Transgenic Expression ofGlud1 (Glutamate Dehydrogenase 1) in Neurons: In Vivo Model of

16 Pages·2009·4.96 MB·English

TheJournalofNeuroscience,November4,2009•29(44):13929–13944•13929
Cellular/Molecular
Transgenic Expression of Glud1 (Glutamate Dehydrogenase 1)
in Neurons: In Vivo Model of Enhanced Glutamate
Release, Altered Synaptic Plasticity, and Selective Neuronal
Vulnerability
XiaodongBao,1,2RanuPal,1,2,3KevinN.Hascup,4YongfuWang,5Wen-TungWang,6WenhaoXu,8DongweiHui,1
AbdulbakiAgbas,1,2XinkunWang,1,2,3MaryL.Michaelis,1,2In-YoungChoi,5,6,7AndreiB.Belousov,5GregA.Gerhardt,4
andEliasK.Michaelis1,2,3
1HiguchiBiosciencesCenter,2DepartmentofPharmacologyandToxicology,and3LifeSpanStudiesInstitute,UniversityofKansas,Lawrence,Kansas
66047,4AnatomyandNeurobiology,CenterforMicroelectrodeTechnology,MorrisK.UdallParkinson’sDiseaseResearchCenterofExcellence,University
ofKentucky,CollegeofMedicine,Lexington,Kentucky40536,5DepartmentofMolecularandIntegrativePhysiology,6HoglundBrainImagingCenter,and
7DepartmentofNeurology,UniversityofKansasMedicalCenter,KansasCity,Kansas66160,and8DepartmentofMicrobiology,UniversityofVirginia
HealthSystem,Charlottesville,Virginia22908
Theeffectsoflifelong,moderateexcessreleaseofglutamate(Glu)intheCNShavenotbeenpreviouslycharacterized.Wecreateda
transgenic(Tg)mousemodeloflifelongexcesssynapticGlureleaseintheCNSbyintroducingthegeneforglutamatedehydrogenase1
(Glud1)underthecontroloftheneuron-specificenolasepromoter.Glud1is,potentially,animportantenzymeinthepathwayofGlu
synthesisinnerveterminals.IncreasedlevelsofGLUDproteinandactivityinCNSneuronsofhemizygousTgmicewereassociatedwith
increasesintheinvivoreleaseofGluafterneuronaldepolarizationinstriatumandinthefrequencyandamplitudeofminiatureEPSCsin
theCA1regionofthehippocampus.DespiteoverexpressionofGlud1inallneuronsoftheCNS,theTgmicesufferedneuronallossesin
selectbrainregions(e.g.,theCA1butnottheCA3region).Invulnerableregions,TgmicehaddecreasesinMAP2Alabelingofdendrites
andinsynaptophysinlabelingofpresynapticterminals;thedecreasesinneuronalnumbersanddendriteandpresynapticterminal
labelingincreasedwithadvancingage.Inaddition,theTgmiceexhibiteddecreasesinlong-termpotentiationofsynapticactivityandin
spinedensityindendritesofCA1neurons.Behaviorally,theTgmiceweresignificantlymoreresistantthanwild-typemicetoinduction
anddurationofanesthesiaproducedbyanestheticsthatsuppressGluneurotransmission.TheGlud1mousemightbeausefulmodelfor
theeffectsoflifelongexcesssynapticGlureleaseonCNSneuronsandforage-associatedneurodegenerativeprocesses.
Introduction neuronstoexcessGluhavebeenstudiedextensively(Aartsetal.,
Glutamate(Glu)isbotharapidlyactingtransmitterandasignal- 2003).However,acutetreatmentswithGluanalogsmaynotfully
ingmoleculewithlong-lastingeffectsonneuronalstructureand replicatetheeffectsofchronic,localized,moderateexcessrelease
function(Cotmanetal.,1988;KomuroandRakic,1993).Exces- ofGluatsynapsesoccurringthroughoutthelifespanofanor-
sive excitation of neurons by Glu causes neurotoxicity (Choi, ganism.Suchreleasemaybeanimportantparameterinthecon-
1992), a process linked to several pathologic states in the CNS trol of synapse structure and function, dendrite and nerve
(Schwarcz and Meldrum, 1985). The molecular and cellular terminalintegrity,orneuronalinjury.Changesinsynapticfunc-
mechanismsofneurodegenerationcausedbyacuteexposureof tion and loss of select glutamatergic neurons or their synapses
occurduringagingorinage-associatedneurodegenerativedis-
eases (Masliah et al., 1993; Morrison and Hof, 1997; Francis,
ReceivedSept.7,2009;acceptedSept.28,2009.
2003), and changes in Glu release or reuptake are considered
ThisworkwassupportedbygrantsfromtheNationalInstituteonAging(AG12993);NationalInstituteonDeaf-
nessandOtherCommunicationDisorders(HD02528);NationalInstituteonAlcoholAbuseandAlcoholism(AA11419, importantfortheobservedsynapticandneuronallosses.How-
AA04732,AA12276);NationalScienceFoundation(DBI-9987807,DBI-0352848);NationalInstituteonDrugAbuse ever,theeffectofchronic,moderate,transientincreasesinextra-
(DA017186,DA015088,TrainingGrantDA022738);NationalInstituteofNeurologicalDisordersandStroke cellular Glu in brain on synaptic morphology and function or
(NS39787);NationalInstituteofMentalHealth(MH58414);KansasTechnologyEnterpriseCorporation;Miller,Hed-
neuronallosshasbeendifficulttoproduceinexperimentalani-
wig,andWilburFund;andUniversityofKansasResearchDevelopmentFund.WethankDr.N.Moschonasfor
providingthemouseGlud1cDNAandDr.J.SutcliffeforthepNSE-LacZvector.ThesupportoftheHiguchiBiosciences malsinvivo.
Centerisacknowledged. TwoanimalmodelsofexcessextracellularGluaccumulation
CorrespondenceshouldbeaddressedtoDr.EliasK.Michaelis,HiguchiBiosciencesCenter,2099ConstantAvenue, andtheresultanteffectsonneuronalstructureandfunctionare
UniversityofKansas,Lawrence,KS66047.E-mail:[email protected].
thenullmutantmicefortheGlutransporterSlc1a2(Eaat2)and
DOI:10.1523/JNEUROSCI.4413-09.2009
Copyright©2009SocietyforNeuroscience 0270-6474/09/2913929-16$15.00/0 thetuberoussclerosiscomplex-1(Tsc1)genes.Thesemicesuffer
13930•J.Neurosci.,November4,2009•29(44):13929–13944 Baoetal.•Glud1TransgenicMice
extensivebraindamageandmarkedreductioninlifespanasa pfupolymeraseandeither100ngofgenomicDNAor1ngofDNAfrom
resultoflossoffunctionofGlutransportersandhighlevelsof thepNSE-GLUD1vector(reactionconditions:32cyclesof95°Cfor30s,
tonic Glu accumulation (1.5- to 32-fold normal levels) in the 53°C for 30 s, 72°C for 1 min). Southern blot analyses were used to
extracellularmedium(Rothsteinetal.,1996;Tanakaetal.,1997; confirm the PCR results. pNSE-GLUD1 and genomic DNA from tail
clippingsweredigestedwithEcoRV.A790bpfragmentfromtheGlud1
Zengetal.,2007).Neitherofthesetwomutantmicewouldbea
ORFwasusedasaprobeforhybridizationwithgenomicDNA.
goodmodelforprobingtheeffectsonneuronsofmoderatein-
EstimationofGlud1genecopynumber.GenomicDNAfromfounder
creasesindepolarization-inducedGlureleasefromnervetermi-
Tgandwild-type(wt)micewasextracted(DNeasytissuekit;Qiagen),
nals across the life span of an organism. To achieve moderate
and the DNA concentration was determined (A /A nm; Bioana-
260 280
increasesinGlureleasebyCNSneuronsandstudytheeffectsof lyzer).QuantitativePCR(qPCR)wasusedtomeasuretheamountof
excess synaptic Glu release on neuronal and synaptic structure Glud1andGrin1DNA.ThevaluesforGrin1ineachsamplewereusedto
andfunction,wecreatedtransgenic(Tg)micewithextracopies standardizetheDNAmeasurementsfortheestimationofGlud1gene
of the gene for Glud1. The protein GLUD1 is a mitochondrial copy numbers in all samples. For measurements of Grin1 DNA, the
enzymethatconvertsGluto2-oxoglutarate(2-OG);the2-OG primersusedwereF ,5(cid:1)-CCAGGCACTACC-3(cid:1),andR ,5(cid:1)-GGCTGCA-
1 1
generatedinmitochondriaiscarriedtothecytosolofnerveter- GATGGAATGATTA-3(cid:1);andtheqPCRprobeforbindingtotheampli-
conswas5(cid:1)-6-FAM-GTGGTTGGGCTGGGCTTTAGATGT-BHQ-1-3(cid:1)
minals, converted back to Glu, and stored in synaptic vesicles
(BiosearchTechnologies).FortheGlud1transgene,primersandprobe
(i.e., feeds into the pool of releasable Glu) (Palaiologos et al.,
were designed according to the sequence of exon 1 of Glud1: F , 5(cid:1)-
1988).Thus,GLUDactivitymayregulateGlutransmitterforma- 1
GGTGCGCGGCATCCT-3(cid:1); R, 5(cid:1)-AACCATGTGTTGAGCCTCTCCT-
tionatnerveendingsand,generally,Gluhomeostasis. 3(cid:1); and probe 5(cid:1)-6-FAM-AG1GATCATCAAGCCCTG-NFQ-MGB-3(cid:1)
WedescribebelowthegenerationandcharacterizationofTg
(AppliedBiosystems).ThepolymerasewasExTaq(Takara),andgene
mice that overexpress the Glud1 transgene in CNS neurons. copynumberswerecalculatedbasedonratiosofGlud1towtCtvalues.
