Behavioral and genetic effects promoted by sleep deprivation in rats submitted to pilocarpine-induced status epilepticus

NeuroscienceLetters515 (2012) 137–140

ContentslistsavailableatSciVerseScienceDirect

Neuroscience

Letters

j o ur na l ho me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / n e u l e t

Behavioral

and

genetic

effects

promoted

by

sleep

deprivation

in

rats

submitted

to

pilocarpine-induced

status

epilepticus

Gabriela

Matos

_,

_Daniel

_A.

_Ribeiro

_,

_Tathiana

_A.

_Alvarenga

_,

_Camila

_Hirotsu

_,

_Fulvio

_A.

_Scorza

_,

Luciana

Le

Sueur-Maluf

_,

_Juliana

_Noguti

_,

_Esper

_A.

_Cavalheiro

_,

_Sergio

_Tufik

_,

_Monica

_L.

_Andersen

a_{DepartamentodePsicobiologia,UniversidadeFederaldeSãoPaulo(UNIFESP),Brazil}

b_{DepartamentodeBiociências,UniversidadeFederaldeSãoPaulo(UNIFESP),Brazil}

c_{DepartamentodeNeurologiaExperimental,UniversidadeFederaldeSãoPaulo(UNIFESP),Brazil}

d_{DepartamentodePatologia,UniversidadeFederaldeSãoPaulo,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received11November2011

Receivedinrevisedform28January2012 Accepted10March2012

Keywords:

Epilepsy Pilocarpine Sleepdeprivation DNAdamage Locomotoractivity

a

b

s

t

r

a

c

t

Theinteractionbetweensleepdeprivationandepilepsyhasbeenwelldescribedinelectrophysiological studies,butthemechanismsunderlyingthisassociationremainunclear.Thepresentstudyevaluatedthe effectsofsleepdeprivationonlocomotoractivityandgeneticdamageinthebrainsofratstreatedwith salineorpilocarpine-inducedstatusepilepticus(SE).After50daysofpilocarpineorsalinetreatment,both groupswereassignedrandomlytototalsleepdeprivation(TSD)for6h,paradoxicalsleepdeprivation (PSD)for24h,orbekeptintheirhomecages.Locomotoractivitywasassessedwiththeopenfieldtest followedbyresectionofbrainforquantificationofgeneticdamagebythesinglecellgelelectrophoresis (comet)assay.Statusepilepticusinducedsignificanthyperactivityintheopenfieldtestandcausedgenetic damageinthebrain.Sleepdeprivationprocedures(TSDandPSD)didnotaffectlocomotoractivityin epilepticorhealthyrats,butresultedinsignificantDNAdamageinbraincells.AlthoughPSDhadthis effectinbothvehicleandepilepticgroups,TSDcausedDNAdamageonlyinepilepticrats.Inconclusion, ourresultsrevealedthat,despitealackofbehavioraleffectsofsleepdeprivation,TSDandPSDinduced geneticdamageinratssubmittedtopilocarpine-inducedSE.

© 2012 Elsevier Ireland Ltd.

1. Introduction

Epilepsy isone of themostcommonneurological disorders, affectingpeopleofallages[7].Inaddition,epilepsyandseizures affectmanyphysiologicalpatternsinhumansandanimals,such assleeparchitecture[5,15].Sleepdisruptionresultsindifferential expressionofmanygenes,includinggenesrelatedtothe sleep-wakecycle[9],andpromotesextensiveDNAbreakageinhealthy rodents[3].Ofnote,DNAfragmentationintherhinalcortexand hippocampusofratswithepilepsyhasbeenreported[12].Beyond molecularalterations,sleeplosshasbeenshowntomodify behav-ioralparameters,suchaslocomotoractivity[1].StewartandLeung [24]demonstratedanincreaseinambulationaftertheinductionof statusepilepticus(SE)inrats.Theauthorssuggestedthatseizures modulatespontaneouslocomotoractivitybydisruptingthenormal functionofthesuprachiasmaticnucleus.Thus,previousresearch

∗Correspondingauthorat:DepartamentodePsicobiologia,RuaNapoleãode Bar-ros,925,VilaClementino–SP04024-002,SãoPaulo,Brazil.Tel.:+551121490155; fax:+551155725092.

