Increase of GABAA receptor-mediated tonic inhibition in DG c(4)

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.