Cortical synaptic transmission and plasticity in acute liver failure are decreased by presynaptic events

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MolNeurobiol(2018)55:1244–1258 DOI10.1007/s12035-016-0367-4

CorticalSynapticTransmissionandPlasticityinAcuteLiverFa ilureAreDecreasedbyPresynapticEvents

MariuszP opek¹^&BartoszB obula²^&JoannaSowa²^&GrzegorzH ess²^&RafałPolowy³^&RobertKub aF ilipkowski³^&MałgorzataFrontczak-

Baniewicz⁴^&BarbaraZ abłocka⁵^&JanAlbrecht¹^&MagdalenaZielińska¹

Received:19September2016/Accepted:28December2016/Publishedonline:23January2017

#TheAuthor(s)2017.ThisarticleispublishedwithopenaccessatSpringerlink.com

AbstractNeurologicalsymptomsofacuteliverfailure(ALF)refle ctdecreasedexcitatorytransmission,butthestatusofALF- affectedexcitatorysynapsehasnotbeencharacterizedindetail.W estudiedtheeffectsofALFinmouseonsynaptictransmissionandp lasticityexvivoanditsrelationtodistributionof(i)synapticvesicle s(sv)and(ii)functionalsynapticproteinswithinthesyn-

apse.ALF-

competentneurologicalandbiochemicalchangeswereinduce dinmicewithazoxymethane(AOM).Electrophysiologica lcharacteristics(long-termpotentiation,whole-

cellrecording)aswellassynapseultrastructurewereeval- uatedinthecerebralcortex.Also,svwerequantifiedinthepresyna pticzonebyelectronmicroscopy.Finally,presynapticproteinsi nthemembrane-enriched(P2)andcytosolic(S2)frac-

tionsofcorticalhomogenateswerequantitatedbyWesternblot.Sli cesderivedfromsymptomaticAOMmicepresentedasetofelectro physiologicalcorrelatesofimpairedtransmitterreleasein- cludingdecreasedfieldpotentials(FPs),increasedpaired-pulse

*MagdalenaZielińskamzie linska@imdik.pan.pl

1 DepartmentofNeurotoxicology,MossakowskiMedicalResearch Centre,PolishAcademyofSciences,Pawińskiego5St,

02-106Warsaw,Poland

2 DepartmentofPhysiology,InstituteofPharmacology,PolishA cademyofSciences,Smętna12St,31-343Cracow,Poland

3 BehaviorandMetabolismResearchLaboratory,MossakowskiMedic alResearchCentre,PolishAcademyofSciences,Pawińskiego5St,02- 106Warsaw,Poland

4 ElectronMicroscopyPlatform,MossakowskiMedicalResearch CentrePolishAcademyofSciences,Pawińskiego5St,

02-106Warsaw,Poland

5 MolecularBiologyUnit,MossakowskiMedicalResearchCentre,P olishAcademyofSciences,Pawińskiego5St,

02-106Warsaw,Poland

facilitation(PPF),anddecreasedfrequencyofspontaneousandmi niatureexcitatorypostsynapticcurrents(sEPSCs/mEPSCs)acco mpaniedbyreductionofthespontaneoustransmitterrelease- drivingprotein,vti1A.Additionally,anincreasednumberofsvper synapseandadecreaseofP2contentand/orP2/S2ratioforsv- associatedproteins,i.e.synaptophysin,synaptotagmin,andMun c18–1,werefound,inspiteofdecreasedcontentofthesv- dockingprotein,syntaxin-1.SlicesfromAOM- treatedasymptomaticmiceshowedimpairedlong-

termpotentiation(LTP)andincreasedPPFbutnochangesintrans mitterreleaseorpresynapticproteincomposition.Ourfindingsde monstratethatadecreaseofsynaptictransmissioninsymptomatic ALFisassociatedwithinefficientrecruitmentofsvproteinsand/or im-pairedsvtraffickingtotransmitterreleasesites.

KeywordsA cuteliverfailure.Presynapticevents.N eurotransmission

Introduction

Neuropsychiatricsymptomsofacuteorchronicliverfailure(ALFo rCLF),collectivelydefinedashepaticencephalopathy(HE),areass ociatedwithadeclineofexcitatoryneurotransmission[1–

3].Presentknowledgeoftheelectrophysiologicalandmolecularco r-relatesofALF-orCLF-

affectedsynapseisconfinedmostlytothepostsynapticeventsrelate dtoalteredGlureceptor-mediatedsig-

nalingpathways[4]and/orsynapticplasticity,i.e.,long-termpotentiation(LTP)andLTD[5],whilethestatusofthepresynapticregi oninvolvedinneurotransmitterreleasehasremainedunat- tended.Afewearlierreportshavedealtwithalterationsinthesynapti ceventsinbrainpreparationstreatedwithammonia,whichisamajo rneurotoxinimplicatedinHE[6],emphasizingtheroleofpostsyna pticchanges[7–

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MolNeurobiol(2018)55:1244–1258 1245

10].However,theobservationswereconfinedtoshort- termeffects(secondstominutes),which

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Con AOM IL-

6(pg/ml)TNF- α(pg/ml)AST(

IU/I)ALT(IU/I )

10.0±2.2 3.1±0.2 172.4±30.6 46.5±6.1

214.5±34.5*

14.7±1.8*

805.4±92.2*

559.0±52.8*

cannotbeconsideredrepresentativefortheprogressionof ALFsymptomslastingforhourstoweeks.Inthisstudy,there- fore,weaddressthequestionwhetherandtowhatextentALFdistur bsthefunctionalandstructuralintegrityofthepresyn-

apticneurotransmitterreleasemachineryinadditiontopost- synapticchanges.Wethereforemeasuredexpressionanddis- tributionofpresynapticproteinsincluding(i)theintegralves- icleproteinssynaptophysinandsynaptotagmin;

(ii)amemberoftheSec1/Munc18-likeproteinfamilyMunc-18- 1;(iii)anintegratedSNAREproteinsyntaxin-

1;and(iv)vt1interactor1a(vti1a)protein,whichcharacterizesvesi clesdrivingspon-taneousrelease[11–

13].TheresponseofpresynapticproteinstoALFwascomparedtot hatoftheconstituentsofthepost-

synapticcomplexactiveinsignaltransduction,NMDAsub- unitNR1,PSD95,andtheneuronalformofnitricoxidesyn- thase(nNOS).TheanalysiswascarriedoutinmiceinwhichALFw asinducedwithahepatotoxin,azoxymethane(AOM),andwaspre cededbythoroughbehavioral,biochemical,andneurophysiologi calcharacterizationofthemodel.TheresultspresentedinTable1a ndFigs.1and2confirmandextendthefindingsbyothersthatthem ouseAOMmodelisavalidmod-elofacuteHEinhumans[14–16].

CerebralcorticalslicesderivedfromAOM-treatedasymp- tomaticandsymptomaticmiceweresubjectedtotheelectro- physiologicalanalysisoffieldpotentials(FPs),includingpaire d-

pulsefacilitation(PPF)ratio,parameterscharacterizingexcitab ilityofpyramidalneurons,andspontaneousexcitatorypostsyna pticcurrents(sEPSCs)aswellasminiatureexcitatorypostsynap ticcurrents(mEPSCs).

Growingevidencethatliverfailureaffectssynapticplastic- ityandthatGlurelease playsacrucialroleindrivingthe postsynapticevents[17]promptedustoassesslong-

lastingactivity-

dependentchangesinsynapticefficiency,knownasLTP.LTPu nderliesmultipleformsofsynapticplasticityinthebrain[18].A nalysisofthemorphologicalandbiochemicalstatusofthesyn apsefocusedontheneurotransmitterreleaseapparatus.Ultrastr ucturalassessmentofthepresynapticzonecomprisedthesizean dcontentofsv.Next,weexaminedthecontentanddistributio nbetweenthecytosolic-andmembrane-

enrichedcompartmentsofthecerebralfrontal

Table1Increasedconcentrationofcytokines,ammonia,andactivityoflive rdamagemarkerenzymesinserumofcontrolandAOMmice

Ammonia(mg/ml)0 .0052±0.00110 .0140±0.0010*

Fig.1Novel-

cageactivityandneurologicalassessment.a Shorterdistancetraveledby AOM-

injectedmiceshortlyafterplacingintoanewcage;**p<0.01,n=9.bDec reasedneurologicalscorefollowingAOMinjection.Asteriskindicatesp<

0.05vs.theresultsoftheprevioussession(2hearlier),andhashtagindicates p<0.05(p<0.01)vs.4-h-earliersession;n=12.Resultsaremeans±SEM

cortexoftheabovementionedrepresentativepresynapticandpo stsynapticproteins.

