反復(fù)應(yīng)激引起前額葉皮層上谷氨酸受體的表達(dá)第1頁(yè)/共35頁(yè)Repeatedrestraintstress:Juvenile(3-to4-week-old)SpragueDawleymaleratswereusedinthisstudy.Forrepeatedrestraintstress,ratswereplacedinair-accessiblecylindersfor2hrdaily(10:00a.m.to12:00p.m.)for5–7days.Thecontainersizewassimilartotheanimalsize,whichmadetheanimalalmostimmobileinthecontainer.第2頁(yè)/共35頁(yè)Results:Totesttheimpactofstressoncognitivefunctions,theymeasuredtherecognitionmemorytaskRU486:GRantagonist第3頁(yè)/共35頁(yè)ToexaminwhetherotherstressorscouldproduceasimilareffectRepeatedunpredictablestressRatsweresubjectedeachdaytotwostressorsthatwererandomlychosenfromsixdifferentstressors,includingforcedswim(RT,30min),elevatedplatform(30min),cagemovement(30min),lightsonovernight,immobilization(RT,1hr),andfoodandwaterdeprivationovernight.第4頁(yè)/共35頁(yè)Objectlocationtask:Totestthespeci?cityofthisstress-inducedmemoryde?cit,wealsosubjectedanimalstotheobjectlocationtask,aparadigmforthePFC-independentmemory第5頁(yè)/共35頁(yè)Openfieldtask:Incontrasttotheimpairedtemporalorderrecognitionmemory,ratsexposedtorepeatedrestraintstressshowednochangesinanxiety-relatedbehaviororlocomotiveactivity第6頁(yè)/共35頁(yè)TemporalOrderRecognitionMemoryTask:To?ndouttheonsetofthedetrimentaleffectsofstressoncognition,weexposedyoungmaleratstovariousdays(1,3,5and7)ofrestraintstress.第7頁(yè)/共35頁(yè)TemporalOrderRecognitionMemoryTask:TotestwhetherglutamatergictransmissioninPFCiscriticalfortheobjectrecognitionmemory,wegaveanimalsastereotaxicinjectionoftheNMDARantagonistAPVandAMPARantagonistCNQXtoPFCprelimbicregionsbilaterally.第8頁(yè)/共35頁(yè)TheconclusionoftheBehavioralTesting:Takentogether,itsuggeststhatrepeatedstresshasadetrimentaleffectonrecognitionmemory,whichmaybeduetothelossofglutamatergictransmissioninPFC.第9頁(yè)/共35頁(yè)TheElectrophysiologicalRecordings:To?ndouttheimpactofrepeatedstressonglutamatergictransmission,weexaminedtheinput/outputcurvesofAMPAR-andNMDAR-mediatedsynapticcurrents(EPSC)inPFCpyra-midalneuronsfromstressed,young(4-week-old)malerats.第10頁(yè)/共35頁(yè)pairedpulseratio(PPR):Totestwhetherthereducedsynaptictransmissionbyre-peatedstressmayresultfromapresynapticmechanism,wemeasuredthepairedpulseratio(PPR)ofAMPAR-andNMDAR-EPSC.PPRwasnotdifferentincontrolversusstressedanimals,suggestingalackofgrosschangeinpresynapticfunction.第11頁(yè)/共35頁(yè)miniatureEPSC(mEPSC):Tofurthercon?rmtheinvolvementofpostsynapticglutamatereceptors,wemeasuredminiatureEPSC(mEPSC),asynapticresponseresultingfromquantalreleaseofsingleglutamatevesicles,inPFCslices.第12頁(yè)/共35頁(yè)Whole-cellioniccurrent:Moreover,wemeasuredwhole-cellioniccurrentelicitedbyAMPA(100mM)orNMDA(100mM)applicationinacutelydissociatedPFCneurons(apurepostsynapticpreparation).第13頁(yè)/共35頁(yè)InjecttheGRantagonistRU486:SystemicinjectionsofRU486LocalinjectionsofRU486tothePFC第14頁(yè)/共35頁(yè)Theralationshipwiththetimeofstress:Tounderstandthecomplexactionsofstresshormones,weexposedanimalstovariousdaysofrestraintstress.第15頁(yè)/共35頁(yè)Totestthedifferentregions:Totesttheregionalspeci?cityoftheeffectofrepeatedstress,wealsoexaminedglutamatergictransmissioninstriatumandhippocampusfromyoungmalerats.第16頁(yè)/共35頁(yè)BiochemicalMeasurementofSurfaceandTotalProteins:WeperformedwesternblottingandsurfacebiotinylationexperimentstodetectthetotalandsurfacelevelsofAMPARandNMDARsubunitsinPFCslicesfromstressed,young(4-week-old)malerats.第17頁(yè)/共35頁(yè)Theamountoftheglutamaterecptors:第18頁(yè)/共35頁(yè)InjecttheGRantagonistRU486:第19頁(yè)/共35頁(yè)Totestthedifferentregions:Nosigni?cantchangeswerefoundinotherbrainareas,includingstriatumandhippocampus.第20頁(yè)/共35頁(yè)Theresultsinvitro:Wenextexaminedwhethertheeffectofrepeatedstressinvivomaybemimickedbycorticosterone(CORT)applicationinvitro.第21頁(yè)/共35頁(yè)InjecttheGRantagonistandMRantagonist:RU486:TheGRantagonist;RU28318:TheMRantagonist第22頁(yè)/共35頁(yè)Measuretheclusterdensity:第23頁(yè)/共35頁(yè)Ubiquitin/Proteasome-dependentDegradation:SincethetotallevelofNR1andGluR1wasreducedinrepeatedlystressedanimals,weexaminedwhetheritcouldbeduetothedecreasedsynthesisorincreaseddegradationofglutamatereceptors.第24頁(yè)/共35頁(yè)Ubiquitin/Proteasome-dependentDegradation:ThelevelofubiquitinatedGluR2,NR2A,orNR2Bsubunitsremainedunchanged.RepeatedstressalsofailedtoaltertheubiquitinationofSAP97(aGluR1bindingprotein)andPSD-95.第25頁(yè)/共35頁(yè)Testotherproteasomeinhibitor:MG132:proteasomeinhibitorLact:anotherproteasomeinhibitorChloro:generallysosomalenzymeinhibitorLeupeptin:lysosomalproteaseinhibitor11R-CS:membrane-permeablecalpainproteaseinhibitor第26頁(yè)/共35頁(yè)Testtheeffectofproteasomeinhibitor:第27頁(yè)/共35頁(yè)ShRNALentiviralKnockdown:Giventheroleofproteasome-dependentdegradationofgluta-matereceptorsinthedetrimentaleffectsofrepeatedstress,wewouldliketoknowwhichE3ubiquitinligasesarepotentiallyinvolvedinthestress-inducedubiquitinationofGluR1andNR1subunitsinPFC.Nedd4-1:anE3ligasenecessaryforGluR1ubiquitinationinresponsetotheagonistAMPA.Fbx2:anE3ligaseintheERthatubiquitinatesNR1subunits.第28頁(yè)/共35頁(yè)ShRNALentiviralKnockdowninvitro:第29頁(yè)/共35頁(yè)ShRNALentiviralKnockdowninvivo:第30頁(yè)/共35頁(yè)ShRNALentiviralKnockdowninvivo:第31頁(yè)/共35頁(yè)Testotherregion:Tounderstandthe