电生理-体外记录课件

ElectroencephalographicrecordingsinhumansandanimalsExtracellularRecordingsinanesthetizedanimalsExtracellularRecordingsinvivoinfree-movinganimalsIntracellularRecordingsinanesthetizedanimals(Current-clamp)VoltageclampinanesthetizedanimalsElectroencephalographicrecordElectroencephalographicrecordingsTheEEGcanhelpdiagnoseseizuredisorderslikeepilepsy,headinjuries,braintumors,encephalitis,somekindsofinfections,metabolicdisturbances,andsleepdisorders.Therecordingisobtainedbyplacingelectrodesonthescalp,usuallyafterpreparingthescalpareabylightabrasionandapplicationofaconductivegeltoreduceimpedance.Eachelectrodeisconnectedtoandifferentialamplifier,whichamplifiesthevoltage(typically1,000–100,000times,or60–100dBofvoltagegain),andthendisplaysitonascreenorinputsittoacomputer.TheamplitudeoftheEEGisabout100µVwhenmeasuredonthescalp,andabout1-2mVwhenmeasuredonthesurfaceofthebrain.ElectroencephalographicrecordEEGhasseverallimitations:Scalpelectrodesarenotsensitiveenoughtopickoutindividualactionpotentials,orwhethertheresultingelectricalactivityisreleasinginhibitory,excitatoryormodulatoryneurotransmitters.Instead,theEEGpicksupsynchronizationofneurons,whichproducesagreatervoltagethanthefiringofanindividualneuron.Secondly,EEGhaslimitedanatomicalspecificitywhencomparedwithotherfunctionalbrainimagingtechniquessuchasfunctionalmagneticresonanceimaging(fMRI).SomeanatomicalspecificitycanbegainedwiththeuseofEEGtopography,whichusesalargenumberofelectrodestotriangulatethesourceoftheelectricalactivity.EEGhasseverallimitations:Eachhorizontaltracingcorrespondstoanelectrodepairplacedonaparticularareaofthepatient'sscalp,formingaregulargrid-likepattern.Bynotingthesetofchannelswhereabnormalwavesoccur(suchasthosemarkedinred),theneurologistisabletoinferthepartsofthebrainwheretheabnormalityislocated.EachhorizontaltracingcorresSteriadeM,TimofeevI.

Neuron.2003Feb20;37(4):563-76SteriadeM,TimofeevI.

