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Comprehensive behavioral analysis of heterozygousSyngap1knockout mice

Citation for published version:Nakajima, R, Takao, K, Hattori, S, Shoji, H, Komiyama, NH, Grant, SGN & Miyakawa, T 2019,'Comprehensive behavioral analysis of heterozygous Syngap1knockout mice', NeuropsychopharmacologyReports. https://doi.org/10.1002/npr2.12073

Digital Object Identifier (DOI):10.1002/npr2.12073

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Neuropsychopharmacology Reports. 2019;00:1–15.  | 1wileyonlinelibrary.com/journal/nppr

Received:4April2019  |  Revised:19June2019  |  Accepted:19June2019DOI: 10.1002/npr2.12073

O R I G I N A L A R T I C L E

Comprehensive behavioral analysis of heterozygous Syngap1 knockout mice

Ryuichi Nakajima1  | Keizo Takao2,3 | Satoko Hattori1  | Hirotaka Shoji1  | Noboru H. Komiyama4 | Seth G. N. Grant5 | Tsuyoshi Miyakawa1,3

ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttribution‐NonCommercial‐NoDerivsLicense,whichpermitsuseanddistributioninanymedium,providedtheoriginalworkisproperlycited,theuseisnon‐commercialandnomodificationsoradaptationsaremade.©2019TheAuthors.Neuropsychopharmacology ReportspublishedbyJohnWiley&SonsAustralia,LtdonbehalfofTheJapaneseSocietyofNeuropsychoPharmacology.

1DivisionofSystemsMedicalScience,InstituteforComprehensiveMedicalScience,FujitaHealthUniversity,Toyoake,Japan2DivisionofAnimalResourcesandDevelopment,LifeScienceResearchCenter,UniversityofToyama,Toyama,Japan3SectionofBehaviorPatterns,CenterforGeneticAnalysisofBehavior,NationalInstituteforPhysiologicalSciences,Okazaki,Japan4CentreforClinicalBrainSciences,ThePatrickWildCentreforResearchintoAutism,FragileXSyndrome&IntellectualDisabilities,TheUniversityofEdinburgh,Edinburgh,UK5GenestoCognitionProgram,CentreforClinicalBrainSciences,UniversityofEdinburgh,Edinburgh,UK

CorrespondenceTsuyoshiMiyakawa,DivisionofSystemsMedicalScience,InstituteforComprehensiveMedicalScience,FujitaHealthUniversity,1‐98Dengakugakubo,Kutsukake‐cho,Toyoake,Aichi470‐1192,Japan.Email:miyakawa@fujita‐hu.ac.jp

Funding informationMinistryofEducation,Culture,Sports,ScienceandTechnology,Grant/AwardNumber:KAKENHIJP16H06276,KAKENHIJP16H06462andKAKENHIJP221S0003;JapanAgencyforMedicalResearchandDevelopment,Grant/AwardNumber:JP18dm0107101;JapanSocietyforthePromotionofScience,Grant/AwardNumber:KAKENHIJP16680015;SimonsInitiativefortheDevelopingBrain,Grant/AwardNumber:R83776

AbstractAims: SynapticRasGTPase‐activatingprotein1(SYNGAP1)regulatessynapticplas‐ticitythroughAMPAreceptortrafficking.SYNGAP1mutationshavebeenfound inhumanpatientswithintellectualdisability(ID)andautismspectrumdisorder(ASD).Almost every individualwithSYNGAP1‐related IDdevelops epilepsy, and approxi‐mately50%haveASD.SYNGAP1‐relatedIDisestimatedtoaccountforatleast1%ofIDcases.InmousemodelswithSyngap1mutations,strongcognitiveandaffectivedysfunctionshavebeenreported,yetsomefindingsareinconsistentacrossstudies.TofurtherunderstandthebehavioralsignificanceoftheSYNGAP1gene,weassessedvariousdomainsofbehaviorinSyngap1heterozygousmutantmiceusingabehavioraltestbattery.Methods: MalemicewithaheterozygousmutationintheSyngap1gene(Syngap1−/+ mice)createdbySethGrant'sgroupweresubjectedtoabatteryofcomprehensivebehavioraltests,whichexaminedgeneralhealth,andneurologicalscreens,rotarod,hotplate,openfield,light/darktransition,elevatedplusmaze,socialinteraction,pre‐pulseinhibition,Porsoltforcedswim,tailsuspension,gaitanalysis,T‐maze,Y‐maze,Barnesmaze,contextualandcuedfearconditioning,andhomecagelocomotoractiv‐ity.TocontrolfortypeIerrorsduetomultiple‐hypothesistesting,P‐valuesbelowthefalsediscoveryratecalculatedbytheBenjamini‐Hochbergmethodwereconsideredasstudy‐widestatisticallysignificant.Results: Syngap1−/+miceshowed increased locomotoractivity,decreasedprepulseinhibition,andimpairedworkingandreferencespatialmemory,consistentwithpre‐cedingstudies. Impairmentofcontext fearmemoryand increasedstartle reflex inSyngap1mutantmicecouldnotbereproduced.Significantdecreases insensitivityto painful stimuli and impairedmotor functionwere observed inSyngap1−/+ mice. Decreasedanxiety‐likebehavioranddepression‐likebehaviorwerenoted,althoughincreasedlocomotoractivityisapotentialconfoundingfactorofthesephenotypes.Increasedhomecagelocomotoractivityindicatedhyperlocomotoractivitynotonlyinspecificbehavioraltestconditionsbutalsoinfamiliarenvironments.

2  |     NAKAJIMA et Al.

1  | INTRODUC TION

SYNGAPisaGTPasehighlyenrichedatexcitatorysynapsesinthebrain 1,2.SeveralmembersoftheRassuperfamilyofGTPases,includ‐ingRap1/2,Ras,andRab53‒6,are inhibitedbySYNGAP.SYNGAPlevels in the dendritic spine are reduced by neuronal activation 7. ThereductioninSYNGAPleadstoRasactivationandAMPArecep‐torincorporationintothemembrane,bothofwhicharerequiredforlong‐termpotentiation6,8,dendriticspineformation9,andneuronaldevelopment10.

De novo SYNGAP1mutationshavebeenfoundinpatientswithID,epilepsy,orASD11‒20.Inalarge‐scaledevelopmentaldisordersstudy, sevenSYNGAP1mutationswere identified in 940patientswith ID; therefore, the frequency of SYNGAP1 mutations is sug‐gested to be ∼0.74% in patients with ID 19. Currently, 0.7%‐1%of ID patients are estimated to have SYNGAP1‐related ID 18. A study,whichrecruited57malepatientswithSYNGAP1mutationsormicrodeletions,reported55casesof ID21.Thesepatientsalsoshowedepilepsy (98%)anddevelopmentaldelays (96%),and53%oftheparticipantswerediagnosedwithASD21.Thesesymptomswereaccompaniedbyseverelanguageimpairments(21%);highpainthreshold(72%);eatingproblemsincludingoralaversion(68%);hy‐potonia(67%);sleepingproblems(62%);ataxiaorgaitabnormalities(51%);andbehavioralproblems(73%)includingaggression,self‐in‐jury,andtantrums21.