MaterialandMethods

AOMModelofAcuteLiverFailureinMice

Allexperimentswereperformedwithagreementoflocalan- imalethicalcommitteeinWarsawinaccordancewithECDir ective86/609/EEC.MaleC57BL/6mice(animalcolonyofthe MossakowskiMedicalResearchCentre,PolishAcademyo fSciencesinWarsaw),bodyweight30.0±5.1g,weresubjectedt oahepatotoxicinsultbysingle,intraperito-

nealAOMinjectionat100mg/kgb.w.Themicehadfreea ccesstowaterandchowandwerehousedinconstanttem- perature,humidity,and12-hlight-darkcycling.Ifnotother- wisestated,experimentswereperformedintheasymptomaticst age,4hafterAOMinjection,andsymptomaticstage,18h afterAOMinjection.Theselectionofthetimepointswas

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*p<0.05vs.Con;n=6 basedontheneurologicalperformanceofmice.

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1246 MolNeurobiol(2018)55:1244–1258

Fig.2 Metabolitea nalysisby¹Hma gneticr esonancesp ectroscopytech niqueandcerebralcortexmicrodialysisofAOMmice.a Spectrometric analysisrevealedALF-

specificchangesincerebralcortexofAOMmice.Asteriskindicatesp<0.05 vs.Con;n=12.AnalyzedmetabolitesAlaalanine,Aspasparticacid,C rcreatine,PCrphosphocreatine,GABAgamma-

aminobutyricacid,Glnglutamine,Gluglutamate,PChphosphocholine,GS Hglutathione,INSmyo-inositol,Laclactate,NAAN-

acetylaspartate,Tautaurine,GPCglycerophosphocholine,NAAGN- acetylaspartylglutamate.b Extracellularglutamateconcentration ,asdeterminedinmicrodialysatesfromfreelymovingmice,wassignific antlyelevatedafterAOMadministration;n =4.Resultsaremeans±SEM

ActivityA ssessment

Foractivityassessment,immediatelyafterAOM(n=11)orsali ne(n=5)injections,themicewereplacedindividuallyinnovelc ages(43×27×15cm),withfreshbedding,coveredbyametal1×1 -

cmgridtoallowobservationfromabove.Theexperimentroom wasilluminatedbydimredlight.Theani-

malswererecordedbyaninfraredacA1300-

60NIRcamera(BasslerAG,Germany)for4h,andtheactivityw asmeasuredusingEthovisionXT10(NoldusInformation Technology,Netherlands).

NeurologicalAssessment

Neurologicaldeclinewasdeterminedinagroupofmice(n=12) bymeasuringthecornealreflex,pinnareflex,vibris-

saereflex,startlereflex,rightingreflex,andposturalreflex[16 ,19],witheachgivenascoreof0(noreflexevident),1

(weakordelayedreflex),or2(regularreflex),resultinginaneur ologicalscoreintherangeof0to12.Thereflexesweremeasured every2hafterAOMinjection.Cornealreflexwasassessedbytou chingtheeyewithsaline-soakedcottonappli-

catorandobservingablinkresponse[14,20].Todeterminepinn areflex,theearlobewastouchedwithacottonrodandearretracti onwasobserved[20,21].Toassessvibrissaeresponse,thewhis kerswerebrushedandreactionalheadmovementwasobserved[

21,22].Startlereflexwastestedbypresentingasudden,unexp ectednoiseandevaluatingthereactionoftheanimal[15,23].Ri ghtingreflexwasdeterminedbyplacingthemiceontheirbac ksandassessinghowfasttheyrightthemselves[20,24].Po sturalreflexwastestedbyplacingthemiceindividuallyina cagewithoutbeddingandrapidlymovingitincardinaldirection sandjudginghowtheanimalstrytokeeptheirbalance[20,25].

BiochemicalBloodAnalyses

EnzymaticDeterminationofLiverEnzymesandAmmoniain Serum

BloodwascollectedintoEppendorfandaftertheformationofacl ot,centrifugedat8000×gfor6min.Thenusingenzymatictestsfo rammonia,alanineaminotransferase(ALT)andaspar- tateaminotransferase(AST),theabsorbanceweremeasuredata wavelengthof340nmaftermixingprobewithreagentandevery 60sfor2minin37°C.Usinganappropriatemodel,basedonthe differenceinabsorbanceintime(Δabs/min),resultsareexpr essedasIU/l.

CytokineIL-6andTNF-αConcentrationsinSerum

BloodwascollectedintoEppendorfandaftertheformationofacl ot,centrifugedat6500×gfor10min.Themicrosphere- basedimmunoassay(cytometricbeadarray(CBA);BDBiosc iences)wasperformedinaccordancetothecommercialprotocol withmodifications[26].Briefly,sixmicrospherepopulations withdistinctfluorescenceintensitiesweredyedwithproprietar ydyes(emission650nm).Thesebeadswerecoatedwithcapture antibodiesagainstcytokinesandmixedwithrecombinantstan dardsorserumandthephycoerythrin(PE)-

conjugatedcytokineantibodies(emission585nm)toform sandwichcomplexes.Theinstrumentsetupwasper- formedwithCaliBRITEbeads,andthecytometersetupbeads(B D)weredoneaccordingtothemanufacturer’sinstructions;2000 eventsweremeasuredandanalyzed.Amonomericmi-

crospherepopulationwasgatedonforwardandsidescatters.Dat awereanalyzedintwo-

colorfluorescencedotplotsrepresentingthedifferentmicrosp herepopulations(emission650nm)andthecytokineconcentr ation(accordingtoPEemission585nm).Meanfluorescencein tensityvalueswere

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collected.Four-

parametriclogisticcalibrationcurveswereused,andresultswe reexpressedaspg/ml.

MetaboliteAnalysisby¹HMagneticResonanceS pectroscopyTechniqueandCerebralCortexMicr odialysisofSymptomaticAOMMice

1HMagneticResonanceSpectroscopyAcquisitiona ndProcessing

Toobtainthespectraofbrainmetabolites,localizedprotonspe ctroscopy(Biospec70/30USR)atshortechowasper-

formedusingPRESSsequence(TR/TE=2000/20ms,512aver ages,2048points,scantime=17min)withVAPORwatersup pression,theoutervolumesuppression,andfrequen-

cydriftcorrection(flipangle5°).Eddycurrentcorrectionwaspe rformedatthescanner.Eachmeasurementwascarriedoutintwo separatedvolumesofinterest(VOI).TheVOI(4×2×1.5m m³)encompassedthefrontalcortex.Linearandsecond- orderglobalshimswereadjustedwithADJ_1st_2nd_ord erprotocol.Afterwards,linearandsecond-

orderlocalshimswereautomaticallyadjustedwithFASTM APinacubicvolumewhichcontainedthevolumeofinterestre gion(4×4×4mm^3forthefrontalcortex).Theunsuppressedwate rlinewidthwastypicallymaintainedataround9–12Hz.

Metaboliteconcentrationsweredeterminedusingalinearco mbinationanalysismethodLCModel[27](http://www.s- provencher.com/pages/lcmodel.shtml).Theunsuppressed watersignalmeasuredfromthesamevolumeofinterestwasuse dasinternalreferenceforabsolutemetabolitequantificat ion.Metaboliteconcentrationsarereportedininstitutionalunit (IU).Thespectrumsignaltonoiseratiowastypicallyataround12 –25.

