What is the main novelty of this article? Why are the results novel? What was previously known, and how do the results expand on previous knowledge?

Provide a brief overview of the main points and conclusions of the article. Only pertinent points should be stated – there is no need to include details of methods unless directly related to a main conclusion. Every result does not need to be included, if not pertinent to the thrust of your discussion. However, there must be sufficient explanation to demonstrate an understanding of the paper. Simply summarizing the abstract is not acceptable.

What is the main novelty of this article? Why are the results novel? What was previously known, and how do the results expand on previous knowledge? Why are these results an addition to the current field? Do they clarify something that was previously unknown? This section MUST incorporate outside research and not just recapitulate the authors’ discussion.

What is/are the outstanding question(s) stemming from this article? This section MUST incorporate outside research that puts the current study in perspective of the field. It must go beyond the discussion of the current article being reviewed, and not just summarize their discussion points. Based on the main novelty of the article, what is the next step? What missing information still needs to be researched? Make sure you focus your discussion of outstanding questions around the main novel results.

I need 2 discussion posts and I included the instructions below. One i based off of the article labeled “neuro”, the second is based off the article labeled “2hearing”. They are separate discusions but use the same three questions and prompts I have shown above.

Requirements: 750 words

Discussion Assignment Instructions

The student will complete 2 Discussions in this course. The student will post one thread of at least 750 words then post 2 replies of at least 350 words. For each thread, students must support their assertions with at least 2 scholarly citations in APA format. Each reply must incorporate at least 1 scholarly citation(s) in APA format. Any sources cited must have been published within the last five years. Acceptable sources include the textbook, the Bible, and peer review journal articles.

ContentslistsavailableatScienceDirectNeuroImage:Clinicaljournalhomepage:www.elsevier.com/locate/yniclNeocorticalmorphometryinHuntington’sdisease:IndicationofthecoexistenceofabnormalneurodevelopmentalandneurodegenerativeprocessesJean-FrancoisMangina,DenisRivièrea,EdouardDuchesnaya,YannCointepasa,VéroniqueGaurab,ChristopheVernyc,PhilippeDamierd,PierreKrystkowiake,Anne-CatherineBachoud-Lévif,PhilippeHantrayeg,PhilippeRemyb,GwenaëlleDouaudh,⁎aUniversitéParis-Saclay,CEA,CNRS,Baobab,Neurospin,Gif-sur-Yvette,FrancebCommissariatàl’EnergieAtomiqueetauxEnergiesAlternatives(CEA),DépartementdesSciencesduVivant(DSV),Institutd’ImagerieBiomédicale(I2BM),MIRCen,FrancecCentrenationalderéférencedesmaladiesneurogénétiques,Servicedeneurologie,CHU,49000Angers,France,UMRCNRS6214–INSERMU1083,FrancedCHUNantes,INSERM,CIC0004,FranceeNeurologie,CHUAmiens-Picardie,FrancefAP-HP,HôpitalHenriMondor,CentredeRéférence-MaladiedeHuntington,FrancegMIRCen,Institutd’ImagerieBiomédicale,DirectiondelaRechercheFondamentale,Commissariatàl’EnergieAtomiqueetauxEnergiesAlternatives,FrancehFunctionalMagneticResonanceImagingoftheBrain(FMRIB)Centre,WellcomeCentreforIntegrativeNeuroimaging,NuffieldDepartmentofClinicalNeurosciences,UniversityofOxford,UnitedKingdomARTICLEINFOKeywords:Huntington’sdiseaseMRICorticalmorphometrySylvianfissureNeurodevelopmentAsymmetryABSTRACTHuntington’sdisease(HD)isaninherited,autosomaldominantdisorderthatischaracteristicallythoughtofasadegenerativedisorder.DespitecellularandmoleculargroundssuggestingHDcouldalsoimpactnormaldevelopment,therehasbeenscarcesystems-leveldataobtainedfrominvivohumanstudiessupportingthishypothesis.Sulcus-specificmorphometryanalysismayhelpdisentanglethecontributionofcoexistingneurodegenerativeandneuro-developmentalprocesses,butsuchanapproachhasneverbeenusedinHD.Here,weinvestigatedcorticalsulcaldepth,relatedtodegenerativeprocess,aswellascorticalsulcallength,relatedtodevelopmentalprocess,inearly-stageHDandage-matchedhealthycontrols.ThismorphometricanalysisrevealedsignificantdifferencesintheHDparticipantscomparedwiththehealthycontrolsbilaterallyinthecentralandintra-parietalsulcus,butalsointheleftintermediatefrontalsulcusandcalcarinefissure.Astheprimaryvisualcortexisnotconnectedtothestriatum,thelatterresultaddstotheincreasinginvivoevidenceforprimarycorticaldegenerationinHD.ThosesulcalmeasuresthatdifferedbetweenHDandhealthypopulationsweremainlyatrophy-related,showingshallowersulciinHD.Conversely,thesulcalmorphometryalsorevealedacrucialdifferenceintheimprintoftheSylvianfissurethatcouldnotberelatedtolossofgreymattervolume:anabsenceofasymmetryinthelengthofthisfissureinHD.Strongasymmetryinthatcorticalregionistypicallyobservedinhealthydevelopment.AstheformationoftheSylvianfissureappearsearlyinutero,andmarkedasymmetryisspecificallyfoundinthisareaoftheneocortexinnewborns,thisnovelfindinglikelyindicatesthefoetaltimingofadisease-specific,geneticinterplaywithneurodevelopment.1.IntroductionCorticalsulcalanalysishasforlongsolelyreliedontheempiricalde-scriptionofthecorticalfoldingsinvestigatedpostmortem(Dareste,1852;Broca,1878).Attheantenatalstage,twofundamentalsteps,nowthoughttobecommontothehigherordermammals,hadbeenobserved:first,theoperculisationoftheinsulaat6months,followedbyaprogressivegyrificationallowingfortheneocorticalsurfacetoincreaseandbecomemorecomplexinthelastthreemonthsofdevelopment.Thesehistoricalobservationsprefiguredatheorythatposesthestabilityofthesesulcal“roots”acrossindividuals,somethingwhichwasfurtherobservedinvivoinnewborns(Regisetal.,2005;Duboisetal.,2008a;Duboisetal.,2008b).Furthermore,ithasbeenknownsincethebeginningofthe19thcenturythatvariousdevelopmentalabnormalitiesleadingtocorticalsulcihttps://doi.org/10.1016/j.nicl.2020.102211Received6June2019;Receivedinrevisedform5February2020;Accepted12February2020⁎Correspondingauthor.E-mailaddress:[email protected](G.Douaud).NeuroImage: Clinical 26 (2020) 102211Available online 13 February 20202213-1582/ © 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).T

