Blocking PirB Up-regulates Spines and Functional Synapses to Unlock Visual Cortical Plasticity and...

1/31/2015 BlockingPirBupregulatesspinesandfunctionalsynapsestounlockvisualcorticalplasticityandfacilitaterecoveryfromamblyopia

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INTRODUCTIONRESULTS

GeneticdeletionofPirBenhancesODplasticityPostnataldeletionofPirBfromexcitatoryneuronsenhancesadultODplasticityBlockadeofPirBligandbindingrapidlyenhancesODplasticitysPirBincreasesspinedensityandfunctionalsynapsesonL5pyramidalneuronssPirBtreatmentafterLTMDenablesrecoveryofspinedensitysPirBinducesrecoveryofvisualacuityafterLTMD

DISCUSSIONsPirBasanacuteregulatorofspineandsynapsedensitysPirBasapotentialtherapyforrecoveryfromamblyopiaPirB:Anendogenoustargetformanipulationsofsynapseandsystemslevelplasticity

MATERIALSANDMETHODSStudydesignMousestrainssPirBproteinproductionOsmoticminipumpimplantationsandsPirBinfusionArcmRNAinductionandinsituhybridizationVEPrecordingsStatisticalanalyses

SUPPLEMENTARYMATERIALSREFERENCESANDNOTES

ScienceTranslationalMedicinestm.sciencemag.org.ezproxy.lib.usf.eduSciTranslMed15October2014:Vol.6,Issue258,p.258ra140Sci.Transl.Med.DOI:10.1126/scitranslmed.3010157

RESEARCHARTICLE

NEUROSCIENCE

BlockingPirBupregulatesspinesandfunctionalsynapsestounlockvisualcorticalplasticityandfacilitaterecoveryfromamblyopiaDavidN.Bochner1,*,RichardW.Sapp1,*,JaimieD.Adelson1,SiyuZhang2,HanmiLee1,MajaDjurisic1,JoshSyken3,YangDan2

andCarlaJ.Shatz1,

AuthorAffiliations

AuthorNotes

Correspondingauthor.Email:[email protected]

Duringcriticalperiodsofdevelopment,thebraineasilychangesinresponsetoenvironmentalstimuli,butthisneuralplasticitydeclinesbyadulthood.ByacutelydisruptingpairedimmunoglobulinlikereceptorB(PirB)functionatspecificages,weshowthatPirBactivelyrepressesneuralplasticitythroughoutlife.WedisruptedPirBfunctioneitherbygeneticallyintroducingaconditionalPirBalleleintomiceorbyminipumpinfusionofasolublePirBectodomain(sPirB)intomousevisualcortex.Wefoundthatneuralplasticity,asmeasuredbydeprivingmiceofvisioninoneeyeandtestingoculardominance,wasenhancedbythistreatmentbothduringthecriticalperiodandwhenPirBfunctionwasdisruptedinadulthood.AcuteblockadeofPirBtriggeredtheformationofnewfunctionalsynapses,asindicatedbyincreasesinminiatureexcitatorypostsynapticcurrent(mEPSC)frequencyandspinedensityondendritesoflayer5pyramidalneurons.Inaddition,recoveryfromamblyopiathedeclineinvisualacuityandspinedensityresultingfromlongtermmonoculardeprivationwaspossibleaftera1weekinfusionofsPirBafterthedeprivationperiod.Thus,neuralplasticityinadultvisualcortexisactivelyrepressedandcanbeenhancedbyblockingPirBfunction.

INTRODUCTION

Duringpostnataldevelopment,thecapacityofthebraintoundergoexperiencedependentchangesinsynapticstrengthandcircuitconnectivityisdynamicallyregulated,withplasticitypeakingduringdevelopmentalcriticalperiodsandthendecreasingwithmaturation(13).Criticalperiodsarekeytimeswhensensoryexperienceisnecessaryfornormalcircuitdevelopmentandwhenabnormalexperiencecangenerateenduringanomaliesinbrainstructureandfunction(2,4).Oculardominance(OD)plasticityisagraphicexampleofexperiencedrivensynapticandcircuitplasticity.Childrenbornwithcongenitalcataractinoneeyewillsufferamblyopiaalossofvisualacuityifnotcorrectedearlyinlife(5,6).Monocularvisualdeprivation(MD)hasbeenusedinanimalmodelsofamblyopiatounderstandunderlying