MicrodialysisonFreelyMovingMice

Micewereanesthetizedwithisoflurane(3.5%inair),placedinas tereotaxicframe,andkeptasleepataflowrateof1.5%.Theheadw asshavedanddecontaminatedby70%ethanol,andtwo1.2- mm-

diameterholesweredrilled,firstatcoordinatesAP−0.5,ML+0 .5,inwhichskullscrewwasinserted,andsecondatcoordinates AP+2.0,ML−0.5,DV−0.5accordingtotheatlas[28],inwhicha guidecannulawasinserted.Guidecannulawasimplantedatadep thof0.5mm.Skullsurfaceandguidecannulaweresecuredwith dentalacryliccement.Beforearousals,antibiotic(Baytril,2.5

%;0.2ml/kgb.w.)andapainkiller(Ketonal,2.5mg/kgb.w.)we resubcutaneous-lyadministered.Animalswereplacedin21- cm-diametercy-

lindricalcageswithadlibitumaccesstofoodandwater.Onedaya ftersurgery,micewereanesthetizedwithisoflurane(3.5%inair )andaprobewasimplanted.Afterpreparingtheprobeinaccord ancewiththeinstructions,ACSFofthe

followingcomposition(inmM):NaCl(130),KCl(5),CaCl2(2.5 ),MgSO4(1.3),KH2PO4(1.25),NaHCO3(26),andD- glucose(10),bubbledwithamixtureof95%O2and5%CO2passe sthroughit.After1hin2.5-

μl/hflow,sampleswerecollectedevery40min(100μl)for4h(si xfractions).Afterthis,AOMwasi.p.injected,andafter16h, sampleswerecollectedevery40min.Allsampleswereimmedi atelyfrozenat−80°C.

High-

PerformanceL iquidChromatographyDeterminationofGluta mate

Glutamateconcentrationinmicemicrodialysateswasmea- suredusingHPLCwithfluorescencedetectionafterderivati- zationina timedreactionwitho-

phthalaldehydeplusmercaptoethanol,exactlyasdescribedea rlier[29].

ElectrophysiologicalStudies

Thebrainswererapidlyremovedfromtheskullsandim- mersedinanice-

coldACSFofthefollowingcomposition(inmM):NaCl(13 0),KCl(5),CaCl2(2.5),MgSO4(1.3),

KH2PO4(1.25),NaHCO3(26),andD-glucose(10),bubbled withamixtureof95%O2and5%CO2.Frontalcorticalslices,400 μmthick,werecutinthecoronalplaneusingavibratingmicroto me(Leica).Sliceswerestoredat32.0±0.5°C.

FPRecordingandLTPInduction

Individualsliceswereplacedintherecordingchamberofaninter facetypewhichwassuperfused(2.5ml/min)withamod- ifiedACSF(temperature32.0±0.5°C)containing(inmM)NaC l(1 32),K Cl(2), C a C l2(2.5),M gSO4(1.3),K H2PO4

(1.25),NaHCO3(26),andD-glucose(10),bubbledwith 95%O2and5%CO2(temperature32.0±0.5°C).Aconcen- tricbipolarstimulatingelectrode(FHC,USA)wasplacedincort icallayerV.Stimuliof0.033-

Hzfrequencyanddurationof0.2mswereappliedusingaconstan t-currentstimulusiso-

lationunit(WPI).GlassmicropipettesfilledwithACSF(2–

5MΩ)wereusedtorecordfieldpotentials.Recordingmicro- electrodeswereplacedincorticallayerII/III.Theresponseswer eamplified(EXT10–2Famplifier,NPI),filtered(1Hz–

1kHz),A/Dconverted(10-

kHzsamplingrate),andstoredonPCusingtheMicro1401interf aceandSignal2software(CED).Astimulus-response(input- output)curvewasmadeforeachslice.Toobtainthecurve,stimu lationintensitywasgraduallyincreasedstepwise(15steps;5–

100μA).Onere-

sponsewasrecordedateachstimulationintensity.Then,stim-

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ulationintensitywasadjustedtoevokeresponsesof30%ofthe maximumamplitude.LTPwasinducedbythetaburststimu lation(TBS).TBSconsistedof10trainsofstimuliat5Hz,rep eated5timesevery15s.Eachtrainwascomposed

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offivepulsesat100Hz.DuringTBS,pulsedurationwasi ncreasedto0.3ms.

Whole-CellRecording

Individualsliceswereplacedintherecordingchambermount edonthestageoftheAxioskop(Zeiss)microscopeands uperfusedat2ml/minwithACSF.Recordingpipetteswerepul ledfromborosilicateglasscapillaries(HarvardApparatus)us ingSutterInstrumentP97puller.Thepipettesolutioncontai ned(inmM)130K-gluconate,5NaCl,0.3

CaCl2,2MgCl2,10HEPES,5Na2-ATP,0.4Na-GTP,and1 EGTA(osmolality290mOsm,pH=7.2).Pipetteshadopentipre sistanceofapproximately6MΩ.Pyramidalcellsweresampled fromthesiteslocatedapproximately2mmlateraltothemidlinea ndapproximately0.3mmbelowthepialsurface.Signalswerere cordedusingtheSEC05LXamplifier(NPI),filteredat2kHz,an ddigitizedat20kHzusingDigidata1440AinterfaceandClam pex10software(MolecularDevices).

Afterobtainingthewhole-

cellconfigurationandsubse-quent10-

minstabilizationperiod,thefiringcharacteristicsoftherecorde dcellswereassessedusingintracellularinjectionsofrectangula rcurrentpulsesofincreasingamplitude(duration400ms)inthec urrentclampmode.Foreachcell,therelation-

shipbetweeninjectedcurrentintensityandthenumberofsp ikeswasplotted.Thegainwasdeterminedasaslopeofthestra ightlinefittedtoexperimentaldata.Thethresholdcurrent(It h)wasdeterminedasacurrentextrapolatedatzerofiringrate.Tor ecordsEPSCs,neuronswerevoltageclampedat−76mVandsy napticeventswererecordedfor4minasinwardcurrents.Tor ecordmEPSCsinsomeexperiments,1μMtetrodotoxin(T TX)wasaddedtotheACSFspontane-

ousandminiatureEPSCsweredetectedofflineusingtheau- tomaticdetectionprotocol(MiniAnalysissoftware,Syna ptosoftInc.)andsubsequentlycheckedmanuallyforac- curacy.Datawereacceptedfortheanalysiswhentheaccessresis tancerangedbetween15and18MΩanditwasstable(<25%ch ange)duringrecording.Thethresholdamplitudeforthedetectio nofanEPSCwassetat5pA.

TransmissionElectronMicroscopy

Theanimalswereanesthetizedandperfusedthroughtheas- cendingaortawith2%paraformaldehydeand2.5%glutaral- dehydein0.1Mcacodylatebuffer,pH7.4.Thecerebralcor- texwasfixedinthesamesolutionfor20h(at4°C)andplacedinam ixtureof1%OsO4and0.8%K4[Fe(CN)6].Electronmicroscopy analysiswasperformedon10sectionsfromeachexperimentala ndcontrolanimals.Synapseswereclassifiedaccordingtotheir morphology.Fortheanalysis,onlyasym-

metric(i.e.,excitatory)synapsesweretakenintoaccount.Svwer equantifiedbycountingtheminallvisiblesynapsesat

eachsection(inallfields).Svwerecountedinspecimensfromco ntrolandsymptomaticAOManimals.Theaveragenumberofth evesicleswascalculatedforeachgroup.

