Synaptic Targets of Medial Septal Projections in the Hippocampus and Extrahippocampal Cortices ...

15 Pages·2015·7.03 MB·English

15812•TheJournalofNeuroscience,December2,2015•35(48):15812–15826
Systems/Circuits
Synaptic Targets of Medial Septal Projections in the
Hippocampus and Extrahippocampal Cortices of the Mouse
XGunesUnal,1AbhilashaJoshi,1XTimJ.Viney,1ViktorKis,1,2andPeterSomogyi1
1MedicalResearchCouncilBrainNetworkDynamicsUnit,DepartmentofPharmacology,UniversityofOxford,OxfordOX13TH,UnitedKingdom,and
2DepartmentofAnatomy,CellandDevelopmentalBiology,Eo¨tvo¨sLora´ndUniversity,1117Budapest,Hungary
Temporalcoordinationofneuronalassembliesamongcorticalareasisessentialforbehavioralperformance.GABAergicprojections
fromthemedialseptumanddiagonalbandcomplexexclusivelyinnervateGABAergicinterneuronsintherathippocampus,contributing
tothecoordinationofneuronalactivity,includingthegenerationofthetaoscillations.Muchlessisknownaboutthesynaptictarget
neuronsoutsidethehippocampus.Torevealthecontributionofsynapticcircuitsinvolvingthemedialseptumofmice,wehaveidentified
postsynapticcorticalneuronsinwild-typeandparvalbumin-Creknock-inmice.Anterogradeaxonaltracingfromtheseptumrevealed
extensiveinnervationofthehippocampusaswellasthesubiculum,presubiculum,parasubiculum,themedialandlateralentorhinal
cortices,andtheretrosplenialcortex.Inallexaminedcorticalregions,manyseptalGABAergicboutonswereincloseappositiontosomata
ordendritesimmunopositiveforinterneuroncell-typemolecularmarkers,suchasparvalbumin,calbindin,calretinin,N-terminalEF-
handcalcium-bindingprotein1,cholecystokinin,reelin,oracombinationofthesemolecules.Electronmicroscopicobservationsre-
vealed septal boutons forming axosomatic or axodendritic type II synapses. In the CA1 region of hippocampus, septal GABAergic
projectionsexclusivelytargetedinterneurons.Intheretrosplenialcortex,93%ofidentifiedpostsynaptictargetsbelongedtointerneu-
ronsandtheresttopyramidalcells.TheseresultssuggestthattheGABAergicinnervationfromthemedialseptumanddiagonalband
complexcontributestotemporalcoordinationofneuronalactivityviaseveraltypesofcorticalGABAergicinterneuronsinbothhip-
pocampalandextrahippocampalcortices.Oscillatoryseptalneuronalfiringatdelta,theta,andgammafrequenciesmayphaseinterneu-
ronactivity.
Keywords: entorhinal;hippocampus;inhibition;interneuron;medialseptum;retrosplenial
SignificanceStatement
DiversetypesofGABAergicinterneuronscoordinatethefiringofcorticalprincipalcellsrequiredformemoryprocesses.During
wakefulnessandrapideyemovementsleep,therhythmicfiringofcorticalGABAergicneuronsplaysakeyroleingoverning
networkactivity.WeinvestigatedsubcorticalGABAergicprojectionsinthemousethatextendfromthemedialseptum/diagonal
bandnucleitoGABAergicneuronsinthehippocampusandrelatedextrahippocampalcorticalareas,includingthemedialento-
rhinalcortex.Theseareascontributetonavigationandshowthetarhythmicactivity.WefoundselectiveGABAergictargetingof
differentgroupsofcorticalGABAergicneurons,immunoreactiveforcombinationsofcell-typemarkers.AsseptalGABAergic
neuronsalsofirerhythmically,theirselectiveinnervationofcorticalGABAergicneuronssuggestsanoscillatorysynchronization
ofneuronalactivityacrossfunctionallyrelatedareas.
Introduction behavioralstates,includingnavigationandrapideyemovement
Thetafrequencyoscillations(4–12Hz)intherodenthippocam- sleep.Lesionorinactivationofthemedialseptum(MS)disrupts
pusandrelatedcorticalareasarehighlycorrelatedwithvigilant thetaoscillationsinthehippocampusandmedialentorhinalcor-
tex(mEC;Rawlinsetal.,1979;Mizumorietal.,1990;Jefferyetal.,
1995;McNaughtonetal.,2006),altersthespatialperiodicityof
ReceivedJuly10,2015;revisedSept.5,2015;acceptedOct.9,2015. mEC grid cells (Brandon et al., 2011; Koenig et al., 2011), and
Authorcontributions:G.U.,A.J.,T.J.V.,V.K.,andP.S.designedresearch;G.U.,A.J.,T.J.V.,V.K.,andP.S.performed
research;G.U.,A.J.,T.J.V.,V.K.,andP.S.contributedunpublishedreagents/analytictools;G.U.,A.J.,T.J.V.,V.K.,and
P.S.analyzeddata;G.U.,A.J.,T.J.V.,V.K.,andP.S.wrotethepaper. Watanabe(DepartmentofAnatomy,HokkaidoUniversityGraduateSchoolofMedicine)forthegiftantibodies.We
ThisworkwassupportedbytheMedicalResearchCouncil(MC_UU_12024/4)andtheResearchCouncilsUK alsothankRayGuillery,Tama´sFreund,BalazsHangya,LindaKatona,andViktorVargaforcommentsonanearlier
(OpenAccess).G.U.issupportedbytheEuropeanBlaschkoVisitingResearchFellowshipoftheDepartmentof versionofthemanuscript.
PharmacologyandLinacreCollege.A.J.issupportedbyaFelixDoctoralScholarship.WethankKristinaWagnerand Theauthorsdeclarenocompetingfinancialinterests.
BenMicklemfortheirexcellenttechnicalassistanceandelectronmicroscopicdata.WearegratefultoMasahiko ThisarticleisfreelyavailableonlinethroughtheJNeurosciAuthorOpenChoiceoption.
Unaletal.•SynapticTargetsofMedialSeptalProjections J.Neurosci.,December2,2015•35(48):15812–15826•15813
impairslearningandspatialmemory(Winson,1978;Chrobaket immunonegativeGABAergicsubpopulationsintheMSandDBC
al.,1989;GivensandOlton,1994;McNaughtonetal.,2006).The thatprojecttothehippocampusandmEC.Toidentifythesyn-
MSanddiagonalbandcomplex(DBC)sendcholinergic(Lewis aptictargetsofseptalGABAergicneurons,weperformedantero-
andShute,1967),glutamatergic(Sottyetal.,2003;Colometal., grade tracing. We have used visualization of pathways by the
2005), and GABAergic (Ko¨hler et al., 1984) projections to the Claritymethod(Chungetal.,2013)toobtainalarge-scaleviewof
hippocampusandneocortex(HennyandJones,2008).Selective theprojectionpatterns.Bycombininganterogradetracingwith
inactivation of the noncholinergic (Dwyer et al., 2007) or immunohistochemistry,wetestedforthetargetcelltypesofin-
GABAergic(Pangetal.,2011)septalprojectionsissufficientto dividualcorticallyprojectingseptalGABAergicneurons.Then,
impairspatialmemory. using electron microscopy, we revealed synaptic targets in the
Intherathippocampus(FreundandAntal,1988)andretro- CA1andretrosplenialcortex.
