Increase of GABAA receptor-mediated tonic inhibition in DG cells after traumatic brain injury

NeurobiologyofDisease38(2010)464–475

ContentslistsavailableatScienceDirect

NeurobiologyofDisease

journalhomepage:/locate/ynbdi

IncreaseofGABAAreceptor-mediatedtonicinhibitionindentategranulecellsaftertraumaticbraininjury

ZakariaMtchedlishvilia,b, ,EkaLepsveridzed,HongXua,ElenaA.Kharlamova,b,BoLua,KevinM.Kellya,b,c

a

CenterforNeuroscienceResearch,Allegheny-SingerResearchInstitute,AlleghenyGeneralHospital,Pittsburgh,PA,USADepartmentofNeurology,DrexelUniversityCollegeofMedicine,Philadelphia,PA,USAc

DepartmentofNeurobiologyandAnatomy,DrexelUniversityCollegeofMedicine,Philadelphia,PA,USAd

IliaChavchavadzeStateUniversity,FacultyofLifeSciences,Tbilisi,Georgia

b

articleinfoabstract

Traumaticbraininjury(TBI)canresultinalteredinhibitoryneurotransmission,hippocampaldysfunction,andcognitiveimpairments.GABAergicspontaneousandminiatureinhibitorypostsynapticcurrents(sIPSCsandmIPSCs)andtonic(extrasynaptic)wholecellcurrentswererecordedincontrolrathippocampaldentategranulecells(DGCs)andat90daysaftercontrolledcorticalimpact(CCI).At34°C,inCCIDGCs,sIPSCfrequencyandamplitudewereunchanged,whereasmIPSCfrequencywasdecreased(3.10±0.84Hz,n=16,and2.44±0.67Hz,n=7,pb0.05).At23°C,300nMdiazepamincreasedpeakamplitudeofmIPSCsincontrolandCCIDGCs,buttheincreasewas20%higherincontrol(26.81±2.2pAand42.60±1.22pA,n=9,p=0.031)comparedtoCCIDGCs(33.46±2.98pAand46.13±1.09pA,n=10,p=0.047).At34°C,diazepamdidnotprolongdecaytimeconstants(6.59±0.12msand6.62±0.98ms,n=9,p=0.12),thelattersuggestingthatCCIresultedinbenzodiazepine-insensitivepharmacologyinsynapticGABAAreceptors(GABAARs).InCCIDGCs,peakamplitudeofmIPSCswasinhibitedby100μMfurosemide(51.30±0.80pAatbaselineand43.50±5.30pAafterfurosemide,n=5,pb0.001),anoncompetitiveantagonistofGABAARswithanenhancedaf nitytoα4subunit-containingreceptors.PotentiationoftoniccurrentbytheGABAARδsubunit-preferringcompetitiveagonistTHIP(1and3µM)wasincreasedinCCIDGCs(47%and198%)comparedtocontrolDGCs(13%and162%),suggestingthepresenceoflargertoniccurrentinCCIDGCs;THIP(1µM)hadnoeffectonmIPSCs.Takentogether,theseresultsdemonstratealterationsinsynapticandextrasynapticGABAARsinDGCsfollowingCCI.

©2010ElsevierInc.Allrightsreserved.

Articlehistory:

Received11November2009Revised10March2010Accepted10March2010

Availableonline18March2010Keywords:

TraumaticbraininjuryDentategranulecellsGABAAreceptorDiazepamFurosemideTHIP

BicucullinemIPSCs

SynapticcurrentsToniccurrents

Introduction

Cognitivede citsareamongthemostenduringandfrequentlyreportedimpairmentsfollowingtraumaticbraininjury(TBI)(Levinetal.,1979;HallandBornstein,1991),whichcanalsoresultindepression,posttraumaticstressdisorder,andposttraumaticepilepsy(PTE).TBIresultsincomplexpathophysiologythatinvolvescascadesofshort-andlong-termchangesatsubcellularandcellularlevels.ThehippocampusisparticularlyvulnerabletoTBI(DeRidderetal.,2006;Pullelaetal.,2006;Saatmanetal.,2006;Tranetal.,2006;Bonislawskietal.,2007),andcanundergolong-termchangesinitsphysiologicalfunctionandcontributetoalterationsinneurotransmission.GABAAreceptor(GABAAR)-mediatedinhibitioniscriticalforkeepinglocalcircuitexcitatoryactivityunderhomeostaticcontrolandisintimatelyinvolvedintheregulationofcognitiveprocesses,especiallyinlearningandmemory(Castellanoetal.,1993;Collinsonetal.,2002;

Correspondingauthor.AlleghenyGeneralHospital,9J9ST.,320EastNorthAvenue,Pittsburgh,PA15212,USA.Fax:+14123596847.

E-mailaddress:zmtchedl@(Z.Mtchedlishvili).

AvailableonlineonScienceDirect()

.0969-9961/$–seefrontmatter©2010ElsevierInc.Allrightsreserved.doi:

10.1016/j.nbd.2010.03.012

Dasetal.,2004).GABAAR-mediatedsignalingcanbeeitherpotenti-atedordecreasedindifferentanimalmodelsofTBI.Enhancementofpaired-pulseinhibitionwasdescribedinthedentategyrusinthe uidpercussioninjury(FPI)modelinrat(Reevesetal.,1997)andinthecontrolledcorticalimpact(CCI)modelinmouse(Huntetal.,2009).However,ef cacyofGABAwasreportedtobereducedindentategranulecells(DGCs)afterFPIduetofunctionalalterationsinthechloride/potassiumpump(Bonislawskietal.,2007).

Littleisknownaboutlong-termalterationsofpostsynapticGABAARsinDGCsafterheadtrauma.GABAregulatessynchronousneuronaloscillationsthatarecriticalforcognitivefunctionssuchasobjectperception,selectiveattentionandworkingmemory,andspatialmemory(EngelandSinger,2001;BuzsakiandDraguhn,2004),aswellasconsciousness(LlinasandRibary,2001).Thus,disruptionsinGABAergicsignalingafterheadtraumaareverylikelytohaveanimpactonmemoryandcognitivecapacities.PositiveallostericmodulatorsofGABAARsimpairmemoryprocessing(ArolfoandBrioni,1991;Mayoetal.,1993;Kantetal.,1996;Krazemetal.,2001;Johanssonetal.,2002;Silversetal.,2003;Turkmenetal.,2006),whereasGABAARblockersorinverseagonistsoftenpotentiatecognitiveandmemoryperformance(BrioniandMcGaugh,1988;Raffalli-Sebilleetal.,1990;Mayoetal.,

Z.Mtchedlishvilietal./NeurobiologyofDisease38(2010)464–475465

1992;Dahhaouietal.,1994;Atacketal.,2006;Collinsonetal.,2006;Dawsonetal.,2006).AlterationsinGABAAR-mediatedtransmissionarealsoamongkeyfactorsthatresultinhyperexcitabilityinthehippocampusandsubsequentepileptogenesis(Otisetal.,1994;Gibbsetal.,1997;Brooks-Kayaletal.,1998;Nusseretal.,1998;Shumateetal.,1998;Pirkeretal.,2003).

GABAARsaremembersofaligand-gatedionchannelsuperfamilyassembledfromsubunitsofsevendifferentclasses:α(1–6),β(1–3),γ(1–3),δ,ε,θ,andπ(SieghartandSperk,2002).Theymaintaintwoformsofinhibition:tonicandphasic.TonicinhibitionresultsfromactivationofextrasynapticGABAARsbylowconcentrationsofambientGABAandisstronglyrepresentedinDGCs(RossiandHamann,1998;StellandMody,2002;ModyandPearce,2004).Conversely,phasic(synaptic)inhibitionismediatedbyactivationofpostsynapticreceptorsbysaturatingconcentrationsofvesicularGABA.TonicinhibitioninDGCsismediatedbyα4andδsubunit-containing,high-af nity,slowlydesensitizing,extrasynapticallylocatedGABAARs(Pengetal.,2002;Weietal.,2003),whereasphasicinhibitioninDGCsismediatedbyα1andγ2subunit-containing,rapidlydesensitizing,synapticallylocatedreceptorswithlowaf nityforGABA(Sunetal.,2004;Manganetal.,2005).BecauseTBIisoftenfollowedbydegenerativechangesinthehippocampus,cognitivedisturbances,andepileptogenesis,wesoughttostudypotentialchronicchangesintonicandphasicGABAAR-mediatedinhibitioninDGCsafterTBI.RatsweresubjectedtoCCIandelectrophysiologicalchangeswerestudiedinvitrousinghippocampalslicepreparationsfromcontrolandbrain-injuredanimals.

MaterialsandmethodsCCImodelofTBIandsurgery

AllproceduresinvolvinganimalswereapprovedbytheInstitu-tionalAnimalCareandUseCommitteeoftheAllegheny-SingerResearchInstitute(ASRI)andwerecarriedoutaccordingtoNIHguidelinesandregulations.AnimalswerehousedindividuallyintheASRIvivarium,maintainedina12hourlight/12hourdarkcycleenvironmentwithcontrolledtemperature(23±2°C),andfoodandwaterweregivenadlibitum.TheCCIprocedurewasperformedaccordingtoDixonetal.(1991)withsomemodi cations.Brie y,maleSprague–Dawleyrats(2–3-moold)wereanesthetizedwithaninitialdoseof4%iso uranemixedwithoxygenandpositionedinastereotaxicframe(DavidKopfInstruments,Tujunga,CA).Theconcentrationofiso uranewasreducedto2–3%aftertheanimalsweredeeplyanesthetized.Bodytemperaturewasmonitoredthrough-outtheprocedureusingarectalprobeandmaintainedat37±2°Cwithaheatingpad(HarvardApparatus).Acraniectomywasperformedovertherightparietalcortexwithintheboundariesofbregmaandlambdawhileleavingtheduraintact.A1.975-cm-diameterpneumaticimpactor,attachedtoadouble-acting,stroke-constrained,pneumaticcylinderwitha5.0cmstroke(PittsburghPrecisionInstruments,Pittsburgh,PA)wasusedtodeliverCCI.Thecylinderwasrigidlymountedinaverticalpositiononacrossbar,whichwasadjustedintheverticalaxisovertheanimal'shead.Theanimal'sheadwassecuredinthestereotaxicframewithearbars,andtheimpactorwaspositioned4–5mmlaterallytothelongitudinalline.Thelowerrodendhadanattached5.5-mmimpactortip(i.e.,thatpartoftheshaftthatcomesintocontactwiththeexposedduramater).Theupperrodendwasattachedtothetransducercoreofalinearvariabledifferentialtransformer.Theimpactorvelocitywasadjustedto4m/s,theimpactdurationwas100ms,andthedepthoftissuedepressionwas2.8mm.FollowingCCIandthecessationofcorticalbleeding,thescalpwassutured,andtheanimalwasreturnedtothevivariumforrecovery.Animalsweresacri ced90daysafterCCIforelectrophys-iologicalandhistologicalstudies.