ImmunoblottingAnalyses

Immediatelyremovedmicebraincortexwasisolatedonice,ho mogenizedinbuffer(20mMTris-

HCl,pH6.8;137mMNaCl;2mMEDTA;1%TritonX- 100;0.5mMDTT;0.5mM

PMSF;PhosphataseInhibitorcocktail1:100;ProteaseInhi bitorCocktail1:200),andcentrifugedat12000×gfor10min.T oseparateP2andS2fractions,cortexwashomog-

enizedinbuffer(15mMTris-

HCl,pH7.6;0.25Msucrose,1mMDTT;0.5mMPMSF,Phosp hataseInhibitorCocktail1:100;ProteaseInhibitorCocktail1:2 00)andcentrifugedat1000×gfor10min(4°C)—

P1fraction.Separatedsupernatantwascentrifugedat14000×gf or20min(4°C)andS2fraction(supernatant)wascollected.Thep ellet,afterbufferaddition,wasfrozenasP2fraction.Protein concentrationsforbothassayswereperformedusingaBCA ProteinAssayfromThermoScientific(Pierce,Rockford,IL,U SA).

Thecontentofsynapticproteinswasassessedbyimmuno- blottingaspreviouslydescribed[30,31].Cortexhomog enates/S2fraction/P2fractionwereloadedwith15–

30μgofproteindilutedinLaemlibufferpereachtissuesam- pleandloadedin10%SDS-

PAGEgels,thentransferredontoPVDFmembrane.Thepurityo fS2andP2fractionswasfur-

theranalyzedbyimmunoblottingusingantibodiesagainstlac- tatedehydrogenase(LDH)andcadherinascytosolicandmemb ranefractionmarkers,respectively.Westernblotmem- braneswereblockedin5%milkandincubatedovernightat4°C withantibodies(againstnNOS,NR1,PSD-

95,synaptophysin,synaptotagmin,syntaxin-1,Munc18–

1,vti1a)inrecommendeddilutionin1%milkandthenfor1hin1

%milkwithsecondaryreagents.Theproteinbandswerevisual- izedwithenhancedchemiluminescence,usingG-

BOX(Syngene).Datawereexpressedasfoldchangeinfluoresce ntbandintensityoftargetantibodydividedbyGAPDH,whichisu sedasaloadingcontrol.Thevaluesofvehicleorcontrolgroup swereusedasabaselineandsettoarelativeproteinexpressionv alueof1.Allbandintensityquantificationswereanalyzedusing GeneTools.

StatisticalAnalyses

AnANOVAfollowedbyDunnettposthoctestwasusedtodetec tintervalsinwhichsignificantchangesoccurredforalldatasets whereaparameterwasmeasuredacrosstimepointswithinatrea tment.Student’sttestortheMann-

WhitneyUtestwasappliedwhentwopopulationsofresponses

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wereexam-

ined.ErrorbarsrepresenttheSDorSEM,whichisspecifical- lyindicated,*p<0.05,**p<0.01,and***p<0.001.

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Results

CharacteristicsoftheAOMModel

Activityassessmentrevealedadifferencebetweencontroland AOM-injectedanimalsinthedistancetraveledfollowingplac- inginanovelcage(Fig.1a).RepeatedmeasuresANOVAsho weda strongeffectoftreatment[F(1,14)= 15.66,p<0.01]an dtestingtime[F(5,70)=23.51,p<0.001]aswellastreatment×ti meeffect[F(5,70)=2.47,p<0.05].Theactivityofbothgrou psgraduallydecreased;however,AOMmicestartedtravelings horterdistancesasearlyas15–

30minpostinjection,asdemonstratedbyplannedcompariso ns(p<0.01foralltimeintervalsafter0–15min).

Theneurologicalscoreateachtimepointwasdefinedasthes ummationofsixreflexes,andtheaveragevaluescanbeseeninFi g.1b.Continuousandsignificantneurologicalde-

clinetowardscomawasobservedstartingfromthesixthhourfoll owingAOMinjection,asrevealedbyFriedmannonpara- metricanalysis(p<0.001)followedbyindividualWilcoxonco mparisonsofdependentgroups.

AOMinjectionledtoasignificantincreaseinthecon- centrationofammoniaandinflammatorycytokines,TNF- αandIL-

6,andactivitiesofliverdamagemarkerenzymes,ALTandAST inplasma(Table1).Invivo¹Hspectrometricanalysisrevealed ALF-

specificchangesincludingincreaseofGlu/Glnratioandmyo- inositolincerebralcortexofAOM-

treatedmice(Fig.2a).ExtracellularGluconcentra-

tion,asdeterminedinmicrodialysatesfromfreelymovingmic e,startedtoincreaseon17hafterAOMadministration(Fig.2b).

StageD ependenceo f E lectrophysiologicalR esponseso fCorticalSlicesfromAOMMice

FPs,PPF,andLTP

AnalysesofFPsrevealednosignificantdifferencesinthesti mulus-

responserelationshipbetweenslicesprepared4hafterAOM administration(AOM4h)andcontrolprepara-

tions(Fig.3a).Incontrast,inbrainslicespreparedfrommicewit hneurologicaldecline(AOM18h),theamplitudeofFPswasmar kedlyloweredoverawiderangeofstimulationinten-

sities(Fig.4b).Parameterscharacterizinginput-

outputcurvesofFPs,calculatedusingtheBoltzmannfits,aresu mmarizedinTable2.ThemeanamplitudeofFPs,measured60 –

75minafterdeliveryofTBS,toinduceLTP,was128.6±4.2%o fbaselineinthecontrolasymptomaticgroup(Fig.3b)and 141.1±6.1%ofintheCon18-

hgroup(Fig.4b).LTPwassignificantlyattenuatedintheslices obtainedfromAOM-

treatedanimalsbothinasymptomaticstage(108.5±6.2%;p<0.

01vs.Con4h;F ig.3b)andi nsymptomatics tage (108.4± 3.6%;p <0.001vs.Con18h group;Fig.4b).

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DespitealackofchangesintheamplitudesofFPs,inslicesobta inedfromAOMmiceatasymptomaticstage,PPFratiowassig nificantlyincreased(Con4h117.6±5.1%vs.AOM4h130.9±

4.4%;p<0.05;Fig.3c1,c2).PPFwasfurtherincreasedinslice sobtained18hafterAOMadministration(Con18h121.5±3.5

%vs.AOM18h158.3±6.9;p<0.001;

Fig.4c1,c2) .

PyramidalN euronM embraneExcitability Whole-

cellrecordingswereobtainedfromlayerII/IIIneuronsexhibiti ngaregularspikingfiringpatterninresponsetoadepol arizingcurrentpulse(Figs.3dand4d).Inslicesprepared4hafte rAOMadministration,therestingmembranepotentialofneur onswassignificantlydifferentfromcontrolneurons(−78.1±

0.8vs.

−73.6±0.6mV,respectively;p<0.001).Asimilardifferencee xistedbetweenneuronsinslicesprepared18hafterAOMinjec tionandcontrolcells(−78.6±2.0vs.

−73.4±0.7mV,respectively;p<0.05).Theinputresistanceofn euronsoriginatingfromAOM4hmicewasnotdifferentfromt hatofthecontrolgroup(143.5±2.8vs.

151.1 ±4.7MΩ,respectively;p=0.36);however,theinputres istanceofneuronsoriginatingfromAOM18-

hmicewassignificantlylowerfromthatoftherespectivecontr olgroup(123.4±11.6vs.167.1±12.0MΩ,respectively;p<0.0 1).

Analysesoftherelationshipbetweentheinjectedcurrenta ndthefiringrate(gain;Figs.3eand4e)demonstratedthatAO Mdidnotmodifytheintrinsicexcitabilityofpyramidalneuro ns.Neitherinslicesprepared4nor18hafterAOMadmi nistration,thedifferencesbetweenexperimentalandcon- trolmiceweresignificant(Figs.3fand4f).However,insliceso btained18hafterAOMadministration,thecalculatedthresh oldcurrentforthegenerationoftheactionpotentialwa ssignificantlyhigherthanintherespectivecontrolmice(Con 18h;344.0± 27.7vs.236.6± 17.2pA;p <0.01;

Fig.4g).Thiseffectappearstoberelatedtoamarkedlylowerme mbraneresistanceofneuronsintheAOM18-

hgroup.Incontrast,theaveragespikethresholdcurrentintheA OM4-hmicewasnotdifferentfromtheCon4-hmice(Fig.3g).