splenial cortex (Freund and Gulya´s, 1991), septal and DBC
GABAergicneuronsexclusivelytargetGABAergicinterneurons. MaterialsandMethods
Theseneuronsareoftenphasecoupledtothehippocampaltheta Allexperimentalproceduresinvolvinganimalswereperformedunder
rhythm(Petscheetal.,1962;Kingetal.,1998;Duqueetal.,2000; approvedprojectandpersonallicensesinaccordancewiththeUKAni-
Borhegyietal.,2004;Hangyaetal.,2009;Hassanietal.,2009). mals(ScientificProcedures)Act,1986.Adult(20–28g)maleC57BL/6J
Theydifferentiallyinnervatediversepopulationsofhippocampal mice (Charles River) and heterozygous PV-Cre mice (B6;129P2-
Pvalbtm1(cre)Arbr/J;JAX#008069;JacksonLaboratory)wereusedforall
interneurons (Gulya´s et al., 1990) that discharge on different
experiments.Animalswerehousedwithlittermatesonanormallight/
phasesofthehippocampalthetarhythmandreleaseGABAonto
darkcycle(lightsonat8:00A.M;lightsoffat8P.M.)at19–21°Cwith
distinctpostsynapticdomainsofthepyramidalcells(Somogyiet (cid:2)55%humidityandhadadlibitumaccesstofoodandwater.
al.,2014).TargetcellselectivityofseptalGABAergicprojections
Allsurgicalprocedureswereperformedunderinhalationanesthesia
inthehippocampusmightalsoapplytotheinterneuronsofthe usingisoflurane.Briefly,wemadesmallsubcutaneousinjectionsofbu-
extrahippocampal cortical areas upstream and downstream of pivacaine (0.125% solution) around the incision sites on the shaved
the hippocampus (Knierim, 2015). Contrary to the finding in scalp.Underasepticconditions,anincisionwasmadeinthescalp,the
rats,arecentretrogradetracingstudyreportedthatinjectionsof skullwasexposed,smallcraniotomiesweremadewithadrilloverthe
a monosynaptically restricted genetically modified rabies virus targetbrainregions,andtheduramaterwascutwithaneedle.Following
into the dorsal CA1 of CaMKIIa-Cre:TVA mice showed that surgery,buprenorphine(Vetergesic,0.08mg/kg)wasadministeredsub-
cutaneouslyasanalgesic.
GABAergic septal neurons provide a significant innervation to
StereotaxiccoordinatesfortheMS,mEC,anddorsalCA1werebased
pyramidalcells(Sunetal.,2014).Incontrast,theseptalGABA
onamousebrainatlas(PaxinosandFranklin,2003).Allcoordinatesare
ergicprojectionwassuggestedtoinnervateselectivelyinterneu-
inmillimeters.Anteroposterior(AP)coordinatesarerelativetobregma;
ronsinthemousemEC(Gonzalez-Sulseretal.,2014).
mediolateral(ML)coordinatesarerelativetothesagittalsuture;dorso-
TargetselectivityofseptalGABAergicprojectionsinthemEC ventral(DV)coordinatesaremeasuredfromthebrainsurface.
may explain how the stellate cells in layer II (LII) have strong Retrogradetracing.Fluorescentlatexmicrospheres(Retrobeads,Lu-
spikephase-lockingtothetafrequencyinputs(Fernandezetal., mafluor)wereinjectedunilaterallyintotheCA1regionofdorsalhip-
2013) while interacting disynaptically only via inhibitory in- pocampus(AP,(cid:3)2.5;ML,(cid:4)1.7;DV(cid:3)1.25;n(cid:5)7)andthedorsocaudal
terneurons(Coueyetal.,2013).SomeofthemECinterneurons mEC(AP,(cid:3)5.02;ML,(cid:4)3;DV(cid:3)1.95;n(cid:5)5)ofC57BL/6Jmice(Apps
targetedbyseptalGABAergicaxonsmayproviderecurrentinhi- andRuigrok,2007).Fouroftheseanimalsreceivedcombinedunilateral
hippocampalandentorhinalinjectionsofRetrobeadsofdifferentcolors
bitionbetweenstellatecells.Assuch,separateindividualseptal
in the same hemisphere (green into the CA1 and red into mEC; 200
GABAergic neurons may independently contribute to hip-
nl/site).Injectionsweremadewithglasspipettes(tipdiameter:10–15
pocampalandentorhinalthetaoscillations,andthiscanexplain (cid:2)m)attachedtothetipof1(cid:2)lsyringes(SGE)atarateof40nl/minusing
how theta phase precession in principal cells of the mEC may
a pressure pump. The pipette was left in place for 10 min after each
occurindependentlyofthehippocampus(Haftingetal.,2008). injectiontominimizedorsaldiffusion.Micewerefixedbyperfusion(see
Manyrhythmiccorticallyprojectingseptalcellsareimmunopo- below)7–16daftertheinjections.Retrogradelylabeledsomatainthe
sitiveforparvalbumin(PV;Borhegyietal.,2004),amarkerfor septalanddiagonalbandnucleiweretestedfortheirimmunoreactivityto
fast-spikingGABAergicseptalneurons(Morrisetal.,1999)that choline acetyltransferase (ChAT), PV, calbindin (CB), and calretinin
coexpress hyperpolarization-activated cyclic nucleotide-gated (CR;seeImmunohistochemistry).Brainsectionsandthepositionsof
channelsinthemouse(Morrisetal.,2004).Thissubpopulation cellswererecordedwithNeurolucidasoftware(MBFBioscience)under
epifluorescenceillumination.
of medial septal GABAergic neurons is also phase coupled to
Anterograde tracing. We injected the anterograde tracer Phaseolus
hippocampal theta in the rat (Simon et al., 2006; Varga et al.,
vulgaris-leucoagglutinin(PHAL;VectorLaboratories;GerfenandSaw-
2008).WehypothesizedthattheseptalGABAergicneurons,in- chenko,1984)intotheMS(AP,(cid:4)0.85;ML,0;DV(cid:3)3.6)offourmice.
cludingthePV-immunopositiverhythmicsubpopulation,exclu-
Thetracerwasdissolvedasa2.5%solutionin0.1Mphosphatebuffer
sively target interneurons in extrahippocampal cortical areas, (PB)andiontophoreticallyinjectedthroughaglasspipette(tipdiameter:
thuscontributingtothetaoscillationsandnetworkcomputations 12–18(cid:2)m)usingpositive5(cid:2)Acurrentpulses,onandoffevery7sfor15
viamechanismssimilartothoseusedinthehippocampus. min.Tominimizetissuedamageanddorsaldiffusion,theelectrodewas
To clarify the nature of septocortical circuits, we first placedintheMS15minbeforethestimulationandretracted5minafter
revealed the distribution of PV-immunopositive and PV- theendofstimulation.Followingrecovery,micewerekeptfor1week
untilperfusionfixation.
WehaveperformedanterogradeCre-dependentadeno-associatedvi-
CorrespondenceshouldbeaddressedtoeitherGunesUnalorPeterSomogyi,MedicalResearchCouncilBrain rus(AAV)injectionsintotheMS(AP,(cid:4)0.85;ML,0;DV,(cid:3)3.6)ofseven
Network Dynamics Unit, University of Oxford, Mansfield Road, Oxford OX1 3TH, UK, E-mail: PV-Cremice.Wepressure-injected400nlofpAAV -EF1a-DIO-EYFP
2
[email protected]@pharm.ox.ac.uk. (VectorBiolabs)in10minusing1(cid:2)lsyringesattachedtoapressure
DOI:10.1523/JNEUROSCI.2639-15.2015 pump.Micewerefixedbyperfusion3weeksafterinjections.
Copyright©2015Unaletal.