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mechanisms.AfterabriefperiodofMDorenucleation(ME)duringjuvenilelife,visuallydrivenresponsesofneuronsinthebinocularzoneofmammalianprimaryvisualcortex(V1)shifttowardtheopeneye,andcorticalterritorycontainingneuronsrespondingtoopeneyestimulationexpands,whereasclosedeyeresponsesweakenandterritoryshrinks(3,7,8).Theseeffectsaremaximalaroundpostnatalday28(P28)inmiceanddecreasethereafterbyadulthood,littleODplasticityresultingfromeyeclosurecanbedetected,particularlywithshorterperiodsofdeprivation(711).Furthermore,alongtermperiodofMD(LTMD)spanningtheentirecriticalperiod(forexample,P19toP47)generatesanenduringlossofacuityandcorticalfunctioninthedeprivedeyeevenifbinocularvisionisrestoredinadulthood(4,12,13).Thisnormaldecreaseinplasticitybyadulthood,althoughimportantforstabilizingneuralcircuits,actsasabarriertorecoveryafterinjurybecauseitlimitscorticalreorganization,canlockineffectsofdysfunctionaldevelopment,andevenopposesacquisitionofnewlearning.Ifadultneuralcircuitscouldbereturnedtoanimmaturestate,criticalperiodsmightbeeffectivelyreopened,facilitatingrecoveryafternervoussystemdamage,leadingtonewtreatmentsforneurodegenerativeordevelopmentaldisorders,orevenenhancinglearninginhealthyindividuals.

Alimitednumberofcandidatemoleculesthatappeartoactasendogenousnegativeregulatorsofcorticalplasticityhavebeenidentified(1418).Onesuchmolecule,pairedimmunoglobulinlikereceptorB(PirB),isexpressedincorticalandhippocampalneuronsaswellasinsomeimmunecells(14).Inthenervoussystem,PirBbindsseveralligands,includingmajorhistocompatibilitycomplex(MHC)classIproteins,NogoA,andmyelincomponents(19,20).Bothinimmunecellsandneurons,ligandbindingrecruitsSHP1andSHP2phosphatases(14,21).SHPrecruitmentrequiresPirBphosphorylationonitsITIM(immunoreceptortyrosinebasedinhibitorymotif)domains(21,22).Inneurons,cofilinisalsorecruitedtoPirB,leadingtochangesintheactincytoskeleton(20).

GermlinePirB/micehaveenhancedODplasticitynotonlyduringthecriticalperiodbutalsobeyond,andtheyrecovermorerapidlyinastrokemodel(14,23,24).PirBanditshumanortholog,leukocyteimmunoglobulinlikereceptor,subfamilyB,member2(LilrB2),bindsolubleamyloidoligomers,andgermlinePirBdeletionrescuesODplasticityandhippocampaldeficitsinamousemodelofAlzheimersdisease(AD)(20).However,itremainsunknownwhethertheenhancedODplasticityandstrokerecoveryingermlinePirB/miceareduetoearlydevelopmentalchanges,orwhetherPirBactsatallagestolimitplasticity,whichwouldmakeitanattractivetherapeutictargetfordrugdevelopment.BecausePirBisareceptor,signalingcanbemodulatedbyconditionalgeneticknockoutorbyinterferingwithligandbinding(19).IfPirBfunctionsthroughoutlife,disruptingPirBshouldenhanceplasticityorfacilitaterecoveryatanyage.

RESULTS

GeneticdeletionofPirBenhancesODplasticity

TodisruptPirBfunctionwithtemporalcontrol,aconditionalalleleofPirBwasgeneratedbyinsertingloxPsitessurroundingexons10to13,whichcontainthetransmembranedomainandfirstITIMdomainofPirB(14)(Fig.1A).Toobtainrobustwidespreaddeletion,thisPirBfloxmouselinewascrossedwithatransgenicmouselineexpressingtamoxifeninducibleCreERT2onaubiquitinCpromoter(25).TheresultingUbcCreERT2PirBflox/floxmicewerebredwithPirBflox/floxmice,producingexperimentalUbcCreERT2PirBflox/floxanimals(henceforthcalledCre+)aswellasPirBflox/flox(Cre)littermatecontrols.TamoxifeninjectionsgiveneitherneonatallyoraftercriticalperiodclosureinducedrobustdeletionofthefloxedallelefromgenomicDNAwithin1week(Fig.1B).PirBproteinlosswasmoregradualforexample,dailytamoxifentreatmentfromP3toP7diminishedPirBproteinintheforebrainby~90%byP27(Fig.1,CandD).AsimilargraduallossofproteinwasseenatP70aftertamoxifentreatmentfromP45toP49(Fig.1,CandE).Thus,tamoxifenadministrationsubstantiallyreducedPirBproteinlevelsbythepeakoftheODcriticalperiodatP28(7),aswellasinadulthoodbyP70.

Fig.1.ATamoxifeninducibleCredependentstrategyfordeletionofPirBwithtemporalcontrol.