ExcitatoryPostsynapticCurrent s

InthecellsoriginatingfromtheAOM4-

hmice,themeanfrequencyofsEPSCswassimilartorespect ivecontrol(Fig.3j).Similarly,themeanamplitudesofsEP SCsintheAOM4-

hmiceandintherespectivecontrolwerenotsignif-

icantlydifferent(Fig.3k).However,inthecellsoriginatingfr omAOM18-

hmice,themeanfrequencyofsEPSCswassignificantlylowe

rincomparisontothatinthecellsfromcontrolanimals(1.

0± 0.1vs.1.5± 0.1Hz;p <0.001;Fig.4j).AOM,actingfor18h,a lsoincreasedthemeanam-

plitudeofsEPSCs(10.4±0.3vs.9.5±0.2pA;p<0.05;Fig.4k ),andadditionally,itsignificantlyincreasedthemean

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1250 MolNeurobiol(2018)55:1244–1258

Fig.3ElectrophysiologyofALF-

inducedchangesincerebrocorticalslicesobtainedfromAOMmiceatasy mptomaticstage.a–

c2TheeffectsofAOMonFPsinslicespreparedfromasymptomaticmic e(AOM4h).aTheamplitudesofFPsofcontrol(Con4h)andAOMmice(

AOM4 h).b ImpairmentofLTP.Insetsina,b showthesuperpositionofa veragedFPsrecordedinrepresentativeexperimentsattimesindicatedbynu mbers.ArrowsinbdenotetheTBS.c1ExamplesofindividualFPsevoked bypairedstimuliinslicesfromcontrol(opencircles)andAOM- treatedmice(filledcircles).c2Summaryquantificationoftheaverag ePPFratio( ±SEM);*p<0.05.d–

gInslicespreparedfromasymptomaticmice(AOM4 h),thebasicelect rophysiologicalpropertiesoflayerII/IIIpyramidalneuronsremainunchan ged(apartfromtherestingmembranepotential,seetext).dAnexampleofre sponseofcontrolpyramidalneuron(lefttrace)andresponse

ofa cellfromAOM-

treated(AOM4h)mouse(righttrace)to adepolarizingc u rrentp ulse(lowert r aces).e T hei n jectedc u rrentv s . spikingraterelationshipi na celloriginat ingfromcontrolm ouse(open

circles)andinaneuronoriginatingfromAOM-

treatedmouse(filledcircles).Solidlinesrepresentthelinearfitstotheexp erimentaldata.fThemeangain(±SEM)andg meanfiringthreshold(±SE M)ofpyramidalneuronspreparedfromcontrol(whitebars;18cells;n=5) andAOM-treatedanimals(blackbars;21cells;n=5).h–

mLackofchangesinsEPSCsinslicesfromasymptomaticmice(AOM 4h).hExamplesofrawrecordsfromcontrolneuron(upperpairoftraces)a ndneuronfromAOMmouse(lowerpairoftraces)recordedbefore(Con4h ,AOM4h)andafteradditionofTTX(Con4h+TTX,AOM4h+TTX).iThes uperpositionofaveragesofallindividualsEPSCsdetectedduring4minbas elinerecordingsfromcontrolneuron(thinline)andaneuronoriginatingfro manAOM-

treatedmouse(thickline).BargraphsillustratealackoftheinfluenceofA OMonjmeanfrequency,kmeanamplitude,lmeanrisetime,andmmeandec aytimeconstantofsEPSCs.Inj–

m,neuronsoriginatingfromcontrolmice,errorbarsrepresenttheSEM(w hitebars;18cells;n=5)andAOM-

treatedanimals(blackbars;21cells;n=5)

decaytimeconstantofsEPSCs(from4.5±0.2to5.9±0.3ms;p<0.0 01;Fig.4m).AOMdidnotalterthemeanrisetimeofsEPSCseither intheAOM4-hmice(Fig.3l)orintheAOM18-hmice(Fig.4l).

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1251 MolNeurobiol(2018)55:1244–1258

TheeffectofNa^+channelblockadeonrecordedEPSCswa sinvestigatedinseparatesamplesofneuronsobtainedfromcon trolandAOM-

treatedmice(Con4hvs.AOM4handCon18hvs.AOM18h).

Theadditionof1μMTTXtoACSF

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Fig.3(continued)

didnotresultinsignificantchangesofthemeanfrequency,mea namplitude,meanrise,andmeandecaytimeconstantofsEPSCs (Table3)eitherincontrolorinexperimentalgroups.Thisfinding indicatesthatinourwhole-

cellrecordings,thecontributionofactionpotential- inducedreleaseofneurotrans-

mitterfrompresynapticterminalsisnegligible.

UltrastructuralAnalysisofCorticalSynapses

Electronmicroscopyanalysisofthecortexfromthesymptom- aticAOMmiceshowedanincreasednumberofsynapses

showingabundanceofsvinthepresynapticzone.Thenum- bersofsvinrandomlyselectedsectionsfromsymptomaticmic ewere(~15%)higherthaninthecontrol(Fig.5a,b).

ExpressionofSynapticProteinsatDifferentStagesof ALF

PresynapticProteins

Themembranetocytoplasmcontentratio(P2/S2)ofpre- synapticproteins,synaptophysinandsynaptotagmin,were

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MolNeurobiol(2018)55:1244–1258

1252

Fig.4ElectrophysiologyofALF-

inducedchangesincerebrocorticalslicesobtainedfromAOMmiceatsym ptomaticstage.a–

c2TheeffectsofAOMonFPsinslicespreparedfromsymptomaticmice(AO M18h).aTheamplitudesofFPsofcontrol(Con18h)andAOMmice(AOM18 h).bImpairmentofLTP.c1ExamplesofindividualFPsevokedbypairedsti muliinslicesfromcontrolandAOM-

treatedmice.c2SummaryquantificationoftheaveragePPFratio(±SEM);

***p<0.001.SymbolsasinFig.3a–c2.d–

gInslicespreparedfromsymptomaticmice(AOM18h),theexcitabilityofl ayerII/IIIpyramidalneuronsisreduced.dAnexampleofresponseofcontrol pyramidalneuron(Con18h)

(lefttrace)andresponseofacellfromsymptomaticmouse(AOM18h) (righttrace)toadepolarizingcurrentpulse(lowertraces).eTheinjectedc urrentvs.spikingraterelationshipinacellfromcontrolmouseandinaneuron fromAOM-

treatedmouse.f Themeangain(±SEM)andg meanfiringthreshold(±SE M)ofpyramidalneuronspreparedfromcontrol(23

cells;n=6)andAOM-

treatedanimals(21cells;n=6);**p<0.01.SymbolsasinFig.3d–g.h–

msEPSCsinslicespreparedfromsymptomaticrats(AOM18h).h Exam plesofrawrecordsfromacontrolneuron(upperpairoftraces)andaneuro nfromAOMmouse(lowerpairoftraces)recordedbefore(Con18h,AOM 18h)andafteradditionofTTX(Con18h + TTX,AOM18h + TTX).i The superpositionofaveragesofallindividualsEPSCsdetectedduring4- minbaselinerecordingsfromacontrolneuron(thinline)andaneuronorigi natingfromanAOM-

treatedmouse(thickline).BargraphsillustratetheeffectofAOMonjmea nfrequency,kmeanamplitude,lmeanrisetime,and mme andecay time constanto fsEPSCs.In j–

m,n eu ronsoriginatingfromcontrolmice,errorbarsrepresenttheSEM(

whitebars;23cells;n=6)andAOM-

treatedanimals(blackbars;21cells;n=6);*p<0.05,**p<0.01,***p<0.0 01

decreasedby~50and~30%,respectively,inthefrontalcortexo fsymptomaticmiceascomparedtothecontrolandthatofMunc 18–1wasincreasedby~70%

(Fig.6b).TheP2fractioncontentofsyntaxin-

1revealedan~20%increase,andthelevelofvti1aprotein,whic

hcharacterizesvesiclesdrivingspontaneousrelease,wassigni ficantly

(16)

MolNeurobiol(2018)55:1244–1258 125 decreased(Fig.6c).IntheAOMmiceattheasymptomaticsta 3

ge,neithertheS2northeP2contentofsynaptophysinwascha nged(Fig.6d).TheS2proteincontentofsynaptophysinin creasedgraduallybetweenasymptomaticandsymptomatics tages(Fig.6d).Thecytosolicandmem-

branefractionmarkersLDHandcadherinwereanalyzed

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Fig.4(continued)

byimmunoblottingandconfirmeda purityofbothfrac- tions(Fig.6a).

