【doc】大鼠脊髓后角内神经降压素对一级传入C纤维突触前抑制效应的形态学证据

【doc】大鼠脊髓后角内神经降压素对一级传入C纤维突触前抑制效应的形态学证据 大鼠脊髓后角内神经降压素对一级传入C 纤维突触前抑制效应的形态学证据 第2期李和等.大鼠脊髓后角内神经降压素对一级传入C纤维进行突触抑制的形 态学证据229 MORPHOLOGICALEVIDENCESFORPRESYNAPTICINHIBITORY EFFECTOFNEUROTENSINONPRIMARYAFFERENTCFIBER INTHESPINALDORSALHORNOFTHERAT HeLi,YinonZhang,MinhaiZhang,ShimingYang.HonglianLi (DepartmentofHistologyandEmbryology,TongjiMedicalUniversity, Wuhan,430030,P.R.ofChina) Theaimofthepresentstudyistorevealwhetherneurotensin(N丁)i,2thespinaZd0,乜Z^0,0 厂f^ ratmightpresynapticallymodulatetheprimaryafferentCfibers.Withuorescencemicroscop e,itwns observedthat,inthelaminaeI一?ofthespinalcord,thedistributionofneurotensin— likeimm",2D口czfv (NTLI)waspartiallyoverlappedwiththatofthebindingofisolectinI-B4fromGriffoniasi什 llicif0la(I— B4).ThefurtherobservationwithconfocallaserscanningmicroscopeshowedthatnfewofiT LI—p0stiue terminalscontactedwithsomeI— B4-labeledterminaIs.Itwasfoundelectronmicroscopicallyinthesupercine layersofthespinalcordoftherattreatedwithsubarachnoidinjectionofcapsaicinthatNTLI— contninng terminalscontactedwithdegeneratedterminalswithand~orwithoutsynapticspecializaf,2s. 丁^PsPres"It indicatethat?丁 maypresynapticallyinhibitthetransmissionofprimaryaffere,2tCfibersVi口口如口z0,2 synapsesand~ornonsynapticcontacts.Additionally,axodendriticsynapsesbetweendegen eratedtermi,2口ls andNTLI-containingdendritesalsoexistinthespinaZdorsaZh0,.,2. KeywordsNeurotensin;Immunohistochemistry;GriffoniasimplicifoliaI-B4;Capsaicin; PrimaryafferentCfiber;Spinaldorsalhorn;Rat Neurotensin(NT),atridecapeptide.isolated originallyformthebovinehypothalamus[,is widelydistributedinthemammaliannervous system['3. .LocalizationofNT—reactive materialL,引andNT.bindingsite[]withinthe superficialIaminaeofthespinaldorsalhorn suggeststhatNTneuronescontributetosensory processatthelevelofthespinalcord.Ithasbeen reportedthatantinOcjceptiveeffectscanbeinduced byintrathecaladministrationofNTatthelumbar levelofthespinalcordoftheratandmice[8,lo]and thatmicroiontophoreticapplicationofNTresults inexcitatory[?andinhibitory[13effectsonthe neuronesinthecatandratspinaIdorsaIhorn. Immunoelectronmicroscopicstudies[,]have showedthatNT—likeimmunoreactive(NTLI) positiveterminalsformasymmetricaland/or symmetricalsynapseswithdendritesand/or somas.ItisthereforesuggestedthatNTmay postsynapticallyactontheneuronesofthespinal dorsalhorn.Todate.therelationshipofNT— containingelementsinthespinaldorsalhorntothe primaryafferentCfibersisnotclear.Afterdorsal rhizotomy,thechangeofNTbindingsitein densityordistributionandtheinteractionofNTLI terminalswithdegeneratedprimaryafferent boutonshasnotbeenfoundintheratspinaldorsal horn[.However.Zhangandhiscoworkers[15] detectedthemRNAofNTreceptorinthedorsal rootganglion(DRG)neuronesoftheratbyusing insituhybridizationandfoundthatNTreceptor mRNAwasexpressedinabout30ofallL4/L5 DRGneurones,mainlyinsmallones.Moreover. theyhavealsofoundNTLIaxoaxoniccontacts withprimaryafferentsinthespinaldorsalhorn. Inthepresentstudy,basedonselective bindingofI—B4isolectinfromtheseedsofGriffonia simplicifoliatosmallprimarysensoryneurones[] andspecificneurotoxicityofcapsaicinto unmyelinatedprimaryafferents[,double 23O中国组织化学与细胞化学杂志第9卷 fluorescencehistochemistry.confocallaser scanningmicroscopyandimmunoelectron microscopyareusedtoexamineifNTLI—containing terminalsareconnectedwiththecentralterminals ofprimaryafferentCfibersinthedorsalhornof theratspinalcord. MaterialsandMethods DoublefluorescencehistochemistryofNTLIandI— B4bindingandconfocallaserscanningmicroscopy FourmaleWistarratsweighing250,,一300g wereperfusedtranscardiallywith0.01mol/L sodiumphosphate—buffered0.85(w/v)saline (PBS;pH7.3),thenwith0.1mol/Lphosphate