Videomonitoring

AsubsetofCCI-injuredanimalsunderwentcontinuousvideomonitoringforoneweekpriortosacri cetoassessforpossiblebehavioralseizureactivity.Animalswerehousedindividuallyinmultiplemonitoringchambersinsatellitevivariaandmaintainedonthe12hourlight/12hourdarkcycle.Animalbehaviorwasmonitoredbyclosed-circuittelevisioncameras,withandwithoutinfraredcapability,thatwereconnectedtovideosplitterunits(AdvancedTechnologyVideo,Inc.,Redmond,Washington).Digitalvideo les(Diva,StellateSystems)wererecordeddirectlytohighcapacityharddiskdrivesusingremovableharddrivebays.Therecordingswerereviewedvisuallyof ineandplayedat2×speedtodetectanybehavioralseizureactivityaccordingtoastandardclassi cationscale(Racine,1972).Electrophysiology

Brainsweredissectedfreeandimmersedincold(2–4°C)arti cialcerebrospinal uid(ACSF)saturatedwith95%O2–5%CO2.TheACSFconsistedofthefollowing(inmM):127.0NaCl,2.0KCl,1.5CaCl2,1.5MgSO4,25.7NaHCO3,1.1KH2PO4,and10.0glucose(osmolarity,300–305mOsm).Thebrainsweremountedonavibratomestage(CamdenInstruments,UK)and350-µm-thickcoronalsectionswerecut.SlicesweremaintainedincontinuouslyoxygenatedACSFat32°Cinaholdingchamberforaminimumof30–45minandthenatroomtemperature(23°C)inarecordingchambermountedonthestageofaNikonFN1microscope(Tokyo,Japan)equippedwitha64×water-immersionobjective,infrareddifferentialinterferencecon-trastoptics,andvideo.Forrecordingsat34°C,thebathtemperaturewasmaintainedbyanSH-27BinlinesolutionheaterandTC-324Btemperaturecontroller(WarnerInstruments,Hamden,CT).Patchelectrodes( nalresistancesof4–6MΩ)werepulledfromborosil-icateglass(GarnerGlass,Clairmont,CA)onahorizontalFlaming–Brownmicroelectrodepuller(modelP-97;SutterInstruments,Novato,CA)usingatwo-stagepullprotocol.Electrodetipswere lledwitha lteredinternalrecordingsolutionconsistingofthefollowing(inmM):153.3CsCl,1.0MgCl2,10.0HEPES,5.0EGTA,3.0ATPMg2+salt,and0.1GTPNa+salt,pH7.2,(withCsOH;osmolarity285–295mOsm).Theextensiveinjurytothehippocampusipsilat-eraltotheCCI-subjectedhemispheremadetheslicescontainingtheipsilateraldentategyrusunsuitableforpatch-clamprecordings.Forthisreason,therecordingswereobtainedfromthelowerbladeofthedentategyrusfromthecontralateralhemisphere.50μMDL-2-amino-5-phosphonovalericacid(AP-5)and20μM6,7-dinitroquinoxaline-2,3-dione(DNQX)wereincludedinACSFforallrecordingstoblockglutamatergictransmission.Themembrane-impermeableNa+chan-nelblockerN-(2,6-dimethylphenylcarbamoylmethyl)triethylam-moniumbromide(QX-314)inthe nalconcentrationof10μMwasincludedintheinternalsolutiontoblockNa+channelsfromtheinsideoftheclampedcellstherebypreventingthecellsfromescapingtheclampduringtherecordingsofspontaneousinhibitorypostsyn-apticcurrents(sIPSCs)andtoniccurrents.Tetrodotoxin(TTX;1μM)wasincludedintheACSFtoblockvoltage-gatedNa+channelsintheslicefortherecordingsofminiatureinhibitorypostsynapticcurrents(mIPSCs).QX-314wasomittedfromtheinternalsolutionforrecordingsofmIPSCs.DiazepamwasdissolvedinDMSOtoa5mMconcentration( rststocksolution)anddilutedto50μMwithACSF(secondstocksolution)onthedayofanexperiment.Thesecondstockwasthandilutedtoa300nM nalconcentrationinoneofthesliceperfusionreservoirs.Furosemidewasdissolvedtoa100mMstocksolutioninmethanol,andwasdissolvedtoa nal100μMconcentrationonthedayofanexperiment.AllsaltswereobtainedfromSigma(St.Louis,MO).AllotherreagentsusedinthisstudywereobtainedfromTocrisBioscience(Ellisville,MO).ControlandCCIDGCswerevisuallyidenti edandvoltage-clampedto 70mVunder

466Z.Mtchedlishvilietal./NeurobiologyofDisease38(2010)464–475

isotonicchlorideconditions(ECl=0mV)withaMulticlamp700Bampli er(MolecularDevices,PaloAlto,CA).Wholecellcapacitanceandseriesresistancewerecompensatedby80%.Recordingswereperformedwhenseriesresistanceaftercompensationwas20MΩorless.Accessresistancewasmonitoredwith10ms, 4mVtestpulses,deliveredonceevery60s.Ifatanytimeduringtheexperimenttheseriesresistanceincreasedby25%,therecordingwasterminatedandthedatawerenotusedforstatisticalanalysis.Currenttraceswere lteredat5kHz,digitizedat10kHzusingaDigidata1442digitizer,andacquiredonacomputerharddriveusingpClamp10.1software(MolecularDevices).

Measurementofsynapticcurrents

sIPSCsandmIPSCswereanalyzedwithMiniAnalysissoftware(Synaptosoft,Leonia,NJ).Thedetectionthresholdwassetat vetimesofrootmeansquare.Afterdetection,peakamplitude,frequency,anddecaytimeconstantswereanalyzed.Followingthepointofpeakamplitude,the0to90%regionofcurrentdecaywasassessedby100iterationsofeacheventbygroupanalysisforbestcurve t.DecaytimeconstantsofmIPSCswere tasamonoexponentialfunction,withMiniAnalysissoftware,usingasimplex-basedalgorithm.Approxi-mately500individualeventswereusedfromeachrecordingforanalysisofpeakamplitudesandfrequencyofsIPSCsandmIPSCs,anddecaytimeconstantsofmIPSCs.Measurementoftoniccurrents

Tomeasuretonicinhibition,the30-sepochimmediatelybeforeapplicationofthedrug(baseline)anda30-sepochafter3minofcontinuousperfusionwithadrugwerecompared.Thebuilt-inGaussianfunctionofClampFit(AxonInstruments)wasusedtodetermineGaussian tsintherawtraces(entiresegmentswithsIPSCspresent).All-pointfrequencydistributionhistogramsusinga2-pAbinsizewerederivedfromthese30-sepochs.

ThecomparisonsofobtainedGaussianmeansofall-pointhistogramdistributionswereperformedwithGraphPadPrism4(MountainView,CA).Gaussianmeans(meanbaselinecurrent)wereusedasmeasuresoftoniccurrent.IncontrolDGCs,Gaussian tsofall-pointhistogramswerecomparedtohistogramsderivedfromthesameepochswithsynapticcurrentsremovedbyacustom-designedalgorithminMATLAB(datanotshown).Nosigni cantdifferencewasfoundbetweentheGaussianmeansofall-pointhistogramsandthosederivedfromtheepochswithsynapticeventsremoved.Therefore,wedeemeditsuf cienttouseall-pointhistogramstostudytoniccurrents.EachDGCwastreatedasanestedunitforstatisticalconsiderations.OneDGCwasrecordedperslicetoavoidpotentialcontaminationofadrugusedinapreviousrecordinginthesameslice.Statisticalconsiderations

Tovalidatethesigni canceoftheeffectsofdiazepamandfurosemideonpeakamplitudesofmIPSCs,cumulativeprobability(fraction)distributionsofpeakamplitudesatbaseline(priortodrugapplication)andduringcontinuousapplicationofthedrugwerecomparedwiththeKolmogorov–Smirnov(KS)testforeachDGCusingthebuilt-inKSanalysisoptioninMinianalysissoftware(Synaptosoft,Leonia,NJ).AllDGCstestedwiththeKStestdemon-stratednon-GaussiandistributionsofmIPSCpeakamplitudes.CumulativefractiondistributionsofmIPSCpeakamplitudesalsowerecomparedbetweencontrolandCCIDGCs.Meansofmedianamplitudes,meanfrequencies,andmeandecaytimeconstantswerecomparedwiththetwo-tailedttest±standarderrorofthemean(SEM).WholecellcapacitancecomparisonswereperformedwiththeMann–Whitneytest±SEM.Statisticalsigni cancewassetatpb0.05.

Perfusionandpreparationoftissuesections

At90dayspost-CCI,someanimalsweredeeplyanesthetizedandtranscardiallyperfusedwithphosphatebufferedsaline(PBS;pH7.4),followedby4%paraformaldehydesolution.Thebrainswereremoved,post- xedinthesamesolution,andcryoprotectedin30%sucrose.Grossexaminationofthebrainswasperformedtocon rmthepresenceofTBI.Forty-micron-thickcoronalandhorizontalsectionswerecutonacryostat,collected,andstoredincryoprotectantsolution(phosphatebuffer/ethyleneglycol/glycerol)at 20°C.Standardhistological(Nissl)techniqueswereusedforsubsequentidenti cationofbraincytoarchitectureandmorphologicalchangesfollowingCCI.Nisslstaining

SectionswerewashedinPBS,mountedongelatin-coatedslides,andair-driedovernightatroomtemperature.Slidesweredehydrated,stainedwith0.5%cresylvioletsolution,rinsedindistilledwater,dehydratedagainthroughgradedalcoholsandxylene,andcoveredwithPermountandglasscoverslips.Results

Generalconsiderations

Twenty-threeCCI-injuredand34non-injured(control)Iperformedinourlaboratoryproducesaseverebraininjuryassociatedwithan～1%mortalityrateduringthe rst24h(unpublisheddata);therewasnoadditionalmortalitybeforeanimalsacri ceat90daysafterCCI.Ofthe23injuredanimalsusedinthestudy,11animalsweremonitoredcontinuouslyforaone-weekperiodpriortosacri ceandsubsequentvoltage-clamprecordings;controlanimalswerenotmonitored.Atotalof1918hofvideorecordingswasobtainedduringtheserecordingsessions,ofwhich1078h(924hlightson/154hlightsoff)wereofadequatetechnicalqualityforreview.Noseizurewasdetectedinanyanimal,similartohistoricalvideo-EEGrecordingsofcontrolSprague–Dawleyratsofsimilarage(Kharlamovetal.,2003).Fig.1showsarepresentativeratbrain(Fig.1A)andstructuralandmorphologicalchangesinthehippocampuswithNissl(Figs.1B,C)90daysafterCCI.PropertiesofsynapticreceptorsinDGCsfollowingCCI

DGCsreceivepowerfulinhibitoryinputfromGABAergicinter-neurons,whichformpredominantlyperisomaticsynapsesonDGCs(TraubandMiles,1991;HalasyandSomogyi,1993;Buhletal.,1994;BuckmasterandSchwartzkroin,1995;SolteszandMody,1995;Milesetal.,1996).Decreasedfeed-forwardinhibitioninthedentategyrushasbeenreportedwithindaysafterFPIandhasbeenattributedtolossofhilarGABAergicinterneurons(Tothetal.,1997).Itisimportanttonotethatpreviousstudieshavefocusedonrelativelyshort-termeffectsofTBIonGABAergictransmissioninthehippo-campus,typicallyassessingtissueonetoafewweekspost-TBI.However,neurologicalconsequencesofheadtrauma,suchascognitiveimpairments,posttraumaticstressdisorder,andPTEoftenmanifestthemselvesaftermonthsorevenyearsfollowingtheinitialinsult.Toinvestigatelong-termeffectsofTBIonGABAergictransmission,wholecellpatch-clamprecordingsofsIPSCsandmIPSCswereperformedinratcontrolandCCIDGCs90daysafterlesioning.SlicesweresuperfusedwithoxygenatedACSFthatcontained50μMDL-APVand20μparisonoffrequencyandpeakamplitudeofsIPSCsincontrolandCCIDGCsdidnotrevealsigni cantchanges.ThefrequencyofsIPSCswas4.95±1.27Hzincontrol(n=27DGCs,6animals),and5.58±1.96HzinCCIDGCs(n=16DGCs,4animals,p=0.93,two-tailedttest).Withinthesamegroupofcells,themeans

Z.Mtchedlishvilietal./NeurobiologyofDisease38(2010)464–475467

Fig.1.Brainmorphology90daysfollowingCCI.(A)Aperfusedbrainofanadultratdemonstratesalossofcorticaltissueandanecroticcavityattheimpactsite.(B,C)ArepresentativeNissl-stainedcoronalsectionshowsevidenceofcelllossintheipsilateralhippocampus(C)inCA1,CA3,anddentategyruscomparedtothecontralateralside(B).Anasterisk(*)marksthenecroticcavityatthecorticalimpactsite(C).Abbreviations:H:hilus;DG:dentategranulecelllayer.

ofmedianamplitudeswerecompared.ThemeanofmedianamplitudeofsIPSCswas60.7±2.74pAincontrol,and58.5±5.17pAinCCIDGCs(p=0.91,two-tailedttest).BecauseapparentindividualsIPSCscanbemultiplesummatedeventsduetoahighprobabilityofsynchronousreleaseoftransmitter,itisdif culttoreliablyinterprettheabsenceofamplitudeanddecaytimechangesofsIPSCsafterCCIasalackofpostsynapticeffect.TofurthertestthepossibilitythatpropertiesofsynapticcurrentsmighthavebeenalteredbyCCI,mIPSCswererecordedinDGCsinthepresenceof1μMTTXinslicescontainingthedentategyrusfromcontrolandCCI-injuredanimals.TTXwasusedtoblockvoltage-gatedNa+channelsandtherebyblockactionpotential-evokedreleaseofthetransmitter.mIPSCsinDGCsaregeneratedbyactionpotential-independentquantalreleaseoftransmitterfrompresynapticGABAergicterminals(Edwardsetal.,1990).Therefore,alterationsofamplitudeanddecaytimeconstantsofmIPSCscanbeinterpretedaspostsynapticeffects.

