大鼠脑啡肽钙结合蛋白双标神经元表达的研究

大鼠脑啡肽钙结合蛋白双标神经元表达的研究

calimate（cab）是一个中期蒙太奇不规则，甚至可以蒙太奇。cal斌din-b28k（cm）、calnin（cr）和paramium（pv）是三个基本的马蹄莲发射光系统的共线性成员。基本信息的传输和改进（cc）；我们的预vi性做法是：（1）cac-u和iii-kan转化为sdh，但只有一个小数量的非修正数量（lam-a）u；（2）部分cai、cr和v-cm授权的区域承包经营权（cd），以及线微信息的随机性（cc）u，随机性。（3）cp也是祈祷。硫斯、itan、pd和cr是区域承包经营权。这是第四种类型的区域承包经营权。GABA-immunoreactive(GABA-IR)andglycine-IR(Gly-IR)terminalsaredenselydistributedinthesuperficiallaminaeoftheMDH,especiallyinlaminaⅡ,andGABAandGlyarecrucialfornoxiousinformationmodulation.Glycinetransporter2(GlyT2)isareliablemarkerforglycinergicneuronalstructures,andGlyT2-IRstructuresarepresentthroughoutthespinalcord,andtheirlaminadistributionsmatchthatofGly-enrichedneurons.5-HTisamajortransmitterinvolvedinthecentralendogenouscontrolofnociception,andthesuperficiallaminaeoftheMDHareheavilyinnervatedby5-HT-IRfibersandterminalsthatmainlyoriginatefromtherostralventromedialmedulla(RVM).Enkephalin(ENK)-IRneuronsandterminalshavebeenfoundthroughoutthedorsalhornSPhasbeenfoundintheunmyelinatedorthinlymyelinatedprimaryafferentfibersthatterminatemainlyinthesuperficiallaminae.NeuronsinthesuperficiallaminaeofthedorsalhorncontactedbySP-ergicprimaryafferentterminalsareconsideredtoreceivenociceptiveinformationdirectlyfromtheperiphery.TheexpressionofFOSproteininducedbythesubcutaneousinjectionofformalinintoperipherytissuesisconsideredtobeausefulneuroanatomicaltooltodemonstratetheactivatedneuronsinvolvedinnociceptiveinformationtransmission.PreviousstudiesalsoshowedthatnoxiousstimulationofdifferentorofacialsitesinducedFOSproteinexpressionintheipsilateralmedullarydorsalhorn,mainlyinlaminaeⅠandⅡ,andadditionallyinlaminaⅢ.ToinvestigatewhetherCB-,CR-andPV-containingneuronsinthesuperficiallaminaeoftheMDHreceiveanorofacialnociceptiveinnervationandarecontactedbythe5-HT-,GABA-,Gly-andENK-IRterminals,immunofluorescencehistochemicaltriple-stainingtechniquewasperformedintheMDHoftherattoexploretheconnectionsbetween5-HT-,GABA-,GlyT2-orENK-containingterminalsandCaBPs/FOSdouble-labeledneurons,andalsotodefinetheconnectionbetween5-HT-,GABA-,GlyT2-orENK-ergicterminals,SP-ergicterminalsandCaBPs-containingneuronsintheratMDH.Thecontactswerefinallyfurtherconfirmedbyimmuno-electronmicroscopy.杏仁杏仁1.phosphwellingingpbTwenty-sixadultmaleWistarratsweighing200-300gwereused.Allsurgicalprocedureswereperformedunderanesthesiabyintraperitonealinjectionofsodiumpentobarbital(40mg/kg,i.p.).Nineoftheratswerelightlyanesthetizedwithethyletherandthenavolumeof0.1mlof5%formalindissolvedindistilledwaterwasinjectedsubcutaneouslyintotheunilateralupperlip.Twohourslater,theratswereperfusedtranscardiallywith100mlof0.025mol/LPBS(pH7.4),followedby500mlof0.1mol/Lphosphatebuffer(PB;pH7.4)containing4%paraformaldehydeand75%picricacid.Aftertheperfusion,thebrainswereremoved,post-fixedinthesamefreshfixativeandplacedin0.1mol/LPBcontaining30%sucroseovernightat4°C.Thelowerbrainstemswerecutinto20-μmthickfrontalsectionsthatwerecollectedas14consecutiveseries(GroupA1-14)andkeptin14dishescontaining0.01mol/LPBS.AllofthesectionswerewashedwithPBS.Elevenoftheratswereperfusedtranscardiallyinthesamewayasmentionedabovebutwithoutformalinstimulation.ThelowerbrainstemscontainingtheMDHwerecutseriallyinto20-μmthickfrontalsectionsthatwerecollectedas23consecutiveseries(GroupB1-23)andkeptin23dishescontaining0.01mol/LPBS.Theothersixratswereusedforimmuno-electronmicroscopicresearch.Theywereperfusedtranscardiallywith100mlof0.9%saline,followedwith500mlof0.1mol/LPB(pH7.4)containing4%paraformaldehyde,0.05%glutaraldehydeand75%saturatedpicricacid.Aftertheperfusion,theirbrainswereremovedimmediately,postfixedfor4to6hrsin0.1mol/LPB(pH7.4)containing4%paraformaldehyde,andthenputinto0.1mol/LPBcontaining30%sucroseovernight(4℃).Thelowerbrainstemswerecutinto50-μmthickfrontalsectionswithamicroslicer(Dosaka)andcollectedin12dishes(GroupC1-12)containing0.025mol/LPBS(pH7.4).2.mo测定常用参数Thefirsttothirdserialsections(groupB1-3)fromtheratswithoutformalin-pretreatmentwereusedforimmunohistochemicalstainingforCB,CRandPV.Thesectionswereincubatedatroomtemperature(RT)sequentiallywith:(1)mouseanti-CB(1∶5000;Sigma),mouseanti-CR(1∶1000;Chemicon)ormouseanti-PV(1∶8000;Sigma)overnight;(2)biotinylateddonkeyanti-mouseIgG(1∶100;Chemicon)for3hrs;and(3)avidin-biotin-horseradishperoxidasecomplex(ABCEliteKit)(1∶100;Vector)for1-3hrs.TheprimaryandthesecondaryantibodiesweredilutedwithPBScontaining0.3%TritonX-100and2.5%normaldonkeyserum(PBS-NDS)(pH7.4),andtheABCcomplexwasdilutedwith0.3%TritonX-100in0.01mol/LPBS(PBS-X)(pH7.4).Subsequently,thesectionsweretreatedwith0.02%diaminobenzidinetetrahydrochloride(DAB)and0.001%H2O2in0.05mol/LTris-HClbuffer(pH7.6).Thefourthserialsections(groupB4)wereusedforNisslstaining.Allsectionsweremountedontogelatin-coatedglassslides,andobservedunderlightmicroscope(Olympus).3.rabcitizat21500Thefifthtoeighthserialsections(GroupB5-8)fromtheratswithoutformalin-pretreatmentwereusedforimmunohistochemicalstainingforGABA,GlyT2,5-HTandENK.ThesectionswereincubatedatRTsequentiallywith:(1)guinea-piganti-GABAantiserum(1∶1000;Chemicon),guinea-piganti-GlyT2(1∶1000;Chemicon),rabbitanti-5-HT(1∶1000;Chemicon)orrabbitanti-ENK(1∶3000;Chemicon)overnight;(2)biotinylateddonkeyanti-guinea-pigorrabbitIgG(Jackson)for3hrs;and(3)ABCcomplex(1∶100;Vector)for1-3hrs.TheprimaryandsecondaryantibodiesweredilutedwithPBS-NDS,andtheABCcomplexwasdilutedwithPBS-X.Subsequently,thesectionsweretreatedwith0.02%DABand0.001%H2O2in0.05mol/LTris-HClbuffer(pH7.6)for20-30minatRT.Theninthserialsections(GroupB9)wereusedforNisslstaining.Thesectionsweremountedontogelatin-coatedglassslidesandexaminedunderanOlympuslightmicroscope.4.ratg3-4.The10thto21stserialsections(GroupB10-21)fromtheratswithoutformalin-pretreatmentwereusedforimmunofluorescencehistochemicaltriple-stainingforCaBPs/GABA/SP,CaBPs/GlyT2/SP,CaBPs/5-HT/SP,CaBPs/ENK/SP,respectively.TheywereincubatedatRTsequentiallywith:(1)Firstantibodies(showedinTab.1)inPBS-NDSfor20-36hrs;(2)10mg/mlbiotinylateddonkeyanti-ratIgG(1∶200;Chemicon)inPBS-NDSfor3-5hrs;(3)10%normalratserumfor30min;and(4)amixtureof5mg/mlTexasRed(TR)-labeledavidin-D(Vector),10μg/mlindodicarbocyanine(Cy5)-labeledgoatanti-guinea-pigorrabbit(Jackson)and10μg/ml5-(4,6-dichlorotriazinylamino)fluorescein(DTAF)-labeledgoatanti-mouseIgG(Chemicon)inPBS-Xfor3hrs.ThesectionswerewashedrepeatedlywithPBS-Xbetweeneverytwosteps.Thesectionsweremountedontocleanglassslides,andcoverslippedwithPBScontaining50%glycerinand2.5%triethylenediamine(anti-fadingreagent).Allsectionswereobservedwithaconfocallaser-scanningmicroscope(LSM410;Zeiss)byusingexcitationlaserbeamsof633,543,and488nmwithappropriateemissionfiltersforCy5(670-810nm),TR(590-610nm),andDTAF(510-525nm).Thesectionsfromthetwenty-secondseries(GroupB22)weremountedontogelatin-coatedglassslidesandusedforNisslstaining.5.普通存出/petctpbs-ndsThefirsttotwelfthserialsections(GroupA1-12)fromtheratswithformalin-pretreatmentwereusedforimmunofluorescencehistochemicaltriple-stainingforGABA/FOS/CaBPs,GlyT2/FOS/CaBPs,5-HT/FOS/CaBPsorENK/FOS/CaBPs,respectively.TheywereincubatedatRTsequentiallywith:(1)Firstantibodies(showedinTab.2)inPBS-NDSfor20-36hrs;(2)10mg/mlbiotinylateddonkeyanti-mouseIgG(1∶200;Chemicon)inPBS-NDSfor3-5hrs;(3)10%normalmouseseruminPBS-Xfor30min;and(4)amixtureof5mg/mlfluoresceinisothiocyanate(FITC)-labeledavidin-D(Vector)/10μg/mlCy5-labeleddonkeyanti-guinea-pigorrabbit(Jackson)and10μg/mlTR-labeleddonkeyanti-sheepIgG(Chemicon)inPBS-Xfor3hrs.ThesectionswerewashedwithPBS-Xbetweeneverytwosteps.Thesectionsweremountedontocleanglassslides,andcoverslippedwithPBScontaining50%glycerinand2.5%triethylenediamine.Thesectionsimmunostainedbythetriple-immunofluorescencehistochemicalmethodwereobservedwithaconfocallaser-scanningmicroscope(LSM410;Zeiss)byusingexcitationlaserbeamsof633,543,and488nmwithappropriateemissionfiltersforCy5(670-810nm),TR(590-610nm),andFITC(510-525nm).Sectionsfromthethirteenthseries(GroupA13)weremountedontogelatin-coatedglassslidesandusedforNisslstaining.6.u2004范围Sectionsfromthefirsttotwelfthsets(GroupC1-12),cutwiththeDosakamicroslicer,wereplacedin0.05mol/LPB(pH7.4)containing25%sucroseand10%glycerolfor30minforcryoprotection,followedbyfreeze-thawingwithliquidnitrogenforenhancementofpenetrationofantibodyintheimmunohistochemicalreaction.Subsequently,thesectionswereincubatedwith0.05mol/LTris-HClbuffered-saline(TBS;pH7.4)containing20%normalgoatserumfor1hrtoblocknon-specificimmunoreactivity,andthenprocessedforimmunohistochemicaldouble-staining.Theimmunogold-silvermethodforCaBPswascombinedwiththeABCmethodfor5-HT,GABA,GlyT2orENK.Thesectionswereincubatedwithfirstantibodies(showedinTab.3)for24hrs.TheincubationmediumwasTBScontaining2%normalgoatserum(TBS-NGS).