[DOC] 诱导成年大鼠海马CA1区长时程压抑的强直刺激型式

[DOC] 诱导成年大鼠海马CA1区长时程压抑的强直刺激型式 诱导成年大鼠海马CA1区长时程压抑的强 直刺激型式 ActaysiologicaSinica,June25,2006,58(3):287-291 http:Hwww.actaps.com.ca Thetetanuspatternsfortheinductionoflong?termdepressioninthe adultrathippocampus CHENLi,JIANGMa-Li,HANTai-Zhen? DepartmentofPhysiologyandPathophysiology,Xi?anJiaotongUniversityS choolofMedicine,Xi?an710061,China Abstract:Previousreportssuggestedthatalow-frequencystimulus(LFS)of1--2Hz(600--900pulses)inducedahomosynapticlong- termdepression(LTD)ofsynapticefficacyinthehippocampalCAIareaofyoungrats(<4-weekold).However,thesestimulation protocolsoftenfailedtoinduceLTDintheadultCAIhippocampus.Inthepresentstudy,weexaminedtheeffectsoftwonoveltetanus patternsonLTDinductioninadultrathippocampalslices.WedeterminedthatthesenovelstimulationprotocolsinducedLTDinthe adulthippocampus,andthatthecharacteristicsofinducedLTDwereparameter-specific,includinglatency(periodfromtheendof tetanustoabeginningofLTD)andtheamplitudeofLTD.Theseresultssuggest thatLFSwithcertainpatternsCaninduceLTDinthe CAIareaofadultrathippocampalslices.andthatthemulti-trainsof2-Hzproto colprovidedmoreeffectiveresponsethanthe5-Hz protoco1. Keywords:hippocampus;long-termdepression;synapticplasticity 诱导成年大鼠海马CA1区长时程压抑的强直刺激型式 陈丽,蒋马莉,韩太真 西安交通大学医学院生理与病理生理学系,西安710061 摘要:低频率连续刺激(1Hz,15min)能够诱导幼年大鼠(<4周) 海马CAI区同突触长时程压抑(1ong.termdepression, LTD),而只有较高频率且持续时间较长的连续刺激才能诱导出成年 动物该部位稳定的LTD.本研究采用成年大鼠海马脑片 标本,电刺激Schaffer侧枝传入纤维,在CAI区锥体细胞层记录群体 锋电位,选用两种新的刺激参数以观测不同刺激型式在 诱导成年大鼠LTD中的作用.诱导LTD的刺激参数为:(1)2Hz,5串, 串长60S,串间隔60S;(2)5Hz,5串,串长 24S,串间隔96S;(3)对照组参数:2Hz,300S.结果显示,对照参数未能诱 导出LTD;而两种频率不同但脉冲总数与 刺激总时程相同的多串刺激,即参数(1)与参数(2),均在成年大鼠海马 CAI区诱导产生了LTD.两种参数所诱导的LTD特征 具有参数特异性,该特征主要表现为LTD诱导潜伏期和LTD的幅度: 参数(1),(2)诱导的LTD的潜伏期分别为15~25min 和30-40min.强直刺激后80min时LTD的幅度分别为(57.5~2.8)%和 (67.7~3.4)%.以上结果表明:特定型式的低频率刺激 能够诱导成年大鼠海马CAI区的LTD,提示LTD的诱导与刺激的组 合型式相关,并且2Hz较5Hz的多串刺激在诱导LTD 中更为有效. 关键词:海马;长时程压抑;突触可塑性 中图分类号:Q42;R338.8 Homosynapticlong—termdepression(LTD)isoneofthe principalformsofuse—dependentsynapticplasticity.LTD wasinvestigatedinitiallyinthecerebellum【l】andisassoci— atedwithcertaintypesoflearningandmemoryrelatedto cerebellaractivities.Inrecentyears,LTDwasobservedin numerousbrainregionsincludingthehippocampus,where itisthoughttoparticipateinsynapticdevelopment.Low— frequencystimulation(LFS,1--2Hz,600,-900pulses) Received2005??09??08Accepted2006??04-09 ThisworkwassupportedbytheNationalNaturalScienceFoundationofChina (No.30170310). „Correspondingauthor.Tel:+86.29-82655274;Fax:+86.29-82655274;E-mail:htzhen@mail. xjtu.edu.ca 288 couldeffectivelyinduceastableuDintheSchaffer-CA1 pathwayinneonatalorjuvenileanimals(<4一weekold), butnotinadults[4].ThephenomenonthatuDinducedbv LFSonlyoccursintheaninlalswiththeageofdwas termedasdevelopmentaldown—regulationof工D【5.6】.In adults,anotherstimulationprotocol,persistentstimuliata frequencyof5~10Hzfor15mini7】couldinducehomo. synapticuDintheCAlregionofthehippocampus.al— thoughstimulusparadigmof5Hzwithshorterduration couldonlyinduce工Dinyounganimals[8.91.Kempandhis