Thetimingandstrengthofsynaptictransmissionareprofoundlydependentontemperature,whichaffectstherateofexocytosis(MichevaandSmith,2005),vesiclepooldepletion(Kushmericketal.,2006),andprobabilityofvesiclerelease(HardinghamandLarkman,1998;Volgushevetal.,2004).Atroom,butnotatphysiologicaltemperature,GABAARaf nitytoGABAcanincreaseinmanytypesofhippocampalandcorticalneuronsbyallostericmodulation(PerraisandRopert,1999;Hájosetal.,2000).TodeterminewhethertherecordingtemperaturecouldalterpossibleeffectsofCCIonmIPSCs,recordingswereconductedat23°Candnear-physiological(34°C)temperaturesandarepresentedinTable1.

At23°C,thefrequencyofmIPSCswassigni cantlydecreasedinDGCsofCCIanimals(1.03±0.18Hz,n=9,4controlanimals,and0.67±0.09Hz,n=10,4CCIanimals;pb0.05,two-tailedttest),inagreementwithaprevious ndingofdecreasedfrequencyofmIPSCsintheFPImodelofTBIatroomtemperature(Tothetal.,1997).No

differenceswerefoundbetweenmeansofmediansofpeakampli-tudes(26.81±2.20pAincontrol,n=9,and33.46±2.98pAinCCIDGCs,n=10,p=0.16,two-tailedttest),aswellasindecaytimeconstantsinthesameneurons(5.12±0.29msincontrol,n=9,and5.48±0.03msinCCIDGCs,n=10,p=0.48,two-tailedttest).

Recordingsat34°CrevealedadecreaseofmIPSCfrequencyintheCCIgroup(3.10±0.84HzincontrolDGCs,n=16,3animals,and2.44±0.67HzinCCIDGCs,n=7,2animals,pb0.05,two-tailedttest).ComparisonofmeansofmedianamplitudesofmIPSCswithinthesamegroupofcellsdidnotdemonstrateanalterationofpeakamplitudeintheCCIgroup(62.10±0.73pAincontrol,n=16,and53.10±0.59pAinCCIDGCs,n=7,p=0.09,two-tailedttest).AnincreaseofpeakamplitudesofmIPSCsinbothgroupsofcellsat34°Ccomparedto23°Cwasinagreementwithaprevious ndingthatincreasingtemperaturefromroomtonear-physiologicaltempera-turecausesanincreaseofchordconductanceoftheGABAARandincreasedamplitudeofmIPSCs(PerraisandRopert,1999).WithinthesamegroupofcontrolandCCIDGCs,wedidnotdetectasigni cantchangeofdecaytimeconstants(5.97±0.07msincontrol,n=12,and6.59±0.12msinCCIDGCs,n=9,p=0.19,two-tailedttest).

Lossofdiazepampotentiationofsynapticcurrentdecay,anddecreasedamplitudepotentiationafterCCI

InDGCs,GABAAα1andγ2subunit-containingreceptorsarepreferentiallylocalizedinsynapses,whereasα4andδsubunit-containingGABAARsarepreferentiallylocalizedintheextrasynapticmembranewheretheymediatetonicinhibition(Nusseretal.,1995;Suretal.,1999;StellandMody,2002;Weietal.,2003;Sunetal.,2004;MtchedlishviliandKapur,2006;Glykysetal.,2008).Theγ2subunitplaysakeyroleinclusteringofGABAARs(Essrichetal.,1998;Kneussel

Table1

SummaryofparametersofmIPSCsandeffectsofdiazepamincontrolandCCIDGCs.mIPSCs

Temperature

ControlBaseline

Frequency(Hz)Peakamplitude(pA)Decaytime(ms)

23°C34°C23°C34°C23°C34°C

1.03±0.183.10±0.84a26.81±2.20b62.10±0.735.12±0.29c5.97±0.07c

a

CCI

300nMdiazepam0.96±0.193.59±1.2242.60±1.22b59.85±0.676.59±0.08c7.22±0.19c

n916916912

Baseline0.67±0.092.44±0.67a33.46±2.98b53.10±0.59b5.48±0.036.59±0.12

a

300nMdiazepam1.21±0.211.42±0.2746.13±1.09b36.06±7.09b5.61±0.086.62±0.98

n107107109

Thevaluesrepresentmean±S.E.M.nindicatesnumberofcells.pb0.05,two-tailedttest.a

Indicatessigni cantdifferencesinfrequencyofmIPSCsat23°Cand34°CunderbaselineconditionsincontrolandCCIgroups.b

Indicatessigni cantdifferencesinpeakamplitudeofmIPSCsat23°CincontrolandCCIDGCsbeforeandafter300nMdiazepam,andat34°CinCCIDGCsbeforeandafter300nMdiazepam.c

Indicatessigni cantdifferencesindecaytimeconstantsofmIPSCsat23°Cand34°CincontrolDGCsbeforeandafter300nM

diazepam.

468Z.Mtchedlishvilietal./NeurobiologyofDisease38(2010)464–475

andBetz,2000;KittlerandMoss,2003;Alldredetal.,2005)andinphasicGABAergicinhibition(NusserandMody,2002).However,someγ2subunitexpressionhasbeendocumentedinextrasynapticlocations(Somogyietal.,1996;Nusseretal.,1998;Sassoè-Pognettoetal.,2000).TheinhibitorynetworkofthedentategyrusundergoessubstantialperturbationsafterTBI,suchaslossofGABAergicinterneuronsandsproutingofexcitatory bers(Santhakumaretal.,2000,2001;Golaraietal.,2001).Anetresultoftheseperturbationsisincreasedneuronalactivityinthetissuesurroundingtheinjurysite.Becauseincreasedneuronalactivityinhippocampalneuronscausesincreasedinternali-zationofγ2subunit-containing,butnotδsubunit-containingGABAARs(Goodkinetal.,2008;Terunumaetal.,2008;JoshiandKapur,2009),wetestedthepossibilitythattonicandphasiccurrentsmightbealtereddifferentiallyafterCCI.γ2subunit-containingreceptorsaresensitivetothebenzodiazepinediazepam,whereasα4/δsubunitsrenderthereceptorsdiazepam-insensitive.

RecordingsofmIPSCsfromcontrolandCCIDGCswereperformedwith300nMdiazepam.TheeffectsofdiazepamonmIPSCfrequency,peakamplitude,anddecaytimeconstantsat23°Cand34°CaresummarizedinTable1.Therewasnosigni canteffectonmIPSCfrequencybydiazepamat23°Cincontrol(1.03±0.18Hzvs.0.96±0.19Hz,n=9,4animals,p=0.43,two-tailedttest)andCCIDGCs(0.67±0.09Hzvs.1.21±0.21Hz,n=10,4animals,p=0.11,two-tailedttest),andat34°Cincontrol(3.10±0.84Hzvs.3.59±1.22Hz,n=16,3animals,p=0.57,two-tailedttest)andCCIDGCs(2.44±0.67Hzvs.1.42±0.27Hz,n=7,2animals,p=0.17,two-tailedttest).PeakamplitudeanddecaytimeconstantsofmIPSCswerealtereddifferentiallybydiazepamincontrolandCCIDGCsat23°CandinCCIDGCsat34°C(Figs.2A–H).At23°C,diazepamincreasedthemeanofmedianpeakamplitudeofmIPSCsinbothcontrolandCCIgroups,buttheincreasewas20%higherincontrolDGCsthaninCCIDGCs.Thesigni canceofthediazepameffectonpeakamplitudewasvalidatedinindividualDGCsbycomparingcumulativefractiondistributionsbytheKStest,beforeandafterdiazepam.TheKStestsuggestedastatisticallysigni canteffectofdiazepam(pb0.05)inallcontrolandCCIDGCs,thusrejectingthenullhypothesisthattheamplitudepopulationswereequalbeforeandafterdiazepam.BecausemIPSCamplitudesareskewed(demonstrateanon-Gaussiandistribution),meansofmedianpeakamplitudesweresubsequentlycompared.IncontrolDGCs,diazepamincreasedtheamplitude(26.81±2.20pAand42.60±1.22pA,n=9,3animals,p=0.031,two-tailedttest)andinCCIDGCs(33.46±2.98pAand46.13±1.09pA,n=10,5animals,p=0.047,two-tailedttest).At34°C,diazepamdidnotincreasepeakamplitudeincontrolDGCs(62.10±0.73pAvs.59.85±0.67pA,n=16,3animals,p=0.11two-tailedttest).However,diazepamdecreasedpeakamplitudeinCCIDGCs(53.10±0.59pAvs.36.06±7.09pA,n=7,2animals,p=0.03,two-tailedttest).

IncontrolDGCs,diazepamprolongeddecaytimeconstantsby21.5%at23°C(5.12±0.29msand6.59±0.08ms,n=9,5animals,p=0.002,two-tailedttest)andby21%at34°C(5.97±0.07msand7.22±0.19ms,n=12,6animals,p=0.001,two-tailedttest).InCCIDGCs,diazepam'sprolongationofdecaytimeconstantswasnotsigni cant:8.9%at23°C(5.48±0.03msand5.61±0.08ms,n=10,5animals,p=0.093,two-tailedttest)andby0.4%at34°C(6.59±0.12msand6.62±0.98ms,n=9,4animals,p=0.12,two-tailedttest).

PotentiationofinhibitionofsynapticcurrentsbyfurosemideafterCCIThediureticfurosemideisanoncompetitiveantagonistofGABAARsthatdemonstratesanenhancedaf nitytoα4subunit-containingrecombinantreceptors(Waffordetal.,1996)anddoesnotdiscriminatebetweenγandδsubunit-containingreceptors(KorpiandLuddens,1997).Recordingswereperformedwithfurosemideat23°Cand34°Candwerequalitativelysimilar;datafromrecordingsat34°Carepresented.TheeffectsoffurosemideontypicalrecordingsofmIPSCsare

presentedinFigs.3A–D.Thesigni canceofthefurosemideeffectonpeakamplitudewasvalidatedinindividualDGCsbycomparingcumulativefractiondistributionsbytheKStest,beforeandafterfurosemide.TheKStestsuggestedastatisticallysigni canteffect(pb0.05)offurosemideinallCCI,butnotincontrol,DGCs.Furosemide(100μM)wasbath-appliedtocontrolDGCsanddidnotalterthemeanofmedianpeakamplitudes(49.80±4.20pAatbaseline,and45.20±4.40pAinthepresenceoffurosemide,n=6,2animals,p=0.32,two-tailedttest),frequency(0.39±0.05Hzatbaseline,and0.36±0.06Hzinthepresenceoffurosemide,n=6,p=0.85,two-tailedttest),ordecaytimeconstants(6.42±0.30msatbaseline,and7.13±0.44msinthepresenceoffurosemide,n=6,p=0.13,two-tailedttest).InCCIDGCs,100μMfurosemidedecreasedthemeanofmedianpeakamplitudes(51.30±0.80pAatbaseline,and43.50±5.30pAinthepresenceoffurosemide,n=5,2animals,pb0.001,two-tailedttest).Furosemidedidnotalterthefrequency(0.42±0.09Hzatbaseline,and0.38±0.11Hzinthepresenceoffurosemide,n=6,p=0.34,two-tailedttest)ordecaytimeconstants(5.26±0.30msatbaselineand5.40±0.20msinthepresenceoffurosemiden=5,p=0.73,two-tailedttest).IncreaseoftonicinhibitioninDGCsafterCCI

InordertoassesspotentialalterationsintoniccurrentsinCCIDGCs,measurementsofwholecellcapacitancewereobtainedtoensurethatpotentialdifferencesintoniccurrentwerenotattribut-abletosigni cantdifferencesincellmembraneareas.MeasurementsofwholecellcapacitancerevealedsimilarvaluesincontrolandCCIDGCs(46.40±6.50pFincontrol,n=36,9animals,and36.30±8.50pF,n=27,8animals,inCCIDGCs,p=0.11,Mann–Whitneytest),suggestingrelativehomogeneityincellmembraneareasincontrolandCCIDGCgroups.

