Differentiation of Apical and Basal Dendrites in Pyramidal Cells and Granule Cells in Dissociated PDF
Preview Differentiation of Apical and Basal Dendrites in Pyramidal Cells and Granule Cells in Dissociated
RESEARCHARTICLE Differentiation of Apical and Basal Dendrites in Pyramidal Cells and Granule Cells in Dissociated Hippocampal Cultures YouKureWu1,KazutoFujishima2,MinekoKengaku1,2* 1 GraduateSchoolofBiostudies,KyotoUniversity,Kyoto,Japan,2 InstituteforIntegratedCell-Material Sciences(WPI-iCeMS),KyotoUniversity,Kyoto,Japan * [email protected] Abstract Hippocampalpyramidalcellsanddentategranulecellsdevelopmorphologicallydistinct dendriticarbors,yetalsosharesomecommonfeatures.Bothcelltypesformalongapical dendritewhichextendsfromtheapexofthecellsoma,whileshortbasaldendritesarede- OPENACCESS velopedonlyinpyramidalcells.Usingquantitativemorphometricanalysesofmousehippo- campalcultures,weevaluatedthedifferencesindendriticarborizationpatternsbetween Citation:WuYK,FujishimaK,KengakuM(2015) pyramidalandgranulecells.Furthermore,weobservedanddescribedthefinalapicalden- DifferentiationofApicalandBasalDendritesin PyramidalCellsandGranuleCellsinDissociated dritedeterminationduringdendriticpolarizationbytime-lapseimaging.Pyramidalandgran- HippocampalCultures.PLoSONE10(2):e0118482. ulecellsincultureexhibitedsimilardendriticpatternswithasingleprincipaldendriteand doi:10.1371/journal.pone.0118482 severalminordendritessothatthecelltypeswerenotreadilydistinguishedbyappearance. AcademicEditor:MakotoSato,OsakaUniversity Whilebasaldendritesingranulecellsarenormallydegradedbyadulthoodinvivo,cultured GraduateSchoolofMedicine,JAPAN granulecellsretainedtheirminordendrites.Asymmetricgrowthofasingleprincipaldendrite Received:September5,2014 harboringtheGolgiwasobservedinbothcelltypessoonaftertheonsetofdendriticgrowth. Accepted:January18,2015 Time-lapseimagingrevealedthatupuntilthesecondweekinculture,finalprincipaldendrite designationwasnotstabilized,butwasfrequentlyreplacedbyotherminordendrites.Before Published:February23,2015 dendriticpolaritywasstabilized,theGolgimoveddynamicallywithinthesomaandwasre- Copyright:©2015Wuetal.Thisisanopenaccess peatedlyrepositionedatnewlyemergingprincipaldendrites.Ourresultssuggestthatpolar- articledistributedunderthetermsoftheCreative CommonsAttributionLicense,whichpermits izedgrowthoftheapicaldendriteisregulatedbycellintrinsicprograms,whileregressionof unrestricteduse,distribution,andreproductioninany basaldendritesrequirescue(s)fromtheextracellularenvironmentinthedentategyrus.The medium,providedtheoriginalauthorandsourceare apicaldendritedesignationisdeterminedfromamongmultiplegrowingdendritesofyoung credited. developingneurons. DataAvailabilityStatement:Allrelevantdataare withinthepaperanditsSupportingInformationfiles. Funding:ThisworkwassupportedbytheNEXT programoftheJapanSocietyforthePromotionof Science(JSPS)andGrant-in-AidforScientific ResearchfromtheMinistryofEducation,Science, Introduction SportsandCultureofJapantoMK.Thefundershad noroleinstudydesign,datacollectionandanalysis, Neuronsofthecentralnervoussystemexhibitenormouslydiversedendriticarborarchitecture, decisiontopublish,orpreparationofthemanuscript. whichdeterminesboththenumberandtypeofsynapticinputsreceived,andhencecritically