Increase of GABAA receptor-mediated tonic inhibition in DG c(8)

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).