co-workers[41determinedthatalowfrequencypaired—pulse 『withtheintervalof50msor200ms]afferentstimulus (PP-LFS)alsoinduceduDinadultrats,butrequireda longerstimulationperiod(15min).Soitisbelievedthat boththefrequencyand/ordurationofstimulationusedin adultstoinduceuDshouldbehigherand/orlongerre— spectivelythanthatusedinyounganimals【71.AUthedata describedabovesuggestthatuDoccurrenceisprobably acommoncharacteristicofbothdevelopmentalandadult animals.However,theconditionsofstimulifor工Din— ductionatdifferentagesalenotidentica1. Inthisstudy,weexaminedtwonovelstimulationprotocols, multi—trainsofLFS.toinvestigatewhethermcanbe inducedintheSchaffer-CA1pathwayofthehippocampus inadultrats.W_ecomparedsynapficefficaciesinducedby single(600pulsesat2I-Iz)andmulti-trainstimulations(600 pulsesat2Hzwitha60一sinter—traininterva1.and600 pulsesat5Hzwitha96一sinter-traininterva1). 1.1MateriaIs MaleSprague—Dawleyratsweighingbetween180~250g werehousedona12一hour/12一hourlight/darkcycle(1ights offat20:00).withfoodandwaterprovidedadlibitum. Thisstudywasconductedwiththeminimumnumberof animalsnecessaryforstatisticalanalysis,andwithmini— malstressordiscomfort.Experimentswerecarriedoutin compliancewiththelaboratoryanimalprotocolutilizedin theSchoolofMedicineofXi?anJiaotongUniversity. 1.2Slicepreparation HippocarIl1)alsliceswerepreparedaspreviouslydescribed[?. Briefly,whenratsweredecapitated,thebrainwasremoved rapidlyandtheleftsideofthehippocampuswasisolated onanice-coldplat.W_ecuttransverseslicesofthehippoc— ampus(400grn)usingatissuechopper,placedthemona nylonmesh,incubatedtheminaninterfacerecordingcham- bermaintainedataconstanttemperature【(31?1).C)】and allowedthemtoequilibrateforatleast1h【0”.Anartificial ActasiologicaSinica,June25,2006,58(3):287-291 cerebrospinalfluid(ACSncontaining(inmmol/L):NaCl 124,KCl5,NaHCo326,MgSO41.3,KHEPO41.2,CaCl2 2.4,glucose10,wasequilibratedwithmixtureof95%o2 and5%C0.. 1.3Electrophysiology W_estimulatedtheSchaffer.collateralfiberswithacon— centricbipolarstainlesssteelelectrode.Populationspikes „S1wererecordedinthepyramidalcelllayeroftheCA1 areawithaglassmicroelectrode舢edwith2mol/LNaCl solution(resistance1—8MQ).Thestimuluswassetto two—foldthethresholdintensityandat0.1一msduration. Teststimuliweredeliveredonceperminuteat0.017Hz, andwereappliedthroughouttheexperiments,exceptdur- ingtetanus.Thestrengthofsynaptictransmissionwas quantifiedbymeasuringtheamplitudeofPS(PSA)and thesizesofPSweremeasuredasthepeaktopeakamplitudes. Baselinerecordingswerecollectedforatleast30min priortotetanusstimulationtoensurethattheresponses hadstabilizedt.Afterobtainingastablebaselinerecording. weappliedtetanusatthetestintensity.TwOnovelLFS protocolswereusedtoinducem.fortheprotocol1:5 trains,eachincluding120pulsesat2Hzandtheinterval betweentrainswas60s:fortheprotocol2:5trains,each including120pulsesat5Hzandtheintervalbetweentrains was96s.Thecontrolparameterwasthesingletrainof 600pulsesat2Hz(Fig.11.Theresponsesaftertetanus wererecordedformorethan80min.Thestandardof uDproductionindicatedthatthePSAaftertetanusde— creasedby20%ormorecomparedtothebaseline