Table2

EffectsofthetreatmentwithAOMonparameterscharacterizingstimu lus-responsecurvesoffieldpotentials

Vmax( Mv) Uh(μA) S Number

Con4h 1.19±0.40 23.86±3.20 9.16±4.21 11 AOM4h 1.13±0.39 22.77±4.50 8.14±4.10 12 Con18h 1.80±0.40 19.38±6.20 8.20±3.59 18 AOM18h 1.05±0.36* 27.77±5.04* 7.59±3.10 25

PostsynapticProteins

AnalysisofpostsynapticcomplexNMDAR/PSD-

95/nNOSrevealed~16,~40,and~35%increaseofitsparticular com-ponents,i.e.,NR1subunit,PSD-

95,andnNOSprotein,re-

spectively,inP2fractionfromthefrontalcortexofsymptom- aticAOM-treatedmice(Fig.7a).Moreover,intheprodromal stageofAOM,thePSD-95,butnotNR1proteinslevel,was

(18)

*p<0.001;AOM18hvs.controlanimals(Con18h) increased(Fig.7b).

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Table3

ComparisonofsEPSCandmEPSCparametersincontrolandAOM- treated(4and18h)mice

Con4h Con18h

sEPSCs mEPSCs sEPSCs

mEPSCsFrequency(Hz) 1.1±0.1 1.1±0.1 1.3±0.1 1.3±0.1

Amplitude(pA) 8.4±0.2 8.4±0.2 9.4±0.2 9.3±0.2 Rise(ms) 1.5±0.1 1.5±0.1 1.4±0.0 1.4±0.0 Tau(ms) 6.2±0.3 6.2±0.3 4.6±0.1 4.6±0.1

AOM4h AOM18h

sEPSCs mEPSCs sEPSCs

mEPSCsFrequency(Hz) 1.1±0.1 1.0±0.1 1.0±0.0 1.0±0.0

Amplitude(pA) 8.9±0.4 8.8±0.4 10.3±0.2 10.2±0.2 Rise(ms) 1.5±0.1 1.5±0.1 1.5±0.1 1.5±0.1 Tau(ms) 6.8±0.2 6.8±0.2 5.9±0.1 5.9±0.1

Discussion

Thepresentstudyprovidedanexhaustivedescriptionofalter- ationsinsynaptictransmission,ultrastructure,andexpressiono fsynaptictransmission-

relatedproteinsinthefrontalcortexofmicewithALF.Inourhand s,theAOMmodelreproducedawidespectrumofchangesinbloo dandbrainbiochemistryaswellasinneurophysiologicalandbe havioralmanifestationsofALF,eachreflectingthosereportedf oracuteHEindiffer-entanimalALFmodels[25,32–

34]andthehealthstatusofpatientswithALF[35–

37].ProgressionofneurologicaldeficitinAOM-

treatedmicesuggestedthatALFcouldaffectand/ormodifyinfo rmationprocessinginmicefrontalcortexandpromptedustoca rryoutdetailedinvestigationsofelectro-

physiologicalpropertiesofpyramidalneuronsandexcitatory

Dataarepresentedasthemean±SEM

Fig.5ElectronmicroscopyofthecerebralcortexofcontrolandAOM- treatedmiceatsymptomaticstage.a Representativesynapsesareindicat

edbyredcircles.bAveragenumberofsynapticvesiclesatsynapsescounte dfrom50randomlyselectedsynapsesofcontrol(n=3)andAOM- treatedmice(n=4);asteriskindicatesp<0.05vs.controlanimals(Con).Res ultsaremeans±SEM

(20)

synapsesattheasymptomaticandsymptomaticstage.The resultsdocumentedthatdisturbancesinneurotransmissionco -

incidewithimpairedneurologicalstatusoftheanimals.Atthea symptomaticstage,nosignificantchangesinmostparame- terscharacterizingsynaptictransmission,suchasFPampli- tudeandthefrequencyandamplitudeofsEPSCs/mEPSCs,w ereevident.However,despitelackofthosechanges,thePPFrat iowaselevatedatthisstage.AsinPPF,anincreaseintheamplitu deoftheresponsetothesecondpulseofapairisdeterm inedbyapresynapticmechanisminvolvingtheresid-

ualcalciumsignalarisingfromcalciumentrythroughvoltage- gatedcalciumchannels[38];thisresultmayindicatethatearly effectsofALF,atasymptomaticstage,includedisturbancesinc alcium-

bufferingmechanismswithinpresynapticterminals.Atthele velofneurologicalstatus,thePPFratiowashigherthanattheas ymptomaticstage;however,thiseffectwasac-

companiedbyothermarkedchangesincludingareductioninth efrequencyofsEPSCs/mEPSCs.

Asthenumberofglutamatergicsynapsesinthefron- talcortexofAOM-

treatedmiceappearedunchanged,theobserveddecreaseins EPSC/mEPSCfrequency(Fig.4j)wasmostlikelyduetoadec reasedprobabilityofreleaseofglutamatequantafrompresyn apticterminals,whichmayalsounderliea decreaseintheamp litudeofFPs.Thisconclusionisfurthersupportedbythefindi ngthatthedistributionofsynaptophysin,synaptotagmin,a ndvti1aproteinswassignificantlydistortedinthesymptom- aticAOM-injectedmice.

Atthelevelofneurologicalstatus,however,anin- creasedamplitudeanda longerdecaytimeconstantofsEPS Cs/mEPSCswerealsoevident.Thesechangesappeartobeof postsynapticorigin,relatedtothereactivityandkineticsofio notropicglutamatereceptorsandmaybere-

latedtoobservedchangesinthepostsynapticcomplexpro- teins.Moreover,inthesymptomaticAOM-

treatedmice,amarkedlylowermembraneresistanceofneuro nswasevi-

dent.Theseeffectswerenotrelatedtoadirectactionofammo niumiononneurons[8]asthesliceshadbeen

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Fig.6Proteincontentofselectedpr esynapticproteins.aPurityofS2an dP2fraction.bSynaptophysin,syn aptotagmin-1,andMunc18–

1proteincontentsinthecerebralco rtexofcontrolandsymptomaticA OM-

treatedmiceshownasmembrane(

P2)tocytosolic(S2)fractionratio(

P2/S2)

(n=8),followedbyrepresentativee lectrophorograms.cSyntaxin- 1andvti1Aproteincontentsinme mbranefraction(P2)incontroland symptomaticAOM-

injectedmice(n=8),followedbyre presentativeelectrophorograms.

dChangesinsynaptophysinprotei ncontentincytosolic(S2)andmem branefraction(P2)atprodromalan dsymptomaticstageofALF (n=6),followedby

representativeelectrophorograms.

Asteriskindicatesp<0.05vs.cont rolanimals(Con).Resultsaremea ns±SEM

incubatedforseveralhoursintheACSFbeforerecordingsbega n.