buffer(PB;pH7.3)containing4(w/v) paraformaldelydeand1(w/v)picricacidunder deepanesthsiabyintraperitonealinjection(i.P.) ofsodiumpentobarbital(100mg/kgbodyweight). Afterperfusion.thelumberenlargementofthe spinalcordwasimmediatelyremovedandplacedin for4h.Subsequently,the thesamefixativeat4? specimenwassaturatedwith25%sucroseinPBS at4?overnight.Thetissueblockswerecutinto crosssections30mthickonafreezing microtome.Thesectionswereincubatedatroom temperaturesequentiallywithamixtureofrabbit anti—NTantiserum(1:3000;Incstar)and1g/ mlbiotinylatedI—B4(Sigma)and2.5~g/ml fluoresceinisothiocvanate(FITC)一labelledavidin D(Vector)for2h.Theincubationmediawere preparedbyusingPBScontaining3(v/v)Triton X一100and2(v/v)normalgoatserum.The sectionswereobservedunderaZeiss epifluorescencemicroscope(Axiophoto)withan appropriatefilterforFITC(excitation450,,一490 nm;emission515,565nm)orTexasRed (excitation530,585nm;emission?615nm). Somedouble—labelledsectionswerethenexamined underaconfocallaserscanningmicroscope(LSM 410:Zeiss)usingexcitationlaserbeamsof488nm and543nmwavelengthsrespectivelywith appropriateemissionfiltersforFITC(510,525 nm)andTexasRed(?570nm). ImmunoelectronmicroscopyofNT FourmaleWistar used,andunderthe rats(200,250g)were generalanesthesiawith sodiumpentobarbital(49mg/kgbodyweight.i. P.),apolyethylenecatheter(PE一10)wasinserted throughthecisternamagnaintospinal subarachnoidspaceoftherostraledgeofthe lumbarenlargement(L1一L2)ofeachratusinga modificationofthemethoddescribed previously[.].Subsequently,20tAof0.25 (w/v)capsaicindissolvedinasterile,osmotically balancedsolution[.containing50(v/v)DMSO wasintrathecallyinjectedviathecatheter.After theinjection.theratwasallowedtosurvivefor3 days,re-anesthetizeddeeplyasdescribedabove, andthenperfusedtranscardiallywithPBS, followedwithPBcontaininig4%(w/v) paraformaldelyde,0.75(w/v)picricacidand. 0.075(w/v)glutaraldehyde.Afterthat,the lumbarenlargementofthespinalcordwas immediatelyremovedandrinsedinPBSfor1,2h, andthencutintocrosssections50--~70mthickon avibratome.Thesectionswereincubated sequentiallyin(1)1:3000rabbitanti—NTserum at4?for35"~40h;(2)10t~g/mlbiotinylatedgoat anti一[rabbitIgG]antibodyatroomtemperaturefor 2h;(3)1:100ABC(Vector)atroom temperaturefor2h.Theincubationmediawere preparedbyusingPBScontaining2(v/v) normalgoatserum.Boundperoxidasewas visualizedbyincubationwith0.05mol/LTris—HCl buffer(pH7.6)containing0.03(w/v)DABand 0.003(v/v)H2O2atroomtemperaturefor15"- 20min.AfterwashinginPBS.thesectionswere osmicated,dehydratedandthenflat—embeddedin Durcupan(Fluka)betweentwosheetsofplastic. Underadissectionmicroscope,thesuperficial layersofthedorsalhornwereremoved.andthen cutintouhrathinsections.Theseuhrathin sectionsweredoublecounterstainedwithuranyl acetateandleadcitrateandthenobservedundera HitiachiH一7100electronmicroscope. Results LightmicroscopicdistributionofNTLIandI—B4 第2期李和等.大鼠脊髓后角内神经降压素对一级传入C纤维进行突触抑制的形态学证据231 binding Inthetransversesectionsofthelumbarspinal cord.NTL1wasconcentratedinthelaminaeI—ill ofthedorsalhornandobservedastwodistinct band(Fig.1a):onebandacrosslaminaIandthe thirdoflamina?(1aminaeI/? outerone— border)andtheotheroneacrosstheinnerone— thirdoflamina?andtheouterhalfoflaminanl (1aminae?