AMPA receptors mediate passive avoidance deficits induced by sleep deprivation

(1)

ContentslistsavailableatScienceDirect

Behavioural

Brain

Research

jo u r n al 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 / b b r

Research

report

AMPA

receptors

mediate

passive

avoidance

deﬁcits

induced

by

sleep

deprivation

Francisco

Paulino

Dubiela

a

_,

_Claudio

_Marcos

_Queiroz

b

_,

_Karin

_Di

_Monteiro

_Moreira

a

_,

Jose

N. Nobrega

c

_,

_Luciane

_Valéria

_Sita

d

_,

_Sergio

_Tuﬁk

a

_,

_Debora

_Cristina

_Hipolide

a,∗

a_Departamento_de_{Psicobiologia,}_Universidade_Federal_de_São_Paulo,_São_Paulo,_Brazil

b_Brain_Institute,_Universidade_Federal_do_Rio_Grande_do_Norte,_Natal,_Brazil

c_Neuroimaging_Research_Section,_Center_for_Addiction_and_Mental_Health,_Toronto,_Canada

d_Departamento_de_Anatomia,_Instituto_de_Ciências_Biomédicas,_Universidade_de_São_Paulo,_São_Paulo,_Brazil

h

i

g

h

l

i

g

h

t

s

•[3_H]AMPA_binding_is_reduced_in_the_hippocampal_formation_by_sleep_deprivation.

•Sleeprecoveryrestored[3_H]AMPA_binding_reduction_induced_by_sleep_deprivation.

•GluR1mRNAlevelsareunaffectedbysleepdeprivation.

•AMPARpotentiatorrevertedsleepdeprivation-inducedimpairmentofmemoryretention.

•AMPARantagonisthadnoeffectonsleepdeprivation-inducedimpairmentofmemoryretention.

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received30July2013

Receivedinrevisedform

18September2013

Accepted21September2013

Available online 28 September 2013 Keywords:

Sleepdeprivation

AMPAreceptor

Passiveavoidancetask

Aniracetam GYKI-52466

a

b

s

t

r

a

c

t

Thepresentstudyaddressedtheeffectsofsleepdeprivation(SD)onAMPAreceptor(AMPAR)binding inbrainregionsassociatedwithlearningandmemory,andinvestigatedwhethertreatmentwithdrugs actingonAMPARcouldpreventpassiveavoidancedeﬁcitsinsleepdeprivedanimals.[3_H]AMPA

bind-ingandGluR1insituhybridizationsignalswerequantiﬁedindifferentbrainregionsofmaleWistar ratseitherimmediatelyafter96hofsleepdeprivationorafter24hofsleeprecoveryfollowing96h ofsleepdeprivation.Anothergroupofanimalsweresleepdeprivedandthentreatedwitheitherthe AMPARpotentiator,aniracetam(25,50and100mg/kg,acuteadministration)ortheAMPARantagonist GYKI-52466(5and10mg/kg,acuteandchronicadministration)beforepassiveavoidancetraining.Task performancewasevaluated2hand24haftertraining.Asigniﬁcantreductionin[3_H]AMPA_binding_was

foundinthehippocampalformationofSDanimals,whilenoalterationswereobservedinGluR1mRNA levels.Thehighestdoseofaniracetam(100mg/kg)revertedSD-inducedimpairmentofpassive avoid-anceperformanceinbothretentiontests,whereasGYKI-52466treatmenthadnoeffect.Pharmacological enhancementofAMPARfunctionmayreverthippocampal-dependentlearningimpairmentsproduced afterSD.WearguethatsucheffectsmightbeassociatedwithreducedAMPARbindinginthehippocampus ofsleepdeprivedanimals.

1. Introduction

Sleeplossisbecominganincreasinglyproblemofmodern

soci-etyresultingindeleteriouseffectsondailyfunctions[1],especially

hinderingcognitiveprocessesinhumans[2].Likewise,animal

stud-ieshavealsorevealedthatsleepdeprivation(SD)resultsinmajor

impairmentsinavarietyofcognitivefunctions,particularlythose

∗ Correspondingauthorat:DepartamentodePsicobiologia,UniversidadeFederal

deSãoPaulo,RuaNapoleãodeBarros,925,SãoPaulo,SP04024-002,Brazil.

Tel.:+551121490155;fax:+551155720754.

E-mailaddress:dchipolide@unifesp.br(D.C.Hipolide).

associatedwithlearningandmemory[3–7].Interestingly,

learn-ingdeﬁcitsobservedafterseveraldaysofsleepdeprivationcanbe

preventedbyashortperiodofsleeprecovery.Thus,previous

stud-ieshaveshownthatdeﬁcitsdisplayedbyratsinpassiveavoidance

(PA)andcontextualfearconditioningtasksafter3–4daysofSDare

preventedifitisfollowedby24hofsleeprecovery[5–7].Despite

theevidencethatunderlyingmnemonicmechanismsalteredby

SDarenormalizedafterashortperiodofsleeprebound,theirexact

identitiesarestillamatterofdebate,asseveralneurotransmitter

systemsareinﬂuencedbySD[8].

