Differentiation of Apical and Basal Dendrites in Pyramidal Cells and Granule Cells in Dissociated

16 Pages·2015·9.82 MB·English

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.
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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
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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
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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
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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
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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).
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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
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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