Tissueprocessing.AlltheRetrobead-injectedandPHAL-injectedmice
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense
CreativeCommonsAttribution4.0International,whichpermitsunrestricteduse,distributionandreproductioninany andfiveAAV-injectedPV-Cremiceweredeeplyanesthetizedwithso-
mediumprovidedthattheoriginalworkisproperlyattributed. diumpentobarbital(50mg/kg,i.p.)andtranscardiallyperfusedatthe
15814•J.Neurosci.,December2,2015•35(48):15812–15826 Unaletal.•SynapticTargetsofMedialSeptalProjections
endoftheirsurvivaltimeswith0.1MPBfollowedbyafixativecontaining chosentominimizespectraloverlap(seebelow).Clarity-processedsec-
0.1MPB,4%depolymerizedparaformaldehyde,and15%v/vsaturated tionswerewashedovernightinPBS-Txandtransferredtothesecondary
picricacid,pH7.4.Theperfusionsolutionwassupplementedwith0.05% antibodysolutionfor2datRT.
glutaraldehydeforPHAL-injectedmiceandwith0.3%glutaraldehyde Thefollowingsecondaryantibodieswereused:donkeyanti-rabbitAl-
fortwoAAV-injectedPV-Cremice(animalsGU41andGU42).Follow- exa Fluor 405 (1:250 dilution; catalog #A31556; Invitrogen); donkey
ingovernightpostfixationinaglutaraldehyde-freefixative,brainswere anti-mouse,anti-goat,oranti-guineapigDyLight405(1:250;715-475-
washedthoroughlyin0.1MPBandsectionedcoronallyorhorizontallyat 151,705-475-147,706-475-148,respectively;JacksonImmunoResearch
60–80(cid:2)mthicknessusingaLeicaVT1000Svibratome(LeicaMicrosys- Laboratories);donkeyanti-chickenoranti-guineapigAlexaFluor488
tems).Sectionswerestoredin0.1MPBwith0.05%sodiumazideat4°C. (1:1000,1:400;703-545-155,706-485-148,respectively;JacksonImmu-
TwoAAV-injectedmice(animalsGU31andGU32)wereprocessed noResearchLaboratories);donkeyanti-rabbitoranti-goatAlexaFluor
withtheClaritymethod(Chungetal.,2013)foruninterruptedobserva- 488 (1:400; A21206, A11055, respectively; Invitrogen); donkey anti-
tion of trajectories of labeled axons in thick sections using epifluo- rabbit,anti-goat,anti-sheep,anti-mouse,oranti-guineapigCy3(1:400;
rescence microscopy. The original Clarity protocol was applied with 711-165-152,705-165-147,713-165-147,715-165-151,706-165-148,re-
acrylamide-containinghydrogelsolutiontobothbrains,whichwerefirst spectively;JacksonImmunoResearchLaboratories);donkeyanti-rabbit,
processedbyelectrophoretictissueclearing(ETC)for48handthencut anti-goat,anti-sheep,anti-mouse,oranti-guineapigCy5(1:250;711-
into500-(cid:2)m-thicksectionsforpassiveclearingofthelipids.Briefly,mice 175-152,705-175-147,713-175-147,715-175-151,706-175-148,respec-
wereperfusedtranscardiallywithPBS(20ml)followedbythehydrogel tively; Jackson ImmunoResearch Laboratories); donkey anti-rabbit,
solution(20ml).After2nightsofpostfixationina50mltubefilledwith anti-goat,oranti-mouseDyLight649(1:250;711-495-152,705-495-147,
20mlofthesamesolutionat4°C,theairinthetubewasreplacedwith 715-495-151, respectively; Jackson ImmunoResearch Laboratories),
nitrogenforpolymerizationat37°C(3–6h).Thepolymerizedhydrogel donkeyanti-sheeporanti-guineapigAlexaFluor647(1:250;713-605-
around the brain was manually removed before washing the tissue 147,706-605-148,respectively;JacksonImmunoResearchLaboratories).
withtheclearingsolution(4%SDSand200mMboricacidindistilled For electron microscopy, we used the following secondary antibodies
H O,pH8.5)overnightatroomtemperature(RT).Thebrainswere fromVectorLabs:biotinylateddonkeyanti-goatIgG(1:100inTBScon-
2
washedagainwiththeclearingsolutionfor48hat37°Candtrans- taining1%NHS),biotinylatedgoatanti-chickenIgG(1:100inTBScon-
ferredtotheETCchamberforactiveremovalofthelipidsinthesame taining1%NGS),andbiotinylatedgoatanti-rabbitIgG(1:100inTBS
solutionfor48h(30(cid:6)4V;37°C).Thebrainswerethenwashedwith containing1%NGS).
PBScontaining0.3%TritonX-100(PBS-Tx)overnightandcutinto Followingsecondaryantibodyincubation,60–80-(cid:2)m-thickimmu-
500-(cid:2)m-thicksections,whichweretransferredtoa37°Cincubator nofluorescencesectionswerewashedthreetimesfor10mineachand
for7–10d.Finally,theClarity-processedthicksectionswerewashed mountedonglassslidesinmethylsalicylate(Retrobeadsectionsonly)or
withPBS-Txovernightandeithermountedin500-(cid:2)m-thickimaging VectaShield.Clarity-processedsectionswerewashedfor2datRTbefore
spacerswithFocusClear(CelExplorerLabs)orstoredinPBS-Txat mounting.
4°Cforsubsequentimmunohistochemistry. Confocal microscopy. Sections were first observed with a wide-field
Immunohistochemistry. Tissue penetration of 60–80-(cid:2)m-thick sec- epifluorescence microscope (Leitz DMRB; Leica Microsystems)
tionsbyantibodieswasachievedbyeitherusingTBScontaining0.3% equippedwithPLFluotarobjectives.Multichannelfluorescenceimages
TritonX-100(TBS-Tx)inallbasesolutionsandwashesorbytreating wereacquiredwithZEN2008softwarev5.0onaZeissLSM710laser
sectionswithtworoundsof“freeze–thaw”(FT),forwhichTBS-Txwas scanningconfocalmicroscope(ZeissMicroscopy),equippedwithDIC
replaced with TBS in all subsequent solutions. For FT, sections were M27 Plan-Apochromat 40(cid:7)/1.3 numerical aperture, DIC M27 Plan-
cryoprotectedin20%sucrosesolutionin0.1MPBfor(cid:3)3hbeforerapidly Apochromat 63(cid:7)/1.4 numerical aperture, and (cid:5) Plan-Apochromat
freezing sections over liquid nitrogen then rapidly thawing in buffer. 100(cid:7)/1.46numericalapertureoil-immersionobjectives.Thefollowing
AfterFT,sectionswerewashed(cid:3)3timesfor10minin0.1MPB.Clarity- channel specifications were used (laser/excitation wavelength, beam
processedsectionswerealwaysincubatedinPBS-Txanddidnotrequire splitter, emission spectral filter) for detection of Alexa405, Alexa488/
tissuepermeabilizationorblocking.The60–80-(cid:2)m-thicksectionswere EYFP,Cy3,andCy5:405-30solid-state405nmwithattenuationfilter
firstblockedfor1hatRTin20%normalhorseserum(NHS)ornormal ND04,MBS-405,409–499nm;argon488nm,MBS-488,493–542nm;
goatserum(NGS),dependingonthespeciesusedtoraisethesecondary HeNe543nm,MBS-458/543,552–639nm;HeNe633nm,MBS-488/
antibody.Afterblocking,mostsectionswereincubatedinprimaryanti- 543/633,637–757nm.Thepinholesizewassetto1Airyunitforthe
body solution containing 1% NHS/NGS for 2–4 d at 4°C; Clarity- shortestwavelengthwhile,tokeepallchannelsatthesameopticalslice
processedsectionswereincubatedfor5datRT.Negativecontrolsforthe thickness,thepinholesizesofthelongerwavelengthchannelsweread-
specificityofthemethodwereprocessedinparallelandlackedthepri- justed to values (cid:8)1 Airy unit. Thus, optical section thickness for all
mary antibodies. Primary antibodies (Table 1) were detected with channelswasbasedonthesetpinholesizefortheshortestwavelength
fluorophore-conjugatedsecondaryantibodiesforwide-fieldepifluores- channel.
cence and confocal microscopy, and with the immunoperoxidase Electronmicroscopy.ObservationsoftheCA1weremadeinmicethat
methodforelectronmicroscopy(seebelow).Molecule,species,dilution, receivedinjectionsofPHAL(animalsAJ10andTV39)orAAV(animals
andmethodfortheprimaryantibodiesareasfollows:calbindin,rabbit, AJ13andGU42).Synapticjunctionsbetweenlabeledboutonsandtheir
1:5000 for immunofluorescence, 1:500 for Clarity; calretinin, rabbit, postsynaptictargetswereidentifiedinstratalacunosum-moleculare,ra-
1:1000forimmunofluorescence,1:100forClarity;N-terminalEF-hand diatum,pyramidale,andoriens.Targetneuronsofcorticallyprojecting
calcium-bindingprotein1(NECAB1),mouse,1:500forimmunofluo- GABAergicMScellsweredocumentedinthegranularretrosplenialcor-
rescence;neuronalnitricoxidesynthase(nNOS),mouse,1:500forim- texoftwoPHAL-injectedanimals(AJ04andTV39),andboutonswere
munofluorescence;PV,goat,1:1000forimmunofluorescence,1:100for identifiedincorticallayersII–VI.