E-mailaddresses:ml.andersen12@gmail.com,mandersen@unifesp.br

(M.L.Andersen).

hasshownthatbothsleepdeprivationandapreclinicalmodelof epilepsycaninduceDNAdamageandhyperactivityinrodents[1,3]. Sleep deprivation is one of the most important triggers of seizuresinepilepsypatients[5,15,26];however,the pathophysio-logicalmechanismsinvolvedinthisharmfulassociationhavebeen unexplored.Inthissense,moreinformation,beyondthefindings ofelectrophysiologicalstudies,couldprovideastartingpointfor futureclinicaltrialsoftreatmentsforepilepsy[16].

Considering the prevalence of sleep deficitsin patientsand animalswithepilepsy,andtheinfluenceofsleepdeprivationon locomotor activity and DNA damage, the aimof present study wastoexaminetheeffectsofsleeploss(totalandselective)on motor behavior and neuronal genotoxicityin ratssubmitted to pilocarpine-inducedSE.

2. Materialsandmethods

All procedures in thepresent studycomplied withthe rec-ommendationsinAnimalModelsasToolsin EthicalBiomedical Research [4]. The study wasapproved by theEthical Commit-tee of Universidade Federal de São Paulo (CEP 1363/10). The

0304-3940© 2012 Elsevier Ireland Ltd. doi:10.1016/j.neulet.2012.03.030

Open access under the Elsevier OA license.

138 G.Matosetal./NeuroscienceLetters515 (2012) 137–140

experimentswereperformedinmaleadultWistar-Hannoverrats aged60daysatthebeginningofthestudy.

2.1. Pilocarpine-inducedstatusepilepticus

Statusepilepticuswasinducedwithasingledoseofpilocarpine (350mg/kg, i.p.) 30min after injection of methylscopolamine (0.1mg/kg,s.c.)(PILOgroup).Thevehiclegroup(SAL)wasinjected withsaline(0.9%NaCl,i.p.).Onlyratsthatdisplayedconvulsiveand intermittentseizuresafterpilocarpineinjectionwereincludedin thisstudy.Seizureactivitywasmonitoredbehaviorallyand termi-natedafter3hofconvulsiveSEwithani.p.injectionofdiazepam (10mg/kg).FiftydaysaftertheSEepisode,pilocarpine-treatedrats wereassigned randomlytototal sleepdeprivation (TSD+PILO), paradoxicalsleepdeprivation(PSD+PILO)orcontrol(keptintheir homecages;CTRL+PILO)groups.RatsintheSALgroupwere dis-tributedintogroupssubmittedtothesameconditions(TSD+SAL, PSD+SAL, and CTRL-SAL). The numbers of animals used were n=11–14fortheopenfield testand n=6forthesinglecellgel electrophoresisassay.

2.2. Totalandparadoxicalsleepdeprivationprocedures

After50 days of SE, TSD was induced bya gentlehandling protocolinTSD+PILOandTSD+SALgroups.Thisprocedurewas performedfor6h(07:00–13:00),andconsistedofeithertouching theanimalswithabrushorgentlymovingtheircageswhenever theyclosedtheireyes.Theanimalswerenotdisturbedduring feed-inganddrinking.

Inparallel,PSDwasinducedfor24husingthemodifiedmultiple platformmethod.Briefly,thePSDprocedureconsistedofplacing ratsinatiledwatertank(106cm×40cm×30cm)containing14 circularplatforms,eachof6.5cmindiameter.Theratscouldthus movearoundinside thetankbyjumpingfrom oneplatformto another.Whentheyreachedtheparadoxicalphaseofsleep,muscle atoniasetin,andtheyfellintothewaterandawoke[2,3].Basedon pilotexperiments,24hwasestablishedasasafeperiodofPSDin ratswithepilepsy.Thecagecontrolgroupwasmaintainedinthe sameroomasthePSDgroup.