affectneuronalconnectivity.PyramidalcellsintheAmmon’shorn(cornuammonis,orCA) CompetingInterests:Theauthorshavedeclared thatnocompetinginterestsexist. andgranulecellsinthedentategyrus(DG)arethetwoprincipalneuronaltypesinthe PLOSONE|DOI:10.1371/journal.pone.0118482 February23,2015 1/16 DifferentiationofDendritesinPyramidalandGranuleCells hippocampalformation,exhibitingdistinctdendriticarborstructures.Pyramidalcellsgiverise toalong,thickapicaldendriteandseveralminorbasaldendritesthatemergefromtheapex andbaseoftheteardrop-shapedsoma,respectively.Theapicalandbasaldendritesareoriented inoppositedirectionsandoccupydifferentlayers,withapicaldendritesextendingtowardthe hippocampalfissurethroughthestratumradiatumandstratumlacunosum-moleculare,and basaldendritesextendingintheoppositedirectionthroughthestratumoriens.Thus,theden- driticarchitectureofpyramidalcellstakeabiconicalshapeandareabletoreceivesynapticin- putsfromdifferentafferentsources.Incontrasttopyramidalcells,dentategranulecellshavea monoconicalarborofapicaldendriteswithallbranchesdirectedtowardthesuperficialregion ofthemolecularlayer.Immaturegranulecellsproducetransientdendritesfromthebasalpor- tionofthesoma,whicharethenretractedbyadulthood,exceptforinasmallpopulationof granulecellsinprimates[1–3]. Apicalandbasaldendritesdifferinvariousproperties,includingsize,geometry,electrical conduction,andresponsivenesstoneurotrophicfactorsorguidancemolecules[4–6].Ithas beenshownthatextensionandbranchingofapicalandbasaldendritesaredifferentiallyregu- latedbythesecretedguidancemolecule,semaphorin3A[7–12].Accumulatingevidencehas alsoimplicatedpreferentiallocalizationoftheGolgiapparatustothebaseoftheapicaldendrite duringestablishmentofdendriticpolarity[13,14].Otherextrinsicandintrinsiccuesinvolved inthecontrolofdendriticpolarityhaveemerged,buttheprecisemechanismofhowpyramidal andgranulecellsinitiate,developandmaintainapicalandbasaldendritesremainelusive[15]. Pyramidalcellsinprimarycultureestablishdendriticpolaritywithasinglethick,elongated dendriteandseveralshortdendritessimilartopyramidalcellsinvivo[16].Primaryculturesof dentategranulecellshavealsobeenestablished,butlessisknownaboutthespecificationof dendritesinculturedgranulecells[17].Here,usinglong-termtime-lapseimagingandquanti- tativemorphometricanalysisinprimarycultures,wecomparativelyanalyzedthedynamicsof dendriticdifferentiationinpyramidalanddentategranulecells. MaterialsandMethods Animals ICRmiceforprimaryhippocampalculturewereobtainedfromJapanSLC,Inc..Allprocedures involvingmicewereperformedinstrictaccordancewiththeinstitutionalguidelinesandap- provedbytheAnimalExperimentationCommitteeofKyotoUniversity(PermitNumber: icems-2-21).Allsurgerywasconductedunderanesthesiausingisofluraneforadults,ordeep hypothermiaforpups,andalleffortsweremadetominimizesuffering. Primaryneuronculture Primaryculturesofhippocampalneuronswerepreparedaspreviouslydescribedwithafew modifications[18,19].Inbrief,hippocampiweredissectedfrommiceagedfromE17toP4. HippocampalneuronsweredissociatedbyusingSUMITOMONerve-CellCultureSystem (SumitomoBakelite)andplatedoncoverslipsorglass-baseddishescoatedwithpoly-D-lysine atadensityof1.0–2.0x105cells/cm2inMEMsupplementedwith10%horseserum(Gibco), 0.6%D-glucose,1mMsodiumpyruvateand1%penicillin-streptomycin.Threehoursafter