TostudytheeffectsofSYNGAP1mutations,Syngap1knockoutmicehavebeengeneratedbyseveralgroups9,10,22‒24.HomozygousSyngap1knockoutmicediewithinaweekofbirth10,22.Inheterozy‐gousSyngap1knockout(Syngap1−/+)mice,robustchangesinbehav‐ioralphenotypeshavebeenreportedbyseveralgroups(seeTableS4).Syngap1−/+miceshowincreasedlocomotoractivity23,25‒29,de‐creasedanxiety‐likebehavior6,23,25,26,28,29,impairedreferencespa‐tialmemory22,26,27,andimpairedworkingspatialmemory23,26,28,29. Inaddition, increased stereotypicbehavior 25, decreases inmotorfunctions in females 26, elevated startle response and a decreaseinprepulseinhibition25,reducedsocialnoveltypreference25,andimpaired cued fear memory 25 have been reported by precedingstudies. However, some observations are inconsistent across thedifferentstudies.InSyngap1−/+mice,impairmentofcontextualfearmemoryhasbeenreportedbytwogroups23,28,whileanotherre‐portfailedtodetectthisbehavioralchange25.Onestudyobserved

decreasedanxiety‐likebehaviorinthenumberofopen‐armentriesin the elevatedplusmaze 26,whereas another report did not ob‐serve similar findings 25.WhilehumanpatientswithSyngap1 mu‐tationshaveahighpain threshold,eatingproblems,ataxiaorgaitabnormalities,hypotonia,andsleepingproblems21,therehavebeennoreportsofsuchbehavioralphenotypesinSyngap1−/+ mice.

Comparedtopreviousreports6,23,25‒30,onlyafewstudieshaveassessed the behavioral phenotypes of the Syngap1−/+mouse linegeneratedbyKomiyama et al22,29. In the present report,we eval‐uated thebehavioralphenotypesofSyngap1−/+micegeneratedbyKomiyamaet al22 onaC57BL/6Jgeneticbackground,whichhavebeenbackcrossedatleast10generationsfromtheoriginalF2MF1geneticbackground.Tostudythebehavioralphenotypesofgenet‐icallymodifiedmice,itisvaluabletogeneratethemwithacommongenetic background of a well‐understood wild‐type phenotype.TheC57BL/6Jgeneticbackground iswidelyadoptedbyknockoutandtransgenicresearches31.ThisisalsoamajorbackgroundoftheSyngap1−/+mouselinesusedinprecedingbehavioralstudies25,28,30.

In this report,weutilizedacomprehensive setofwell‐definedbehavioral tests 31‒38 and investigated behavioral phenotypes in‐cluding the sensorimotor functions and the cognitive functionsoftheSyngap1−/+mice generated byKomiyama et al on aC57BL/6Jgeneticbackground.

2  | METHODS

2.1 | Animals and experimental design

Syngap1−/+miceweregeneratedaspreviouslydescribed22. The mice werebackcrossed to theC57BL/6Jmice (Charles river,MA,USA),foratleasttengenerations,whichisalsoexpectedtominimizege‐neticdrift.Wild‐type(WT)andSyngap1−/+miceweregeneratedbycrossingmaleSyngap1−/+miceandWTfemalemice.Thesamepopu‐lationofmalemiceolderthan53weeksweresequentiallysubjectedtodifferentbehavioraltests(fortheageofthemiceandorderofthetests,seeTableS1).Micewerehousedtwotofourpercage(onetothreeforeachgenotype)inaroomwitha12‐hourlight/darkcycle(lightsonat7:00am),withaccesstofoodandwateradlibitum.Roomtemperaturewaskeptat23±2°C.Behavioraltestswereperformedbetween9:00am and6:00pm.Before the tests,micewere left inthetestingroomforatleast30minutestoallowacclimation,unless

Conclusion: In Syngap1−/+mice,wecouldreproducemostofthepreviouslyreportedcognitiveandemotionaldeficits.Thedecreasedsensitivitytopainfulstimuliandim‐pairedmotorfunctionthatwefoundinSyngap1−/+miceareconsistentwiththecom‐moncharacteristicsofpatientswithSYNGAP‐relatedID.WefurtherconfirmedthattheSyngap1heterozygotemouserecapitulatesthesymptomsofIDandASDpatients.

K E Y W O R D S

autismspectrumdisorder,intellectualdisability,motorfunction,nociception,SYNGAP1

     |  3NAKAJIMA et Al.

otherwisenoted.Aftereachtest,thetestingapparatuswascleanedwith superhypochlorouswater toprevent abiasdue toolfactorycues,unlessotherwisenoted.

2.2 | Behavioral tests

Unless otherwise noted, most of the behavioral tests were per‐formedaspreviouslydescribed39‒41.

2.3 | Neurological screen and neuromuscular strength test

Therighting,whiskertwitch,andeartwitchreflexeswereevaluated.Physical features, including the presence ofwhiskers or bald hairpatches,werealsorecorded.Agripstrengthmeter(O’HARA&Co.)wasused toassess forelimbgrip strength.Thepeak forceappliedbytheforelimbsofthemousewasrecordedinNewtons(N).Eachmousewastestedthreetimes,andthegreatestvaluemeasuredwasusedfordataanalysis.Inthewirehangtest,themousewasplacedon awiremesh thatwas then slowly inverted, so that themousegrippedthewireinordernottofalloff.Latencytofallwasrecorded,witha60secondscutofftime.

2.4 | Rotarod test

Motor coordination and balance were tested using the rotarodtest. This test,which uses an accelerating rotarod (UGOBasile),wasperformedbyplacingmiceonrotatingdrums(3cmdiameter),made of polyvinyl chloride (PVC), and measuring the time eachanimalwasabletomaintain itsbalanceontherod.Thespeedoftherotarodwasacceleratedfrom4to40rpmovera5‐minutepe‐riod.All themiceweresubjectedtothetestwithoutanypretesttraining.

2.5 | Hot plate test

Thehotplatetestwasusedtoevaluatesensitivitytoapainfulstimu‐lus.Micewereplacedona55.0°Chotplatewithblackanodizedalu‐minumfloor(ColumbusInstruments),andlatencytothefirstfore‐orhindpawresponsewasrecordedwitha15secondscutofftime.Thepawresponsewasdefinedaseitherapawlickorafootshake.

2.6 | Open field test

Eachmousewasplacedinthecorneroftheopenfieldapparatus(40×40×30cm;AccuscanInstruments)whichconsistsofwhiteplasticfloorandtransparentPlexiglaswall.Theapparatuswasil‐luminatedat100l×.Totaldistancetraveled(cm),verticalactivity,timespentinthecenterarea(20×20cm),andbeam‐breakcountsfor stereotypedbehaviorswere recorded. Immediately after themicewere placed in the arena, their behaviorwas recorded for120minutes.

2.7 | Light/dark transition test

Alight/darktransitiontestwasconductedaspreviouslydescribed42.Theapparatusconsistedofacagewithawhite floormadeofPVC(21×42×25cm)dividedintotwosectionsofequalsizebyapartitionwithadoor(O’HARA&Co.,Tokyo,Japan).Onecham‐berwasbrightlyilluminated(390lux),whereastheotherchamberwasdark (two lux).Micewereplaced into thedark chamberandallowedtomovefreelybetweenthetwochamberswiththedooropen for 10minutes. The total number of transitions, latency tofirstenterthelitchamber,distancetraveled,andtimespentineachchamberwererecordedbyImageLDsoftware(seeSection,“DataAnalysis2.17”).Incaseswiththemicedidnotenterthelightcom‐partmentduringtheentire10‐minutessession,thelatencytolightwasconsideredas600seconds,andthedatawereincludedinthestatisticalanalysis.