Clarity;PV,guineapig,1:5000forimmunofluorescence,1:500forClar- Following FT and blocking (as above), sections were incubated in
ity; PV, rabbit, 1:1000 for immunofluorescence; special AT-rich primary antibody solution for 2 nights at 4°C, followed by overnight
sequence-bindingprotein-1(SATB1),goat,1:200forimmunofluores- incubationinsecondaryantibodysolutionand2dwitha1:100dilution
cence;SATB1,rabbit,1:1000forimmunofluorescence;vesicularGABA of avidin/biotin complex (Vector Labs) in TBS at 4°C. Sections were
transporter(VGAT),guineapig,1:500forimmunofluorescence;VGAT, reactedwithasolutionof0.05%diaminobenzidineand0.002%hydro-
rabbit,1:500forimmunofluorescence(Vineyetal.,2013,theirsupple- genperoxideinTrisbufferfor(cid:2)10mininthedark.Afterwashingin
mentaryTable2).Forimmunofluorescencelabeling,60–80-(cid:2)m-thick 0.1MPBseveraltimestostopthereaction,sectionsweretreatedwith1%
sectionsweresimultaneouslyprocessedwith(cid:4)4primaryantibodies.Af- osmiumtetroxidesolutionin0.1MPBfor1h.Afterwashingseveral
terprimaryantibodyincubation,sectionswerewashedthreetimesfor10 timesinPB,sectionsweredehydratedinagradedseriesofalcohol(70,
min and transferred to a secondary antibody solution containing 1% 90,95,and100%)followedbypropyleneoxide.Uranylacetate(1%)was
NHS/NGSfor4hatRTorovernightat4°C.Antibodyfluorophoreswere addedtothe70%alcoholfor35minforcontrastenhancement.Dehy-
Unaletal.•SynapticTargetsofMedialSeptalProjections J.Neurosci.,December2,2015•35(48):15812–15826•15815
Table1.Primaryantibodiesa
Dilutionand
Molecule Hostandantibody procedure Source Antigen Specificityinformation
CB Goat,polyclonal 1:1000,IF FrontierInstitute;code: Recombinantmouse Immunoblotdetectsasingleprotein
Calbindin-Go-Af104 calbindin bandat28kDa(manufacturer’s
technicalinformation)
CCK Guineapig 1:1000,IF Dr.M.Watanabe,Hokkaido Cysteine-taggedCCK-8 Labelingpatternaspublishedwith
University,Japan (CDYMGWMDF) otherantibodies
coupledto
keyholelimpet
hemocyanin
Rabbit 1:500,IF Dr.M.Watanabe,Hokkaido Cysteine-taggedC-ter- Labelingpatternaspublishedwith
University,Japan minal9aaofpro- otherantibodies
CCK(CSAEDYEYPS)
coupledtokeyhole
limpethemocyanin
ChAT Goat 1:500,IF; Chemicon(MerckMillipore); HumanplacentalChAT Westernblot(manufacturer’stech-
1:50,CL code:AB144P nicalinformation)
CR Goat 1:1000,IF Swant;code:CG1 Recombinanthuman AntibodydoesnotlabelthebrainofCR
calretinin knock-outmice(manufacturer’s
technicalinformation)
GFP Chicken,polyclonal 1:500,IF,EM; AvesLabs;code:GFP-1020 RecombinantGFP Antibodydoesnotlabelthebrainof
1:200,CL wild-typecontrolmice
Insulin-likegrowthfactor Goat,polyclonal 1:100,IF R&DSystems;code:AF578 Recombinantmouse Westernblot(manufacturer’stech-
bindingprotein5 myelomacellline nicalinformation)
NS0-derivedinsu-
lin-likegrowth
factorbinding
protein5
PHAL Goat,polyclonal 1(cid:2)g/ml,IF, Vector:code:AS-2224 Purifiedlectin BothreactwithPhaseolus
EM vulgariserythroagglutininand
Rabbit,polyclonal 1(cid:2)g/ml,IF, Vector,code:AS-2300 Purifiedlectin leucoagglutinin
EM
Reelin Mouse 1:1000,IF Chemicon(MerckMillipore); Recombinantreelin deBergeycketal.,1998
code:MAB5364 aminoacids
164–496
Somatostatin Mouse 1:400,IF GeneTex;code:GTX71935 Humansomatostatin Labelingpatternaspublishedwith
conjugatedto otherantibodies
proteincarrier
Vesicularacetylcholine Goat,polyclonal 1:400,IF Chemicon(MerckMillipore); Keyholelimpethemo- Manufacturer’stechnical
transporter code:ABN100 cyanin-conjugated information
linearpeptide
correspondingto
theCterminusof
ratvesicularacetyl-
cholinetransporter
Vesicularglutamate Guineapig, 1:250,IF SynapticSystems;code: Recombinantrat Westernblot(manufacturer’stech-
transporter2 polyclonal 135404 vesicularglutamate nicalinformation)
transporter2amino
acids510–582
WFS1 Rabbit,polyclonal 1:500,IF Proteintech;code: Recombinanthuman Takedaetal.,2001
11558-1-AP WFS1(wolframin)
aminoacids1–134
Abbreviations:IF,Immunofluorescencefortriton-treated60–80-(cid:2)m-thicksections;EM,electronmicroscopy(freeze–thaw-treated60–80-(cid:2)m-thicksections);CL,Clarity-processed500-(cid:2)m-thicksections.
aInformationonsources,dilutions(foreachprocedureused),andspecificityofprimaryantibodiesthathavenotbeenlistedinVineyetal.,2013,theirsupplementaryTable2,wherereferencesarecited.