4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol(THIP)isalowaf nityagonistofGABAARswithpreferentialaf nityforδsubunits(Adkinsetal.,2001;Brownetal.,2002).1and3μMTHIPwereusedforpotentiationand20μMbicucullinewasusedforblockadeoftoniccurrents.Therecordingswereperformedat34°C.Quantita-tivemeasurementoftoniccurrentwasperformedbycomparingGaussiandistributionsofall-pointhistogramsof30-sepochsimmediatelybeforetheopeningoftheTHIP-containingreservoir(baseline)and3minafteropeningthereservoir.Bothconcentra-tionsofTHIPincreasedbaselinenoiseandshiftedholdingcurrentinbothcontrolandCCIDGCs.ThedataoftoniccurrentalterationarepresentedinTable2.1μMTHIPshiftedholdingcurrentincontrolDGCsfrom98.7±30.4pAto114.7±20.4pA(13%increase,n=13,6animalsp=0.0005,two-tailedttest)whereasinCCIDGCs,THIPshiftedholdingcurrentfrom119.8±34.6pAto176.3±42.2pA(47%increase,n=9,4animals,p=0.0003,two-tailedttest).Tocon rmthat1μMTHIPcausedagreaterpotentiationoftoniccurrentinCCIDGCs,werepeatedthisexperimentbyincreasingtheconcentrationofTHIPbyahalf-log.3μMTHIPshiftedholdingcurrentincontrolDGCsfrom88.9±20.6pAto233.7±50.4pA(162%increase),n=11,3animals,p=0.0005,two-tailedttest,Figs.4A,B),whereasinCCIDGCs,holdingcurrentwasshiftedfrom111.6±30.8pAto334.0±69.6pA)(198%increase,n=7,2ani-mals,p=0.0001,two-tailedttest,Figs.4C,D).AsteeperrisetimeofTHIP-inducedcurrentinCCIDGCssuggeststhat,comparedtocontrolDGCs,activationofalargernumberofreceptorsbyTHIPresultsinahigherrateofcurrentamplitudeincrease.

InordertocorroboratethepresenceoflargertoniccurrentinCCIDGCs,20μMbicucullinewasbath-appliedtothesameDGCswhereholdingcurrentwaspotentiatedby1and3μMTHIP.WefoundthatsubstantialamountsoftimewererequiredforTHIP-inducedcurrenttoequilibratetoasteadylevel,likelyduetoaveryslowdesensitizationrate.Inmanycases,thequalityoftherecordingduringprotractedapplicationofTHIPwouldoftendeterioratetosuchanextentthatapplicationofbicucullinewasnotfeasible.Ouraimwastodetectthe

Z.Mtchedlishvilietal./NeurobiologyofDisease38(2010)464–475469

Fig.2.LossofdiazepamsensitivityinDGCsafterCCI.TypicalexamplesofmIPSCsincontrolandCCIDGCs(averagesfromsingleneurons)andcumulativeprobabilityplotsofpeakamplitudeanddecaytimeconstantsatbaseline(solidline)andinthecontinuouspresenceof300nMdiazepam(dottedline).DGCswerevoltage-clampedto 70mVandrecordedat23°Cinthepresenceof50μMAP-5,20μMDNQX,and1μMTTX.(A)AnaveragedtracefromacontrolDGCatbaseline(black)andafterapplicationof300nMdiazepam(grey).Eachtraceisobtainedfrom～500individualmIPSCsinthisandsubsequenttraces.(B)AnaveragedtracefromaCCIDGCatbaseline(black)andafterapplicationof300nMdiazepam(grey)Notethestrongerpotentiationofpeakamplitudeby300nMdiazepaminthecontrolDGCcomparedtotheCCIDGC.(C,D)CumulativeprobabilityplotsofthepeakamplitudesofmIPSCsrecordedfromthesameneurons(A,B),respectively.NotethelargerrightwardshiftofthecurveinthepresenceofdiazepaminthecontrolDGC(C)comparedtotheCCIDGC(D).(E,F)ForthesameDGCsasin(A)and(B),thepeakamplitudesofmIPSCswerenormalizedtotheamplitudesofthetracesrecordedundercontinuousapplicationof300nMdiazepamtodemonstratetheeffectsofdiazepamondecaytimesincontrolandCCIDGCs.Notethelossofpotentiationofdecaytimeby300nMdiazepaminmIPSCsrecordedintheCCIDGC.(G,H)CumulativeprobabilityplotsofdecaytimesofmIPSCsrecordedfromthesamecontrolandCCIDGCs(E,F)NotethelargerrightwardshiftofthecurveinthepresenceofdiazepaminthecontrolDGC(G)comparedtotheCCIDGC(H).

relative,ratherthantheabsolute,differenceintoniccurrent.Forthesereasons,weconsidereditessentialthatbicucullinewastestedshortlyafterTHIP-inducedcurrentreacheditspeak.

Slowinwardcurrentoccursinresponsetobicucullineapplicationaccompaniedbylossofsynapticcurrentsandreductionofbaselinenoise.Reductionofbaselinenoiseoccursbecausetheclosureofchloridechannelscausesanincreaseofmembraneresistance.SeveralstudieshaveusedtheshiftofholdingcurrentbyGABAARantagoniststomeasuretonicinhibition(Baietal.,2001;Hamannetal.,2002;NusserandMody,2002;ModyandPearce,2004;Manganetal.,2005;MtchedlishviliandKapur,2006).Bicucullineblockedmostsynapticevents,decreasedbaselinenoise,andshiftedholdingcurrentin

both

470Z.Mtchedlishvilietal./NeurobiologyofDisease38(2010)464–475

Fig.3.PotentiationoffurosemideinhibitionofmIPSCsinDGCsafterCCI.TypicalexamplesofmIPSCsincontrolandCCIDGCs(averagesfromsingleneurons)andcumulativeprobabilityplotsofpeakamplitudeatbaseline(solidline)andinthecontinuouspresenceof100μMfurosemide(dottedline).DGCswerevoltage-clampedto 70mVandrecordedat23°Cinthepresenceof50μMAP-5,20μMDNQX,and1μMTTX.Averagedtracesfromacontrol(A)andCCIDGC(B)beforeandafterapplicationof100μMfurosemide.Eachaveragedtraceisobtainedfrom～500individualmIPSCs.Theaveragedbaselinetraceisshowninblack,andtheaveragedtracerecordedinthepresenceoffurosemideisshowningrey.Notethedecreaseofpeakamplitudeby100μMfurosemideintheCCIDGC.(C,D)CumulativeprobabilityplotsofthepeakamplitudesofmIPSCsrecordedfromthesameneurons.NotethelargerleftwardshiftofthecurveinthepresenceoffurosemideintheCCIDGC.

control(Fig.4A)andCCIDGCs(Fig.4C).However,itcausedagreaterinhibitionofthecurrentsrecordedinCCIDGCsthanincontrolDGCs(Table2).IntheDGCsrecordedinthepresenceof1μMTHIP,bicucullinedecreasedtheholdingcurrentcomparedtothebaselineincontrolDGCsfrom98.7±30.4pAto54.7±14.5pA(54%decrease,n=13,pb0.0001,two-tailedttest),whereasinCCIDGCs,bicucullinedecreasedholdingcurrentfrom119.8±34.6pAto21.2±7.9pA(83%decrease,n=9,pb0.0001,two-tailedttest).Inthecurrenttracesacquiredfromtherecordingswith3μMTHIP,thedecreaseofholdingcurrentbybicucullinewassimilartothatobservedinthetracesobtainedfrom1μMTHIPrecordings.BicucullinedecreasedholdingcurrentcomparedtobaselinelevelincontrolDGCsfrom88.9±20.6pAto41.1±6.8pA(45%decrease,n=11,pb0.0001,two-tailedttest),andinCCIDGCsfrom111.6±30.8pAto29.2±6.0pA(74%decrease,n=7,p=0.0004,two-tailedttest).

Propertiesofsynapticreceptorsdonotcontributetotheincreaseoftonicinhibition

PotentiationoftonicinhibitionafterTBI,revealedbyTHIPinDGCsfromanimals90daysafterCCI,suggestedthattherewasanincreasednumberofhigh-af nity,andpossiblyδsubunit-containing,GABAARs.Wequestionedwhethertheincreasednumberofhigh-af nityGABAARswasrestrictedtoextrasynapticreceptors,orwhethertheinjurymightcauseabnormalinsertionofδsubunit-containingreceptorsintosynapsesinDGCsafterCCI.

mIPSCswererecordedincontrolandCCI-injuredDGCsvoltage-clampedat 70mV.1μMTHIPwasbath-appliedtoslicesafterstablerecordingswereachieved.IncontrolDGCs,1μMTHIPdidnotalterthebaselinefrequencyofmIPSCs(0.95±0.21Hzatbaseline,and0.82±0.14HzinthepresenceofTHIP,n=4,1animal,p=0.54,two-tailedttest,Fig.5A).InthesamegroupofcontrolDGCs,comparisonofthemeansofmedianamplitudesofmIPSCsbeforeandafterapplicationofTHIPdidnotdemonstrateastatisticallysigni cantalterationofpeakamplitudeby1μMTHIP(50.78±2.4pAatbaseline,and53.70±1.93pAafterapplicationofTHIP,n=4,p=0.37,two-tailedttest,Fig.5C).Inaddition,1μMTHIPdidnotalterthedecaytimeconstantsofmIPSCs(5.24±0.08msatbaseline,and5.65±0.09msafterapplicationofTHIP,n=4,p=0.54,two-tailedttest,Fig.5E).

Next,westudiedtheeffectsof1μMTHIPonsynapticcurrentsinCCIDGCs.1μMTHIPdidnotalterthefrequencyofmIPSCs(1.09±0.12Hzatbaseline,and1.63±1.13HzinthepresenceofTHIP,n=7;1animal,p=0.54,two-tailedttest,Fig.5B).InthesamegroupofCCIDGCs,comparisonofthemeansofmedianamplitudesofmIPSCsbeforeandafterapplicationof1μMTHIPdidnotdemonstrate

a

Table2

Summaryofeffectsof1and3μMTHIPand20μMbicucullineontoniccurrentsincontrolandCCIDGCs.

ControlBaseline

Toniccurrent(pA)

98.7±30.4Baseline

Toniccurrent(pA)

88.9±20.6

1μMTHIP114.7±20.43μMTHIP233.7±50.4

Bicuculline54.7±14.5Bicuculline41.1±6.8

n13n11

CCIBaseline119.8±34.6Baseline111.6±30.8

1μMTHIP176.3±42.23μMTHIP334±69.6

Bicuculline21.2±7.9Bicuculline29.2±6.0

n9n7

ThevaluesrepresentGaussianmean±S.E.M.nindicatesnumberofcells.

Z.Mtchedlishvilietal./NeurobiologyofDisease38(2010)464–475471

Fig.4.IncreaseoftonicinhibitioninDGCsafterCCI.Thelowaf nityGABAAreceptoragonist3µMTHIPcausedalargerpotentiation,andtheGABAARantagonist20μMbicucullinecausedalargerinhibition,oftheholdingcurrentinaCCIDGCcomparedtoacontrolDGC.Typicalrecordingsat34°CfromaDGCfromacontrol(A)andaCCI(C)animal90daysafterlesioning.DGCswerevoltage-clampedto 70mVandbaselineepochswereselectedimmediatelypriortoopeningthereservoircontaining3μMTHIP(arrows),andanepochcorrespondingtoTHIPselected3minafterthevalveopening.THIPcausedanoutwardshiftoftheholdingcurrentandpotentiationofbaselinenoiseinbothcontrol(A)andCCI(C)DGCs.NotethelargershiftoftheholdingcurrentintheDGCfromaCCIanimal.Bicucullineinhibitedtoniccurrentinbothcells,butcausedalargershiftofholdingcurrentinCCIDGCs(shownbydottedline).(B–D)Summaryoftheeffectsof3µMTHIPand20µMbicucullineontonicinhibitionincontrolandCCIDGCs.(B)IncontrolDGCs,3µMTHIPcauseda162%increaseofholdingcurrentcomparedtothebaseline.20µMbicucullinecauseda54%decreaseofholdingcurrentcomparedtothebaseline.(D)InCCIDGCs,3µMTHIPcauseda198%increaseofholdingcurrentcomparedtothebaseline.20µMbicucullinecausedan83%decreaseofholdingcurrentcomparedtothebaseline.ThebarsrepresentGaussianmeansof30-sepochsacquiredimmediatelybeforeTHIPapplication(baseline),atthepeakofTHIP-evokedcurrent(THIP),and5minafterapplicationof20µMbicuculline(bicuculline).Errorbarsaremean±S.E.M.AugmentationofholdingcurrentbyTHIPandinhibitionbybicucullineweresigni cantwithincontrol(n=11),andCCIDGCs(n=7,pb0.05,pairedttest).AnenhancedeffectofTHIPandbicucullineinCCIDGCscomparedtocontrolDGCswassigni cant(pb0.05,two-tailedttest).

statisticallysigni cantalterationofpeakamplitudeby1μMTHIP(61.9±2.2pAatbaseline,and82.1±4.4pAinthepresenceof1μMTHIP,p=0.08,two-tailedttest,Fig.5D).1μMTHIPdidnotchangethedecaytimeconstantsofmIPSCs(7.8±1.1msincontrol,and7.4±0.7msintheCCIgroup,p=0.63,two-tailedttest,Fig.5F).Theseresults,corroboratedbyour ndingthatδsubunitimmunoreactivityisnotpresentinsynapticlocationsincontrolandCCIDGCs(unpublisheddata),suggestthatsynapticGABAARsdonotcontributetotoniccurrentmediatedbyδsubunit-expressingGABAARs.