2.3. Openfieldtest

Immediately after the sleep deprivation protocols (13:00–15:00), the open field test was applied to assess gen-eralmotorbehavior.Ratswereplacedinacirculararena(96cm diameter),thefloorof which wasdividedinto19 squares. Fre-quencyof peripherallocomotion (numberofentrancesintothe floorunitsclosetothewallsofthebox),centrallocomotion (num-berofentrancesintothefloorunitsnotclosetothewalls),total locomotion(numberoffloorunitsentered),andrearing(number oftimestheanimalsstoodontheirhindlegs)werequantifiedby countingthe number of inter-square lines crossed. Immobility (totaltimeoflackofmovement)wasalsomeasured.

2.4. Singlecellgelelectrophoresis(comet)assay

After behavioral analysis, the animals were euthanized by decapitation,arapidandpainlessprocedurecarriedoutlessthan 1min in adjacentroom. Brains wererapidly removed,and the cerebralcortexwasdissectedandmincedin0.9%NaCl.The super-natantwasremovedandcellularsuspensionswereusedinsingle cellgelelectrophoresis(comet)assaysconductedaccordingSasaki and colleagues [20] with some modifications. Cell suspensions (10␮L)wereaddedto120␮Lof0.5%low-melting-pointagarose

at37◦_{C,layeredontoaslidepre-coatedwith1.5%regularagarose,} andcoveredwithacoverslip.Afterrapidagarosesolidificationin arefrigerator,thecoverslipswereremovedandtheslideswere immersed in lysis solution (2.5M NaCl, 100mM EDTA, 10mM Tris–HCl buffer, pH 10, 1% sodium sarcosinate with 1% Triton X-100 and 10% DMSO) for approximately 1h. The slides were placed in alkaline buffer (pH>13) for 20min and then elec-trophoresedfor20minat0.7V/cm,300mA.Afterelectrophoresis, theslides wereneutralized in 0.4MTris–HCl (pH 7.5),fixedin absoluteethanolandstoreduntilanalysis.

2.5. Genotoxicitydataanalysis

Followingthecometassayprocedure,a totalof50randomly capturedcometsperanimal(25cellsoneachoftwoslides)[10] wereexaminedblindlybyanexpertobserverat400× magnifica-tionunderafluorescencemicroscope(Olympus).Themicroscope wasconnectedtoanimageanalysissystem(CometAssayII, Per-ceptiveInstruments,Haverhill,Suffolk,UK)calibratedpreviously according to the manufacturer’s instructions. Undamaged cells haveanintactnucleuswithoutatail,whereasdamagedcellshave theappearanceofacomet.Inthepresentstudy,DNAdamagewas analyzedbasedonthetailmomentparameter(theproductofthe taillengthandthefractionofDNAinthecomettail)[10].

2.6. Statisticalmethods

Allvariableswerefirsttestedfornormalityandnoneshoweda normaldistribution(p>0.05intheShapiro-Wilktest).Thus,the datawereconverted intoz-score. Oncethere were2 factorsin thecurrentstudy:Sleep(CTRL,TSDandPSD)andTreatment(SAL andPILO),thevariableswerefurtheranalyzedwith2-wayANOVA followedbyTukeyposthoctestwhennecessary.Thelevelof sig-nificancewassetat5%.Dataarereportedasmeans±SEM.

3. Results

3.1. Openfieldtest

The PILO group showed significant increase in peripheral (F1,54=17.12; P<0.001),central (F1,54=10.64; P<0.01) and total locomotion(F1,54=22.09;P<0.001),andareductioninimmobility (F1,54=24.75;P<0.0001)comparedwiththeSALgroup(Table1). TSD and PSD did not affect locomotion or immobility in the

Table1

Behavioraldatafromopenfieldtest.