ThesemiceexhibitedincreasedGlureleaseafterdepolarization, ImmunoblotestimatesofGLUD1andNMDAreceptorsubunitlevels.To
increasedfrequencyandamplitudeofminiatureEPSCs(mEPSCs) measurethelevelsofGLUD1proteininwtandTgmousebrains,whole
inhippocampus,relativeresistancetoanestheticagentsknownto brainsfrom6-to8-month-oldmicewereexcisedandhomogenized,and
suppressGluneurotransmission,age-associatedneuronallosses 35(cid:1)gofproteinfromeachhomogenatewasanalyzedbySDS-PAGEand
in select brain regions, age-related alterations in dendrite and immunoblotting(Paletal.,2003).Anti-GLUD1antibodies(polyclonal;
NovusBiologicals)wereusedat1:1000dilution.Relativelevelsofexpres-
synaptic structures, altered dendrite spine density, and dimin-
sionwereestimatedbydensitometry.Homogenateswereobtainedfrom
ishedlong-termpotentiation(LTP)ofsynapticactivity.
sex-matchedlittermatepairsofwtandTg,andthesamplesprocessedat
thesametime.TomeasurethelevelsofNMDAreceptorsubunitsNR1
MaterialsandMethods andNR2A,thehippocampus,striatum,andfronto-parietalcortexwere
GenerationofGlud1andEgfpTgmice.Experimentalproceduresrelated dissectedfromthreemale6-month-oldwtandTgmice.TheP2fraction
toanimalsfollowedtheproceduresoftheInstitutionalAnimalCareand wasisolatedfromhomogenatesfromthesethreeregionsasdescribed
UseCommitteeoftheUniversityofKansasandthoseoftheNational below.EqualamountsofP2protein(50(cid:1)g)wereanalyzedbyimmuno-
InstitutesofHealth.Tgmiceweregeneratedbymicroinjectingfertilized blotassaysasdescribedaboveforGLUD1.ImmunelabelingwithNR1
oocytesfromsuper-ovulatingC57BL6/SJLhybridmicewithlinearized andNR2A(bothpolyclonal;Millipore)andactin(monoclonal;Milli-
DNAcontainingthecDNAofmouseGlud1(TzimagiorgisandMoscho- pore)antibodies(1:1000dilutionforall)wasperformed.Actinwasused
nas,1991).ThecDNAwasplacedunderthecontroloftheNsepromoter. asaloadingcontrol,andthelevelsofeachreceptorsubunitinthesamples
ThispromoterwasexcisedfrompNSE-LacZvector(Forss-Petteretal., wereestimatedastheratioofimmunereactionoverthatoftherespective
1990)bydigestingtheSV40polyAtail(BamHIandEcoRI),bluntingand actincontrol(Paletal.,2003).
cloningitintopGEM-7ZbetweenSmaIandBamHI.ThentheNsepro- MitochondriaandsynaptosomepreparationandmeasurementofGLUD
moterwasexcisedfrompNSE-LacZ(BamHIandSphIdigestion)and activityinvitroandinsitu.BraintissuefromwtandTgmicewasexcised
clonedintothemodifiedpGEM-7Z.Alinkercontainingmultiplerestric- andplacedinsucrosebuffercontaining(inmM)320sucrose,0.5MgSO4,
tion sites (BglII/EcoRV/HindIII/MluI/XbaI/KpnI; sense sequence, 5(cid:1)- 10KCl,1dithiothreitol,14-(2-aminoethyl)benzenesulfonylfluoridehy-
AGATCTGATATCAAGCTTACGCGTCTAGAGGTAC-3(cid:1)) was cloned drochloride,1sodiumorthovanadate,10HEPES,pH7.4andamixture
betweenSphI(blunted)andKpnIofpGEM-7Z,whichcontainedtheNse ofproteaseinhibitors(1:100dilution;Sigma).Bothacrudemitochon-
promoter and SV40 polyA tail (pNSE-GEM-7Z). The open reading drial(P2)andapurifiedsynaptosomefractionwereisolated(Michaelis
frame (ORF) of Glud1 cDNA was excised from pUC19 [by SacI 5(cid:1)- etal.,1983).Thepelletedorganelleswereresuspendedin1–2mlofbuffer
untranslated region (UTR)/HpaI 3(cid:1)-UTR] and subcloned into the (0.32Msucrose,10mM(cid:2)-aminocaproicacid,0.1mMEGTA,1:100di-
Eco72IsiteofareconstructedpSindRep5Sindbisvirusvector,intowhich lutedproteaseinhibitormixture,pH7.5),aliquotedinsmallvolumes,
apolylinker(BclI/XhoI/SacI/NotI/PvuI)wasintroduced.Theorienta- andstoredat(cid:2)70°C.
tionoftheGlud1ORFinsertwasconfirmedbysequencing;theinsertwas GLUDactivityinP2fractions(50–100(cid:1)lsamples)wasmeasuredafter
digested (XbaI/XhoI) and cloned into pNSE-GEM-7Z to create the thawing the samples in 1 ml (final volume) of 50 mM Na-phosphate
pNSE-GLUD1vector.ThisvectorwasdigestedwithEcoRI,andtheDNA buffer, pH 7.4, containing 1 mM potassium cyanide and 0.4% Triton
wasmicroinjectedintothepronucleiof215fertilizedmouseoocytesand X-100.Thesuspensionwasincubatedfor5minat37°Candcentrifuged
transferredtotheoviductsofpseudopregnantmice.Verysimilarpro- at16,000(cid:3)gfor20min,andtheclearsupernatantcontainingextracted
cedurestothoseforGlud1vectorconstructionwereusedtogenerate mitochondrialGLUDwasusedformeasurementsofenzymeactivity.
the vector for microinjection of pNSE-EGFP. A total of 80 oocytes Proteincontentwasmeasured(Michaelisetal.,1983),andGLUDen-
were injected with the linearized NSE-EGFP vector to generate the zymeassayswereperformedineitherthedirectionofGludeamination
EgfpTgmice. (Plaitakisetal.,2000)orthedirectionof2-OGamination(Mazon,1978).
Forty-eight pups were born after transfer of oocytes microinjected ThedeaminationofGluwasmeasuredat37°Casfollows:30(cid:1)gsample
withthepNSE-GLUD1construct.Ofthesepups,fourhadthetransgene proteinwasaddedtothereactionbuffer(inmM:50Na-phosphatebuffer,
forGlud1.GenotypingwasperformedongenomicDNAextractedfrom pH7.4,1ADP,and0.2NAD(cid:4))andpreincubatedfor2minat37°C,and
tailbiopsiesobtainedfrom3-to4-week-oldpups.TheDNAwassub- thereactionwasinitiatedbyaddingL-Glu.TheNADHproducedwas
jectedtoSouthernblotandPCRanalysis.Fortheidentificationofthe monitored at 340 nm from 0 to 10 min, and the initial velocity was
Glud1 transgene by PCR, the primers used were as follows: F , 5(cid:1)- calculated.Enzymeactivitiesweremeasuredbyvaryingtheconcentra-
1
GATATCGGGTGCATCTGAG-3(cid:1);andR1,5(cid:1)-GGTTTATGAGGACAC- tionsofeitherGlu(0.05–20mM)orNAD(cid:4)(0–200(cid:1)M),whilekeepingthe
AGAGG-3(cid:1)(900bpproduct).PCRamplificationwasperformedusing concentrationoftheallostericactivatorADPconstant.TheK andV
M max
Baoetal.•Glud1TransgenicMice J.Neurosci.,November4,2009•29(44):13929–13944•13931
values(cid:5)SEMwerecalculatedusingSigmaPlot(Zaidietal.,1998).The MAP2Aantibodieswerethosedescribedpreviously(Kimetal.,2007;
measurement of time-dependent kinetics of amination of 2-OG in Matsubayashietal.,2008),withsomemodifications.Thesectionswere
freshlypreparedsynaptosomeswasperformedusingsimilarmethodsto obtainedfrommiceperfusedwith2%paraformaldehyde–PBS.Afterad-
thosefortheP2fractionandareactionmixturecontaining(inmM)100 ditionalfixationofthebraininthesamefixative(4hat23°C),vibratome
potassiumphosphate,pH7.8,50NH Cl,0.2NADH,and2.5potassium sections(100(cid:1)m)werecut,DiIcrystalsweredelivered,andthesections
4
2-OG.TheNADHconversiontoNAD(cid:4)wasdeterminedbasedonthe wereincubatedinPBSfor14hat23°Ctoallowdyediffusiontooccur.DiI
decreaseinabsorbanceat340nm.TheintegratedMichaelisequation crystalswereappliedtobrainsectionsusingmicropipettes(Kimetal.,
V (cid:1)t(cid:6)([S ](cid:2)[S])(cid:4)(K (cid:1)ln([S ]/[S]))(WhartonandSzawelski, 2007).TopreparetheDiImicropipettes,adropletofDiIdissolvedin
app 0 M 0
1982)wasusedtoestimateV andK . N,N-dimethylformamide(0.5%w/v)wasplacedonthetipofeachglass
app M
GLUDactivityinbraincellsinsituwasmeasuredintissuesectionsas pipette and dried (3–12 h, 60°C), and the crystals were delivered by
described previously (Kugler and Baier, 1992). In brief, 20-(cid:1)m-thick touchingthebrainsections.AfterDiIdiffusion,thesectionswerebriefly
cryotomesectionsfromGlud1andwtbrainswererapidlyfrozeninliquid incubatedin3%gelatin–PBS,followedbyincubationfor30minwith
N ,mountedonslides,airdriedat23°C,andincubatedfor20minina digitonin(0.5mg/mlinPBS).Labelingwithanti-MAP2Aantibodiesand
2
mediumcontaining50mMNa-Glu,5mMNAD(cid:4),10mMNaN3,5mM imagingbyconfocalmicroscopyfollowedasdescribedabove.
