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BlockingPirBupregulatesspinesandfunctionalsynapsestounlockvisualcorticalplasticityandfacilitaterecoveryfromamblyopia

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Home>ScienceJournals>ScienceTranslationalMedicineHome>15October2014>Bochneretal.,6:(258):258ra140

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stm.sciencemag.org.ezproxy.lib.usf.edu
SciTranslMed15October2014:
Vol.6,Issue258,p.258ra140
Sci.Transl.Med.DOI:10.1126/scitranslmed.3010157
RESEARCHARTICLE

NEUROSCIENCE

BlockingPirBupregulatesspinesandfunctional
synapsestounlockvisualcorticalplasticityandfacilitate
recoveryfromamblyopia
DavidN.Bochner1, *,RichardW.Sapp1, *,JaimieD.Adelson1,

INTRODUCTION
RESULTS
GeneticdeletionofPirB
enhancesODplasticity
PostnataldeletionofPirB
fromexcitatoryneurons
enhancesadultODplasticity

SiyuZhang2,HanmiLee1,MajaDjurisic1,JoshSyken3,YangDan2

BlockadeofPirBligand

andCarlaJ.Shatz1,

bindingrapidlyenhancesOD
plasticity

AuthorAffiliations

AuthorNotes

Correspondingauthor.Email:cshatz@stanford.edu
Duringcriticalperiodsofdevelopment,thebraineasilychanges
inresponsetoenvironmentalstimuli,butthisneuralplasticity
declinesbyadulthood.Byacutelydisruptingpaired
immunoglobulinlikereceptorB(PirB)functionatspecificages,
weshowthatPirBactivelyrepressesneuralplasticity
throughoutlife.WedisruptedPirBfunctioneitherbygenetically
introducingaconditionalPirBalleleintomiceorbyminipump
infusionofasolublePirBectodomain(sPirB)intomousevisual
cortex.Wefoundthatneuralplasticity,asmeasuredby
deprivingmiceofvisioninoneeyeandtestingocular
dominance,wasenhancedbythistreatmentbothduringthe
criticalperiodandwhenPirBfunctionwasdisruptedin
adulthood.AcuteblockadeofPirBtriggeredtheformationof
newfunctionalsynapses,asindicatedbyincreasesinminiature
excitatorypostsynapticcurrent(mEPSC)frequencyandspine
densityondendritesoflayer5pyramidalneurons.Inaddition,
recoveryfromamblyopiathedeclineinvisualacuityandspine
densityresultingfromlongtermmonoculardeprivationwas
possibleaftera1weekinfusionofsPirBafterthedeprivation
period.Thus,neuralplasticityinadultvisualcortexisactively
repressedandcanbeenhancedbyblockingPirBfunction.
INTRODUCTION

sPirBincreasesspinedensity
andfunctionalsynapseson
L5pyramidalneurons
sPirBtreatmentafterLTMD
enablesrecoveryofspine
density
sPirBinducesrecoveryof
visualacuityafterLTMD
DISCUSSION
sPirBasanacuteregulatorof
spineandsynapsedensity
sPirBasapotentialtherapy
forrecoveryfromamblyopia
PirB:Anendogenoustarget
formanipulationsofsynapse
andsystemslevelplasticity
MATERIALSANDMETHODS
Studydesign
Mousestrains
sPirBproteinproduction
Osmoticminipump
implantationsandsPirB
infusion
ArcmRNAinductionandin
situhybridization
VEPrecordings

BacktoTop

Statisticalanalyses

SUPPLEMENTARY
Duringpostnataldevelopment,thecapacityofthebraintoundergo
MATERIALS
experiencedependentchangesinsynapticstrengthandcircuit
REFERENCESANDNOTES
connectivityisdynamicallyregulated,withplasticitypeakingduring
developmentalcriticalperiodsandthendecreasingwithmaturation(13).
Criticalperiodsarekeytimeswhensensoryexperienceisnecessaryfornormalcircuitdevelopmentandwhen
abnormalexperiencecangenerateenduringanomaliesinbrainstructureandfunction(2,4).Oculardominance
(OD)plasticityisagraphicexampleofexperiencedrivensynapticandcircuitplasticity.Childrenbornwith
congenitalcataractinoneeyewillsufferamblyopiaalossofvisualacuityifnotcorrectedearlyinlife(5,6).
Monocularvisualdeprivation(MD)hasbeenusedinanimalmodelsofamblyopiatounderstandunderlying

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BlockingPirBupregulatesspinesandfunctionalsynapsestounlockvisualcorticalplasticityandfacilitaterecoveryfromamblyopia
mechanisms.AfterabriefperiodofMDorenucleation(ME)duringjuvenilelife,visuallydrivenresponsesof
neuronsinthebinocularzoneofmammalianprimaryvisualcortex(V1)shifttowardtheopeneye,andcortical
territorycontainingneuronsrespondingtoopeneyestimulationexpands,whereasclosedeyeresponsesweaken
andterritoryshrinks(3,7,8).Theseeffectsaremaximalaroundpostnatalday28(P28)inmiceanddecrease
thereafterbyadulthood,littleODplasticityresultingfromeyeclosurecanbedetected,particularlywithshorter
periodsofdeprivation(711).Furthermore,alongtermperiodofMD(LTMD)spanningtheentirecriticalperiod
(forexample,P19toP47)generatesanenduringlossofacuityandcorticalfunctioninthedeprivedeyeevenif
binocularvisionisrestoredinadulthood(4,12,13).Thisnormaldecreaseinplasticitybyadulthood,although
importantforstabilizingneuralcircuits,actsasabarriertorecoveryafterinjurybecauseitlimitscortical
reorganization,canlockineffectsofdysfunctionaldevelopment,andevenopposesacquisitionofnewlearning.If
adultneuralcircuitscouldbereturnedtoanimmaturestate,criticalperiodsmightbeeffectivelyreopened,
facilitatingrecoveryafternervoussystemdamage,leadingtonewtreatmentsforneurodegenerativeor
developmentaldisorders,orevenenhancinglearninginhealthyindividuals.
Alimitednumberofcandidatemoleculesthatappeartoactasendogenousnegativeregulatorsofcortical
plasticityhavebeenidentified(1418).Onesuchmolecule,pairedimmunoglobulinlikereceptorB(PirB),is
expressedincorticalandhippocampalneuronsaswellasinsomeimmunecells(14).Inthenervoussystem,PirB
bindsseveralligands,includingmajorhistocompatibilitycomplex(MHC)classIproteins,NogoA,andmyelin
components(19,20).Bothinimmunecellsandneurons,ligandbindingrecruitsSHP1andSHP2phosphatases
(14,21).SHPrecruitmentrequiresPirBphosphorylationonitsITIM(immunoreceptortyrosinebasedinhibitory
motif)domains(21,22).Inneurons,cofilinisalsorecruitedtoPirB,leadingtochangesintheactincytoskeleton
(20).
GermlinePirB /micehaveenhancedODplasticitynotonlyduringthecriticalperiodbutalsobeyond,andthey
recovermorerapidlyinastrokemodel(14,23,24).PirBanditshumanortholog,leukocyteimmunoglobulinlike
receptor,subfamilyB,member2(LilrB2),bindsolubleamyloidoligomers,andgermlinePirBdeletionrescues
ODplasticityandhippocampaldeficitsinamousemodelofAlzheimersdisease(AD)(20).However,itremains
unknownwhethertheenhancedODplasticityandstrokerecoveryingermlinePirB /miceareduetoearly
developmentalchanges,orwhetherPirBactsatallagestolimitplasticity,whichwouldmakeitanattractive
therapeutictargetfordrugdevelopment.BecausePirBisareceptor,signalingcanbemodulatedbyconditional
geneticknockoutorbyinterferingwithligandbinding(19).IfPirBfunctionsthroughoutlife,disruptingPirBshould
enhanceplasticityorfacilitaterecoveryatanyage.
RESULTS

