反复应激引起前额叶皮层上谷氨酸受体的表达

反复应激引起前额叶皮层上谷氨酸受体的表达第1页/共35页Repeatedrestraintstress:Juvenile(3-to4-week-old)SpragueDawleymaleratswereusedinthisstudy.Forrepeatedrestraintstress,ratswereplacedinair-accessiblecylindersfor2hrdaily(10:00a.m.to12:00p.m.)for5–7days.Thecontainersizewassimilartotheanimalsize,whichmadetheanimalalmostimmobileinthecontainer.第2页/共35页Results:Totesttheimpactofstressoncognitivefunctions,theymeasuredtherecognitionmemorytaskRU486:GRantagonist第3页/共35页ToexaminwhetherotherstressorscouldproduceasimilareffectRepeatedunpredictablestressRatsweresubjectedeachdaytotwostressorsthatwererandomlychosenfromsixdifferentstressors,includingforcedswim(RT,30min),elevatedplatform(30min),cagemovement(30min),lightsonovernight,immobilization(RT,1hr),andfoodandwaterdeprivationovernight.第4页/共35页Objectlocationtask:Totestthespecificityofthisstress-inducedmemorydeficit,wealsosubjectedanimalstotheobjectlocationtask,aparadigmforthePFC-independentmemory第5页/共35页Openfieldtask:Incontrasttotheimpairedtemporalorderrecognitionmemory,ratsexposedtorepeatedrestraintstressshowednochangesinanxiety-relatedbehaviororlocomotiveactivity第6页/共35页TemporalOrderRecognitionMemoryTask:Tofindouttheonsetofthedetrimentaleffectsofstressoncognition,weexposedyoungmaleratstovariousdays(1,3,5and7)ofrestraintstress.第7页/共35页TemporalOrderRecognitionMemoryTask:TotestwhetherglutamatergictransmissioninPFCiscriticalfortheobjectrecognitionmemory,wegaveanimalsastereotaxicinjectionoftheNMDARantagonistAPVandAMPARantagonistCNQXtoPFCprelimbicregionsbilaterally.第8页/共35页TheconclusionoftheBehavioralTesting:Takentogether,itsuggeststhatrepeatedstresshasadetrimentaleffectonrecognitionmemory,whichmaybeduetothelossofglutamatergictransmissioninPFC.第9页/共35页TheElectrophysiologicalRecordings:Tofindouttheimpactofrepeatedstressonglutamatergictransmission,weexaminedtheinput/outputcurvesofAMPAR-andNMDAR-mediatedsynapticcurrents(EPSC)inPFCpyra-midalneuronsfromstressed,young(4-week-old)malerats.第10页/共35页pairedpulseratio(PPR):Totestwhetherthereducedsynaptictransmissionbyre-peatedstressmayresultfromapresynapticmechanism,wemeasuredthepairedpulseratio(PPR)ofAMPAR-andNMDAR-EPSC.PPRwasnotdifferentincontrolversusstressedanimals,suggestingalackofgrosschangeinpresynapticfunction.第11页/共35页miniatureEPSC(mEPSC):Tofurtherconfirmtheinvolvementofpostsynapticglutamatereceptors,wemeasuredminiatureEPSC(mEPSC),asynapticresponseresultingfromquantalreleaseofsingleglutamatevesicles,inPFCslices.第12页/共35页Whole-cellioniccurrent:Moreover,wemeasuredwhole-cellioniccurrentelicitedbyAMPA(100mM)orNMDA(100mM)applicationinacutelydissociatedPFCneurons(apurepostsynapticpreparation).第13页/共35页InjecttheGRantagonistRU486:SystemicinjectionsofRU486LocalinjectionsofRU486tothePFC第14页/共35页Theralationshipwiththetimeofstress:Tounderstandthecomplexactionsofstresshormones,weexposedanimalstovariousdaysofrestraintstress.第15页/共35页Totestthedifferentregions:Totesttheregionalspecificityoftheeffectofrepeatedstress,wealsoexaminedglutamatergictransmissioninstriatumandhippocampusfromyoungmalerats.第16页/共35页BiochemicalMeasurementofSurfaceandTotalProteins:WeperformedwesternblottingandsurfacebiotinylationexperimentstodetectthetotalandsurfacelevelsofAMPARandNMDARsubunitsinPFCslicesfromstressed,young(4-week-old)malerats.第17页/共35页Theamountoftheglutamaterecptors:第18页/共35页InjecttheGRantagonistRU486:第19页/共35页Totestthedifferentregions:Nosignificantchangeswerefoundinotherbrainareas,includingstriatumandhippocampus.第20页/共35页Theresultsinvitro:Wenextexaminedwhethertheeffectofrepeatedstressinvivomaybemimickedbycorticosterone(CORT)applicationinvitro.第21页/共35页InjecttheGRantagonistandMRantagonist:RU486:TheGRantagonist;RU28318:TheMRantagonist第22页/共35页Measuretheclusterdensity:第23页/共35页Ubiquitin/Proteasome-dependentDegradation:SincethetotallevelofNR1andGluR1wasreducedinrepeatedlystressedanimals,weexaminedwhetheritcouldbeduetothedecreasedsynthesisorincreaseddegradationofglutamatereceptors.第24页/共35页Ubiquitin/Proteasome-dependentDegradation:ThelevelofubiquitinatedGluR2,NR2A,orNR2Bsubunitsremainedunchanged.RepeatedstressalsofailedtoaltertheubiquitinationofSAP97(aGluR1bindingprotein)andPSD-95.第25页/共35页Testotherproteasomeinhibitor:MG132:proteasomeinhibitorLact:anotherproteasomeinhibitorChloro:generallysosomalenzymeinhibitorLeupeptin:lysosomalproteaseinhibitor11R-CS:membrane-permeablecalpainproteaseinhibitor第26页/共35页Testtheeffectofproteasomeinhibitor:第27页/共35页ShRNALentiviralKnockdown:Giventheroleofproteasome-dependentdegradationofgluta-matereceptorsinthedetrimentaleffectsofrepeatedstress,wewouldliketoknowwhichE3ubiquitinligasesarepotentiallyinvolvedinthestress-inducedubiquitinationofGluR1andNR1subunitsinPFC.Nedd4-1:anE3ligasenecessaryforGluR1ubiquitinationinresponsetotheagonistAMPA.Fbx2:anE3ligaseintheERthatubiquitinatesNR1subunits.第28页/共35页ShRNALentiviralKnockdowninvitro:第29页/共35页ShRNALentiviralKnockdowninvivo:第30页/共35页ShRNALentiviralKnockdowninvivo:第31页/共35页Testotherregion:Tounderstandthe