Peripherallocomotion Centrallocomotion Totallocomotion Immobility Rearing

CTRL+SAL 21.2±2.3 5.0±1.5 26.2±3.2 20.8±3.0 12.7±1.5

CTRL+PILO 89.7±17.5* _15.3

±3.5* _105.1

±17.0* _2.1

±1.4* _7.9

±2.7

TSD+SAL 22.0±5.6 6.1±1.7 28.1±6.3 17.7±3.3 12.4±3.0

TSD+PILO 91.4±18.8* _16.8±3.7* _108.2±19.5* _0.2±0.1* _14.0±3.1

PSD+SAL 23.5±5.4 7.3±3.1 30.8±7.3 16.0±2.5 11.7±2.5

PSD+PILO 92.8± 24.7* _19.2

±4.8* _112.1

±25.1* _6.4

±5.4 7.0±1.5 Dataareexpressedasmean±SEM.

G.Matosetal./NeuroscienceLetters515 (2012) 137–140 139

Fig.1.DNAdamageexpressedastailmomentinbraincells.CTRL+SAL:SALrats keptintheirhomecages;CTRL+PILO:PILOratskeptintheirhomecages;TSD+SAL: SALratssubmittedtoTSD;TSD+PILO:PILOratssubmittedtoTSD;PSD+SAL:SAL ratssubmittedtoPSD;PSD+PILO:PILOratssubmittedtoPSD.*P<0.05when com-paredtorespectivecontrolgroup.#_{P<0.05whencomparedwithvehiclegroup.} CTRL:homecagecontrols;PILO:rats submittedtopilocarpine-inducedstatus epilepticus,PSD:paradoxicalsleepdeprivation,SAL:vehiclegroup,TSD:totalsleep deprivation.

normaland epilepticgroups(F1,54<0.85;P>0.43).Furthermore, rearingbehaviordidnotdiffersignificantlybetweenthegroups (F1,53=1.47;P=0.23).

3.2. Cometassay

Thegroupsinjectedwithpilocarpinedemonstratedextensive genotoxicwhencompared totheSALanimalsinallprocedures (F1,23=47.70;P<0.001)(Fig.1).Sleepdeprivationinduced signifi-cantincreaseinthenumberofdamagedcellsinbothSALandPILO rats(F1,23=11.51;P<0.001).TheposthoctestsrevealedthatPSD, butnot TSD,inducedsignificantDNA damageintheSALgroup (P=0.03vs.P=0.22,respectively),whereasbothTSD(P=0.02)and PSD(P=0.003)inducedsignificantneuronalgenotoxicityinPILO rats.

4. Discussion

Inthepresentstudy,weshowedthatacutesleepdeprivation causesDNAdamageinthebrainsofratssubmittedto pilocarpine-inducedSE.Althoughpilocarpineratsexhibitedhyperactivity,this appearednottobeinfluencedbyacutesleepdeprivation.

The epilepsy-induced hyperactivity reported in the current studyis consistentwithearlier findingsshowingan augmenta-tion of locomotor activityduring thelight period in rats with epilepsy [24]. In the present study, ambulation was measured between1PMand3PM.Thehyperactivityobservedinourstudy couldbepartially dueto anabnormalsleep-wake cycle inrats withepilepsy,oncetheseanimalspresentedanincreaseofactive wakefulnessduringtheafternoon[15].Infact,ratswithepilepsy showalteredsleeparchitectureandanabnormaldistributionof sleep-wakephasescomparedwithhealthyrats[15].Inaddition toepilepsy-inducedsleep-wakealterations,lesionsinmanybrain areas,includingthepiriformcortex,hippocampalformationand thalamus,couldpotentiallyexplaintheimpairmentsinlocomotor activityandotherbehaviors[25].Furthermore,ourdata demon-stratedthatthepathologicalmechanismsinvolvedinlocomotor hyperactivityinratssubmittedtopilocarpine-inducedSEwerenot affectedbyacute sleepdeprivation.In fact,TSD didnot induce hyperactivityinmiceandlongerperiodsofPSDwererequiredto producebehavioralalterations[1,19].Itisimportanttomention

that,evenwiththeabsenceofeffectofsleepdeprivation; locomo-toractivityisafinalparameterresultingfromacomplexprocess andmaynotbesufficientlysensitiveforthisapproach.