BacktoTop

GeneticdeletionofPirBenhancesODplasticity
TodisruptPirBfunctionwithtemporalcontrol,aconditionalalleleofPirBwasgeneratedbyinsertingloxPsites
surroundingexons10to13,whichcontainthetransmembranedomainandfirstITIMdomainofPirB(14)(Fig.
1A).Toobtainrobustwidespreaddeletion,thisPirBfloxmouselinewascrossedwithatransgenicmouseline
expressingtamoxifeninducibleCreERT2onaubiquitinCpromoter(25).TheresultingUbcCreERT2PirBflox/flox
micewerebredwithPirBflox/floxmice,producingexperimentalUbcCreERT2PirBflox/floxanimals(henceforth
calledCre+)aswellasPirBflox/flox(Cre)littermatecontrols.Tamoxifeninjectionsgiveneitherneonatallyorafter
criticalperiodclosureinducedrobustdeletionofthefloxedallelefromgenomicDNAwithin1week(Fig.1B).PirB
proteinlosswasmoregradualforexample,dailytamoxifentreatmentfromP3toP7diminishedPirBproteinin
theforebrainby~90%byP27(Fig.1,CandD).AsimilargraduallossofproteinwasseenatP70aftertamoxifen
treatmentfromP45toP49(Fig.1,CandE).Thus,tamoxifenadministrationsubstantiallyreducedPirBprotein
levelsbythepeakoftheODcriticalperiodatP28(7),aswellasinadulthoodbyP70.
Fig.1.ATamoxifeninducibleCredependentstrategy
fordeletionofPirBwithtemporalcontrol.

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(A)SchematicofPirBproteinstructure(top)andfloxed
PirBallele(bottom)beforeandafterCremediated
excision.(B)Dailytamoxifengivenviainjectionofnursing
mother(P3toP7)inducesdeletionofthefloxedregionat
P21asdetectedbypolymerasechainreaction(PCR).(C)
WesternblotsforPirBproteininforebrainatages(left)of
tamoxifen(TAM)administrationandWesternblotting.(D)
QuantificationofPirBproteininforebrainaftertamoxifen
administration(P3toP7),normalizedtoaverageCre
levelsacrossallagesassayed:CreP21:n=4mice
versusCre+P21:n=5,P=0.02,Utest.CreP27:n=5
versusCre+P27:n=4,P=0.02,Utest.(E)
QuantificationofPirBproteininforebrainatP70(adult)
aftertamoxifeninjectionfromP45toP49.CreP70:n=
4versusCre+P70:n=4,P=0.03,Utest.*P<0.05.

ToassesswhetherODplasticityduringthecriticalperiodisincreasedbyacutepostnatalremovalofPirB,mice
receivedMEfromP28toP32.AtP32,weusedthemethodofArcmRNAinductiontoassesshowmuchthe
functionalrepresentationofthesparedeyehadexpandedwithinvisualcortex(8).Arcisanimmediateearlygene

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BlockingPirBupregulatesspinesandfunctionalsynapsestounlockvisualcorticalplasticityandfacilitaterecoveryfromamblyopia
inducedwithinminutesofvisualstimulation,andtheupregulatedmRNAcanbedetectedincorticalneurons
functionallydrivenbythestimulatedeye(26).WemeasuredthehorizontalextentoftheArcmRNAinsitu
hybridizationsignalalongL4ofvisualcortexipsilateraltothespared,stimulatedeye(Fig.2,AtoC,andfig.S1).
ThisexpansioninwidthofArcmRNAsignalisareliablemeasureofopeneyestrengtheningaftervisual
deprivationandcorrelateswellwithothermethodsusedtoassessODplasticityincludingsingleunit
electrophysiology(7,8,27,28),visualevokedpotentials(VEPs)(8,11,29),orintrinsicsignalimaging(8,24,
3033).ThewidthofArcmRNAinductiondoesnotexpandintransgenicmiceknowntolackODplasticityas
measuredbyothermethods(20,28,33),whereasthereisanincreaseinwidthofArcmRNAsignalinmiceknown
tohaveincreasedODplasticity(14,15,24).
Fig.2.TimedgeneticdeletionofPirBenhancesOD
plasticity.
(A)Schematicofmousevisualsystem.Eachretina(right:
red,left:blue)projectsprimarilycontralaterallytothe
lateralgeniculatenucleus(LGN),whichprojectstovisual
cortex(V1).Asmallbinocularzone(BZ,purple)inV1
receivesinputfrombotheyesinresponsetodeprivation
ofoneeye(forexample,left),therepresentationofthe
open(rightipsilateral)eyeexpands(arrows).(B)Timeline
ofinducibleknockoutofPirBandassessmentofOD
plasticityviaArcmRNAinduction.(C)Example
micrographsofinsituhybridizationsforArcmRNA
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inducedinBZofvisualcortexafteropeneyestimulation.
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Eachblackdotisacell.MEfromP28toP32resultsin
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expansionoftheipsilateral(open)eyerepresentation
(betweenredasterisks),ascomparedwithnormalrearing
(NR).Cre=PirBflox/flox.Cre+=UbCCreERT2PirB
flox/flox.WidthofArcsignalinL4wasmeasured(seefig.S1).Corticallayersindicatedatleftscale
bar,500m.CP,criticalperiod.(D)CumulativehistogramsofwidthofArcmRNAsignalbyindividual
section.NRCre:n=41sectionsNRCre+:n=44MECre:n=39MECre+:n=52.(E)Graphof
datain(D),withmeanandSEMbyanimal:deletionofPirBduringthecriticalperiodenhancesOD
plasticity.NRCre:n=7miceversusNRCre+:n=7,P=0.65MECre:n=7versusMECre+:n=
7,****indicatesP<0.0001,twowayANOVAwithTukeyposthoctest.(F)Timelineofinducible
deletionofPirBandassessmentofODplasticityinadults.(G)ExampleArcmRNAinsitu
hybridizationmicrographsatP74,asin(C).(H)CumulativehistogramsofwidthofArcmRNA
inductionfromindividualsections.NRCre:n=23sectionsNRCre+:n=20MECre:n=41ME
Cre+:n=51.(I)Graphofdatashownin(G)(meanandSEMbyanimal):deletionofPirBinadulthood
enhancesODplasticity.NRCre:n=5miceversusNRCre+:n=5,P=0.99MECre:n=7versus
MECre+:n=8,P=0.013MECreversusNRCre:P=0.18MECre+versusNRCre+:P=
0.0004,bytwowayANOVAwithTukeyposttest.*P<0.05,***P<0.001.
Asexpectedduringthecriticalperiod,4daysofMEineitherCre+orCremicecausedsubstantialexpansionin
widthofArcmRNAsignalascomparedtonormallyrearedcontrols(Fig.2,BtoE).However,inmicelackingPirB
(Cre+),thesparedeyerepresentationexpanded21%morethancontrol(Cre)littermates,whereasinnormally
rearedmice,therewasnodifferencebetweengenotypesinthewidthofArcmRNAsignalinducedbystimulation
oftheipsilateraleye(Fig.2E).Atwowayanalysisofvariance(ANOVA)confirmedasignificantinteractioneffect
betweenvisualmanipulationandgenotype(P<0.0001).Tofurtherfacilitatecomparisonsbetweengenotypes,a
plasticityindexwascalculatedbynormalizingthewidthofArcmRNAinductionafterMEtothenormallyreared
valueforeachgenotype.TheplasticityindexinCre+miceis23%higherthanthatinCremice(fig.S2A),
consistentwiththeabsenceofPirB.TheseobservationsimplythatPirBdoesnotactonlyearlyinfetallife,but
ratheractivelyrepressesODplasticityduringthecriticalperiod.
Indevelopingwildtypeanimals,ODplasticitydecreasesbetweenP35andP40(theendofthecriticalperiod),and
shortperiodsofMDorME(3to4days)thereafterhavelittleeffectonOD(711).Todeterminewhether
decreasingPirBfunctionmightenhanceplasticityinadulthood,tamoxifenwasadministeredfromP45toP49(Fig.
2F),whichresultedinanalmostcompletelossofPirBproteinbyP70(Fig.1,CandE).Then,theseadultmice
receivedMEfromP70toP74,weeksafterthecriticalperiodhasnormallyclosed.Aswithjuvenilemice,atP74,
therewasnosignificantdifferenceinbaselinewidthofArcmRNAsignalbetweengenotypesinnormallyreared
controls(Fig.2,GtoI).TherewasalsonosignificantdifferenceinwidthofArcmRNAsignalafterMEfromP70
toP74inCrecontrols,asexpected,giventhebrief4dayperiodofdeprivationatthisolderage.However,we
observedasignificantexpansionofopeneyerepresentation(P=0.0004)inCre+mice(whichlackedPirB)with4
daysofME(Fig.2,HandI,andfig.S2B).Inadults,asinjuveniles,atwowayANOVAconfirmedthattherewas
asignificantinteractionbetweenvisualmanipulationandgenotype(P=0.04).Althoughthemagnitudeofadult
ODplasticitywasreducedinallgenotypesascomparedtothatobservedduringthecriticalperiod,deletionofPirB
inadultswassufficienttoenhanceODplasticityandtocauseasignificantexpansioninopeneyerepresentation
notobservedinadultwildtypelittermatecontrolswithnormalPirBlevels.
PostnataldeletionofPirBfromexcitatoryneuronsenhancesadultODplasticity
Intheexperimentsabove,aubiquitouslyexpressedCrerecombinasewasusedtoachieverobustdeletionofPirB