IncreasedtonicinhibitioninCCIDGCs

Inthepresentstudy,wehaveshownanincreaseofGABAAR-mediatedtoniccurrentsinDGCs90daysafterCCI.Severalmechanisms,bothpre-andpostsynaptic,canaccountforincreasedtonicconductanceinCCIDGCs.ElevationofambientlevelsofGABAcanincreasetoniccurrentbyincreasedspilloverofvesicularGABAcausedbyincreased ringofGABAergicinterneurons(Kanedaetal.,1995;Brickleyetal.,1996;WallandUsowicz,1997;Hamannetal.,2002;Brightetal.,2007),releaseofGABAfromglialsources(Liuetal.,2000;Kozlovetal.,2006),orimpairmentofGABAtransporters(RichersonandWu,2003).LargerTHIP-evokedtoniccurrentinCCIDGCssuggestsanincreaseddensityofδsubunit-containingperi-andextrasynapticGABAARs.Theδsubunit-containingGABARspossessdistinctbiophysicalandpharmacologicalproperties.Thesereceptorshavehighaf nitytoGABA(Brownetal.,2002;Sundstrom-Poromaaetal.,2002)andaslowrateofdesensitiza-tion(BianchiandMacdonald,2002;Brownetal.,2002).Thesepropertiesallowδsubunit-containingGABARsto“sense”ambientlevelsofGABAinasubmicromolarandlowmicromolarrange(Glykysetal.,2008).InDGCs,α5βxγ2GABAARscancontributetotoniccurrent,whichisincreasedafterstatusepilepticus(ZhanandNadler,2009).Itispossiblethatdifferentialpatternsoftraf ckingofreceptorsubunitsplayaroleinanincreasedexpressionofδ-containingGABAARs,whichremainonthemembranesurface,whereasγ2-containingGABAARsareinternalizedunderconditionsofincreasedneuronalactivity(Goodkinetal.,2008)resultinginunchanged(Goodkinetal.,2005)orincreased(Nayloretal.,2005)toniccurrent.

IthasbeenestablishedthatTBIinducesupregulationofbrain-derivedneurotrophicfactor(BDNF)inthetissuesurroundingtheinjurysite(Hicksetal.,1997).BDNFplaysacrucialroleinneuronalmaturationbyregulatingintracellularCl levelsandalteringGABAergicsignalingfromdepolarizingtohyperpolarizing(Hübneretal.,2001;Riveraetal.,2002)andimprovingmemory(Falkenbergetal.,1992).In

injured

Discussion

Theprincipal ndingsinDGCs90daysafterCCIwere:1)anincreaseofGABAAR-mediatedtoniccurrents;2)adecreasedfrequen-cyofmIPSCs;and3)alossofdiazepampotentiationandanincreaseoffurosemideinhibitionofsynapticGABAARs.

AnumberofexperimentalmodelshavebeendevelopedoverthelastfewdecadesthatsimulatedifferentaspectsoftheclinicalconditionofTBIwithvaryingdegreesofaccuracy.TwoofthemostcommonlyusedmodelsareFPIandCCI.TheadvantageoftheFPImodelisitsrelativesimplicityanditsabilitytoproducesigni cantinjuryinthebrain,includingaxonalinjuryandintraparenchymalhemorrhages(Povlishocketal.,1983).However,the uidpulseofFPIentersthecalvariumanddispersesdiffuselywithintheepiduralspace,asdemonstratedwithhighspeedcineradiography(Dixonetal.,1988),makingtissuedisplacementdif culttoquantify.AnadvantageoftheCCImodelisthatitcanproduceamorepreciseandreproducibleinjuryasdemonstratedbyexperimentsmeasuringtheeffectthatthevelocityanddepthofimpacthaveontheseverityofinjury(Dixonetal.,1991).RecentreportsindicatethattheCCImodelcanbesuccessfullyappliedtoinducePTEinmice(Huntetal.,2009)andinrats(Statleretal.,2009).

472Z.Mtchedlishvilietal./NeurobiologyofDisease38(2010)464–475

Fig.5.Low-af nityGABAagonistdoesnotaffectsynapticreceptorsinDGCsfromcontrolanimals.RecordingsincontrolDGCswereperformedat23°C.(A)1μΜTHIPdidnotalterthefrequencyofmIPSCs(0.95±0.21Hzatbaseline,and0.68±0.07HzinthepresenceofTHIP,n=4,p=0.54,two-tailedttest).(C)ComparisonofmeansofmedianamplitudesofmIPSCsbeforeandafterapplicationofTHIPdidnotdemonstrateastatisticallysigni cantalterationofpeakamplitudeby1μMTHIP(50.78±2.4pAinthebaseline,and53.70±1.93pAafterapplicationofTHIP,n=4,p=0.37,two-tailedttest).(E)1μMTHIPdidnotalterthedecaytimeconstantsofmIPSCs(5.24±0.08msand5.65±0.09ms,n=4,p=0.54,two-tailedttest).THIPdidnotaltersynapticcurrentsinDGCsafterCCI.(B)THIPdidnotalterthefrequencyofmIPSCs(1.09±0.12Hzinthebaseline,and1.63±1.13HzinthepresenceofTHIP,n=7,p=0.54,two-tailedttest).(D)ComparisonofmeansofmedianamplitudesofmIPSCsbeforeandafterapplicationof1μMTHIPdidnotdemonstrateastatisticallysigni cantalterationofpeakamplitudeby1μMTHIP(61.9±2.2pAinthebaseline,and82.1±4.4pAinthepresenceof1μMTHIP,n=7p=0.08;two-tailedttest).(F)1μMTHIPdidnotchangethedecaytimeconstantsofmIPSCs(7.8±1.1msand7.4±0.7ms;n=7,p=0.63,two-tailedttest).

tissue,increasedlevelsofBDNF(Yangetal.,1996)anditsreceptortrkB(Binderetal.,1999)arebelievedtopromoteexcitatoryaxonalsproutingandthereforeareconsideredepileptogenicafterbraininjury(Dinocourtetal.,2006)andinkindling(Ernforsetal.,1991;Merlioetal.,1993;Elmeretal.,1998).ExogenousBDNFinhibitsinternalizationandsigni cantlypromotessurfaceexpressionofδsubunit-containingGABAARsinorganotypichippocampalneurons(JoshiandKapur,2009).ElevatedBDNFinthehippocampusafterheadtrauma,inadditiontopersistentlyincreasedexcitatorysignalingduetomossy bersprouting,maycausealargersurfaceexpressionofδsubunit-containingGABAARsinDGCs,whichresultsinlargertonicGABAAR-mediatedcurrents.

Alteredpropertiesofsynapticreceptors

UnchangedamplitudesofsynapticcurrentsafterCCIsuggestthatlevelsofγ2subunit-containingGABAARsareeitherunalteredordecreasedacutelyafterinjuryandarerestoredtocontrollevels90daysfollowinginjury.InCCIDGCs,benzodiazepine-insensitivepharmacologyofsynapticreceptorsisconsistentwithco-expressionofα4andγsubunitsinthesynapse.

Undernormalcircumstances,α4isco-expressedwithδsubunitsinextrasynapticreceptors(Nusseretal.,1998;Sunetal.,2004),butitcanbeexpressedsynapticallyinDGCsinepilepsy(Payneetal.,2006;Sunetal.,2007).DecreaseofallostericmodulationofGABAARsofDGCsbydiazepam,characterizedbydecreasedprolongationofdecaytimeconstantsandpotentiationofpeakamplitudehasbeencharacterizedunderpathologicalconditions,suchasfollowingacuteseizures(KapurandMacdonald1997;Goodkinetal.,2005;Nayloretal.,2005,Fengetal.,2008),aswellasinchronicepilepsy(Cohenetal.,2003;Pengetal.,2004).LossofpotentiationofdecaytimeconstantsandreducedpotentiationofpeakamplitudesofmIPSCsinCCIDGCssuggestthatthesynapticGABAARsundergochangesleadingtobenzodiazepineinsen-sitivitysimilartothosefoundinepilepsy.Thepossibilityofthepresenceofα4subunit-containingreceptorsinGABAergicsynapsesinDGCswasfurthersuggestedbyincreasedfurosemideinhibitionofGABAAR-mediatedmIPSCsinCCIDGCs.Furosemideisanα4-preferringnoncompetitiveantagonistofGABAARs(Waffordetal.,1996)anddoesnotdiscriminatebetweenandγandδsubunit-containingreceptors(KorpiandLuddens,1997).Furosemideselectivelyinhibitstheamplitudeoffast,butnotslow,mIPSCsinCA1pyramidalneurons(Banksetal.,1998),suggestingacontributionofα4-containingGABAARstotheseevents.

Factorsotherthansubunitvariationsthatcouldaffectdiazepamsensitivitymightincludephosphorylationstateand/ormutationsofGABAARsubunits.PhosphorylationofGABAARsbyproteinkinaseChasbeenshowntoreducediazepampotency(GaoandGreen eld,2005;Qietal.,2007),andmutationsoftheγ2subunitcaneliminate(Wallaceetal.,2001)orreduce(Bowseretal.,2002;Eugèneetal.,2007)benzodiazepinesensitivity.Arecentreportsuggeststhatγ2subunitmutationsattheα1γ2interface(Arg43andGlu117),wherethebenzodiazepinebindingsiteislocated,resultinalossofbenzodiazepinesensitivity(Goldschen-Ohmetal.,2010).Mutationsofhistidineresiduesintheα1subunithavebeenlinkedtodiazepam

insensitivity

Z.Mtchedlishvilietal./NeurobiologyofDisease38(2010)464–475473

(RudolphandMöhler,2006).Inaddition,our ndingofdecreasedfrequencyofmIPSCs,butnotsIPSCs,mightsuggestdecreasedprobabilityofrelease.SimilarobservationswerereportedinDGCsintheFPImodelofTBI(Tothetal.,1997).

PotentialpathophysiologicalconsequencesofincreasedtonicinhibitionafterCCI

AnincreaseofGABAergictonicinhibitioninDGCs90daysafterCCImaysuggesttheexistenceofanovelmechanismbywhichTBIcontributestotheimpairmentofcognitiveandmemoryfunctions.IthasbeenrecognizedthatincreasedGABAAR-mediatedtonicinhibitionisnegativelycorrelatedwithcognitiveandmemoryfunctions(Caraiscosetal.,2004),whereaspositivemodulatorsofGABAARs,suchasbenzodiazepines,haveamnesiceffectsandadverselyaffectmemory(Maubach,2003).TheroleofGABAergictoniccurrentmediatedbyδsubunit-containingreceptorsinmemoryandcognitionisbeginningtoemerge.Arecent ndingsuggeststhatpotentiationoftonicinhibitioninareaCA3interneuronsmediatedbyδsubunit-containingGABAARscansuppressCA3gamma-frequencyoscillations,whicharecriticalformemoryencodingandretrieval(MannandMody,2009).IncreasedlevelsoftheGABAsynthesizingenzymeGAD67intheprefrontalcortexhasbeencorrelatedwithdeclineofworkingmemory(Koborietal.,2006).InthependulummodelofTBI,impairedspatialmemorywasimprovedafterblockadeofGABAARsandwasattributedtodisinhibitionofpresynapticcholinergictransmission(O'DellandHamm,1995).Takentogether,theseresultssuggestthatconcomitantalterationsintonicandphasicinhibitioninDGCsmayoccurafterTBI.