recording,andthisdepressionlastedformorethan30min. AttheendoftheuDrecording.wetestedthehippocam- p旧dslicebyapplyingasingletetanustrain[highfrequency 2Hz300S(600pulses) “ 60s Fig.1.Thetctanizingandcontrolstimulationadoptedinthisstudy. Protocol1wascomposedof5l~ins,eachincluding120pulsesat2 Hz.withaninter-trainintervalof60s.Protocol2consistedof5 wains,eachincluding120pulsesat5Hz.Controlwascomposedof asingleburstof600pulsesat2Hz.StimuliwereaUat0.1-ms duration. CHENLietal:TetanusPatternsInducedLTDinAdultRatHippocampus stimulationaWS),100Hz,1s;30-60,0.1一msduration】 totheSchaffer-collateralfibers. 1.4Dataanalysis LTDrelatedvflueswereexpressedasmeans~SEM.In eachcase,?n?signifiedthenumberofhippocampalsfices producingtheLTD.Weobtainedeachshcefromasepa— rateratandusedtwo—tailedindependentt-testtocalculate thestatisticaldifferences.SignificancewassetatP<0.05. 2.1Trainstimulationsatlowfreiluenciesinduced stablem Inthisstudy,wedeterminedthatsingleWainof600pulses at2HzappliedintheSchaffer-collateralfibersfailedto induceuDofsynapticefficiencyinslicesfromadultrats 【(92.9~2_3)%,n=8,measuredat80minaftertetanus]. However,themulti.trainstimulir5trainswithaconstant inter-Wainintervalof60s1atthesamefrequency(protocol l1resultedinLTDwithin15-,25minaftertetanus(the PSAmeasuredat20minand80minpost—tetanuswere (78.1~3.1)%and(57.5~2.8)%,respectively,n=10),as showninFig.2. 2.2DistinctpropertiesoftheL1IDinducedbytwo protocols Todeterminetheeffectsofthealtematelowfrequency trainprotocolsontheinductionofm.weadoptedpro- tocol2(5trains,eachincluding120pulsesat5Hz,the a) .三 面 „I, ? 0 ,一 《 ? la. rimefmin) Fig.2.LTDinducedbyprotocol1inthehippocampalCA1area. LTDwassignifiedbythepercentageofbaseline(PSA)andthese dataweremeasuredonlyfromthoseslicesproducingLTD.The parameterusedinthecontrolgroup(n=8)was2Hz,600pulses.The protocol1group(n=l0)wasstimulatedwith5trains,eachincluding 120pulsesat2Hz,withaninter?stimulusintervalof60s.The horizontalbarindicatesthetimewhentetanusorLFSwasapplied. inter-stimulusintervalwas96s1intheseexperiments.耵le resultsrevealedthattheprotocol2stimulusalsoproduced astableIrDwithin30minaftertetanus(Fig_31.Com- paringthedevelopmentalprocessesofmproducedby thestimulationofprotocols1and2.wefoundthatthere weretwodistinctproperties.First,thelatency(defmedas theperiodfromtheendoftetanustothetimewhenPSA decreasedby20%ofbasdine)ofuDinducedbyproto— col1stimulus【(78.1~3.1)%,n=10,measuredat20min post—protocol】wasshorterthanthatinducedbyprotocol 2stimulusf(78.3~5_3)%,n=8,measuredat35minpost- 要 邑 la. rimofmin) Fig.3.DistinctpropertiesofuDinducedbythestimulationoftwo protocols.L1][)wassignifiedbythepercentageofbaseline(PSA) andthesedatawererecordedonlyfromthosesficesproducinguD. Parametersmonitoredforprotocol2wei?e5Hz.5仃ainswithall inter-trainintervalof96s(,l=8).nIehorizontalbarindicatesthe timewhentetanuswasappfied. ControlProtocol1Protocol2 Time(min) Fig.4.Differentpercentagesofbaselineinducedbythreeprotocols. ThesedatawererecordedonlyfromthoseslicesproducingLTD. Measuredat80minpost-tetanus,thepercentageofbaseline(PSA) forcontrolandprotocolsl,2were(92.9?2-3)%(,l=8),(57.5~2.8)% (n=10)and(67.7~3.4)%(,l=8),respectively.”P<O.01control group.P<0.05vsprotocol1group. ????