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BlockingPirBupregulatesspinesandfunctionalsynapsestounlockvisualcorticalplasticityandfacilitaterecoveryfromamblyopia
inallcells.Next,weinvestigatedwhetherlossofPirBspecificallyinforebrainexcitatoryneuronswassufficientto
enhanceODplasticity.PirBflox/floxmicewerecrossedwithaCamKIIaCreline,whichexpressesCreexclusively
inforebrainexcitatoryneurons(34,35),generatingCamKIIaCrePirBflox/floxconditionalknockouts,and
CamKIIaCrePirB +/+littermatecontrols.PCRgenotypingofbrainandearconfirmsbrainspecificdeletionofthe
floxedregionofPirB(Fig.3A).ToconfirmthespatialpatternofCredeletion,CamKIIaCremicewerealso
crossedtotheAi14TdTomatoreporterline(36).ResultsshowfaithfulCreactivityatP30inpyramidalneuronsof
hippocampusandcortex(Fig.3B).PreviousstudieshaveshownthatexcisionoffloxedregionsofDNAinthisCre
lineisgradual,withcompletedeletionoccurringaround3monthsofage(35),permittingustoexamineeffectsof
PirBdeletioninadulthood.
Fig.3.CremediateddeletionofPirBfromforebrain
excitatoryneuronsenhancesadultODplasticity.
(A)Genotypingofsamplesfromearandcerebralcortex
fromP100CamKIIaCrePirBflox/flox(cKO)orCamKIIa
CrePirBWT(wildtype),showingdeletionoffloxedPirB
incortexbutnotear.(B)CamKIIaCrePirBflox/+
breederswerecrossedwiththeAi14TdTomatoreporter
line,generatingredfluorescenceinthepresenceofCre.
Sagittalsectionthroughvisualcortex(layersindicatedat
right)andhippocampusofaP30mouseshowsCre
presentinpyramidalneurons.(C)GraphsofwidthofL4
regionactivatedbystimulationofipsilateral(open)eyein
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visualcortex,assessedusingArcmRNAinduction.
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DeletionofPirBfromforebrainexcitatoryneurons
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increasesopeneyeexpansioninadultmiceafterMEfrom
P100toP110.NRWT:n=5miceversusNRcKO:n=4,
P=0.91.MEWT:n=8miceversusMEcKO:n=5,P=
0.006.NRversusMEWT:P=0.39,NRversusMEcKO:P=0.0002,bytwowayANOVAwithTukey
posthoctest.**P<0.01,***P<0.001.
CamKIIaCrePirBflox/floxconditionalknockouts,andCamKIIaCrePirB +/+littermatecontrolsreceived10days
ofMEfromP100toP110,followedbyassessmentofODplasticity.Openeyeexpansioninvisualcortexin
CamKIIaconditionalknockoutmicewas~13%greaterthaninlittermatecontrols(Fig.3Candfig.S2C),
suggestingthatpostnataldeletionofPirBfromexcitatoryneuronsissufficienttoincreaseODplasticitybyP110.
AtwowayANOVAconfirmedsignificantinteractionbetweenvisualmanipulationandgenotype(P=0.007).
Furthermore,thisincreaseissimilarinmagnitudetopreviousobservationsofenhancedODplasticityinadultmice
withPirBgermlinedeletion(14),implyingthatlossofPirBfunctioninexcitatoryneuronsmaybelargely
responsiblefortheobservedPirB /phenotype.
BlockadeofPirBligandbindingrapidlyenhancesODplasticity
ThegeneticapproachesusedaboveexcisePirBfromthegenomepostnatally,buttheensuinglossofPirBprotein
isgradualandwidespread.TheseexperimentsestablishthatPirBactivelyfunctionsthroughouttheanimalslives.
Next,amolecularpharmacologicalapproachwasusedasaproofofconceptforatherapeuticreagentthatblocks
PirBfunctionandtoachieverapidandlocaldisruptionofPirBfunctionwithinvisualcortex.Wegenerateda
solublePirBectodomain(sPirB)protein,containingthefirstsiximmunoglobulinG(Ig)likedomainsandHisand
Myctagsforpurificationanddetection(Fig.4A).Solubleectodomainsofreceptorsactasdecoystosequester
endogenousligands,thusreducingligandbindingandsubsequentreceptorsignaling.Theyhavebeenused
frequentlyinexperimentalcontexts,andseveralarecurrentlyonthemarketastherapeutics(3739).Previous
studieshavedemonstratedthatsolubleversionsofthefullPirBectodomainwithsimilarsequencetotheoneused
herecanbindknownPirBligandsincludingMHCclassI(14,21,40)myelincomponentsNogoA,OMgp,and
MAG(19)andamyloidoligomers(20).However,thePirBectodomainhasneverbeenusedtherapeutically.
sPirBwouldbeexpectedtoblockPirBbindingtoendogenousligandsandthereforereducedownstreamsignaling.
Fig.4.BlockadeofPirBbindingenhancesOD
plasticityinWTvisualcortex.