(A)SchematicofPirBproteinstructure(top)andfloxedPirBallele(bottom)beforeandafterCremediatedexcision.(B)Dailytamoxifengivenviainjectionofnursingmother(P3toP7)inducesdeletionofthefloxedregionatP21asdetectedbypolymerasechainreaction(PCR).(C)WesternblotsforPirBproteininforebrainatages(left)oftamoxifen(TAM)administrationandWesternblotting.(D)QuantificationofPirBproteininforebrainaftertamoxifenadministration(P3toP7),normalizedtoaverageCre

levelsacrossallagesassayed:CreP21:n=4miceversusCre+P21:n=5,P=0.02,Utest.CreP27:n=5versusCre+P27:n=4,P=0.02,Utest.(E)QuantificationofPirBproteininforebrainatP70(adult)aftertamoxifeninjectionfromP45toP49.CreP70:n=4versusCre+P70:n=4,P=0.03,Utest.*P





inducedwithinminutesofvisualstimulation,andtheupregulatedmRNAcanbedetectedincorticalneuronsfunctionallydrivenbythestimulatedeye(26).WemeasuredthehorizontalextentoftheArcmRNAinsituhybridizationsignalalongL4ofvisualcortexipsilateraltothespared,stimulatedeye(Fig.2,AtoC,andfig.S1).ThisexpansioninwidthofArcmRNAsignalisareliablemeasureofopeneyestrengtheningaftervisualdeprivationandcorrelateswellwithothermethodsusedtoassessODplasticityincludingsingleunitelectrophysiology(7,8,27,28),visualevokedpotentials(VEPs)(8,11,29),orintrinsicsignalimaging(8,24,3033).ThewidthofArcmRNAinductiondoesnotexpandintransgenicmiceknowntolackODplasticityasmeasuredbyothermethods(20,28,33),whereasthereisanincreaseinwidthofArcmRNAsignalinmiceknowntohaveincreasedODplasticity(14,15,24).

Fig.2.TimedgeneticdeletionofPirBenhancesODplasticity.

(A)Schematicofmousevisualsystem.Eachretina(right:red,left:blue)projectsprimarilycontralaterallytothelateralgeniculatenucleus(LGN),whichprojectstovisualcortex(V1).Asmallbinocularzone(BZ,purple)inV1receivesinputfrombotheyesinresponsetodeprivationofoneeye(forexample,left),therepresentationoftheopen(rightipsilateral)eyeexpands(arrows).(B)TimelineofinducibleknockoutofPirBandassessmentofODplasticityviaArcmRNAinduction.(C)ExamplemicrographsofinsituhybridizationsforArcmRNAinducedinBZofvisualcortexafteropeneyestimulation.Eachblackdotisacell.MEfromP28toP32resultsinexpansionoftheipsilateral(open)eyerepresentation(betweenredasterisks),ascomparedwithnormalrearing(NR).Cre=PirBflox/flox.Cre+=UbCCreERT2PirB

flox/flox.WidthofArcsignalinL4wasmeasured(seefig.S1).Corticallayersindicatedatleftscalebar,500m.CP,criticalperiod.(D)CumulativehistogramsofwidthofArcmRNAsignalbyindividualsection.NRCre:n=41sectionsNRCre+:n=44MECre:n=39MECre+:n=52.(E)Graphofdatain(D),withmeanandSEMbyanimal:deletionofPirBduringthecriticalperiodenhancesODplasticity.NRCre:n=7miceversusNRCre+:n=7,P=0.65MECre:n=7versusMECre+:n=7,****indicatesP







inallcells.Next,weinvestigatedwhetherlossofPirBspecificallyinforebrainexcitatoryneuronswassufficienttoenhanceODplasticity.PirBflox/floxmicewerecrossedwithaCamKIIaCreline,whichexpressesCreexclusivelyinforebrainexcitatoryneurons(34,35),generatingCamKIIaCrePirBflox/floxconditionalknockouts,andCamKIIaCrePirB+/+littermatecontrols.PCRgenotypingofbrainandearconfirmsbrainspecificdeletionofthefloxedregionofPirB(Fig.3A).ToconfirmthespatialpatternofCredeletion,CamKIIaCremicewerealsocrossedtotheAi14TdTomatoreporterline(36).ResultsshowfaithfulCreactivityatP30inpyramidalneuronsofhippocampusandcortex(Fig.3B).PreviousstudieshaveshownthatexcisionoffloxedregionsofDNAinthisCrelineisgradual,withcompletedeletionoccurringaround3monthsofage(35),permittingustoexamineeffectsofPirBdeletioninadulthood.

Fig.3.CremediateddeletionofPirBfromforebrainexcitatoryneuronsenhancesadultODplasticity.