plating,mediawasreplacedbyNeurobasalmedium(Gibco)supplementedwithB-27supple- ment(Gibco),0.5mML-glutamineand1%penicillin-streptomycin.Allneuronsweremain- tainedat37°Cin5%CO .NeuronsweretransfectedwithLipofectamine2000(Invitrogen) 2 accordingtothemanufacturer’sinstructions.pCAGGS-GRASP65-GFPwastransfectedat DIV4.OtherconstructsweretransfectedatDIV2–4. PLOSONE|DOI:10.1371/journal.pone.0118482 February23,2015 2/16 DifferentiationofDendritesinPyramidalandGranuleCells DNAconstructsandantibodies pCA-EGFPandpAAV-CAG-TdTomatowerepreviouslydescribed[20,21].pAcGFP1-Golgi wasobtainedfromClontech.ForconstructionofpCAGGS-GRASP65-GFP,cDNAencoding GRASP65wasclonedbyPCRfromacDNAlibrary.ThePCRproductwassubclonedinto pEGFP-N1vector(Clontech),andtheEGFP-fusedGRASP65wassubclonedintopCAGGS vector.Antibodiesusedforimmunostainingwereasfollows:rabbitanti-Math2,anti-Prox1 andanti-SynapsinI,chickanti-NeuN(Abcam);mouseanti-GM130(BDBiosciences);rabbit anti-Calbindin,anti-GFAPandmouseanti-GAD67(Chemicon);mouseanti-PSD95(Funa- koshi);mouseanti-Prox1(Millipore);goatanti-AnkyrinG(SantaCruz);rabbitanti-Calretinin (Swant);Alexa405-,Alexa488-,Alexa568-,Alexa633-orAlexa647-conjugatedanti-chick,anti- goat,anti-mouseoranti-rabbitIgG(MolecularProbes). Immunofluorescenceandmorphologicalanalysis Cellswerefixedwith4%paraformaldehydeinPBSandpermeabilizedwith0.3%TritonX-100 inPBS.Cellswerethenblockedwithblockingsolution(2%skimmilk,0.1%Tween20inPBS) andincubatedwiththeprimaryantibodiesat4°Covernight.AfterwashingwithPBS,cellswere incubatedwithsecondaryantibodiesat4°CovernightandstainedwithDAPIatroomtempera- turefor10minutes.Morphologiesofimmunostainedcellswereanalyzedbyalaser-scanning confocalmicroscopeFV1000(Olympus)witha20×dryobjective(N.A.0.75,Olympus),40×dry objective(N.A.0.95)anda100×oil-immersionobjective(N.A.1.4).Detailedmethodsforimmu- nostainingandconfocalanalysesoflabeledneuronsweredescribedpreviously[18]. Axon/dendritespecificationwasidentifiedbyimmunostainingwithanti-AnkyrinGandthe morphologicalcriteriadescribedpreviously[22,23].Cellsthatwereclearlyfreefromother transfectedcellswereselectedandanalyzedbyvirtueofsolubleGFPorTdTomatofill.Den- dritesweretracedwiththeaidofNeurolucidasoftware(MBFBioscience)andprocessedfor quantitativeanalysisusingNeurolucidaExplore(MBFBioscience)andImageJ(NIH).The principaldendritewasdefinedasthelongestdendriteofaneuron.Toanalyzethelocalization oftheGolgiapparatus,thenucleuscenterofmasswassetasapolarcoordinateorigin,andthe directiontothebaseoftheprincipaldendritewasdefinedasφ=0.Aneuronwasseparated intothreeregionsbyφinterval:(–π/4,π/4]astheapicalregion,(π/4,3π/4]and(-3π/4,-π/4]as thelateralregion,andtheremainingintervalasthebasalregion.Theareaoccupiedwiththe GM130signalineachregionrelativetothewholeGM130areainthesomawascalculated.All dataareexpressedasmean±SEM. Time-lapseimaging Fortime-lapseimaging,neuronsfromP0micewereplatedonaglass-baseddishandtrans- fectedwithindicatedplasmidsencodingfluorescentmarkers.Labeledcellswereobserved every15or30minuteswithaspinning-discconfocalmicroscopeCV1000(Yokogawa)through a20×dryobjective(N.A.0.75,Olympus)ora40×oil-immersionobjective(N.A.1.3,Olym- pus).Allneuronswereimagedat37°Cwithconstantgasflowwith5%CO and95%air. 2 Results Morphologiesofneuronsintheprimarycultureofthemouse hippocampalformation Theprimarycultureofhippocampalneuronswaspreparedfromthehippocampalformation