ThesectionsthenwerewashedinTBSandincubatedfor14-16hrsatRTwithamixtureofgoatanti-mouseIgGconjugatedto1.4nmgoldparticles(1∶100;Nanoprobes)andbiotinylateddonkeyanti-guinea-pigIgG(1∶200;Jackson)oranti-rabbitIgG(1∶200;Jackson).EachantibodywasdilutedwiththeTBS-NGS.Subsequently,thesectionsweretreatedasdescribedelsewhere.Briefly,thesectionswereprocessedfor:(1)postfixationwithglutaraldehyde,(2)silverenhancementwithHQSilverKit(Nanoprobes),(3)incubationwithABCEliteKit(1∶50;Vector),(4)visualizationof5-HT-,GABA-,GlyT2-orENK-immunoreactivitywith0.02%DABand0.0003%H202dissolvedin0.05mol/LTB(pH7.6),(5)osmification,(6)counterstainingwithuranylacetate,and(7)flat-embeddinginEpon812afterdehydrationandmountingontosilicon-coatedglassslides.Thenthesuperficialpartsofthemedullarydorsalhorn,containingdense5-HT-,GABA-,GlyT2-orENK-immunoreactiveterminalsandCaBPs-IRneurons,wereselectedandpreparedforelectronmicroscopy.Thetissuesamplesoftheselectedregionswerecutintoultrathinsectionsonanultramicrotome(LKB).Thesesectionsweremountedontosingle-slotgridscoatedwithpioloformmembrane(AgarScientific)andexaminedwithanelectronmicroscope(CM100,Philips).7.peri中小型项目ThesectionsfromGroupA14andGroupB23wereusedforcontrolexperimentsfortheimmunofluorescencehistochemicalstaining.WhenoneoftheprimaryantibodieswasomittedorreplacedwithnormalIgG,noimmunoreactivityfortheomittedorreplacedantibodywasfound.产品系统1.pv-irneuronson,u.4,3.3.4和3.5.3.3.4.3.3.4和3.5.5.3.3.3.5.5.5.5.3.3.3.4和3.5.5.5.5.5.5.3.5.5.3.5.3.3.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.3.5.5.5.5.5.3.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.3.5.5.5.5.5.5.5.5.5.5.3.5.5.5.5.5.5.5.5.5.5.5.3.3.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.NeuronalcellbodiesshowingCB-,CR-orPV-immunoreactivitywerefoundinlaminaeⅠ-Ⅲofthemedullarydorsalhorn(MDH)(Fig.1).ThemajorityoftheseCB-,CR-orPV-IRneuronsweredistributedinlaminaⅡ,especiallyinitsinnerpart(laminaⅡi),andasmallernumberwerelocatedinlaminaeⅠandⅢ.PV-IRneuronalcellbodies(Fig.1c)werelessnumerousthanCB-orCR-IRneurons(Figs.1a,b)inalllayersoftheMDH,andtheratioofCB-,CR-andPV-IRMDHneuronswasroughly3∶2.5∶1.CB-,CR-,andPV-IRneuronsindifferentlayershowedsimilarmorphologicalfeatures.Theyusuallyhadround,fusiformortriangularneuronalcellbodieswithamaximaldiameterof10-15μm.Additionally,asmallnumberoflargerCB-,CR-,andPV-IRneuronalcellbodieswereoccasionallyobservedinlaminaeⅠandⅢoftheMDH.TheselargerMDHneuronshadfusiform,triangularormultipolarshapedcellbodieswithamaximaldiameterof20-35μm.ThesuperficiallaminaeoftheMDHwerealsodenselypackedwithpunctuateprofilesforCB-andCR-IRfibersandterminals,whereasdensePV-IRfibersandterminalswereprincipallyencounteredinlaminaⅡi(Fig.1).2.glyt2-ir持续过错设计laminealamina15-HT-IRfibersandterminals,whichshowedbeadedstructures,weremainlylocatedinlaminaeⅠandⅡoftheMDH,andweredenserinlaminaⅠthanthatinlaminaⅡ.InlaminaⅢ,5-HT-IRfibersandterminalsweresparse(Figs.2a,a’).GABA-IRterminalswerepresentdenselyintheneuropiloflaminaeⅠandⅡ.ThedensityofpositivestructuresgraduallydecreasedfromcaudaltorostralMDHandfromthesuperficialparttothedeeperpart(Figs.2b,b’)whereGABA-IRfiberswereseldomseen(Figs.2b,b’).GlyT2-IRterminalsintheneuropilweresparseinlaminaeⅠ-ⅢofMDH,somewhatdenserinthesuperficialpartthanthatinthedeeperpartoflaminaⅢ(Figs.2c,c’).ENK-IRfibersandterminalsweredenselylocatedinlaminaeⅠandⅡofMDH.InlaminaⅢ,onlysparselydistributedENK-IRfibersandterminalswereencountered(Figs.2d,d’).SP-IRfibersandterminalsweremainlylocatedinlaminaeⅠ-ⅢofMDH.ThedensityofSP-IRfibersandterminalsinlaminaeⅠandⅢwaslowerthanthatinlaminaⅡ(Fig.4).InlaminaⅢ,onlysparselydistributedSP-IRfibersandterminalswerefound.3.相关和同基亚甲基的风基因子Afterinjectionofformalinintotheupperlip,formalininjectioninducedFOSproteinexpressionwasobservedinnucleioftheneuronsinthesuperficiallaminaeoftheMDH.FOS-IRneuronsweremainlyobservedintheMDHipsilateraltotheformalininjection,andweremostfrequentlyconfinedwithinlaminaⅡ,althoughsomeFOSproteinpositiveneuronswerealsofoundinlaminaeⅠandⅢ(Figs.3a-f)andcontrolateralMDH.NeuronalcellbodiesshowedimmunopositivestainingforCBandFOS(CB/FOSdouble-labeledneurons),CRandFOS(CR/FOSdouble-labeledneurons)weremainlyfoundinlaminaeⅠandⅡwithoval,triangular,andfusiform-shapedneuronalcellbodies(Figs.3a-d),whereasPV/FOSdouble-labeledneuronswerechieflyfoundinlaminaⅡandⅢwithoval,triangular,andfusiform-shapedneuronalcellbodies(Figs.3e,f).Noparticularmorphologicalcharacteristicswerefoundamongthedoublyimmunostainedneurons.4.cr/fosoUndertheconfocallaser-scanningmicroscope,5-HT-,GABA-,GlyT2-andENK-IRterminalswerefoundtomakeclosecontactswithsomeoftheCB/FOS,CR/FOSorPV/FOSdouble-labeledneuronsinthesuperficiallaminaeoftheMDH,especiallyinlaminaⅡ(Figs.3a-f).5.体现原理即初产物2-,lma-4cInthesuperficiallaminaeoftheMDH,someSP-IRterminals,5-HT-,GABA-,GlyT2-,orENK-IRterminalsmadecloseappositionswiththeneuronalcellbodiesanddendritesofCB-,CR-orPV-IRneuronsinlaminaⅡ(Figs.4a,b,d).Afew5-HT-,GABA-,GlyT2-,orENK-IRterminalsalsohadclosecontactswithCB-andCR-IRneuronalcellbodiesanddendritesinlaminaeⅠandⅢ(Fig.4c).6.synap研磨法dex,ws5-HT-,GABA-,GlyT2-andENK-IRaxonterminalswerecharacterizedbythepresenceofelectrondenseDABreactionproductsadheringtotheoutersurfaceofcellorganellessuchasmitochondria,synapticvesiclesandtheinnersurfaceoftheplasmamembrane(Figs.5a-d).CB-,CR-andPV-IRneuronalcellbodiesanddendriticprofileslabeledbysmallirregularblackgold/silvergrainsusuallylocatedinthecytoplasmoronthecellmembrane(arrowsinFig.5).5-HT-,GABA-,GlyT2-andENK-IRaxonterminalscontainedpleomorphicsynapticvesicleswerefoundfrequentlyontheCB-,CR-andPV-IRdendriticprofilesandneuronalcellbodiesandprincipallytomakesymmetricsynapticconnections(trianglesinFig.5).cr-orpv-immotoreactityofficici三维建模Themedullarydorsalhorn(MDH)istheprincipalterminationsiteoftheprimarynociceptiveafferentfibersthattransmitnociceptiveinformationfromtheorofacialregiontothecentralnervoussystem(CNS).Descendingfibersfromthemidbrainperiaqueductalgray,brainstemraphenucleiandrelatedstructuresofthereticularformation,whichareinvolvedininhibitionofthenociceptiveinformationtransmissionfromtheperipherytotheCNS,alsoterminatepredominantlyinthesuperficiallaminae(laminaeⅠandⅡ)oftheMDH.TheseresultssuggestthattheMDHplaysimportantrolesinnociceptionandantinociception.PreviousimmunohistochemicalandimmunofluorescencehistochemicalstudieshavedemonstratedthatimmunoreactivityforeachoftheCaBPs,CB-,CR-orPV-immunoreactivityislocatedindifferentsubsetsofneuronsindifferentlaminaeoftheratMDH.TheseresultsindicatethatneuronsexpressingdifferentCaBPsmayhavedifferenteffectsontheneuronalactivitiesrelatedtonoxiousinformationtransmissionandmodulation.ThereisconsiderableevidencefortheinvolvementofSPintheprocessingofnociceptiveinformationintheregionofthefirstsensorysynapseinthesuperficiallaminaeofthemedullaryandspinaldorsalhorns.TheneuronsinthesuperficiallaminaeofthedorsalhornincontactwithSP-ergicprimaryafferentterminalsareconsideredtoreceivenociceptiveinformationdirectlyfromtheperiphery.ThenociceptivenatureofneuronsintheMDHcanbeidentifiedbyexpressionofFOS-immunoreactivityafterchemicalirritationoftheupperlips.AlthoughFOS-proteinisnotconsideredasanexclusivemarkerforcentralnociceptiveneurons,ithasoftenbeenusedforinvestigationintosomatotopyintrigeminalnociceptivepathways.GABAandglycine(Gly)havebeenobservedinneuronsofthesuperficialpartofthedorsalhornandconsideredastwoclassicalinhibitorytransmitters.IontophoresisofGABAorGlyinhibitsneuronalactivities,notonlyonspinaldorsalhornintrinsicneuronsbutalsoonspinothalamictractneurons.PreviousstudieshavedemonstratedthatGABAorGlyplayimportantrolesinthemodulationofprimarynociceptivetransmission.Previousdatahavesuggestedthatglycintransporter2(GlyT2)isexpressedinpresynapticelementsofglycinergicneuronsandplaysamajorroleintheterminationoftheinhibitoryeffectofGlyinthebrainstemandspinalcordofvertebrates.Thus,GlyT2-immunoreactivityisconsideredasareliablemarkerforterminalsofglycinergicneurons.Serotonin(5-HT)isamajortransmitterinvolvedinthecentralendogenouscontrolofnociception.TheMDHisheavilyinnervatedby5-HT-IRfibersandterminals,whichmainlyoriginatefromtherostralventromedialmedulla(RVM),includingthenucleusraphemagnus(NRM)anditssurroundingreticularformation.5-HT-IRterminalsmakeaxo-somaticandaxo-dendriticsynapseswithnociceptiveneuronsthatprojecttothethalamusandlocalcircuitneuronsintheMDH.Inthespinaldorsalhorn,5-HTdepressestheexcitatoryeffectsevokedbyglutamateandnociceptivepinchstimulationanddecreasestheneuronalreactiontonociceptivestimulation.Enkephalin(ENK)isaninhibitoryneurotransmitteroriginatingfromtheendogenouspaincontrollingsystemandfrominhibitorylocalcircuitneuronswithintheMDH.ENKcanexertitsinhibitoryeffectthroughboth