2.8 | Elevated plus maze test

Anelevatedplusmazetestwasconductedaspreviouslydescribed43.Theelevatedplusmazeconsistedoftwoopenarms(25×5cm)andtwoenclosedarmsofthesamesizewith15cmhightransparentwalls,andthearmswereconnectedbyacentralsquare(5×5cm)(O’HARA&Co.,Tokyo,Japan).Theopenarmsweresurroundedbyaraisedledge(3mmthickand3mmhigh)toavoidmicefallingoffthearms.Thearmswereelevated55cmabovethefloor.Armsofthesametypewerelocatedoppositefromeachother.Eachmousewasplacedinthecentralsquareofthemaze,facingoneoftheenclosedarms.AllthearmsandwallsweremadeofPVC.Thenumberofen‐triesintotheopenandenclosedarmsandthetimespentintheopenor enclosed arms were recorded during a 10‐minute test period.Percentageof entries intoopen arms, time spent in open arms(s),numberof totalentries,and totaldistance traveled (cm)werecal‐culated.Whenamousefallsfromthemaze,thedatawereexcludedfromthestatisticalanalysisfordistancetraveled,entriesintoopenarms, timeonopenarms,andnumberofentries.Dataacquisitionandanalysiswereperformedautomatically,usingImageEPsoftware(seeSection,“Dataanalysis2.17”).

2.9 | Social interaction test in a novel environment

Inthesocialinteractiontest,twomiceofidenticalgenotypesthatwerepreviouslyhousedindifferentcageswereplacedinawhitePVCplasticboxtogether(40×40×30cm)(O’HARA&Co.)andallowedtoexplorefreelyfor10minutes.Behaviorwasrecordedandanalyzedautomaticallyusing ImageSIprogram(seeSection,“Dataanalysis2.17”).Thetotalnumberofcontacts,totaldurationofactivecontacts,totalcontactduration,meandurationpercon‐tact,andtotaldistancetraveledweremeasured. If thetwomicecontactedeachotherandthedistancetraveledbyeithermousewas longer than10cm, thebehaviorwasclassifiedasan“activecontact.”

4  |     NAKAJIMA et Al.

2.10 | Crawley's sociability and social novelty preference test

Thistestisawell‐designedmethodtoinvestigatetheeffectofcom‐plexgeneticsonsociabilityandpreferenceforsocialnovelty44,45. Thetestingapparatusconsistedofarectangular,three‐chamberedPVCplasticboxandalidwithaninfraredvideocamera(O’HARA&Co.).Eachchamberwas20×40×47cm,andthedividingwallsweremadefromclearPVCplastic,withsmallsquareopenings(5×3cm)allowing access into each chamber.Wemodified themethodde‐scribedbyref.45asfollows:Ahabituationsessionwasperformedintheapparatusfor10minutesthedaybeforethesociabilitytest,andthewirecagesinthelateralcompartmentswerelocatedinthecornersofeachcompartment.Inthesociabilitytest,anunfamiliarC57BL/6Jmalemouse(stranger)thathadnopriorcontactwiththesubjectmicewasplacedinoneofthesidechambers.Thelocationofthestrangermouse(strangerside)intheleftvsrightsidechamberwassystematicallyalternatedbetweentrials.Thecagewas11cminheight,withabottomdiameterof9cm,andverticalbars0.5cmapart.The subjectmousewas firstplaced in themiddlechamberandallowedtoexploretheentiretestboxfora10‐minutesession.Theamountof timespent ineachchamberanddistancetraveledweremeasuredwithacamerafittedontopofthebox. Intheso‐cialnoveltypreferencetest,eachmousewastestedina10‐minutesessiontoquantifysocialpreferenceforanewstranger.Afterthefirst10‐minute session, a secondunfamiliarmousewasplaced inthechamberthathadbeenemptyduringthefirst10‐minuteses‐sion.Thissecondstrangerwasalsoenclosed inan identical smallwirecage.Theamountoftimespentineachchamberanddistancetraveled during the second 10‐minute sessionweremeasured asdescribedabove.Dataacquisitionandanalysiswereperformedau‐tomaticallyusingImageCSI(seeSection,“Dataanalysis2.17”).

2.11 | Startle response/prepulse inhibition (PPI) test

Astartlereflexmeasurementsystem(O’HARA&Co.)wasusedtomeasureacousticstartleresponseandPPI.Beforethistest,micewerekeptinasoundproofroomseparatefromthetestingroom.AtestsessionbeganbyplacingamouseinatransparentPVCplas‐ticcylinderwhere itwas leftundisturbed for10minutes.Whitenoise (40ms)wasusedas thestartle stimulus forall trial types.Thestartle responsewas recorded for400msstartingwith theonsetofthestartlestimulus.Thebackgroundnoiselevelineachchamberwas70dB.Atestsessionconsistedofsixtrialtypes(ie,two types for startle stimulus‐only trials, and four types forPPItrials). The intensity of the startle stimuluswas 110 or 120 dB.The prepulse sound was presented 100 ms before the startlestimulus,anditsintensitywas74or78dB.Fourcombinationsofprepulse and startle stimuliwere used (74‐110, 78‐110, 74‐120,and78‐120dB).Sixblocksofthesixtrialtypeswerepresentedinapseudo‐randomorder, suchthateachtrial typewaspresentedoncewithin a block. The average inter‐trial intervalwas15 sec‐onds(range10‐20seconds).

2.12 | Porsolt forced swim test

APlexiglascylinder(20cmheight×10cmdiameter)filledwithwater(21‐23°C)uptoaheightof7.5cmwasputinawhiteplasticchamber(31×41×41cm)(O’HARA&Co.).Micewereplacedintothecylinder,andbothimmobilityandthedistancetraveledwererecordedovera10‐minutetestperiod.Imageswerecapturedat2‐framepersec‐ond.Foreachpairofsuccessiveframes,theamountofarea(pixels)withinwhichthemousemovedwasmeasured.Whentheamountofareawasbelowacertainthreshold,mousebehaviorwasclassifiedas “immobile.” Immobility lasting for<2 secondswasnot includedintheanalysis.Dataacquisitionandanalysiswereperformedauto‐matically,usingImageTSsoftware(seeSection,“Dataanalysis2.17”).

2.13 | Gait analysis

Weanalyzedgaitofthemiceduringwalk/trotlocomotionbyventralplane videography as described 46,47 using DigiGait Imaging System(MouseSpecificsInc).Thissystemenablesmicetowalkonamotorizedtransparenttreadmillbelt,andthesoftwareautomaticallyidentifiesthestanceandswingcomponentsofstrideandcalculatesstancewidth,stridelength,stepangle,andpawangle.Briefly,weplacedthemiceonatreadmillbeltthatmovesataspeedof24.7cm/s.Wecollecteddigitalvideoimagesoftheundersideofmiceat150framespersecond.

2.14 | Tail suspension test

The tail suspension testwas performed for a 10‐minute test ses‐sion.Miceweresuspended30cmabovethefloorofawhiteplasticchamber(31×41×41cm)(O’HARA&Co.),andthebehaviorwasre‐cordedovera10‐minutetestperiod.AssimilartothePorsoltforcedswim test, immobility (%)was judged by the application program.Data acquisition and analysiswere performed automatically usingImageTSsoftware(seeSection“Dataanalysis2.17”).