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(A)SchematicofsolublePirBMycHis(sPirB)fusion
protein,indicatingextracellularIglikedomainsplusMyc
andHistags.(B)Westernblotofculturesupernatantfrom
sPirBtransfectedHEK293cells,detectingMyctagand
PirBectodomainMycHistaggedalkalinephosphatase
(APMycHis)isapositivecontrol.(C)PirB
phosphorylationisdecreasedafter7days(P21toP28)of
sPirBinfusionintoWTmousecortex,asshownby
phosphotyrosineIPandPirBWesternblotofcortical
lysatesfrominfused(sPirBinfused),uninfused(sPirB
uninf.)hemispheres,oruntreatedlittermatecontrols.(D)
SectionofvisualcorteximmunostainedwithantiMyc
antibodyafter11days(P21toP32)ofsPirBorBSA

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BlockingPirBupregulatesspinesandfunctionalsynapsestounlockvisualcorticalplasticityandfacilitaterecoveryfromamblyopia
infusion(1mg/ml).Scalebar,1mm.(EandF)Minipump
infusionsofsPirBduringcriticalperiod(CP).Timelineasshown.(E)ExampleArcmRNAinsitu
hybridizationmicrographsofvisualcortexafterBSA(top)orsPirB(bottom)treatment.Scalebar,500
m.RedasterisksindicatebordersofArcmRNAsignalinducedbystimulatingtheipsilateral(open)
eyeinlayer4.(F)GraphscomparingwidthofArcmRNAsignalinL4afteropeneyestimulation.
WidthofterritoryactivatedbyopeneyestimulationafterMEisgreateraftersPirBinfusionthanwith
BSA.NRBSA:n=4mice,NRsPirB:n=4,MEBSA:n=5versusMEsPirB:n=6,P<0.0001,by
twowayANOVAandTukeyposthoctestforallcomparisonsindicated.(GtoI)sPirBinfusionsinto
adultWTvisualcortextimelineasshown.(G)ExampleofArcmRNAsituhybridizationmicrographs
atP74.Scalebar,500m.(H)GraphscomparingwidthofArcmRNAsignalinL4afterstimulationof
theipsilateral(open)eye.sPirBinfusionfromP63toP74enhancesopeneyeexpansionafterME.NR
BSA:n=4miceversusNRsPirB:n=4,P=0.99.MEBSA:n=4versusMEsPirB:n=5,P=
0.0004.NRversusMEBSA:P=0.88,NRversusMEsPirB:P<0.0001.(I)sPirBinfusioncoupled
with3daysofMDalsoenhancesODplasticity.(J)sPirBinfusionhasnoeffectonODplasticitywhen
infusedintovisualcortexofPirB /mice.MEPirB /BSA:n=5miceversusMEPirB /sPirB:n=
5,P=0.95,MEPirB /BSAversusMEWTBSA:P=0.034.MDBSAversusMDsPirB:n=4mice
pergroup,P=0.036.*P<0.05,***P<0.001,****P<0.0001,bytwowayANOVAandTukeypost
hoctest.

WetransfectedaplasmidcodingforsPirBintohumanembryonickidney(HEK)293cells,andtherecombinant
proteinwasthensecretedintoculturesupernatant.WedetectedsPirBbyWesternblottingagainstboththeMyc
tagandthePirBectodomain(Fig.4B).sPirBwasthenproducedatlargescaleandpurifiedviaNiHiscolumn.
Next,eithersPirBorbovineserumalbumin(BSA)wasinfusedintovisualcortex(V1)ofwildtypemiceduringthe
criticalperiodviaosmoticminipumps.ToassessefficacyofsPirBinfusions,weimplantedminipumpsatP21.At
P28,corticaltissuewasharvestedposteriortotheimplantationsiteintheinfusedandcontralateralhemispheres,
aswellasinuninfusedlittermates.PirBphosphorylation(14)wasdecreasedinvisualcortexposteriortothe
infusionsitecomparedtoboththecontralateralhemisphereanduntreatedlittermatecontrols(Fig.4C).Eleven
daysafterimplantation,extensivediffusionofsPirBcanbedetectedbyantiMycimmunostainingofsections(Fig.
4D)acrossvisualcortexasfaras2mmposteriortotheinfusionsite(fig.S3B)noantiMycstainingwasdetected
inBSAinfusedcontrolbrains(Fig.4Dandfig.S3C).
MinipumpinfusionofsPirBintovisualcortexofwildtypemicefor11daysduringthecriticalperiod(P21toP32),
combinedwithMEfromP28toP32,resultedinapronouncedexpansioninwidthofvisualcortexcontaining
neuronsfunctionallydrivenbytheopen(ipsilateral)eye,asassessedwithArcmRNAinduction(Fig.4E).Bythis
measure,ODplasticitywas24%greaterwithsPirBinfusionthanincontrolsinfusedwithanequivalent
concentrationofBSA(Fig.4Fandfig.S2D)atwowayANOVAconfirmedsignificantinteractionbetweenvisual
manipulationandtreatment(P<0.0001).Becausetheinfusionwaslocalandlimitedtoan11dayperiod
beginningattheonsetofthecriticalperiod(7,8),theresultsofthisexperimenthelptonarrowconsiderablythe
spatiotemporalwindowinwhichPirBactstosuppressplasticity.TogetherwiththetamoxifeninduciblePirB
knockoutresultspresentedabove,ourdatasuggestthatPirBactivelysuppressesplasticitylocallyinvisualcortex
duringthecriticalperiodforODplasticity.
SubstantialODplasticitycouldberestoredtothevisualcortexofadultwildtypemiceafterminipumpinfusionsof
sPirBfromP63toP74.MEfromP70toP74resultedinsignificantexpansionofthefunctionalrepresentationof
theopeneyeinthepresenceofsPirB,butnotBSA(Fig.4,GandH,andfig.S2E).ThiseffectofsPirBwas
twiceaslargeasthatoftamoxifendrivenPirBexcisioninadultmice(compareFig.2I).Incontrast,therewasno
significanteffectof11dayminipumpinfusionsofeitherBSAorsPirBonthewidthofArcmRNAinductionafter
stimulationoftheipsilateraleyeinmicerearedwithnormalvision(Fig.4H,NRsPirB).Consistentwiththeidea
thatsPirBaffectstheexpansionratherthanthebaselinewidthofArcinduction,atwowayANOVAdetectsa
significantinteractioneffectbetweenvisualdeprivationandtreatment(P=0.001).
WefurtherassessedtheeffectofsPirBonadultODplasticityusingMD(asopposedtoME).Inadultmice,abrief
periodofMDsuchas3daysdoesnotusuallyproduceashiftinODfavoringtheopeneye(conferFig.4,HandI)
(7,10,11,41).Therefore,weexaminedwhetherMDfromP70toP73,coupledwithsPirBtreatment,wouldresult
inopeneyeexpansion.SimilartoresultswithME,sPirBtreatedadultmicereceivingMDhadasignificant
increaseinopeneyeexpansion,withnochangeinBSAtreatedcontrols(Fig.4Iandfig.S2F).Therefore,inadult
visualcortex,sPirBtreatmentduringbriefperiodsofeitherMDorMEgeneratesdetectableODplasticity.
PirBbindsmultipleligands(19,40),whichthemselvescanbindtootherreceptors(19,40),possiblyaccountingfor
theobservationthatsPirBhasalargereffectonODplasticitythangeneticallyinduceddeletionofPirB.Totest
thisidea,weinfusedsPirBorBSAbyminipumpsintoadultgermlinePirB /mice,whichalsoreceived4dayME
(P70toP74).GermlinePirB /miceimplantedwithBSAminipumpsdemonstratedtheexpectedincreaseinOD
plasticity(14,24)whencomparedtowildtypemicereceivingBSA(Fig.4,HandJ).However,therewasno
additionaleffectofsPirBinfusioncomparedtoBSAinfusioninPirB /mice(Fig.4J).ThislackofeffectofsPirB
infusioninadultPirB /visualcortexsuggeststhatendogenousPirBisrequiredtoenhanceODplasticityandthat
thepharmacologicalblockadeisPirBspecific.TogetherwithresultsfromthetamoxifeninduciblePirBmice,these
findingsindicatethatacute,specificmanipulationsthatdeleteorblockPirBfunctionaresufficienttoenhanceOD
plasticityevenwhenadministeredwellbeyondtheendofthecriticalperiod.
sPirBincreasesspinedensityandfunctionalsynapsesonL5pyramidalneurons
ThereisasignificantincreaseinspinedensityontheapicaltuftsofL5pyramidalneuronsinthevisualcortexof