malformationareassociatedwithsensorimotor,cognitiveorbehaviouraldisorders(Bruce,1889;Cameron,1907).Investigatingsulcalmorpho-metrymightthuscaptureabnormalitiesemergingduringneocorticalde-velopment,eitherchronologicallycoincidingtotheformationofsulcalroots,orlaterduringcorticalmaturationprocess.Huntington’sdisease(HD)isafatalautosomaldominant,neurode-generativedisorderresultingfromanexpansionofaCAGrepeatwithintheIT15geneonchromosome4.Whilethestriatumisthemostatro-phiedstructureinHD,thereisevidencethatthecorticalatrophyismorewidespreadthanpreviouslythoughtbasedonpostmortemob-servations,thislossofvolumesometimesappearingevenbeforetheonsetofsymptoms(Rosasetal.,2002;Thiebenetal.,2002;Rosasetal.,2005;Douaudetal.,2006;Rosasetal.,2008).Importantly,tworecentinvivostudiesofglobalanthropometricmeasuresinasymptomaticsubjectscarryingthemutatedgenealsopointatadevelopmentalaspectinHD(Nopoulosetal.,2011;Leeetal.,2012).Theseare,toourknowledge,theonlyhumanstudiesshowingresultssupportingthethought-provokingideathatdegenerationinsomedisordersofpossiblegeneticaetiology,includingHDandAlzheimer’sdisease,mightbetheconsequenceofabnormaldevelopment,withcertainpopulationsofneuronalcellsmademorevulnerabletolatelifestressors(MehlerandGokhan,2000;Moleroetal.,2009;MarderandMehler,2012).Here,wecarriedoutforthefirsttimeinHDasulcalmorphometryanalysisusingatoolthatautomaticallyreconstructsandlabelssulcifromT1-weightedimages(Riviereetal.,2002;Manginetal.,2004).Thisapproachhasrevealedforinstancesignificantphylogeneticdif-ferencesinalanguage-relatedsulcalarea(Leroyetal.,2015),oral-terationsinsulcalshapeinageing(Kochunovetal.,2005)–with,forinstance,areducedsulcaldepthrelatedtoadjacentgyralatrophy–aswellasinmildcognitiveimpairmentandAlzheimer’sdisease(Reineretal.,2012;Hamelinetal.,2015).Furthermore,differencesinsulcallengthhavebeenrecentlyconsistentlyrelatedto(abnormal)developmentalprocesses(Auziasetal.,2014;Cachiaetal.,2014;Muellneretal.,2015).Wethusexpectedthatsulcalmorphometryanalysismightrevealevidenceforcoexistingabnormaldegenerativeanddevelopmentalprocesses,inlinewiththeduality,observedforthemutantprotein,ofbothgain-of-functionandloss-of-function(effectswhichareinturnthoughttoplayadistinctroleinbraindegenerationandabnormaldevelopmentrespectively)(MarderandMehler,2012).Asthisex-ploratory,yetregion-of-interestbasedapproachprovidesinformationontheshapeofsulcicomplementarytoinformationobtainedwithvoxelwisetechniques,weanticipatedthatitshouldinparticulardetectsubtleabnormalitiesnotidentifiedusinganapproachsuchasVBM(Manginetal.,2004;Douaudetal.,2006)andthatitmight,crucially,revealnovelabnormalitiesrelatedtoalteredneurodevelopmentinHD.2.MethodsThisstudywaspartoftheMIG-HDproject(MulticentricIntracerebralGraftinginHuntington’sDisease)andwasapprovedbytheethicscommitteeofHenriMondorHospitalinCréteil.Allsubjectsgavewritteninformedconsent.2.1.ParticipantsTwenty-threeHDpatients(14males,9females,2left-handed,aged42±8years,range25–54)wereincludedfromfourdifferenthospitals(Nantes,Angers,LilleandCréteil).Allwerescannedusingthesamescanner,inthesameimagingcentreinOrsay.Tomeetinclusioncri-teria,allhadgeneticallyprovenHD,withanabnormalnumberofCAGrepeatsrangingfrom40to57(46±4).NonehadjuvenileHD.Theyallhadclinicalsymptomsforatleast1yearand15wereatstageIofthediseaseaccordingtotheirtotalfunctionalcapacityscore(TFC≥11)(ShoulsonandFahn,1979),i.e.,theywereautonomousandcouldfunctionfullybothatworkandathome(onaverage10.9±1.4,range8–13).18healthycontrols(HC,14males,4females,2left-handed)matchedforage(41±8years)totheHDpatientsunderwentthesameimagingprotocol.EachHDpatientwasexaminedusingtheUnifiedHuntington’sDiseaseRatingScale(UHDRS,1996)ineachhospitalandthescoresforeachsubscale(motor,behavioural,functionalandneu-ropsychological)werecollected(Table1).2.2.DataacquisitionWhole-brainanatomicalMRIwasacquiredinall41participantswitha1.5TSignaimager(GeneralElectricHealthcare,Milwaukee,WI)withastandard3DT1-weightedinversionrecoveryfastspoiledgra-dientrecalled(IR-FSPGR)sequencewiththefollowingparameters:axialorientation,matrix256×256,124slicelocations,0.9375×0.9375mm2in-planeresolution,slicethickness1.2mm,TI/TE/TR(inversion/echo/repetitiontime)600/2/10.2ms,flipangle(α)10°,readbandwidth(RBW)12.5kHz.2.3.ImageprocessingHereisabriefdescriptionofthemainstepsimplementedinBrainVISAforthereconstructionofthesulcihttp://brainvisa.info(Manginetal.,2004).First,T1-weightedimageswerecorrectedforinhomogeneitiesandabrainmask(greymatterGMandwhitematterWM)wascreatedforeachimage,basedontheanalysisofthehistogramandamorphologicalopening,beforebeingsegmentedintoleftandrighthemispheres,aswellascerebellum.Next,thecomplementofthewhitematter,definedasthespacebetweenthebrainenvelope(identifiedusingamorpho-logicalclosing)andtheGM/WMboundary(identifiedfromthein-tensitiesofthetwotissues),wasskeletonisedtocreatea3Dprintofeachsulcus.Wethusobtainedthe3Dreconstructionofsulciforeachofthe23HDpatientsand18healthycontrols.Varioussulcalfeaturescanthenbeanalysed,butherewefocusedontwothatareeasilyinterpretable:depthandlengthofthesulcus.Decreaseofdepthofsulcihasbeenconsistentlyreportedincaseofneurodegeneration(withhealthyageingandAlzheimer’sdisease),asthesulcibecomemoreshallowasadjacentgyridegenerate(Kochunovetal.,2005;Reineretal.,2012;Hamelinetal.,2015).Incontrast,differencesinlengthofthesulciarethoughttorelatetoab-normaldevelopmentalprocesses(Auziasetal.,2014;Cachiaetal.,2014;Muellneretal.,2015).Asthereisasubstantialinter-subjectvariabilityintheshapeandlocationofthesulci,makinganon-linearwarpingtostandardspaceapproachnotappropriate,thestrategyherewastousetheautomaticrecognitionofthesulcibasedonsupervisedlearningfromadatabasecreatedbyneurosurgeonsandusingneuralnetworks(Riviereetal.,2002).Thisprocessreliesonenergyminimisationandinthisspecificcasethreesuccessiveannealings,whereweselectedtheonewhichTable1ClinicalvariablesfortheHDparticipants.ClinicalVariableMean±stdRangeCAGrepeat46±440–57TotalFunctionalCapacity11±18–13DiseaseBurden409±73239–538MotorUHDRS35±1416–61BehaviouralUHDRS12±100–36FunctionalAssessment27±225–31IndependenceScale88±970–100VerbalFluency(P,R,V)–1min27±107–43VerbalFluency(P,R,V)–2min37±1314–62DigitSymbol26±914–48Stroop(Words)63±2129–103Stroop(Colour)46±1524–76Stroop(Interference)26±910–43J.-F.Mangin,etal.NeuroImage: Clinical 26 (2020) 1022112

minimisedbestthesystem’senergy.Tocreateanadditionalvariable,wemanuallydelineatedthestriatalregionsoneachaxialplaneofeachindividualT1-weightedscan,afteralltheimageswererigidlyreorientedsothattheanteriorandposteriorcommissureswerelocatedinthesameaxialplane(Douaudetal.,2006).Theaccuracyofdelineationwasfurthercheckedinbothsagittalandcoronalplanes,andeachstriatalregionwasreconstructedin3Dtocontrolfortheshapeofeachvolumecreated.Wethencalculatedtheasymmetryindexofthestriatalregionstofurthercorrelatewithpos-sibleresultsshowingamarkedunilateraleffect.2.4.StatisticalanalysisWecarriedoutanANCOVAtocomparesulcibetweenthetwopo-pulations,withdiagnosis,age,andagebydiagnosisinteractionascovariatestomaketheresultseasilycomparablewithapreviousvoxel-basedstudyinthispopulation(18outof23HDpatientsincommon)(Douaudetal.,2006).ResultswereconsideredsignificantforP<0.05(two-tailed),correctedforfalsediscoveryrate(FDR)acrossallsulci(n=57).Weadditionallycheckedthatoursulcalresultsheldwhen:1.addingsexandhandednessasadditionalcovariates,2.normalisingforin-tracranialvolumebycalculatingtheresidualsfordepthandlengthafterthelinearcontributionoftheintracranialvolumetothepower1/3wasremoved(Sanfilipoetal.,2004).Wefurtherensuredthatourresultsshowingdifferencesinthelengthofthesulci–presumablyofdevelopmentalnature–wereinfactnotassociatedwithdiseaseburden((nCAG-35.5)×age)ordiseasestage(TFC).Tothiseffect,wecalculatedthecorrelationcoefficientwithintheHDgroupbetweenthesetwoclinicalmeasuresandourimagingmeasuresoflengthshowingsignificantgroupdifferences.Inaddition,weinvestigatedwithintheHDgroupwhetheranyofoursignificantfindingsmightbecorrelatedaposterioriwiththeirbe-haviouralandclinicalscores(Table1)usingPearsoncorrelation(withandwithoutageaddedasacovariateofnointerest),aswellaswiththeirstriatalvolumetricasymmetryforasymmetricfinding.Toaccountformulticollinearityofthesescores,wereducedthesetofclinicalscorestothosethatdidnotsharemorethan50%ofexplainedvariance.NormalityofthedatawastestedinRforeverystatisticalanalysis(usingtheDatamindsoftwareofBrainVISA)(Duchesnayetal.,2007).3.ResultsSeveralsulciweresignificantlyabnormalintheHDpatients(Table2,Fig.1,FDR-corrected).Whilemostofthemeasuresthatdif-feredbetweenthetwopopulationswereatrophy-related,showingshallowersulciinHD(8outof9ofthesignificantfindings),onemeasurecouldnotberelatedtolossofgreymattervolumeseeninthisneurodegenerativedisorder:thelengthoftheleftposteriorSylvianfissure.Theseresultsheldwhenaddingsexandhandednesstothestatisticalmodel,aswellasafternormalisingforintracranialvolume3.1.Resultsconsistentwithvoxel-basedfindingsofcorticalatrophyInlinewiththeliteratureandourpreviousvoxel-basedresultsbasedonthesameHDpopulation(18outof23HDparticipantsincommon)(Douaudetal.,2006),wefoundthestrongestdifferenceintheleftcentralsulcus,withasignificantreductionofdepthofmorethan8.8%intheHDpatients(seeTable2,Figs.1and2).TherightcentralsulcusdepthwasalsofoundsignificantlyreducedinHD(−6.6%).Theothersulcussignificantlydifferentbilaterallyinthepa-tientscomparedwiththehealthycontrolswastheintra-parietalsulcus,whichwasshallowerontheleftby8.3%,andontherightby8.7%(Table2,Figs.1and2).3.2.FurthercorticalatrophyfindingsThisindividualmeasureapproachfurtherrevealedsignificantlyshallowerleftintermediatefrontalsulcus,decreasedby12.9%inthepatients,inlinewiththeconsistentcorticalpostmortemobservationofdorso-lateralprefrontalcortexatrophy(Table2,Figs.1and3).Thedepthofrightsubparietalsulcus(intheprecuneus)andleftsuperiortemporalsulcuswerealsofoundsignificantlydecreasedinthepatients(Table2).Remarkably,wealsofoundastrongdecreaseindepthoftheleftcalcarinefissureof20.6%intheHDpatients,despitethiscorticalareanotprojectingontothebasalganglia(Table2,Figs.1and3).3.3.EvidenceforabnormalityofneurodevelopmentinHDBeyondtheseconsistentfindingsofreducedsulcaldepthpointingatcorticaldegeneration,thesulcalanalysisalsorevealedastrongdiffer-enceinthelengthoftheposteriorSylvianfissure,withalengthin-creasedby18.9%fortheHDparticipantscomparedwithhealthycon-trols(Table2,Figs.1and4).Thismeasureofthesulcallength,onthecontrarytothatofsulcaldepthwhichisaprobablymarkerofcoloca-lisedatrophy,ismorelikelythehallmarkofanaltereddevelopmentalprocessduringtheformationofthesulcalroots(Auziasetal.,2014;Cachiaetal.,2014;Muellneretal.,2015).InvestigatingfurtherthemeasureoflengthintheSylvianfissure,itisclearthat,inhealthycontrols,thisfissureisinfactconsiderablyshorterinthelefthemispherethantheright(Fig.5).RatherthansimplyseeingthefindingintheleftSylvianfissureasamerelongersulcusinthepatients,itcanthusbeinterpretedmoreappropriatelyasanalmostcompleteabsenceofasymmetryforthissulcusinHD,asymmetrythatisnormallyfoundinhealthyparticipants(Fig.5).TheleftSylvianfissureisindeedshorterthantherightby18.8%inthehealthyparticipants,comparedwithonly5.5%inHD.Thisabsenceofasymmetryisfurthermaintainedatthesingle-subjectlevel,astheasymmetryindexbetweenleftandrightSylvianfissurelength,AI=(R-L)/0.5*(R+L),issig-nificantlydecreased(towards0)intheHDgroup(P=0.02,n=41).Table2Allsignificant(FDR-corrected)sulcaldifferencesbetweenHD(n=23)andhealthycontrols(HC,n=18).SulciSideFeatureHCMean±stdHDMean±stdP-valueP-value*(Sex+Handedness)P-value⁎⁎(ICV)CentralSulcusLDepth22.6±1.321.2±1.14.0×10−41.9×10−31.8×10−2RDepth23.0±1.221.0±1.34.6×10−63.0×10−52.1×10−3Intra-parietalsulcusLDepth25.1±1.523.0±1.82.9×10−51.3×10−45.2×10−4RDepth24.4±1.822.2±1.53.0×10−44.4×10−42.1×10−3IntermediatefrontalsulcusLDepth17.1±2.114.9±1.53.5×10−52.6×10−23.2×10−2CalcarinefissureLDepth31.4±5.924.9±6.41.5×10−41.1×10−31.2×10−2SubparietalsulcusRDepth14.3±2.911.4±2.82.0×10−45.6×10−35.5×10−3SuperiortemporalsulcusLDepth24.7±2.422.7±4.07.3×10−41.6×10−32.6×10−2Sylvian(lateral)fissureLLength282.7±42.3335.9±58.13.2×10−48.9×10−41.4×10−3⁎Sameanalysescarriedoutaddingsexandhandednessastwoadditionalcovariatesofnointerest.⁎⁎Sameanalysescarriedoutontheresidualsobtainedafterpartiallingouttheeffectofintracranialvolume(ICV).J.-F.Mangin,etal.NeuroImage: Clinical 26 (2020) 1022113