/nlborder).ThebandofNTLIatthe I/?borderwasdenserthanthatattheI/? border,andthezonebetweentwobandsexpressed fewimmunoreactivities.NONTL1wasobservedin thedorsolateraltractofLissauer.Thebinding reactivityofI—B4wasalsodistributedinthe superficiallayersofthespinaldorsalhorn, restrictedinthelaminaeI—nl(Fig.1b). Althoughthebindingreactivitywassomewhat moreintenseinlamina?thaninlaminaI.no distinctiveband—likelabellingpatternwasseen.In contrastwithNTLI,thebindingofI—B4wasalso presentinLissauerstract. Underconfocallaserscanningmicroscope, mostofpresumedaxonalterminalslabelledwith TexasRedorFITCshowedsinglefluorescence, theredorthegreen.However,afewof componentsweredetectedtoexhibityellow fluorescence,themixtureofredandgreen fluorescence(Fig.2). Featuresofdegeneratedterminals Underelectronmicroscope,thespecific degeneratednerveterminalswereseeninthe superficiallayersofthedorsalhornoftherats treatedwithcapsaicin.Thesedegenerated terminalsweremainlyfoundinlaminaeI—n1. Theydisplayedmarkedand. evenlyincreasing electrondensity,obscurestructuresofthe organelles,e.g.irregularvesicles.Someofthe degeneratedterminalsconstitutedsynaptic glomeruliwithseveralsurroundingdendritesand/ oraxonalterminals(Fig.3.4). UltrastructurallocalizationofNTLI Underelectronmicroscope,NTL1was prominentlypresentinaxonsandaxonalterminals throughouttheneuropilofthesuperficialdorsal horn,thepositivereactionproductsbeingmainly localizedontheoutersurfacesofsmallclear vesiclesandalsoinlargedensevesicles(Fig.5, 6).TheseNTLIlabelledterminalsformed asymmetricalandsymmetricalsynapseswith unlabelleddendrites(Fig.5,6)orwithunlabelled somas.SomeNTLI—labelleddendriteswerealso seeninlaminaeI—nl,inwhichNTLI—positive productswereassociatedwiththemicrofilaments, microtubulesandmitochondria(Fig.7). UltrastructuralrelationshipofNTLI—components withthedegeneratedterminals Inthespinaldorsalhornoftherattreated withsubarachnoidinjectionofcapsaicin,mostof NTLI-containingcomponentswereconnectedwith unlabelleddendrites,somasorterminalsbutnot withdegeneratedterminals.Insomeultrathin sections,axoaxonalcontactsexistedbetween NTLI—containingterminalsandthedegenerated terminals,however,mostofthemdidnotshow synapticspecialization.Occasionally,NTLI— positivenerveterminals,whichcontainedmany synapticvesicles,madedistinctaxoaxonal synapsesonthedegeneratedterminals(Fig.8).In addition,afewofaxodendriticsynapsesformedby thedegeneratedterminalsandNTLI—labelled dendriteswerefoundinthesameregionsofthe spinalcord;theseaxodendriticsynapseswere alwaysasymmetrical(Fig.9). Discussion Thesuperficiallaminaeofthespinaldorsal hornarethemainregionsofthecentral terminationofunmyelinatedafferentsofCfibers derivedfromsmallsensoryneuronesofthedorsal rootganglion[....Theresultsinthepresentstudy byimmunofluorescenceobservationshowedthat NTL1wasrestrictedlydistributedinthesuperficial layersofthespinaldorsalhornastwodistinct bandsoffiber—andterminal—likefluorescence, whichwascoincidentwiththeprevious reports[.?.?引.Thefluorescencedouble—labelling examinationrevealedthat,inthesameregions, thedistributionofNTLIoverlappedwiththatof 232中国组织化学与细胞化学杂志第9卷 thebindingsitesforI—B4.I—B4.anisolectinfrom theseedsofGriffoniasimplicifolia,hasbeen knowntoselectivelybindtoasub—populationof smalldiametersensoryganglionicneurones,the greatmajorityofwhichsuppliesnociceptors[. ThecharacteristicdistributionofNTLIandthe partialdistributivematchbetweenNTLIandI-B4 bindingreactivitystronglysuggeststhepossible correlation. betweenNTLIcomponentsandCfibers whichinvolveintheinputofnociception. Thefurtherobservationofthefluorescence double—labelledsectionswithconfocallaser scanningmicroscopeidentifiedsomeelementswith yellowfluorescenceformedbythemixingofred andgreenfluorescence.NT—containingaxonsin thespinaldorsalhornwasthoughttobeoriginated notfromprimaryafferentneuronesbutfrom intrinsicneurones.sinceontheconditionofdorsal rhizotomy,treatmentwithcapsaicinonneonatal animals,orspinalhemisection,nodetectable depletionofNTwascausedinthespinaldorsal horn[''''.Thus.themixedfluorescence observedinthepresentstudyhintsthepossibility thatthereareclosecontactsbetweenNTLI— containingaxonicterminalsandcentralterminals