2.15 | T‐maze test

Thespontaneousalternation taskwasconductedusinganauto‐maticT‐mazeapparatus (O’HARA&Co.)aspreviouslydescribed48. ItwasconstructedofwhitePVCplastic runwayswith25‐cmhighwalls.Themazewaspartitionedoff intosixareasbyslidingdoorsthatcanbeopeneddownward.ThestemoftheTwascom‐posedofareaS2(13×24cm),andthearmsofTwerecomposedofareasA1andA2 (11.5×20.5cm).AreasP1andP2were theconnectingpassagewaysfromtherespectivearm(areaA1orA2)tothestartcompartment(areaS1).Miceweresubjectedtoaspon‐taneousalternationprotocolforfivesessions,withatleast1day(2 days maximum) of session‐to‐session intervals. One sessionconsistsof10trialswitha50‐minutecutoff time.Eachtrialhadfirstandsecondruns.Onthefirstrun,themousewasforcedtochooseoneofthearmsoftheT(areaA1orA2).Afterthemousestayedmore than 10 seconds, the door that separated the arm(areaA1orA2) and the connecting passageway (areaP1 or P2)

     |  5NAKAJIMA et Al.

wouldbeopened,andthemousecouldreturntothestartingcom‐partment(areaS1)viatheconnectingpassageway.Themousewasthengivena3‐seconddelayinareaS1,followedbyafreechoicebetweenbothTarms.Thepercentageoftrialsinwhichmiceen‐teredthearmoppositetotheirforced‐choicerunduringthefreechoicerunwascalculated.Thelocationofthesamplearm(leftorright)variedpseudo‐randomlyacrosstrialsusingtheGellermannschedule so that mice received equal numbers of left and rightpresentations.Dataacquisition,controlofslidingdoors,anddataanalysiswereperformedbyImageTMsoftware(seeSection,“DataAnalysis2.17”).

2.16 | Y‐maze test

Y‐mazetestwasperformedaspreviouslydescribed49.ExploratoryactivitywasmeasuredusingaY‐mazeapparatus(armlength:40cm,armbottomwidth: 3 cm, armupperwidth: 10 cm, height ofwall:12cm).ThefloorofthemazeismadeofwhitePVCplastic,andthewallismadeoftransparentPVCplastic.EachsubjectwasplacedinthecenteroftheY‐mazefield.Thenumberofentriesandalterationswas recorded using amodified version of the ImageYM software.Datawerecollectedforaperiodof10minutes.

2.17 | Barnes maze test

TheBarnesmazetaskwasconductedon“dryland,”1.0mindiam‐eter,with12holesequallyspacedaroundtheperimeter(O’HARA&Co.).Themaze ismadeofPVCplastic.AblackPlexiglasescapebox(17×13×7cm),whichhadpapercagebeddingonitsbottom,waslocatedunderoneoftheholes.Theholeabovetheescapeboxrepresentedthetarget.Thelocationoftheescapebox(target)wasconsistentforagivenmousebutrandomizedacrossmice.Themazewasrotateddaily,withthespatiallocationofthetargetunchangedwithrespecttothedistalvisualroomcues,topreventabiasbasedon olfactory or proximal cueswithin themaze.One trial per daywasconductedforthefirstfivetrials.Fromthesixthtrial,twotrialswereperformedperday.Eachtrialendedwhenthemouseenteredtheescapeboxorafter5minuteshadelapsed.Thenumberofer‐rors (definedby theanimalplacing itsnose inahole thatdidnotleadtotheescapebox),theamountoftimethatthemicetooktoenterthebox,totaldistancetraveledtotargethole,andthenumberofomissionerrors (definedbythevisit to thetargetholewithoutsubsequententryintothetargethole)wererecordedbyImageBMsoftware.Onday7,aprobetestwasconductedwithouttheescapebox, to assessmemorybasedondistal environmental roomcues.Anotherprobetrialwasconducted1monthafterthelasttrainingsessiontoevaluatememoryretention.Thetimespentaroundthetargetholewasrecordedintheseprobetestsbythesoftware.

2.18 | Contextual and cued fear conditioning test

Contextual and cued fear conditioning test was performed aspreviously described 50‒52. Before this test, mice were kept in a

soundproofroomseparatefromthetestingroom.Toassess fear‐relatedlearningandmemory,eachmousewasplacedinanacrylicchamberconsistingofwhite(side)andtransparent(front,rear,andtop)PVCplasticwalls(33×25×28cm)withastainless‐steelgridfloor(0.2cmdiameter,spaced0.5cmapart;O’HARA&CO.),andwasallowedtoexplorefreelyfor2minutes52.Subsequently,acon‐ditionedstimulus(CS;55dBwhitenoise)waspresentedfor30sec‐onds, followed by a mild foot shock (2 seconds, 0.3 mA), whichservedastheunconditionedstimulus(US).TwomoreCS‐USpairingswerepresentedwith2‐minuteinterval.Contexttestwasconducted1dayafterconditioninginthesamechamberfor300secondsoneachmouse.Acuedtestwithanalteredcontextwasconductedinatriangularchamberatleast100minutesafterthecontexttestonthesameday(33×29×32cm;madeofwhiteacrylicplasticwallsand floor,whichwas located in a different room).After a 3‐min‐utefree‐movingperiodinthetriangularchamber,tonestimulusforthecuedtest(55dBwhitenoise)wasappliedfor180seconds.Ineachtest,freezingpercentageanddistancetraveled(cm)werecal‐culatedautomaticallyusing ImageFZsoftware (seeSection,“Dataanalysis2.17”).Aftereachtrialintheconditioningtest,thewallsandgridsof the chamberwerewipedwith superhypochlorouswaterand65%ethanol,respectively.Inthecuedtest,thewallsandfloorwerecleanedwithsuperhypochlorouswater.

2.19 | Social interaction in home cage

Tomonitorsocialbehaviorbetweentwomiceinafamiliarenviron‐ment,asystemthatautomaticallyanalyzessocialbehaviorinhomecagesofmicewasusedaspreviouslydescribed53.Twogeneticallyidentical mice that had been housed separately were placed to‐getherinahomecage(seeSection,“locomotoractivitymonitoringin home cage2.16”). Their social behaviorwas thenmonitored for7days.Outputsfromthevideocameraswerefedintoacomputer.Images fromeach cagewere captured at a rate of one framepersecond.Socialinteractionwasmeasuredbycountingthenumberofparticles ineachframe:Twoparticles indicatedthemicewerenotincontactwitheachother;andoneparticledemonstratedcontactbetween the twomice.Wealsomeasured locomotoractivitydur‐ingtheseexperimentsbyquantifyingthenumberofpixelschangedbetweeneachpairofsuccessiveframes.

2.20 | Locomotor activity monitoring in home cage

Locomotoractivitymonitoringinhomecagewasperformedwithasys‐temthatautomaticallyanalyzesthelocomotoractivityofmiceintheirhomecage53.Thesystemcontainsahomecage(29×18×12cm),afilteredcagetop,andaninfraredvideocamerawhichisattachedtothetopofastand.Eachmousewasindividuallyhousedineachhomecage,andtheirlocomotoractivitywasmonitoredforaweek.Outputsfromthevideocameraswerefed intoacomputer. Imagesfromeachcagewerecapturedatarateofoneframepersecond,anddistancetrave‐ledwasmeasuredautomaticallyusingImageHAsoftware(seeSection,“Dataanalysis2.17”).

6  |     NAKAJIMA et Al.