ADP,20mMNaCl,0.15mMphenazinemethosulfate,5mMnitroblue Microelectrodemeasurementsofdepolarization-inducedGlureleaseand
tetrazoliumchloride,0.05mMMgSO4,and0.1(cid:3)10(cid:2)3zincacetate,in50 reuptakeinstriatum.Glud1Tg(10–12months;n(cid:6)4)andwt(7–12
mM HEPES buffer, pH 7.5, plus 22% polyvinyl alcohol. Background months; n (cid:6) 9) male and female mice were group housed in a 12 h
stainingwasobtainedinparallelsectionsincubatedintheabsenceofGlu. light/darkcyclewithfoodandwateradlibitum.Enzyme-basedmicro-
After rinsing the sections in ice-cold water, they were air dried, and electrodearrays(MEAs)withplatinum(Pt)recordingsurfaceswerefab-
microscopicimageswereobtainedwithin12hoftheendofthereaction. ricated,assembled,andcalibratedforinvivomouserecordingsaccording
Magneticresonancespetroscopymeasurementsofbrainaminoacids.1H topublishedliterature(Burmeisteretal.,2000).TheMEAassembliesfor
magnetic resonance spetroscopy (MRS) data were acquired from the invivorecordingsconsistedofanenzyme-basedworkingelectrodeanda
hippocampus and striatum of 28 Glud1 Tg and 13 wt mice (age, 9 single-barrelglassmicropipette(internaltipdiameter,12–15(cid:1)m)po-
months)usingaVarian9.4Tsystem.Duringtheexperiments,theani- sitionedatanequaldistancefromthefourrecordingelectrodesites
malswereanesthetized(1:1air:oxygenplus1–2%isoflurane),andtheir (BurmeisterandGerhardt,2001;Burmeisteretal.,2002).Eitheraniso-
coretemperaturemaintainedat37°C.Magneticfieldhomogeneitywas tonicsolutionof70mMKCl(70mMKCl,79mMNaCl,and2.5mMCaCl2,
adjustedusingafast,automaticshimmingtechniquebymappingalong pH7.4)ora5mML-Glusolution(inphysiologicalsaline,pH7.4)was
projections(Gruetter,1993).Spinecho,full-intensityacquiredlocalized loadedintothesingle-barrelglassmicropipette.Glud1andwtmicewere
spectroscopy (echo time, 3 ms; repetition time, 4 s) (Mlynarik et al., anesthetized(1.25g/kgurethane,i.p.)andplacedinastereotaxicframe
2006)wasusedtoacquire 1HMRSdatafromvoxels((cid:7)5(cid:1)l)inhip- fittedwithamouseadaptorinamannersimilartothatdescribedrecently
pocampusandstriatum.ThenumberoftransientsforMRspectrawas (Burmeisteretal.,2002).A200-(cid:1)m-diameterAg/AgClreferenceelec-
320–480forthehippocampusand480–640forthestriatum.Thecon- trodewasimplantedintothefrontalcortexatasiteremotefromthe
centrationsoftheneurochemicalsalanine,GABA,glutamine,andGlu recordingareas.TheMEAassemblieswereloweredintothestriatumin
werequantifiedusingtheLCModel(LinearCombinationofModelspec- stepsof500(cid:1)m(FriedemannandGerhardt,1992)usingthecoordinates
tra)analysis(Provencher,1993). for mouse brain [from bregma: anteroposterior, (cid:4)1.3; mediolateral,
Histology, immunohistochemistry, and labeling with 1,1(cid:1)-dioctadecyl- (cid:5)1.5;dorsoventral(DV),(cid:2)2.25to(cid:2)3.75](FranklinandPaxinos,1997).
3,3,3(cid:1),3(cid:1)-tetramethylindocarbocyanine. Mouse brains were immersion SolutionswereejectedfromtheglassmicropipetteusingaPicospritzerII
fixed in 4% paraformaldehyde in PBS for 72 h at 4°C, cryoprotected (ParkerHannifin).Thevolumeejectedwasmonitoredusingastereomi-
(incubationin30%sucrose–PBS),andfrozenbyexposurefor30sto croscopefittedwithareticule(FriedemannandGerhardt,1992).Con-
2-methyl-butaneatliquidN temperatures.Coronalsections(24(cid:1)m stant voltage amperometry was performed using a Fast Analytical
2
thickness)werecutinacryotomeandtransferredtogelatin-coatedglass Sensing Technology (FAST)-16 electrochemistry instrument (Quan-
slides. For staining with methylene blue, the sections were air dried, teon,L.L.C.)andFASTsoftwareforfour-channelsimultaneousrecord-
stainedwith0.5%(w/v)dyesolution(5minat23°C),rinsedindistilled ings(BurmeisterandGerhardt,2001).Allmicewerekilledattheendof
H O, dehydrated through graded ethanol, cleared with xylene, and arecordingsessionbyanoverdoseofanesthetic.
2
mounted with permount. For immunofluorescence staining, sections Dose–responsestudieswereperformedineachstriatalrecordingsite
wereblockedwith3%(w/v)gelatininPBS(1hat37°C),treatedwith todeterminethemaximumK(cid:4)-evokedresponsesinwtandGlud1mice.
0.1%TritonX-100inPBS(15minat23°C),andreacted(overnightat Amperometricdata,time,andpressureejectionmarksforeachindivid-
4°C,thenfor1hat23°C)withthefollowingprimaryantibodies:anti- ualPtrecordingsiteweresavedintherecordingsystem.AmodifiedExcel
GLUD1 (1:2000), anti-microtubule-associated protein 2A (MAP2A; spreadsheet,inconjunctionwithindividualelectrodecalibrationcon-
monoclonal; 1:500 or 1:1000; Millipore), anti-synaptophysin (poly- stants,wasusedtoanalyzetheL-Glucurves.Themaximumsignalam-
clonal;1:1000;Millipore),anti-neuron-specificenolase(NSE;monoclo- plitude(peakGluconcentrationinmicromolars)wasdeterminedfor
nal; 1:250; Millipore), anti-ubiquitin (monoclonal; 1:500; Millipore), eachsignalandaveragedacrosseachmousetype.Approximately10sig-
andanti-vimentin(polyclonal;1:400;Millipore).AfterrinsinginPBS, nalsperrecordingsitewereobtainedfromeachDVpositionoftheMEA
thesectionswereincubated(2hat37°C)withfluorescentdye-labeled inthestriatum.Thesesignalswereaveragedintoasingledatapoint,thus
secondaryantibodies(Alexa568goatanti-rabbitorAlexa488goatanti- generatingonedatapointforeachDVpositionandamaximumoffourdata
mouse; 1:1000 dilution in PBS; Invitrogen) and processed (Pal et al., pointsperpassinthestriatumofeitherhemisphere.Determinationofthe
2003).SomesectionsweredoublelabeledwithSYTOXblue(Invitrogen) averageamplitudeofGlureleasedrequiredthatthevolumeofKClejectedbe
plus one of the antibodies. SYTOX labeling was performed after the carefullymatchedacrossexperiments.Signalsobtainedfrom70mMKCl
reactionwiththesecondaryantibody.Thesectionswererinsedfirstin (100–200nl)ejectionsinwtandGlud1micewereusedforanalysis.