(A)GenotypingofsamplesfromearandcerebralcortexfromP100CamKIIaCrePirBflox/flox(cKO)orCamKIIaCrePirBWT(wildtype),showingdeletionoffloxedPirBincortexbutnotear.(B)CamKIIaCrePirBflox/+breederswerecrossedwiththeAi14TdTomatoreporterline,generatingredfluorescenceinthepresenceofCre.Sagittalsectionthroughvisualcortex(layersindicatedatright)andhippocampusofaP30mouseshowsCrepresentinpyramidalneurons.(C)GraphsofwidthofL4regionactivatedbystimulationofipsilateral(open)eyeinvisualcortex,assessedusingArcmRNAinduction.DeletionofPirBfromforebrainexcitatoryneuronsincreasesopeneyeexpansioninadultmiceafterMEfromP100toP110.NRWT:n=5miceversusNRcKO:n=4,P=0.91.MEWT:n=8miceversusMEcKO:n=5,P=

0.006.NRversusMEWT:P=0.39,NRversusMEcKO:P=0.0002,bytwowayANOVAwithTukeyposthoctest.**P



infusion(1mg/ml).Scalebar,1mm.(EandF)MinipumpinfusionsofsPirBduringcriticalperiod(CP).Timelineasshown.(E)ExampleArcmRNAinsituhybridizationmicrographsofvisualcortexafterBSA(top)orsPirB(bottom)treatment.Scalebar,500m.RedasterisksindicatebordersofArcmRNAsignalinducedbystimulatingtheipsilateral(open)eyeinlayer4.(F)GraphscomparingwidthofArcmRNAsignalinL4afteropeneyestimulation.WidthofterritoryactivatedbyopeneyestimulationafterMEisgreateraftersPirBinfusionthanwithBSA.NRBSA:n=4mice,NRsPirB:n=4,MEBSA:n=5versusMEsPirB:n=6,P



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germlinePirBknockoutmicerearedwithnormalvision.IthasbeenproposedthattheseextraspinesrepresentapreexistingstructuralsubstratethatisthenrecruitedforthemorerapidandrobustODplasticityobservedinthesemice(24).Indeed,previoussensoryexperienceinvisualorauditorysystemsincreasesplasticity,andisaccompaniedbyincreasedstructuralconnectivity(4144).WetestedwhetherPirBmightcontributetothesestructuralchanges.

sPirBinfusionmighttriggeranincreaseinspinedensityevenwithoutvisualdeprivation.Totestthishypothesis,visualcortexofnormallyrearedwildtypeThy1YFPHtransgenicmice(45),inwhichcorticalL5pyramidalneuronsareyellowfluorescentprotein(YFP)labeled,receivedminipumpinfusionsofeithersPirBorBSAfromP63toP74(Fig.5,AandB).SpinesonapicaldendritesofL5pyramidalneuronswereexaminedinthebinocularzoneatadistanceposteriortotheinfusionsitecomparabletothatstudiedaboveforassessmentofODplasticity.InthisregionaftersPirBinfusion,pyramidalneuronsomata,dendrites,andspinesappearedintactandhealthy,withoutfragmentationorblebbing(Fig.5A).SpinedensityonL5apicaldendritictuftsofanimalsrearedwithnormalvisionwas38%greaterinthepresenceofsPirBthanofBSA(Fig.5B).SpinedensityonL5neuronsintheuninfusedhemispherewasnotaltered,andatwowayANOVAconfirmsasignificantinteractioneffectbetweenhemisphereandtreatment(P=0.03).TheobserveddensityincreasecouldariseifsPirBactsonasubclassofdendriticspines.However,afteran11dayinfusionofeithersPirBorBSA,therewasnosignificantdifferenceintheproportionofspinesclassifiedasmushroom,thin,orstubby(46)(fig.S4B).Together,resultsshowthatinadultvisualcortex,itispossibletogeneratealocalincreaseinspinedensityonL5neuronsbyinfusingsPirB,evenintheabsenceofavisualmanipulationordeprivation.

Fig.5.sPirBincreasesspinedensityandfunctionalsynapsesonL5pyramidalneuronsofnormallyrearedmice.

(A)Timelineofminipumpinfusions[BSA(1mg/ml)orsPirBfromP63toP74]andexampledendritesofYFPlabeledL5pyramidalneuronsinbinocularzoneofvisualcortexinWTThy1YFPHanimalsrearedwithnormalvisualexperience.Scalebar,10m.(B)HistogramsofspinedensityonapicaltuftsofL5neuronsinsPirBinfusedversusintheuninfused(unif.)contralateralhemisphere,orinBSAcontrols:BSAinfused:n=5miceversussPirBinfused:n=5,P=0.01,onetotwocellsperanimal.BSAuninf.:n=5versussPirBuninf.:n=5,P=0.96,BSAinf.versusuninf.:P=0.99,sPirBinf.versusuninf.:P=0.016,bytwowayANOVAandTukeyposthoc

test.(C)ExampletracesofmEPSCresponsesrecordedfromvisualcorticalslices(P70toP77)fromL5pyramidalneuronsafterBSAorsPirBinfusion,asin(A).(D)IncreasedmEPSCfrequencywithsPirBinfusion:BSA:n=12neuronsversussPirBn=13,P=0.046,byMannWhitneyUtest.(E)NochangeinmEPSCamplitude:BSA:n=12neuronsversussPirBn=13,P=0.70,byMannWhitneyUtest.