One potential mechanism that may be altered by SD is

thefunctionofglutamatergicalpha

amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR). These receptors

(2)

190 F.P.Dubielaetal./BehaviouralBrainResearch257 (2013) 189–196

mediatethemajorityoffastexcitatorytransmissioninthecentral

nervoussystem,andarecloselyinvolvedinmnemonicprocesses

andhippocampalsynapticplasticity[9–11].AMPARaretetramers

composedoffoursubunits(GluR1–GluR4)[12],andareinﬂuenced

bychangesinglutamatergictransmissioninducedeither

pharma-cologicallyorbybraininjury(e.g.,cerebralischemia)[13–15].In

thiscontext,sleepdeprivation hasbeenshown toincrease

glu-tamatergictransmissionin thecortex and hippocampusofrats

[16,17],whichmaytriggerAMPARplasticity.Previousstudies

how-everhaveproducedconﬂictingevidenceregardingSDeffectson

AMPARfunction.Vyazovskiyetal.[18]reportedincreased

expres-sionof the AMPARsubunit GluR1 in corticaland hippocampal

regionsaftertotalsleepdeprivation,whileMcDermottetal.[19]

foundnochanges inAMPAR currentsrecordedin hippocampal

outside-outpatchesfromratsdeprivedofsleepfor72h.Finally,

Ravassardetal.[20]havereporteddecreasedlevelsof

hippocam-palGluR1withinthesameSD period.Althoughmuchattention

hasbeengiventoSDeffectsonAMPARfromthehippocampal

for-mation,thereisascarcityofdataaboutotherlimbicstructures.

Wereasonedthatacomprehensivewide-rangeanalysisofAMPAR

bindingin brain regions associated withlearning and memory

mightcontributetoelucidatethisissue.

AnotherapproachtoexaminetheinvolvementofAMPARinthe

learningdeﬁcitsinducedbySDispharmacologicalmanipulationof

thesereceptors,anapproachwhichhasbeenwidelyusedto

inves-tigatememorymechanisms[10,21].AMPARpotentiators,suchas

aniracetamandampakines,producepositiveallostericeffectson

theirsites [22,23]. Because of these effects, this class of drugs

hasbeenusedtopreventmemorydeﬁcitsinducedbya variety

ofconditions, suchas brainlesions,aging and pharmacological

amnesia[24–26].ConcerningSD-inducedmemorydeﬁcits,ithas

beenshownthattheampakineCX717isabletoameliorate

cog-nitivedeﬁcitsproducedbylackofsleepinmonkeysandhumans

[27,28].AnotherlineofevidenceshowsthatAMPARantagonists,

suchasGYKI52466,amelioratememorydeﬁcitsinanimal

mod-elsofbrainischemia[29,30].Thus,wehypothesizedthatasimilar

treatmentmightreverseSDdeleteriouseffectsonlearning

perfor-mance,asSDandbrainischemiaseemtosharesimilaritieswith

respecttoincreases in glutamatergiclevelsand AMPAR

modu-lation[15,31,32].Moreover,chronictreatmentwithGYKI52466

hasbeenshowntoincreaseAMPARfunctionbyinducingan

up-regulationofAMPARbothincorticalandhippocampalregions[13].

Altogether,thesereportssuggestthatmanipulationofAMPARby

pharmacologicalagentsmaypreventlearningdeﬁcitsinducedby

SD.

Therefore, the objectives of the present work were (1) to

examine AMPAR binding after sleep deprivation and recovery

in brain regions involved in learning and memory processes,

along with an analysis of GluR1 mRNA expression in regions

wherebindingwasaffectedbySD;and(2)toevaluateiflearning

deﬁcitsinducedbySDcouldbepreventedbyAMPARmediation

througheitheraniracetamorGYKI52466treatment.We

hypoth-esizedthat [3_H]AMPA _binding_would _be _reduced _after_SD, _and

thatpharmacologicaltreatmentsaimedatrestoringAMPAR

func-tionwouldattenuatedeﬁcitsofpassivelearningassociatedwith

SD.

2. Materialsandmethods

2.1. Subjects

MaleWistarrats,aged3months(250–350gofbodyweight),

wereobtainedfromtheCEDEMEanimalfacilityatUNIFESP.

Ani-malswerekeptingroupsof5inwire-meshcagesinaroomunder

controlledtemperature(23±2◦C)and12hlight/darkcycle(lights

onat07:00),withfoodandwateradlibitum.Procedureswere

con-ductedinconformitywiththeguidelinesforthecareanduseof

laboratoryanimalsoftheBrazilian Societyof Neuroscienceand

Behavior,andwereapprovedbythebytheEthicsCommitteeof

UNIFESP(CEP#2075/07).

2.2. Sleepdeprivationprocedure

Ratsweresleepdeprivedusingthemodiﬁedmultipleplatform

method[33].Sleepdeprivationwasconductedbyplacingratsin

alargewatertank(145cm×44cm×45cm)thatcontained

nar-rowplatforms(6cmindiameter).Thisprocedure,whichrelieson

themuscleatoniathataccompaniesparadoxicalsleep,completely

abolishesthissleepstageandalsodecreasesslow-wavesleepto

someextent[34].Thepresenceofmultipleplatformsmitigatesthe

movementrestrictionandisolationassociatedwithearlier

tech-niques ofsleep deprivation.Sleep-deprived animals(SD group)

weresubjectedtosleepdeprivationfor96h.Animalsinthecage

control(CC)groupremainedintheirhomecagesinthesameroom

wheresleepdeprivationprocedurestookplace.

2.3. [3_H]AMPA_receptor_binding

Ratsweresacriﬁcedbydecapitationeitherimmediatelyafter

thesleepdeprivationprocedureoraftersleepdeprivationfollowed

by24hofsleeprecovery(SR).Brainswereimmediatelyremoved,

frozenoverdryiceandstoredat−80◦_C_for_[3_H]AMPA_binding_and

GluR1insituhybridizationessays,followingproceduresofNobrega

etal.[35] andGitaíet al.[36],respectively,withmodiﬁcations.

