Increase of GABAA receptor-mediated tonic inhibition in DG c(2)

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