PBS(5minat23°C)andthenwithTris-EDTA(TE)buffer(10mMTris Forthecalculationofuptakeratekinetics(micromolarspersecond),
and1mMEDTA,pH7.5;5min),incubatedwithdye(1:1000dilutionin onlyamplitudesofL-Glureleasedrangingbetween3and13(cid:1)Mwere
TE;30minat23°C),mounted,andviewedusingeitheraZeissLSM510 used in the analyses, regardless of the amount of stimulus needed to
Metaconfocal(Paletal.,2003)oraspinningdiscOlympus1X81micro- evoketherelease.L-Gluuptakefollowedfirst-order-ratekinetics.There-
scope.ImageswereprocessedusingSlidebook4.1,ImageJ,andPhoto- fore,theuptakerateconstant(k(cid:2)1)wasthelogarithmicslopeofL-Glu
shopforquantificationofpixeldensity,dendritelengthdetermination, concentrationversustime(sec(cid:2)1)estimatedbyuseofregressionanaly-
spinecharacterizationanddensity,andthree-dimensionalreconstruc- ses(R2(cid:3)0.9).Multiplicationofk(cid:2)1withthemaximumamplitudeof
tionofimages. Glusignalyieldedtheuptakerate(micromolarspersecond).
The methods used to stain neurons with both 1,1(cid:1)-dioctadecyl- Slicepreparationandelectrophysiologicalrecordings.Acutebrainslices
3,3,3(cid:1),3(cid:1)-tetramethylindocarbocyanine(DiI)dye(Invitrogen)andanti- werepreparedfrom2.5-to3-month-oldwtandTgmiceasdescribed
13932•J.Neurosci.,November4,2009•29(44):13929–13944 Baoetal.•Glud1TransgenicMice
previously(BelousovandvandenPol,1997).Briefly,animalswereanes- hocTukey’stestandthetwo-tailedStudent’sttest(paired,whenpossi-
thetized with isoflurane and decapitated. The hippocampi from both ble)withInStatsoftware(GraphPadSoftware)andClampfit10software
hemisphereswereremoved,andcoronalhippocampalslices(400(cid:1)m (MolecularDevices).Alldataarepresentedasmeans(cid:5)SEM.
thick)wereprepared(at2–4°C)andkept(at20–22°C)inartificialCSF
(ACSF)thatcontained(inmM)124NaCl,3.0KCl,2.0CaCl2,2.0MgCl2, Results
1.23NaH2PO4,26NaHCO3,and10D-glucose(aerated,pH7.3).Aflow DevelopmentofGlud1Tgmice
pipe perfusion system was used for slice perfusion at 4–5 ml/min
TheTgmiceweregeneratedbypronuclearmicroinjectionsof
(BelousovandvandenPol,1997).
mouseoocytes(C57BL6/SJLhybrid)withaconstructcontain-
MeasurementsofmEPSCswereperformedinCA1pyramidalneurons
usingwhole-cellvoltage-clamprecordinginthepresenceof1(cid:1)Mtetro- ing the cDNA for mouse Glud1 under the control of the Nse
dotoxin(avoltage-gatedsodiumchannelblocker)inACSF.Aglassre- promoter.TheNsegeneisexpressedonlyinneuronsofbrain
cordingelectrodewasfilledwithaninternalsolutionthatincluded(in and spinal cord (Peeletal.,1997).Inpreliminarystudies,pri-
mM)145KMeSO4,10HEPES,2MgCl2,0.1CaCl2,1.1EGTA,2Na-ATP, mary neuronal cultures transfected with either pNSE-lacZ or
0.3Na-GTP,and5QX314bromide(ablockerofvoltage-activatedso- pNSE-EGFP vector exhibited expression of (cid:5)-galactosidase
diumchannels;pH7.2,3–7M(cid:8)).mEPSCswererecordedinagap-free (lacZ)orenhancedgreenfluorescentprotein(EGFP)inneuronal
modeataholdingmembranepotentialof(cid:2)65mVusingtheMulticlamp
cellsbutnotingliapresentinthesecultures(datanotshown).In
amplifierandClampexsoftware.ThefrequencyandamplitudeofmEP-
addition,inTgmicethathadthetransgeneforEgfpunderthe
SCs were analyzed during 10 min recordings using Mini Analysis 5.0
control of the Nse promoter, we observed high EGFP fluores-
software(Synaptosoft).AnmEPSCeventwasconsideredifitsamplitude
was(cid:9)5pA. cenceinregionsenrichedinneuronalcellbodies,notinmyelin-
atedaxonaltracks(datanotshown).
EvokedfieldEPSPs(fEPSPs)weremeasuredusingextracellularre-
cordings.Aglassrecordingelectrodefilledwith3MNaCl(2–3M(cid:8))was FourfounderTgmicewereidentifiedbygenotypingashaving
placedinthestratumradiatum(SR)oftheCA1hippocampalregion.A the Glud1 transgene. The Glud1 gene-copy number in the
stimulatingelectrodemadefromapairofTeflon-coated,90%platinum/ founder mice was estimated by qPCR to be either six or eight
10%iridiumwires(50(cid:1)minnerdiameter)wasusedtoevokefEPSPsby copies(8.1(cid:5)2.1,8.5(cid:5)2.2,5.7(cid:5)2.3,and6.4(cid:5)2.4infounders
stimulatingtheSchaffercollaterals(S48stimulator;GrassTechnologies). 1–4,respectively).ThefoundermicewerematedwithC57BL/6,
EvokedfEPSPswererecordedasdescribedaboveformEPSCs.Forthe andgenotypingrevealedanormalMendeliantransmissionpattern
analysisoffEPSPs,a30minbaselinerecordingwasestablishedusing
ofthetransgene(supplementalFig.1,availableatwww.jneurosci.
low-frequencystimulation(0.033Hz;0.05msimpulseduration)andthe
orgassupplementalmaterial).Allmiceusedinthepresentstud-
adjustedintensitythatinducedfEPSPswith(cid:7)40%ofthemaximalfEPSP
ies were hemizygous for the transgene, were from the third to
amplitude.LTPwasinducedusinghigh-frequencystimulation(HFS)
10thgenerationofcrossbreeding,andwerederivedfromallfour
consistingof0.05msimpulseduration,100impulses,100Hz,withthe
same stimulation intensity as that used for baseline recordings. After founders. No consistent differences were seen in physiological
HFS, the background electrical stimulation was continued using the andbiochemicalmeasuresbetweenmicederivedfromfounders
stimulus parameters that were used for the baseline stimulation. The with eight and those with six extra Glud1 genes; therefore, the
slope of fEPSPs was determined from the rise time period between data from the Tg mice were not segregated on the basis of the
theinitial10%andthefinal90%ofincreaseinfEPSPamplitudeandwas foundermicefromwhichtheoffspringwerederived.
normalizedrelativetotheaveragefEPSPslopeobtainedduringbaseline Totalbrainhomogenatefrommice(6–8monthsofage)was
recording.StatisticalanalysisoffEPSPslopeswasdoneusingthedata
subjectedtoimmunoblotstudiestodetermineGLUD1levelsin
froma10minbaselinerecording(conductedimmediatelybeforeHFS) brain.GLUD1levelsinthreeTgmicewere28.5(cid:5)2.6%higher
anda10minpost-HFSrecording(conductedbetween50and60min thaninthreelittermatewtmice(p(cid:6)0.01;df(cid:6)4;ttest;n(cid:6)3
afterHFS).Signalsweredigitizedat20kHz,filteredat5kHz,andstored
pairs).BiochemicalmeasurementsofGLUDenzymeactivityin
onapersonalcomputer.Thetestswereconductedblindlytothegeno-
typeoftheexperimentalanimal. whole-brainhomogenatesatasinglesubstrate(5mMGlu)and
Measurementoflossofrightingreflexandrecoveryfromanesthesia.A cofactor (200 (cid:1)M NAD(cid:4)) concentration paralleled the protein
groupofwtandTgmice(8–10monthsold)wereusedtodetermine immunereactivitymeasures.Theenzymeactivitywas35–40%
sensitivitytothecombinationofanestheticsketamineplusxylazine.The higherinTgcomparedwithwtlittermates(datanotshown).The
miceweretestedusingtwodifferentdosesoftheanestheticsadminis- increasesinGLUD1levelsandGLUDactivityinbrainhomoge-
tered7dapart.Inthefirststudy,200mg/kgketamineand13.3mg/kg nateswerelikelyanunderestimateoftheincreasesinthisenzyme
xylazinewereadministeredintraperitoneally,andinthesecondstudy, inneuronsbecausehomogenatescontainbothglialandneuronal
132mg/kgketamineand8.8mg/kgxylazinewereinjected.Immediately
GLUD, whereas the Glud1 transgene under the Nse promoter
aftertheinjections,eachanimalwasplacedonaheatingpad,andthe
would be expressed in neurons. Because of this consideration,
timetoonsetoflossofrightingreflex(LORR)aftertheinjectionandthe
additionalanalysesofenzymeactivitywereperformedontheP2
durationofLORRwererecorded.LORRwasdefinedasthetimeafter
fractionisolatedfrombrainhomogenates.Thisfractioncon-
theinjectionofanestheticswheneachmouselostitsabilitytoright
itselffromasupinepositiontostandingonallfourpaws.Durationof tainsmitochondriaderivedfromneuronalandglialcellbod-
LORRwasdefinedasthetimebetweenlossandrecoveryoftheright- ies, as well as those from nerve endings (synaptosomes).