AsthelengthoftheleftposteriorSylvianfissureseemedtobeahallmarkofabnormalasymmetryinHD,wefurtherinvestigatedwithinthisgroupifitwasassociatedwiththeirstriatalvolumetricasymmetry,asmeasuredusingcarefulmanualsegmentationofthesubcorticalstructures(Douaudetal.,2006).Wefoundthatitwassignificantlycorrelatedwithsuchsubcorticalasymmetry(r23=0.49,24%ofvar-ianceexplained,P=0.017,SupplementaryFigure1).Finally,weestablishedthattheabnormallengthoftheSylvianfis-sureinHDwasnotcorrelatedwitheitherdiseaseburden(r21=0.08,P=0.38)orTFC(r20=0.19,P=0.21).3.4.Post-hoccorrelationswithclinicalscoresWefirstreducedthesetofscorestothosethatdidnotsharemorethan50%ofexplainedvariance(r>0.70).ThisallowedustoassesscorrelationsbetweenthesignificantsulcalfindingsofdepthandlengthwiththeMotorUHDRS,BehaviouralUHDRS,StroopInterference(highlycorrelatedwithStroopWordandColour,andDigitSymbol),FunctionalAssessment(highlycorrelatedwithIndependenceScale),TFCandSumFluency(wherewesummedthetworuns,andwhichwashighlycorrelatedwiththeMATTIS).AssociationsaresummarisedinFig.1.VisualrepresentationofsomeofthesulcifoundthemostdifferentbetweenhealthyandHDparticipants(5of7).Weshowthesulciinthelefthemisphereofonerandomlyselectedhealthycontrol:left,opaquecortex;right,partiallytransparentcortextovisualisethe3Dconformationofthesulci,andthoseonthemedialsurface.Thecentralsulcusappearsinred,theintra-parietalsulcusingreen,theposteriorlateralfissureindarkblue,theintermediatefrontalsulcusinlightblue,andbytransparency,thecalcarinefissureinbrown.Whiletheresultsinthecentralsulcusandintra-parietalsulcuswerebilateral,differencesintheposteriorlateralfissure,intermediatefrontalsulcusandcalcarinefissurewereleft-lateralised.Fig.2.BilateralresultsconsistentwithcorticalatrophyinHD:shallowercentralandintra-parietalsulcusinHD.Top:CentralSulcus.Left,3Drenderingoftheleftcentralsulcusinonehealthysubject.MiddleandRight,maximaldepthoftherightandleftcentralsulcusinthehealthycontrols(HC,n=18,inbluecircles,averageindarkblue),andintheHDparticipants(n=23,inmagentatriangles,averageindarkmagenta)(a.u.).Bottom:Intra-ParietalSulcus.Samerepresentationasabove.J.-F.Mangin,etal.NeuroImage: Clinical 26 (2020) 1022114

SupplementaryTable1,butbriefly:theseshowedanassociationbe-tweenStroopInterferenceanddepthoftherightintra-parietalsulcus(r20=0.40,16%ofvarianceexplained,P=0.04),FunctionalAssessmentanddepthoftheleftintermediatefrontalsulcus(r20=−0.46,21%ofvarianceexplained,P=0.02),andBehaviouralUHDRSanddepthoftheleftcalcarinefissure(r20=−0.52,28%ofvarianceexplained,P=0.009).Afterregressingageout,wealsofoundanassociationbetweenthesumofthefluencyscoresandthedepthoftheleftintermediatefrontalsulcus(SupplementaryTable1).4.DiscussionThisisthefirststudyofsulcalmorphologycarriedoutinHD.Themotivationforthisstudywastwo-fold.First,itwaspromptedbyastringofpublishedevidencethathasestablishedearlycorticaldegenerationinHD,whetherinthesameearlyHDpopulation(18outof23incommon):(Douaudetal.,2006),oreveninpremanifestHD:(Thiebenetal.,2002;Rosasetal.,2005).ApreviousglobalmorphologicalstudyfoundaglobaldecreaseofsulcaldepthinHD(Nopoulosetal.,2007).Here,ourresultsmightexplainthisglobaleffectbyshowingaclear,localiseddecreaseindepthofthecentralandintra-parietalsulcusinbothhemispheres,rightsub-parietalsulcus,andleftintermediatefrontalsulcus,calcarinefissureandsuperiortemporalsulcus.Second,asthissulcalmorphometryap-proachmaybeabletodifferentiateunderlyingdegenerativeanddevel-opmentalprocesses,itwasfurthermotivatedbytworecentinvivostudiesingenecarriersshowingthefirstsignsofabnormaldevelopmentusinganthropometricmeasurements(Nopoulosetal.,2011;Leeetal.,2012).Remarkably,oursulcalanalysisrevealedasubstantialdifferenceintheimprintoftheposteriorSylvianfissure,namelyanabsenceofasymmetryintheHDpopulationbetweenleftandrighthemispheres,suggestingaveryearlyinsulttothedevelopingneocortex.AshallowercentralsulcusinourHDparticipantscanbeeasilyrelatedtothemostconsistentlossofcorticalgreymatterintheprecentralandpostcentralgyriobservedinameta-analysisinHD(Doganetal.,2013),Fig.3.Additionalleft-lateralisedresultsconsistentwithcorticalatrophyinHD:shallowerintermediatefrontalsulcusandcalcarinefissure.Top:IntermediateFrontalSulcus.Left,3Drenderingoftheleftintermediatefrontalsulcusinonehealthysubject.Right,maximaldepthoftheleftintermediatefrontalsulcusinthehealthycontrols(HC),andintheHDpatients(a.u.).Bottom:CalcarineFissure.Samerepresentationasabove.J.-F.Mangin,etal.NeuroImage: Clinical 26 (2020) 1022115

