反復(fù)應(yīng)激引起前額葉皮層上谷氨酸受體的表達(dá)

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，課件共35頁，創(chuàng)作于2023年2月Introduction:Adrenalcorticosterone,themajorstresshormone,throughtheactivationofglucocorticoidreceptor(GR)andmineralocorticoidreceptor(MR),caninducelong-lastingin?uencesoncognitiveandemotionalprocesses.Mountingevidencesuggeststhatinappropriatestressresponsesactasatriggerformanymentalillnesses.Ithasbeenproposedthatglutamatereceptor-mediatedsynaptictransmissionthatcontrolsPFCneuronalactivityiscrucialforworkingmemoryInthisstudy,wesoughttodeterminewhetherrepeatedstressmightnegativelyin?uencePFC-mediatedcognitiveprocessesbydisturbingglutamatergicsignalinginjuvenileanimals第2頁，課件共35頁，創(chuàng)作于2023年2月Repeatedrestraintstress:Juvenile(3-to4-week-old)SpragueDawleymaleratswereusedinthisstudy.Forrepeatedrestraintstress,ratswereplacedinair-accessiblecylindersfor2hrdaily(10:00a.m.to12:00p.m.)for5–7days.Thecontainersizewassimilartotheanimalsize,whichmadetheanimalalmostimmobileinthecontainer.第3頁，課件共35頁，創(chuàng)作于2023年2月Results:Totesttheimpactofstressoncognitivefunctions,theymeasuredtherecognitionmemorytaskRU486:GRantagonist第4頁，課件共35頁，創(chuàng)作于2023年2月ToexaminwhetherotherstressorscouldproduceasimilareffectRepeatedunpredictablestressRatsweresubjectedeachdaytotwostressorsthatwererandomlychosenfromsixdifferentstressors,includingforcedswim(RT,30min),elevatedplatform(30min),cagemovement(30min),lightsonovernight,immobilization(RT,1hr),andfoodandwaterdeprivationovernight.第5頁，課件共35頁，創(chuàng)作于2023年2月Objectlocationtask:Totestthespeci?cityofthisstress-inducedmemoryde?cit,wealsosubjectedanimalstotheobjectlocationtask,aparadigmforthePFC-independentmemory第6頁，課件共35頁，創(chuàng)作于2023年2月Openfieldtask:Incontrasttotheimpairedtemporalorderrecognitionmemory,ratsexposedtorepeatedrestraintstressshowednochangesinanxiety-relatedbehaviororlocomotiveactivity第7頁，課件共35頁，創(chuàng)作于2023年2月TemporalOrderRecognitionMemoryTask:To?ndouttheonsetofthedetrimentaleffectsofstressoncognition,weexposedyoungmaleratstovariousdays(1,3,5and7)ofrestraintstress.第8頁，課件共35頁，創(chuàng)作于2023年2月TemporalOrderRecognitionMemoryTask:TotestwhetherglutamatergictransmissioninPFCiscriticalfortheobjectrecognitionmemory,wegaveanimalsastereotaxicinjectionoftheNMDARantagonistAPVandAMPARantagonistCNQXtoPFCprelimbicregionsbilaterally.第9頁，課件共35頁，創(chuàng)作于2023年2月TheconclusionoftheBehavioralTesting:Takentogether,itsuggeststhatrepeatedstresshasadetrimentaleffectonrecognitionmemory,whichmaybeduetothelossofglutamatergictransmissioninPFC.第10頁，課件共35頁，創(chuàng)作于2023年2月TheElectrophysiologicalRecordings:To?ndouttheimpactofrepeatedstressonglutamatergictransmission,weexaminedtheinput/outputcurvesofAMPAR-andNMDAR-mediatedsynapticcurrents(EPSC)inPFCpyra-midalneuronsfromstressed,young(4-week-old)malerats.第11頁，課件共35頁，創(chuàng)作于2023年2月pairedpulseratio(PPR):Totestwhetherthereducedsynaptictransmissionbyre-peatedstressmayresultfromapresynapticmechanism,wemeasuredthepairedpulseratio(PPR)ofAMPAR-andNMDAR-EPSC.PPRwasnotdifferentincontrolversusstressedanimals,suggestingalackofgrosschangeinpresynapticfunction.