ingreflex. Substrate-dependent kinetics of GLUD activity in P2 revealed
Statisticalanalyses.Student’sttest(two-tailed)statisticswereusedto thattheV forGludeaminationinTgmouseP2wassignifi-
max
compareproteinlevelsandenzymeactivitiesinvolvingasinglemeasure cantly(65%)higherthaninthewt(Tg,2.25(cid:5)0.26(cid:1)mol(cid:1)mg(cid:2)1
ofsuchlevelsoractivities.Allcomparisonswerebetweenlittermatemale vs. wt, 1.36 (cid:5) 0.2 (cid:1)mol(cid:1)mg(cid:2)1; p (cid:6) 0.04, t test; n (cid:6) 3). In
orfemalewtandTgmice.Thesmallestsampleswerebetweengroupsof
contrast, the K values did not differ significantly between Tg
threelittermates.KineticdatapointsofenzymeactivityorGlurelease andwtmiceforMeitherthesubstrateGlu(Tg,6.0(cid:5)0.9mMvs.wt,
aonfdrergeiuopntsa,kaen,dasawgeeslloafswmtualtnidplTegcommipcea,riwsoernesaancraolyszsebdrabinyAreNgiOonVsA,s.tPraotsat 4.0(cid:5)1.1mM;n(cid:6)3pairs;p(cid:6)0.1)orthecofactorNAD(cid:4)(wt,
hocanalysesbetweengroupswereperformedusingeitherBonferroni’sor 124.4(cid:5)11.1(cid:1)Mvs.Tg,86.5(cid:5)3.5(cid:1)M;p(cid:6)0.06,ttest;n(cid:6)4).The
Tukey’s post hoc test. Statistical significance was defined as p (cid:4) 0.05. observed KM values were in the range of those reported previ-
Statisticalanalysisofthesensitivitytoanestheticagentswasbymeansof ously(Shashidharanetal.,1994;Plaitakisetal.,2000;Zhangetal.,
thettest.fEPSPdatawereperformedusingANOVAfollowedbythepost 2004).ThegreaterdifferencebetweenwtandTgintheV for
max
Baoetal.•Glud1TransgenicMice J.Neurosci.,November4,2009•29(44):13929–13944•13933
thanwtmiceacrossthethreestrataofCA1
and 52.9 (cid:5) 1.6% higher in Tg than wt
across all strata of CA3. Statistical com-
parison of overall Tg versus wt levels of
GLUD1 immunoreactivity in CA1 and
CA3indicatedsignificantdifferencesbe-
tween Tg and wt mice in both regions
(ANOVA;CA1:df(cid:6)1,40;F(cid:6)10.1;p(cid:6)
0.003; CA3: df (cid:6) 1, 28; F (cid:6) 25.4; p (cid:10)
0.0005).Therewasnosignificanteffectof
region(CA1orCA3)onGLUD1immu-
noreactivity (ANOVA with Bonferroni
posthoccomparisons;df(cid:6)1,60;F(cid:6)2.3;
p(cid:6)0.14)norofthethreestrata,SO,stra-
tumpyramidale(SP),andSR(p(cid:6)0.5–0.9).
Inotherwords,GLUD1immunoreactiv-
ity was higher in Tg compared with wt
mouse hippocampus in all strata and all
regionsofthehippocampus.Significantly
increasedlevelsofGLUD1immunoreactiv-
ityinTgversuswtmice(51.8%higherin
Tg)werealsodetectedinneuronsofthece-
rebralcortex,oflayersIVandV(ANOVA;
df (cid:6) 1, 10; F (cid:6) 6.0; p (cid:6) 0.034), and of
otherbrainregions(datanotshown).The
biochemical and immunocytochemical
datawereindicativeofoverallincreasesin
GLUD1levelsandactivityinthebrainsof
Figure1. ImmunelabelingofGLUD-immunoreactivesitesinCA3(A,B)andCA1(C,D)ofthehippocampusof12-month-oldTg Tgmice,andtheincreaseswerenotlocal-
andwtmice.Sectionswereimmunolabeledwithanti-GLUDpolyclonalantibodies.Scalebars,50(cid:1)m. izedtoselectgroupsofcells.
GLUDintheP2fractionvs.thatinwhole-brainhomogenatewas AminoacidconcentrationsinTgandwthippocampusand
mostlikelyattributabletotherelativeenrichmentofP2with striatuminvivo
synaptosomes and associated intraneuronal mitochondria. Glud1expressioninneuronsofthebrainmayleadtoincreasesin
This assumption was confirmed by estimates of the V for Glu formation (Yudkoff et al., 1991) and possibly detectable
max
2-OGaminationtoGluinisolatedsynaptosomepreparations. changesinwhole-tissueconcentrationsofGluintheCNS.Also,
Based on kinetic analyses of enzyme activity using the inte- because Glu in the cytoplasm of neurons may function as the
gratedMichaelisequation(WhartonandSzawelski,1982),the precursor for the synthesis of GABA, it is possible that some
V was153%higherinTgthanwtbrainsynaptosomes(Tg, changeintheconcentrationofGABAmightoccurininhibitory
max
21.7(cid:1)M(cid:1)min(cid:2)1vs.wt,8.58(cid:1)M(cid:1)min(cid:2)1).Thus,intraneuronal neurons.Inaddition,theuptakeofreleasedGluintogliacould
mitochondria of Tg mice appeared to be substantially en- produceincreasesinglutaminelevelsinTgmousebrain(Yudkoff
richedinGLUDactivity. etal.,1989),whereastheexcess2-OGformedinneuronalmito-
Theresultsofinvitrobiochemicalassayswereconfirmedbyin chondriaandtransportedintothecytoplasmmightleadtode-
situhistochemicaldetectionofGLUDactivityinbrain(Kugler creasesinalanineasaresultoftransaminationreactionsinmice
andBaier,1992).Thedepositionoftetrazoliumdyeinthepyra- thatoverexpressGlud1(Pengetal.,1991;Erecinskaetal.,1994).
midalcelllayeranddendriticfieldsofthestratumoriens(SO) Toobtainsimultaneousmeasurementsoftheconcentrationof
andSRofthehippocampusCA1andCA3(supplementalFig.2, severalaminoacidsinthebrainsoflivingmice,weusedtheMRS
available at www.jneurosci.org as supplemental material) re- approach for neurochemical measurements in (cid:7)5 (cid:1)l volume
gions,andinthemolecularlayerofthedentategyrus,wasgreater voxelsofhippocampusandstriatum.AscanbeseeninTable1,in
inTgthanwtmouse.Underhighmagnification,theincreased bothhippocampusandstriatum,theconcentrationofGluwas
GLUDactivityinSOandSRappearedaslabelingwithinnerve significantly increased, whereas the levels of GABA and glu-
terminalfields.MarkeddifferencesbetweenTgandwtmicein tamineinthesamebrainregionswerenotsignificantlydifferent
theinsituGLUDactivitywerealsodetectedinthestriatumand betweenwtandTgmice.Incontrast,thelevelsofalaninewere
cortex(datanotshown). significantlydecreasedinbothregions.Theconcentrationsofall
Immunohistochemicalstudiesalsoshowedhigherlabelingby fouraminoacidsmeasuredbyMRSinwtmousehippocampus
anti-GLUD1 antibodies in neurons of all brain regions of Tg andstriatumwereclosetothosepreviouslydeterminedinwhole
comparedwithwtmice(e.g.,inhippocampalpyramidalneurons mousebrainusingstandardbiochemicalmeasurements(Leviet
ofbothCA1andCA3)(Fig.1).Densitometricmeasurementsof al.,1967;Agrawaletal.,1968).BasedontheMRSmeasurements,
GLUD1immunoreactivityoverequal-sizedareas(3128(cid:1)m2)of theexpressionofGlud1inneuronsbroughtaboutmoderatein-
eachhippocampalregionandeachstratumofCA1andCA3from creasesinGluconcentrationinbrainwithoutincreasingGABA
fivetosevenpairsof6-month-oldwtandTgmicewereusedto levels in inhibitory neurons. The lack of changes in glutamine
quantifythedifferentiallevelsofGLUDlabelinginTgversuswt levelsmightindicatearelativelysmallcontributiontothepoolof
mice. GLUD1 immunoreactivity was 53 (cid:5) 7.1% higher in Tg mostlyglialglutaminebytheextraGluformed,possiblyincom-
13934•J.Neurosci.,November4,2009•29(44):13929–13944 Baoetal.•Glud1TransgenicMice
Table1.Aminoacidlevelsinadult(9-month-old)mousehippocampusand nl;Tg,134(cid:5)8nl;p(cid:6)0.57;df(cid:6)50),yettheaveragemaximum
striatumofwtandTgmicemeasuredbyMRS amplitudeforGlureleasewassignificantlygreaterinTgthanwt
Aminoacidconcentration((cid:1)mol/g) mice(wt,6.3(cid:5)0.7(cid:1)M;Tg,9.9(cid:5)1.4(cid:1)M;p(cid:6)0.016;df(cid:6)50).