NeuronTracesofelectroencephalographicrecordingsofmiceduringseizures.Nicotine-inducedseizuresinwildtype(WT)miceresultinspike-wavedischarges,whereasseizuresinhomozygous(Hom)mutantdon’t.Amecanotransducerattachedtothebottomofthecagedetectsfrequencyandintensityofmovement.WTmicedonothaveseizureswheninjectedwith2mg/kgnicotine.TracesofelectroencephalograpTheintact,functioningbrainisreadilyexploredwithmicroelectrodesinanesthetizedanimals.Inthisapproach,theanimalisanesthetized,mostcommonlywithabarbiturate.Theanimalisthenplacedinastereotaxicinstrumentwhichpositionstheskullinanexactpositionandorientationwithrespecttosubmillimeterscalesinthreedimensionsontheinstrument.Bypositioningthemicroelectrodetipatadesiredcoordinatealongthesescales,determinedbyreferencetoastereotaxicatlasofthebrainofthatspecies,anysitewithinthebraincanbefoundandcellularactivityrecorded.X-rayormagneticresonanceimagingmethodsmayalsobeusedforthispurposeinhumanstudies.UnitExtracellularrecordingsinanesthetizedanimalsTheintact,functioningbrainIntheseexperiments,impulseactivityofneuronsistypicallyrecordedextracellularly.Inextracellularrecordings,thetipofamicroelectrode(typically1–10mmindiameter)ispositionedimmediatelyadjacentto,butoutsideof,aneuron.Whenincloseproximitytotheneuron,currentfieldsgeneratedbyactionpotentialsinthatcellaredetectedbythemicroelectrodeassmallvoltagedeflections(typically0.1–1mV).Theadvantagesofinvivoelectrophysiologycomparedtotheinvitromethodsareobviouslyduetothemoreintactpreparationinvivo.Withtheseinvivomethods,onecanstudybrainregionsorneuronsintheirintactstatewithitsnormalcomplementofinputsandtargets,andintheirnaturalmilieuofcirculatinghormonesandfactors.Thecellsbeingstudiedusuallyhavenotbeenseveredordamaged,asisalmostalwaysthecasewithslicestudies,andhavedevelopednormallyintheintactorganism,incontrasttotheculturepreparation.Theseconsiderationslendadditionalcredibilityandfewercaveatstoresultsconcerningneuronalactivityinvivo.Intheseexperiments,impulseUnitExtracellularrecordingsinanesthetizedanimalsUseglassormetalelectrodes.Theanimalisfixedtoansteretoaxicapparatus.Givesinformationregardingthefrequencyandmodeoffiringandcellularresponsestodrugapplicationorelectricalstimulation.Recordsfromonesingleneuron.UnitExtracellularrecordingsThereareseveralexperimentalquestionsthatrequireanintactorganism,andtheycannotbepursuedinvitro.Forexample,tomimictheclinicalsituationitisimportanttodeterminetheeffectofasystemicallyadministereddrug(e.g.,abuseddrugslikeethanoloropiates)uponactivityinaparticularbrainregion.Inthiswayevenifthedrughasseveralsitesofactioninthebrain,oneseesthe"neteffect"ofhuman-likedrugexposureontheneuronsofinterest.Theintactinvivopreparationisalsonecessaryfordeterminingtheeffectofcertainphysiologicalmanipulationsonparticularneurons(e.g.,effectsofchangesincardiovascularactivityorsteroidlevels).Similarly,theeffectsoffunctionallydefinedinputstypicallymustbeexaminedintheintactorganism(e.g.,sensoryorpainfulstimuli).Finally,asignificantadvantageoftheinvivopreparationforelectrophysiologyisthatitismorereadilycorrelatedwithanatomicalstudiesthanininvitromodels.ThereareseveralexperimentalIontophoresisIontophoresisHowever,therearealsoseveraldisadvantagesofinvivopreparations:Inadditiontotherelativedifficultyinperformingmanyoftheintracellularstudies(andthereforeinobtainingdataonmembranemechanismsofdrugaction),theresearcherdoesnothaveasmuchknowledgeasintheinvitropreparationsofactualdrugconcentrationsatthecellunderstudy.Therefore,drugandtransmitterresponsesarelessconfidentlyidentifiedwithaspecificreceptororchannel.Inaddition,theremaybeotherconfounds,suchasthepresenceofanesthetics(orinawakeanimals,immobilizationstress)thatcouldalterthenormalelectrophysiologicalresponsestodrugsandtransmitters.However,therearealsoseveraInadditiontointeractionsbetweenindividualneurons,thereareother,morecomplexorganizationsinthenervoussystem.Neuronsaretypicallyassociatedinfunctionallyrelatedgroupsandcircuits.Functionatthebehaviorallevelisaproductoftheseneuronalnetworksratherthansimplytheproductofpropertiesofindividualneurons.Therearenetworksandcircuitsspecializedforsensoryandmotorfunctions,andothersspecializedforassociativeactivities.Itseemshighlylikelythattheelementsofsuchneuronalnetworkshaveevolvedwithinthecontextofnetworkfunction(s)tohavespecificandperhapsuniquepropertiestailoredforthatnetwork.

UnitExtracellularRecordingsinBehavingAnimalsInadditiontointeractionsbeAshasbeenstatedaboveforinvivotechniques,manyquestionsconcerningneuropsychopharmacologyrequireexperimentsintheintactanimal.Thisisperhapsmosttrueforquestionsregardingcognition.Whilemolecularandcellularexperimentsareimportantforunderstandingdetailsofprocessesinvolvedinmentaldysfunctionordrugresponses,theyareunabletointegratesuchresultstoultimatelyandcompletelyexplaincognitivefunctionssuchasattention,perception,emotion,ormemory.Hence,moststudiesintheelectrophysiologyofcognitiveprocessesinvolverecordingsingleneuronsinbehavinganimals.Thesemethodsrelyonthesameprinciplesasdescribedabovewithsomemodificationsforbehavinganimals.Moststudiesemployextracellularrecordingsfromametalmicroelectrodeheldinaminiaturemicropositionerontheanimal'shead.Themicropositioneristypicallyattachedtoachronicallyimplantedsteelchamberorcylinderthatisstereotaxicallypositionedandpermanentlycementedtotheskullinapriorsurgery.