第12頁，課件共35頁，創(chuàng)作于2023年2月miniatureEPSC(mEPSC):Tofurthercon?rmtheinvolvementofpostsynapticglutamatereceptors,wemeasuredminiatureEPSC(mEPSC),asynapticresponseresultingfromquantalreleaseofsingleglutamatevesicles,inPFCslices.第13頁，課件共35頁，創(chuàng)作于2023年2月Whole-cellioniccurrent:Moreover,wemeasuredwhole-cellioniccurrentelicitedbyAMPA(100mM)orNMDA(100mM)applicationinacutelydissociatedPFCneurons(apurepostsynapticpreparation).第14頁，課件共35頁，創(chuàng)作于2023年2月InjecttheGRantagonistRU486:SystemicinjectionsofRU486LocalinjectionsofRU486tothePFC第15頁，課件共35頁，創(chuàng)作于2023年2月Theralationshipwiththetimeofstress:Tounderstandthecomplexactionsofstresshormones,weexposedanimalstovariousdaysofrestraintstress.第16頁，課件共35頁，創(chuàng)作于2023年2月Totestthedifferentregions:Totesttheregionalspeci?cityoftheeffectofrepeatedstress,wealsoexaminedglutamatergictransmissioninstriatumandhippocampusfromyoungmalerats.第17頁，課件共35頁，創(chuàng)作于2023年2月BiochemicalMeasurementofSurfaceandTotalProteins:WeperformedwesternblottingandsurfacebiotinylationexperimentstodetectthetotalandsurfacelevelsofAMPARandNMDARsubunitsinPFCslicesfromstressed,young(4-week-old)malerats.第18頁，課件共35頁，創(chuàng)作于2023年2月Theamountoftheglutamaterecptors:第19頁，課件共35頁，創(chuàng)作于2023年2月InjecttheGRantagonistRU486:第20頁，課件共35頁，創(chuàng)作于2023年2月Totestthedifferentregions:Nosigni?cantchangeswerefoundinotherbrainareas,includingstriatumandhippocampus.第21頁，課件共35頁，創(chuàng)作于2023年2月Theresultsinvitro:Wenextexaminedwhethertheeffectofrepeatedstressinvivomaybemimickedbycorticosterone(CORT)applicationinvitro.第22頁，課件共35頁，創(chuàng)作于2023年2月InjecttheGRantagonistandMRantagonist:RU486:TheGRantagonist;RU28318:TheMRantagonist第23頁，課件共35頁，創(chuàng)作于2023年2月Measuretheclusterdensity:第24頁，課件共35頁，創(chuàng)作于2023年2月Ubiquitin/Proteasome-dependentDegradation:SincethetotallevelofNR1andGluR1wasreducedinrepeatedlystressedanimals,weexaminedwhetheritcouldbeduetothedecreasedsynthesisorincreaseddegradationofglutamatereceptors.第25頁，課件共35頁，創(chuàng)作于2023年2月Ubiquitin/Proteasome-dependentDegradation:ThelevelofubiquitinatedGluR2,NR2A,orNR2Bsubunitsremainedunchanged.RepeatedstressalsofailedtoaltertheubiquitinationofSAP97(aGluR1bindingprotein)andPSD-95.第26頁，課件共35頁，創(chuàng)作于2023年2月Testotherproteasomeinhibitor:MG132:proteasomeinhibitorLact:anotherproteasomeinhibitorChloro:generallysosomalenzymeinhibitorLeupeptin:lysosomalproteaseinhibitor11R-CS:membrane-permeablecalpainproteaseinhibitor第27頁，課件共35頁，創(chuàng)作于2023年2月Testtheeffectofproteasomeinhibitor:第28頁，課件共35頁，創(chuàng)作于2023年2月ShRNALentiviralKnockdown:Giventheroleofproteasome-dependentdegradationofgluta-matereceptorsinthedetrimentaleffectsofrepeatedstress,wewouldliketoknowwhichE3ubiquitinligasesarepotentiallyinvolvedinthestress-inducedubiquitinationofGluR1andNR1subunitsinPFC.Nedd4-1:anE3ligasenecessaryforGluR1ubiquitinationinresponsetotheagonistAMPA.Fbx2:anE3ligaseintheERthatubiquitinatesNR1subunits.第29頁，課件共35頁，創(chuàng)作于2023年2月ShRNALentiviralKnockdowninvitro:第30頁，課件共35頁，創(chuàng)作于2023年2月ShRNALentiviralKnockdowninvivo:第31頁，課件共35頁，創(chuàng)作于2023