Brainregion/genotype Alanine GABA Glutamine Glutamate ThekineticsofGlureuptakeweredeterminedonthebasisof
the temporal dynamics of changes in KCl-evoked Glu release.
Hippocampusa
SinceneurotransmitteruptakefollowsMichaelis–Mentenkinet-
wt 0.79(cid:5)039 1.85(cid:5)0.25 3.02(cid:5)0.46 8.99(cid:5)1.19
Tg 0.56(cid:5)0.14 1.85(cid:5)0.30 3.08(cid:5)0.42 9.76(cid:5)0.93 ics(Nicholson,1995)andisaffectedbytheamountofavailable
(p(cid:6)0.02)b (p(cid:6)0.99) (p(cid:6)0.65) (p(cid:6)0.03) substrate,Gluuptakewasanalyzedbyselectingasubsetofsignals
Striatuma withmaximumreleasevaluesintherangeof3–13(cid:1)M.Thisrange
wt 1.40(cid:5)0.84 2.81(cid:5)0.54 3.83(cid:5)0.73 8.01(cid:5)1.46 was chosen because peak Glu values in this range were easily
Tg 1.01(cid:5)0.33 2.45(cid:5)0.61 4.17(cid:5)0.42 8.87(cid:5)0.79 obtainedinbothwtandTgmice.Withinthissubsetofsignals
(p(cid:6)0.04) (p(cid:6)0.11) (p(cid:6)0.08) (p(cid:6)0.03) (n(cid:6)35signalsforwtandn(cid:6)20signalsforTg),reuptakeratesof
aAtotalof13wtand28Tgmicewereusedtomeasureaminoacidconcentrationsinthehippocampus.Aminoacid stimulus-evokedGlureleasewereessentiallyequalforTg(1.9(cid:5)
cmoincceewnterraetiionnclsuidnetdhienstthrieactuamlcuwlaetiroenmoefaasluanreindeinco1n0coefntthraeti1o3nwinttmheicheiapnpdocinamalplu2s8boafstehdeoTngmmeiceet.inDgattahefrcormite2r0iaTogf 0.3(cid:1)M/s)andwt(1.8(cid:5)0.2(cid:1)M/s)mice(df(cid:6)53;p(cid:6)0.66).
theLCModelforconsistencyandclarity.Thevaluesfortheconcentrationofeachaminoacidarepresentedasthe GlutransportintoneuronsorgliainthevicinityoftheMEAs
mean(cid:5)SD. wasalsoestimatedbymeasuringGluclearanceafterlocalappli-
bStatisticalsignificanceofdifferencesbetweenwtandTgmousebrainmeasurementsisindicatedbythepvalue cationofnanolitervolumesof5mML-Glu.Pressureejectionsof
(unpairedStudent’sttest).
5mML-GluintothestriatumofTgandwtmiceproducedrepro-
ducibleGluresponsessimilartothoseseenwith70mMKClde-
polarization.Maximumamplitudesranged,onceagain,between
3and13(cid:1)Mforwt(n(cid:6)29signals)andTg(n(cid:6)13signals)mice.
Clearance of exogenously applied L-Glu did not differ signifi-
cantlybetweenTg(2.5(cid:5)0.5(cid:1)M/s)andwt(2.9(cid:5)0.2(cid:1)M/s)mice
(df(cid:6)40;p(cid:6)0.40).TheestimatedratesofGluuptakeofendog-
enously released Glu were lower than those for the uptake of
exogenouslyintroducedGlu.ThedifferentialratesofGluuptake
were probably attributable to the electrogenic nature of Glu
transportersasGluuptakeisdrivenbyelectrochemicalgradients
acrossthecellmembrane(Takahashietal.,1997;Doble,1999).
Undertheconditionsofstimulus-evokedGlureleasebythelocal
applicationof70mMKCl,glialandneuronalmembraneswould
bedepolarized,andthustherateofGluuptakedecreasedcom-
Figure2. Depolarization-inducedGlureleaseinthestriatumofTgandwtmice.Stimulus- paredwiththeclearancerateofexogenouslyappliedGlu.Never-
evokedGlureleaseinTgandwtmicewasmeasuredusingMEAsafterpressureejectionof theless,thekeyobservationwasthatbothendogenouslyreleased
100–200nlof70mMKClatthetimesshown.ReproduciblesignalsofGlureleasewereobtained andexogenouslyintroducedGluhadverysimilarratesofclear-
frombothTgandwtmice.Conc.,Concentration. anceinTgastheydidinwtmice.Therefore,theexcessGlurelease
afterKCl-induceddepolarizationwasnotlikelytheresultofdi-
binationwithagreaterrateofconversionofneuronalglutamine minishedGlutransportinTgcomparedwithwtmice.
toGluinTgmice.Finally,thedecreasedalaninelevelsmighthave Anesthetics,suchastheurethanethatwasusedinthepresent
been the result of increases in the transamination reaction be- studies,exerttheireffectsbyenhancinginhibitorysynapticneu-
tweenalanineandexcess2-OGinTgmice,areactionthatleadsto rotransmissionaswellasbyinhibitingexcitatoryneurotransmis-
theformationofGlu. sion(HaraandHarris,2002).RestingGlulevelsaresignificantly
attenuatedinurethane-anesthetizedrats(Rutherfordetal.,2007)
Increasesinstimulus-evokedGlureleasefromstriatal and, although both wt and Tg mice received the same dose of
neuronsinvivo urethane(finaldoseof1.25g/kg),theurethaneanesthesiamight
To determine whether Glud1 overexpression leads to not only have had differential effects on neuronal release of Glu in Tg
excessGluformationbutalsoincreasedL-Glureleasedynamicsin comparedwithwtmice.Forthisreason,anotherapproachwas
vivo,aself-referencingMEAforthemeasurementofGlurelease pursuedtoassesspossibledifferentialratesofGlureleasebyCNS
andreuptakeinvivowasused(BurmeisterandGerhardt,2001; neuronsinwtversusTgmice:recordingmEPSCsinvitrofrom
Burmeisteretal.,2002;Thomasetal.,2009).Measurementswere knownexcitatory,glutamatergicsynapsesinthehippocampus.
obtainedfromthestriatumofanesthetizedwtandTgmice.Glu
releasewasevokedbyanisotonicsolutionof70mMKCltosim- IncreasesinsynapticreleaseofGluatglutamatergicsynapses
ulateburstfiringofGluprojections.Therecordedsignalswere ofthehippocampus
analyzed for maximum L-Glu amplitude and rate of uptake of IftheenhancedreleaseofGluafterneuronaldepolarizationde-
released Glu. Local application of 70 mM KCl for 1–2 s in the scribedaboverepresentedsynapticGlurelease,thenthismightbe
striatum of Tg and wt mice produced robust, reproducible reflected in increases in the amplitude of mEPSCs or both the
changesinextracellularL-Glu(Fig.2);GlureleaseelicitedbyKCl amplitudeandfrequencyofmEPSCsinglutamatergicsynapses.
applicationswasconsistentlyhigherinTgmice(Fig.2).Forcon- InDrosophila,elevatedcontentofGluinsynapticvesiclesafter
sistencyindataanalysis,onlyGlusignalselicitedbyejectionsof overexpressionofavesicularGlutransporterledtoincreasesin
100–200nlof70mMKClwereanalyzedtoobtaintheaverage bothamplitudeandfrequencyofmEPSCsatglutamatergicsyn-
maximum amplitudes in wt (n (cid:6) 35 signals) and Tg (n (cid:6) 18 apses (Daniels et al., 2004). Thus, if synaptic vesicle filling is
signals) mice. Ejection volumes of KCl eliciting these signals increasedinGlud1miceasaresultofincreasedcytoplasmiclevels
were,onaverage,nearlyidenticalinwtandTgmice(wt,140(cid:5)6 ofL-Glu,thenbothmEPSCfrequencyandamplitudemightbe
Baoetal.•Glud1TransgenicMice J.Neurosci.,November4,2009•29(44):13929–13944•13935
Figure3. ExpressionofmEPSCsinCA1pyramidalneurons.Representativerecordingsof
mEPSCsinneuronsfromwt(a)andTg(b)miceareshown.Thebottomtraceisanexpanded
fragmentoftherecordingthatisindicatedinthetoptrace.Calibration:ordinate,10pA;ab-
scissa,1min(toptrace)and60ms(bottomtrace).