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germlinePirBknockoutmicerearedwithnormalvision.Ithasbeenproposedthattheseextraspinesrepresenta
preexistingstructuralsubstratethatisthenrecruitedforthemorerapidandrobustODplasticityobservedinthese
mice(24).Indeed,previoussensoryexperienceinvisualorauditorysystemsincreasesplasticity,andis
accompaniedbyincreasedstructuralconnectivity(4144).WetestedwhetherPirBmightcontributetothese
structuralchanges.
sPirBinfusionmighttriggeranincreaseinspinedensityevenwithoutvisualdeprivation.Totestthishypothesis,
visualcortexofnormallyrearedwildtypeThy1YFPHtransgenicmice(45),inwhichcorticalL5pyramidal
neuronsareyellowfluorescentprotein(YFP)labeled,receivedminipumpinfusionsofeithersPirBorBSAfrom
P63toP74(Fig.5,AandB).SpinesonapicaldendritesofL5pyramidalneuronswereexaminedinthebinocular
zoneatadistanceposteriortotheinfusionsitecomparabletothatstudiedaboveforassessmentofODplasticity.
InthisregionaftersPirBinfusion,pyramidalneuronsomata,dendrites,andspinesappearedintactandhealthy,
withoutfragmentationorblebbing(Fig.5A).SpinedensityonL5apicaldendritictuftsofanimalsrearedwith
normalvisionwas38%greaterinthepresenceofsPirBthanofBSA(Fig.5B).SpinedensityonL5neuronsinthe
uninfusedhemispherewasnotaltered,andatwowayANOVAconfirmsasignificantinteractioneffectbetween
hemisphereandtreatment(P=0.03).TheobserveddensityincreasecouldariseifsPirBactsonasubclassof
dendriticspines.However,afteran11dayinfusionofeithersPirBorBSA,therewasnosignificantdifferencein
theproportionofspinesclassifiedasmushroom,thin,orstubby(46)(fig.S4B).Together,resultsshowthatin
adultvisualcortex,itispossibletogeneratealocalincreaseinspinedensityonL5neuronsbyinfusingsPirB,
evenintheabsenceofavisualmanipulationordeprivation.
Fig.5.sPirBincreasesspinedensityandfunctional
synapsesonL5pyramidalneuronsofnormally
rearedmice.
(A)Timelineofminipumpinfusions[BSA(1mg/ml)or
sPirBfromP63toP74]andexampledendritesofYFP
labeledL5pyramidalneuronsinbinocularzoneofvisual
cortexinWTThy1YFPHanimalsrearedwithnormal
visualexperience.Scalebar,10m.(B)Histogramsof
spinedensityonapicaltuftsofL5neuronsinsPirB
infusedversusintheuninfused(unif.)contralateral
hemisphere,orinBSAcontrols:BSAinfused:n=5mice
View larger version:
versussPirBinfused:n=5,P=0.01,onetotwocellsper
In this page In a new window
animal.BSAuninf.:n=5versussPirBuninf.:n=5,P=
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0.96,BSAinf.versusuninf.:P=0.99,sPirBinf.versus
uninf.:P=0.016,bytwowayANOVAandTukeyposthoc
test.(C)ExampletracesofmEPSCresponsesrecordedfromvisualcorticalslices(P70toP77)from
L5pyramidalneuronsafterBSAorsPirBinfusion,asin(A).(D)IncreasedmEPSCfrequencywith
sPirBinfusion:BSA:n=12neuronsversussPirBn=13,P=0.046,byMannWhitneyUtest.(E)No
changeinmEPSCamplitude:BSA:n=12neuronsversussPirBn=13,P=0.70,byMannWhitney
Utest.

Toexaminewhethertheincreaseinspinedensityrepresentsnewfunctionalsynapses,miniatureexcitatory
postsynapticcurrents(mEPSCs)wererecordedfromL5pyramidalneuronsinslicesofvisualcortex(P70toP77),
after7to11daysofsPirBminipumpinfusioninvivo,inmicerearedwithnormalbinocularvision(Fig.5C).
mEPSCfrequencywassignificantlygreateraftersPirBtreatmentthaninBSAlittermates(Fig.5D),withno
changeinmEPSCamplitude(Fig.5E).ThisfindingisconsistentwiththeideathatsPirBinfusioncausesan
increaseinsynapticconnectivity,suggestingthatnewlyformedspinesrepresentsitesoffunctionalsynapses.
sPirBtreatmentafterLTMDenablesrecoveryofspinedensity
LTMDisawellstudiedanimalmodelofamblyopiabecauseitinvolvesanexperiencedependentdevelopmental
lossoffunctioninthedeprivedeye(47,48).Inrodents,LTMDprofoundlydecreasesvisualacuity,aswellasthe
numberofcorticalneuronsvisuallydrivenbythedeprivedeye.Thereislittle,ifany,recovery,evenafter
restorationofbinocularvision(4,12,13,17,49).Ithasbeenproposedthatadecreaseindendriticspinedensity
underliesthesefunctionaldeficits(4,49).Forexample,LTMDgeneratesasignificantdeclineinspinedensityon
basolateraldendritesofL5pyramidalneuronscontralateraltothedeprivedeye(49).
GiventherapidandgenerativeeffectofsPirBonspinedensityandmEPSCfrequencydescribedaboveinnormal
visualcortex,wewonderedwhethersPirBtreatmentmightgenerateaspinedensityincreasethatcouldfacilitate
recoveryfromLTMD.Thy1YFPwildtypemicewereeithernormallyrearedorreceivedLTMDspanningtheentire
criticalperiodforODplasticity(P19toP47).AtP47,thedeprivedeyewasreopenedtorestorebinocularvisionfor
1week.ThenatP54,minipumpscontainingeithersPirBorBSAwereimplantedinthevisualcortexcontralateral
tothedeprivedeyeuntilP61(Fig.6A),atwhichtime,spinedensityonL5basolateraldendriteswasmeasured.
Fig.6.sPirBallowsstructuralandfunctionalrecoveryfromamblyopiaafterLTMD.
(A)Experimentaltimeline:LTMDfromP19toP47,eyereopeningatP47,andminipumpinfusion
fromP54toP61.(B)RepresentativeYFPlabeledL5cellsomaandbasolateral(arrow)dendritesin
visualcortexofWTThy1YFPHmice.Scalebar,50m.(C)Bargraphsshowingchangesin
basolateraldendriticspinedensity:LTMDcausesa