ofneonatalmiceatpostnatalday0(P0).Cultureswerefixedafter2daysinvitro(DIV2)for immunofluorescenceusingcell-typespecificmarkers,whichincludepyramidalcellmarker PLOSONE|DOI:10.1371/journal.pone.0118482 February23,2015 3/16 DifferentiationofDendritesinPyramidalandGranuleCells Math2[24,25],granulecellmarkerProx1[26,27],interneuronmarkerGAD67[28]and GFAPforglialcells[29].TheexpressionofMath2andProx1wasmutuallyexclusive(S1Fig.). Approximately25%oftheculturedcellswereMath2-positivepyramidalcellsandanother25% wereProx1-positivegranulecells.GAD67-positiveinterneuronsaccountedforlessthan10% ofcellsintheculture.GFAP-positivegliaconstitutedabout50%ofcellsintheculture. Bythesecondweekinvitro,pyramidalcellshavealargesomawithanaverageareaof 154.5±38.6μm2(n=40)whilegranulecellshaveasmallersomaof87.5±19.1μm2inarea (n=40)(Fig.1Aand1B).Pyramidalandgranulecellsexhibitedsimilarmorphologywitha singlelong,thickprimarydendrite(referredtoasprincipaldendritehereafter)andseveral shorterdendrites.Weconfirmedthatthesedendritesweredistinctfromtheaxonexpressing anaxonalmarkerAnkyrinG(Fig.1C).Bothprincipalandminordendritesborenumerous protrusionsexpressingapost-synapticmarkerPSD95(Fig.1D).Thesedendriticprotrusions werefrequentlyjuxtaposedwithpunctatesignalsofapre-synapticmarkerSynapsinI,sug- gestingthattheneuronsinthedissociatedculturedevelopedmaturedendriteswithfunction- alsynapses.Theoverallsizeofthedendriticarborwaslargerinpyramidalcellsthangranule cells,asindicatedbythetotallengthofdendriticbranchesandthenumberofprimaryden- dritesandbranchterminals(Fig.1E-1G).Theasymmetryofdendriticarborswasmore prominentingranulecellssuchthatthelongestdendritecontributed67%ofthetotalden- driticlengthingranulecells,incontrastto53%inpyramidalcells(Fig.1H). Incontrasttopyramidalandgranulecells,GAD67-positiveinterneuronsextendedseveral equivalentdendriteswithnocleardendriticpolarityaspreviouslyreported(Fig.1A)[16]. Differentiationofpyramidalandgranulecellsinculture Dentategranulecellsandhippocampalpyramidalcellsoriginatefromadjacentbutdistinctregions intheneuroepitheliuminthedorsaltelencephalon,alsocalledthemedialpallium,withtheprimor- diumoftheDGlyinginthemedial-mostregion,followedbytheCAfieldsandthesubicularcortex [30–32].Ithasbeenshownthatdifferentiationofpyramidalandgranulecellsstartsalmostsimulta- neouslyatembryonicday10(E10)inthemousehippocampalformation[33].Thegenerationof pyramidalcellscompletesataroundE18,whilegranulecellproductioncontinuesuntiladultstages. Wethusquestionediftheratioofpyramidalandgranulecellschangedinculturespreparedfrom hippocampiatdifferentages.Wefoundthattheproportionofpyramidalcellswashigherincul- turespreparedfromyoungermice(Fig.2A).InculturespreparedfromE17mice,Math2-positive pyramidalcellsaccountedfor53%ofallneurons,while38%wereProx1-positivegranulecells.Py- ramidalcellssharplydecreasedinculturespreparedfrompostnatalbrainandwerenotobservedin culturespreparedfromP4mice.Incontrast,theproportionofProx1-positivegranulecellsslightly increasedwithage,reaching62%inculturesfromP4mice. Wenextexaminedneuronalmaturationinculture.Ithaspreviouslybeenshownthatim- maturegranulecellsexpresscalretinin,whilematuregranulecellsexpresscalbindinandse- quentiallydownregulatecalretinin[34].Weanalyzedtheexpressionofthesedifferentiation markerstogetherwithProx1intheprimaryhippocampalculturepreparedfromP0mice.At