Quantificationofsvandanalysisofchangesinthedistributiono fsynapticproteinsfurtherconfirmedthecontributionofpresyn- apticmodificationstoALF-inducedchangesinneurotransmis- sioninthemodel.Thenumberofsvpersynapsewasfoundincreas ed,whichisconsistentwiththedecreaseofmembrane-

tosynaptoplasmcontentratioofsynaptophysinandsynaptotag- min,suggestingadecreasedefficiencyofvesicletraffickingtothe membrane.Itistemptingtospeculatethatincreasedassocia- tionwiththemembranesofthefusionproteinMunc-18-1andthe

dockingproteinsyntaxin-1mayreflecttheirresponseofthede- ficientsupplyofvesiclesforinteractionwiththeseproteins.Howe ver,analysisoftheexpressionandpositioningofmanyothercompo nentsofthedockingmachinery,notconsideredhere,includingMu nc-13,calcium-dependentactivatorproteinforse-cretion,SNAP- 25,andsynaptobrevin-

2areneededtoverifythisspeculation.Thevps10ptailinteractor1a (vti1a)protein,actingtogetherwithvesicle-

associatedmembraneprotein7,character-

izesvesiclesdrivingspontaneousrelease[11].Sincethepostsyn- apticcurrentsmeasuredcorrespondedtomEPSCs,aproductofacti vity-independent,spontaneousreleaseofglutamatefrom

(22)

Fig.7Proteincontentofselectedpo stsynapticproteins.aPSD- 95,nNOS,andNR1proteinconten tsinmembranefraction(P2)incont rolandsymptomaticAOM- treatedmice(n=8),followedbyrep resentativeelectrophorograms.bC hangesinPSD-

95andNR1proteincontentinmem branefraction(P2)atprodromalan dsymptomaticstageofALF (n=6),followedby

representativeelectrophorograms.

Asteriskindicatesp<0.05vs.cont rolanimals(Con).Resultsaremea ns±SEM

presynapticterminals,theALF-relateddecreaseofvti1aexpres- sionmaythereforebeconsideredasalikely,albeitperhapsnottheo nlycauseoftheinhibitionofmEPSCs.

Inourstudy,slicesfromAOM- treatedmicewithearly,

minorneurologicaldeficitsandfromthesymptomaticanimalss howedimpairedLTP.ImpairmentofLTPhasbeenrepeatedlyde monstratedinchronicliverfailure[4,39],thestudiesmost- lyfocusingontheroleofmodulationoftheNMDA-sGC- nNOS-NO-

cGMPpathway[5,40,41].Thepresentstudydocuments,toour knowledgeforthefirsttime,thatal-

thoughtheexpressionofproteinscriticalforpostsynapticacti vity(theNR1/PSD-95/nNOScomplex)wasin-

creased,LTPwasneverthelessimpaired.Thus,mecha- nismsoperatingbeyondthatcomplexappeartounderlietheobs ervedeffect.Itisconceivablethattheymayin-

volveobservedincreaseinthelevelsofproinflammatorycytok ines,IL-6andTNF-

α,asactivationofthecytokinenetworkinthebrainhasbeensho wntobeinvolvedinLTP[42].

Inconclusion,theherepresenteddatademonstratethats ymptomaticALFisassociatedwithdecreasedsynaptictrans- missionwhichappearstoberelatedtoderangementsofpre- synapticproteins,resultingininefficientsvdockingtothesy napticmembrane.Bycontrast,thecontentofpostsynapticprote insisenhanced,whichmayreflectaresponsetode- creasedpresynapticactivity.Evidently,thepostsynapticmod-

ifications,occurringduringsymptomaticALF,appearinsuffi- cienttoprovideeffectivecompensationforthedeclineofLTP.

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Authors’ContributionsMZ/JAconceivedtheidea,de- signedtheprotocol,andwrotethemanuscript.

MP/BB/JS/MF- B/RPperformedalltheanalysesforthestudy.RKF/GH/BZhelp edwritethemanuscriptandintellectuallyrefinetheprotocol.T heauthorswanttogivespecialthankstotheSmallAnimalMag neticResonanceLaboratoryteamfor helpwithcarryingouttheMRImeasurements.

Compliancewithethicalstandard s

FinancialSupportTheresearchstudyhasreceivedfundingfromthePolis h-

NorwegianResearchProgramoperatedbytheNationalCentreforRese archandDevelopmentundertheNorwegianFinancialMechanism2 009–2014intheframeofProjectContractNo.Pol-

Nor/196190/23/2013(MP,JS,BB,GH,JA,MZ).MPwas additionallysupportedbytheLeadingNationalResearchCentre (KNOW)program,andRKFwasfinancedbytheNationalScienceCentr egrant2014/14/M/NZ4/00561.ProjectwascarriedoutwiththeuseofCe PTinfrastructurefinancedbytheEuropeanUnion–

theEuropeanRegionalDevelopmentFoundintheOperationalProgra mmeBInnovativeEconomy^for2007-2013.

ConflictofInterestTheauthorsdeclarethattheyhavenoconflictofintere st.

OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCo mmo nsAt tribu t ion4. 0In t ernat iona lLicen se(ht tp://creativeco mmons.org/licenses/by/4.0/),whichpermitsunrestricteduse,distributi on,andreproductioninanymedium,providedyougiveappro-

priatecredittotheoriginalauthor(s)andthesource,providealinktotheCre ativeCommonslicense,andindicateifchangesweremade.

(24)

References

1. AlbrechtJ,JonesEA(1999)Hepaticencephalopathy:molecularme chanismsunderlyingtheclinicalsyndrome.J NeurolSci170(2):13 8–146

2. AlbrechtJ,ZielinskaM,NorenbergMD(2010)Glutamineasame diatorofammonianeurotoxicity:acriticalappraisal.BiochemPhar macol80(9):1303–1308.doi:10.1016/j.bcp.2010.07.024 3. PrakashR,MullenKD(2010)Mechanisms,diagnosisandmanage-

mentofhepaticencephalopathy.NatRevGastroenterolHepatol7(

9):515–525.d oi:10.1038/nrgastro.2010.116

4. MonfortP,MunozMD,ElAyadiA,KosenkoE,FelipoV(2002)Effe ctsofhyperammonemiaandliverfailureonglutamatergicneu- rotransmission.MetabBrainDis17(4):237–250

5. MunozMD,MonfortP,GazteluJM,FelipoV (2000)Hyperam monemiaimpairsNMDAreceptor-dependentlong-

termpotentiationintheCA1ofrathippocampusinvitro.Neurochem Res25(4):437–441

6. JayakumarAR,T ongXY,CurtisKM,Ruiz-

CorderoR,ShamaladeviN,AbuzamelM,JohnstoneJ,GaidoshG etal(2014)Decreasedastrocyticthrombospondin-

1secretionafterchronicammoniatreatmentreducesthelevelofsyna pticproteins:invitroandinvivostudies.J Neurochem131(3):333 –347.doi:10.1111/jnc.12810

7. FanP,LavoieJ,LeNL,SzerbJC,ButterworthRF(1990)Neuroche micalandelectrophysiologicalstudiesontheinhibitoryeffectofam moniumionsonsynaptictransmissioninslicesofrathippocampus:e videnceforapostsynapticaction.Neuroscience37(2):327–334 8. FanP,SzerbJC(1993)Effectsofammoniumionsonsynaptictr

ansmissionandonresponsestoquisqu alate andN-methyl- D-

aspartateinhippocampalCA1pyramidalneuronsinvitro.BrainRes 632(1–2):225–231

9. RaabeW(1992)Ammoniumionsabolishexcitatorysynaptictrans- missionbetweencerebellarneuronsinprimarydissociatedtissuecul ture.JNeurophysiol68(1):93–99

10. RaabeW,LinS(1985)Pathophysiologyofammoniaintoxication.

ExpNeurol87(3):519–532

11. CrawfordDC,KavalaliET(2015)Molecularunderpinningsofsyn- apticvesiclepoolheterogeneity.Traffic16(4):338–

364.doi:10.1111/tra.12262

12. ImigC,MinSW,KrinnerS,ArancilloM,RosenmundC,SudhofTC, RheeJ,BroseNetal(2014)Themorphologicalandmolecularnatureo fsynapticv e siclep rimingatp resynapticactivezo nes.Neuron84(2 ):416–431.d oi:10.1016/j.neuron.2014.10.009

13. MichelK,MullerJA,OprisoreanuAM,SchochS(2015)Thepre- synapticactivezone:adynamicscaffoldthatregulatessynapticeff icacy.ExpCellRes335(2):157–

164.doi:10.1016/j.yexcr.2015.02.011

14. BelangerM,CoteJ,ButterworthRF(2006)Neurobiologicalchar- acterizationofanazoxymethanemousemodelofacuteliverfailure.N e u r o c h e mI n t4 8( 6–7 ) : 4 3 4–