Toexaminewhethertheincreaseinspinedensityrepresentsnewfunctionalsynapses,miniatureexcitatorypostsynapticcurrents(mEPSCs)wererecordedfromL5pyramidalneuronsinslicesofvisualcortex(P70toP77),after7to11daysofsPirBminipumpinfusioninvivo,inmicerearedwithnormalbinocularvision(Fig.5C).mEPSCfrequencywassignificantlygreateraftersPirBtreatmentthaninBSAlittermates(Fig.5D),withnochangeinmEPSCamplitude(Fig.5E).ThisfindingisconsistentwiththeideathatsPirBinfusioncausesanincreaseinsynapticconnectivity,suggestingthatnewlyformedspinesrepresentsitesoffunctionalsynapses.

sPirBtreatmentafterLTMDenablesrecoveryofspinedensity

LTMDisawellstudiedanimalmodelofamblyopiabecauseitinvolvesanexperiencedependentdevelopmentallossoffunctioninthedeprivedeye(47,48).Inrodents,LTMDprofoundlydecreasesvisualacuity,aswellasthenumberofcorticalneuronsvisuallydrivenbythedeprivedeye.Thereislittle,ifany,recovery,evenafterrestorationofbinocularvision(4,12,13,17,49).Ithasbeenproposedthatadecreaseindendriticspinedensityunderliesthesefunctionaldeficits(4,49).Forexample,LTMDgeneratesasignificantdeclineinspinedensityonbasolateraldendritesofL5pyramidalneuronscontralateraltothedeprivedeye(49).

GiventherapidandgenerativeeffectofsPirBonspinedensityandmEPSCfrequencydescribedaboveinnormalvisualcortex,wewonderedwhethersPirBtreatmentmightgenerateaspinedensityincreasethatcouldfacilitaterecoveryfromLTMD.Thy1YFPwildtypemicewereeithernormallyrearedorreceivedLTMDspanningtheentirecriticalperiodforODplasticity(P19toP47).AtP47,thedeprivedeyewasreopenedtorestorebinocularvisionfor1week.ThenatP54,minipumpscontainingeithersPirBorBSAwereimplantedinthevisualcortexcontralateraltothedeprivedeyeuntilP61(Fig.6A),atwhichtime,spinedensityonL5basolateraldendriteswasmeasured.

Fig.6.sPirBallowsstructuralandfunctionalrecoveryfromamblyopiaafterLTMD.

(A)Experimentaltimeline:LTMDfromP19toP47,eyereopeningatP47,andminipumpinfusionfromP54toP61.(B)RepresentativeYFPlabeledL5cellsomaandbasolateral(arrow)dendritesinvisualcortexofWTThy1YFPHmice.Scalebar,50m.(C)Bargraphsshowingchangesin

basolateraldendriticspinedensity:LTMDcausesa





significantdeclineinspinedensity(BSALTMD)thatcanbefullyreversedwithsPirBinfusion(sPirBLTMD)(BSANR:n=5miceversusBSALTMD:n=4,P=0.02.sPirBLTMD:n=5,sPirBversusBSALTMD,P=0.001,sPirBNR:n=5animals,onetotwocellsperanimal,sPirBversusBSANR:P=0.003).*P



showntoproducerapidincreasesinspinedensity,sPirBtreatmentproducesalargermagnitudechangeanddoessointheabsenceofnovelstimuliortraining.Together,ourobservationsimplythattargetinganddisruptingPirBfunctionincreasesynapticconnectivityandplasticity,evenafterthecriticalperiod.BecausePirBisexpressedbypyramidalneuronsthroughouttheneocortex(14),ourresultsmayalsoapplytocorticalareasotherthanthevisualsystem.

