白藜芦醇对下丘脑脑片室旁核神经元放电的影响

白藜芦醇对下丘脑脑片室旁核神经元放电的影响

resversion（3.4），5-再生，自然发生池，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，生命促进计划，促进计划，促进政策，母细胞糖、发芽肌、微丝马蹄莲和亚麻仁碱母活力。不同的东西，中部亚麻碱发生区的u25.。Itiswellknownthatthehypothalamicparaventricularnucleus(PVN)playsanimportantroleincardiovascularintegration,anditispossiblethatresveratrolmayexertitscardiovascularactionthroughthisarea.Therefore,toexaminetheeffectsofresveratrolonthespontaneousdischargeofPVNneuronswillcontributetounderstandingtheeffectsofresveratrolontheCNS.TheaimofthepresentstudywastoexaminetheeffectsofresveratrolontheelectricalactivityinratPVNneuronsinhypothalamicslicepreparationsusingextracellularrecordingtechniqueandtoelucidatethemechanisminvolved.1杏仁杏仁1.1SlicepreparationAllexperimentswereperformedonPVNneuronsinhypothalamicslicespreparedfrommaleSprague-Dawleyrats(18-24d).Ratswererapidlydecapitated,andtheforebrainswereremovedandimmersedintoice-coldoxygenatedartificialcerebrospinalfluid(ACSF).ThecompositionofACSFwas(inmmol/L)NaCl124,KCl3.5,KH2PO41.24,MgSO41.3,CaCl21.5,NaHCO318andglucose20;pH7.40-7.45.Thehypothalamuswasdissectedaccordingtoanatomicsigns(Fig.1)andthensliced(thicknessof350-500μm).Thesliceswerepre-incubatedinACSFaeratedwith95%O2and5%CO2for60-90minatroomtemperature(25oC)beforerecording.Asingleslicewasthentransferredandplacedonnylonnetinachamber(1mL)andimmobilizedwithnylonnetontheuppersurfaceoftheslice.Thepreparationwascompletelysubmergedintheperfusatewhichmaintainedat33-35oCandperfusedconstantlyatarateof2mL/min.1.2trodequeh-n相关定义Extracellularsingle-unitrecordingsfromPVNneuronsinhypothalamicsliceswereobtainedwithglassmicroelectrodesfilledwith0.5mol/Lsodiumacetatecontaining2%pontamineskyblue.Microelectrodeswerepulledfrommicropipetteswithaninsidediameterof1mm.Withthehelpofmicroscope,microelectrodeswereperpendicularlyinsertedinextracellulargapofPVNneurons.Thecuspidatediameterofmicroelectrodeswaslessthan1µm.Directcurrentresistanceofthemicroelectrodewas5-10MΩ.Electricalsignalswereamplifiedbyamicroelectrodeamplifier(MEZ-8201,NihonKohden),displayedonadualbeamoscilloscope(VC-9,NihonKohden),andanalyzedbyaprogramforhistogram(exploitedbyourdepartment)onacomputer.Columniationofthefrequencyhistogramwasengenderedevery10s.1.3ationsotechrasedAfterastablespontaneousdischargehadbeenobtainedforatleast10minandrecordedfor3-5minascontrol,achemicalinfusionstarted.Neuronswereclassifiedas“excited”or“inhibited”ifthefiringrateafterapplicationofchemicalsincreasedordecreasedbymorethan20%.Theprotocolsweredividedintothefollowinggroups:(1)resveratrol(n=29):Superfusionwithresveratrol(0.05,0.5,5.0μmol/L)tosuperfusatefor2min;(2)L-glutamate+resveratrol(n=8):AfterpretreatmentwithL-glutamate(0.2mmol/L),resveratrol(5.0μmol/L)wasadministeredfor2mininthepresenceofL-glutamate;(3)BayK8644+resveratrol(n=8):AfterpretreatmentwithBayK8644(0.1μmol/L),resveratrol(5.0μmol/L)wasadministeredfor2mininthepresenceofBayK8644;(4)L-NAME+resveratrol(n=8):AfterpretreatmentwithL-NAME(50μmol/L),resveratrol(5.0μmol/L)wasappliedfor2mininthepresenceofL-NAME.1.4ach&mertb等的so岩与sols制备sof-sofsreficidisence,l-glut4,龙-关于4,5,4,7,4,7,4,7,4,4,7,4,7,7,4,4,7,4-dige-l-so,7,4,7,7,7,7,7,7,7,7,7,7,7,7,7,7,7,7,7,7,7,7,7.4.,7.4.3.,7.4.,7.4.,7.4.3.,7.4.,7.4.,7.4.,7.4.,7.4.,7.4.,7.4.,7.4.3.,7.4.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4和3.4.3.4.3.4.3.4.3.4.3.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.3.3.3.4和3.4.3.3.3.3.3.3.3.3.3.3.3.3.3.3Resveratrol,BayK8644andL-NAMEwerepurchasedfromSigmaCompany.L-glutamatewaspurchasedfromShanghaiZhengxiangChemicalInstitute.Foreachexperiment,allchemicalsandsolutionswerefreshlyprepared.L-glutamateandL-NAMEweredissolvedbyACSF.Resveratrolwasfirstdissolvedindimethylsulfoxide(DMSO)anddilutedinACSF;thefinalconcentrationofDMSOintheperfusingsolutionwaslessthan0.004%.BayK8644wasfirstdissolvedin99%ethylalcoholanddilutedinACSF,andthefinalconcentrationofethylalcoholintheperfusingsolutionwaslessthan0.05%.1.5t.4etrafter-t.5回生statityeart-pcrstatre兴co治理/st-tosstatingquertwrackrewellingspsosAlldatawerepresentedasmeans±SEM.ThedifferencesoftheparametersbetweenbeforeandafterchemicalapplicationwereanalyzedbypairedStudent’st-test.Differencesbetweengroupswereassessedbyone-wayANOVAandfurtheranalyzedusingqtest.StatisticalanalyseswereperformedusingSPSS11.0softwareforWindows.P<0.05wasconsideredstatisticallysignificant.2pvnneurespartgeFifty-threespontaneouslyactivesingle-unitswererecordedinPVNneuronsfrom53slicepreparations.Thepatternsofdischargewereirregular.MeandischargerateofPVNneuronswas(2.93±0.05)Hz.2.1hztoret选择In9single-unitsofhypothalamicslices,afterapplicationofresveratrol(0.05μmol/L)tothesuperfusatefor2min,thedischargerateof8/9(88.8%)slicesdecreasedsignificantlyfrom(3.01±0.12)Hzto(1.96±0.08)Hz(P<0.01),whilethatof1/9(11.1%)neuronsshowednochange.Afterapplicationofresveratrol(0.5μmol/L)tothesuperfusatefor2min,thedischargerateofall10neuronsdecreasedsignificantlyfrom(2.83±0.07)Hzto(1.22±0.04)Hz(P<0.01).In10slices,afterapplicationofresveratrol(5.0μmol/L)tothesuperfusatefor2min,thedischargerateofall10/10(100%)slicesdecreasedsignificantlyfrom(2.95±0.07)Hzto(0.45±0.04)Hz(P<0.001).Theinhibitoryeffectsofresveratrolonspontaneousdischargerate(SDR)inhypothalamicsliceswereconcentration-dependent(Fig.2).2.2回生的requentraftn7.In8hypothalamicslices,pretreatmentwithL-glutamate(0.2mmol/L)ledtoamarkedincreaseintheSDRinallslicesfrom(2.59±0.12)Hzto(5.14±0.24)Hz(P<0.01)inanepileptiformpattern(highlyfrequentandrepeateddischarge,fulminantdischargewithdisconnectedoutbreakorapackofvariousdischarge).Resveratrol(5.0μmol/L)significantlyattenuatedtheincreasedSDRinall8slicesfrom(5.14±0.24)Hzto(2.71±0.13)Hz(P<0.01)(Fig.3).2.3restitetity国际专利基因循环/回归系统sraceda4.120.12.AsshowedinFig.4,perfusionoftheselectiveL-typecalciumchannelagonist,BayK8644(0.1μmol/L),inducedasignificantincreaseinthedischargerateinall8slicesfrom(2.73±0.10)Hzto(4.12±0.12)Hz(P<0.01).Theincreaseddischargesinall8slicesweresignificantlydeclinedbyapplicationofresveratrol(5.0μmol/L)from(4.12±0.12)Hzto(2.05±0.11)Hz(P<0.001).2.4perfus穿透力Applicationofthenitricoxidesynthase(NOS)inhibitorL-NAME(50μmol/L)intheperfusatesignificantlyincreasedtheSDRinall8slicesfrom(2.44±0.07)to(3.59±0.11)Hz(P<0.01).Resveratrol(5.0μmol/L)significantlyreducedtheincreaseddischargerateinall8slicesfrom(3.59±0.11)Hzto(2.16±0.12)Hz(P<0.01)(Fig.5).3whichinciutity法律ratneuron,whichindiphinsicipalsThepresentstudydemonstratesthatresveratrolinhibitsSDRofPVNneuronsinhypothalamicslicesinaconcentration-dependentmanner,andattenuatesL-glutamateinducedandBayK8644-inducedincreaseindischargeofPVNneurons.ThisinhibitoryeffectofresveratrolcannotbeaffectedbythepresenceofL-NAME.ItisknownthatL-glutamateisamajorexcitatoryaminoacidandexcessiveL-glutamateinducesinjuryofneuronsintheCNS.L-glutamatehaseffectsontheN-methyl-D-aspartate(NMDA)andnon-NMDAsubtypereceptorsbutmainlyactivatestheNMDAreceptorsandincreasescalciuminfluxandtheNMDAreceptor-mediatedelevationincalciuminfluxwasshowntoinduce“epilepsy”inculturedratneurons..Muchevidenceindicatesthatresveratrolmayparticipateinneuronalprotection,suchasprotectingdopaminergicneurons,andattenuatingexcitotoxicityinducedbykainicacid,whichisknowntocausesevereconvulsionasaconsequenceofglutamatereceptorstimulation.OurpreviousstudieshaveshownthatresveratrolinhibitsL-glutamate-inducedincreaseindischargeinthehippocampalandsubfornicalorganslicepreparations.Inthepresentstudy,resveratrolsignificantlyreducedepileptiformdischargesinducedbyL-glutamateandreversedtheincreaseddischargesinducedbyBayK8644.ConsideringBayK8644isaselectiveL-typevoltage-gatedcalciumchannelagonist,theinhibitoryeffectofresveratrolmightbemainlyduetotheblockadeofL-typevoltage-gatedcalciumchannelandsubsequentinhibitionofcalciuminflux.TheseresultssuggestthatresveratrolreducesL-glutamateinducedandBayK8644-inducedincreaseindischargeofPVNneuronsinhypothalamicslicesandexertsneuroprotectiveactionstosomeextent.NOisanovelmessengerintheCNS,whichinhibitsCa2+influxandtheactivityofneurons.PreviousstudiessuggestthatNOisreleasedfromthehippocampalneuronsinrestingstateandmayinhibittheactivityofhippocampus.Resveratrolinhibitstheelect