Increase of GABAA receptor-mediated tonic inhibition in DG c(9)

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