sPirBasanacuteregulatorofspineandsynapsedensity

InfusionofsPirBdecreasesPirBdownstreamsignaling(Fig.4C),consistentwithitspreviouslydemonstratedsequestrationofendogenousPirBligands(14,19,20,23).Inadulthood,acuteblockadewithsPirBresultsinenhancedODplasticityandproducesarapidincreaseinspinedensityandmEPSCfrequency,evenintheabsenceofalteredvision.ManyinterventionsthataffectsynapticconnectivityandspinedynamicsalsoenhanceODplasticity,includingtransplantationofinhibitoryneuronprogenitors(53)ordisruptionofNgR1NogoAfunction(16,54,55).Spinedensityincreasesalsocorrelatewithenhancedsubsequentplasticity(41,44):newspinesgeneratedduringaninitialMDcanbecooptedformorerobustODplasticityduringasubsequentMD(24,41,44).Furthermore,ingermlinePirB/mice,enhancedODplasticityisassociatedwithalargeincreaseinspinedensityonL5neuronsandanincreaseinthemagnitudeofL4toL2/3longtermpotentiation(LTP)invisualcortex(24).Collectively,theseexperimentsconnectanincreaseinspinedensityandfunctionalconnectivitytoenhancedsynapticplasticity.Thus,sPirBmaygenerategreaterODplasticitybycreatingamorehighlyinterconnectedstructuralsubstratethatcanbeaccessedformorerapidandrobustsynapticchange.

sPirBasapotentialtherapyforrecoveryfromamblyopia

LTMDthroughoutthecriticalperiod,usedhereasananimalmodelofamblyopia,leadstoaprofoundlossofvisualacuity,aswellastolossofvisualresponsivenessofcorticalneuronstostimulationofthedeprivedeyebotharehighlyresistanttorecoveryevenwhenbinocularvisionissubsequentlyrestored(4,12,13,17,49).DecreasesinspinedensityonbothL2/3pyramidalcells(4)andpyramidalneuronsthroughoutcortexhavealsobeenreportedafterLTMDorchronicMD(49).Althoughreversalofthisspinelossoncorticalpyramidalneuronshasbeenseen,reversalrequiredthattheformerlyopeneyebesuturedclosedincombinationwithfairlydisruptivetreatmentssuchaschondroitinaseABCtodigestextracellularmatrix(4),or10daysofdarkexposurefollowedbyvisualstimulation(49,56).RecoveryofspinesinbothofthesecaseswasaccompaniedbyrobustrecoveryofVEPacuity.Inourstudy,wefoundthatsPirBinfusion,combinedwithbinocularvision,wassufficientbyitselftobringspinedensityvaluesandVEPacuityestimatesclosetonormal.VisualacuityasmeasuredbyVEPspredictsphysiologicallyrelevantrecoveryofvisualfunctioninthedeprivedeye,indicatingthatvisionthroughthedeprivedeyeinsPirBtreatedmicehasgreatlyimproved(4,43,44).Togetherwiththedataonspinerecovery,theseresultssuggestthatsPirBcanenablesignificantstructuralandfunctionalrecoveryfromamblyopiaafterLTMDwithinjust7daysoftreatment.

TheseobservationsimplythatsPirBasolublereceptorectodomainisapotentialtherapeuticagent,andtheyprovideproofofconceptforgeneratingotherPirBblockingreagents.Thestandardtreatmentforhumanamblyopicpatientsmandatesearlyinterventionduringadevelopmentalcriticalperiodandinvolvesalternatingpatchingbetweenthetwoeyestostrengthentheamblyopiceye,butthistreatmentinterfereswithdevelopmentofbinoculardepthperception(6).ThereareseveralPirBhomologsinhumans(LilrBs),andLilrB2proteinisexpressedinthehumanbrain(25).TargetingLilrB2orothermembersoftheLilrBreceptorfamilymightpermitrecoveryfromamblyopiawithoutrequiringeyepatching,asimpliedbytheresultsoftheLTMDexperimentsinmice.

Thereareanumberoflimitationsandissuestoconsiderintranslatingourfindings.First,itwouldbeimportanttodeterminewhethertheincreaseinspinedensityandfunctionalrecoveryfromamblyopiapersistsstablybeyondtheperiodofsPirBinfusion.Second,itispossiblethatalongerinfusionorhigherconcentrationofsPirBwouldproduceamorerobustrecoveryforallanimals.Inaddition,ratherthanminipumpinfusions,itwouldbepreferabletodevelopasmallmoleculedrugthatcancrossthebloodbrainbarrier.Finally,asmentionedabove,LilrB2ispresentinhumanbrain,butbecausethereareotherfamilymembers,itwillbeimportanttocharacterizetheirexpressionandfunctioninhumancentralnervoussystem.