Coronalbrainsections(20␮mthick)wereseriallycutat−20◦_C,

mountedonslides,anddried.On thedayofthebindingassays

(N=8/group),sectionswerepreincubatedforremovalof

endoge-nousligandswith50mMTris–acetatebuffer(pH7.2)containing

100mMKSCNfor30minat4◦C.AMPAreceptorswerelabeledwith

10nM [3_H]AMPA _{(Perkin-Elmer} _Life_Sciences) _in _{preincubation}

bufferfor45minat4–8◦Cwithorwithout10mMquisqualateas

unlabeledligandforthedeterminationofnon-speciﬁcortotal

bind-ing,respectively.Thisprocedurewasfollowedbyfourrinsesinthe

buffer(4seach)at4–8◦C.Finally,thesectionsweredippedtwice

into100mlacetonecontaining2.5mlglutaraldehyde,driedunder

astreamofcoldair,andexposedtoX-rayﬁlms(KodakBioMax

MR-1,AmershamPharmaciaBiotech)for6weeksalongwithcalibrated

[3_H] _standards. _After_ﬁlm _development, _{densitometric} _analyses

wereperformed using an M2MCID system(Imaging Research,

MCID,St.Catharines,Ontario).Calibrationcurvesconstructedfrom

[3_H]_standards_allowed_the_conversion_of_integrated_optical_density

intotissueradioactivityvalues(␮Ci/g).

2.4. InsituhybridizationofGluR1subunit

Mounted sectionsof CC, SD and SR animals (N=6–7/group)

weregradually warmedtoroom temperature,post-ﬁxed in 4%

formaldehyde,pH7.4andrinsedin10mmol/LPBS,pH7.4,after

whichtheyweretreatedfor30minwithproteinaseK(0.5␮g/ml;

LifeTechnologies, Gaithersburg,MD, USA), EDTA(0.05, pH8.0)

andTris–HCl(0.1M,pH8.0)atroomtemperature.Thiswas

fol-lowedby10minofacetylationwithtriethanolamine–HCl(Avocado

Research,Heysham,UK),rinsesin 2×SSCanddehydrationin a

gradedethanolseries.Hybridizationwasperformedusing35_S-UTP

labeledriboprobescomplementarytothesequencesofinterest,

generatedbyinvitrotranscription(Promega,Madison,USA).

Dur-ing transcription a cDNA sample was ampliﬁed by PCR using

compoundprimersmadeupofconsensuspromotersequencesfor

eitherSP6RNApolymerase(ATTTAGGTGACACTATAGAA)attached

atthe5endoftheleftprimerand/orforT7RNApolymerase

(3)

Fig.1.SchematicrepresentationofdesignusedtoaddressAMPARmediationonpassiveavoidanceaftersleepdeprivation.CC:cagecontrolgroup,SD:sleepdeprivation

group,V:vehicletreatment.

complementarytothetargetgene.Thelabeledriboprobesprepared

forGluR1werecomplementarytoGenbank#NM031608(bases

108–405),andwerediluted(106cpm/ml)inhybridizationsolution.

Thesolutionconsistedof50%formamide,1%sodiumthiosulfate

and2%5Mdithiothreitol.Thehybridizationsolution(120␮l)and

acoverslipwereappliedtoeachslide,andthesectionswere

incu-batedat57◦Covernight.SectionswerethensoakedinRNaseA

(20_␮g/ml;USB,Cleveland,OH,USA)for30min,afterwhichthey

werewashedfor30mininRNasebuffer(5Mof10%NaCl,1Mof

4%Tris,pH8.0,and0.5Mof0.2%EDTA,pH8.0,insterilewater).

TheywerethenrinsedindecreasingconcentrationsofSSC,

dehy-dratedandplacedinX-rayﬁlmcassetteswithBMR-2ﬁlm(Eastman

Kodak,Rochester,NY,USA)for4days.Subsequently,slideswere

dippedin NTB2photographicemulsion (EastmanKodak), dried

andstoredwithdesiccantinfoil-wrappedslideboxesat4◦Cfor

28 days. Slides were developed withD-19 developer(Eastman

Kodak),counterstainedwithThionin,dehydrated,clearedinxylene

andcoverslippedwithDPX.Controlsectionsincubatedwithsense

probesshowednosignal.GluR1mRNAexpressionwasevaluatedby

integratedopticaldensityperformedonemulsion-coatedsections

underdark-ﬁeldillumination,usingtheImage-ProPlussoftware

(MediaCybernetics,SilverSpring,MD,USA).Thehybridization

sig-nalwasobtainedfromaconstantarea(0.15mm2₎_of_CA1_and_CA3

stratumpyramidaleandDGgranulecelllayer,normalizedagainst

anadjacentareathatdoesnotexpressGluR1mRNA.

2.5. AMPARmediationofpassiveavoidance

InordertoexamineAMPARinvolvementinSD-induced

learn-ingdeﬁcits,aseparatesetofanimalswassubjectedto96hsleep

deprivationasdescribedaboveandsubmittedtodifferentprotocols

ofdrugtreatment(N=8–14/group,Fig.1).Animalswereacutely

treatedwitheithervehicle,theAMPARpotentiatoraniracetam(25,

50and100mg/kg;p.o.)ortheAMPARantagonistGYKI52466(5

and10mg/kg;i.p.)1hbeforebeingsubmittedtoastep-through

passiveavoidancetask,appliedimmediatelyafterSDprocedure.

InordertoevaluateAMPARplasticityduringSD, anothergroup

ofanimalsweretreatedwitheithervehicleorGYKI52466(5and

10mg/kg; i.p.), twicea day(1 administrationevery 12h) for 4

days(8injectionsintotal).Inthisexperiment,thePAtaskstarted

immediatelyafterthe96hofSDand19hafterthelast

adminis-trationoftheAMPARantagonist.In allexperimentsaniracetam

(Sigma–Aldrich,Brazil)wassuspendedina0.25%carboxymethyl

cellulosesolutioncontainingafewdropsofTween80.GYKI52466

(Sigma–Aldrich, Brazil) was dissolved in 0.1N HCl and diluted

indistilledwater.Bothdrugswereadministeredinavolumeof

2ml/kg.