TBIisamajorriskfactorforthedevelopmentofPTE.Ithasbeensuggestedthatthedentategyrusfunctionsasa“gate”inpreventingthereverberationofexcessiveexcitatorydrivethroughthetrisynapticcircuitformedbythemossy bertoCA3toCA1pyramidalneuron.Breakdownofthisgatecouldbeacriticaleventinthedevelopmentofseizureactivitywithinthetemporallobe(Collinsetal.,1983;StringerandLothman,1989;Heinemannetal.,1992).AlterationsofGABAergicsignalinginDGCsafteranepileptogenicinsulthavebeenlongrecognizedasanimportantpartofawidearrayofmolecular,cellular,andcircuitlevelchangesinthedentategyrus.Althoughwedidnotdocumentseizuresinthe11CCI-injuredanimalsthatunderwentlimitedvideomonitoringbeforesacri ceandelectrophysiologicalstudies,itispossiblethattheseanimalsdemonstratedconvulsiveactivitybeforetheyweremonitoredorhadnon-convulsiveseizures,whichwouldhavebeenundetectablebyvideorecordingswithoutconcomitantEEG.Inaddition,itispossiblethatthe12CCI-injuredanimalsthatwerenotmonitoredmighthavehadundetectedconvulsiveornon-convulsiveseizuresatanypointintimefollowingcorticalimpact.Ifanimalshadbecomeepilepticduringthe90-dayperiodfollowinglesioning,theresultsofthepresentstudywouldneedtobeinterpretedinapotentiallydifferentcontextthanthatofTBI-inducedchangesalone.

Insummary,thepresent ndingssupportdifferentialalterationsoftonicandphasicGABAAR-mediatedinhibitioninDGCsafterasingleepisodeofCCI.Thenetresultofthesedifferentialalterationsisthattheamplitudeoftoniccurrentwasincreasedandbenzodiazepinesensitivityofsynapticreceptorswaslost.These ndingsdemonstrateforthe rsttimethattonicinhibitioncanbealteredafterCCIinDGCs.AdditionalstudiesarerequiredtoelucidatetherelationshipbetweenTBI,increasedtonicinhibitioninthedentategyrus,cognitiveperformance,andthedevelopmentofPTE.Acknowledgments

ThestudywassupportedbyHealthResearchFormulaFundRFA01-07-26,PennsylvaniaDepartmentofHealth(ZM),aResearchGrantfromtheEpilepsyFoundation(ZM),andagrantfromtheGeorgian

NationalScienceScholarsProgram(EL).WethankTeresaM.Hentoszforassistanceinthepreparationofthemanuscript.

References

Adkins,C.E.,Pillai,G.V.,Kerby,J.,Bonnert,T.P.,Haldon,C.,McKernan,R.M.,Gonzalez,J.E.,

Oades,K.,Whiting,P.J.,Simpson,P.B.,2001.alpha4beta3deltaGABA(A)receptorscharacterizedby uorescenceresonanceenergytransfer-derivedmeasurementsofmembranepotential.J.Biol.Chem.276,38934–38939.

Alldred,M.J.,Mulder-Rosi,J.,Lingenfelter,S.E.,Chen,G.,Lüscher,B.,2005.Distinctgamma2

subunitdomainsmediateclusteringandsynapticfunctionofpostsynapticGABAAreceptorsandgephyrin.J.Neurosci.25,594–603.

Arolfo,M.P.,Brioni,J.D.,1991.DiazepamimpairsplacelearningintheMorriswater

maze.Behav.Neural.Biol.55,131–136.

Atack,J.R.,Bayley,P.J.,Seabrook,G.R.,Wafford,K.A.,McKernan,R.M.,Dawson,G.R.,2006.L-655,708enhancescognitioninratsbutisnotproconvulsantatadoseselectiveforalpha5-containingGABAAreceptors.Neuropharmacology51,1023–1029.

Bai,D.,Zhu,G.,Pennefather,P.,Jackson,M.F.,MacDonald,J.F.,Orser,B.A.,2001.Distinct

functionalandpharmacologicalpropertiesoftonicandquantalinhibitorypostsyn-apticcurrentsmediatedbygamma-aminobutyricacid(A)receptorsinhippocampalneurons.Mol.Pharmacol.59,814–824.

Banks,M.I.,Li,T.B.,Pearce,R.A.,1998.ThesynapticbasisofGABAA,slow.J.Neurosci.18,

1305–1317.

Bianchi,M.T.,Macdonald,R.L.,2002.SlowphasesofGABA(A)receptordesensitization:

structuraldeterminantsandpossiblerelevanceforsynapticfunction.J.Physiol.544,3–18.

Binder,D.K.,Routbort,M.J.,McNamara,J.O.,1999.Immunohistochemicalevidenceof

seizure-inducedactivationoftrkreceptorsinthemossy berpathwayofadultrathippocampus.J.Neurosci.19,4616–4626.

Bonislawski,D.P.,Schwarzbach,E.P.,Cohen,A.S.,2007.Braininjuryimpairsdentate

gyrusinhibitoryef cacy.Neurobiol.Dis.25,163–169.

Bowser,D.N.,Wagner,D.A.,Czajkowski,C.,Cromer,B.A.,Parker,M.W.,Wallace,R.H.,

Harkin,L.A.,Mulley,J.C.,Marini,C.,Berkovic,S.F.,Williams,D.A.,Jones,M.V.,Petrou,S.,2002.AlteredkineticsandbenzodiazepinesensitivityofaGABAAreceptorsubunitmutation[gamma2(R43Q)]foundinhumanepilepsy.Proc.Natl.Acad.Sci.U.S.A.99,15170–15175.

Brickley,S.G.,Cull-Candy,S.G.,Farrant,M.,1996.Developmentofatonicformofsynaptic

inhibitioninratcerebellargranulecellsresultingfrompersistentactivationofGABAAreceptors.J.Physiol.497,753–759.

Bright,D.P.,Aller,M.I.,Brickley,S.G.,2007.SynapticreleasegeneratesatonicGABA(A)

receptor-mediatedconductancethatmodulatesburstprecisioninthalamicrelayneurons.J.Neurosci.27,2560–2569.

Brioni,J.D.,McGaugh,J.L.,1988.Post-trainingadministrationofGABAergicantagonists

enhancesretentionofaversivelymotivatedtasks.Psychopharmacology(Berl.)96,505–510.

Brooks-Kayal,A.R.,Shumate,M.D.,Jin,H.,Rikhter,T.Y.,Coulter,D.A.,1998.Selective

changesinsinglecellGABA(A)receptorsubunitexpressionandfunctionintemporallobeepilepsy.Nat.Med.4,1166–1172.

Brown,N.,Kerby,J.,Bonnert,T.P.,Whiting,P.J.,Wafford,K.A.,2002.Pharmacological

characterizationofanovelcelllineexpressinghumanalpha(4)beta(3)deltaGABA(A)receptors.Br.J.Pharmacol.136,965–974.

Buckmaster,P.S.,Schwartzkroin,P.A.,1995.Physiologicalandmorphologicalheterogene-ityofdentategyrus-hilusinterneuronsinthegerbilhippocampusinvivo.Eur.J.Neurosci.7,1393–1402.

Buhl,E.H.,Halasy,K.,Somogyi,P.,1994.Diversesourcesofhippocampalunitaryinhibitory

postsynapticpotentialsandthenumberofsynapticreleasesites.Nature368,823–828.Buzsaki,G.,Draguhn,A.,2004.Neuronaloscillationsincorticalnetworks.Science304,

1926–1929.

Caraiscos,V.B.,Elliott,E.M.,You-Ten,K.E.,Cheng,V.Y.,Belelli,D.,Newell,J.G.,Jackson,M.F.,

Lambert,J.J.,Rosahl,T.W.,Wafford,K.A.,MacDonald,J.F.,Orser,B.A.,2004.TonicinhibitioninmousehippocampalCA1pyramidalneuronsismediatedbyalpha5subunit-containinggamma-aminobutyricacidtypeAreceptors.Proc.Natl.Acad.Sci.U.S.A.101,3662–3667.

Castellano,C.,Cestari,V.,Cabib,S.,Puglisi-Allegra,S.,1993.Strain-dependenteffectsof

post-trainingGABAreceptoragonistsandantagonistsonmemorystorageinmice.Psychopharmacology(Berl.)111,134–138.

Cohen,A.S.,Lin,D.D.,Quirk,G.L.,Coulter,D.A.,2003.DentategranulecellGABAAreceptors

inepileptichippocampus:enhancedsynapticef cacyandalteredpharmacology.Eur.J.Neurosci.17,1607–1616.

Collins,R.C.,Tearse,R.G.,Lothman,E.W.,1983.Functionalanatomyoflimbicseizures:

focaldischargesfrommedialentorhinalcortexinrat.BrainRes.280,25–40.

Collinson,N.,Kuenzi,F.M.,Jarolimek,W.,Maubach,K.A.,Cothliff,R.,Sur,C.,Smith,A.,

Out,F.M.,Howell,O.,Atack,J.R.,McKernan,R.M.,Seabrook,G.R.,Dawson,G.R.,Whiting,P.J.,Rosahl,T.W.,2002.EnhancedlearningandmemoryandalteredGABAergicsynaptictransmissioninmicelackingthealpha5subunitoftheGABAAreceptor.J.Neurosci.22,5572–5580.

Collinson,N.,Atack,J.R.,Laughton,P.,Dawson,G.R.,Stephens,D.N.,2006.Aninverse

agonistselectiveforalpha5subunit-containingGABAAreceptorsimprovesencodingandrecallbutnotconsolidationintheMorriswatermaze.Psychopharmacology(Berl.)188,619–628.

Dahhaoui,M.,Caston,J.,Stelz,T.,1994.EffectsofinhibitionoftheGABAergicsystemsby

picrotoxinonretentionofanociceptiveexperienceintherat,withspecialreferencetothein uenceofcerebellarcortexoutput.Physiol.Behav.56,257–264.

474Z.Mtchedlishvilietal./NeurobiologyofDisease38(2010)464–475

Das,P.,Bell-Horner,C.L.,Huang,R.Q.,Raut,A.,Gonzales,E.B.,Chen,Z.L.,Covey,D.F.,Dillon,

G.H.,2004.InhibitionoftypeAGABAreceptorsbyL-typecalciumchannelblockers.Neuroscience124,195–206.

Dawson,G.R.,Maubach,K.A.,Collinson,N.,Cobain,M.,Everitt,B.J.,MacLeod,A.M.,Choudhury,

H.I.,McDonald,L.M.,Pillai,G.,Rycroft,W.,Smith,A.J.,Sternfeld,F.,Tattersall,F.D.,Wafford,K.A.,Reynolds,D.S.,Seabrook,G.R.,Atack,J.R.,2006.Aninverseagonistselectiveforalpha5subunit-containingGABAAreceptorsenhancescognition.J.Pharmacol.Exp.Ther.316,1335–1345.

DeRidder,M.N.,Simon,M.J.,Siman,R.,Auberson,Y.P.,Raghupathi,R.,Meaney,D.F.,2006.

Traumaticmechanicalinjurytothehippocampusinvitrocausesregionalcaspase-3andcalpainactivationthatisin uencedbyNMDAreceptorsubunitcomposition.Neurobiol.Dis.22,165–176.

Dinocourt,C.,Gallagher,S.E.,Thompson,S.M.,2006.Injury-inducedaxonalsproutinginthe

hippocampusisinitiatedbyactivationoftrkBreceptors.Eur.J.Neurosci.24,1857–1866.Dixon,C.E.,Lighthall,J.W.,Anderson,T.E.,1988.Physiologic,histopathologic,and

cineradiographiccharacterizationofanew uid-percussionmodelofexperimentalbraininjuryintherat.J.Neurotrauma5,91–104.

Dixon,C.E.,Clifton,G.L.,Lighthall,J.W.,Yaghmai,A.A.,Hayes,R.L.,1991.Acontrolledcortical

impactmodeloftraumaticbraininjuryintherat.J.Neurosci.Methods39,253–262.Edwards,F.A.,Konnerth,A.,Sakmann,B.,1990.Quantalanalysisofinhibitorysynaptic

transmissioninthedentategyrusofrathippocampalslices:apatch-clampstudy.J.Physiol.430,213–249.

Elmer,E.,Kokaia,Z.,Kokaia,M.,Carnahan,J.,Nawa,H.,Lindvall,O.,1998.Dynamicchanges

ofbrain-derivedneurotrophicfactorproteinlevelsintheratforebrainaftersingleandrecurringkindling-inducedseizures.Neuroscience83,351–362.

Engel,A.K.,Singer,W.,2001.Temporalbindingandtheneuralcorrelatesofsensory

awareness.TrendsCogn.Sci.5,16–25.

Ernfors,P.,Bengzon,J.,Kokaia,Z.,Persson,H.,Lindvall,O.,1991.Increasedlevelsof

messengerRNAsforneurotrophicfactorsinthebrainduringkindlingepileptogenesis.Neuron7,165–176.