dratedsectionswereembeddedinDurcupanresin(Fluka)andpolym- WeidentifiedputativeglutamatergicandGABAergicsynapticjunc-
erizedat60°Cfor2d.Targetareaswerecutoutfromtheresin-embedded tionsrespectivelyastypeI(oftencalledasymmetrical)andtypeII(often
60–80-(cid:2)m-thicksectionsandre-embeddedforultramicrotomesection- calledsymmetrical)basedontheirfinestructure:typeIsynapsespossess
ing.Serial60-nm-thicksectionswerecutandmountedonsingle-slot, athickpostsynapticdensity,whereastypeIIsynapsesarecharacterized
pioloform-coatedcoppergrids.SectionswereobservedwithaPhilips byathinpostsynapticdensity(Gray,1959).Electronmicroscopiciden-
CM100 transmission electron microscope and electron micrographs tificationofdendriticprofilesinthehippocampuswasmadeaccording
wereacquiredwithaGatanUltraScan1000CCDcamera.AllPHAL- toestablishedcriteria(Gulya´setal.,1999;Megíasetal.,2001)byfollow-
containingorenhancedyellowfluorescentprotein(EYFP)-containing ingpostsynapticdendritesin(cid:4)30consecutivesections.Briefly,pyrami-
boutonscutatthesectionplanewerefollowedinserialsectionstolocate dal cells of the CA1 region have spiny dendrites and receive type I
synapticjunctions. synapsesontotheirdendriticspines,whereastheirdendriticshaftsare
15816•J.Neurosci.,December2,2015•35(48):15812–15826 Unaletal.•SynapticTargetsofMedialSeptalProjections
Figure1. Distributionofretrogradelylabeledseptalanddiagonalbandneuronsprojectingtothehippocampusand/orthemEC.A,GreenRetrobeadinjectionsite(arrow)inthedorsalCA1region
(mouseGU06,coronalsection).B,RedRetrobeadinjectionsite(arrow)inthedorsocaudalmEC(mouseGU07,coronalsection).C–E,Confocalmicroscopicimagesofretrogradelytransportedgreen
(C)orred(D)RetrobeadsinsomataandtheircolocalizationinanhDBneuron(E,arrow).F,AsingleconfocalmicroscopicopticalsectionofCA1-projectinggreenRetrobead-labeledneurons(arrows)
inthemedialseptumwithimmunohistochemistryforPV(magenta).APV-immunopositiveretrogradelylabeledneuron(framedarea)isshownintheinset(scalebar,10(cid:2)m).G,H,Singleconfocal
microscopicimagesdepictingcolocalizationofCB(cyan)andChAT(orange)insome(yellow,arrows)butnotallneuronsintheposteriorMS–vDBborder.I–M,Tworetrogradelylabeled
mEC-projectingMS/vDBneurons(arrows)arebothimmunoreactiveforChAT(K)butonlyoneofthemisimmunoreactiveforCB(L).N–P,Neurolucidadrawingsoftheseptalanddiagonalbandarea
atthreerostrocaudallevelsmarkedwithpositionsofretrogradelylabeledCA1-projecting(green)andmEC-projecting(red)neurons.ArrowheadinOpointstoa(Figurelegendcontinues.)
Unaletal.•SynapticTargetsofMedialSeptalProjections J.Neurosci.,December2,2015•35(48):15812–15826•15817
innervatedalmostexclusivelybytypeIIsynapses.Incontrast,theover- Table2.NumberandproportionofCA1-projectingormEC-projectingMS/vDBand
whelmingmajorityofhippocampalinterneuronshaveaspinyorsparsely hDBneuronstestedforChATandPVimmunoreactivitya
spinydendrites,whichreceivebothtypeIandtypeIIsynapses(Taka´cset ChAT-negativecells
al.,2012).FreundandGulya´s(1991)reportedthatthesedifferencesin AllcellstestedforChAT testedforPV
thedendriticsynapticinputsarepreservedintheretrosplenialcortexof Neurons Location N ChAT(cid:4) (%) N PV(cid:4) (%)
therat,wherepyramidalcellshavespinydendritesandreceivethebulk
oftheirexcitatoryinputthroughaxospinoussynapses,whereasthema- CA1-projecting MS/vDB 161 57 (35) 83* 8 (10)
jorityofinterneuronshavevaricoseandaspinydendrites.Furthermore, hDB 156 97 (62) 59 3 (5)
pyramidalcelldendritesintheratretrosplenialcortexarecharacterized Subtotal 317 154 (49) 142 11 (8)
bymanyparallelandevenlydistributedmicrotubules,relativelysmall mEC-projecting MS/vDB 78 46 (59) 32 3 (9)
mitochondria,andcytosolicproteinaggregates.Incontrast,interneuron hDB 68 59 (87) 9 1 (11)
dendritesoftencontainmoreandlargermitochondria,andfreeribo- Subtotal 146 105 (72) 41 4 (10)
Total 463 259 (56) 183 15 (8)
somes(FreundandGulya´s,1991).
AdobePhotoshopCS3(AdobeSystems)wasusedtocroptheoriginal aNumberandproportionofallretrogradelylabeledCA1-projectingormEC-projectingneuronsinMS/vDBandhDB
imagesandtoapplybrightnessandcontrastadjustmentstothewhole thatweretested(N)forChATimmunoreactivity;theChAT-immunonegativecellsweretestedforPV.
frame.AdobeIllustratorCS3wasusedtoaddlabelsandscalebars.Sta- *Notethatonly83of104ChAT-immunonegativecellsweretestedforPV.
tisticalanalyseswereperformedinMatlab(MathWorks).Meanvalues
arereportedasmean(cid:6)SD.Medianvaluesarereportedwithinterquar-
tilerange(IQR).
We tested 463 retrogradely labeled somata for ChAT
Results immunoreactivity(Fig.1)intheMSanddiagonalbandnuclei
Immunohistochemicalidentificationofseptalanddiagonal (Table2).TheChAT-negative,putativeGABAergic,andgluta-
bandneuronsprojectingtothedorsalCA1and/ormEC matergicprojectioncells(n(cid:5)183)weretestedforthecalcium-
AllRetrobeadinjectionsresultedinrestrictedlabelinginthetar- bindingproteinPVimmunoreactivity(Table2;Fig.1F).Overall,
getregions,theCA1(Fig.1A)andthemEC(Fig.1B),withmin- 8% of the ChAT-immunonegative projection neurons showed
imal diffusion to neighboring areas. Both the dorsal CA1 and PVimmunoreactivity.
dorsocaudalmECinjectionsrevealedretrogradelylabeledcellsin Wehavealsotested248ChAT-immunopositivecellsforCB
the MS, and the vertical and horizontal diagonal band (vDB, immunoreactivityintheMS,vDB,andhDBofthreemice,and
hDB) nuclei (n (cid:5) 863 cells from 8 mice). Injections into the foundthat15(cid:6)6%ofChAT-immunopositivecellsexpressde-
dorsalCA1labeled330neurons(n(cid:5)7mice)intheMSandvDB, tectable,butoftenlowlevelsofCB(Fig.1G,H).Wethentested
and273neurons(n(cid:5)5mice)inthehDBnucleiofbothhemi- immunoreactivity for CB in the retrogradely labeled ChAT-
spheres.FourmicereceivedinjectionsofdifferentlycoloredRet- immunoreactive neurons and found that 9 of 138 (7%) of the
robeadsinthetwoareas.Thedistributionoflabeledcellsshows CA1-projecting neurons and 3 of 66 (5%) of the mEC-projec-
that the hippocampal injection sites received a substantial, but tingneuronsintheDBCwerealsoimmunopositiveforCB(Fig.
significantlysmaller(Wilcoxonsigned-ranktest,p(cid:8)0.05)pro- 1I–M).WedidnotobserveanyretrogradelylabeledCBimmu-
portionoftheirhDBinputfromthecontralateralside(median(cid:5) nopositivecellsthatwereimmunonegativeforChAT.Finally,no
7cells,IQR(cid:5)16)comparedtotheipsilateralside(median(cid:5)21 retrogradelylabeledcell(n(cid:5)58testedcells)wasimmunoposi-
cells,IQR(cid:5)28;n(cid:5)273cellsfrom7mice).InjectionsofRet- tiveforCRinanyseptalorDBCregion(fortheratbasalfore-
robeads into the mEC resulted in fewer labeled neurons than brain, see Zaborszky et al., 1999). Many of the CR-positive
thosefoundtoprojecttoCA1(160cellsintheMSandvDB,and GABAergicneuronsprojecttothesupramammilarynucleusin
109cellsinthehDB,n(cid:5)5animals).SimilartotheCA1,unilat- therat(Leranthetal.,1999).