DIV3,thelargemajorityofProx1-positivegranulecellsexpressedcalretinin(Fig.2Band2C). Althoughcalretininexpressiongraduallydecreasedinolderculture,expressionwasstilldetect- ablebyDIV16in31%ofProx1-positivecells,unlikeinmaturegranulecellsinvivo.Calbin- din-positivegranulecellswerefirstobservedbyDIV7andrapidlyincreasedwithagein culture,reaching80%byDIV16.Thus,developinggranulecellsintheprimaryhippocampal cultureundergotime-dependentchangesingeneexpressionsimilartothoseobservedinvivo. Wenextcompareddendriticdevelopmentinpyramidalandgranulecellsintheprimary culturepreparedfromP0mice.Exceptfortheapparentdifferenceinsize,overallbranch PLOSONE|DOI:10.1371/journal.pone.0118482 February23,2015 4/16 DifferentiationofDendritesinPyramidalandGranuleCells Fig1.ThemorphologyofpyramidalandgranulecellsatDIV13indissociatedculture.(A)Typicalmorphologyofpyramidalcells,granulecellsand interneurons.ThecelltypewascharacterizedbyimmunostainingwithMath2,Prox1(magenta)andGAD67,respectively.Pyramidalandgranulecellswere visualizedbyGFPtransfection.InterneuronswerevisualizedbyimmunostainingwithGAD67.Scalebar:40μm.(B)Histogramdistributionofsomalsizein pyramidalandgranulecellsofDIV10–13.n=40foreachcelltype.(C)Axon/dendritespecificationasdemarcatedbyAnkyrinGexpression.Cellswere immunostainedwithAnkyrinG(magenta)andMath2orProx1(blue)atDIV7.Asterisksindicateaxon.Scalebar:20μm.(D)Expressionofsynapticmarkers atDIV16.Boxedregionsintheleftpanelareshownintherightpanels.PSD95(red)waslocalizedinprotrusionsinbothprincipal(upper)andminor(lower) dendrites.SomePSD95signalsindendriticprotrusions(asterisks)wereapposedtoSynapsinIsignals(green).DendriteswerevisualizedbyGFP expression.Scalebar:20μm(left),2μm(right).(E-G)Quantificationoftotaldendriticlength(E),thenumberofprimarydendrites(F)andbranchterminals (G).n=20.Student'st-test:*P<0.05,**P<0.03,***P<0.001.(H)Ratiooflengthofeachdendritearbortothelongestdendrite.Alldendritearborsina neuronwererankedfromthelongesttotheshortest.n=20. doi:10.1371/journal.pone.0118482.g001 patternandmorphologyweresimilarinpyramidalandgranulecellsatDIV7(Fig.3A).Bothcell typeshadasingleprincipaldendritewithnumerousobliquebranchesandmultipleminorden- dritesthatwerelessbranched.Granulecellslookedlikeminiaturesofpyramidalcellsatthisearlier stage.DifferenceinbranchpatternsbecamemoreprominentinolderculturebyDIV16(Fig.3B). Pyramidalcellshadanelaboratelybranchedprincipaldendriteandseveralminordendriteswith PLOSONE|DOI:10.1371/journal.pone.0118482 February23,2015 5/16 DifferentiationofDendritesinPyramidalandGranuleCells Fig2.Populationofpyramidalandgranulecellsindissociatedculture.(A)Proportionofneuronal speciesinculturesfromdifferentagedpups.Wholeneurons,pyramidalcellsandgranulecellswereidentified asNeuN-,Math2-andProx1-positivecells,respectively.n=722(E17),508(P0),634(P4).(B)Maturationof granulecells.Eachpointshowstheratioofeachmarker-positivecellstoProx1-positivecells.n(DIV3,DIV7, DIV11,DIV16)=(371,202,258,95)forcalretinin,(395,186,208,123)forcalbindin.(C)Maturationmarker expressionofgranulecells.Cellswereimmunostainedwithcalretininorcalbindin(green)andProx1 (magenta).Scalebar:30μm. doi:10.1371/journal.pone.0118482.g002 Fig3.Morphologychangeinpyramidalandgranulecells.