电生理-体外记录

•Electroencephalographicrecordingsinhumansandanimals•ExtracellularRecordingsinanesthetizedanimals•ExtracellularRecordingsinvivoinfree-movinganimals•IntracellularRecordingsinanesthetizedanimals(Current-clamp)•VoltageclampinanesthetizedanimalsElectroencephalographicrecordingsTheEEGcanhelpdiagnoseseizuredisorderslikeepilepsy,headinjuries,braintumors,encephalitis,somekindsofinfections,metabolicdisturbances,andsleepdisorders.Therecordingisobtainedbyplacingelectrodesonthescalp,usuallyafterpreparingthescalpareabylightabrasionandapplicationofaconductivegeltoreduceimpedance.Eachelectrodeisconnectedtoandifferentialamplifier,whichamplifiesthevoltage(typically1,000–100,000times,or60–100dBofvoltagegain),andthendisplaysitonascreenorinputsittoacomputer.TheamplitudeoftheEEGisabout100µVwhenmeasuredonthescalp,andabout1-2mVwhenmeasuredonthesurfaceofthebrain.EEGhasseverallimitations:1)Scalpelectrodesarenotsensitiveenoughtopickoutindividualactionpotentials,orwhethertheresultingelectricalactivityisreleasinginhibitory,excitatoryormodulatoryneurotransmitters.Instead,theEEGpicksupsynchronizationofneurons,whichproducesagreatervoltagethanthefiringofanindividualneuron.2)Secondly,EEGhaslimitedanatomicalspecificitywhencomparedwithotherfunctionalbrainimagingtechniquessuchasfunctionalmagneticresonanceimaging(fMRI).SomeanatomicalspecificitycanbegainedwiththeuseofEEGtopography,whichusesalargenumberofelectrodestotriangulatethesourceoftheelectricalactivity.Eachhorizontaltracingcorrespondstoanelectrodepairplacedonaparticularareaofthepatient'sscalp,formingaregulargrid-likepattern.Bynotingthesetofchannelswhereabnormalwavesoccur(suchasthosemarkedinred),theneurologistisabletoinferthepartsofthebrainwheretheabnormalityislocated.SteriadeM,TimofeevI.Neuron.2003Feb20;37(4):563-76Tracesofelectroencephalographicrecordingsofmiceduringseizures.Nicotine-inducedseizuresinwildtype(WT)miceresultinspike-wavedischarges,whereasseizuresinhomozygous(Hom)mutantdon’t.Amecanotransducerattachedtothebottomofthecagedetectsfrequencyandintensityofmovement.WTmicedonothaveseizureswheninjectedwith2mg/kgnicotine.Theintact,functioningbrainisreadilyexploredwithmicroelectrodesinanesthetizedanimals.Inthisapproach,theanimalisanesthetized,mostcommonlywithabarbiturate.Theanimalisthenplacedinastereotaxicinstrumentwhichpositionstheskullinanexactpositionandorientationwithrespecttosubmillimeterscalesinthreedimensionsontheinstrument.Bypositioningthemicroelectrodetipatadesiredcoordinatealongthesescales,determinedbyreferencetoastereotaxicatlasofthebrainofthatspecies,anysitewithinthebraincanbefoundandcellularactivityrecorded.X-rayormagneticresonanceimagingmethodsmayalsobeusedforthispurposeinhumanstudies.UnitExtracellularrecordingsinanesthetizedanimalsIntheseexperiments,impulseactivityofneuronsistypicallyrecordedextracellularly.Inextracellularrecordings,thetipofamicroelectrode(typically1–10mmindiameter)ispositionedimmediatelyadjacentto,butoutsideof,aneuron.Whenincloseproximitytotheneuron,currentfieldsgeneratedbyactionpotentialsinthatcellaredetectedbythemicroelectrodeassmallvoltagedeflections(typically0.1–1mV).Theadvantagesofinvivoelectrophysiologycomparedtotheinvitromethodsareobviouslyduetothemoreintactpreparationinvivo.Withtheseinvivomethods,onecanstudybrainregionsorneuronsintheirintactstatewithitsnormalcomplementofinputsandtargets,andintheirnaturalmilieuofcirculatinghormonesandfactors.Thecellsbeingstudiedusuallyhavenotbeenseveredordamaged,asisalmostalwaysthecasewithslicestudies,andhavedevelopednormallyintheintactorganism,incontrasttotheculturepreparation.Theseconsiderationslendadditionalcredibilityandfewercaveatstoresultsconcerningneuronalactivityinvivo.UnitExtracellularrecordingsinanesthetizedanimalsUseglassormetalelectrodes.Theanimalisfixedtoansteretoaxicapparatus.Givesinformationregardingthefrequencyandmodeoffiringandcellularresponsestodrugapplicationorelectricalstimulation.Recordsfromonesingleneuron.Thereareseveralexperimentalquestionsthatrequireanintactorganism,andtheycannotbepursuedinvitro.Forexample,tomimictheclinicalsituationitisimportanttodeterminetheeffectofasystemicallyadministereddrug(e.g.,abuseddrugslikeethanoloropiates)uponactivityinaparticularbrainregion.Inthiswayevenifthedrughasseveralsitesofactioninthebrain,oneseestheneteffectofhuman-likedrugexposureontheneuronsofinterest.Theintactinvivopreparationisalsonecessaryfordeterminingtheeffectofcertainphysiologicalmanipulationsonparticularneurons(e.g.,effectsofchangesincardiovascularactivityorsteroidlevels).Similarly,theeffectsoffunctionallydefinedinputstypicallymustbeexaminedintheintactorganism(e.g.,sensoryorpainfulstimuli).Finally,asignificantadvantageoftheinvivopreparationforelectrophysiologyisthatitismorereadilycorrelatedwithanatomicalstudiesthanininvitromodels.IontophoresisHowever,therearealsoseveraldisadvantagesofinvivopreparations:1)Inadditiontotherelativedifficultyinperformingmanyoftheintracellularstudies(andthereforeinobtainingdataonmembranemechanismsofdrugaction),theresearcherdoesnothaveasmuchknowledgeasintheinvitropre