2.6. Passiveavoidancetask

Thepassiveavoidanceapparatusconsistedoftwoacrylicboxes,

each measuring21cm×26cm×27.5cm, connectedbya sliding

door.One of theboxes wasthe safecompartment, withwhite

walls,whereastheotherboxwastheaversivecompartment,with

blackwalls.Theﬂooroftheapparatusconsistedofparallelmetallic

rods,each0.4cmindiameter,1.2cmapart,connectedtoan

elec-tricshockgenerator(AVSProjetosEspeciais,SãoPaulo,Brazil).PA

performancewasevaluatedusingonetrainingsessionandtwo

sub-sequentretentiontestsessions.Inthetrainingsession,eachanimal

wasplacedinthesafecompartmentoftheapparatus,withthe

slid-ingdoorclosed.Tensecondslater,thedoorwasopenedand,as

soonastheanimalcrossedtotheaversivecompartmentthedoor

wasclosed,latencytoenterwasrecorded,andtheanimalreceived

asinglefootshock(0.8mA/1s).Fifteensecondslater,theanimal

wasremovedfromtheapparatusandmovedtoitshomecage.Two

hourslater,theﬁrstretentiontesttookplace.Eachanimalwasagain

placedinthesafecompartmentoftheapparatus,andthelatency

tocrosstotheaversivecompartmentwasrecordedwithoutany

shockpresentation.Eachanimalwasallowed300stocrossoverto

theaversivecompartment.Asinthetrainingsession,acrossingwas

deﬁnedasthepresenceoffourpawsintheaversivecompartment.

Ifitdidnotdoso,theanimalwasremovedfromtheapparatusand

alatencyof300swasassigned.Twenty-fourhoursaftertraining,a

secondretentiontestwasconductedinthesameawayasdescribed

above.

2.7. Statisticalanalyses

[3_H]AMPA_binding_and_GluR1_in_situ_{hybridization}_data_were

analyzedbyseparateone-wayANOVAsforeachbrainregion.In

thepassiveavoidancetask,latenciestocrosstothedarkchamber

werecomparedusingtwo-wayrepeatedmeasureANOVAs,with

groupasthebetweensubjectfactorandsessionasthewithin

(4)

192 F.P.Dubielaetal./BehaviouralBrainResearch257 (2013) 189–196 Table1

[3_H]AMPA_binding_after_sleep_deprivation_and_recovery.

Controlcage(n=8) Sleepdeprivation(n=8) Sleeprecovery(n=8) Cortex

Frontalassociationcortex 0.663±0.04 0.569±0.03 0.631±0.03

Prelimbiccortex 0.646±0.02 0.713±0.05 0.714±0.03

Cingulatecortex,area1 0.621±0.03 0.603±0.04 0.612±0.06

Cingulatecortex,area2 0.627±0.03 0.586±0.03 0.600±0.06

Infralimbiccortex 0.683±0.02 0.732±0.06 0.743±0.05

Piriformcortex 0.691±0.03 0.677±0.03 0.723±0.03

Entorhinalcortex 0.650±0.03 0.606±0.03 0.622±0.02

Lateralseptalnucleus

Dorsalpart 0.812±0.03 0.757±0.06 0.709±0.04 Intermediatepart 0.416±0.05 0.433±0.04 0.410±0.03 Ventralpart 0.412±0.03 0.391±0.04 0.369±0.02 Caudateputamen Dorsomedial 0.348±0.02 0.380±0.03 0.326±0.03 Dorsolateral 0.361±0.02 0.394±0.04 0.336±0.03 Ventrolateral 0.459±0.04 0.464±0.04 0.448±0.04 Posterior 0.336±0.01 0.340±0.02 0.327±0.02

Accumbensnucleus,core 0.617±0.04 0.623±0.04 0.592±0.05

Accumbensnucleus,shell 0.655±0.04 0.688±0.03 0.621±0.05

Amygdala Basolateralnucleus 0.607±0.05 0.571±0.04 0.615±0.02 Centralnucleus 0.274±0.03 0.263±0.03 0.275±0.02 Medialnucleus 0.411±0.04 0.339±0.03 0.370±0.01 Hippocampalformation CA1 1.115±0.03 0.998±0.03* _1.076_±_0.03 CA3 0.687±0.04 0.538±0.04* _0.596_±_0.02 Dentategyrus 1.087±0.05 0.925±0.04* _1.013_±_0.03

Valuesareexpressedasmean±SEMin␮Ci/goftissue.

* _P_<_0.05_vs._control_cage_animals_(Duncan_test).

Table2

HippocampalGluR1mRNAinsituhybridizationaftersleepdeprivationandrecovery.

Controlcage(n=6) Sleepdeprivation(n=7) Sleeprecovery(n=7)

CA1 28,761±5152 26,315±10,153 54,530±9014

CA3 37,658±7232 40,189±15,621 76,375±14,595

Dentategyrus,dorsalblade 31,977±4126 34,576±7625 43,670±6899

Dentategyrus,ventralblade 29,701±3531 36,048±7531 50,038±4453

Valuesareexpressedasmean±SEMoftheintegratedopticaldensityinarbitraryunits.

analyseswerecarriedoutusingDuncanmultiplerangetest,with

thelevelofsigniﬁcancesetatP<0.05.