notleastinthesamepatients(Douaudetal.,2006).ThedepthoftheintraparietalsulcuswasalsosignificantlydecreasedbilaterallyintheHDparticipants,particularlysointhelefthemisphere(Fig.3).Theleftin-traparietalsulcusis,togetherwiththepremotorandprimarysensorimotorcortex,thecorticalregionfoundtoalsodiscriminatebestbetweenpre-manifestandmanifestHDinameta-analysis(Doganetal.,2013).Inad-dition,therightintraparietalsulcusdepthcorrelatedinthepatientswiththeStroopInterference(r20==0.40,SupplementaryTable1),ameasureofselectiveattentionwhosefunctionalnetworkiscentredontheintra-parietalsulcus(Heddenetal.,2012).Whenwealsoinvestigated,asanadditionalanalysis,thesurfacemeasureofthesulci,wefoundthatthestrongestdifferenceswerefoundbilaterallyintheintraparietalsulcus(SupplementaryTable2).Whilemainlyredundant(andlesssensitive)thanthemeasureofsulcaldepth,thesurfaceofsulcisolelyrevealedasignificantdifferenceintheleftolfactorysulcus,whichmightbelinkedtothesmelldeficitsconsistentlyobservedinHD(Paulsenetal.,2017).Findingsofaleft-lateraliseddegenerationaroundtheintermediatefrontalsulcusconcurwiththewealthofpostmortemevidenceontheinjurytothedorso-lateralprefrontalcortexe.g.,(Hedreenetal.,1991;Hallidayetal.,1998).AsitwasnotdetectedusingVBM(Douaudetal.,2006),thissuggeststhatthemethodusedheremightbesensitivetodetectveryearlysignsofprefrontaldegeneration,whicharetypicallyseenatlaterstagesofHD(Rosasetal.,2008).Forinstance,totalfunctionalcapacityscore(TFC)rangingfrom1to13wasfoundtocorrelatewithleftprefrontalareas(Rosasetal.,2008).Inourpre-dominantlystageIHDpopulation,whereTFCrangewasmorelimited,Fig.5.EvidenceforabnormalityofneurodevelopmentinHD:absenceofasymmetryintheposteriorSylvianfissureinHD.A.ThereisanaturalasymmetrybetweenleftandrightlengthoftheposteriorSylvianfissureinhealthycontrols(HC)(a.u.).InHC,theleftfissureisshorteronaveragebyalmost20%.Bycontrast,thereisalmostnodifferenceonaverageintheHDpatients.Forthepatients,theleftSylvianfissureisonlyshorterbylessthan6%onaverage.B.Thisabsenceofasymmetryisalsofoundatthesingle-subjectlevel:theasymmetryindexoftheSylvianfissurelengthiscloseto0intheHDpatients.Fig.4.EvidenceforabnormalityofneurodevelopmentinHD:longerleftposteriorSylvian(lateral)fissureinHD.Left,3DrenderingoftheleftposteriorSylvianfissureinonehealthysubject.Right,lengthoftheleftposteriorSylvianfissureinthehealthycontrols(HC,n=18,turquoisecircles),andintheHDparticipants(n=23,mauvetriangles)(a.u.).J.-F.Mangin,etal.NeuroImage: Clinical 26 (2020) 1022116