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significantdeclineinspinedensity(BSALTMD)thatcan
befullyreversedwithsPirBinfusion(sPirBLTMD)(BSA
NR:n=5miceversusBSALTMD:n=4,P=0.02.sPirB
LTMD:n=5,sPirBversusBSALTMD,P=0.001,sPirB
NR:n=5animals,onetotwocellsperanimal,sPirB
versusBSANR:P=0.003).*P<0.05,**P<0.01,by
twowayANOVAandTukeyposthoctest.(D)Averaged
corticalVEPresponseamplitudes(microvolts)tostimuliat
arangeofspatialfrequencies(cyclesperdegree)after
LTMDinmicereceivingminipumpinfusionofeithersPirB
View larger version:
orBSA.Dottedline:Semilogarithmicregressionofvisual
In this page In a new window
responses.Inset:Populationaveragetracesat0.05cycle
Download PowerPoint Slide for Teaching
perdegree.(E)Bargraphsshowingspatialacuityafter
LTMDplusinfusionofeitherBSAorsPirB.Grayshaded
regionindicatesmeanacuitySEMofnormallyreared(NR)controls.Measurementsfromindividual
miceareplotted(circles).LossofacuitywithLTMDisreversedafterjust1weekofsPirBinfusion(NR,
n=6miceversusBSALTMD,n=5mice,P=0.004.sPirBLTMD,n=4miceversusBSALTMD,P
=0.016).*P<0.05,**P<0.01,byUtest.

LTMDcauseda28%decreaseinspinedensityalongL5basolateraldendritesinBSAtreatedcontrolsdespite2
weeksofsubsequentbinocularvision(Fig.6,BandC),asexpectedfrompreviousstudies(4,49).Incontrast,the
spinelossaccompanyingLTMDwasalmostentirelyreversedbyminipumpinfusionofsPirB.Therewasa57%
greaterbasolateraldendriticspinedensityinsPirBtreatedanimalsthaninLTMDBSAtreatedcontrols,essentially
restoringspinedensitytolevelsofBSAtreatedcontrolsrearedwithnormalvision(Fig.6C).Therewasno
detectablechangeinoveralldistributionofbasolateralspinetypes(fig.S4D),similartoobservationsaboveforL5
apicaldendrites.Inaddition,innormallyrearedlittermatesinfusedwithsPirB,spinedensityincreasedon
basolateraldendritescomparedtonormallyrearedBSAinfusedcontrols,demonstratingthatsPirBinfusioncan
increasespinedensitynotonlyonapicaltuftsofL5pyramidalneurons(compareFig.5B)butalsoontheir
basolateraldendrites(Fig.6C).Together,theseresultsdemonstratethatsPirBtreatmentjustfor1weekcan
reversespinelossresultingfromLTMDevenwheninfusedafteradelayfollowingeyereopening.
sPirBinducesrecoveryofvisualacuityafterLTMD
Toassesswhethertherecoveryinspinedensitycouldmediaterecoveryofvisualfunction,VEPrecordingswere
madefromcorticallayer4/5tomeasurespatialfrequencyacuity.Thismethodiswellestablished,ishighly
sensitive,andtightlycorrelateswithbehavioralandsingleunitmeasurementsofvisualrecoveryafterLTMD(4,
12,17,49,50).Moreover,becausetheeffectsofsPirBminipumpinfusionsarerestrictedtoalocalregionof
visualcortex(Fig.5Bandfig.S3B),VEPsarewellsuitedtoassessrecoveryofdeprivedeyefunctionspecifically
withintheinfusedregion.Visualacuityismeasuredbyvaryingthespatialfrequencyofvisualstimuliand
monitoringtheamplitudeofVEPresponses(Fig.6D)(4,12).
InnormallyrearedThy1YFPwildtypemice,visualacuitymeasurementsweresimilartopreviouslyreported
values,about0.550.1cyclesperdegree(Fig.6E,shadedregion)(17,29,50).After4weeksofLTMDfromP19
toP47,BSAtreatedcontrolanimalsexperiencedamarkedlossofvisualacuity,withresponsesabovenoiselevel
onlytoamaximumspatialfrequencyof0.05cycleperdegree(Fig.6,DandE).Thissevere(85to90%)reduction
inacuity(Fig.6E)persistedfor2weeksevenafterrestorationofbinocularvision,including1weekofBSA
minipumptreatment.Thelackofrecoveryisconsistentwithpreviousresults,suggestingthatbinocularvision
aloneisnotsufficientforrecoveryofvisualfunctionafterthecriticalperiod(4,12,13,17,49).
Incontrast,therewassignificantimprovementinvisualacuityinmicereceivinga1weekminipumpinfusionof
sPirB(Fig.6,DandE).Althoughthedegreeofrecoverywasvariableamonganimals,50%ofthesPirBtreated
micerecoveredvisualacuityto0.4cycleperdegreeorhigher,nearlythenormalrangeforunmanipulatedmice
(Fig.6E).EventheremainingsPirBtreatedmiceregainedacuityhigherthanthatmeasuredinthehighestLTMD
BSAtreatedcontrols.Theseresultssuggestthatafterjust1weekofsPirBtreatment,therewassignificant
functionalrecoveryofvisualacuityinthedeprivedeyeandthat,insomecases,visualfunctionrapidlyrecoveredto
nearlynormallevels.
DISCUSSION

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Thereareseveralmajorfindingsfromthisstudy.First,byacutelydisruptingthefunctionoftheendogenous
receptorPirB,wefindthatODplasticityinvisualcortexofadultmicecanbeenhancedlongaftercriticalperiod
closure.Twoindependentbutcomplementarymethodswereused:geneticdeletionofPirBwithtemporalcontrol
andblockadeofligandbindingwithasPirBdecoyprotein.BothmethodsgeneratedenhancedODplasticity,not
onlyduringthecriticalperiodbutalsoinadulthood,phenocopyinggermlinePirBknockouts(14,24).These
observationssuggestthatendogenousPirBactivelyactstorepresscorticalplasticitythroughoutlife,validating
PirBasatargetfortherapeuticinterventionsthatcouldimproverecoveryfrominjury,correctdysfunctional
developmentalplasticity,orperhapseventemporarilyenhancelearninginnormalindividuals.
Second,wereportthatinamodelofamblyopia,itispossibletoreversethelossofboththespinesandthevisual
acuityincortexafterLTMDbyrestoringbinocularvisioncoupledwithaninfusionofsPirB.Last,sPirBinfusioninto
thevisualcortexofnormallyrearedmiceproducedrapidincreasesinspinedensityandfunctionalsynapsesonL5
pyramidalneurons.Althoughmotorlearning(51,52),sensoryenrichment(52),orbriefMD(44)allhavebeen