3. Results

3.1. Effectsofsleepdeprivationandsleepreboundon[3_H]AMPA

bindingandhippocampalGluR1expression

Sleepdeprivationproducedsigniﬁcantreductionin[3_H]AMPA

binding in localized brain regions in comparison to controls

(Table1).Majordifferenceswereobservedinthehippocampus,

e.g.,dentategyrus,CA3andCA1(Fig.2aandb).Ontheotherhand,

otherlimbicstructures(e.g.,amygdala)didnotdifferfromCCand

SRgroups(Fig.2candd).

Interestingly,24-hofsleepreboundwassufﬁcienttopartially

restorehippocampalAMPAbindingtocontrollevels,sinceSRdid

notdifferfrombothCCorSDanimals(P>0.11).

Regarding GluR1 expression, no major differences were

observedinbetweencontrols,SDandSRgroupsinallhippocampal

areas(Table2),althoughanon-signiﬁcantincreasewasobserved

inSRanimals.

3.2. EffectsofAMPAmediationonpassiveavoidanceaftersleep

deprivation

3.2.1. Acuteaniracetamtreatment

SDanimalsdisplayedsigniﬁcantlowerlatenciesinthepassive

avoidancetestthanCCanimals2hand24haftertraining(group

effectF[7,70]=10.16,P<0.05,N=8–10/group;two-way ANOVA;

Fig.3).Thelatencytocrosstotheothercompartmentdidnot

dif-ferinthetrainingsessionforbothgroups(P>0.05;NS;Fig.3,left

panel).Onlythehigherdoseofaniracetam(100mg/kg)increased

the latency in the test performed 2h and 24h after training

(interactionbetweengroupvs.treatmentF[14,140]=6.15,P<0.05;

two-wayANOVA;Fig.3).Aniracetamadministrationdidnotchange

the performance of CC animals (P>0.05; NS), irrespectively of

dose.

3.2.2. AcuteGYKI52466treatment

Results are shown in Fig. 4. Two-way repeated measures

ANOVAindicatedsigniﬁcantmaineffectsofgroup(F[5,46]=17.046,

P<0.05, N=8–10/group) and session (F[2,92]=160.63, P<0.05),

and a signiﬁcant interaction (F[10,92]=8.3627, P<0.05). Post

hocanalysesrevealedthat allanimalsbehaved similarly inthe

training session (P>0.05; NS);while SD animalsdisplayed

sig-niﬁcant lower latencies in the passive avoidance test than CC

animals2hand24haftertraining(P<0.05)irrespectivelyofthe

treatment.

3.2.3. ChronicGYKI52466treatment

Results are shown in Fig. 5. Two-way repeated measures

ANOVAindicatedsigniﬁcantmaineffectsofgroup(F[5,54]=11.676,

P<0.05, N=8–14/group)andsession (F[2,108]=114.36,P<0.05),

and a signiﬁcantinteraction between them (F[10,108]=6.4066,

(5)

Fig.2. Illustrationofdown-regulationofAMPARinthehippocampusofsleepdeprivedrats.(a)[3_H]AMPA_binding_in_hippocampal_regions._(b)_Columns_showing_[3_H]AMPA

bindingofCA1,CA3anddentategyrusinCC,SDandSRgroups.(c)[3_H]AMPA_binding_in_amygdaloid_areas._(d)_Columns_showing_[3_H]AMPA_binding_of_basolateral_(BLA),

central(CeA)andmedial(MeA)amygdale.Columnsindicatemeans±SEMin␮Ci/g;*P<0.05(Duncantest).

Fig.3.Effectsofacuteaniracetamtreatmentonpassiveavoidanceperformanceinsleepdeprivedanimals.CC:cagecontrolgroup,SD:sleepdeprivationgroup,V:vehicle

treatment.N=8–10/group.Dataaremeans±SEMinseconds;*P<0.05vs.respectivecagecontrolgroup(Duncantest).

similarlyinthetrainingsession(P>0.05;NS);whileSDanimals

displayedsigniﬁcantlowerlatenciesinthepassiveavoidancetest

thanCCanimals2hand24haftertraining(P<0.05)irrespectively

oftreatment.

4. Discussion

Sleepdeprivation resultedin impairment of emotional- and

(6)

Fig.4. EffectsofacuteGYKI52466treatmentonpassiveavoidanceperformanceinsleepdeprivedanimals.CC:cagecontrolgroup,SD:sleepdeprivationgroup,V:vehicle

Fig.5.EffectsofchronicGYKI52466treatmentonpassiveavoidanceperformanceinsleepdeprivedanimals.CC:cagecontrolgroup,SD:sleepdeprivationgroup,V:vehicle

previousreports[3–7].InconsonancewithstudiesfromIzquierdo

etal.(forreview,see[37]),theuseoftworetentiontestsinthePA

taskdidnotinﬂuencethelongtermretentiontest,asthe

perfor-manceofbothcontrolandsleepdeprivedanimalsdidnotchangein

betweentestsessions.Accordingly,apaststudyusingthePAtask

withshortandlongtermretentiontestsinseparateexperiments

yieldedsimilarresultsinrespecttothelearningdeﬁcitinduced

bySD[7].Thepresentsetofexperimentsshowedthathighdoses

(100mg/kg)ofaniracetam,anAMPARpotentiator,prevents

learn-ingdeﬁcitsofsleepdeprivedanimals.Interestingly,animalsfrom

thecontrolgroupdidnotshowPAperformanceimprovementafter

aniracetam,whichmayhavebeenduetoaceilingeffectduringthe

retentiontests.Infact,aniracetamhasbeenreportedtoenhance

learningonlyincasesofimpairment[24–26].Ontheotherhand,the

AMPARantagonistGYKI52466wasunabletomodifySDinduced

learningdeﬁcitsorimpair learningin animalsfromthecontrol

group,insingleorrepeatedadministrations.Theseobservations

suggestamorecomplexpicturethanonlyup-anddown-regulation

ofreceptortranscriptionandtranslation.Wediscusstheﬁndings

andlimitationsofthepresentstudyinlightofmediationofAMPAR

functioninsleeplossconditions.

