachusetts, USA: The MIT Press Location; 2007.

16. Compte A, Sanchez-Vives MV, McCormick DA, Wang XJ: Cellularand network mechanisms of slow oscillatory activity (


47. Hong SI, Kwon SH, Hwang JY, Ma SX, Seo JY, Ko YH, Kim HC,Lee SY, Jang CG: Quinpirole increases melatonin-augmentedpentobarbital sleep via cortical ERK, p38 MAPK, and PKC inmice. Biomol. Ther. (Seoul) 2016, 24:115-122.

48.��

Funato H, Miyoshi C, Fujiyama T, Kanda T, Sato M, Wang Z, Ma J,Nakane S, Tomita J, Ikkyu A et al.: Forward-genetics analysis ofsleep in randomly mutagenized mice. Nature 2016, 539:378-383.

Forward genetics screening in mice identified sleep-regulating genes.SIK3 kinase promotes NREM sleep, and the voltage-independent, non-selective cation channel NALCN inhibits REM sleep. The phosphorylationlevel at a specific site (T221) of SIK3 increased in response to sleepdeprivation, although the upstream kinase is unknown.

49. Vecsey CG, Baillie GS, Jaganath D, Havekes R, Daniels A,Wimmer M, Huang T, Brown KM, Li XY, Descalzi G et al.: Sleepdeprivation impairs cAMP signalling in the hippocampus.Nature 2009, 461:1122-1125.

50. Havekes R, Bruinenberg VM, Tudor JC, Ferri SL, Baumann A,Meerlo P, Abel T: Transiently increasing cAMP levelsselectively in hippocampal excitatory neurons during sleepdeprivation prevents memory deficits caused by sleep loss. J.Neurosci. 2014, 34:15715-15721.

51. Havekes R, Park AJ, Tudor JC, Luczak VG, Hansen RT, Ferri SL,Bruinenberg VM, Poplawski SG, Day JP, Aton SJ et al.: Sleepdeprivation causes memory deficits by negatively impactingneuronal connectivity in hippocampal area CA1. eLife 2016, 5.

52. Hellman K, Hernandez P, Park A, Abel T: Genetic evidence for arole for protein kinase A in the maintenance of sleep andthalamocortical oscillations. Sleep 2010, 33:19-28.

53. Huber R, Ghilardi MF, Massimini M, Ferrarelli F, Riedner BA,Peterson MJ, Tononi G: Arm immobilization causes corticalplastic changes and locally decreases sleep slow-waveactivity. Nat. Neurosci. 2006, 9:1169-1176.

54. Huber R, Esser SK, Ferrarelli F, Massimini M, Peterson MJ,Tononi G: TMS-induced cortical potentiation duringwakefulness locally increases slow-wave activity duringsleep. PLoS One 2007, 2:e276.

55. Huber R, Ghilardi MF, Massimini M, Tononi G: Local sleep andlearning. Nature 2004, 430:78-81.

56. Suzuki A, Sinton CM, Greene RW, Yanagisawa M: Behavioral andbiochemical dissociation of arousal and homeostatic sleepneed influenced by prior wakeful experience in mice. Proc.Natl. Acad. Sci. U. S. A. 2013, 110:10288-10293.

57. Iftinca MC, Zamponi GW: Regulation of neuronal T-type calciumchannels. Trends Pharmacol. Sci. 2009, 30:32-40.

58. Blesneac I, Chemin J, Bidaud I, Huc-Brandt S, Vandermoere F,Lory P: Phosphorylation of the Cav3.2 T-type calcium channeldirectly regulates its gating properties. Proc. Natl. Acad. Sci. U.S. A. 2015, 112:13705-13710.

59. Graves LA, Hellman K, Veasey S, Blendy JA, Pack AI, Abel T:Genetic evidence for a role of CREB in sustained corticalarousal. J. Neurophysiol. 2003, 90:1152-1159.

60. Hendricks JC, Williams JA, Panckeri K, Kirk D, Tello M, Yin JC,Sehgal A: A non-circadian role for cAMP signaling and CREBactivity in Drosophila rest homeostasis. Nat. Neurosci. 2001,4:1108-1115.

61. Joiner WJ, Crocker A, White BH, Sehgal A: Sleep in Drosophila isregulated by adult mushroom bodies. Nature 2006, 441:757-760.

62. Stavropoulos N, Young MW: Insomniac and Cullin-3 regulatesleep and wakefulness in Drosophila. Neuron 2011, 72:964-976.

63. Flourakis M, Kula-Eversole E, Hutchison AL, Han TH, Aranda K,Moose DL, White KP, Dinner AR, Lear BC, Ren D et al.: Aconserved bicycle model for circadian clock control ofmembrane excitability. Cell 2015, 162:836-848.


64. Belle MD, Diekman CO, Forger DB, Piggins HD: Daily electricalsilencing in the mammalian circadian clock. Science 2009,326:281-284.

65. Whitt JP, Montgomery JR, Meredith AL: BK channel inactivationgates daytime excitability in the circadian clock. Nat. Commun.2016, 7:10837.

66. Roffwarg HP, Muzio JN, Dement WC: Ontogenetic developmentof the human sleep-dream cycle. Science 1966, 152:604-619.

67. Perez-Otano I, Larsen RS, Wesseling JF: Emerging roles ofGluN3-containing NMDA receptors in the CNS. Nat. Rev.Neurosci. 2016, 17:623-635.

68. Wong HK, Liu XB, Matos MF, Chan SF, Perez-Otano I, Boysen M,Cui J, Nakanishi N, Trimmer JS, Jones EG et al.: Temporal andregional expression of NMDA receptor subunit NR3A in themammalian brain. J. Comp. Neurol. 2002, 450:303-317.