Essrich,C.,Lorez,M.,Benson,J.,Fritschy,J.M.,Luscher,B.,1998.Postsynapticclusteringof

majorGABAAreceptorsubtypesrequirestheγ2subunitandgephyrin.Nat.Neurosci.1,563–571.

Eugène,E.,Depienne,C.,Baulac,S.,Baulac,M.,Fritschy,J.M.,LeGuern,E.,Miles,R.,

Poncer,J.C.,2007.GABA(A)receptorgamma2subunitmutationslinkedtohumanepilepticsyndromesdifferentiallyaffectphasicandtonicinhibition.J.Neurosci.27,14108–14116.

Falkenberg,T.,Mohammed,A.K.,Henriksson,B.,Persson,H.,Winblad,B.,Lindefors,N.,

1992.Increasedexpressionofbrain-derivedneurotrophicfactormRNAinrathippocampusisassociatedwithimprovedspatialmemoryandenrichedenvironment.Neurosci.Lett.138,153–156.

Feng,H.J.,Mathews,G.C.,Kao,C.,Macdonald,R.L.,2008.AlterationsofGABAA-receptorfunctionandallostericmodulationduringdevelopmentofstatusepilepticus.J.Neurophysiol.99,1285–1293.

Gao,L.,Green eld,L.J.,2005.ActivationofproteinkinaseCreducesbenzodiazepine

potencyatGABAAreceptorsinNT2-Nneurons.Neuropharmacology48,333–342.GibbsIII,J.W.,Sombati,S.,DeLorenzo,R.J.,Coulter,D.A.,1997.Physiologicaland

pharmacologicalalterationsinpostsynapticGABA(A)receptorfunctioninahippocam-palculturemodelofchronicspontaneousseizures.J.Neurophysiol.77,2139–2152.Glykys,J.,Mann,E.O.,Mody,I.,2008.WhichGABA(A)receptorsubunitsarenecessaryfor

tonicinhibitioninthehippocampus?J.Neurosci.28,1421–1426.

Golarai,G.,Greenwood,A.C.,Feeney,D.M.,Connor,J.A.,2001.Physiologicalandstructural

evidenceforhippocampalinvolvementinpersistentseizuresusceptibilityaftertraumaticbraininjury.J.Neurosci.21,8523–8537.

Goldschen-Ohm,M.P.,Wagner,D.A.,Petrou,S.,Jones,M.V.,2010.Anepilepsy-related

regionintheGABA(A)receptormediateslong-distanceeffectsonGABAandbenzodiazepinebindingsites.Mol.Pharmacol.77,35–45.

Goodkin,H.P.,Yeh,J.L.,Kapur,J.,2005.Statusepilepticusincreasestheintracellular

accumulationofGABAAreceptors.J.Neurosci.25,5511–5520.

Goodkin,H.P.,Joshi,S.,Mtchedlishvili,Z.,Brar,J.,Kapur,J.,2008.Subunit-speci ctraf cking

ofGABA(A)receptorsduringstatusepilepticus.J.Neurosci.28,2527–2538.

Hájos,N.,Nusser,Z.,Rancz,E.A.,Freund,T.F.,Mody,I.,2000.Celltype-andsynapse-speci c

variabilityinsynapticGABAAreceptoroccupancy.Eur.J.Neurosci.12,810–818.

Halasy,K.,Somogyi,P.,1993.SubdivisionsinthemultipleGABAergicinnervationof

granulecellsinthedentategyrusoftherathippocampus.Eur.J.Neurosci.5,411–429.Hall,S.,Bornstein,R.A.,1991.Therelationshipbetweenintelligenceandmemory

followingminorormildclosedheadinjury:greaterimpairmentinmemorythanintelligence.J.Neurosurg.75,378–381.

Hamann,M.,Rossi,D.J.,Attwell,D.,2002.Tonicandspilloverinhibitionofgranulecells

controlinformation owthroughcerebellarcortex.Neuron33,625–633.

Hardingham,N.R.,Larkman,A.U.,1998.Rapidreport:thereliabilityofexcitatorysynaptic

transmissioninslicesofratvisualcortexinvitroistemperaturedependent.J.Physiol.507,249–256.

Heinemann,U.,Beck,H.,Dreier,J.P.,Ficker,E.,Stabel,J.,Zhang,C.L.,1992.Thedentate

gyrusasaregulatedgateforthepropagationofepileptiformactivity.EpilepsyRes.7,273–280.

Hicks,R.R.,Numan,S.,Dhillon,H.S.,Prasad,M.R.,Seroogy,K.B.,1997.AlterationsinBDNF

andNT-3mRNAsinrathippocampusafterexperimentalbraintrauma.BrainRes.48,401–406.

Hübner,C.A.,Stein,V.,Hermans-Borgmeyer,I.,Meyer,T.,Ballanyi,K.,Jentsch,T.J.,2001.

DisruptionofKCC2revealsanessentialroleofK–Clcotransportalreadyinearlysynapticinhibition.Neuron30,515–524.

Hunt,R.F.,Scheff,S.W.,Smith,B.N.,2009.Posttraumaticepilepsyaftercontrolledcortical

impactinjuryinmice.Exp.Neurol.215,243–252.

Johansson,I.M.,Birzniece,V.,Lindblad,C.,Olsson,T.,Backstrom,T.,2002.Allopregna-noloneinhibitslearningintheMorriswatermaze.BrainRes.934,125–131.

Joshi,S.,Kapur,J.,2009.SlowintracellularaccumulationofGABA(A)receptordeltasubunit

ismodulatedbybrain-derivedneurotrophicfactor.Neuroscience164,507–519.

Kaneda,M.,Farrant,M.,Cull-Candy,S.G.,1995.Whole-cellandsingle-channelcurrents

activatedbyGABAandglycineingranulecellsoftheratcerebellum.J.Physiol.485,419–435.

Kant,G.J.,Wylie,R.M.,Vasilakis,A.A.,Ghosh,S.,1996.Effectsoftriazolamanddiazepamon

learningandmemoryasassessedusingawatermaze.Pharmacol.Biochem.Behav.53,317–322.

Kapur,J.,Macdonald,R.L.,1997.Rapidseizure-inducedreductionofbenzodiazepineand

Zn2+sensitivityofhippocampaldentategranulecellGABAAreceptors.J.Neurosci.17,7532–7540.

Kharlamov,E.A.,Jukkola,P.I.,Schmitt,K.L.,Kelly,K.M.,2003.Electrobehavioralcharacter-isticsofepilepticratsfollowingphotothromboticbraininfarction.EpilepsyRes.56,185–203.

Kittler,J.T.,Moss,S.J.,2003.ModulationofGABAAreceptoractivitybyphosphorylationand

receptortraf cking:implicationsfortheef cacyofsynapticinhibition.Curr.Opin.Neurobiol.13,341–347.

Kneussel,M.,Betz,H.,2000.Receptors,gephyrinandgephyrin-associatedproteins:

novelinsightsintotheassemblyofinhibitorypostsynapticmembranespecializa-tions.J.Physiol.525,1–9.

Kobori,N.,Clifton,G.L.,Dash,P.K.,2006.Enhancedcatecholaminesynthesisinthe

prefrontalcortexaftertraumaticbraininjury:implicationsforprefrontaldysfunction.J.Neurotrauma23,1094–1102.

Korpi,E.R.,Luddens,H.,1997.FurosemideinteractionswithbrainGABA(A)receptors.

Br.J.Pharmacol.120,741–748.

Kozlov,A.S.,Angulo,M.C.,Audinat,E.,Charpak,S.,2006.Targetcell-speci cmodulationof

neuronalactivitybyastrocytes.Proc.Natl.Acad.Sci.U.S.A.27,10058–10063.

Krazem,A.,Borde,N.,Beracochea,D.,2001.Effectsofdiazepamandbeta-CCMonworking

memoryinmice:relationshipswithemotionalreactivity.Pharmacol.Biochem.Behav.68,235–244.

Kushmerick,C.,Renden,R.,vonGersdorff,H.,2006.Physiologicaltemperaturesreducethe

rateofvesiclepooldepletionandshort-termdepressionviaanaccelerationofvesiclerecruitment.J.Neurosci.26,1366–1377.

Levin,H.S.,Grossman,R.G.,Rose,J.E.,Teasdale,G.,1979.Long-termneuropsychological

outcomeofclosedheadinjury.J.Neurosurg.50,412–422.

Liu,Q.Y.,Schaffner,A.E.,Chang,Y.H.,Maric,D.,Barker,J.L.,2000.Persistentactivationof

GABA(A)receptor/Cl( )channelsbyastrocyte-derivedGABAinculturedembryonicrathippocampalneurons.J.Neurophysiol.84,1392–1403.

Llinas,R.,Ribary,U.,2001.Consciousnessandthebrain.Thethalamocorticaldialoguein

healthanddisease.Ann.N.Y.Acad.Sci.929,166–175.

Mangan,P.S.,Sun,C.,Carpenter,M.,Goodkin,H.P.,Sieghart,W.,Kapur,J.,2005.Cultured

hippocampalpyramidalneuronsexpresstwokindsofGABAAreceptors.Mol.Pharmacol.67,775–788.

Mann,E.O.,Mody,I.,2009.Controlofhippocampalgammaoscillationfrequencyby

tonicinhibitionandexcitationofinterneurons.Nat.Neurosci.13,205–212.

Maubach,K.,2003.GABA(A)receptorsubtypeselectivecognitionenhancers.Curr.Drug

TargetsCNSNeurol.Disord.2,233–239.

Mayo,W.,Dellu,F.,Cherkaoui,J.,Chapouthier,G.,Dodd,R.H.,Le,M.M.,Simon,H.,1992.

Cognitiveenhancingpropertiesofbeta-CCMinfusedintothenucleusbasalismagnocellularisoftherat.BrainRes.589,109–114.

Mayo,W.,Dellu,F.,Robel,P.,Cherkaoui,J.,Le,M.M.,Baulieu,E.E.,Simon,H.,1993.Infusion

ofneurosteroidsintothenucleusbasalismagnocellularisaffectscognitiveprocessesintherat.BrainRes.607,324–328.

Merlio,J.P.,Ernfors,P.,Kokaia,Z.,Middlemas,D.S.,Bengzon,J.,Kokaia,M.,Smith,M.L.,

Siesjo,B.K.,Hunter,T.,Lindvall,O.,Persson,H.,1993.IncreasedproductionoftheTrkBproteintyrosinekinasereceptorafterbraininsults.Neuron10,151–164.

Micheva,K.D.,Smith,S.J.,2005.Strongeffectsofsubphysiologicaltemperatureonthe

functionandplasticityofmammalianpresynapticterminals.J.Neurosci.25,7481–7488.Miles,R.,Tóth,K.,Gulyás,A.I.,Hájos,N.,Freund,T.F.,1996.Differencesbetweensomatic

anddendriticinhibitioninthehippocampus.Neuron16,815–823.

Mody,I.,Pearce,R.A.,2004.DiversityofinhibitoryneurotransmissionthroughGABA(A)

receptors.TrendsNeurosci.27,569–575.

Mtchedlishvili,Z.,Kapur,J.,2006.High-af nity,slowlydesensitizingGABAAreceptors

mediatetonicinhibitioninhippocampaldentategranulecells.Mol.Pharmacol.69,564–575.

Naylor,D.E.,Liu,H.,Wasterlain,C.G.,2005.Traf ckingofGABA(A)receptors,lossof

inhibition,andamechanismforpharmacoresistanceinstatusepilepticus.J.Neurosci.25,7724–7733.

Nusser,Z.,Mody,I.,2002.Selectivemodulationoftonicandphasicinhibitionsindentate

gyrusgranulecells.J.Neurophysiol.87,2624–2628.

Nusser,Z.,Roberts,J.D.,Baude,A.,Richards,J.G.,Somogyi,P.,1995.Relativedensitiesof

synapticandextrasynapticGABAAreceptorsoncerebellargranulecellsasdeterminedbyaquantitativeimmunogoldmethod.J.Neurosci.15,2948–2960.

Nusser,Z.,Hajos,N.,Somogyi,P.,Mody,I.,1998.IncreasednumberofsynapticGABA(A)

receptorsunderliespotentiationathippocampalinhibitorysynapses.Nature395,172–177.

O'Dell,D.M.,Hamm,R.J.,1995.ChronicpostinjuryadministrationofMDL26,479

(Suritozole),anegativemodulatorattheGABAAreceptor,andcognitiveimpairmentinratsfollowingtraumaticbraininjury.J.Neurosurg.83,878–883.