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showntoproducerapidincreasesinspinedensity,sPirBtreatmentproducesalargermagnitudechangeanddoes
sointheabsenceofnovelstimuliortraining.Together,ourobservationsimplythattargetinganddisruptingPirB
functionincreasesynapticconnectivityandplasticity,evenafterthecriticalperiod.BecausePirBisexpressedby
pyramidalneuronsthroughouttheneocortex(14),ourresultsmayalsoapplytocorticalareasotherthanthevisual
system.
sPirBasanacuteregulatorofspineandsynapsedensity
InfusionofsPirBdecreasesPirBdownstreamsignaling(Fig.4C),consistentwithitspreviouslydemonstrated
sequestrationofendogenousPirBligands(14,19,20,23).Inadulthood,acuteblockadewithsPirBresultsin
enhancedODplasticityandproducesarapidincreaseinspinedensityandmEPSCfrequency,eveninthe
absenceofalteredvision.Manyinterventionsthataffectsynapticconnectivityandspinedynamicsalsoenhance
ODplasticity,includingtransplantationofinhibitoryneuronprogenitors(53)ordisruptionofNgR1NogoAfunction
(16,54,55).Spinedensityincreasesalsocorrelatewithenhancedsubsequentplasticity(41,44):newspines
generatedduringaninitialMDcanbecooptedformorerobustODplasticityduringasubsequentMD(24,41,
44).Furthermore,ingermlinePirB /mice,enhancedODplasticityisassociatedwithalargeincreaseinspine
densityonL5neuronsandanincreaseinthemagnitudeofL4toL2/3longtermpotentiation(LTP)invisualcortex
(24).Collectively,theseexperimentsconnectanincreaseinspinedensityandfunctionalconnectivitytoenhanced
synapticplasticity.Thus,sPirBmaygenerategreaterODplasticitybycreatingamorehighlyinterconnected
structuralsubstratethatcanbeaccessedformorerapidandrobustsynapticchange.
sPirBasapotentialtherapyforrecoveryfromamblyopia
LTMDthroughoutthecriticalperiod,usedhereasananimalmodelofamblyopia,leadstoaprofoundlossof
visualacuity,aswellastolossofvisualresponsivenessofcorticalneuronstostimulationofthedeprivedeyeboth
arehighlyresistanttorecoveryevenwhenbinocularvisionissubsequentlyrestored(4,12,13,17,49).Decreases
inspinedensityonbothL2/3pyramidalcells(4)andpyramidalneuronsthroughoutcortexhavealsobeenreported
afterLTMDorchronicMD(49).Althoughreversalofthisspinelossoncorticalpyramidalneuronshasbeenseen,
reversalrequiredthattheformerlyopeneyebesuturedclosedincombinationwithfairlydisruptivetreatmentssuch
aschondroitinaseABCtodigestextracellularmatrix(4),or10daysofdarkexposurefollowedbyvisualstimulation
(49,56).RecoveryofspinesinbothofthesecaseswasaccompaniedbyrobustrecoveryofVEPacuity.Inour
study,wefoundthatsPirBinfusion,combinedwithbinocularvision,wassufficientbyitselftobringspinedensity
valuesandVEPacuityestimatesclosetonormal.VisualacuityasmeasuredbyVEPspredictsphysiologically
relevantrecoveryofvisualfunctioninthedeprivedeye,indicatingthatvisionthroughthedeprivedeyeinsPirB
treatedmicehasgreatlyimproved(4,43,44).Togetherwiththedataonspinerecovery,theseresultssuggestthat
sPirBcanenablesignificantstructuralandfunctionalrecoveryfromamblyopiaafterLTMDwithinjust7daysof
treatment.
TheseobservationsimplythatsPirBasolublereceptorectodomainisapotentialtherapeuticagent,andthey
provideproofofconceptforgeneratingotherPirBblockingreagents.Thestandardtreatmentforhumanamblyopic
patientsmandatesearlyinterventionduringadevelopmentalcriticalperiodandinvolvesalternatingpatching
betweenthetwoeyestostrengthentheamblyopiceye,butthistreatmentinterfereswithdevelopmentofbinocular
depthperception(6).ThereareseveralPirBhomologsinhumans(LilrBs),andLilrB2proteinisexpressedinthe
humanbrain(25).TargetingLilrB2orothermembersoftheLilrBreceptorfamilymightpermitrecoveryfrom
amblyopiawithoutrequiringeyepatching,asimpliedbytheresultsoftheLTMDexperimentsinmice.
Thereareanumberoflimitationsandissuestoconsiderintranslatingourfindings.First,itwouldbeimportantto
determinewhethertheincreaseinspinedensityandfunctionalrecoveryfromamblyopiapersistsstablybeyondthe
periodofsPirBinfusion.Second,itispossiblethatalongerinfusionorhigherconcentrationofsPirBwould
produceamorerobustrecoveryforallanimals.Inaddition,ratherthanminipumpinfusions,itwouldbepreferable
todevelopasmallmoleculedrugthatcancrossthebloodbrainbarrier.Finally,asmentionedabove,LilrB2is
presentinhumanbrain,butbecausethereareotherfamilymembers,itwillbeimportanttocharacterizetheir
expressionandfunctioninhumancentralnervoussystem.
PirB:Anendogenoustargetformanipulationsofsynapseandsystemslevelplasticity
Ourobservationsaddtoagrowingbodyofresearchthathasunmaskedactiverolesformoleculesinthebrain
actingasnegativeregulatorsoffunctionalandstructuralplasticitybothindevelopmentandinadulthood(17,50,
54).InthecaseofPirB,thisnegativeregulationmayalsobehijacked,asinAD,whereamyloidoligomersbind
toPirB/LilrB2withnanomolaraffinity,resultinginlossofODplasticityanddeficitsincorticalandhippocampal
synapticplasticity(20).Thus,sPirBandhumansolublereceptorhomologsmightevenbeviabletherapeuticsfor
AD.BygeneratingarecombinantsPirBprotein,wehavedemonstratedauseforselectivelyblockingPirBreceptor
interactionwithendogenousligands.TheseresultsfurthersupportthevalueofcreatingPirB/LilrBantagoniststhat
crossthebloodbrainbarrier,enhancingplasticityandincreasingsynapseandspinedensityincasesofdisease,
dysfunction,injury,orevenforcognitiveenhancementinnormalindividuals.
MATERIALSANDMETHODS

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Studydesign
TheobjectiveofthisstudywastodevisemethodstodeletePirBfunctionacutely,thenmonitortheeffectson
measuresofsynapticandODplasticity,andrecoveryfromLTMD.Twomethodswereused:tamoxifeninduced
PirBdeletionviaaPirBconditionalallele,orsPirBminipumpinfusion.BecauseODplasticityisinducedby
changesinvisualexperience,experimentsweredesignedtocaptureaninteractioneffectbetween