AshasbeenstatedaboveforiFreeMovingFreeMoving电生理-体外记录ppt课件电生理-体外记录ppt课件IntracellularRecordingsinvivoUse“sharp”glasselectrodes(tipopening~1µM,resistance25-150MΩ).Measurechangesinvoltage(seecurrent-clamptechniques).IntracellularRecordingsinviintra1intra2intra1intra2Leeetal,Neuron2006Leeetal,Neuron2006CurrentClamp/VoltageClamp

CurrentClamp VoltageClamp Measurevoltage Measurecurrent Changecurrent ChangevoltageK+channelcurrentsK+K+K+K+K+K+K+WholecellrecordingsinvitroCurrentClamp/VoltageClamp Extracellular/Intracellular/PatchExtracellular(fieldorsingleunit)IntracellularPatchClampExtracellular/Intracellular/PaFieldRecordingsThelocalfieldpotentialrepresentsthesumofsynapticactivityacrosscellsFieldRecordingsThelocalfiel/LCMS/LCMS/LCMS/Topic/About/File/PCMov.htmlSinglechannelrecordingWholecellcurrentrecording/PerforatedPatchRecordingUsedtopreventdialysisoftheintracellularcontentsoftherecordedneuronPerforatedPatchRecordingUsedChannelRecordings-heterologousexpressionsystemsChannelRecordings-heterologTissuecultureTissuecultureRecordingfromtissueslicesRecordingfromtissueslicesSlicecultureGenegunSlicecultureGenegunSliceculture/co-cultureSliceculture/co-cultureInvitropitfalls

(Thereisnosuchthingasaperfectpreparation)InvitroisnotinvivoIssomethingmissing?LossofextrinsicinputsInvitropitfalls

(ThereisnoInvivoInvitroInvivoInvitrosinglemicroelectrodepenetratesthecell.Thevoltagerecordedisthesumofthevoltagedrop(Ve)acrosstheelectrodeandthemembranepotential(Vm).Thevoltageisbufferedbyahigh-impedance,low-bias-current,bufferamplifierandthenappliedtoasample-and-holdamplifier.Thesample-and-holdamplifierpreservesforthewholeofthecycleinterval(T1plusT2)thevalueoftherecordedvoltage(Vms)thatispresentatthemomentlabeledsampleinthefigure.Vmsiscomparedtothecommandvoltage(Vcmd)inthedifferentialamplifier.Thedifferencevoltage,ε,isamplifiedbythedifferentialamplifierandappliedtothecurrent-passinginputoftheelectronicswitch.Theelectronicswitchalternatestheinputpathtoavoltage-controlledcurrentsource.Thefunctionofthevoltage-controlledcurrentsourceistogenerateaspecifiedcurrentintheelectrode.Duringthecurrentpassinginterval(T1)thevoltage-controlledcurrentsourcepassesacurrentintotheelectrodethatisproportionaltotheoutputofthedifferentialamplifier,withamagnitudedeterminedbythetransconductanceGT.Inthisexample,atthebeginningofthecyclethecurrentisadepolarizingcurrentpulse.Thesquarepulseofcurrentintheelectrodecausestheelectrodevoltagetoriseataratedeterminedinanon-simplefashionbytheseriesresistanceoftheelectrode,theinputimpedance(mostlycapacitance)ofthecell,thecapacitancethroughthewalloftheelectrode,straycapacitancestogroundandthecapacitanceattheinputtothebufferamplifier.Forthesakeofsimplicity,thechangeintheelectrodevoltageisshownasifitwereasimpleexponential.Thecurrentpulseintervalisnormallyafewelectrodetimeconstantsbut,importantly,itismuchshorterthanthemembranetimeconstant.Thereisnoneedforthevoltageontheelectrodeduringthecurrentpulsetoreachsteadystateandinmostcasesinordertomaximizetheswitchingratethecurrentpulseisterminatedbeforesteady-stateconditionsarereached.BecausethedurationofthecurrentpulseismuchshorterthanthetimeconstantofthecelltheVmchangeshowninFigu