4 4 0 .d o i :1 0 . 1 0 1 6 / j .neuint.2005.11.022

15. MatkowskyjKA,MarreroJA,CarrollRE,DanilkovichAV,GreenR M,BenyaRV(1999)Azoxymethane-

inducedfulminanthepaticfailureinC57BL/6Jmice:characterizatio nofanewanimalmodel.AmJPhys277(2Pt1):G455–G462 16. McMillinM,GalindoC,PaeHY,FramptonG,DiPatrePL,QuinnM,

WhittingtonE,DeMorrowS(2014)Gli1activationandprotec- tiona gainsthepaticencephalopathyissuppressedbycirculatingtra nsforminggrowthfactorbeta1inmice.JHepatol61(6):1260–

1266.doi:10.1016/j.jhep.2014.07.015

17. WenS,SchroeterA,KlockerN(2013)Synapticplasticityinhepaticen cephalopathy—

amolecularperspective.ArchBiochemBiophys536(2):183–

188.d oi:10.1016/j.abb.2013.04.008

18. MalenkaRC,BearMF(2004)LTPandLTD:anembarrassmentofrich es.Neuron44(1):5–21.doi:10.1016/j.neuron.2004.09.012 19. MilewskiK,HilgierW,FreskoI,PolowyR,PodsiadlowskaA,Zolo

cinskaE,GrymanowskaAW,FilipkowskiRKetal(2016)Carnosin ereducesoxidativestressandreversesattenuationofrightingandpo sturalreflexesinratswiththioacetamide-

inducedliverfailure.NeurochemRes41(1–2):376–

384.doi:10.1007/s11064-015-1821-9

20. CrawleyJN(2007)What’swrongwithmymouse:behavioralphe- notypingoftra nsgenicandk no ck outmice.Wiley-

Interscience,Hoboken,N.J

21. CorneSJ,PickeringRW,WarnerBT(1963)Amethodforassessingth eeffectsofdrugsonthecentralactionsof5-

hydroxytryptamine.BrJPharmacolChemother20:106–120 22. OssatoA,VigoloA,TrapellaC,SeriC,RimondoC,SerpelloniG

,MartiM(2015)JWH-

018impairssensorimotorfunctionsinmice.Neuroscience300:174 –188.doi:10.1016/j.neuroscience.2015.05.021

23. PapadopoulosMC,VerkmanAS(2005)Aquaporin-4genedisrup- tioninmicereducesbrainswellingandmortalityinpneumococcalme ningitis.JBiolChem280(14):13906–

13912.doi:10.1074/jbc.M413627200

24. JaholkowskiP,MierzejewskiP,ZatorskiP,ScinskaA,Sienkiewicz- JaroszH,KaczmarekL,SamochowiecJ,FilipkowskiRKetal (2011)IncreasedethanolintakeandpreferenceincyclinD2knock- outmice.GenesBrainBehav10(5):551–556.doi:10.1111/j.1601- 183X.2011.00692.x

25. RangrooThraneV,ThraneAS,WangF,CotrinaML,SmithNA,Che nM,XuQ,KangNetal(2013)Ammoniatriggersneuronaldisinhibit ionandseizuresbyimpairingastrocytepotassiumbuffer-

ing.NatMed19(12):1643–1648.doi:10.1038/nm.3400

26. CookEB,StahlJL,LoweL,ChenR,MorganE,WilsonJ,VarroR,Chan Aetal(2001)Simultaneousmeasurementofsixcytokinesinasinglesa mpleofhumantearsusingmicroparticle-basedflowcy-

tometry:allergicsvs.non-allergics.JImmunolMethods254(1–

2):109–118

27. ProvencherSW(2001)Automaticquantitationoflocalizedinvivo1H spectrawithLCModel.NMRBiomed14(4):260–264

28. PaxinosG,FranklinKBJ(2004)Themousebraininstereotaxiccoo rdinates.ElsevierAcademicPress

29. ZielinskaM,HilgierW,LawRO,GorynskiP,AlbrechtJ(1999)Effe ctsofammoniainvitroonendogenoustaurineeffluxandcellvolumei nratcerebrocorticalminislices:influenceofinhibitorsofvolume- sensitiveaminoacidtransport.Neuroscience91(2):631–638 30. MilewskiK,HilgierW,AlbrechtJ,ZielinskaM (2015)Thedimethy

larginine(ADMA)/nitricoxidepathwayinthebrainandperipheryo fratswiththioacetamide-

inducedacuteliverfailure:modulationbyhistidine.NeurochemInt 88:26–31.doi:10.1016/j.neuint.2014.12.004

31. ZielinskaM,MilewskiK,SkowronskaM,GajosA,ZieminskaE,Be resewiczA,AlbrechtJ(2015)Inductionofinducibleni-

tricoxidesynthaseexpressioninammonia-

exposedculturedastrocytesiscoupledtoincreasedargininetranspo rtbyupreg-

ulatedy(+)LAT2transporter.JNeurochem135(6):1272–

1281.doi:10.1111/jnc.13387

32. HermenegildoC,MonfortP,FelipoV(2000)ActivationofN- methyl-D-

aspartatereceptorsinratbraininvivofollowingacuteammoniaintox ication:characterizationbyinvivobrainmicrodial-

ysis.Hepatology31(3):709–715.doi:10.1002/hep.510310322 33. HilgierW,OlsonJE(1994)Brainionandaminoacidcontentsd

uringedemadevelopmentinhepaticencephalopathy.J Neuroc hem62(1):197–204

34. HilgierW,OlsonJE,AlbrechtJ(1996)Relationoftaurinetransportan dbrainedemainratswithsimplehyperammonemiaorliver

(25)

failure.JNeurosciRes45(1):69–74.doi:10.1002/(SICI)1097- 4547(19960701)45:1<69::AID-JNR6>3.0.CO;2-F 35. GorgB,QvartskhavaN,BidmonHJ,Palomero-GallagherN,

KircheisG,ZillesK,HaussingerD (2010)Oxidativestressmarkersi nthebrainofpatientswithcirrhosisandhepaticencephalopathy.H epatology52(1):256–265.doi:10.1002

/hep.23656

36. HaussingerD,SchliessF (2008)Pathogeneticmechanismso fh ep ati ce ncep h alop ath y.Gu t5 7 (8 ): 115 6–

11 65 .doi:10.1136/gut.2007.122176

37. PoordadFF(2007)Reviewarticle:theburdenofhepaticen- cephalopathy.AlimentPharmacolTher25(Suppl1):3–

9.doi:10.1111/j.1746-6342.2006.03215.x

38. CarterAG,VogtKE,FosterKA,RegehrWG(2002)Assessingt heroleofcalcium-inducedcalciumreleaseinshort-

termpresynapticplasticityatexcitatorycentralsynap- ses.J Neurosci22(1):21–28

39. FelipoV(2013)Hepaticencephalopathy:effectsofliverfailureonb rainfunction.NatRevNeurosci14(12):851–

858.doi:10.1038/nrn3587

40. MonfortP,CauliO,MontoliuC,RodrigoR,LlansolaM,PiedrafitaB, ElMliliN,BoixJetal(2009)Mechanismsofcognitivealter- ationsinhyperammonemiaandhepaticencephalopathy:thera peuticalimplications.NeurochemInt55(1–3):106–

112.doi:10.1016/j.neuint.2009.01.021

41. MonfortP,PiedrafitaB,FelipoV(2009)TransportofAMPArecept orsduringlong-

termpotentiationisimpairedinratswithhepaticencephalopathy.

NeurochemInt55(7):514–

520.doi:10.1016/j.neuint.2009.05.006

42. delReyA,BalschunD,WetzelW,RandolfA,BesedovskyHO(201 3)Acytokinenetworkinvolvingbrain-borneIL-1beta,IL- 1ra,IL-18,IL-6,andTNFalphaoperatesduringlong-termpotenti- ationandlearning.BrainBehavImmun33:15–

23.doi:10.1016/j.bbi.2013.05.011