2.21 | Data analysis

Behavioral data were obtained automatically through applicationsbasedontheImageJprogram,andtheyweremodifiedforeachtestbyTsuyoshiMiyakawa(availablethroughO’HARA&Co.).TheImageJpl‐ugins,andtheprecompiledpluginsforlight/darktransitiontest(ImageLD),elevatedplusmaze(ImageEP),openfieldtest(ImageOF),fearcon‐ditioningtest(ImageFZ),andT‐maze(ImageTM)arefreelyavailableonthewebsiteof“MousePhenotypeDatabase”(http://www.mouse‐phenotype.org/software.html) 54. Statistical analysis was conducted

usingStatView(SASInstitute).Datawereanalyzedusingtwo‐tailedt test,one‐wayANOVA,two‐wayrepeatedmeasuresANOVA,andchi‐squaredtest.Valuesingraphsareexpressedasmean±SEM.Tocon‐trolfortypeIerrorsduetomultiple‐hypothesistesting,wecalculatedthefalsediscoveryrate(FDR)bytheBenjamini‐Hochbergmethod55. We defined “study‐wide significance” as the statistical significancethatsurvivedFDRcorrection.“Nominalsignificance”wasdefinedastheonethatachievedastatisticalsignificanceinanindex(P<.05)butdidnotsurvivethiscorrection.

3  | RESULTS

StatisticaldatafortheseresultsarepresentedinTablesS2andS3.In the results section,P‐valueswith a study‐wide significance arelabeledwithasterisks(*P<.05,**P<.01,and***P<.001).P‐valueswith“#”indicateanominalsignificance.

3.1 | GeneralcharacterizationofSyngap1−/+ mice

TherewerenosignificantdifferencesbetweentheSyngap1−/+andWTmiceintermsofbodyweight(Figure1A,P = .959),bodytemperature(Figure1B,P = .6062),gripstrength(Figure1C,P = .3232),orlatencytofalloffthewiregrid(Figure1D,P = .4633).AsshowninFigure1E,themutantmiceshowedanimpairedmotorfunction(P = .003**),asassessedbytherotarodtest.Theinteractionbetweengenotypeand

F I G U R E 1  Generalhealthandneurologicalscreen,motorlearning,andpainsensitivitybetweengenotypes.A,Bodyweight;B,bodytemperature;C,gripstrength;D,latencytofallinwirehangtest;E,latencytofallintherotarodtest;andF,latencyofthefirstfore‐orhindpawresponseinthehotplatetest.Datarepresentthemean±SEM.TheP‐valuesindicategenotypeeffectsinaone‐wayANOVA(A‐D,andF),ortwo‐wayrepeatedmeasuresANOVA(E)

(E)

(A) (B)

(C) (D)

Hot plate test

General health and neurological screen

Rotarod test

(F)

Bod

y w

eigh

t (g)

Bod

y te

mpe

ratu

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°C)

Grip

str

engt

h (N

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Wire

Han

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tenc

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Late

ncy

(s)

Late

ncy

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Controls (n = 16)Mutants (n = 22)

Controls (n = 16)Mutants (n = 22)

Controls (n = 16)Mutants (n = 22)

P = 0.959 P = 0.6062

P = 0.3232 P = 0.4633

P = 0.003 P = 0.0004

30

35

34

33

32

31

0

50

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F I G U R E 2   IncreasedlocomotoractivityofSyngap1−/+ mice in openfieldtest.A,Totaldistancetraveled;B,verticalactivity;C,timespentinthecenterarea;D,stereotypicbehaviorcountsarerepresented.Datarepresentthemean±SEM.TheP‐valuesindicategenotypeeffectsintwo‐wayrepeatedmeasuresANOVA

Open field test(A) (B)

(C) (D)

Dis

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e tr

avel

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cm)

Ver

tical

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ivity

Cen

ter

time

(s)

Ste

reot

ypic

cou

nts

Time (min) Time (min)

Time (min) Time (min)

P < 0.0001 P = 0.0055

P = 0.041 P = 0.0032

Controls (n = 16)Mutants (n = 22)

     |  7NAKAJIMA et Al.

trialintherotarodtestwasnotsignificant(P = .064).GaitanalysistestdidnotrevealanydifferencebetweenSYNGAP1−/+andcontrolmice(dataareavailableintheMousePhenotypeDatabasedescribedinthedataanalysis2.17section).Inthehotplatetest,Syngap1−/+miceexhib‐iteddecreasedpainsensitivity.(Figure1F,P = .0004**).

3.2 | IncreasedlocomotoractivityofSyngap1−/+ mice intheopenfieldtest

Intheopenfieldtest,Syngap1−/+miceexhibitedsignificantincreasesinthetotaldistancetraveled (Figure2A,P< .0001***),numberofvertical activities (Figure 2B, P = .0055*), center time (Figure 2C,P = .041#),andstereotypiccounts(Figure2D,P = .0032**)comparedwiththoseinthecontrolmice.

3.3 | Normallight/darktransitionofSyngap1−/+ mice

The light/dark transition test detected no significant differencesbetween themutantmice andWTmice in the distances traveledbetweenthe light/darkcompartments (Figure3A, light,P = .1189;dark,P = .2648),numberoftransitionsbetweenlight/darkcompart‐ments(Figure3B,P = .7704),latencytoenterthelightcompartment

(Figure 3C, P = .1025), or time spent in the light compartment(Figure3D,P = .4639).ThereweretwoSyngap1−/+micewhichdidnotenterthelightcompartmentduringtheentire10‐minutesession.

3.4 | Increasesinlocomotoractivityandopen‐armtimeofSyngap1−/+miceintheelevatedplusmaze

Intheelevatedplusmazetest,Syngap1−/+miceshowedasignificantin‐crease inthetotaldistancetraveled inarms(Figure3E,P< .0001***).Therewasnosignificantdifferenceinpercentageofentriesintotheopenarms(Figure3F,P = .0945)betweengenotypes.Percentageoftimespentinopenarmsandthetotalnumberofentriesweresignificantlyincreasedinthemutantmice(Figure3G,P = .0052*;Figure3H,P = .0001***).Therewere 10 Syngap1−/+miceandtwocontrolmicewhichfellfromthemazeofwhichdatawereexcludedfromthestatisticalanalysis(Figure3E‐H).Themutantmiceshowedasignificantlyhigherincidenceofafallfromthemazethanthecontrolmice(Figure3I,P = .0309#).

3.5 | SocialbehaviorinSyngap1−/+ mice

Inthesocialinteractiontest,Syngap1−/+miceexhibitedasignificantdecreaseintotaldurationofcontacts(Figure4A,P = .0001**).There

F I G U R E 3  Anxiety‐relatedbehaviorsofSyngap1−/+miceobservedinelevatedplusmazeandlight/darktransitiontest.(A‐D)Light/darktransitiontest:A,distancetraveledinthelight/darkcompartments;B,numberoflight/darktransitions;C,latencytoenterthelightcompartment;andD,timespentinthelightcompartment.(E‐H)Elevatedplusmazetest:E,distancetraveled;F,percentageofentriesintoopenarms;G,percentageoftimespentinopenarms;H,numberofarmentries;I,percentageofmicedroppedfromthemaze.Datarepresentthemean±SEM.TheP‐valuesinpanels(A‐I)indicategenotypeeffectsinone‐wayANOVA.TheP‐valueof(I)wasevaluatedusingachi‐squaretest.Onlythedataofthemicethatcompletedthesessionwithoutfalling(controls,n=14;mutants,n=12)wereincludedinthestatisticalanalysisinE‐H

Light/dark transition test

Elevated plus maze test

0

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(A) (B) (C) (D)

(E) (F) (G) (H) (I)

8  |     NAKAJIMA et Al.

was no difference betweenSyngap1−/+mice andWTmice in thetotalnumberofcontacts(Figure4B,P = .8005).Meandurationpercontactwassignificantlydecreasedinthemutantmice(Figure4C,P = .0001**).Themutantmicetraveledalongerdistancethanthecontrolmice(Figure4D,P = .0107*).ThemicewerealsosubjectedtoaCrawley'ssociabilityandsocialnoveltypreferencetestwhichiscomposedofasociabilitytestandasocialnoveltypreferencetest.Inthesociabilitytest,socialbehaviorcanbeassessedbasedonthetimespentaroundawirecagewithanunfamiliarmouse(strangerside)vs the timespentaroundanemptycage (empty side).BoththemutantandWTmicespentmoretimearoundthestranger‐sidecagethantheempty‐sidecage(Figure4E,WT:P = .0044*,mutant:P = .0097*).Comparedtomutants,WTmicestayedlongeraroundthestrangerside(Figure4E,P = .0007**).Themutantmiceshowedasignificantincreaseinthetotaldistance(Figure4F,P<.0001***).Inthesocialnoveltypreferencetest,bothWTandSyngap1−/+ mice tended to spend longer time around the stranger 2‐sided cage;however, they were not statistically significant (Figure 4G, WT:P = .1278,Syngap1−/+: P = .3556).WTmice stayed longer aroundthestrangersideofthecage(Figure4G,P<.0007**)thandidthe

mutants. Themutantmice showed a significant increase in totaldistance(Figure4H,P = .0007**).