PirB:Anendogenoustargetformanipulationsofsynapseandsystemslevelplasticity

Ourobservationsaddtoagrowingbodyofresearchthathasunmaskedactiverolesformoleculesinthebrainactingasnegativeregulatorsoffunctionalandstructuralplasticitybothindevelopmentandinadulthood(17,50,54).InthecaseofPirB,thisnegativeregulationmayalsobehijacked,asinAD,whereamyloidoligomersbindtoPirB/LilrB2withnanomolaraffinity,resultinginlossofODplasticityanddeficitsincorticalandhippocampalsynapticplasticity(20).Thus,sPirBandhumansolublereceptorhomologsmightevenbeviabletherapeuticsforAD.BygeneratingarecombinantsPirBprotein,wehavedemonstratedauseforselectivelyblockingPirBreceptorinteractionwithendogenousligands.TheseresultsfurthersupportthevalueofcreatingPirB/LilrBantagoniststhatcrossthebloodbrainbarrier,enhancingplasticityandincreasingsynapseandspinedensityincasesofdisease,dysfunction,injury,orevenforcognitiveenhancementinnormalindividuals.

MATERIALSANDMETHODS

Studydesign

TheobjectiveofthisstudywastodevisemethodstodeletePirBfunctionacutely,thenmonitortheeffectsonmeasuresofsynapticandODplasticity,andrecoveryfromLTMD.Twomethodswereused:tamoxifeninducedPirBdeletionviaaPirBconditionalallele,orsPirBminipumpinfusion.BecauseODplasticityisinducedbychangesinvisualexperience,experimentsweredesignedtocaptureaninteractioneffectbetween

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genotype/treatmentandvisualmanipulationfourgroupsandatwowayANOVAdesignwereusedtotestforinteractions.Allexperimentswereperformedblindtogenotypeand/ortreatment.Littermateswereusedtocontrolforgeneticvariation,andmicewererandomlyassignedtodifferentvisualmanipulationsandtreatmentswithinalitter.Todetectgenotypeeffectssimilarorgreaterthanthosepreviouslyreported,samplesizeswerechosenonthebasisofastatisticalpowerof80%withanvalueof0.05(14,24).Thenumberofreplicatemeasurementsandanimalsisgivenineachfigurelegend.

Mousestrains

PirB/andPirBflox/floxmiceweregeneratedasdescribed(14).APirBWTlinewasmaintainedonthesamebackgroundandusedforallminipumpinfusionexperimentsperformedduringthecriticalperiod(P21toP32).Foradultminipumpexperiments(P63toP74),PirBWTandPirB/micewerecrossedwiththeThy1YFPHtransgenicline(JAX#003782),whichexpressesYFPinasubsetofL5pyramidalneurons(45).Forinducibleknockoutexperiments,UbCCreERT2mice(JAX#007001)(25)werebredwithPirBfloxmicetogenerateUbCCreERT2PirBflox/floxmiceandPirBflox/floxlittermates.Forconditionalknockoutexperiments,CamKIIaCrePirBflox/+mice(57)werebredwithPirBflox/+micetogenerateCamKIIaCrePirBflox/floxmiceandCamKIIaCrePirB+/+littermates.CamKIIaCremicewerealsobredwithAi14TdTomatoreportermice(36).AllexperimentswereperformedinaccordancewithprotocolsapprovedbyStanfordUniversityAnimalCareandUseCommitteeinkeepingwiththeNationalInstitutesofHealthsGuidefortheCareandUseofLaboratoryAnimals.

sPirBproteinproduction

TocreateasPirBmimic,thePirBectodomainwasclonedintoaplasmidcontainingaHistagforpurificationandaMyctagforantibodydetectionwithasequenceidenticaltopreviouspublications(14,19,20,40).Forminipumpinfusions,InvitrogenCustomServicesproducedsPirBinlargerquantitiesinFreeStyleHEK293cellsandpurifieditonanickelcolumn(NiHis,Invitrogen).

OsmoticminipumpimplantationsandsPirBinfusion

Craniotomieswereperformed,andminipumps(ALZETmodel10020.25l/hour,100lcapacity)containingeithersPirB(1mg/ml)orBSA(1mg/ml)(VWREM2930)in0.1Mphosphatebufferedsalinewereimplantedsubcutaneously,connectedtoacannula.Thecannulawasinsertedjustanteriortoprimaryvisualcortex(2.5mmlateraland3mmposteriortobregma).

ArcmRNAinductionandinsituhybridization

ArcmRNAwasinducedbyplacingmiceovernightintotaldarkness(16to18hours),followedbybrightilluminationfor30mintopermitvisionthroughtheopeneyebeforeeuthanasiaviaisofluraneanesthesiaanddecapitation(8).AdigoxigeninlabeledArcantisensemRNAprobewasusedforcolorimetricinsituhybridizationsperformedonbrainsections(8,33).ImageswereacquiredviabrightfieldmicroscopyandanalyzedusingtheLineScanfunctionofNeuroLenssoftwaretomeasurethewidthoftheArcmRNAhybridizationsignalipsilateraltotheopen(nondeprived)eyealongL4ofthevisualcortex,atthe3to4border(fig.S1).Multiplesectionswerescannedandaveragedperanimal(forexample,Fig.2).