(A-B)TypicalmorphologyofpyramidalandgranulecellsatDIV7(A)andDIV16(B)ofcultures preparedfromP0mice.CellswerevisualizedbyGFPtransfection.CelltypeswereidentifiedbyMath2andProx1immunostaining(magenta).Theprincipal dendriteandaxonwereindicatedbywhitearrowheadandasterisk,respectively.Scalebar:40μm.(C-E)Quantificationoftotaldendriticlength(C),the numberofbranchterminals(D)andprimarydendrites(E)duringdendritedevelopment.n=20.Student'st-test:*P<0.05,**P<0.03,***P<0.001. doi:10.1371/journal.pone.0118482.g003 PLOSONE|DOI:10.1371/journal.pone.0118482 February23,2015 6/16 DifferentiationofDendritesinPyramidalandGranuleCells increasedlengthandcomplexity.Incontrast,granulecellsatDIV16hadawell-developedprincipal dendrite,whileotherminordendritesremainedshortandboreonlyfewbranches. Forquantitativecomparisonofdendriticdevelopment,thenumberandlengthofallbranches inMath2-positivecellsandProx1-positivecellsweremeasuredfromDIV7to16.Thetotalden- driticlengthgraduallyincreasedinparallel,withpyramidalcelldendritesbeinglongerthangran- ulecelldendritesbyapproximately2-foldatallstagesexamined(Fig.3C).Thenumberofbranch terminalswasalsoconsistentlygreaterinpyramidalcells,withaslightincreaseinbothpyramidal andgranulecellsfromDIV7toDIV16(Fig.3D).Pyramidalcellshadonaverage5ormorepri- marydendritesatallstagesexamined(Fig.3E).Incontrast,granulecellshadanaverageof4or5 primarydendritesupuntilDIV13,whichdecreasedto3or4primarydendritesbyDIV16.Den- driticpolarityofgranulecellsbecameprominentbythisstageinculture. Dendritegrowthdynamicsinpyramidalandgranulecells Usingtime-lapseimagingofdevelopingneuronsinculture,wenextobservedthedynamic structuralplasticityofdendriticarborsinpyramidalandgranulecellsatvariousstages.The primarycultureswerepreparedfromP0mice.AtDIV7,bothpyramidalandgranulecellsun- derwentextensionsandretractionsofdendrites(Fig.4Aand4B,S1Movie).Formationand eliminationofminordendriteswerefrequentlyobservedatthisstage.Weoccasionallyob- servedretractionofprincipaldendritesandsubstitutionbyothergrowingdendrites.Dendrites ofpyramidalcellsbecamelessdynamicbyaroundDIV10.Primarydendritesincludingthe Fig4.Dynamicsofdendritedevelopment.(A)TypicalmorphologychangeinpyramidalandgranulecellsculturedfromP0mice.Bluelinesindicatenewly extendeddendrites,redlinesindicateretracteddendriteswithina12hrperiodofobservation.(B)Thenumberofdendritesduringdendriticdevelopment. Eachbarindicatesthenumberofstable,appearedordisappeareddendriteswithina12hrperiodofobservationin30min-intervalmovies.n=10(DIV7,10), 16(DIV13). doi:10.1371/journal.pone.0118482.g004 PLOSONE|DOI:10.1371/journal.pone.0118482 February23,2015 7/16 DifferentiationofDendritesinPyramidalandGranuleCells principaldendriteandotherminordendritesappearedtobefixed,whiledendritictipsand higherorderedbranchescontinuedtogrowwithrepeatedextensionandretraction.Thesewere mostlystabilizedbyDIV13(S2Movie).Incontrast,granulecelldendritesremainedhighlydy- namicatDIV10.Principaldendritesappearedtobedetermined,whilehigherordered branchesandminorprimarydendritescontinuedextensionandretractionuntilDIV13 (Fig.4Aand4B,S2Movie).Thus,granulecelldendritesmaintainedstructuralplasticityuntil laterstagescomparedtopyramidalcelldendrites. Specificationoftheapicaldendriteinpyramidalcellsandgranulecells Previousstudiesusingratneuronshaveindicatedthatdendritesarenotpolarizedbutequiva- lentinlengthuntilDIV8[13,16].Incontrast,inourculturepreparedfromP0mice,dendritic