wefoundsimilarassociationsspecificallybetweenthedepthoftheintermediatefrontalsulcusandtheUHDRSmeasuresoFunctionalAs-sessment(r20=−0.46),andatatrendlevelwithTFC(r20=0.31)(SupplementaryTable1).Decreaseindepthoftheleftcalcarinefissurecouldseemsurprisingatfirst,asthispartofthebrainisnotconnectedtothestriatum.Butitisinfactaresultconsistentwithinvivosurface-basedstudiesofHD,wheredegenerationwasfoundintheoccipitallobeandinparticulararoundtheleftcalcarinefissure(Rosasetal.,2002;Rosasetal.,2008),aswellaswithpostmortemstudies(Hallidayetal.,1998).Indeed,whilecorticaldegen-erationinHDhadbeeninitiallythoughttobeasecondaryeventduetothestriataldegeneration,itismorelikelythatbothprimaryandsecondarydegenerativeprocessesco-existinthecortex(Rubetal.,2015).ThisisfurthersupportedbyhistopathologicalfindingsshowingdamagetolayerVIofthecortexthatdoesnotprojecttothestriatum(Hedreenetal.,1991).Ofnote,thedecreaseindepthinthecalcarinefissureisthestrongestintermsofeffectsize(morethan20%)comparedwithallothersulcifoundshallowerinHD.Intriguingly,thedepthoftheleftcalcarinefissurewascorrelatedwiththebehaviouralUHDRSscore(r20=−0.52,SupplementaryTable1).Thisassociationmightperhapsberelatedtotheassociationobservedbetweenbehaviouralsymptoms–visualhallucina-tionsanddepression–andthisspecificregionofthebrainalsoseeninParkinson’sdisease(Matsuietal.,2006;Huetal.,2015).Interestingly,thissulcalanalysisalsorevealedanincreaseofnearly20%inthelengthoftheleftposteriorSylvianfissureinHDcomparedwiththehealthyparticipants.Theconsistentdecreaseofdepthfoundinvarioussulciareconsistentwithaneurodegenerativeprocess,andthusmainlyconsistentwithvolume-basedandsurface-basedfindings.Asignificantdifferenceinthelengthofonesulcus,onthecontrary,ismoredifficulttobeinterpreted,especiallyinlightoftheabsenceofcolocalisedatrophy,andthelackofassociationwithdiseasestageorburden,andage.Assuch,itismorelikelyrelatedtoanaltereddevelopment.Thisleftperi‑Sylvianregionisforinstancewellknowntobeassociatedwithfunctionallan-guagelateralisationandspecialisation,althoughitdidnotcorrelatewithverbalfluency(Table1),theonlylanguage-relatedmeasureavailableinourHDpopulation.Morphologicalanomaliesinthisbrainregionhavebeenfoundinpopulationwithneurodevelopmentaldisorders,suchasstutteringandinchildrenwithdyslexia(Foundasetal.,2004;Kibbyetal.,2004;Cykowskietal.,2008).Itisalsoconnectedbywhitemattertractsthataretheonlyfibrebundlesshowingtheeffectofgeneticassociationswithhandedness(Wibergetal.,2019).However,asshownintheResultssection,healthydevelopmenttypicallyleadstoastrongasymmetrybe-tweenthetwohemispheres–infactthestrongestasymmetryfoundacrosstheentirecortex,asdemonstratedforinstanceinpretermnewborns(Duboisetal.,2010).OurresultintheposteriorSylvianfissurethereforedemonstratesanabsenceofasymmetryinHD,comparedwithnormalde-velopment.Interestingly,differencesinsulcalasymmetryhaverecentlydemonstratedtobekeyinunderstandingdifferencesindevelopmentalprocesses(Kloppeletal.,2010;Cachiaetal.,2014;Leroyetal.,2015).ThisaltogethersuggeststhattheabnormallengthoftheleftposteriorSylvianfissureinHDmightbearthehallmarkofanearly,altereddevel-opmentalprocess.AstheformationoftheSylvianfissureappearsearlyinutero,andmarkedasymmetryisspecificallyfoundinthisregioninpre-termnewborns(Duboisetal.,2010),thislikelyindicatesthefoetaltimingofadisease-relatedgeneticinterplaywithneurodevelopment.InourHDpopulation,thelengthoftheleftposteriorSylvianfissurewasfurthersignificantlyassociatedwiththestriatalvolumetricasymmetry,asmea-suredusingcarefulmanualsegmentationofthesubcorticalstructures(r23=0.49,24%ofvarianceexplained,P=0.017,SupplementaryFigure1)(Douaudetal.,2006).SuchstriatalasymmetryinturnexplainsasubstantialpartofthevarianceintwofundamentalUHDRSmeasuresinourcohort:TFC(r20=−0.49,24%ofvarianceexplained,P=0.027)andIndependenceScale(r19=−0.59,35%ofvarianceexplained,P=0.0075).ItcouldthusbethatthesubcorticalvolumeasymmetryseeninthestriatumofHDpatientsisbothacombinationofdevelopmentalanddegenerativeprocesses.ComparedwithatechniquesuchasVBM,thisspecificsulcalap-proachcannotshowpreciselywheresomeoftheabnormalitiesmightbelocalisedalongasulcus(e.g.,dorsalvs.ventralpartofthecentralsulcus).Newestdevelopmentsmightbeabletoresolvetheselimitations(Coulonetal.,2015).Inanycase,itrevealedinthesameHDpopulation(18outof23incommon),andusingthesamestatisticalmodel,sig-nificantdifferencesinareaswheretheVBManalysishadfailedtodetectalossofvolumeormorphology:therightprecuneus,aswellastheleftdorso-lateralprefrontalcortex,primaryvisualcortex,superiortemporalcortexandperi‑Sylvianregion(Douaudetal.,2006).OtherVBMstu-dies,possiblybecauseoflargersamplesizeormoreadvancedHDpo-pulation,haveinsomecasesdemonstratedvoxelwisedifferencesinthosecorticalregionswhereonlyoursulcalapproachrevealedab-normalities(Muhlauetal.,2007;Scahilletal.,2013;Minkovaetal.,2018).Thesamplesizeofthisstudyisalsolimited,butwemadesuretoonlypresentinthemainmanuscriptsulcalgroupdifferencessurvivingcorrectionformultiplecomparisons(asanindication,top20resultsinSupplementaryTable3).Thisrelativelysmallsamplesizealsomeantlargeeffectsizesforoursignificantresults,suchasadifferenceof19%inlengthoftheSylvianfissure,orof21%inthedepthofthecalcarinefissure.Finally,anotherclearlimitationisthatourparticipantswerealreadysymptomatic.EspeciallyforthefindingsintheSylvianfissure,astudyon(ideallyyoung)genecarriersfarfromtheonsetofsymptoms-suchasdoneinLeeetal.(2012)-shouldconfirmthepre-existingnatureofthissulcalabnormality,andinparticularofitsdistinctiveasymmetryinHD.Insummary,weusedforthefirsttimeadetailedanalysisofsulcalmorphologyinHD.Thisapproach,whichpreciselytargetscorticalfeatures,offerscomplementarysourcesofinformation,notonlytoconventionalvoxel-andvertex-wiseapproaches,butalsoinhowtheyrelatetodifferentunderlyingphysiopathologicalprocesses,andcouldhelpdetectsubtleneurodevelopmentalabnormalitiesthatwouldotherwisegounnoticedinotherdegenerativedisorderswithageneticsusceptibility.ItrevealedinHDabnormalitiesconsistentwithaneu-rodegenerativeprocess,butalsoimportantlywithanalteredneurode-velopment.WhiletheatrophyfoundintheleftvisualcortexaddstotheincreasingwealthofdataindicativeofaprimarycorticaldegenerationinHD,thisstudyprovides,tothebestofourknowledge,thefirstinvivoindicationofaninterplaybetweendiseaseandneocorticaldevelop-ment.CRediTauthorshipcontributionstatementJean-FrancoisMangin:Conceptualization,Formalanalysis,Software,Writing-originaldraft.DenisRivière:Formalanalysis,Software,Writing-originaldraft.EdouardDuchesnay:Formalana-lysis,Software.YannCointepas:Software.VéroniqueGaura:Resources.ChristopheVerny:Resources.PhilippeDamier:Resources.PierreKrystkowiak:Resources.Anne-CatherineBachoud-Lévi:Resources,Fundingacquisition.PhilippeHantraye:Supervision,Fundingacquisition.PhilippeRemy:Supervision,Resources,Fundingacquisition.GwenaëlleDouaud:Conceptualization,Resources,Investigation,Formalanalysis,Fundingacquisition,Writing-originaldraft,Writing-review&editing.DeclarationofCompetingInterestTheauthorsdeclarenocompetingfinancialinterests.AcknowledgementsandFundingG.D.issupportedbytheUKMedicalResearchCouncil(MR/K006673/1).ThisstudywaspartoftheMIG-HDtrialcoordinatedbyA.-C.B.-L.(Principalinvestigator)andgrantedthroughPHRCsAOM00139andAOM04021fromtheDRCD(AssistancePublique-HôpitauxdeParis).Wewouldliketothankthepatientsandtheirfamilies.J.-F.Mangin,etal.NeuroImage: 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ContentslistsavailableatScienceDirectBehaviouralBrainResearchjournalhomepage:www.elsevier.com/locate/bbrHearinglossasariskfactorforcognitiveimpairmentandlossofsynapsesinthehippocampusMunyoungChanga,1,HaengJunKimb,1,InheeMook-Jungb,c,Seung-haOhc,d,⁎aDepartmentofOtorhinolaryngology-HeadandNeckSurgery,Chung-AngUniversityCollegeofMedicine,102Heukseok-ro,Dongjak-gu,Seoul,06973,RepublicofKoreabDepartmentofBiomedicalSciences,SeoulNationalUniversityCollegeofMedicine,103Daehak-ro,Jongno-gu,Seoul,03080,RepublicofKoreacDepartmentofBiochemistry,SeoulNationalUniversityCollegeofMedicine,103Daehak-ro,Jongno-gu,Seoul,03080,RepublicofKoreadDepartmentofOtolaryngology-HeadandNeckSurgery,SeoulNationalUniversityCollegeofMedicine,103Daehak-ro,Jongno-gu,Seoul,03080,RepublicofKoreaARTICLEINFOKeywords:HearinglossAlzheimer’sdiseaseDementiaAmyloid-βHippocampusABSTRACTAlthoughepidemiologicalstudieshaveidentifiedanassociationbetweenhearinglossandcognitiveimpairment,thereisalackofbiologicalevidencedetailingthemechanismsunderlyingthisassociation.Thepresentstudyinvestigatedtheeffectsofhearinglossoncognitiveimpairmentusinganat-riskmodel.Inthisanimalmodel,amyloid-β(Aβ)wasadministeredtothebraintosuchanextentthatitdidnotcausecognitiveimpairmentsbutmadethebrainvulnerabletoriskfactors.ThisstudyincludedfourexperimentalgroupsbasedonhearinglevelandAβadministration.Behavioraltestswereconductedtoevaluatecognitivefunction,andsynapticproteinlevelsweremeasuredinthehippocampusandprefrontalcortex.ThegroupwithhearinglossandAβadmin-istrationshowedsignificantlygreaterdeficitsoncognitivetestsassociatedwiththehippocampusthantheotherthreegroups(onlyAβadministration,onlyhearingloss,andwithouthearinglossorAβadministration).ThehearinglossandAβadministrationgroupalsohadsignificantlylowerlevelsofsynapticproteinsinthehippo-campusthantheothergroups.ThepresentresultssuggestthathearinglossmayactasariskfactorforcognitiveimpairmentinAlzheimer’sdisease.Additionally,thepresentfindingsindicatehearinglossmaycausehippo-campalsynapsestobemorevulnerabletoAβ-induceddamage.1.IntroductionIn2016,approximately43.8millionpeoplesufferedfromdementiaworldwide.Furthermore,theworldwidedeathrateassociatedwithdementiawas2.4millionpeople,whichmadeitthefifthleadingcauseofdeath[1].TheleadingcauseofdementiaisAlzheimer’sdisease(AD)[2]and,therefore,thereisanurgentneedforthedevelopmentoftreatmentsforAD.Althoughmuchresearchhasbeenconductedinthisarea,thecurrentlyavailabletreatmentsforADhaveyettoachievesignificantclinicalefficacyinthattheycanpartiallystabilizethesymptomsofthisdiseasebutnotcorrectit[3].ItisalsoimportanttoidentifyriskfactorsforAD,asthisinformationwillallowustodevelopmethodspreventingADdevelopmentorslowingdiseaseprogression.Age,familyhistory,andheredityarethemostimportantriskfactorsofAD[4]andcanbeusedtopredictitsoccurrence.However,thesefactorscannotbemodifiedand,thus,cannotcontributetothepreventionofAD.Recentepidemiologicalevidencesuggeststhatthereisanassociationbetweenhearinglossandcognitiveimpairment[5–8]andotherstudieshaveshownthathearinglossmaybeapotentiallymodifiableriskfactorofAD[9].Approxi-matelyone-thirdofelderlypeople65yearsofageandolderhavehearingloss,whichcanbeamelioratedbyhearingaidsandcochlearimplants.Therefore,ifhearinglossisariskfactorofcognitiveim-pairmentanditsmechanismscanbeidentified,thenthetreatmentofhearinglosscancontributetothepreventionofAD.However,thecausalrelationshipbetweenhearinglossandADremainscontroversial.Forexample,ithasbeensuggestedthattheassociationbetweenhearinglossandADexistsduetodifficultiesincognitivefunctionteststhatpatientswithhearinglossexperienceduetopoorverbalcommunica-tion.Furthermore,thebiologicalmechanismsthatunderliethisasso-ciationhaveyettobeelucidated.Thus,thepresentstudyemployedanimalmodelstoinvestigatehttps://doi.org/10.1016/j.bbr.2019.112069Received1May2019;Receivedinrevisedform9June2019;Accepted1July2019Abbreviations:Aβ,amyloid-β;AD,Alzheimer’sdisease;OPT,object-in-placetask;OLT,objectlocationtask;NOR,novelobjectrecognitiontask;ABR,auditorybrainstemresponse;NH-SA,normalhearing-subthresholdamyloid-β;deaf-SA,deaf-subthresholdamyloid-β;NH-NA,normalhearing-nonamyloid-β;deaf-NA,deaf-nonamyloid-β⁎Correspondingauthor.E-mailaddress:[email protected](S.-h.Oh).1Theseauthorscontributedequallytothiswork.Behavioural Brain Research 372 (2019) 112069Available online 02 July 20190166-4328/ © 2019 Published by Elsevier B.V.T