eralinjectionsintothemECresultedinbilateralretrogradelabel-
inginhDBnuclei(AlonsoandKo¨hler,1984). SeptalGABAergicinnervationofextrahippocampal
Themajorityoftheretrogradelylabeledseptalcellswereme- corticalareas
diallylocatedinoraroundthePV-denseregionoftheMS(Fig. Iontophoretic injections of PHAL into the MS of C57BL6j
1N–P).Negligiblenumbersofretrogradelylabeledcellswereob- miceresultedinneuronallabelingalongtherostrocaudalsep-
servedinorbeyondtheCR-richlayerofthelateralseptum(Wei talaxis.TheMLandDVdiffusionofthetracerwasminimal;
etal.,2012)andallofthemwereChATandPVimmunonegative labelingwasmostlyrestrictedtotheMSanddorsal-mostpart
(Fig.1O).Only9of453cellsprojectedtoboththeCA1andthe ofthevDBwithfewlabeledneuronsobservedintheCR-rich
mECasrevealedwhengreenandredRetrobeadswerecolocalized layer around the MS. The densest somatic labeling was ob-
inthesamesomata(Fig.1C–E;n(cid:5)4mice).Allofthesedouble- servedatthecenteroftheinjectionsite,correspondingtothe
labeled neurons were located in the diagonal band nuclei and middle of the rostrocaudal extent of the MS in each animal
wereimmunopositiveforChAT.Thisdoesnotruleouttheexis- (Fig.2A).
tenceofmultiplearea-targetingnoncholinergicseptalneuronsas TotracetheaxonsofthesubpopulationofGABAergicsep-
thetwoinjectionsitesmaynothavebeenattopographicallyre- tal neurons that express PV, we injected pAAV -EF1a-DIO-
2
latedpositions. EYFP virus into the septum of B6;129P2-Pvalbtm1(cre)Arbr/J
mice, resulting in Cre-dependent expression of EYFP. These
injectionsproducedrestrictedsomaticlabelingatthedelivery
4 sites with numerous EYFP-labeled axons projecting dorsally
toward the fornix and fimbria (Fig. 2B). They produced few
(Figurelegendcontinued.) retrogradelylabeledcellinthelateralseptum.Thecentralout-
labeledneuronsoutsidethearearichinPV-positivecellsofthe
linedregionindicatestheareacontainingallthePV-immunoreactivesomataandlabeledpro-
MS (Fig. 2C). The expression of EYFP was restricted to PV-
cesses.Q–S,ProportionsandnumbersofretrogradelylabeledneuronscountedinMS/vDBor
hDBforallCA1injections(Q,green)andmECinjections(R,red),whichincludethedoubleCA1 positivecells,asconfirmedbyimmunohistochemistryforPV
andmECinjections(S).DG,Dentategyrus;V1,primaryvisualcortex;ac,anteriorcommissure;cc, (Fig. 2D–F). Accordingly, all the EYFP-immunolabeled so-
corpuscallosum;v,lateralventricle;B,bregma. mata (Fig. 2D, arrows) were immunoreactive for PV (Fig.
15818•J.Neurosci.,December2,2015•35(48):15812–15826 Unaletal.•SynapticTargetsofMedialSeptalProjections
Figure2. AnterogradetracerinjectionsitesandimmunohistochemicaltestingofEYFPlabelingresultingfromAAVinjectionsinthemedialseptumofPV-Cremice.A,PHALinjectionintheMS
(mouseTV39,wildtype),visualizedbywide-fieldimmunofluorescenceofPHAL(green).B,Three-dimensionalClarity-processed500-(cid:2)m-thicksectionimageofaPV-Cremouse(GU31)showingthe
dorsalprojectionoftheEYFP-immunolabeledaxonstowardthefimbria/fornixafteranAAVinjectionintotheMS.C,EpifluorescenceimageofanAAVinjectionintotheMSofaPV-Cremouse(AJ12).
D–F,SingleopticalsliceimagesoftheinjectionsiteshowninCwithimmunohistochemistryforEYFP(D,green)andPV(E,magenta)showingthatallEYFP-immunolabeledneuronalcellbodies
(arrows)areimmunoreactiveforPV(F).NotethevariablelevelsofPVimmunoreactivityandlackofvirallabelinginsomePV-immunopositiveneurons(F,asterisks).ac,Anteriorcommissure;cc,
corpuscallosum;v,lateralventricle;LS,lateralseptum.
2E,F), but not all PV-immunopositive neurons expressed labeled and EYFP-labeled axons. Strata radiatum, pyramidale,
EYFP(Fig.2F). andorienswereheavilyinnervated.Fewlabeledseptalaxonswere
Cortical target regions were delineated by immunohisto- observed in the stratum lacunosum-moleculare. Similarly, the
chemistry for molecular markers. The entorhinal cortex and molecular layer of the dentate gyrus was innervated less than
parasubiculumweremarkedrespectivelybystrongandweakim- thegranulecellandthepolymorphiclayers(Fig.3C).Consistent
munoreactivityforNECAB1.Thismarkerwasusedtovisualize anterograde axonal labeling was obtained in the cortical areas
thebordersoftheentorhinalcortexaswellastheborderbetween describedbelowbybothPHAL-labeledprojections,presumably
thepresubiculumandparasubiculum(Fig.3A).Reelinimmuno- includingthecholinergic,GABAergic,andglutamatergicaxons,
labelingclearlydifferentiatedtheentorhinalcortexbydensela- andtheEYFP-labeledaxonsfromPV-immunopositiveGABAe-
beling of stellate cells and interneurons in LII (Fig. 3B). rgiccells.Asdiscussedbelow,themajordifferencebetweenthe
Immunohistochemistryfortheinsulin-likegrowthfactorbind- twomethodsconcernedthetypesofseptalaxonslabeled.
ingprotein5delineatedthepresubiculumbylabelingthalamic ThemainaxonsofthePHAL-labeledneuronsshowedmorevari-
axons(Stenversetal.,1994).Confirmingearlierobservationsof ability in appearance than those originating only from PV-
insituhybridizationforWolframsyndrome1homolog(WFS1; expressing neurons. They could be divided into two groups:
Ding,2013),immunohistochemistryfortheproteinWFS1(wol- relatively thick and smooth main axons with large boutons, and
framin)revealedtheparasubiculumandLIIofthemEC;inboth substantiallythinnermainaxonswithsmallerboutons.Wesampled
areasitwasfoundinLIIpyramidalcells(Takedaetal.,2001). 30thick/smoothaxonsbranchingandformingatotalof128bou-
Thehippocampusreceivedmuchdensermedialseptalinput tonsandalloftheseboutonstestedbyconfocalmicroscopywere
comparedwithothercorticalareasasobservedbybothPHAL- immunopositiveforVGAT(n(cid:5)3animals;Fig.4A,B).Thus,the
Unaletal.•SynapticTargetsofMedialSeptalProjections J.Neurosci.,December2,2015•35(48):15812–15826•15819
Figure3. IdentificationofthetargetregionsofEYFP-labeledseptalaxonsbyimmunohistochemistryinthehippocampusandextrahippocampalareas.A,B,Epifluorescentimagesshowing
demarcation(dashedlines)oftheparasubiculum(PaS)andentorhinalcortex(EC)byimmunohistochemistryforNECAB1(blue),andtheECbyreelin(red)inhorizontalbrainsectionsofaPV-Cre
mousesuperimposedwithEYFP-immunofluorescentseptalaxons(green).TheECandPaSarerespectivelydifferentiatedbydenseandlighterNECAB1immunoreactivity(A;caseGU41s47).