polaritywasalreadyprominentbyDIV7inbothpyramidalandgranulecells.Wethusques- tionedwhendendriticpolaritywasestablishedinculturedmousehippocampalneuronsby usingtime-lapseimaging. Bothpyramidalandgranulecellsexhibitedapolarizedshapewithasingleprincipalden- dritebyDIV4(Fig.5A).However,principaldendritedesignationwasnotfixed;rather,allden- driteswerehighlydynamicwithextensivegrowthandretractionatthisstage.Theprincipal dendriteoccasionallyreceded,andinturn,oneoftheotherminordendritesrapidlyextended tobecomeanewprincipaldendrite(Fig.5Band5C,S3Movie).Neuronsthusmaintaineda unipolarshapeatanypointofobservation.ByDIV10,theprincipaldendritewasmostlyfixed andrarelyreplacedbyotherdendrites(Fig.5Dand5E).Frequencyofreplacementoftheprin- cipaldendritedesignationwasgraduallydiminishedoverthecultureperiod,reachingalmost zerobyDIV10(Fig.5F).Theseresultssuggestthattheprincipaldendriteisdeterminedseveral daysafterdendriticpolarityisestablishedbothinculturedpyramidalandgranulecells. LocalizationoftheGolgiapparatusingrowingdendrites IthasbeenshownthattheGolgiapparatusispolarizedtowardtheprincipaldendriteinculture andtheapicaldendriteinpyramidalcellsinvivo[13].ToconfirmwhethertheGolgipolarizes towardtheprincipaldendrite,weanalyzedthedistributionofcis-Golgilabeledwithanti- GM130antibodyinthesoma[35](Fig.6A).Consistentwithpreviousreports,thesomatic Golgistackswerelocalizednearthebaseoftheprincipaldendriteinbothpyramidalandgran- ulecells(Fig.6Band6C).TheorientationoftheGolgitowardtheprincipledendritewasmore pronouncedatDIV7,whenthedendriticpolaritywasnotyetfullyestablished,comparedto DIV16,bywhichdendriticidentitieswerealreadystabilized. IthasbeendemonstratedthatthepolarizedorientationoftheGolgidirectssecretorytraf- fickingpredominantlytowardtheapicaldendritefortheestablishmentandmaintenanceof dendriticpolarityinpyramidalcells[13].ThisraisesapossibilitythattheGolgidynamically moveswithinthesomabeforethefinalprincipaldendritedesignationisdetermined.Totest this,welabeledtheGolgibytransfectingAcGFPfusedwiththeN-terminal81aminoacidsof humanbeta-1,4-galactosyltransferase(pAcGFP1-Golgi),andobservedthedynamicbehavior oftheGolgiinlivehippocampalneuronsatDIV5.Consistentwiththeimmunofluorescence resultsusinganti-GM130,AcGFP1-GolgiwasabletolabeltheGolgistacksandwasoftenlocat- edatthebaseoftheprincipaldendrite(Fig.7A).TheGolgiactivelychangeditsshapewhileit remainedneartheprincipaldendrite,anditwouldoccasionallyinvadeintotheprincipalden- drite,orexpandingtocoverawideareainthesoma.Whentheprincipaldendriteregressed, theGolgirapidlyrelocatedtothenewlyemergedprincipaldendritethatbecamethelongest amongall(Fig.7B).ThefrequencyofsimultaneousGolgirelocationtotheprincipaldendrite wasapproximately80%(Fig.7C).Thus,theestablishmentoftheprincipaldendriteispreceded PLOSONE|DOI:10.1371/journal.pone.0118482 February23,2015 8/16 DifferentiationofDendritesinPyramidalandGranuleCells Fig5.Theprincipaldendritedetermination.(A)TypicalmorphologyofthepyramidalandgranulecellatDIV4.CellswereisolatedfromP0hippocampi andvisualizedbyGFPtransfection.CelltypeswereidentifiedbyMath2andProx1immunostaining(magenta).Theprincipaldendriteandaxonwere indicatedbywhitearrowheadandasterisk,respectively.Scalebar:20μm.