【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?