VEPrecordings

Animalswereanesthetizedwithurethane(0.6to1.2g/kgSigma)andchlorprothixene(5mg/kgSigma),andatincisionswithlidocaine(2%,SparhawkLaboratories),andthenthescalpwasexposedandtheminipumpwascannularemoved.AfteracraniotomycenteredoverV1,aglasspipettefilledwithACSF(artificialcerebrospinalfluid)wasinsertedtorecordlocalfieldpotentialsatadepthof450to600m.Responsestosinusoidalgratingstimuliwereaveragedoverstimulusblocks,andapeakresponseamplitudewithina500mswindowafterstimulusonsetwasdetermined.Visualacuitywasestimatedbyfindingthexinterceptofasemilogarithmicregressionofresponseamplitudesacrossdifferentspatialfrequencies(11,29).

Statisticalanalyses

AllstatisticalanalyseswereperformedwithPrismsoftware(Graphpad).Whenonlytwogroupswereinvolved,twosamplettestswereused,withWelchscorrectionforunequalvariancesappliedwhereappropriate.DataforwhichanormaldistributioncouldnotbeassumedwereanalyzedwithMannWhitneyUtests.Incaseswherebothtreatment/genotypeandvisualmanipulationorhemispherewerevaried,atwowayANOVAwasconducted,withTukeyposthoctestsforindividualpairsofcolumns.

SUPPLEMENTARYMATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/6/258/258ra140/DC1

MaterialsandMethods

Fig.S1.ExamplelinescanmeasurementsofArcmRNAinsituhybridizationsignalinvisualcortexinducedbystimulationoftheipsilateraleye.

Fig.S2.PlasticityindicesforgeneticorpharmacologicaldisruptionofPirBfunction.

Fig.S3.CharacterizationofsPirBminipumpinfusionareaandeffectonODplasticity.

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Fig.S4.EffectofminipumpinfusionsofsPirBorBSAondendriticspinesbycellsandbyspinetype.

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58. Acknowledgments:WethankN.SoteloKury,P.Kemper,andC.Chechelskiforlogisticsandmousebreeding,andG.Vidalformicroscopyadviceandtraining.WethankB.Q.ZhuangandL.HsiehWilsonattheCaliforniaInstituteofTechnologyforthesPirBplasmid,andJ.Schafferforillustrating(Fig.2A.WealsothankL.Luo,E.Knudsen,andT.Clandininforhelpfulfeedback.Funding:ThisprojectwassupportedbyNIHgrantsEY02858andMH07166,theMathersCharitableFoundation,andtheRosenbergFamilyFoundationtoC.J.S.NIHgrantEY018861toY.D.NationalScienceFoundationGraduateResearchFellowshipstoD.N.B.andJ.D.A.andNationalDefenseScienceandEngineeringFellowshiptoJ.D.A.R.W.S.receivedaBioXSummerUndergraduateResearchFellowship.Authorcontributions:D.N.B.andC.J.S.proposedandoutlinedtheexperimentalplan.D.N.B.performedallexperimentsinvolvingtamoxifeninduceddeletionD.N.B.andR.W.S.performedminipumpimplantationsurgeriesandsubsequentanalysisJ.D.A.,R.W.S.,andM.D.performedandanalyzedthestudiesofCamKIIaCrePirBflox/floxvisualcortex.S.Z.performedtheVEPrecordingsandanalysis,andY.D.supervisedthatcollaboration.H.L.performedwholecellrecordingsofmEPSCsinL5neurons.M.D.characterizedanewbatchofthePirBantibodyusedhereandmadesubstantialintellectualcontributionstotheproject.J.S.createdthefloxedPirBandPirB/mouseintheShatzlaboratory.D.N.B.andC.J.S.wrotethemanuscriptandreviewedalldatacollectionandanalysis.Competinginterests:C.J.S.andJ.S.areinventorsonU.S.Patentapplication12/087799assignedtothePresidentandFellowsofHarvardCollegeonCompositionsandmethodsforenhancingneuronalplasticityandregeneration.Theotherauthorsdeclarethattheyhavenocompetinginterests.

Receivedforpublication24July2014.Acceptedforpublication17September2014.Copyright2014,AmericanAssociationfortheAdvancementofScience

Citation:D.N.Bochner,R.W.Sapp,J.D.Adelson,S.Zhang,H.Lee,M.Djurisic,J.Syken,Y.Dan,C.J.Shatz,BlockingPirBupregulatesspinesandfunctionalsynapsestounlockvisualcorticalplasticityandfacilitaterecoveryfromamblyopia.Sci.Transl.Med.6,258ra140(2014).

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