约O —ouIIo??D一《? protocol】(Fig.3).Second,theamplitudeofLTDproduced byprotocol1wassignificantlyhigherthanthatproduced byprotocol2(尸<0.05).AsshowninFig.4,thepercent- ageoftIleirbaseline(PSA)measuredat80minafterteIta? nusinthecontrol,protocols1and2were(92.9?2.3)% (=8),(57.5~2.8%)(=10)and(67.7~3.4)%(=8), respectively. Inthisstudywepresentevidencethatadultratsrespond similarlytohomosynapticmasdoyoungrats.though underdifferentinductionconditions.Forthesedeterminations, weusedtrainpatterntetanusadministeredintwodifferent protocolsatlowfrequenciesandundervariabletrainstimuli. W_efoundthatdifferentstimulusfrequencieswiththesalTle totalpulsesproducedminadultrathippocampiwith differentproperties.includinglatencyandamplitudeofU][). Asaresult,weadoptedthemulti.trainof2Hz.LFSfor higheffective?induction. 3.1SfimulationprotocolsandthresholdforIrDin. duction Mockereta1.[13】reportedthattwotrainsofstimuluswith 10-minitueintervalsat1Hz.couldinduceuDinthehip- pocampusofadultanimals,andproposedthatthefirst trainprobablyfacilitatedthegene?sexpressionresponseto thesubsequenttrain.Somedataindicatedtl1atsynaptic stimulationswhichactivateN-methy1.D-aspartate(NMDA) receptorsmightincreasetheinductionthresholdoflong. termpotentiation(LTP)sufficiently,andthusdecreasethe thresholdofLTDinthehippocampus[14].Inourpresent study.thetetanusofacontinuousstimulusat2HzwitIl 6()0totalpulsescouldnotinduceuDinadultrats.other- wisethestablemcouldbeproducedinadultsbythe traintetanus(5trains,inter-trainintervalof60s1withthe samefrequencyandthesaI/leamountofpulses.Eachwain stimulusmightproduceamini.activationofsynapsesand decreasethethresholdofLTDinductiongradually. Therefore.the?wouldnotoccurimmediatelyafterthe lasttrainandwouldrequirealatencyperiod.Berrettaand Cherubini【?】proposedthatthestimulusfrequencyand/or durationusedtoinduceinadultratsmustbehigher and/orlonger,respectively,thanthatusedtoinducem indevelopinganimals.Otherresearchersproposedthatthe lowfrequency.inducedmprocessescouldbedivided intoinitiationandinductionphases,whichovedappedin theirdurations.Theinitiationphaseestablishedthebasis fortheinductionphase.inwhichmwasexpressedo3]. ActaPhysiologicaSinica,June25,2006,58(3):287—291 TheresultsofOUrstudyvalidatedthisdua1.phasetheory, sincetheminductionsthatwerecordedresultedfrom multi-trainratherthansingletrainapplications.Tllepresent datasuggestthatthe?inductionmayrequireacritical pre—state,orinitiationstate,whichestablishesahigherprob- abilityforuDformation.andthatthetrainpatternswe usedtostimulatetetanusmayrepresentthenovelproto. colsforestablishingthisstateinhippocampalneurons. Thedistinctpropertiesofthemproducedbythetwo protocolsmaybeduetothedifferentdurationofeach traininthetetanus.Itwasreportedthatdurationofthe inductionparadigmisanimportantparameterformi1l adults阴.Therefore.weproposethatthelongertrainmight activateneuronstoestablishthecriticalinductionstatefor mformationmoreefficientlythantheshorterone. 