Ourresultsrevealedthatepilepsyinducesgeneticdamagein brain cells, and that sleep loss potentiates this damage. These findings demonstrate that genetic changes in the healthy and epilepticbrainscanbeginevenafterjustafewhourswithoutsleep. Pilocarpine-inducedSEhasbeenshowntocauseneuronaldeathin thebrain[8,22,23]andinternucleosomalDNAfragmentationhas beenobservedinnecroticcellsat24and72hfollowingSE[8].The resultsofthepresentstudyshowthatepilepsyalsoincreases vul-nerabilitytoDNAdamage.Inaddition,ourobservationofgenotoxic effectsofsleepdeprivationisconsistentwiththeresultsof previ-ousstudiesreportingDNAdamageinthebrainsof healthyrats after24hofPSD[3].Moreover,nitricoxide-mediatedregulationof sleepphases[21]andoxidativedamageinmicedeprivedofsleep [13]maypotentiallyberelatedtosleepdeprivation-inducedDNA damage[3].

Epilepsyclearlyincreasedtheamountofgenotoxicdamageafter TSDandPSD.However,onlyPSDinducedadetrimentaleffectin healthy rats.TSD and epilepsycombined produced detrimental effects,possiblyasaresultoftheabnormalsleep-wakecyclein animalswithepilepsy[15].For instance,ratswithspontaneous recurrentseizuresshowincreasedNREMsleepduringthe morn-ing[15],theperiodduringwhichratsweresubmittedtoTSD.As aconsequence,ratsinthePILOgroupwereexposedtoahigher pressureforsleepcomparedwiththevehiclegroup.Infact, dur-ingtheperiodofTSD,epilepticratsshowednoexploratoryactivity andthevastmajorityofanimalswithepilepsyshowedextreme drowsinessthroughouttheentireexperimentalperiod(whereas healthyanimalsweremorealert).However,itispossiblethatother variable,suchasthehighlevelsofcorticosteronepresentinrats withepilepsy[26]and/orthestressinducedbygentlehandling [27]couldcorroboratedwithaforementionedresult.

Fewstudieshaveinvestigatedthedifferencesinthemolecular effectsoftotalversusselectivesleeploss.Leeetal.[14]reported thatneitherTSDnorPSDaffectedthestabilityofreferencegenes inbrainandblood.Incontrast,behavioraltests,suchastestsof plus-mazediscriminativeavoidanceorthepassiveavoidancetask, revealeddifferencesbetweentheeffectsofPSDandTSDon state-dependentlearninginmice[17].

Inconclusion,weshowthatepilepsyinducesmarkedlocomotor hyperactivityandDNAdamageinrats.However,thedetrimental effectofsleeplossonbraincellswasnotaccompaniedbybehavioral changes.InviewofthefactthatDNAdamageisanimportantstep intheeventsleadingtogenomicinstability,ourresultsemphasize thepotentialhealthrisksassociatedwithacutesleepdeprivation inpatientswithepilepsy.

Acknowledgements

Theauthorsgratefullyacknowledgetheinvaluableassistanceof MarinaF.Aguiar,ElizaY.SonodaandWaldemarksLeite.Thiswork wassupportedbyAssociac¸ãoFundodeIncentivoàPesquisa(AFIP), CNPq,Fundac¸ãodeAmparoàPesquisado Estado deSão Paulo (CEPID#98/14303-3toS.T.,#10/15110-8toG.M.,#09/01030-5to T.A.A.,#10/50129-1toC.H.andCInAPCeprogram)andMCT (INCT-InstitutoNacionaldeNeurociênciaTranslacional).M.L.A.,S.T.,F.A.S. andE.A.C.arerecipientsoftheCNPqfellowship.

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