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genotype/treatmentandvisualmanipulationfourgroupsandatwowayANOVAdesignwereusedtotestfor
interactions.Allexperimentswereperformedblindtogenotypeand/ortreatment.Littermateswereusedtocontrol
forgeneticvariation,andmicewererandomlyassignedtodifferentvisualmanipulationsandtreatmentswithina
litter.Todetectgenotypeeffectssimilarorgreaterthanthosepreviouslyreported,samplesizeswerechosenon
thebasisofastatisticalpowerof80%withanvalueof0.05(14,24).Thenumberofreplicatemeasurements
andanimalsisgivenineachfigurelegend.
Mousestrains
PirB /andPirBflox/floxmiceweregeneratedasdescribed(14).APirBWTlinewasmaintainedonthesame
backgroundandusedforallminipumpinfusionexperimentsperformedduringthecriticalperiod(P21toP32).For
adultminipumpexperiments(P63toP74),PirBWTandPirB /micewerecrossedwiththeThy1YFPH
transgenicline(JAX#003782),whichexpressesYFPinasubsetofL5pyramidalneurons(45).Forinducible
knockoutexperiments,UbCCreERT2mice(JAX#007001)(25)werebredwithPirBfloxmicetogenerateUbC
CreERT2PirBflox/floxmiceandPirBflox/floxlittermates.Forconditionalknockoutexperiments,CamKIIaCre
PirBflox/+mice(57)werebredwithPirBflox/+micetogenerateCamKIIaCrePirBflox/floxmiceandCamKIIa
CrePirB +/+littermates.CamKIIaCremicewerealsobredwithAi14TdTomatoreportermice(36).All
experimentswereperformedinaccordancewithprotocolsapprovedbyStanfordUniversityAnimalCareandUse
CommitteeinkeepingwiththeNationalInstitutesofHealthsGuidefortheCareandUseofLaboratoryAnimals.
sPirBproteinproduction
TocreateasPirBmimic,thePirBectodomainwasclonedintoaplasmidcontainingaHistagforpurificationand
aMyctagforantibodydetectionwithasequenceidenticaltopreviouspublications(14,19,20,40).Forminipump
infusions,InvitrogenCustomServicesproducedsPirBinlargerquantitiesinFreeStyleHEK293cellsandpurifiedit
onanickelcolumn(NiHis,Invitrogen).
OsmoticminipumpimplantationsandsPirBinfusion
Craniotomieswereperformed,andminipumps(ALZETmodel10020.25l/hour,100lcapacity)containing
eithersPirB(1mg/ml)orBSA(1mg/ml)(VWR EM2930)in0.1Mphosphatebufferedsalinewereimplanted
subcutaneously,connectedtoacannula.Thecannulawasinsertedjustanteriortoprimaryvisualcortex(2.5mm
lateraland3mmposteriortobregma).
ArcmRNAinductionandinsituhybridization
ArcmRNAwasinducedbyplacingmiceovernightintotaldarkness(16to18hours),followedbybright
illuminationfor30mintopermitvisionthroughtheopeneyebeforeeuthanasiaviaisofluraneanesthesiaand
decapitation(8).AdigoxigeninlabeledArcantisensemRNAprobewasusedforcolorimetricinsituhybridizations
performedonbrainsections(8,33).ImageswereacquiredviabrightfieldmicroscopyandanalyzedusingtheLine
ScanfunctionofNeuroLenssoftwaretomeasurethewidthoftheArcmRNAhybridizationsignalipsilateraltothe
open(nondeprived)eyealongL4ofthevisualcortex,atthe3to4border(fig.S1).Multiplesectionswerescanned
andaveragedperanimal(forexample,Fig.2).
VEPrecordings
Animalswereanesthetizedwithurethane(0.6to1.2g/kgSigma)andchlorprothixene(5mg/kgSigma),andat
incisionswithlidocaine(2%,SparhawkLaboratories),andthenthescalpwasexposedandtheminipumpwas
cannularemoved.AfteracraniotomycenteredoverV1,aglasspipettefilledwithACSF(artificialcerebrospinal
fluid)wasinsertedtorecordlocalfieldpotentialsatadepthof450to600m.Responsestosinusoidalgrating
stimuliwereaveragedoverstimulusblocks,andapeakresponseamplitudewithina500mswindowafterstimulus
onsetwasdetermined.Visualacuitywasestimatedbyfindingthexinterceptofasemilogarithmicregressionof
responseamplitudesacrossdifferentspatialfrequencies(11,29).
Statisticalanalyses
AllstatisticalanalyseswereperformedwithPrismsoftware(Graphpad).Whenonlytwogroupswereinvolved,two
samplettestswereused,withWelchscorrectionforunequalvariancesappliedwhereappropriate.Dataforwhich
anormaldistributioncouldnotbeassumedwereanalyzedwithMannWhitneyUtests.Incaseswhereboth
treatment/genotypeandvisualmanipulationorhemispherewerevaried,atwowayANOVAwasconducted,with
Tukeyposthoctestsforindividualpairsofcolumns.
SUPPLEMENTARYMATERIALS

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MaterialsandMethods
Fig.S1.ExamplelinescanmeasurementsofArcmRNAinsituhybridizationsignalinvisualcortexinduced
bystimulationoftheipsilateraleye.
Fig.S2.PlasticityindicesforgeneticorpharmacologicaldisruptionofPirBfunction.
Fig.S3.CharacterizationofsPirBminipumpinfusionareaandeffectonODplasticity.

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Fig.S4.EffectofminipumpinfusionsofsPirBorBSAondendriticspinesbycellsandbyspinetype.

REFERENCESANDNOTES

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58. Acknowledgments:WethankN.SoteloKury,P.Kemper,andC.Chechelskiforlogisticsandmouse
breeding,andG.Vidalformicroscopyadviceandtraining.WethankB.Q.ZhuangandL.HsiehWilsonat
theCaliforniaInstituteofTechnologyforthesPirBplasmid,andJ.Schafferforillustrating(Fig.2A.Wealso
thankL.Luo,E.Knudsen,andT.Clandininforhelpfulfeedback.Funding:Thisprojectwassupportedby
NIHgrantsEY02858andMH07166,theMathersCharitableFoundation,andtheRosenbergFamily
FoundationtoC.J.S.NIHgrantEY018861toY.D.NationalScienceFoundationGraduateResearch
FellowshipstoD.N.B.andJ.D.A.andNationalDefenseScienceandEngineeringFellowshiptoJ.D.A.
R.W.S.receivedaBioXSummerUndergraduateResearchFellowship.Authorcontributions:D.N.B.and
C.J.S.proposedandoutlinedtheexperimentalplan.D.N.B.performedallexperimentsinvolvingtamoxifen
induceddeletionD.N.B.andR.W.S.performedminipumpimplantationsurgeriesandsubsequentanalysis
J.D.A.,R.W.S.,andM.D.performedandanalyzedthestudiesofCamKIIaCrePirBflox/floxvisualcortex.
S.Z.performedtheVEPrecordingsandanalysis,andY.D.supervisedthatcollaboration.H.L.performed
wholecellrecordingsofmEPSCsinL5neurons.M.D.characterizedanewbatchofthePirBantibodyused
hereandmadesubstantialintellectualcontributionstotheproject.J.S.createdthefloxedPirBandPirB /
mouseintheShatzlaboratory.D.N.B.andC.J.S.wrotethemanuscriptandreviewedalldatacollectionand
analysis.Competinginterests:C.J.S.andJ.S.areinventorsonU.S.Patentapplication12/087799
assignedtothePresidentandFellowsofHarvardCollegeonCompositionsandmethodsforenhancing
neuronalplasticityandregeneration.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,BlockingPirBup
regulatesspinesandfunctionalsynapsestounlockvisualcorticalplasticityandfacilitaterecoveryfromamblyopia.Sci.Transl.Med.6,
258ra140(2014).

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