69. Rice RA, Berchtold NC, Cotman CW, Green KN: Age-relateddownregulation of the CaV3.1 T-type calcium channel as amediator of amyloid beta production. Neurobiol. Aging 2014,35:1002-1011.

70. Pimentel D, Donlea JM, Talbot CB, Song SM, Thurston AJ,Miesenbock G: Operation of a homeostatic sleep switch.Nature 2016, 536:333-337.

71. Kume K, Kume S, Park SK, Hirsh J, Jackson FR: Dopamine is aregulator of arousal in the fruit fly. J. Neurosci. 2005, 25:7377-7384.

72. Cirelli C, Bushey D, Hill S, Huber R, Kreber R, Ganetzky B,Tononi G: Reduced sleep in Drosophila Shaker mutants. Nature2005, 434:1087-1092.

73.��

Liu S, Liu Q, Tabuchi M, Wu MN: Sleep drive is encoded byneural plastic changes in a dedicated circuit. Cell 2016,165:1347-1360.

Drosophila lines expressing dTrp1 under various GAL4 drivers werescreened to identify a neuron subset (called R2 neurons). InactivatingR2 neurons caused the flies to become more resilient to sleep deprivationwithout changing the basal level of sleep duration. Sleep deprivationincreased the excitability, intracellular Ca2+ level, and NR1-gene expres-sion levels in R2 neurons. Conversely, knocking down the IP3 receptor inR2 neurons reduced the amount of recovery sleep needed, suggestingthat the ER-mediated regulation of intracellular Ca2+ levels is involved inregulating sleep homeostasis.

74. Vanderheyden WM, Gerstner JR, Tanenhaus A, Yin JC, Shaw PJ:ERK phosphorylation regulates sleep and plasticity inDrosophila. PLoS One 2013, 8:e81554.

75. Tomita J, Mitsuyoshi M, Ueno T, Aso Y, Tanimoto H, Nakai Y,Aigaki T, Kume S, Kume K: Pan-neuronal knockdown ofcalcineurin reduces sleep in the fruit fly, Drosophilamelanogaster. J. Neurosci. 2011, 31:13137-13146.

76. Nakai Y, Horiuchi J, Tsuda M, Takeo S, Akahori S, Matsuo T,Kume K, Aigaki T: Calcineurin and its regulator sra/DSCR1 areessential for sleep in Drosophila. J. Neurosci. 2011, 31:12759-12766.

77.�

Bushey D, Tononi G, Cirelli C: Sleep- and wake-dependentchanges in neuronal activity and reactivity demonstrated in flyneurons using in vivo calcium imaging. Proc. Natl. Acad. Sci. U.S. A. 2015, 112:4785-4790.

Calcium imaging of Kenyon cells in the mushroom body with single-cellresolution during wake, sleep, and recovery sleep periods revealed thatKenyon cells in an individual fly respond heterogeneously to the samestimulus when the fly has been awake for a long period. This phenomenonmay be related to local sleep in mammals.

78.��

Tomita J, Ueno T, Mitsuyoshi M, Kume S, Kume K: The NMDAreceptor promotes sleep in the fruit fly, Drosophilamelanogaster. PLoS One 2015, 10:e0128101.

Knocking down an NMDA receptor (Nmdar1) decreased the sleepamount. This result was further confirmed by the finding that adminis-tering MK-801 also decreased the sleep amount. This fruit-fly study wasthe first to demonstrate the effect of MK-801 on sleep duration.


http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0235http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0235http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0235http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0235http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0240http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0240http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0240http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0240http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0245http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0245http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0245http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0245http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0250http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0250http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0250http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0250http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0250http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0255http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0255http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0255http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0255http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0260http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0260http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0260http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0265http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0265http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0265http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0265http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0270http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0270http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0270http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0270http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0275http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0275http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0280http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0280http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0280http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0280http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0285http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0285http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0290http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0290http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0290http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0290http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0295http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0295http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0295http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0300http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0300http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0300http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0300http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0305http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0305http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0305http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0310http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0310http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0315http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0315http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0315http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0315http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0320http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0320http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0320http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0325http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0325http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0325http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0330http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0330http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0335http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0335http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0335http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0340http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0340http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0340http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0340http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0345http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0345http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0345http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0345http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0350http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0350http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0350http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0355http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0355http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0355http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0360http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0360http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0360http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0365http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0365http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0365http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0370http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0370http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0370http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0375http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0375http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0375http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0375http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0380http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0380http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0380http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0380http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0385http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0385http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0385http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0385http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0390http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0390http://refhub.elsevier.com/S0959-4388(16)30167-2/sbref0390

Fast and slow Ca2+-dependent hyperpolarization mechanisms connect membrane potential and sleep homeostasisIntroductionCa2+-dependent hyperpolarization of cortical-membrane potential during non-rapid eye movement (NREM) sleepFast dynamics: the Ca2+-dependent hyperpolarization mechanism acts through ion channels and pumps during NREM sleepSlow dynamics: the homeostatic regulation of sleep/wake cycles through Ca2+-dependent phosphorylation cascadesConnecting fast and slow dynamics: Ca2+-dependent phosphorylation cascades homeostatically regulate the Ca2+-dependent hyp...Evolutionary conservation of cell-intrinsic sleep regulationConclusionReferences and recommended readingConflict of interest statementAcknowledgements

Documents

Fast and slow Ca2+-dependent hyperpolarization ...sys-pharm.m.u-tokyo.ac.jp/pdf/Ode_2017b.pdfFast and slow Ca2+-dependent hyperpolarization mechanisms connect membrane potential and