whetherhearinglosswouldbeariskfactorforADandtoassessthemechanismsbywhichhearinglossmayactasariskfactor.Becauseseveralempiricalcasesandotherevidenceindicatesthathearinglossalonedoesnotleadtocognitiveimpairment[10],asubthresholdamyloid-β(Aβ)modelofAD[11]wasusedinthepresentstudy.Inthismodel,Aβisadministeredtothebraintosuchanextentthatitdoesnotcausecognitiveimpairmentsbutmakesthebrainvulnerabletoriskfactorssothatitmightbepossibletoverifywhetherhearinglosswouldbeariskfactorforcognitiveimpairment.2.Methods2.1.ExperimentaldesignThisstudywasapprovedbytheInstitutionalAnimalCareandUseCommitteeofChung-AngUniversity(2016-00086)andSeoulNationalUniversityHospital(16-0133-C1A0)andallexperimentswerecon-ductedinaccordancewithrelevantguidelinesandregulations.Seven-week-oldmaleWistarrats(200–250g)wereusedandallanimalswereadaptedtolaboratoryconditionsfor1weekpriortothestartoftheexperimentandhousedinatemperature-andhumidity-controlledroomwitha12-hlight:darkcyclewithfoodandwateravailableadlibitum.Auditorybrainstemresponse(ABR)recordingsandsurgicalprocedureswereperformedunderanesthesiainducedbythein-traperitonealadministrationofketaminehydrochloride(100mg/kg;Ketamine®,YuhanCo.;Seoul,Korea)mixedwithxylazine(10mg/kg;Rompun®,Bayer-Korea;Seoul,Korea).Thepresentstudyconsistedoftwostages:determiningthetimecourseofcognitivedeclinefollowinghearinglossandthenevaluatingchangesincognitivefunctionandsynapticproteinlevelsafterinduc-tionofthehearingloss(Fig.1).Inthefirststage,10ratswererandomlydividedintotwogroups:apilot-normalhearing-subthresholdAβgroup(pilot-NH-SA;n=5)thatunderwentashamsurgeryandtheinfusionofsubthresholdAβandapilot-deaf-subthresholdAβgroup(pilot-deaf-SA;n=5)thatunderwentbilateralcochlearablationandinfusionofsub-thresholdAβ.TheinfusionofsubthresholdAβfor2weeksbegan3weeksaftersurgeryandtheY-mazetestwasperformedevery2weeksinallratsstartingat7weeksafterthesurgery.Theresultsofthefirststagewereusedtodeterminethetimepointsatwhichhearinglossin-ducedasignificanteffectoncognitiveimpairment.Inthesecondstage,26ratswererandomlydividedintofourex-perimentalgroups:anormalhearing-nonAβgroup(NH-NA;n=6)thatunderwentashamsurgerybutnotinfusionofsubthresholdAβ,anormalhearing-subthresholdAβgroup(NH-SA;n=6)thatunderwentashamsurgeryandtheinfusionofsubthresholdAβ,adeaf-nonAβgroup(deaf-NA;n=7)thatunderwentbilateralcochlearablationbutnotinfusionofsubthresholdAβ,andadeaf-subthresholdAβgroup(deaf-SA;n=7)thatunderwentbilateralcochlearablationandtheinfusionofsubthresholdAβ.TheinfusionofsubthresholdAβfortwoweeksbegan9weeksaftersurgeryandcognitivetestsincludingtheY-mazetest,object-in-placetask(OPT),objectlocationtask(OLT),andnovelobjectrecognitiontask(NOR),wereperformedtoallrats11weeksaftersurgery.Afterthecognitivefunctiontests,tissuesampleswereharvestedfromthehippocampusandprefrontalcortex.Onean-imalinthedeaf-SAgroupexhibitedposturalasymmetrywhenpickedupafterthebilateralcochlearablationandwasexcludedfromtheex-periment.Duringthebreedingperiod,oneanimalintheNH-SAgroupandoneanimalinthedeaf-SAgroupdied.Ultimately,theNH-NA,NH-SA,deaf-NA,anddeaf-SAgroupsconsistedof6,5,7,and5animals,respectively.WeperformedanadditionalexperimentusinganothernineratstoassesswhethertheanimalspreferredfamiliarornovelobjectsintheNOR.2.2.ABRrecordingsABRrecordingswereconductedinallratsbeforesurgeryand1week,6weeks,and11weeksaftersurgerytomeasurehearinglevels.ABRsontheleftsidewererecordedwithsubdermalneedleelectrodesbetweentheleftmastoidandthenapeoftheneckwiththerightmastoidasthereturnwhileABRsontherightsideweremeasuredbyreversingthedirectionoftheelectrodes.ABRswererecordedwithhigh-frequencytransducers(HFT9911–20–0035)andsoftware(ver.2.33)Fig.1.Experimentalflowofthefirst(a)andsecond(b)stage.Aβ,amyloid-β;NH-SA,normalhearing-subthresholdamyloid-β;deaf-SA,deaf-subthresholdamyloid-β;NH-NA,normalhearing-nonamyloid-β;deaf-NA,deaf-nonamyloid-β.M.Chang,etal.Behavioural Brain Research 372 (2019) 1120692

fromSmartEP(IntelligentHearingSystems;GlenvarHeights,FL,USA)andtheresponseswereamplified(100,000×),bandpass-filtered(100–1500Hz),andaveragedover512stimulusrepetitions.Tonepipsof8,16,and32kHzwereusedassoundstimuli(5-msduration,cosshaping,21Hz)andstimulusintensitywasreducedin5dBSPLde-crements.Tworesearchers,blindtotheexperimentalconditions,de-terminedtheloweststimulusintensitythatevokedarecognizablere-sponse,andthatwasregardedasthethreshold.2.3.CochlearablationCochlearablationwasperformedonbothsidesaspreviouslyde-scribed[12].Briefly,afteraretroauricularincision,theexternalaudi-torycanalwasopenedandthetympanicmembraneandossicles,exceptforthestapes,wereremoved.Then,asmallholewasmadeonthebonywallofthecochleaandthecontentsofthecochleawereablatedwithadentalpick.Asmallamountofsofttissuewaspackedintothesmallholeonthebonywallofthecochlea.Intheshamsurgery,thesameoperativeprocedurewasperformedbeforethepointofopeningtheexternalauditorycanal.2.4.BehavioraltestsforvestibulardeficitsToexcludetheeffectsofvestibularfunctiondeteriorationduringcochlearablation,thebehavioraltestforvestibulardeficitswasper-formedthedayandweekaftersurgeryaspreviouslydescribed[13].Briefly,thebehavioralscoringforvestibulardeficitsconsistedofthreecomponents:posturalasymmetry,headrolltilt,andnystagmus(TableS1).Ifanydeficitswerefoundinanyofthesethreecomponents,theanimalwasexcludedfromtheexperiment.2.5.InfusionofsubthresholdAβTheAβpeptidesolutionwascontinuouslyadministeredintotheintracerebroventricularspace(160pmol/day)for2weeksusingabraininfusioncannula(BrainInfusionKit2,Alzet;Cupertino,CA,USA)thatwasconnectedtoamini-osmoticpump(OsmoticPump2002,Alzet).Theinfusioncannulawasimplantedintotherightcerebrallateralventricle(AP:−0.3,L:1.2,V:4.5)accordingtothecoordinatesofPaxinosandWatson(2006)[14].ThecompositionoftheAβpeptidesolution,whichdoesnotinducecognitiveimpairment,hasbeende-scribedpreviously[11].Briefly,aAβ1-42peptidesolution(AnaSpecInc.;SanJose,CA,USA)wasdissolvedin35%acetonitrile/0.1%tri-fluoroaceticacid.Themini-osmoticpumpwasremoved2weeksafterimplantation,andtheremainingvolumeofAβ1-42peptidesolutionmeasuredtoconfirmthattheexpectedvolumehadbeendelivered;wesubtractedtheresidualfromtheinitialvolume.2.6.Cognitivetesting2.6.1.Y-mazetestCognitivefunctionwasassessedbyrecordingspontaneousalterna-tionbehaviorinasinglesessionintheY-maze;theprotocolforthistaskhasbeenpreviouslyreported[15].Briefly,eacharmofthemazewas40cmlong,30cmhigh,and15cmwideandconvergedinacentraltrianglearea.NoneoftheanimalshadeverexperiencedaY-mazebe-fore.Allarmswerebrushedwith10%ethanolpriortoeachsessiontoremovethepossibleeffectsofodorcuesandtheexperimenterwasnotintheroomduringtesting.EachratwasplacedononearmtipoftheY-mazeandthenallowedtowalkaroundthemazefor7minwithoutrestriction.EachsessionintheY-mazewasvideorecordedandanalyzedlater.Theratwasconsideredtohaveenteredthearmwhenitshindpawsenteredthearmandalternationwasdefinedassuccessiveentriesintothreearmsbasedonoverlappingtriplets.Thealternationpercen-tagewascalculatedasfollows:actualalternations/possiblealterna-tions(totalnumberofarmentriesminustwo).2.6.2.OPT,OLT,andNORTheOPT,OLT,andNORwereconductedbymodifyingapreviouslyreportedmethod[16].Beginning4daysbeforethetests,theratswereplacedinanopenfieldbox(58×42×35cm)withoutstimulifor10–15mindaily.Eachsessionconsistedoffamiliarizationandtestphasesandeitherthetypeorlocationofthestimulusobjectsinthetestphasewasdifferentfromthatinthefamiliarizationphase.Inthefa-miliarizationphase,theratsexploredstimulusobjectsintheopenfieldboxfor5minandwerethenreturnedtotheirhomecageforafixedamountoftime(5minforOPTandOLTand3hforNOR).Then,theratswereplacedintheboxagainandallowedtoexplorethestimulusobjectsduringthetestphase.Theexperimentwasvideorecordedinaroomwithouttheexperimenterandtherecordedvideowasanalyzedlater.Exploratorybehaviorwasdefinedasdirectingthenosetowardanobjectatadistanceoflessthan2cmortouchingtheobjectwiththenoseorpaws.Adiscriminationratiowascalculatedasfollows:(ex-plorationtimewiththechangedobject-explorationtimewiththeunchangedobject)/(totalexplorationtimewiththechangedandun-changedobject).Whenexplorationtimewasshorterthan15sduringthefamiliarizationphaseorshorterthan10sinthetestphase,thedatawereexcludedfromtheanalysis.ThetestconditionsareshowninFig.S1.FortheOPTfamiliarizationphase,fourdifferentstimulusobjectswereplacedinthecornersofthebox(10cmfromthewall).DuringtheOPTtestphase,thepositionsoftwooftheobjects(whichwerebothontheleftorrightofthebox)wereswitched.FortheOLTfamiliarizationphase,twoidenticalobjectswereplacedinthecornersofthebox.DuringtheOLTtestphase,oneobjectwasrepositionedtothecorneradjacenttoitsoriginalposition;thus,thetwoobjectswerediagonaltoeachother.FortheNORfamiliarizationphase,twoidenticalobjectswereplacedinthecornersofthebox.DuringtheNORtestphase,oneobjectwaschangedtoanovelobject.BeforetheNOR,weperformedanadditionalexperimenttoassessob-jectbias.Afteradaptationtotheopenfieldbox,anothernineratsex-ploredthetwoobjects(afamiliarandanovelobject)tobeusedintheNORtestphasefor5min.Thedurationsoftimespentexploringeachobjectweremeasuredandcompared.2.7.WesternblotanalysisAftercompletionofthebehavioraltests,allanimalswereeu-thanizedandbraintissuesampleswereharvestedfromthehippo-campusandprefrontalcortexbasedonthecoordinatesofPaxinosandWatson(2006)[14].FortheWesternblotanalyses,tissuesfromthehippocampusandprefrontalcortexofallgroupswerelysedinaradioimmunoprecipitationassaybuffer(RIPA)buffer(iNtRONBio-technology;Seoul,Korea)containingaproteaseinhibitorcocktail(Sigma;St.Louis,MO,USA),proteinphosphataseinhibitorcocktail(AGScientific;SanDiego,CA,USA),andphenyl-methylsulfonylfluoride(PMSF;Sigma).Then,thebrainlysatesweresonicatedtoensurethor-oughlysis.TheconcentrationsoftheproteinlysatesweredeterminedwithaBCAassayandanidenticalamountofproteinfromeachsamplewaselectrophoreticallyseparatedbysodiumdodecylsulphatepoly-acrylamidegelelectrophoresis(SDS-PAGE)in4–12%Bis–Trisgelsandthentransferredtopolyvinylidenedifluoride(PVDF)membranes.Themembraneswereblockedin5%non-fatdrymilkinTris-bufferedsaline(TBS)and0.1%Tween-20(TBS-T)andthenincubatedwiththefol-lowingprimaryantibodiesat4℃overnight:postsynapticdensitypro-tein95(PSD95;ab18258,Abcam;Cambridge,UK),synaptophysin(mab268,Millipore;Burlington,MA,USA),Ca2+/calmodulin-depen-dentproteinkinaseII(CaMKII;ab52476,Abcam),phosphorylatedCAMKII(pCaMKII;3361s,CellSignalingTechnology;Danvers,MA,USA),N-methylD-aspartatereceptorsubtype2B(NR2B;06–600,Mil-lipore),andα-tubulin(05–829,Millipore).Next,themembraneswerewashedwithTBS-Tfor30minandincubatedwithsecondaryIgG-HPantibodiesagainsteachprimaryantibodyfor1h.Then,themembraneswerewashedwithTBS-TandincubatedwithanECLchemiluminescentM.Chang,etal.Behavioural Brain Research 372 (2019) 1120693