Toourknowledge,thepresentworkistheﬁrstinvestigation

ontheeffects ofsleepdeprivation onAMPARinmultiplebrain

regions,includingseveralthoughttobeinvolvedinlearningand

memory.Ourﬁndingswith[3_H]AMPA_binding_revealed_a_selective

decreaseinthehippocampalformationafter96hofSD,whileother

limbicregionsassociatedwithaversivememoryremained

unaf-fected,includingtheentorhinalcortexandbasolateralamygdala

[38,39].ThehippocampalbindingofAMPARwasnormalizedafter

24hofsleeprecovery,aphenomenonthatmayrelatetoresults

from previous studies showing an amelioration of SD induced

deﬁcitsinhippocampus-dependentmemorytasks,whenanimals

aregiventheopportunitytosleepbeforelearning[5–7].Asimilar

patternisobtainedregardingsynapticplasticity:while72hSD

pre-ventshippocampallongtermpotentiation,eithershort(150min)

or long (24h) periods of sleep recovery reverses this effect

[5,20].

WedidnotﬁndsigniﬁcantdifferencesinGluR1mRNA

expres-sionamongcontrol,SDandSRanimalsinanyhippocampalregion,

whichisinagreementwiththeobservationsofRavassardetal.[20].

TheseinvestigatorsdescribedreducedGluR1proteinexpression,

butnotGluR1mRNA,inthedorsalhippocampusofratsdeprived

ofsleepfor72h.ItisthuslikelythatdecreasedhippocampalAMPAR

functionafterSDisduetodownstreamtranscriptionmechanisms,

aspreviousreportshaveshownreducedphosphorylationofGluR1

inthehippocampusofsleepdeprivedmiceandrats[20,40].GluR1

phosphorylation,animportantstepforAMPARincorporationinto

synapticmembranes[41]isaugmentedafterPAlearning[11]thus

suggestingthatSDeffectsonGluR1phosphorylationmayhave

(7)

Based on previous studies showing antiamnesic effects of

AMPARantagonistsina rodentmodelofbrainischemia[29,30]

andconsideringsimilareffectsinducedbythismodelandbySDon

theglutamatergicsystem[15,31,32],wesoughttoverifyif

treat-mentwithGYKI52466couldpreventthelearningdeﬁcitsinduced

bysleepdeprivation.Contrarytoourhypothesis,chronicoracute

treatments with this drug didnot improve PA performance in

sleepdeprivedanimals,suggestingthatdifferentmechanismsmay

underliethememorydeﬁcitsinducedbybrainischemiaandsleep

deprivation.Likewise,whilethereisevidencethatchronic

admin-istrationofGYKI52466augmentsAMPARbindingincorticaland

hippocampalregionsinnaïveanimals[13],theunderlying

mech-anismbywhichthisoccursremainselusive;itispossiblethatit

dependsonspeciﬁcconditions,i.e.SDanimalsmayberesistant

tothisparticularAMPARupregulationeffect.Furtherstudiesare

requiredtoaddressthisissue.

ThepresentevidenceofdecreasedhippocampalAMPAR

bind-inginsleepdeprivedanimalsseemstoindicatethathighdosesof

AMPARpotentiatorsarerequiredinordertoovercomelow

avail-abilityoftheirsitesandthereforeameliorateSDinducedcognitive

deﬁcits.ThisisinagreementwithrecentevidencefromBoyleetal.

[28],whichshowedthatcognitiveimpairmentdisplayedbysleep

deprivedhumanswereonlycounteractedwiththehighestdose

oftheampakineCX717.Although lowtointermediate dosesof

aniracetamareknowntoproduceimprovementinseveral

mod-elsofmemoryimpairment[24–26],the25 and50mg/kgdoses

used inthe current study didnot blockthe deleterious effects

ofSD.Itisplausiblethatthisinefﬁcacycouldbeduetothelow

availabilityofhippocampalAMPARafter96hSD.Interestingly,a

similarresultwasobservedintheGABA–benzodiazepinesystem,

inwhichthepromnesicactionof␤-CCM(abenzodiazepineinverse

agonist)wasnotobservedinsleepdeprivedanimals,alongwith

evidenceofdown-regulationofitsbindingsitesintheentorhinal

cortex[7].

Concerning the mechanisms by which aniracetam improves

learning deﬁcitsin sleepdeprived animals, besidesits positive

modulationofAMPAR[22,23],aniracetamisalsoknowntoactivate

proteinkinasesincludingPKC[41,42].Theactivityofthis

particu-larenzymeiscrucialtoGluR1phosphorylationandensuingAMPAR

incorporationintohippocampalsynapsesduringlongterm

poten-tiation(LTP)[43],whichsuggestsapotentialtreatmenttoreverse

bothAMPARbindingreductionandimpairedLTPinducedbysleep

deprivation[5,19,20].Furtherstudiesarerequiredtoaddressthis

issue.

Our datacorroborate and expand thenotionthat prolonged

SDinducesAMPARhypofunctionspeciﬁcallyinthehippocampal

formation,whileotherregionsassociatedwithmemoryare

unaf-fected.TheseeffectswerenotaccompaniedbychangesinGluR1

mRNAexpression,andwerenormalizedafter24hofsleep

recov-ery.TheAMPARpotentiatoraniracetamwasabletocounteractPA

learningdeﬁcitsinducedbySD,whereastheAMPARantagonist

GYKI52466didnotaffecttheperformanceofSDanimalsinthetask.