ImmunolabelingforreelinvisualizesmECLII(largedashedlines)bylabelingthestellatecellsandinterneurons(B;caseGU41s20).Thewhitematter(wm)islabelednonspecifically.Bshows,ina
confocalmicroscopicimage(maximumintensityprojection,5.14mz-stack),anterogradelylabeledseptalaxons(green)nearreelin-positiveinterneurons(red,fromtheframedareainB).C,
DifferentialinnervationoflayersofthehippocampusbyEYFP-labeledmedialseptalaxons;maximumintensityconfocalmicroscopicprojectionofa350-(cid:2)m-thickz-stackofaClarity-processedthick
section.SolidlinemarksthehippocampalfissurebetweentheCA1regionanddentategyrus(caseGU31hpc).D,AnterogradelylabeledseptalPV-expressingGABAergicaxonsatthreerostrocaudal
levelsintheretrosplenialcortex(animalGU42).Dashedlinesindicateestimatedbordersbetweentheagranular(RSA)andgranular(RSG)retrosplenialcortices.Scalebars,200(cid:2)m.Medianfilterwas
applied(x,y:radius,1pixel)inC.DG,dentategyrus;PrS,presubiculum;Sub,subiculum;LI,layerI;so,stratumoriens;sp,stratumpyramidale;sr,stratumradiatum;slm,stratumlacunosum-
moleculare;ml,molecularlayerofthedentategyrus;g,granulecelllayer;pl,polymorphiclayerofthedentategyrus;cc,corpuscallosum;R,rostral;C,caudal;M,medial;L,lateral;B,bregma.
relatively thick and smooth type of main axons originated from boutonsinthehippocampusaswellasintheextrahippocampal
GABAergicseptalneurons.Incontrast,thePHAL-labeledmainax- corticalareaswassimilarinthetwomouselinesshowingsimilar
onswithsmallerdiameterandboutonswereVGATimmunonega- labeling efficacy. Accordingly, the following conclusions are
tive.WehaverestrictedouranalysisinthePHAL-labeledmaterialto based on observations of the pooled data. The high density of
theaxonspossessingVGAT-immunoreactiveterminalsandthose innervationofthehippocampusproperbyGABAergicaxonswas
that displayed similar boutons. These axons contributed both en alsopresentinthesubiculumatalllevelsalongtheseptotemporal
passant(Fig.4B)andterminalvaricositiesinallthetargetregions axis.TheparasubiculumreceivedthedensestseptalGABAergic
examined.Onlyaminorityofboutonswasinappositiontoneu- projectionamongotherextrahippocampalstructures(Fig.3A),
ronalsomata;therestpresumablyinnervateddendrites.Target similar to the dense projections to mEC LII and layer IV in
somata were often encapsulated by numerous boutons of the the rat (Alonso and Ko¨hler, 1984). A lower density of PHAL-
samePHAL-labeledaxon(FreundandBuzsa´ki,1996),forminga immunolabeledandEYFP-immunolabeledaxonswasobserved
basket-likemesh(Fig.4C).Thesamemannerofinnervationwas in the presubiculum, mEC, and granular retrosplenial cortex.
observedalsofortheEYFP-immunolabeledaxons(Fig.5A). Togetherwiththehippocampusandparasubiculum,thesecorti-
The relative density of PHAL-immunolabeled or EYFP- calregionsreceivedthemajorityofseptocorticalGABAergicin-
immunolabeled axons with VGAT-immunopositive terminal put(Fig.3A,B).
15820•J.Neurosci.,December2,2015•35(48):15812–15826 Unaletal.•SynapticTargetsofMedialSeptalProjections
Figure4. InnervationofdifferentinterneuronsbyPHAL-immunopositiveseptalaxons(green)intheentorhinalcortex.A,ConfocalimageofPV-immunopositivesomata(magenta)inlayersII/III
ofthemEC.Thesomainthelowerrightcornerisinnervated(caseAJ04b;singleopticalsection,0.4(cid:2)mthick).Theboutons(arrows)innervatingthesomaareVGATpositive(A(cid:2),red).B,A
CR-immunopositivesoma(white)inappositiontoenpassantPHAL-labeledVGAT-positive(B(cid:2),red)septalboutons(arrows;caseAJ10g)inlayersII/IIIofthelateralentorhinalcortex;singleoptical
sectionof0.43(cid:2)m.C,OneofthetwoCB-immunopositivesomata(blue)isinnervatedbyPHAL-labeledseptalboutonsinlayerIVofthemEC(caseAJ10c;singleopticalsection,0.49(cid:2)mthick).This
somaissurroundedbyboutonsofthesameaxonformingadensebasket-likemesh(C(cid:2);confocalimagestack,9.27(cid:2)m).
Anunevendistributionofanterogradelylabeledmedialseptal splenial cortex, interneurons immunopositive for PV, CB, CR,
GABAergicaxonsintheentorhinalcortexandretrosplenialcor- NECAB1,CCK,orreelinwerefoundinclearappositiontoseptal
texprobablywasduetoatopographicalorganization.Originat- GABAergicboutonsformingputativeaxosomatic(Fig.5A,B)or
ingfromourinjectionsites,moreseptalaxonswerefoundinthe axodendritic (Fig. 5C–F) synapses. In the retrosplenial cortex,
medialandmostcaudalportionsoftheentorhinalcortex,with onlyPV,CB,CR,andreelinwerefoundinputativeinterneuron
thedorsocaudalmECineachanimalreceivingthemostlabeled target somata. Combinations of molecular markers were ob-
axonsandboutons.Wehaveobservedrelativelyfewlabeledax- served in 28 target neurons. For instance, 47% of the PV-
onsinthelateralentorhinalcortexandevenfewerintheperirhi- immunopositive neurons (Fig. 5D) were also immunoreactive
nal cortex. In the retrosplenial cortex, the densest labeling was forCB(Fig.5E;n(cid:5)99)inthedepictedareaofparasubiculum
foundinthecaudalandmost-medialportions,correspondingto (Fig.5D,E).
thegranularregion.Themorelateralanddorsalpartofthecor-
ThepossibilitythatseptalGABAergicaxonsselectivelytarget
tex, known as the five-layered, agranular region, received sub-
certaininterneurontypeswasassessedbyfollowingsinglelabeled
stantiallylessseptalGABAergicinput.Thedensityoflabelingalso axons(n(cid:5)16)inconsecutivesectionsorthrough500-(cid:2)m-thick
decreasedinmoreanteriorlevelswheretheretrosplenialcortex
Clarity-processedtissueimmunoreactedforinterneuronmolec-
fuseswiththecingulatecortex.
ular markers. The limited number of antibodies and fluoro-
phores that could be used in the same section allowed the
Diversetypesofcorticalinterneuroninnervatedbyseptal
visualizationofonlyfewofthetargetsofeachsingleaxon.Nev-
GABAergicprojections
ertheless, we often observed a single labeled axon innervating
We used immunohistochemistry to identify somata and den-
target cells labeled for the same molecule, such as PV (n (cid:5) 4
dritesoftheinterneuronstargetedbyanterogradelylabeledseptal
axons, 2–3 cells each; Fig. 5A), or NECAB1 and CCK (n (cid:5) 2
axons.ImmunohistochemistryforPV,CB,CR,NECAB1,CCK,
axons,2and4cells).Theseseptalaxonswithanapparenttarget
SATB1,nNOS,orreelinrevealednumerousimmunopositivetar-
selectivityalsoformedboutonsaroundcellularprofilesthatwere
getprofilesinclearappositiontotheboutonsimmunolabeledfor
notlabeledbyanyofthetestedmarkers,includingthemolecule
PHAL(Fig.4)orEYFP(Fig.5).Wehavefoundnodifferencesin
thatwasfoundinthenearbytargetedcells(Fig.5A,B).Thus,we
thetargetsoftheGABAergicseptalaxonsvisualizedbyimmuno-
havenotobservedstricttargetcell-typeselectivitybylabeledsep-
labelingforPHALorEYFPintheentorhinalcortex,compared
withtheabovesomatodendriticinterneuronmarkersormarker talaxons.Onthecontrary,ninesingleseptalaxonswereinappo-
combinationsin(cid:3)3animalseach.Therefore,inotherextrahip- sition to profiles that were immunopositive for different set of
pocampalcortices,wehaveanalyzedonlythetargetsoftheEYFP- molecularmarkers.Forexample,boutonsofasingleseptalaxon
immunolabeledseptalGABAergicaxons. inthesubiculumwereinappositiontoaninterneuronimmuno-
Immunohistochemistry for the aforementioned molecular positive for PV and SATB1, as well as to a nearby NECAB1-
markersrevealedthatthediversityoftheinterneurontargetsof immunopositive, but PV-immunonegative, neuron that was
EYFP-immunolabeled septal axons observed in the entorhinal also immunoreactive for SATB1. In another case, a single
cortex(Figs.4,5B,F)appliedtothesubiculum,presubiculum, EYFP-immunolabeledaxoninsubiculuminnervatedasomaim-
parasubiculum,andretrosplenialcortex(Table3;n(cid:5)7animals). munoreactiveforCCKandNECAB1aswellasanotherNECAB1-
In each of these extrahippocampal areas, excluding the retro- immunopositivebutCCK-immunonegativeneuron(Fig.5G).