3.6 | DecreasedprepulseinhibitionoftheacousticstartleresponseinSyngap1−/+ mice

In theprepulse inhibition test, therewasno significantdifferencebetween the Syngap1−/+ and WT mice in the startle amplitude(Figure5A,P = .3613).Syngap1−/+mice showed a significantly de‐creasedprepulse inhibitionof thestartle responsecomparedwithWTmice(Figure5B,110dB,P=.0004**;120dB,P=.0037**).

3.7 | DecreasedimmobilityofSyngap1−/+miceinthetestsfordepression‐likebehavior

InthePorsoltforcedswimtest,Syngap1−/+miceexhibitedasignif‐icantlydecreased immobility timeonday1 andday2 (Figure6A,P=.0051*andP=.0002**,respectively).Likewise,inthetailsuspen‐siontest,Syngap1−/+miceshowedasignificantlydecreasedimmobil‐itytime(Figure6B,P <.00001***).

F I G U R E 4  SociabilityandsocialnoveltypreferenceofSyngap1−/+mice.(A‐D)Socialinteractiontestinnovelenvironments:A,totaldurationofcontacts;B,numberofcontacts;C,meandurationpercontact;andD,totaldistancetraveled.(E‐J)Crawley'ssociabilityandsocialnoveltypreferencetest:E,timespentaroundthecage,andF,totaldistancetraveledinthesociabilitytest;G,timespentaroundthecage,andH,totaldistancetraveledinthesocialnoveltypreferencetest.Datarepresentthemean±SEM.TheP‐valuesin(A‐D,F,andH)indicategenotypeeffectsinone‐wayANOVA.TheP‐valuesinpanels(E)and(G)representthegenotypeeffects(controlsvsmutants)orsideeffects(emptysidevsstranger1side,orstranger1sidevsstranger2side)inone‐wayANOVA

(A) (B) (C)

(E)

(G)

Tim

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arou

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(s)

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(D)

(F)

(H)

Social interaction test in novel environment

Tota

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Con

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)

Controls Mutants(n = 16) (n = 22)

P = 0.0001 P = 0.8005 P = 0.0001 P = 0.0107

Num

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Mea

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Controls (n = 7)Mutants (n = 11)

Crawley’s sociability and social novelty preference test

Controls (n = 16)Mutants (n = 22)

Controls (n = 16)Mutants (n = 22)

P = 0.0007

P = 0.0044

P = 0.0097

Controls Mutants(n = 16) (n = 22)

Empty sideStranger 1 side

Stranger 1 sideStranger 2 side

P = 0.0007

P = 0.1278

P = 0.3556

Dis

tanc

e tr

avel

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cm)

Dis

tanc

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avel

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cm)

     |  9NAKAJIMA et Al.

3.8 | IncreasedlocomotoractivityandimpairedworkingmemoryofSyngap1−/+miceintheT‐maze

AT‐mazespontaneousalternationtaskwasperformedtocomparetheworkingmemorybetweentheWTandSyngap1−/+ mice 48. The percentageofcorrectresponsesofSyngap1−/+micewaslowerthanthatoftheWTmice(Figure7A,P=.0031**).Syngap1−/+miceandWTmiceshowednoobviousdifferencesinlatencytocompleteasession(Figure7B,P=.0785).Syngap1−/+micetraveledalongerdistancetocompleteasession(Figure7C,P=.0003**)thantheWTmice.

In the Y‐maze, Syngap1−/+mice demonstrated an increased num‐berofentries(Figure7D,P=.0001**)andtotalalternations(Figure7E,P=.0006**).Percentageofalternationsinthetotalnumberofentrieswasnotdifferentbetweenthegenotypes (Figure7F,P= .632).Totaldistance was significantly increased in Syngap1−/+ mice (Figure 7G,P=.0005**).

3.9 | ImpairedspatialreferencememoryinSyngap1−/+ mice

IntheBarnesmazetest,Syngap1−/+miceshowedanincreaseinthefollow‐ingindices:numberoferrorsbeforereachingthetargethole(Figure8A,P=.002**),latencytoreachthetargethole(Figure8B,P=.0204*),dis‐tancetoreachthetargethole(Figure8C,P=.0002**),andnumberofomissionerrorsbeforereachingthetargethole(Figure8D,P=.0282#).Probetrialswhereintheescapeboxwasremovedwereperformed1dayaftertrainingandamonthafterthelastdayoftraining.Syngap1−/+ mice spentlesstimearoundthetargetduringtheseprobetests(Figure8E;1day,P=.0022**;1month,P=.0156*)thantheWTmice.

3.10 | DecreasedfreezingofSyngap1−/+ mice duringconditioningincontextualandcuedfearconditioningtest

TherewasnosignificantdifferencebetweenSyngap1−/+andWTmiceinthedistancetraveledbefore,during,oraftereachfootshockduring

theconditioningperiod(Figure9A,footshock1,P=.0774;footshock2,P=.5818;andfootshock3,P=.5153).Syngap1−/+miceexhibitedasignificantdecreaseinthepercentageoffreezingduringconditioning(Figure9B,P=.0001***).During2nddayoftesting,therewerenodif‐ferencesbetweengenotypesinthepercentageoffreezing(Figure9Ctop,P=.5696)orinthedistancetraveled(Figure9Cbottom,P=.494).Inthecuedtestonday2,Syngap1−/+miceshoweddecreasedfreez‐ingduringthesoundrepresentation(Figure9Dtop,P=.0357#).Therewasnodifferenceindistancetraveled(Figure9Dbottom,P=.573)be‐tweenthegenotypes.Inthecontexttesting30daysafterthefearcon‐ditioning,therewerenosignificantdifferencesinfreezing(Figure9Etop,P= .1378)or indistancetraveled (Figure9Ebottom,P= .1661)between the genotypes. In the cued testing after 30days, freezing(Figure9F top,P = .1302) anddistance traveled (Figure9Fbottom,P=.1012)duringthetonerepresentationwerenotsignificantlydiffer‐entbetweenthegenotypes.