increasedinglutamatergicsynapsesofGlud1mice.Patch-clamp
measurementsofmEPSCsinCA1neuronsofthehippocampus
of Glud1 and wt mice indicated highly significant increases in
both frequency and amplitude of mEPSCs in Glud1 versus wt
mice (Fig. 3). A fivefold increase in mEPSC frequency (wt:
0.012(cid:5)0.004,n(cid:6)3;Tg:0.059(cid:5)0.011,n(cid:6)4;p(cid:6)0.017)anda Figure4. TimetoLORRanddurationofLORRinwtandTgmice.Themean((cid:5)SEM)time
twofoldincreaseinamplitudeofmEPSCs(wt:6.319(cid:5)0.19,n(cid:6) periodtoLORRafterinjectionwiththecombinationofketamineplusxylazine(A)andthemean
((cid:5)SEM)durationofLORRbeforerecovery(B)areshown.Twodifferentdosecombinationsof
3; Tg: 11.776 (cid:5) 1.51, n (cid:6) 4; p (cid:6) 0.029) were observed, thus
ketamineplusxylazinewereusedasshown.Statisticallysignificantdifferencesdeterminedby
supportingtheideathattheincreasesinGlureleaseafterneu- unpairedStudent’sttestareshown(n(cid:6)5wtandn(cid:6)6Tg;*p(cid:6)0.05;**p(cid:6)0.005).
ronaldepolarization(Fig.2)occurredatexcitatorysynapsesin
theCNS.
depotforanestheticagentsthusalteringpharmacokineticprop-
Sensitivitytoanestheticagentsandneurological ertiesinGlud1mice,becausetheTgmiceusedinthesestudies
manifestationsinGlud1mice weighedalittlelessthanwtmice(wt,29.5(cid:5)0.6g;Tg,26.2(cid:5)
Induction of anesthesia by both intravenous and volatile anes- 0.6g;p(cid:6)0.02;df(cid:6)11).
theticagents,suchasthiopental,propofol,ketamine,ketamine–
xylazine,andenflurane,involvessuppressionofGlureleaseand NeuronalstructureinthebrainsofTgmice
ofpostsynapticGlureceptoractivation(Orseretal.,1995;Buggy InitialexaminationofneuronalstructureinthebrainsofTgand
etal.,2000;Rozzaetal.,2000;ChengandKendig,2003).Itwas wtmicewasperformedbystainingneuronswithmethyleneblue.
reasonedthatiftheGlud1miceproducedandreleasedexcessGlu VariousregionsofthebrainsfromfivepairsofTgandwtmice
in glutamatergic synapses, then they might exhibit differential (12–20 months of age) were examined. Sections from the hip-
sensitivitytoanestheticagentssuchasketamineorthecombina- pocampusrevealedsomethinningofthepyramidalcelllayerin
tionofketamine–xylazine.Anassessmentofanestheticsensitiv- the CA1 field of the hippocampus (Fig. 5A,B) and the ventral
itywasundertakenusingmaleandfemalewtandTgmice.Two granule cell layer of the dentate gyrus (Fig. 5C,D). Counts of
measureswereobtained,timetoLORRanddurationofLORR. stainedneuronsrevealedsignificantdecreasesinthenumbersof
Thesemeasureswereobtainedusingtwodifferentdosesofthe neurons in the CA1 region of the hippocampus of Glud1 mice
combinationofketamineandxylazine,200and13.3mg/kgket- comparedwithwtmice(cellsper1100(cid:1)2;wt:194.7(cid:5)2.3,n(cid:6)4;
amineandxylazine,respectively,and130and8.8mg/kg.Glud1 Glud1:167.3(cid:5)1.5,n(cid:6)4;p(cid:6)0.005)aswellasinthegranulecell
miceexhibitedsignificantdifferencesfromwtmiceinbothtime layerofthedentategyrus(cellsper6162(cid:1)2;wt:406.7(cid:5)20.3,n(cid:6)
toLORR(longerinGlud1mice)(Fig.4A)andtotaltimebefore 4;Glud1:321.3(cid:5)16.6,n(cid:6)4;p(cid:6)0.05).Inaddition,neuronsin
recovery of righting reflex (shorter in Glud1 mice) (Fig. 4B). boththeCA1fieldandthedentategyrusappearedshrunkenand
ThesedifferencesbetweenTgandwtmiceweresignificantunder darklystained(pyknotic).Thepyknoticappearanceofpyramidal
bothdosecombinationsofketamineandxylazine.Thedifferen- neurons in CA1 and granule neurons in the dentate gyrus of
tialsensitivitytoanestheticagentscouldnotbeascribedtoin- Glud1micewassimilartothatobservedaftertheinjectioninto
creasedfattytissueinTgmice,whichmighthavefunctionedasa thehippocampusofaninhibitorofbrainGlutransporters(Arias
13936•J.Neurosci.,November4,2009•29(44):13929–13944 Baoetal.•Glud1TransgenicMice
etal.,1997).Thesimilaritiesinmorpho-
logical changes observed in Glud1 mice
and those produced by the acute injec-
tionsofaGlutransporterinhibitormight
be viewed as indirect evidence that en-
hancedexpressionofGlud1alteredneu-
ronal structure in hippocampus and
dentate gyrus as a result of increases in
extracellularGlulevels.
Besides the neuronal changes in hip-
pocampus, loss of large pyramidal cells
was apparent in layers III–V of the pri-
marymotor/somatosensorycortexofTg
mice (Fig. 5E,F). The numbers of neu-
rons in layers III–V were indicative of a
significant decrease in the total popula-
tionofstainedneuronsinsomatosensory
cortex layers III–V of Glud1 mice com-
paredwithwtmice(Fig.6).Unlikecorti-
calandhippocampalneurons,otherlarge
neurons,suchasthePurkinjeneuronsof
thecerebellarcortex,didnotappeartobe
affectedbytheoverexpressionofGlud1in
theTgmice(datanotshown).
Beyondtheapparentneuronallossesde-
scribedabove,thereweremarkeddecreases
in dendrite labeling by anti-MAP2A anti-
bodiesinGlud1mouseneurons;thesede-
creases were most apparent in the SR of
the CA1 (Fig. 7). Sections from pairs of
Glud1 and wt mouse brains were pro-
cessed in parallel and fluorescence mi-
croscopy using identical laser intensity
and signal amplification for Tg and wt
mice.Thedifferencesinpixeldensitybe-
tweenTgandwtMAP2Alabelinginthe
SRofCA1(densityper3128(cid:1)m2)were
significant(ANOVA;df3,8;F(cid:6)11.6;p(cid:6)
0.003).However,unlikethedifferencesin
MAP2A labeling of CA1 neurons, those
for CA3 neurons were not statistically
significant(ANOVA;F(cid:6)3.4;p(cid:6)0.24).
DoubleimmunelabelingofCA1neurons
with anti-MAP2A and anti-synaptophysin
antibodies revealed marked decreases in Figure5. HistologicalsectionsthroughthehippocampusCA1(A,B),dentategyrus(C,D),andcerebralcortex(E,F)of16-
labeling of nerve terminals in Glud1 month-oldTg(A,C,E)andwt(B,D,F)mice.Sectionswerestainedwithmethyleneblue,andallimageswereobtainedatthesame
mousehippocampusbytheanti-synapto- magnification.Scalebars,10(cid:1)m.GrCD,Granulecelllayer,dorsal;GrCV,granulecelllayer,ventral;DG,dentategyrus.Large
pyramidal(Pyr.)neuronsinlayersIV–Vofcerebralcortexinwt(F,arrow)andtheabsenceofsuchneuronsinGlud1(E,square)are
physinantibodycomparedwiththatinwt
pointedout.
hippocampus(Fig.7B,D,F,H)(i.e.,there
wereconcurrentdecreasesinnervetermi-
decreased, despite the fact that in this brain region the nerve
nalanddendritelabelinginTgversuswtmousehippocampus).
terminallabelingbyanti-synaptophysinantibodiesshowedlittle
The decreases in anti-MAP2A labeling in dendrites of CA1
changefromthatobservedinwtmice(Fig.8).Becausethemi-
pyramidal neurons of Glud1 mice were similar to decreases in
MAP2Alabelinginhippocampusobservedafterchemicalinhi- croanatomicalstructureofthestriatumismorecomplexthanthe
bition of Glu transporters and elevations of extracellular Glu layeredstructureofthehippocampus,z-planestackedviewsof
(Ariasetal.,1997).Thus,thedecreasesinMAP2Alabelinginthe thestriatumwereobtained,andthree-dimensionalreconstruc-
Glud1mousehippocampusmighthavebeentheresultofexcess tionwasusedtoallowforamoreclearvisualizationoftheanti-
releaseofGluintotheextracellularspaceofthebrain.Because MAP2Aandanti-synaptophysinlabelingtobestudied(Fig.8).
increases in Glu concentration and in depolarization-induced Also,becauseofthecomplexarchitectureofthestriatum,pixel
GlureleaseinGlud1miceweremeasuredinthestriatum(Fig.2), density measurements over a predominantly dendritic field
thatregionofthebrainwasalsoexaminedforpossiblechangesin couldnotbeobtained.Therefore,measurementsofMAP2Apixel
MAP2Alabeling(Fig.8).TheoveralllabelingofMAP2Ainboth densitywerebasedontheoveralllabelingofneuronsandneuro-
dendritesandcellbodiesofstriatalneuronsofGlud1micewas nalprocessesinthestriatum.Areasrepresentinglargecorticospi-
Baoetal.•Glud1TransgenicMice J.Neurosci.,November4,2009•29(44):13929–13944•13937
Figure6. NeuroncellcountsinsensorimotorcortexofwtandTgmice.Methyleneblue-
stainedsectionssimilartothoseinFigure5wereanalyzedtoestimatethetotalnumberof
neuronsinasetarea(7225(cid:1)2)oflayersIII–IVofsensorimotorcortex.Neuronswerecountedas
thetotalnumberofneuronsandaslargepyramidalcells.Thecountsshownrepresentthemean
((cid:5)SEM)foreachcellpopulationandwereobtainedfromfourwtandfourTgage-matched
mice.Statisticallysignificantdifferences(pairedStudent’sttest)arealsoshown;*p(cid:6)0.05.