Takentogether,ourresultssuggestthatAMPARmediateaversive

memoryimpairmentinducedbysleepdeprivation.

Acknowledgments

This work was supported by Conselho Nacional de

Desen-volvimento Cientíﬁco e Tecnológico – CNPq; FAPESP (Grants

#1998/1403-3,#2007/53176-8,#2011/09816-8);Coordenac¸ãode

Aperfeic¸oamento de Pessoal de Nível Superior – CAPES and

Associac¸ãoFundodeIncentivoaPesquisa.Theauthorsareindebted

to Joelcimar Martins da Silva, Diva Maria Lima, Danilo Carlos

MachadoandJoséBernardodaCostafortheirinvaluabletechnical

assistance.

References

[1]ColtenHR,AltevogtBM.Sleepdisorderandsleepdeprivation:anunmetpublic healthconcern.Washington,DC:NationalAcademiesPress;2006.

[2]LimJ,DingesDF.Ameta-analysisoftheimpactofshort-termsleepdeprivation oncognitivevariables.PsycholBull2010;136:375–89.

[3]BuenoOF,LoboLL,OliveiraMG,GuglianoEB,PomaricoAC,TuﬁkS.Dissociated paradoxicalsleepdeprivationeffectsoninhibitoryavoidanceandconditioned fear.PhysiolBehav1994;56:775–9.

[4]YoungbloodBD,ZhouJ,SmaginGN,RyanDH,HarrisRB.Sleepdeprivation bytheﬂowerpottechniqueandspatialreferencememory.PhysiolBehav 1997;61:249–56.

[5]McDermottCM,LaHosteGJ,ChenC,MustoA,BazanNG,MageeJC.Sleep depri-vationcausesbehavioral,synaptic,andmembraneexcitabilityalterationsin hippocampalneurons.JNeurosci2003;23:9687–95.

[6]DubielaFP, OliveiraMG, Moreira KM,NobregaJN,Tufik S,Hipolide DC. Learningdeficitsinducedbysleepdeprivationandrecoveryarenot associ-atedwithaltered[(3_)H]muscimol_and_[(3_{)H]flunitrazepam}_binding._Brain_Res

2005;1037:157–63.

[7]DubielaFP,OliveiraMG,MoreiraKM,NobregaJN,TuﬁkS,HipolideDC.Inverse benzodiazepineagonistbeta-CCMdoesnotreverselearningdeﬁcitinducedby sleepdeprivation.NeurosciLett2010;469:169–73.

[8]LongordoF,KoppC,LüthiA.Consequencesofsleepdeprivationon neurotrans-mitterreceptorexpressionandfunction.EurJNeurosci2009;29:1810–9.

[9]CammarotaM,IzquierdoI,WolfmanC,LevideSteinM,BernabeuR,Jerusalinsky D,etal.Inhibitoryavoidancetraininginducesrapidandselectivechangesin [3_H]AMPA_receptor_binding_in_the_rat_hippocampal_formation._Neurobiol_Learn

Mem1995;64:257–64.

[10]IzquierdoLA,BarrosDM,ViannaMR,CoitinhoA,deDavideSilvaT,ChoiH, etal.Molecularpharmacologicaldissectionofshort-andlong-termmemory. CellMolNeurobiol2002;22:269–87.

[11]WhitlockJR,HeynenAJ,ShulerMG,BearMF.Learninginduceslong-term poten-tiationinthehippocampus.Science2006;313:1093–7.

[12]MonaghanDT,WentholdRJ,editors.Theionotropicglutamatereceptors.New Jersey:Humana;1997.

[13]SinhaAK,MirzaAH,LiuX,ChiOZ,WeissHR.EffectofupregulationofAMPA glutamatereceptorsoncerebralO(2)consumptionandbloodﬂowinrat.Brain Res1999;842:230–2.

[14]SinhaAK,AzevedoR,ChiOZ,WeissHR.Down-regulationofAMPA gluta-matereceptorsreducescerebrocorticalmetabolicresponsetostimulation. NeurochemRes2004;29:1425–30.

[15]Dos-Anjos S, Martínez-Villayandre B, Montori S, Regueiro-Purri ños MM, Gonzalo-OrdenJM,Fernández-LópezA.Globalischemia-inducedmodifications intheexpressionofAMPAreceptorsandinflammationinratbrain.BrainRes 2009;1287:20–7.

[16]BettendorffL,Sallanon-MoulinM,TouretM,WinsP,MargineanuI, Schoffe-nielsE.Paradoxicalsleepdeprivationincreasesthecontentofglutamateand glutamineinratcerebralcortex.Sleep1996;19:65–71.

[17]MohammedHS,AboulEzzHS,KhadrawyYA,NoorNA.Neurochemicaland electrophysiologicalchangesinducedbyparadoxicalsleepdeprivationinrats. BehavBrainRes2011;225:39–46.

[18]VyazovskiyVV,CirelliC,Pﬁster-GenskowM,FaragunaU,TononiG.Molecular andelectrophysiologicalevidencefornetsynapticpotentiationinwakeand depressioninsleep.NatNeurosci2008;11:200–8.

[19]McDermottCM,HardyMN,BazanNG,MageeJC.Sleepdeprivation-induced alterationsinexcitatorysynaptictransmissionintheCA1regionoftherat hippocampus.JPhysiol(London)2006;570:553–65.

