微量注射cnqx對大鼠臂旁核感官反應的影響

微量注射cnqx對大鼠臂旁核感官反應的影響

在roped，中央nuk膝（cea）是馬蹄蓮的一個發(fā)射包的發(fā)射包的一個發(fā)射包的一個發(fā)射包的信息，從pe面向sultan，從u體制中的一個分配信息，從u體制中的一個分配信息到另一個分配信息。這也是為什么分配的。從遺傳因素到非正統(tǒng)階段，有一個或兩個生命體征，三個特征的痕跡，一個或兩個生命體征的特征，三個特征的痕跡，一個或兩個生命體征的特征，以及三個微帶中的一個或兩個微帶的特征。這也表明，這是一個評估精度的過程，而不是努力或行為的過程。此外，這一過程中的一些跡象也表明，這是一個過程。體制改革或預算活動中的非物質絲沒有形成，這是可以接受的。Inthecentralnervoussystem,glutaminergictransmissionplaysanimportantroleintheneuralmechanismoftasteformation.Ithasbeenshownthatglutamateisanexcitatoryneurotransmittermediatingchordatympani(CT)gustatoryafferentinputtothetaste-responsiveNSTareas,andthatthisneurotransmitterprimarilyactsonAMPAreceptors.Intheinsulargustatorycortex(IGC),thenormalexcitatorytransmissionoftasteafferentsisalsomediatedbyAMPAreceptors.Immunohistochemicalstudieshaveshownthattherearemanyglutamateimmunoreactiveneuronsintheamygdaloidcomplex,includingtheCeA.Thereisevidencethattheseneuronsareinvolvedintheformationofconditionedtasteaversion(CTA),andthatAMPAreceptorsareindispensablefortheacquisitionandexpressionoftheCTA.ThesefindingssuggestthatglutamatereceptorsareinvolvedintheexcitatorytransmissionoftasteintheCeA.However,noreportshavesofarbeenmadetoinvestigatetheroleofglutamatereceptorswithintheCeAinmodulatingthegustatoryresponsesofthePBN.Theaimofthepresentstudywastoassesstheeffectsofmicroinjectionof6-cyano-7-nitro-quinoxaline-2,3-dione(CNQX),aselectiveantagonistofAMPAreceptor,intotheCeAonthetasteresponsesofthePBNneurons.WerecordedresponsesofsinglePBNneurontogustatorystimulibeforeandaftertheipsilateraladministrationofCNQXtotheCeAandfoundthattheresponsesofthePBNneuronstoHClandQHClweresignificantlyinhibitedafterdrugapplication.1杏仁杏仁1.1ualiinstiagesin網(wǎng)絡MaleSprague-Dawleyratsweighing220～290gwerehousedindividuallyinstainlesssteelcageswithlaboratorychowandtapwatercontinuouslyavailableexceptontheexperimentday.Roomlightswereon12hperdayandtemperaturewasmaintainedat(25±1)℃.1.2應用因子trepbn體的細胞系統(tǒng)見表3Theratswereanesthetizedwithethylcarbamate(urethane)(1.4～1.5g/kg,i.p.),andadditionalanestheticwasgivenasneededduringthecourseofeachexperiment.Theanimalwassecuredinastereotaxicinstrumentbyusingnonpunctureearbarsandabitebar.ForaccesstotheCeA,a2-mmtrephineholewascentered3.8～4.4mmlateraltothemidline,2.1～2.8mmposteriortobregma,asdescribedinPaxinosandWatson.AguidecannulawasimplantedintotheCeA.ForipsilateralPBNpenetration,thetrephinewascenteredat9.0～9.7mmposteriortobregmaand1.5～2.1mmlateraltothemidline.ThetransversesinusoverlyingthePBNwasligatedandretracted,andtheexposedtissuewasbathedwithwarmmineraloil.Theelectrocardiogramwascontinuouslymonitoredandtherectaltemperaturewasmaintainedatabout37℃byusingaheatingpadthroughouttheexperiment.1.4抗混合物的定義ExtracellularactivityfromPBNneuronwasisolatedusingaglassmicroelectrodefilledwith2%pontamineskybluein0.5mol/Lsodiumacetate(5～10M?).Duringthesearchprocedure,thegustatoryneuronwasidentifiedbyapplyingtothetonguewithamixtureoffourstandardtastants.Actionpotentialswererecordedthroughconventionalphysiologicalequipmentsconsistingofapreamplifier(MEZ-8201,NihonKohden,Japan),cathode-rayoscilloscope(VC-10,NihonKohden,Japan),recordingsystem(PowerLab/4SP,ADInstruments,Australia),andmicrocomputer(iMac,USA).Uniformamplitudeandwaveformwereusedasthecriteriaforisolationofsingleunit.Onceacellwasidentifiedasatasteresponsiveneuron,gustatoryresponsesweretestedrepeatedlybeforeandaftertheantagonistmicroinjection,andagainafterrecovery.Peri-stimulustimehistograms(PSTHs)weremadebymeansofacomputersystem.1.6規(guī)訓程序4:respese,etity,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,tuning,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,ets,eta的合成,以respityNeuralresponsetoatastestimuluswascalculatedbysubtractingthe5sdischargeratetowaterfromits5sdischargeratetoeachstimulus,beginningwiththeonsetofastimulusinfusion.Anincreaseinfiringrateinthefirst5softhestimuluspresentationthatexceededthespontaneousspikebyatleast50%wasdefinedasaresponse.Tocomparetheeffectsofthepharmacologicalmanipulations,thetasteresponsesfollowingthedrugapplicationwerecomparedwiththatbeforeapplication.Theneuronswereclassifiedasinhibitedoraugmentediftheseresponsemeasuresdifferedfromtheircorrespondingcontrolratesby<20%or>20%,respectively.Basedonitsresponsetoastandardconcentrationofeachofthefoursapidchemicals,eachneuronwascategorizedaccordingtoitsbeststimulus.Sincethemainresponseinbest-stimuluscategoryplaysamoreimportantroleintastecoding,theeffectsofCNQXmicroinjectedintotheCeAontheresponseinbest-stimuluscategorieswereexamined.Toassesstheacross-unitdifferencesamongstimuli,thePearsonproduct-momentcorrelationsacrossstimuliweredeterminedforPBNunitswithandwithoutCNQXmicroinjections.Asanindicationofthebreadthoftuning,anuncertaintymeasurewascalculatedwiththefollowingformula:H=-1.661ΣpIlogpI,wherepIistheproportionoftheresponsetoeachofthestimuliagainstthetotalresponsetoallthestimuli.One-wayANOVAwasappliedwhereitwasappropriateforcomparisonofneuralresponses.Allaverageswereindicatedasthemean±SEM.DataanalysiswasmadebyusingSPSS,andP<0.05wasconsideredsignificant.1.7非生物c.n.相關因子Aftertherecordingsession,acathodeDCcurrent(10～20μA,10～20min)waspassedthroughtherecordingelectrodeinordertodepositadyemark.Thelociofthedyespotswereestablishedhistologically.Theextentsofthedrugadministrationwereobservedfromanterior,medialandposteriorsections.Toidentifythelocusoftheinjectioncannulas,0.5μlofasolutionofPontamineSkyBluedyewasinfusedthroughasimilarinjectioncannulabeforeratswerekilled.TheratswerethengivenanoverdoseofurethanandtranscardiallyperfusedwithPBSfollowedby10%bufferedformalin.Thebrainswereremoved,cutcoronallyin40μmsectionswithafreezingmicrotome,andstainedwithneutralred.2wreng-reefficivumTheexperimentaldatawerefinallyanalyzedfrom33animals,inwhichthemicroinjectionsiteswerefoundtobecorrectlyplacedwithintheCeA.Thedataof21ratswereexcludedbecauseofwrongplacementofthemicroinjectionsites.Figure1illustratestheseinjectionsites.TasteresponsiveneuronswererecordedfromthemedialandlateralPBNandwithinthebrachiumconjunctivum.2.1inpch-pcr反應Theapplicationof0.5μgCNQXinhibitedtheresponsestothecurrentstimulationoftonguein4outof10PBNneuronstested.Thedurationofdrugeffectwasabout10minintheneurons,andtheycompletelyrecoveredin20min(Fig.2B).Acrossthe4cells,themeanfiringrateovera10-minperiodbeforedruginjectionwas(15.19±1.78)Hz,whichsignificantlydecreasedto(9.92±1.03)Hz(F1,7=6.58,P<0.05).Theother6neuronsrecordedshowednochangesafterCNQXinjectionsandPBSinjectionhadnoeffect(Fig.2A).2.2inpch-pcr-4,3,4.3,4.3,4.3,4.3,4.3,4.3,3.4.3,3.4.3,3.4.3,3.4.3,3.4.3,3.4.3,3.4.3,3.4.3,3.4.3,3.4.3,3.4.3,3.4.3,3.4.3,....,................3.3,............................Amongthe33tasteneurons,thespontaneousactivitiesof8neuronswereinhibitedafterCNQXadministration.Meanspontaneousfiringrate[(2.91±0.47)Hz]ofthePBNunitsafterCNQXmicroinjectionwassignificantlylowerthanthat[(4.59±0.64)Hz]beforeinjection(F(1,65)=4.46,P<0.05).Ofthe33PBNtasteneuronsobserved,30respondedtoNaCl,25toHCl,18toQHCl,and13tosucrose.Themeanspike[(10.85±0.82)Hz]tofourbasictastantsfollowingCNQXinjectionsignificantlyreducedcomparedwiththat[(14.36±1.14)Hz]beforedruginjection(F(1,263)=6.23,P<0.05).AfterCNQXapplication,theresponsestoHCl[(12.25±1.37)Hz]andtoQHCl[(6.77±0.85)Hz]wereinhibitedwiththemagnitudessignificantlylowerthanthose[(17.36±2.12)Hzand(10.63±1.65Hz)]obtainedbeforeinjections(P=0.047andP=0.042,respectively).HowevertherewasnosignificantdifferencebetweentheresponsestoNaClandsucrose(Fig.3).Byusingtheuncertaintymeasurement,itindicatedthatthebreadthoftuningwasnotsignificantchangedbefore(0.71±0.05)andafter(0.68±0.04)CNQXinjection.Todeterminetherecoverycourse,thegustatoryresponsesofPBNneuronswerealsoobservedin25minafterdruginjection.Theresultsshowedthatin25minafterCNQXadministration,allactivitiesofPBNtasteneuronsrecovered.Fig.4showsanexampleofonePBNtasteneuronbefore,immediatelyafter,and25minafterCNQXinjectionintotheCeA.Itcanbeseenthatallresponsemagnitudestofourbasictastestimulirecoveredtothebasiclevelintheperiodof25minafterdrugapplication.Basedontheirlargestresponsestothefoursapidstimuli,ofthe33PBNtasteneurons,15wereNaCl-best,10HClbest,5QHCl-bestand3sucrose-best.Accordingtothebest-stimuluscategory,afterCNQXinjection40%NaClbest(6/15),30%HCl-best(3/10)and20%QHCl-best(1/5)neuronsdecreasedtheirresponsestoatleastonebasictastestimulusafterAMPAreceptorswereblocked.ThegustatoryresponsesofNaCl-,HCl-,QHCl-,andsucrosebestneuronsbeforeandafterCNQXinjectionswerecalculatedrespectively(Table1).InHCl-bestandQHCl-bestneruons,themainresponseswereinhibitedwiththefiringratesignificantlyreducingfrom(23.68±2.74)Hzand(25.92±3.41)Hzto(13.94±1.55)Hzand(12.56±1.92)Hz(P<0.01),respectively.Toassessthesimilarityoftheneuronalresponsepatternsevokedbythetastestimuli,thePearsonproductmomentinterstimuluscorrelationswereanalyzed.AsshowninTable2,thecorrelationsbetweentheNaClandtheotherthreetastantsdecreasedafterCNQXinfusionintotheCeAInterstimuluscorrelationsamongtheothertastantswerecomparablebeforeandafterCNQXinjection.3u3000cea標準Inthepresentstudy,thespontaneousactivityandthetasteresponsewereinhibitedinsomePBNneuronsafterCNQXmicroinjectionintotheCeA,andtheresponsestoHClandtoQHCldecreasedwiththemagnitudesbeingsignificantlylowerthanthosebeforeinjection.Accordingtothebest-stimuluscategory,afterCNQXinjection40%NaCl-best,30%HCl-bestand20%QHCl-bestneuronsdecreasedtheirresponsesafterAMPAreceptorswereblocked.InHCl-bestandQHCl-bestneruons,themainresponseswereinhibitedrespectivelyafterdrugmicroinjection.AnalysisofPearsonproduct-momeninterstimuluscorrelationsrevealedthatcorrelationsbetweenNaClandtheotherthreetastantsdecreasedafterCNQXinfusionintotheCeA,suggestingthattheCeAinjectionmakestheresponsetoNaClmoresalientamonggustatoryresponses.TheresultsofthepresentstudysuggestthatglutamateisanexcitatoryneurotransmitterintheprocessingoftasteinformationbetweentheCeAandPBN.PreviousstudieshavedemonstratedthatCeAcanmodulatethegustatoryresponsesinthePBNbyelectricalstimulationandelectrolyticlesionsoftheCeA.Immunohistochemicalstudieshaveshownmanyglutamateimmunoreactiveneuronsintheamygdaloidcomplex,includingtheCeA.Thereissubstantialevidencethatglutaminergictransmissioninthecentralnervoussystemplaysanimportantroleintheneuralmechanismoftasteformation.AMPAreceptorsplayanimportantroleininformationprocessingandtherebycontributetotheworkingtastefunction.ThepresentresultssuggestthatthedescendingmodulationfromCeAtoPBNmaybemediatedpartlythroughAMPAreceptorHowever,wecannotexcludethepossibilitythattheexcitatoryafferentintheCeAwasblockedfollowingCNQXapplication.Inthepresentstudy,thespontaneousandtaste-evokedactivitiesinpartofthePBNneuronsreducedfollowingtheinjectionofCNQXintotheCeA.Itsuggeststhattheremaybeglutamate-medicatedexcitatoryinputfromtheCeAtothePBN,and/orsomesynapsetransmissionswithintheCeAwereblockedsothattheresponsesofthePBNneuronswereaffected.OurpreviousstudyshowedthattheCeAstimulationmainlydecreasedtheresponsesofPBNneurontoaversivetastestimuli,whiletheCeAlesionsmainlyincreasedtheseresponses.However,wealsoobservedsomeoppositeeffects.Forexample,CeAstimulationexcitedafewofPBNneurons.SointheCeAtheremaybeexistdirectorindirectexcitatoryprojectionstoPBNortheCeAinfluencesthePBNneuronsviasomeotherforebrainregionsbecausetheamygdalaconnectsnotonlywiththePBNbutalsowithothertaste-responsiveregions,suchasIGCandhypothalamus.ThesenucleicanindirectlymodulateorinfluencethegustatoryresponsesofthePBN.Additionally,anindirecteffectmightbeappliedtothePBNviaforebraininputtoNST.CNQX(Sigma-AldrichCompany),aselectiveAMPAreceptorantagonist,wasdissolvedin99.5%dimethylsulfoxide(DMSO)(Sigma-AldrichCompany),andthendilutedwithPBS(pH=6.8)toachieveafinalconcentrationof0.5μg/0.5μlCNQX.Thetipofthemicroinjectiontubeprotruded1.0mm