reagent.Finally,peroxidaseactivitywasdetectedwithLAS4000(GEHealthcareLifeScience;Marlborough,MA,USA);theopticaldensitieswerenormalizedwithastandardprotein.2.8.StatisticalanalysesIBMSPSSsoftwareversion21.0(IBM;NewYork,NY,USA)wasusedforallstatisticalanalyses.ABRthresholdswereanalyzedwithone-wayanalysisofvariance(ANOVA)tests.ScoresonthecognitivetestsinthefirststageoftheexperimentwereanalyzedwithrepeatedmeasuresANOVAtestsandscoresateachtimepointwereanalyzedwithunpairedtwo-tailedStudent’st-test.Scoresonthecognitivetestsinthesecondstageoftheexperimentwereanalyzedwithone-wayANOVAsandpairedt-tests.TheresultsoftheWesternblotanalyseswereanalyzedwithone-wayANOVAs.AllposthoctestingwasperformedusingTukey’stests.3.Results3.1.ABRrecordingsPriortosurgery,thebaselineABRthresholdsat8,16,and32kHzrangedfrom20to35dBSPLinallanimals;thesevaluesdidnotdiffersignificantlyamongthegroups(p>0.05).At1week,6weeks,and11weeksaftersurgery,theABRthresholdsat8,16,and32kHzrangedfrom20to35dBSPLintheNHgroupbutwerehigherthan80dBSPLinthedeafgroup(Fig.2).3.2.DoseofAβ1-42peptidesolutiondeliveredThedailyvolumesofdeliveredAβ1-42peptidesolutionrangedfrom12.0to11.9μL,correspondingto161.0to159.0pmoL/dayoftheAβ1-42peptide,similartotheanticipatedvolumes.Thedailydosesdidnotdiffersignificantlybetweenthegroups(p>0.05).3.3.TimecourseofcognitivedeclinefollowinghearinglossInthefirststageoftheexperiment,thetimecourseofcognitivedeclinefollowinghearinglosswasevaluatedusingtheresultsoftheY-mazetest(Fig.3a).TheinfluenceofhearinglosswasexploredwitharepeatedmeasuresANOVAusingtheY-mazescoresacrosstimeasarepeatedmeasure(7,9,and11weeksaftersurgery)andthegroupsasfixedfactors.Mauchly’stestofsphericityindicatedthattheassumptionFig.2.ABRthresholdsbeforesurgeryand1week,6weeks,and11weeksaftersurgery.(a)pilot-NH-SAgroup.(b)pilot-deaf-SAgroup.(c)NH-NAgroup.(d)NH-SAgroup.(e)deaf-NAgroup.(f)deaf-SAgroup.Errorbarsindicatestandarddeviation.ABR,auditorybrainstemresponse;SPL,soundpressurelevel;NH,normalhearing;SA,sub-amyloid-β;NA,non-amyloid-β.Fig.3.Cognitivetestresults.(a)Timecourseofcognitivedeclinefollowinghearingloss.Y-mazescoresweresignificantlylowerinthepilot-deaf-SAgroupcomparedtothepilot-NH-SAgroupat11weeksaftersurgery.(b)IntheY-maze,OPT,andOLTtests,thedeaf-SAgrouphadsignificantlylowerscoresthantheotherthreegroupsinthesecondstageoftheexperiment.Alldataarepresentedasamean±SEM.(a)Unpairedtwo-tailedStudent’st-testateachtimepoint.(b,c)One-wayANOVAfollowedbyTukey’spost-hoctest.*P<0.05,**P<0.01,***P<0.001.SA,sub-amyloid-β;NH,normalhearing;OPT,object-in-placetask;OLT,objectlocationtask;NA,non-amyloid-β.M.Chang,etal.Behavioural Brain Research 372 (2019) 1120694