Otis,T.S.,De,K.Y.,Mody,I.,stingpotentiationofinhibitionisassociatedwithan

increasednumberofgamma-aminobutyricacidtypeAreceptorsactivatedduringminiatureinhibitorypostsynapticcurrents.Proc.Natl.Acad.Sci.U.S.A.91,7698–7702.Payne,H.L.,Donoghue,P.S.,Connelly,W.M.,Hinterreiter,S.,Tiwari,P.,Ives,J.H.,

Hann,V.,Sieghart,W.,Lees,G.,Thompson,C.L.,2006.AberrantGABA(A)receptorexpressioninthedentategyrusoftheepilepticmutantmousestargazer.J.Neurosci.26,8600–8608.

Z.Mtchedlishvilietal./NeurobiologyofDisease38(2010)464–475

475

Peng,Z.,Hauer,B.,Mihalek,R.M.,Homanics,G.E.,Sieghart,W.,Olsen,R.W.,Houser,C.R.,

2002.GABA(A)receptorchangesindeltasubunit-de cientmice:p.Neurol.446,179–197.

Peng,Z.,Huang,C.S.,Stell,B.M.,Mody,I.,Houser,C.R.,2004.Alteredexpressionoftheδ

subunitoftheGABAAreceptorinamousemodeloftemporallobeepilepsy.J.Neurosci.24,8629–8639.

Perrais,D.,Ropert,N.,1999.EffectofzolpidemonminiatureIPSCsandoccupancyof

postsynapticGABAAreceptorsincentralsynapses.J.Neurosci.19,578–588.

Pirker,S.,Schwarzer,C.,Czech,T.,Baumgartner,C.,Pockberger,H.,Maier,H.,Hauer,

B.,Sieghart,W.,Furtinger,S.,Sperk,G.,2003.IncreasedexpressionofGABA(A)receptorbeta-subunitsinthehippocampusofpatientswithtemporallobeepilepsy.J.Neuropathol.Exp.Neurol.62,820–834.

Povlishock,J.T.,Becker,D.P.,Cheng,C.L.,Vaughan,G.W.,1983.Axonalchangeinminor

headinjury.J.Neuropathol.Exp.Neurol.42,225–242.

Pullela,R.,Raber,J.,Pfankuch,T.,Ferriero,D.M.,Claus,C.P.,Koh,S.E.,Yamauchi,T.,Rola,

R.,Fike,J.R.,Noble-Haeusslein,L.J.,2006.Traumaticinjurytotheimmaturebrainresultsinprogressiveneuronalloss,hyperactivityanddelayedcognitiveimpair-ments.Dev.Neurosci.28,396–409.

Qi,Z.H.,Song,M.,Wallace,M.J.,Wang,D.,Newton,P.M.,McMahon,T.,Chou,W.H.,Zhang,

C.,Shokat,K.M.,Messing,R.O.,2007.ProteinkinaseCepsilonregulatesgamma-aminobutyratetypeAreceptorsensitivitytoethanolandbenzodiazepinesthroughphosphorylationofgamma2subunits.J.Biol.Chem.282,33052–33063.

Racine,R.J.,1972.Modi cationofseizureactivitybyelectricalstimulation.II.Motor

seizure.Electroencephalogr.Clin.Neurophysiol.32,281–294.

Raffalli-Sebille,M.J.,Chapouthier,G.,Venault,P.,Dodd,R.H.,1990.Methylbeta-carboline-3-carboxylateenhancesperformanceinamultiple-triallearningtaskinmice.Pharmacol.Biochem.Behav.35,281–284.

Reeves,T.M.,Lyeth,B.G.,Phillips,L.L.,Hamm,R.J.,Povlishock,J.T.,1997.Theeffectsof

traumaticbraininjuryoninhibitioninthehippocampusanddentategyrus.BrainRes.757,119–132.

Richerson,G.B.,Wu,Y.,2003.Dynamicequilibriumofneurotransmittertransporters:

notjustforreuptakeanymore.J.Neurophysiol.90,1363–1374.

Rivera,C.,Li,H.,Thomas-Crusells,J.,Lahtinen,H.,Viitanen,T.,Nanobashvili,A.,Kokaia,Z.,

Airaksinen,M.S.,Voipio,J.,Kaila,K.,Saarma,M.,2002.BDNF-inducedTrkBactivationdown-regulatestheK+–Cl cotransporterKCC2andimpairsneuronalCl extrusion.J.CellBiol.159,747–752.

Rossi,D.J.,Hamann,M.,1998.Spillover-mediatedtransmissionatinhibitorysynapses

promotedbyhighaf nityalpha6subunitGABA(A)receptorsandglomerulargeometry.Neuron20,783–795.

Rudolph,U.,Möhler,H.,2006.GABA-basedtherapeuticapproaches:GABAAreceptor

subtypefunctions.Curr.Opin.Pharmacol.6,18–23.

Saatman,K.E.,Feeko,K.J.,Pape,R.L.,Raghupathi,R.,2006.Differentialbehavioraland

histopathologicalresponsestogradedcorticalimpactinjuryinmice.J.Neurotrauma23,1241–1253.

Santhakumar,V.,Bender,R.,Frotscher,M.,Ross,S.T.,Hollrigel,G.S.,Toth,Z.,Soltesz,I.,

2000.Granulecellhyperexcitabilityintheearlypost-traumaticratdentategyrus:the‘irritablemossycell’hypothesis.J.Physiol.524,117–134.

Santhakumar,V.,Ratzliff,A.D.,Jeng,J.,Toth,Z.,Soltesz,I.,2001.Long-termhyperexcitability

inthehippocampusafterexperimentalheadtrauma.Ann.Neurol.50,708–717.

Sassoè-Pognetto,M.,Panzanelli,P.,Sieghart,W.,Fritschy,J.M.,2000.Colocalizationof

p.Neurol.420,481–498.

Shumate,M.D.,Lin,D.D.,GibbsIII,J.W.,Holloway,K.L.,Coulter,D.A.,1998.GABA(A)

receptorfunctioninepileptichumandentategranulecells:comparisontoepilepticandcontrolrat.EpilepsyRes.32,114–128.

Sieghart,W.,Sperk,G.,2002.Subunitcomposition,distributionandfunctionofGABA(A)

receptorsubtypes.Curr.Top.Med.Chem.2,795–816.

Silvers,J.M.,Tokunaga,S.,Berry,R.B.,White,A.M.,Matthews,D.B.,2003.Impairmentsin

spatiallearningandmemory:ethanol,allopregnanolone,andthehippocampus.BrainRes.BrainRes.Rev.43,275–284.

Soltesz,I.,Mody,I.,1995.Ca(2+)-dependentplasticityofminiatureinhibitorypostsynaptic

currentsafteramputationofdendritesincentralneurons.J.Neurophysiol.73,1763–1773.

Somogyi,P.,Fritschy,J.M.,Benke,D.,Roberts,J.D.,Sieghart,W.,1996.Thegamma2

subunitoftheGABAAreceptorisconcentratedinsynapticjunctionscontainingthealpha1andbeta2/3subunitsinhippocampus,cerebellumandglobuspallidus.Neuropharmacology35,1425–1444.

Statler,K.D.,Scheerlinck,P.,Pouliot,W.,Hamilton,M.,White,H.S.,Dudek,F.E.,2009.A

potentialmodelofpediatricposttraumaticepilepsy.EpilepsyRes.86,221–223.Stell,B.M.,Mody,I.,2002.Receptorswithdifferentaf nitiesmediatephasicandtonic

GABA(A)conductancesinhippocampalneurons.J.Neurosci.22,RC223.

Stringer,J.L.,Lothman,E.W.,1989.Modelofspontaneoushippocampalepilepsyinthe

anesthetizedrat:electrographic,[K+]0,and[Ca2+]0responsepatterns.EpilepsyRes.4,177–186.

Sun,C.,Sieghart,W.,Kapur,J.,2004.Distributionofalpha1,alpha4,gamma2,anddelta

subunitsofGABAAreceptorsinhippocampalgranulecells.BrainRes.1029,207–216.

Sun,C.,Mtchedlishvili,Z.,Erisir,A.,Kapur,J.,2007.Diminishedneurosteroidsensitivityof

synapticinhibitionandalteredlocationofthealpha4subunitofGABA(A)receptorsinananimalmodelofepilepsy.J.Neurosci.27,12641–12650.

Sundstrom-Poromaa,I.,Smith,D.H.,Gong,Q.H.,Sabado,T.N.,Li,X.,Light,A.,Wiedmann,M.,

Williams,K.,Smith,S.S.,2002.Hormonallyregulatedalpha(4)beta(2)deltaGABA(A)receptorsareatargetforalcohol.Nat.Neurosci.8,721–722.

Sur,C.,Farrar,S.J.,Kerby,J.,Whiting,P.J.,Atack,J.R.,McKernan,R.M.,1999.Preferential

coassemblyofalpha4anddeltasubunitsofthegamma-aminobutyricacidAreceptorinratthalamus.Mol.Pharmacol.56,110–115.

Terunuma,M.,Xu,J.,Vithlani,M.,Sieghart,W.,Kittler,J.,Pangalos,M.,Haydon,P.G.,Coulter,

D.A.,Moss,S.J.,2008.De citsinphosphorylationofGABAAreceptorsbyintimatelyassociatedproteinkinaseCactivityunderliecompromisedsynapticinhibitionduringstatusepilepticus.J.Neurosci.28,376–384.

Toth,Z.,Hollrigel,G.S.,Gorcs,T.,Soltesz,I.,1997.Instantaneousperturbationof

dentateinterneuronalnetworksbyapressurewave-transientdeliveredtotheneocortex.J.Neurosci.17,8106–8117.

Tran,L.D.,Lifshitz,J.,Witgen,B.M.,Schwarzbach,E.,Cohen,A.S.,Grady,M.S.,2006.

Responseofthecontralateralhippocampustolateral uidpercussionbraininjury.J.Neurotrauma23,1330–1342.

Traub,R.D.,Miles,R.,1991.Multiplemodesofneuronalpopulationactivityemergeafter

modifyingspeci csynapsesinamodeloftheCA3regionofthehippocampus.Ann.N.Y.Acad.Sci.627,277–290.

Turkmen,S.,Lofgren,M.,Birzniece,V.,Backstrom,T.,Johansson,I.M.,2006.Tolerance

developmenttoMorriswatermazetestimpairmentsinducedbyacuteallopregna-nolone.Neuroscience139,651–659.

Volgushev,M.,Kudryashov,I.,Chistiakova,M.,Mukovski,M.,Niesmann,J.,Eysel,U.T.,

2004.Probabilityoftransmitterreleaseatneocorticalsynapsesatdifferenttemperatures.J.Neurophysiol.92,212–220.

Wafford,K.A.,Thompson,S.A.,Thomas,D.,Sikela,J.,Wilcox,A.S.,Whiting,P.J.,1996.

Functionalcharacterizationofhumanγ-aminobutyricacidAreceptorscontainingtheα4subunit.Mol.Pharmacol.50,670–678.

Wall,M.J.,Usowicz,M.M.,1997.Developmentofactionpotential-dependentand

independentspontaneousGABAAreceptor-mediatedcurrentsingranulecellsofpostnatalratcerebellum.Eur.J.Neurosci.9,533–548.

Wallace,R.H.,Marini,C.,Petrou,S.,Harkin,L.A.,Bowser,D.N.,Panchal,R.G.,Williams,

D.A.,Sutherland,G.R.,Mulley,J.C.,Scheffer,I.E.,Berkovic,S.F.,2001.MutantGABA(A)receptorgamma2-subunitinchildhoodabsenceepilepsyandfebrileseizures.Nat.Genet.28,49–52.

Wei,W.,Zhang,N.,Peng,Z.,Houser,C.R.,Mody,I.,2003.Perisynapticlocalizationof

deltasubunit-containingGABA(A)receptorsandtheiractivationbyGABAspilloverinthemousedentategyrus.J.Neurosci.23,10650–10661.

Yang,K.,Perez-Polo,J.R.,Mu,X.S.,Yan,H.Q.,Xue,J.J.,Iwamoto,Y.,Liu,S.J.,Dixon,C.E.,

Hayes,R.L.,1996.Increasedexpressionofbrain-derivedneurotrophicfactorbutnotneurotrophin-3mRNAinratbrainaftercorticalimpactinjury.J.Neurosci.Res.44,157–164.

Zhan,R.Z.,Nadler,J.V.,2009.EnhancedtonicGABAcurrentinnormotopicandhilarectopic

dentategranulecellsafterpilocarpine-inducedstatusepilepticus.J.Neurophysiol.102,670–681.

本文来源：https://www.bwwdw.com/article/x154.html