Unaletal.•SynapticTargetsofMedialSeptalProjections J.Neurosci.,December2,2015•35(48):15812–15826•15821
Table3.Molecularcellmarkersandtheircombinationsfoundinseptaltarget
neuronsa
Lateral
Retrosplenial entorhinal
Subiculum Presubiculum Parasubiculum cortex cortex mEC
PV (cid:4) (cid:4) (cid:4) (cid:4) (cid:4) (cid:4)
CB (cid:4) (cid:4) (cid:4) (cid:4) (cid:4) (cid:4)
CR (cid:4) (cid:4) (cid:4) (cid:4) (cid:4) (cid:4)
NECAB1 (cid:4) (cid:4) (cid:4) n.o. (cid:4) (cid:4)
CCK (cid:4) (cid:4) (cid:4) n.o. (cid:4) (cid:4)
Reelin (cid:4) (cid:4) (cid:4) (cid:4) (cid:4) (cid:4)
nNOS (cid:4) n.o. (cid:4) n.o. n.o. (cid:4)
PV(cid:4)CB (cid:4) (cid:4) (cid:4) n.o. n.t. (cid:4)
PV(cid:4)SATB1 (cid:4) (cid:4) (cid:4) (cid:4) n.t. n.o.
CB(cid:4)reelin (cid:4) n.o. n.o. (cid:4) n.o. n.o.
CB(cid:4)nNOS (cid:4) n.t. n.t. n.o. n.t. n.t.
NECAB1(cid:4)CCK (cid:4) n.o. (cid:4) n.t. n.t. n.t.
NECAB1(cid:4) (cid:4) n.t. (cid:4) n.t. n.t. n.t.
SATB1
PV(cid:4)SATB1(cid:4) (cid:4) n.t. n.t. n.o. n.t. n.o.
nNOS
aImmunohistochemicalmolecularmarkersormarkercombinationsfoundininterneuronsinclearappositiontothe
EYFP-immunolabeledseptalGABAergicboutonsineachextrahippocampalcorticalarea.(cid:4),Observed;n.o.,not
observedwhensearchedin(cid:9)4sections;n.t.,nottested.
SeptalGABAergicprojectionsinthemEC,retrosplenial
cortex,andtheCA1innervateinterneurons
We have tested the postsynaptic targets of septal axons in the
cerebralcortexbytwomethods.First,weimmunolabeledpyra-
midal cells in LII of the mEC by immunohistochemistry for
WFS1andCB,andstellatecellsforreelin.Atdifferentrostrocau-
dallevelsinLIIoftheentorhinalcortex,wefollowedsinglela-
beled GABAergic axons of PV-expressing septal neurons
immunolabeledforEYFPandsearchedforputativetargetpro-
filesimmunopositiveforWFS1,CB,orreelin(n(cid:5)4animals).No
labeledseptalboutonswerefoundinclearappositiontoWFS1/
CB-immunopositive somata or proximal dendrites or reelin-
positive stellate cell somata (Fig. 6A, asterisks). Some other
smaller reelin-positive somata were found in apposition to the
EYFP-labeledaxonalvaricositiesinalllayersofthemEC(Fig.6),
whicharelikelytobeinterneurons(Chinetal.,2007).
Second,toinvestigatetherangeofsynaptictargets,weana-
lyzedwithanelectronmicroscopePHAL-labeled(Fig.7A,C–E)
orEYFP-expressing(Fig.7B)axonalboutons.Theyformedtype
IIsynapsesandtargetedmostlydendriticshaftsintheCA1region
(Fig.7A,B)andretrosplenialcortex(Fig.7C–E).Weidentifieda
Figure5. InnervationofinterneuronsbyEYFP-labeledseptalaxons(green)inextrahip-
total of 33 synaptic junctions formed by PHAL-immunolabe-
pocampalcorticalareasofPV-Cremice.A,B,InnervationoftwoPV-immunopositive(magenta)
led boutons in the strata radiatum, pyramidale, lacunosum-
somatabythesameaxoninpresubiculum(A;caseAJ14ab),andanotherneuronindeeplayers
moleculare,andoriensofCA1(n(cid:5)2animals).One-thirdofthe
ofthelateralentorhinalcortex(B;caseAJ13a).C,JuxtapositionofseptalboutonswithaCB-
immunopositive(blue)dendriticprofileinpresubiculum;singleopticalslices(thickness:1(cid:2)m) synapseswereaxosomaticandtheremainingwereaxodendritic
areenlarged(C(cid:2))andoverlaidwiththelabeledaxon(C(cid:3))showingputativeaxodendriticcon- (Fig. 7A). All the postsynaptic profiles were identified as in-
tacts(arrows;caseAJ14j).D,E,Twoseparatelabeledaxonsinparasubiculuminnervatetwo terneuronal,astheyreceivedtypeIsynapsesfromunlabeledbou-
targetcellsimmunopositiveforPV(D,magenta).Theupperneuronisalsoimmunoreactivefor tonsandnospineswereobservedoriginatingfromthedendritic
CB(E,blue).Asingleopticalslice(thickness:0.36(cid:2)m)oftheproximaldendriteofthiscell shafts.WealsoexaminedthesynaptictargetsofPV-expressing
(framed)isshowninE(cid:2)andoverlaidwiththeseptalaxons(E(cid:3)),demonstratingputativeaxo- septalGABAergicneuronsinthestratumpyramidaleofCA1.Of
dendritic contacts (arrows, case GU41jk). F, A CR-immunopositive (white) and a CB-
the15axodendriticand5axosomatic(Fig.7B)typeIIsynapses
immunopositive(blue)somaareinnervatedbyseparatelabeledaxonsinthelateralentorhinal identified,allbelongedtointerneurons(n(cid:5)2animals).Hence,
cortex.ArrowspointtoboutonsoverlappingwiththedendriteoftheCB-immunopositiveneu-
inthesesamples,nolabeledboutonsthatformedtypeIIjunc-
ron(caseAJ12eeff).G,FourNECAB1-immunopositive(blue)neuronsaretargetedbyseptal
tions and originated from identified or presumed GABAergic
axonsinthedorsalsubiculum(caseAJ13s14).TwotargetsstronglyimmunopositiveforNECAB1
arealsopositiveforCCK(pink),whereasweaklyNECAB1-immunoreactivesomataareCCKim- medial septal axons made synapses with pyramidal cells in the
munonegative(asterisks).Onelabeledseptalaxon(left)innervatesbothkindsofcells. CA1region.Finally,weanalyzedthesuperficialanddeeplayersof
Medianfilterwasapplied(x,y:radius,1pixel)inAandB.Maximumintensityprojections thegranularretrosplenialcortexandrecordedinserialsections
ofconfocalimagestacks:A,21.58(cid:2)m;B,12.07(cid:2)m;C,22(cid:2)m;DandE,2.49(cid:2)m;F, 47typeIIsynapticjunctionsformedbyPHAL-immunolabeled
10.38(cid:2)m;G,8.19(cid:2)m. boutons(n(cid:5)2animals).Weidentified18postsynapticdendritic