3.2lnductionofrDisrelatedtotheextentofthe postsynapticactivation Nakanoeta1.【】proposedthatthepostsynapticactivation extentislikelyimportantforminduction.TIlis??extent?? mightberepresentedbythepostsynapticmembranevolt- ageandthepostsynapticcalciumionconcentrationt16-18].It isnecessaryforminductiontoreachasuitablelevelof thepostsynapticactivation.Inaddition,theextentof postsynapticactivationmaybeaffectedbythestimulus frequency【.Inshort.amoreappropriatepostsynaptic extentincreasesthelikelihoodofaneasieruDinduction. Fromabove.thereasonwhytheamplitudeof?pro. ducedby2..Hztetanuswashigherthanthatby5..Hzteta- nusisprobablyduetothattheactivationextentproduced bytheformerfrequencyismoreappropriatethanthatby thelaterone. Therearetwomechanismsinvolvedinthe?induc. tionintheCA1aI1eaofhippocampus:oneisNDArecep- tor-dependent[z3a9]andanotherisinositol1,4,5.tdphosphate (IP3)一sensitivecalciumstorage—dependent??川.TheII)3 sensitivitymechanismisalsoknownasthemetabotropic glutamate(mGluR)一dependentmechanismbecausethe levelintheceUsofhippocampuscouldincreaseaftertype ImGluRsareactivatedandtheintraceUularCa2+concen. trationincreasessubsequentlyt22.231.Itwasproposedthat boththemechanismsdescnbedaboveexistedinthepro- cessofIDformationinducedbyLFSat0.5Hz.and NDAreceptor-dependentmechanismalonewaspresented inthefeasibleactivationextent,however,the一sensitive calciumstoragemechanismalsoplayedaroleunderthe otheractivationconditions[ts].Accordingtothereportde. scribedabove,wepresumethatinpresentstudythemecha- nismofminductionmightbeinvolvedinthedifferent CHENLietal:TetanusPatternsInducedLTDinAdultRatHippocampus :cI暑p畋Iorintercellularsignaling:2-Hztetanus-LTDmight beNMDAreceptor—dependent.and5一Hztetanus.ITD mightbeassociatedwithIP3-calciumsignaling. Inconclusion,ourfindingssuggestthatthestimulus patternofafferentsisimportantfortheinductionofLTD inadultrats.Twodifferentcellularmechanismsmightbe involvedinL]巾producedbydistinctstimuluspatterns. thoughadditionalexperimentswouldbeneededtodemon- stratetheLTDmechanismsandcellularsignalingpath— waysactivatedbythevariousprotocols. 1It0M.Long-termdepression.AnnaRevNeurosci1989;12:85— 102. 2DudekSM,BearNF.Homosynapticlong—termdepressionin areaCA1ofhippocampusandeffectsofN-methyl-D—aspartate receptorblockade.ProcNatlAcadSciUSA1992;89:4363— 4367. 3MulkeyRM,MalenkaRC.Mechanismsunderlyinginduction ofhomosynapticlong-termdepressioninareaCA1ofthe hippocampus.Neuron1992;9:967—975. 4KempN,McQueenJ,FaulkesS,BashirZI.Differentformsof LTDintheCA1regionofthehippocampus:roleofageand stimulusprotoco1.EurJNeurosci2000;12:360—366. 5AbrahamWC,Mason-ParkerSE,LoganB.Low-ffequencystimu- lationdoesnotreadilycauselong-termdepressionordepoten— tiationinthedentategymsofawakerats.BrainRes1996;722: 217—221. 6BashirZI,CollingridgeGL.Aninvestigationofdepotentiation oflong-termpotentiationintheCA1regionofthehippocampus. 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