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.