ofsphericityfortimehadbeenviolated(p=0.453)and,therefore,theresultsfortimearereportedusingtheGreenhouse-Geissercorrection(ε=0.832).TheY-mazescoreschangedovertime(p=0.046)andtherewasasignificantinteractionbetweentimeandgroup(p=0.032);thus,themaineffectsforgrouparereportedateachtimepoint.TheY-mazescoresofthepilot-NH-SAandpilot-deaf-SAgroupsdidnotsig-nificantlydifferat7or9weeksaftersurgery(p=0.624andp=0.208,respectively)buttheY-mazescoresofthepilot-deaf-SAgroupweresignificantlylowerthanthoseofthepilot-NH-SAgroupat11weeksaftersurgery(p=0.014).3.4.CognitivefunctionandsynapticmakerproteinlevelsafterhearinglossThecognitivetestingresultsinthesecondstageoftheexperimentaredisplayedinFig.3bandTableS2andS3.Thetimespentbytheanimalsinexplorationexceeded15sduringthefamiliarizationphasesand10sduringthetestphasesoftheOPT,OLT,andNOR.Noanimalwasexcludedfromtheanalysis.ThetotaltimespentexploringobjectsduringthefamiliarizationandtestphasesoftheOPT,OLT,andNORdidnotdifferamongthegroups(TableS2).DuringthefamiliarizationphasesoftheOPTandOLT,nosignificantdifferencesinthetimespentexploringobjectsthatwereswitchedandthosenotswitchedduringthetestphaseswereapparent(TableS3).ThiswasalsothecasefortheadditionalexperimentoftheNOR(21.8±3.3and21.6±4.0sre-spectively,p=0.852,pairedt-test).IntheY-maze,OPT,andOLTtests,thedeaf-SAgrouphadsig-nificantlylowerscoresthantheotherthreegroups(p<0.05,Fig.3b).TherewerenosignificantdifferencesamongtheotherthreegroupsonthosethreetestsandnosignificantdifferencesamongallfourgroupsintheNORtest.Thepresentstudyalsoinvestigatedmolecularchangesinthehip-pocampusandprefrontalcortexofallgroupsbyquantifyingsynapticproteinlevelswithWesternblotanalyses.Inthehippocampus,thereweresignificantdecreasesinNR2BandPSD95,whicharepost-synapticmarkers,andsynaptophysin,whichisapre-synapticmarker,levelsinthedeaf-SAgroup(Figs.4a–dandS2)butnosignificantchangesintheotherthreegroups.Additionally,therewerenosignificantchangesinthephosphorylationlevelsofCaMKII(Fig.4aande).Intheprefrontalcortex,PSD95levelssignificantlydecreasedinthedeaf-SAgroupcomparedtotheNH-NAanddeaf-NAgroups(Fig.4fandi).Synapto-physinlevelssignificantlydecreasedintheNH-SAanddeaf-SAgroupscomparedtotheNH-NAanddeaf-NAgroupshoweddecreasingtrends(Fig.4fandh).ThephosphorylationlevelsofCaMKIIdecreasedinallothergroupscomparedtotheNH-NAgroup(Fig.4fandj).NR2Blevelsintheprefrontalcortexdidnotsignificantlydifferamongthegroups(Fig.4fandg).4.DiscussionAlthoughseveralepidemiologicalstudieshavesuggestedthathearinglossisariskfactorforcognitivedecline[6–8,17],theunder-lyingmechanismsremainunclear.Threerepresentativehypotheseshavebeenpresented;theyinvolvetheeffectsofhearingimpairmentsoncognitiveloadandbrainstructureanddecreasedsocialengagement[18].Thecognitiveloadhypothesissuggeststhatauditoryperceptualprocessingrequiresmorecognitiveresourceswhentheauditorysignalisdegraded,whichresultsinthedegradationofothercognitivepro-cesses,suchasworkingmemory.Anotherhypothesisproposesthatimpairedauditorysignalsandreducedstimulationfromanimpairedcochleacausechangesinbrainstructure.Thiswouldmakethebrainmorevulnerabletobrainpathology-causingfactors,suchasAβaccu-mulation,neurofibrillarytangles,andmicrovasculardisease,andleadtoanincreasedriskofdementia.Thethirdhypothesissuggeststhatcognitivefunctionisdegradedbysocialisolationduetohearingloss.However,fewstudieshaveprovidedevidencesupportingthesehy-potheses.Thus,thepresentstudyattemptedtodeterminewhetherhearinglosswouldactasariskfactorforADandtoidentifytheme-chanismsunderlyingthisassociation.Whenplanningthepresentexperiments,itwasimportanttocon-siderthatcognitivedysfunctionwillnotbeinducedwhenonlyhearinglossispresent.Theresultsofafollow-upstudyinvestigatingcognitivefunctioninsubjectswhodevelopedhearinglossinchildhoodreportedthatlong-termsensoryimpairmentalonehasanegligibleeffectonone’soveralllevelofcognitivefunction[10].Therefore,thepresentstudycomparedcognitivefunctioninanimalswithhearinglossandnormalhearingusingamodelofsubthresholdAβ,whichhasbeenpublishedunderthenameoftheat-riskmodel[11].ThismodelisintendedtorepresentindividualswithapredispositionforAβbuildupbutnormalcognitivefunction.Thus,itispossibletoinvestigatewhethercertainfactorsmayberiskfactorsofAD.Inthepresentstudy,fourexperimentalgroupsbasedonhearinglevelandthesubthresholdadministrationofAβwereformedandcognitivetestsknowntoberelatedtospecificbrainregionswerecon-ducted.Cognitivetestsassociatedwiththehippocampus,suchastheY-maze,OPT,andOLT[16],revealedsignificantdecreasesincognitivefunctioninthedeaf-SAgroupafterhearingloss,ascomparedtotheothergroups.However,therewerenosignificantdifferencesamongthegroupsintheNOR.ThehippocampusmayaffectNORresultswhenthetimebetweenthefamiliarizationandtestphasesisextended[19,20].However,othershavereportedthatthehippocampusdoesnotinflu-enceNORresultsregardlessofthetimeintervalbetweenthetwophases[16,21–24].ThediscrepanciesmaybeattributabletodifferencesinthemethodsusedtoeliminatehippocampalfunctionandtheexperimentalconditionsunderwhichNORwasperformed.AstudythatevaluatedNORexactlyaswedidreportedthatthehippocampusdidnotaffecttheresults[16].Therefore,inourexperiment,thehippocampusmaynotaffectNORresults.Takentogether,theseresultssuggestthathearinglossaffectedthehippocampusandmaybeariskfactorforcognitiveimpairment.ComparisonsofsynapticproteinlevelsinthehippocampusbetweentheNH-NAandNH-SAgroupsrevealednosignificantdifferences.TheseresultsindicatethatthesubthresholdadministrationofAβdidnotaf-fectsynapticproteinlevelsinthehippocampusinnormalhearingan-imals.Thechangesinsynapticproteinlevelsinthehippocampusafterhearinglossmirroredtheresultsofthecognitivetesting:thedeaf-SAgroupexhibitedasignificantdecreaseinsynapticproteinscomparedtotheotherthreegroups.ThesedataindicatethatcognitiveimpairmentmaybeacceleratedbythesynergisticeffectsofhearinglossandAβduetosynapticloss.Inthecaseofprefrontalcorticalsynapticproteinlevels,someproteinsinthedeaf-SAgroupexhibitedareductionbutthesechangeswerenotconsistentandwerenotlikelytobeaffectedbyhearingloss.ThepresentstudydemonstratedthathearinglossmightactasariskfactorforcognitiveimpairmentinADpatientsandthathearinglossmaycausehippocampalsynapsestobemorevulnerabletobrainpa-thology.Thisfindingindicatesthatthereareconnectionsbetweenthecentralauditorypathwayandthehippocampus,whichhasbeenpro-posedinpreviousstudies.Forexample,therearechangesinthehip-pocampusfollowingsoundexposure[25–29]andtheuseofante-rogradetracersrevealedthatthehippocampusreceivessignalsfromtheauditorycortexviatheentorhinalcortex[30].Therefore,degenerationinthecentralauditorypathwayinducedbyhearingloss[31,32]maycausethedegenerationofhippocampalsynapsesormakethesesy-napsesmorevulnerabletodamage.Thishypothesisissupportedbyfindingsshowingthatfocalcorticalinfarctionofbrainregionsthatareremotebutconnectedtothehippocampusinduceneuronallossinthehippocampus[33].Furtherstudiesareneededtoobtainsolidconclu-sions.Thepresentstudyhasseverallimitationsthatshouldbenoted.First,thedevelopmentofhearinglossandAβdepositionintheanimalmodelsusedinthisstudydifferfromthoseinactualhumans.Inmosthumans,hearinglossandAβdepositionprogressslowlyand,therefore,M.Chang,etal.Behavioural Brain Research 372 (2019) 1120695

itwillbenecessarytodevelopanovelanimalmodelinwhichhearinglossandAβdepositionprogressinamannersimilartothatofhumans.Second,thepresentstudyshowedthattherewasadecreaseinhippo-campalsynapsesfollowinghearingloss.However,thelocationsandrolesofthedegeneratedsynapsescouldnotbeidentifiedandfurtherresearchwillbenecessarytoclarifythesefindings.5.ConclusionsThepresentstudyshowedthathearinglossmayactasariskfactorforcognitiveimpairmentinAD.Furthermore,hearinglossmaymakesynapsesinthehippocampusmorevulnerabletodamagethatcanresultinbrainpathology.AcknowledgementsThisworkwassupportedbytheNationalResearchFoundationofKorea(NRF)grantfundedbytheKoreagovernment(MinistryofScienceandICT)(No.NRF-2016R1C1B2007131toM.C.).References[1]E.Nichols,C.E.I.Szoeke,S.E.Vollset,N.Abbasi,F.Abd-Allah,Global,regional,andnationalburdenofAlzheimer’sdiseaseandotherdementias,1990-2016:asys-tematicanalysisfortheGlobalBurdenofDiseaseStudy2016,LancetNeurol.18(2019)88–106.[2]C.Ballard,S.Gauthier,A.Corbett,C.Brayne,D.Aarsland,E.Jones,Alzheimer’sdisease,Lancet377(2011)1019–1031.[3]J.Cao,J.Hou,J.Ping,D.Cai,AdvancesindevelopingnoveltherapeuticstrategiesforAlzheimer’sdisease,Mol.Neurodegener.13(2018)64.[4]F.Panza,V.Solfrizzi,G.Logroscino,Age-relatedhearingimpairment-ariskfactorandfrailtymarkerfordementiaandAD,Nat.Rev.Neurol.11(2015)166–175.[5]S.Behrman,L.Chouliaras,K.P.Ebmeier,Consideringthesensesinthediagnosisandmanagementofdementia,Maturitas77(2014)305–310.[6]J.Gallacher,V.Ilubaera,Y.Ben-Shlomo,A.Bayer,M.Fish,W.Babisch,P.Elwood,Auditorythreshold,phonologicdemand,andincidentdementia,Neurology79(2012)1583–1590.[7]K.M.Kiely,B.Gopinath,P.Mitchell,M.Luszcz,K.J.Anstey,Cognitive,health,andsociodemographicpredictorsoflongitudinaldeclineinhearingacuityamongolderadults,thejournalsofgerontology,SeriesA,Biologicalsciencesandmedicalsci-ences67(2012)997–1003.[8]F.R.Lin,K.Yaffe,J.Xia,Q.L.Xue,T.B.Harris,E.Purchase-Helzner,S.Satterfield,H.N.Ayonayon,L.Ferrucci,E.M.Simonsick,Hearinglossandcognitivedeclineinolderadults,JAMAIntern.Med.173(2013)293–299.[9]G.A.Gates,J.H.Mills,Presbycusis,Lancet366(2005)1111–1120.[10]M.Vernon,Fiftyyearsofresearchontheintelligenceofdeafandhard-of-hearingchildren:areviewofliteratureanddiscussionofimplications,J.DeafStud.DeafEduc.10(2005)225–231.[11]T.T.Tran,M.Srivareerat,K.A.Alkadhi,Chronicpsychosocialstresstriggerscog-nitiveimpairmentinanovelat-riskmodelofAlzheimer’sdisease,Neurobiol.Dis.37(2010)756–763.[12]S.K.Mun,K.H.Han,J.T.Baek,S.W.Ahn,H.S.Cho,M.Y.Chang,Losartanpreventsmaladaptiveauditory-somatosensoryplasticityafterhearinglossviatransforminggrowthfactor-betasignalingsuppression,Clin.Exp.Otorhinolaryngol.12(2019)33–39.[13]M.Y.Chang,S.Park,J.J.Choi,Y.K.Kim,M.W.Suh,J.H.Lee,S.H.Oh,M.K.Park,MicroR.N.As218a-5p,219a-5p,and221-3pregulatevestibularcompensation,Sci.Fig.4.SynapticmarkerproteinsarealteredbyhearinglossandAβinfusionintheratbrain.Pre-andpost-synapticmarkerproteinlevelsinthehippocampusdecreasedfollowinghearinglossandAβinfusion.(a)Representativeimagesand(b–e)quantificationalgraphs(n=5–7).Somepre-andpost-synapticmarkerproteinlevelsintheprefrontalcortexdecreasedfollowinghearinglossandAβinfusion.(f)Representativeimagesand(g–j)quantificationalgraphs(n=5–7).Alldataarepresentedasamean±SEM.One-wayANOVAfollowedbyTukey’spost-hoctest.*P<0.05,**P<0.01,***P<0.001.NA,non-amyloid-β;SA,sub-amyloid-β;Aβ,amyloid-β.M.Chang,etal.Behavioural Brain Research 372 (2019) 1120696

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