(B-E)Time-lapseimagingofdendriticdevelopmentinGFP-transfectedpyramidal andgranulecells.CelltypeswereidentifiedbyMath2andProx1immunostaining(magenta)afterimaging.Asterisksindicateaxon;arrowsandarrowheads indicatetheprincipaldendritesatthetimepointsofobservation.Scalebar:30μm.(F)Thefrequencyofreplacementoftheprincipaldendriteobservedfrom DIV4.Thereplacementoftheprincipaldendritewascountedevery10hrin30min-intervalmovies.n=22. doi:10.1371/journal.pone.0118482.g005 byaperiodinwhichtheGolgilocalizationwasinconstantandtransientlypolarizedtowarda tentativeprincipaldendriteamongmultiplehighlydynamicprimarydendrites. WenextaskedifthechangeinGolgilocalizationinterferedwiththepolarizationoftheprinci- paldendrites.IthasbeenshownthatoverexpressionoftheGolgiproteinGRASP65leadstodis- persaloftheGolgiintomultipledendritesanddisruptionofapicaldendritespecification[13, 36].Consistentwiththepreviousreports,overexpressionofGRASP65-GFPcausedvesiculation anddispersaloftheGolgiandreductionindendritepolarization(Fig.7Dand7E).Time-lapse PLOSONE|DOI:10.1371/journal.pone.0118482 February23,2015 9/16 DifferentiationofDendritesinPyramidalandGranuleCells Fig6.LocalizationoftheGolgiapparatus.(A)ImmunofluorescenceoftheGolgiapparatuswithanti-GM130inpyramidalandgranulecellstransfectedwith GFPatDIV7.CellmorphologyandtheGolgiwerevisualizedbyGFPexpression(green)andanti-GM130immunostaining(magenta).Celltypeswere identifiedbyMath2andProx1immunostaining(cyan).Scalebar:10μm.(B-C)GolgidistributionatDIV7andDIV16inpyramidal(B)andgranulecells(C). Definingthecentroidofthenucleusastheoriginofapolarcoordinatesystem,thesomawasseparatedintothreeregionsasshown.n=10. doi:10.1371/journal.pone.0118482.g006 observationrevealedthatoverexpressionofGRASP65-GFPdidnotdecreasethemotilityofden- drites,butnoneofthembecamepredominantduringDIV5–7whencontrolneuronsconstantly exhibitedapolarizedshapewithasingleprincipaldendrite(Fig.7FandS4Movie).Theseresults indicateacriticalroleofGolgilocalizationintheprincipaldendritespecification. Discussion Morphologicaldifferencesbetweenpyramidalandgranulecellsinculture Weidentifieddifferentcelltypesincludedintheconventionalhippocampalcultureandcon- ductedquantitativeassessmentofdendriticmorphogenesisinpyramidalandgranulecellsby immunofluorescenceandlive-cellimaging.Despiteslightbutsignificantdifferencesinsome morphometricanddynamicproperties,overalldendriticmorphologyofpyramidalandgran- ulecellsinculturewasremarkablyanalogoustoeachother,especiallyinyoungerculturesbe- foreDIV13.Unlikeinvivo,granulecellsincultureretainedmultipleminordendritesin additiontoasingleprincipaldendrite.Althoughtheaveragesizeofthesomataanddendritic arborsweresignificantlydifferent,aconsiderableproportionofcellshadanintermediatesize, withcelltypeidentificationonlydistinguishablebymolecularmarkerexpression. Theprimarymousehippocampalcultureutilizedinthisstudyhasbeenwidelyusedfor analysesofelectrophysiologicalandmorphometricpropertiesofpyramidalcells.However,we haveshownthatgranulecellswereconsistentlyincludedinculturespreparedfromdifferent agedmice.Thisisconsistentwithpreviousfindingsthatinmice,pyramidalandgranulecells emergesimultaneouslybyE10,whileinratsthegenerationofthetwocelltypesbeginatdiffer- enttimepointsduringembryonicdevelopment[33,37].Itwasalsodifficulttocompletely PLOSONE|DOI:10.1371/journal.pone.0118482 February23,2015 10/16
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