3.11 | HomecageactivitiesofSyngap1−/+ mice

Inthesocialinteractiontestinhomecage,anincreasedmeannumberofparticlesofSyngap1−/+micewereobservedduringnight(Figure10A;wholeperiod,P= .2343;day,P= .7405;night,P= .0431#), thoughthisdidnotsurviveFDRcorrection.Syngap1−/+miceexhibitedmore

F I G U R E 5  DecreasedprepulseinhibitionofSyngap1−/+mice.A,StartleamplitudeandB,percentofprepulseinhibitionweretested.Datarepresentthemean±SEM.TheP‐valuesindicategenotypeeffectsintwo‐wayrepeatedmeasuresANOVAthatwasseparatelyperformedinexperimentwithdifferentstartlesoundlevel

Prepulse inhibition test

110 1200

0.5

1

Sta

rtle

Am

plitu

de

Sound Level (dB) Prepulse Sound Level (dB)

P = 0.3613 P = 0.0004 P = 0.0037

(A) (B)

Pre

puls

e in

hibi

tion

(%)

110 dBstartle

120 dBstartle

74 78 74 78

Controls (n = 16)Mutants (n = 22)

F I G U R E 6  DecreasedimmobilityofSyngap1−/+miceinthetestsfordepression‐likebehavior.A,Percentageofimmobilitytimeonday1andday2inaPorsoltforcedswimmingtest.B,Percentageofimmobilitytimeinthetailsuspensiontest.Datarepresentthemean±SEM.TheP‐valuesindicategenotypeeffectsintwo‐wayrepeatedmeasuresANOVA

(A)

1 2 3 4 5 6 7 8 9 10Time (min)

1 2 3 4 5 6 7 8 9 10

(B)

Porsolt forced swim test

Tail suspension test

Day 1P = 0.0051

Day 2P = 0.0002

P < 0.0001

Controls (n = 16)Mutants (n = 22)

Imm

obili

ty (

%)

Imm

obili

ty (

%)

Time (min)1 2 3 4 5 6 7 8 9 10

Controls (n = 16)Mutants (n = 19)

10  |     NAKAJIMA et Al.

locomotor activity during the night (Figure 10B; whole period,P=.0028;day,P=.1247;night,P=.0022**)thantheWTmice.Inthehomecageactivitytestwithsinglemouseinacage,Syngap1−/+ mice showed increasedactivity levelduring thenight (Figure10C;wholeperiod,P=.0021;day,P=.809;night,P=.0006**).

4  | DISCUSSION

In this study,we subjectedmaleSyngap1−/+miceon aC57BL/6Jgenetic background to a comprehensive behavioral test battery.Inagreementwithpreviousbehavioralstudieswhichareusingdif‐ferent Syngap1−/+ mouse lines, we have reproducedmost of thepreviously reportedbehavioralphenotypes: increased locomotoractivity23,25‒29;decreasedprepulseinhibition25;impairedworking23,26,28,29; and reference spatialmemory 22,26,27. Similar to a pre‐ceding report25,heterozygousSyngap1knockoutmiceshowedadecreaseincuedfearmemoryinourstudy,eventhoughthisfailedto reach a study‐wide significance. While weakened contextualfear memory 23,28 and increased startle reflex 25 of the mutantmicehavebeenpreviouslyreported,wefailedtoreproducethesephenotypes. Inaddition,wefoundthatthesemiceshowedade‐creasedsensitivitytopainfulstimuliandimpairedmotorfunction(seeTableS4).

ThedecreasedsensitivitytopainfulstimuliofSyngap1−/+ mice is consistentwith a previous studywhich reported a high pain

thresholdin72%ofSYNGAP‐relatedIDpatients21.Ontheotherhand, two preceding studies failed to detect altered thermalnociception inSyngap1−/+ mice 26,30.Duarteet al30 showed thatcapsaicin‐induced thermal hypernociception occurred at lowerdosesofcapsaicininSyngap1−/+micethaninWTmice.However,thisstudydidnotdetectalterednociceptioninthesemicewith‐outthe injectionofcapsaicin.Differences inthetimecourseofheatapplicationmayhaveledtotheinconsistenciesinpaw‐with‐drawal latency in Syngap1−/+ mice between other studies andours 26,30,56.Duarte et al30 andMuhia et al26 used instrumentswhichapplygraduallyincreasingheatstimuli56‒58.Ontheotherhand, our hot plate provides immediate heat at 55℃. We alsofound that male Syngap1−/+ mice have impairedmotor functionasassessedbytheacceleratingrotarodtest.Learningeffects inthemutantmicewerenotdetectedinthesametest.Thelossofmotorfunctionandthedifficultyinmotorlearningofthesemu‐tantmicemaycorrespondtoataxiaorgaitabnormalitiessimilarto human SYNGAP1‐related ID patients 13,16,18,21,59,60, althoughthepossibleconfoundingeffectofhyperlocomotoractivitycan‐notbeexcludedinthisapparentperformancedeficitofthemu‐tantsintherotarodtest.Ontheotherhand,Muhiaetal26didnotfind suchmotor dysfunctions inmalemutantmice assessed bytheacceleratingrotarodtest.SuchinconsistenciesacrossstudiesmaybeduetovariationsinthedeletionsiteoftheSyngapgene,geneticbackground,and/orageofthemice.DuarteetalusedaSyngap mutantmice on a C57BL/6J background 10,30, of which

F I G U R E 7   ImpairedworkingmemoryandincreasedlocomotoractivityofSyngap1−/+miceobservedinY‐mazeandT‐maze.(A‐C)T‐mazespontaneousalternationtest:A,percentageofcorrectresponses;B,latencytocompleteasession;C,distancetraveledtocompleteasession.(D‐G)Y‐mazetest:D,numberofentries;E,totalalterations;F,numberofalterationsasapercentageoftotalentries;andG,totaldistancetraveled.Datarepresentthemean±SEM.TheP‐valuesindicategenotypeeffectsintwo‐wayrepeatedmeasuresANOVA(A‐C),orone‐wayANOVA(D‐G)

1000

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4000(D) (E) (F) (G)

(A) (B) (C)

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Y-maze test

T-maze test Controls (n = 16)Mutants (n = 19)

Controls (n = 16)Mutants (n = 19)

P = 0.0001 P = 0.0006 P = 0.632 P = 0.0005

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     |  11NAKAJIMA et Al.

exon cassette containing the first commonmethionine presentin Syngap‐c gene was chosen for deletion 10. Muhia et al em‐ployedamutantmouselineonaC57BL/6background(substrainnotspecified)9,26,whereinexons4to9withintheSyngapgenewerecompletelydeleted. Inour study,weusedmutantswithaC57BL/6J background, lacking the codon for arginine 312 (or470)oftheSyngapgene1,2,22.Inadditiontothevariationsinthemutationsite,thereisadifferenceintheageoftheanimalsstud‐ied.Muhiaetal26startedthebehavioraltestswhenthemicewere10‐12weeksold.Ontheotherhand,ourmicewere53‐56weeksoldatthebeginningofthetestbattery.Basedonareportdemon‐strating the effects of age on various behavioral domains inC57BL/6Jmice32,possibleage‐dependencyofthephenotypesinthemutantmiceshouldbetakenintoconsideration.Inthisstudy,femaleSyngap1−/+micewerenottested.Severalpreviousstudieshavereportedthatmanybehavioralphenotypesweresharedbe‐tweenmaleandfemaleSyngap1−/+ mice 6,22,23,25‒27,whileMuhiaetal26reportedadecreasedlatencytofallintherotarodtestonlyinfemales.FurtherstudiesarenecessarytoclarifytheeffectofsexonthephenotypesoftheSyngap1−/+mouselinethatweusedinthepresentstudy.