LargePyr.Neur.,Largepyramidalneurons.
naltractsthroughthestriatumwereavoidedinthequantification
of MAP2A pixel density (no immune staining in these tracts).
The measurements obtained revealed significant decreases in
MAP2AlabelinginGlud1mice(pixeldensityper9930(cid:1)2;wt,
37.7(cid:5)2.1;Glud1,21.9(cid:5)1.6;p(cid:6)0.005).
Despitethesignificantlossofimmunelabelingindendritesof
pyramidalcellsinthehippocampusofTgmice,itwasnotedthat
the cell bodies of these neurons were generally well labeled by
MAP2Aantibodies(Fig.7)(i.e.,therewascontinuedsynthesisof
MAP2AinneuronalcellsofGlud1mice).ThepatternofMAP2A
labelingobservedinGlud1miceshowedsomesimilaritiestothat
seenaftertransient,excessivestimulationofNMDAreceptorsin
brainslicesexvivo(Hoskisonetal.,2007),namelydecreasesin
labelingofdendritesandconcurrentincreasesinlabelingofneu-
ronal cell bodies in CA1 pyramidal neurons. However, the ap-
pearance of the dendrites in the CA1 region of Glud1 mice
differedfromthatofhippocampusslicesacutelytreatedwitha
largedoseofNMDA,asMAP2AlabelinginGlud1micewasless
suppressedthanthatinslicestreatedforashortperiodoftime
with high concentrations of NMDA (Hoskison and Shuttle-
worth,2006;Hoskisonetal.,2007).Furthermore,thedendrites
of CA1 neurons in Glud1 mice did not exhibit the varicosities
observedinneuronstreatedacutelywithNMDA.
To determine the overall structure of dendrites of CA1 and
corticalneurons,brainsectionsweredoublelabeledbythecar-
bocyaninedyeDiIandbyanti-MAP2Aantibodies.BothDiIand
Figure7. ImmunelabelingofMAP2A(green)andsynaptophysin(red)reactivesitesinCA1
anti-MAP2Alabelinginneuronsfromwtmicewascontinuous
ofhippocampusof12-month-oldwt(A–D)andTg(E–H)mice.C,G,Enlargedviewsofdendrite
throughout the length of primary dendrites (Fig. 9). For the
labelingbyanti-MAP2AantibodiesintheSOofCA1pyramidalneurons.D,H,Superimposed
Glud1mouseCA1region,labelingbyDiIindicatedthatthepri- imagesofMAP2A-andsynaptophysin-immunoreactivesites.Scalebars,10(cid:1)m.
marydendriteshaftsinTgmicewereasintactandcontinuousin
theirstructureasthoseinwtmice(Fig.9)(i.e.,noclearindica-
tionsofdendritevaricositiesordisruptionofstructure).How- Effectsofageondendritestructureandneuronalnumbersin
ever,MAP2Alabelingwithintheshaftsofthesedendritesshowed brainsofTgmice
decreasedimmunereactivityandapatternofdiscontinuities Ifsynapticandneuronallossesduringtheagingprocessarethe
inlabelingdescribedabove.Itwasunlikelythatthedisconti- resultofabnormallevelsoractivityofGluinbrain,wewondered
nuitiesinMAP2Alabelingresultedfromthehandlingofthe whetherthedecreasesinMAP2AlabelingindendritesofTgmice
tissuesections,suchasdifferentialtemperaturesduringtissue wouldbecomemorepronouncedwithadvancingage.Toaddress
preparationandfixation(Zhangetal.,2007),becausethehip- thisquestion,thelossofMAP2Alabelingindendriteswasmon-
pocampifromawtandaTgmousebrainwerealwaysprocessedin itoredacrossageinTgandwtmice(Fig.10A).MAP2Aimmune
parallelandunderidenticalconditions.Nevertheless,thewthip- labelinginCA1dendritesofwtandTgmouselittermates(age,
pocampusdidnotexhibitdiscontinuitiesinor“beading”ofMAP2A 6–20 months) indicated a significant decline in labeling in Tg
labeling. miceacrossage(ANOVA;df(cid:6)1,25;F(cid:6)5.3;p(cid:6)0.03),butnot
13938•J.Neurosci.,November4,2009•29(44):13929–13944 Baoetal.•Glud1TransgenicMice
Figure8. Three-dimensionalreconstructionofimmunelabelingofMAP2A(green)andsyn-
aptophysin(red)reactivesitesinthestriatumof12-month-oldTg(A)andwt(B)mice.z-plane
stackedimages(0.1(cid:1)mthickness)obtainedbyspinningdiscmicroscopywereusedforthree-
dimensionalreconstructionandquantificationofMAP2A-andsynaptophysin-immunoreactive
sites.ThearrowinBpointstogreenfluorescence(GreenFluoresc.)ofMAP2Aindendritesof
striatalneurons.Scalebars,10(cid:1)m.
inwtmice(df(cid:6)1,25;F(cid:6)1.63;p(cid:6)0.21).Althoughtherewasa
trendtowarddecreasedMAP2Alabelingintheoldestgroupofwt
mice(20months),ANOVAdidnotrevealasignificantageeffect
norwasthedecreaseinMAP2Aimmunelabelingat20months
significantlydifferentfromthatat11monthsinwtmice(Bon-
ferroniposthocanalysis).ComparisonsbetweenTgandwtmice
ateachage(6,11,and20months)revealedsignificantdifferences
foreachagegroupcomparison(Bonferroniposthocanalysis,p(cid:6)
0.0005).Thus,significantlylowerlevelsofMAP2Alabelingwere
alreadypresentinTgmiceat6monthsofage,andMAP2Alevels
decreasedsignificantlywithadvancingageinTgmice.Itshould
benoted,though,thatfrom11to20months,theratesofdecrease
inbothwtandTgmouseCA1werenearlyparallel,anindication
thattheeffectofGlud1overexpressionondendritelabelingwas
superimposedonatrendofanage-dependentdecreaseinlabel-
inginwtmouseCA1.
ThepreservationofMAP2Alabelinginhippocampusneuro-
nalcellbodiesofbothTgandwtmiceaffordedtheopportunityto
use images similar to those in Figure 7, to count cell numbers
within equal-sized areas of CA1 and CA3. Some sections were
Figure9. DuallabelingofpyramidalneurondendritesintheCA1regionofhippocampusby
doublestainedwithSYTOXnuclearstaintoconfirmtheaccuracy DiIandMAP2Aantibodies.Hippocampussectionsfrom12-month-oldTg(A–D)andwt(E–H)
of the cell counts. The counts were obtained from the mid- micewerefixedandlabeledbyDiI(red)andanti-MAP2Aantibodies(green)asdescribedin
hippocampus CA1 and CA3 regions (Fig. 10B). There was a MaterialsandMethods.A,E,MAP2A-immunoreactivesites.B,F,DiIlabelingofneurons.C,G,
highlysignificanteffectofGlud1overexpressiononcellnum- SuperimposedMAP2AandDiIlabeling.D,H,EnlargedimagesofCandG.Scalebars,10(cid:1)m.
bersinCA1(ANOVA;df(cid:6)3,8;F(cid:6)12.1;p(cid:6)0.002).Posthoc
analyses indicated significant differences between Tg and wt neuronal numbers between 11- and 20-month-old wt and Tg
cell numbersinCA1ateveryagetested(p(cid:6)0.001–0.028).A mice might indicate again that the effect of overexpression of
statistically significant effect of age on CA1 cell numbers was Glud1inTgmicewasanaddedeffectonabackgroundtrendof
obtainedforTg(df(cid:6)2,23;F(cid:6)9.1;p(cid:6)0.001)butnotforwt age-associated,butnotyetsignificant,neuronallossinwtmice.
(p(cid:6)0.057)mice.However,therewasatrendtowarddecreased NosignificantdifferencesbetweenTgandwtmicewerede-
cellnumberswithadvancingageinwtmice,andthesedecreases tectedforneuronsoftheCA3region,norwasthereasignificant
approximated significant levels for an age effect on neuronal effectofageoneitherwtorTgmouseCA3neuronalnumbers
numbersinwtCA1.Furthermore,thenearlyparalleldecreasein (Fig.10B).SincethelevelsofGLUDimmunoreactivityinCA3