ofCfibersidentifiedbyI—B4一bindingreactivity. Whetherthecontactsaresynapsesornotisstill uncertainowingtothelimitationoftheresolution ofconfocallaserscanningmicroscope. Ithasbeengenerallyacceptedthatcapsaicin exertsaspecificneurotoxicactiononCfibersof primaryafferents[.Consistentwiththeprevious studies[172324],itwasobservedunderelectron microscopethatintrathecalinjectionofcapsaicinin adultratselectivelyinduceddegenerationof primaryafferentCfibers.Theterminalsof degeneratedprimaryafferentCfibersinthe substantiagelatinosaappearedcharacteristic electronmicroscopicimages,dispalyingmarked highelectrondensityandoftenformingsynaptic glomeruliwithsurroundingdendrites.The observationthatthecentralterminalboutonsof unmyelinatedorthinmyelinatedprimary afferents[]isfurthersupportedbyourdata. IntrathecaladministrationofNTresultedin analgesiainavarietyofbehavioralassays[.,.t..] butdidnotblockpainbehavior[t]inducedby intrathecalsubstanceP,aneuropeptideinvolvedin nociception.ItisthereforepossiblethatNT analgesiamayoccurviapresynapticinhibitionof primaryafferentneurones.Thishypothesishas beensupportedbyZhangandcoworkerswork[] inwhichtheyhavedemonstratedthatsmall neuronesindorsalrootganglionoftherat expressedmRNAforNTreceptorandthatNTLI— containingterminalscontactednon—synapticallyon thecentralterminalsofprimaryafferentsinthe spinaldorsalhorn.However,theydidnotidentify whethertheprimaryafferentstheterminalsof whichwerecontactedonbyNTLI?-positive terminalswereCfibersintheirstudy.Ithasbeen observedinourconfocallaserscanningmciroscopic andimmunoelectronmicroscopicworkthatNTLI— positiveaxonicterminalsnotonlyformednon— synapticaxoaxoniccontactsbutalsoformed definiteaxoaxonicsynapseswithCfiberterminals whichwereidentifiedbytheselectivityofI—B4's bindingtoCfiberandthespecificityofcapsaicin's endamagingCfiber,whichfurtherprovidedmore m0rphologicalevidencesforthepossibilitythatNT mighthavepresynapticallyinhibitoryeffecton primaryafferentCfibersviaaxoaxonicsynapses and/ornon—synapticaxoaxoniccontacts.Themost ofaxoaxoniccontactsfoundinourworkdidnot showsynapticspecialization,thetypicalsynapses wereveryfew.Infact,itislikelythat neuropeptidesarereleasedanddiffusednon— synaptically[.Thus.NTinthespinaldorsal hornmaybemainlynon—synapticallyreleasedto exertadirectactiononprimaryCafferents. 一 Similartothepriorreports[.'.the asymmetricalaxodendriticsynapsesbetweenNTLI axonicterminalsandunlabelleddendritesaremore frequentlyseenthansymmetricalonesinthe presentstudy.Theasymmetricalaxodendritic synapsesareexcitatory.NTismorelikelytobean excitatoryneuropeptide,whichhasbeenindicated bythepreviouselectrophysiologicalfindingsthat 第2期李和等.大鼠脊髓后角内神经降压素对一级传入C纤维进行突触抑制的形 态学证据233 NTpredominantlyexertsexcitatoryeffectsonthe spinaldorsalneurones[..?.andisfurther supportedbyourresultthattheasymmetricaxo ~NTLI]一dendritic(unlcobelled)synapsesaremore oftenobservedthan mentionedabove,NThas asymmetricones.As apparentanalgesiaatthe levelofthespinalcordandtheaxoaxonicsynaptic andnon—synapticcontactsarerare,the postsynapticactionofNTisthereforelikelytobe moreimportantthanitspresynapticone.Ifthe excitatoryeffectofNTonthespinaldorsal neuronesisinvolvedinanalgesia,theanalgesia wouldbemediatedviaaninhibitoryinterneuron. Additionally,itwasfoundthatNTL1was alsolocalizedwithinsomedendritesinthespinal dorsalhorn,moreover,someNTLI—containing dendriticprofileswereshowntobepostsynapticto thedegeneratedterminals.Thistypeofsynaptic connectionmayberelatedtotheincreaseofNT releaseinresponsetoperipheralnerve stimulation[引. Acknowledgments Theauthorsaregratefulforthephotographic helpofMr.XuejunKangatDepartmentof HistologyandEmbryology,TongjiMedical University,P.R.ofChina.This?