[20]RavassardP,PachoudB,ComteJC,Mejia-PerezC,Scoté-BlachonC,GayN, etal.Paradoxical(REM)sleepdeprivationcausesalargeandrapidlyreversible decreaseinlong-termpotentiation,synaptictransmission,glutamate recep-torproteinlevels,andERK/MAPKactivationinthedorsalhippocampus.Sleep 2009;32:227–40.

[21]LynchG.Memoryandthebrain:unexpectedchemistriesandanew pharma-cology.NeurobiolLearnMem1998;70:82–100.

[22]ItoI,TanabeS,KohdaA,SugiyamaH.Allostericpotentiationofquisqualate receptorsbyanootropicdruganiracetam.JPhysiol(London)1990;424:533–43.

[23]JinR, ClarkS,WeeksAM,Dudman JT,GouauxE,PartinKM.Mechanism of positive allosteric modulators acting on AMPA receptors. J Neurosci 2005;25:9027–36.

[24]CuminR,BandleEF,GamzuE,HaefelyWE.Effectsofthenovelcompound aniracetam(Ro13-5057)uponimpairedlearningandmemoryinrodents. Psychopharmacology(Berl)1982;78:104–11.

[25]BartoliniL, CasamentiF, PepeuG.Aniracetamrestoresobjectrecognition impairedbyage,scopolamine,andnucleusbasalislesions.PharmacolBiochem Behav1996;53:277–83.

[26]LuY,WehnerJM.Enhancementofcontextualfear-conditioningbyputative (+/−)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptormodulatorsandN-methyl-d-aspartate(NMDA)receptorantagonists inDBA/2Jmice.BrainRes1997;768:197–207.

[27]PorrinoLJ,DaunaisJB,RogersGA,HampsonRE,DeadwylerSA.Facilitation oftaskperformanceandremovaloftheeffectsofsleepdeprivationbyan ampakine(CX717)innonhumanprimates.PLoSBiol2005;3:e299.

[28]BoyleJ,StanleyN,JamesLM,WrightN,JohnsenS,ArbonEL,etal.Acute sleepdeprivation:theeffectsoftheAMPAKINEcompoundCX717onhuman cognitive performance, alertness and recovery sleep. J Psychopharmacol 2012;26:1047–57.

(8)

[29]BlockF,SchwarzM.Correlationbetweenhippocampalneuronaldamageand spatiallearningdeﬁcitduetoglobalischemia.PharmacolBiochemBehav 1997;56:755–61.

[30]Gyertyan I, Gigler G, Simo A. The neuroprotective and hypothermic effectofGYKI-52466,anoncompetitive alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionicacid-antagonistonhistologicalandbehaviouralvariables inthegerbilglobalischemiamodel.BrainResBull1999;50:179–86.

[31]WesterbergE,MonaghanDT,CotmanCW,WielochT.Excitatoryaminoacid receptorsandischemicbraindamageintherat.NeurosciLett1987;73:119–24.

[32]Sommer C, Fahrner A, Kiessling M. [3_H]Muscimol _binding _to

gamma-aminobutyricacid(A)receptorsisupregulatedinCA1neuronsofthegerbil hippocampusintheischemia-tolerantstate.Stroke2002;33:1698–705.

[33]SucheckiD,TuﬁkS.Socialstabilityattenuatesthestressinthemodiﬁed multi-pleplatformmethodforparadoxicalsleepdeprivationintherat.PhysiolBehav 2000;68:309–16.

[34]MachadoRB,HipolideDC,Benedito-SilvaAA,TufikS.Sleepdeprivationinduced bythemodifiedmultipleplatformtechnique:quantificationofsleeplossand recovery.BrainRes2004;1004:45–51.

[35]NobregaJN,RaymondR,BarlowK,HamannM,RichterA.ChangesinAMPA receptorbindinginananimalmodelofinbornparoxysmaldystonia.ExpNeurol 2002;176(2):371–6.

[36]Gitaí DL, Martinelli HN, Valente V, Pereira MG, Oliveira JA, Elias CF, etal.IncreasedexpressionofGluR2-ﬂipinthehippocampusoftheWistar

audiogenic rat strain after acute and kindled seizures. Hippocampus 2010;20(1):125–33.

[37]Izquierdo I, Medina JH, Vianna MR, Izquierdo LA, Barros DM. Separate mechanismsforshort-andlong-termmemory.BehavBrainRes1999;103: 1–11.

[38]UekiA,MiwaC,MiyoshiK.Impairmentintheacquisitionofpassiveandactive avoidancelearningtasksduetobilateralentorhinalcortexlesions.JNeurolSci 1994;125:14–21.

[39]McGaughJ,Intorini-CollisonA,NagaharaA,CahillL.Involvementofthe amyg-daloidcomplexinneuromodulatoryinﬂuencesonmemorystorage.Neurosci BiobehavRev1990;14:425–31.

[40]HagewoudR,HavekesR,NovatiA,KeijserJN,VanderZeeEA,MeerloP.Sleep deprivationimpairsspatialworkingmemoryandreduceshippocampalAMPA receptorphosphorylation.JSleepRes2010;19:280–8.

[41]BoehmJ,KangMG,JohnsonRC,EstebanJ,HuganirRL,MalinowR.Synaptic incorporationofAMPAreceptorsduringLTPiscontrolledbyaPKC phosphory-lationsiteonGluR1.Neuron2006;51:213–25.

[42]NishizakiT,MatsumuraT.Theaniracetammetabolite2-pyrrolidinoneinduces along-termenhancementinAMPAreceptorresponsesviaaCaMKIIpathway. BrainResMolBrainRes2002;98:130–4.

[43]SmithAM,WehnerJM.Aniracetamimprovescontextualfearconditioningand increaseshippocampalgamma-PKCactivationinDBA/2Jmice.Hippocampus 2002;12:76–85.