生后早期聽覺剝奪對大鼠聽皮層nr2b mrna年齡——依賴性表達模式的影響

1、TheeffectofearlyauditydeprivationontheagedependentexpressionpatternofNR2BmRNAinrataudityctexCaixiaBiYileiCuiYutingMaoJinghongXuFengXuJipingZhangXindeSunResearchCentrefBrainScienceEastChinaNmalUniversityShanghai200062Chin

2、aAbstractUsinginsituhybridization(ISH)presentresearchminutelyinvestigatedtheagedependentexpressionpatternofNR2BmRNAinrataudityctex(AC)duringpostnataldevelopmenttheeffectofearlyauditydeprivation.Ftheratsinthenmalgroupthem

3、RNAexpressionofNR2Bwashighestatbirth(postnatalday1)butdeclinedrapidlyuntillowlevelofadulthoodduringwhichtherewasarisingperiodwithatransientpeakonpostnatalday21(P21)ftheratsinthedeprivedgroupthewholedecliningasagingtrendo

4、fNR2BmRNAexpressionwassimilartothatofnmalgroupbuttheexpressionlevelfromP15toP27weremuchlowerthanthatofnmalgroupwiththegreatestreductionfromP21toP23thetransientpeakonP21disappeared.InbothgroupsthedistributionpatternofNR2B

5、mRNApositiveneuronsin6layersdsalmedialventralsubdistrictsofaudityctexwasalsoexamined.Thereisnosignificanteffectontheexpressionpatternbetweennmaldeprivedgroup.Ourresultsindicatedthattheearlyauditydeprivationdecreasedtheag

6、edependentexpressionpatternofNR2BmRNAsuggestingthatNR2Bmayplayanimptantroleinthedevelopmentalplasticityofaudityfunctioninrats.Keywds:AudityCtexAudityDeprivationInsituhybridization(ISH)NR2BmRNAExpression1.IntroductionDuri

7、ngasensitiveperiodinpostnataldaysanimalsshowafmofexperiencedependentdevelopmentplasticityincentralnervoussystem.IthasbeenproposedthattheNmethylDasparate(NMDA)receptplayacrucialroleduringthiscriticalperiod.Withagethechang

8、esofNMDARfunctionmayberelatedtothevarietyofthesubunitcompositionwhichincludetheNR2BNR2A[3040].IntheneoctextheNMDAreceptiscomposedofNR1NR2subunits.NR2ANR2BthetwoNR2subunitshaveattractedfocusesrespectively.Someresearchprop

9、osedthatNR2BmightpromoteplasticitymethandoestheNR2Asubunit[414344].NakanishihavedemonstratedthatmRNAencodingNR2Bwashighlyexpressedthroughoutthefebrainwithespeciallyhighlevelsintheolfactybulbshippocampusctex.Levelswereund

10、etectableintheponsmedullacerebellum[32].In﹡Crespondingauth.XindeSunEmail:.severalareasofcerebellumbrainstemtheexpressionleveldecreasedrapidlyin7daysafter:www.paper.11.3InsituhybridizationInsituhybridizationwasusedwiththe

11、signdetectionreagentboxesboughtfromtheBoehringerCompany.TheDiglabeledoligonucleotideprobefNR2BmRNAwassynthesizedbyShanghaiBioengineeringCompanywiththesequencesasfollows:5GGGCCTCCTGGCTCTCTGCCATCGGCTAGGCACCTGTTGTAAACC3ddUT

12、PDig.Atleasttwoanimalsofeachdetectionagewereusedfinsituhybridization.SDratsweredeeplyanaesthetizedwithinjectedPentobabital(50mgkg.BW)perfusedwith0.9%Naclthen4%Parafmaldehyde(PFA)in0.1MPhosphatebufferedsaline(PBS).Thebrai

13、nthatobtainedafterdecapitationwasimmediatelyfixedin4%PFAPBSf2hoursthenwasimmersedinto20%30%SucrosePBSrespectivelyfdehydrationtillthebrainpedonthebottom.Subsequentlyconarysectionsof30mwerecutbyafreezingmicrotomethawmounte

14、donto1%glutincoatedslidesthendriedin40Cdesiccationboxovernight.Brainsectionswerepretreatedwith0.1MPBSGlyPBSTritonPBSf10minutesatroomtemperature(RT)respectivelythenincubatedintoproteinaseK(1gml)TEbufferf30minutesat37C4%PF

15、Af5minat4C.Slideswerethenincubatedwith170lprehybridizationsolution[47%FmaeDeionized2salinesodiumcitrated(SSC)0.05Mphosphatebuffer(PB)]f2hoursat37C.Afterawashin2SSCf5minutesslideswerecoveredwith170lhybridizationsolution[4

16、7%FmaeDeionized2SSC0.05MPB1Denhard’ssolution10%dextransulfate(DS)0.05MDithiothreitol(DTT)0.25mgmlyeasttRNA0.5mgmldenaturalizedsalmonspermicDNA2gmlDiglabledoligonucleotideprobefNR2BmRNA]f18hoursat37C.Followingarewashesof2

17、SSC1SSC0.25SSCrespectivelyf2timesof15minutesat37CBufferⅠf2timesof10minutesatRTtreatingofBlockingbufferf30minutesatRTincubationwithantibodysolutionf2hoursat37CwashesofBufferⅠBufferⅡf2timesof10minutesrespectively.Thenslide

18、swerecoveredby170lNBTBCIPsolutionf18hoursindarkboxatRT.BufferⅢwasusedtostopthecolationreactionthenbrainsectionswererestainedbyfastgreenf2minutesfollowedbywashesofwaterf2timesof10minutesdehydrationofdescendingethanolthefi

19、nalsealing.Thecontrolexperimentsftheprobe’sNR2Bspecificitywereperfmedthesamewayexceptthattheprobewasexcludedfromthehybridizationsolution.TheexaminationcountingofpositiveneuronsinACwasperfmedthroughopticalmicroscopesusing

20、thepositiveneuronsdensityasanindexfcomparison.TheratACcontains6layersthatcanbedividedintoventralmedialdsalareas(Fig.1.a)accdingtotherelevantdatareference[15].Atleast20consecutiveslicesinthesamebrainareaofeachanimalweresu

21、rveyed.Weexaminedtheaudityctexunderhighpowermicroscope.16gridsweresetas1mm2thepositivecellsineachgridwascountedthenthedensityofpositiveneuronsdensitycanbeobtained(Nmm2).ThedatawasexpressedasMeanSEwiththestatisticalsignif

22、icancedeterminedbyttest.2.Results2.1TheexpressionofNR2BmRNAinaudityctexToinvestigatetheNR2BexpressionweanalyzedthemRNAleveleverydayfrompostnatal1to21thenevery3daysafterP18.Fig.2showesthedramaticdecreaseofNR2BmRNAinACof:w