Intheelevatedplusmaze,Syngap1−/+micestayedontheopenarmforasignificantlylongertimethanWTmice,whichisnormally

interpretedasadecreasedanxiety‐likebehavior61.Severalgroupshave also reported increased open‐arm stay time for Syngap1−/+ miceintheelevatedplusmaze6,23,25,26,28,29.MuhiaetalandGuoetal25,26 investigated theconfoundingeffectofelevated locomotoractivityontheincreasedopen‐armtimeintheelevatedplusmazeandclaimedthatmutantmicehaveabnormalanxietylevels.Muhiaetal26analyzedthefirst2minutesoftheelevatedplusmazetest,inwhichactivitylevelsdidnotdifferbetweenthetwogenotypes,andspeculatedthattheobservedincreaseinentriesandtimespentintheopenarmsbythemutantmicewerenotconfoundedbyen‐hancedlocomotoractivity.Guoetal25alsoarguedthatmutantmicedidnotproperlyperceivedanger,andtheincreasedopen‐armtimewasnotrelatedtoageneralizedornovelty‐inducedhyperactivity,becausetherewerenodifferencesbetweengenotypesinthenum‐berofopenor totalarmentries.On theotherhand,Kilincetal6 claimedthatitwasuncleariftheincreasedtimeintheopenarmsofthemutantmicereflectsreducedanxietyoran increasedexplor‐atorydrive,orboth.Overall, it isstillunclearwhetherSyngap1−/+ micehaveadecreasedanxiety.However,astudyreportedanxiousbehaviorinpatientswithSyngap1mutations62.Someresearchershavespeculated that the increasedexplorationof theopenarmsmayreflectanincreasedpanic‐likeescaperesponsetostressand/or a higher level of anxiety 32,53,63‒65. For example, Schunurri‐2

F I G U R E 8   ImpairedspatialreferencememoryofSyngap1−/+miceintheBarnesmaze.A,Numberoferrorsbeforereachingthetargethole;B,latencytoreachthetargethole;C,distancetoreachthetargethole;andD,numberofomissionerrorsbeforereachingthetargetholeareshown.E,Timespentaroundeachholeintheprobetrialconducted1d(left)and1mo(right)afterthelasttrainingsession.TheP‐valuesindicategenotypeeffectsintwo‐wayrepeatedmeasuresANOVA(A‐D),orone‐wayANOVA(E)

(B)(A) (C)

(E) Probe test (1 mo after training)Probe test (1 d after training)

Barnes maze test(D)

2 4 6 8 10 12 140

1

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Num

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mis

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Controls (n = 16)Mutants (n = 22)

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Tim

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Distance (angle) from target Distance (angle) from target

P = 0.002 P = 0.0204 P = 0.0002

2 4 6 8 10 12 142 4 6 8 10 12 142 4 6 8 10 12 140

100200

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P = 0.0022

P = 0.0156

Controls (n = 16)Mutants (n = 22)

Controls (n = 16)Mutants (n = 20)

12  |     NAKAJIMA et Al.

knockout mice, which lack a major histocompatibility complexbindingprotein,showincreasedopen‐armexplorationinadditiontohigherplasmacorticosteronelevels32,64.Furtherinvestigationsarethereforenecessarytoclarifythe linkbetweenSyngap1genemutationsandanxiety.

While many studies have shown elevated locomotor activitiesofSyngap1−/+miceinnovelenvironmentsinvariousbehavioraltests23,25‒29, activity in familiar environment has not yet been tested 6. In thepresentstudy,weobserved thehomecage locomotoractiv‐ityofthesemiceandfoundthattheyhaveasignificantly increased

F I G U R E 9  ContextualandcuedfearmemoryinSyngap1−/+mice.(A)Shocksensitivitymeasuredbythedistancetraveledduringtheshock.Percentageoffreezingtimeduring:(B)conditioning,(C,top)contexttesting,(D,top)cuedtestingwithalteredcontext,(E,top)contexttestingafter30d,and(F,top)cuedtestingwithalteredcontextafter30d.Eachdatapointinthefigurepanels(B)and(C‐F,top)indicatespercentageoffreezingineach1‐minbin.Distancetraveledin:(C,bottom)contexttesting,(D,bottom)cuedtestingwithalteredcontext,(E,bottom),contexttestingafter30d,and(F,bottom)cuedtestingwithalteredcontextafter30d.Eachdatapointinfigurepanels(C‐F,bottom)indicatesthedistancetraveledineach1‐minbin.Datarepresentthemean±SEM.TheP‐valuesindicategenotypeeffectsintwo‐wayrepeatedmeasuresANOVA.Thehorizontalblackbarsindicatethetimeduringwhichthetonestimuliwereadministered

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     |  13NAKAJIMA et Al.

locomotor activity at night, which indicates that these mice showhyperlocomotor activity not only in novel, but also in familiarenvironments.

Collectively, we confirmed that the Syngap1−/+ mouse reca‐pitulatesthesymptomsofIDandASDinpatientswithSYNGAP1 mutations. A reduction in Syngap1 levels dramatically affectedlocomotoractivity,cognitivefunctions,emotion,painsensation,andmotorfunction.However,theassociationbetweenSYNGAPandanxietyneeds tobe reconsidered.These findingsalsopro‐videcluestophysiologicalrolesofSYNGAP‐regulatedpathways.Our analysis of Syngap1−/+ mice can prove to be an invaluablemodel for further investigations of ID and ASD patients withSYNGAP1mutations.

ACKNOWLEDG MENTS

WethankKeikoToyama,MikaTanaka,YoshihiroTakamiya,andNaoHirata for their technical support in this study. Thisworkwas sup‐ported by the following grants: MEXT KAKENHI Grant NumbersJP221S0003, JP16H06462, JP16H06276; JSPS KAKENHI GrantNumberJP16680015;AMEDunderGrantNumberJP18dm0107101;andSimonsInitiativefortheDevelopingBraingrantR83776.

CONFLIC T OF INTERE S T

Theauthorshavenoconflictsofinteresttodeclare.

DATA REPOSITORY

The rawdata of the behavioral tests and the information abouteach mouse are accessible on the public database “MousePhenotypeDatabase”(http://www.mouse‐phenotype.org/).

ANIMAL S TUDIE S

AllthebehavioraltestswerecarriedoutintheSectionofBehaviorPatterns,CenterforGeneticAnalysisofBehavior,NationalInstitutefor Physiological Sciences. All the experimental protocols wereapprovedby theAnimalCareandUseCommitteeof theNationalInstituteforPhysiologicalSciences.

ORCID

Ryuichi Nakajima https://orcid.org/0000‐0002‐0024‐4422

Satoko Hattori https://orcid.org/0000‐0002‐1047‐6454

Hirotaka Shoji https://orcid.org/0000‐0003‐4843‐6949

F I G U R E 1 0  ElevatedlocomotoractivityanddecreasedsocialactivityinSyngap1−/+miceinhomecage.(A‐B)Socialinteractioninhomecageasindicatedby(A)meannumberofparticles,and(B)activitylevels.(C)Homecagelocomotoractivityofsinglemouse.Datarepresentthemean±SEM.TheP‐valuesindicategenotypeeffectsintwo‐wayrepeatedmeasuresANOVA.ThethreeP‐valuesineachpanel(A‐C)representthegenotypeeffects(controlsvsmutants)intwo‐wayrepeatedmeasuresANOVAfortheactivitylevelsofwholeday,day,ornight,fromtoprow

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P = 0.0021Day P = 0.809Night P = 0.0006

Controls (n = 7 pairs)Mutants (n = 9 pairs)

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SUPPORTING INFORMATION

Additional supporting information may be found online in theSupportingInformationsectionattheendofthearticle.

How to cite this article:NakajimaR,TakaoK,HattoriS,etal.ComprehensivebehavioralanalysisofheterozygousSyngap1 knockoutmice.Neuropsychopharmacol Rep. 2019;00:1–15. https://doi.org/10.1002/npr2.12073