Olfactory Bulb Deep Short-Axon Cells Mediate Widespread Inhibition of Tufted Cell Apical Dendrites

22 Pages·2017·11.77 MB·English

TheJournalofNeuroscience,February1,2017•37(5):1117–1138•1117
Systems/Circuits
Olfactory Bulb Deep Short-Axon Cells Mediate Widespread
Inhibition of Tufted Cell Apical Dendrites
XShawnD.Burton,1,2GregLaRocca,3XAnnieLiu,2,4XClaireE.J.Cheetham,1andXNathanielN.Urban1,2,3,4
1DepartmentofBiologicalSciences,CarnegieMellonUniversity,Pittsburgh,Pennsylvania15213,2CenterfortheNeuralBasisofCognition,Pittsburgh,
Pennsylvania15213,and3DepartmentofNeurobiologyand4CenterforNeuroscience,UniversityofPittsburgh,Pittsburgh,Pennsylvania15213
Inthemainolfactorybulb(MOB),thefirststationofsensoryprocessingintheolfactorysystem,GABAergicinterneuronsignalingshapes
principalneuronactivitytoregulateolfaction.However,alackofknownselectivemarkersforMOBinterneuronshasstronglyimpeded
cell-type-selectiveinvestigationofinterneuronfunction.Here,weidentifythefirstselectivemarkerofglomerularlayer-projectingdeep
short-axoncells(GL-dSACs)andinvestigatesystematicallythestructure,abundance,intrinsicphysiology,feedforwardsensoryinput,
neuromodulation,synapticoutput,andfunctionalroleofGL-dSACsinthemouseMOBcircuit.GL-dSACsarelocatedintheinternal
plexiformlayer,wheretheyintegratecentrifugalcholinergicinputwithhighlyconvergentfeedforwardsensoryinput.GL-dSACaxons
arborizeextensivelyacrosstheglomerularlayertoprovidehighlydivergentyetselectiveoutputontointerneuronsandprincipaltufted
cells.GL-dSACsarethuscapableofshiftingthebalanceofprincipaltuftedversusmitralcellactivityacrosslargeexpansesoftheMOBin
responsetodiversesensoryandtop-downneuromodulatoryinput.
Keywords: acetylcholine;chrna2;inhibition;interneuron;olfaction;olfactorybulb
SignificanceStatement
Theidentificationofcell-type-selectivemolecularmarkershasfosteredtremendousinsightintohowdistinctinterneuronsshape
sensoryprocessingandbehavior.Inthemainolfactorybulb(MOB),inhibitorycircuitsregulatetheactivityofprincipalcells
preciselytodriveolfactory-guidedbehavior.However,selectivemarkersforMOBinterneuronsremainlargelyunknown,limiting
mechanisticunderstandingofolfaction.Here,weidentifythefirstselectivemarkerofanovelpopulationofdeepshort-axoncell
interneuronswithsuperficialaxonalprojectionstothesensoryinputlayeroftheMOB.Usingthismarker,togetherwithimmu-
nohistochemistry,acutesliceelectrophysiology,andoptogeneticcircuitmapping,werevealthatthisnovelinterneuronpopula-
tionintegratescentrifugalcholinergicinputwithbroadlytunedfeedforwardsensoryinputtomodulateprincipalcellactivity
selectively.
Introduction (Kubota,2014;WesterandMcBain,2014;Letzkusetal.,2015).In
The mammalian brain contains a vast diversity of GABAergic themainolfactorybulb(MOB),inhibitorycircuitsregulatethe
inhibitoryinterneurons.Theidentificationofcell-type-selective activityofprincipalmitralandtuftedcells(M/TCs)precisely
molecular markers has fostered tremendous insight into how to drive olfactory-guided behavior (Fukunaga et al., 2012,
distinct interneurons shape sensory processing and behavior 2014;Gschwendetal.,2015).However,selectivemarkersfor
manyMOBinterneuronshavenotbeenidentified(Eyreetal.,
2009),impedinginvestigationofcell-type-selectiveinterneu-
ReceivedSept.13,2016;revisedNov.14,2016;acceptedDec.3,2016.
ronfunction.
Authorcontributions:S.D.B.andN.N.U.designedresearch;S.D.B.,G.L.,A.L.,andC.E.J.C.performedresearch;
S.D.B.,G.L.,andA.L.analyzeddata;S.D.B.,G.L.,A.L.,C.E.J.C.,andN.N.U.wrotethepaper. Sensory input to the MOB is organized into odorant-
ThisworkwassupportedbytheNationalInstitutesofHealth–NationalInstituteonDeafnessandOtherCommu- receptor-specific glomeruli, where olfactory sensory neuron
nicationDisorders(GrantF31DC013490toS.D.B.,GrantR01DC005798toN.N.U.,andGrantR01DC011184toN.N.U.) (OSN)axonsandexternaltuftedcells(ETCs)drivesensoryacti-
andthePennsylvaniaDepartmentofHealthCommonwealthUniversalResearchEnhancementProgram(N.N.U.).
vationofM/TCapicaldendritictuftsinthesuperficiallayersof
WethanktheMellonInstituteCentralizedVivariumstaffforexcellenttechnicalassistance;RachelS.Bouchardand
ArynH.GittisforassistancewithAi3mice;andCharlesR.Gerfen,LauraKus,andmembersoftheUrban,Gittis,Seal, theMOB(Nagayamaetal.,2014).DeeperintheMOB,thein-
Kuhlman,andOswaldlaboratoriesforhelpfuldiscussions. framitral layers contain a large class of GABAergic deep short-
Theauthorsdeclarenocompetingfinancialinterests. axon cell (dSAC) interneurons, the functional roles of which
CorrespondenceshouldbeaddressedtoNathanielN.Urban,DepartmentofNeurobiology,Universityof
remainalmostentirelyunknown.dSACscanbesubdividedinto
Pittsburgh,E1440BSTWR,200LothropStreet,Pittsburgh,PA15213.E-mail:[email protected].
granulecelllayer(GCL)-,externalplexiformlayer(EPL)-,and
DOI:10.1523/JNEUROSCI.2880-16.2016
Copyright©2017theauthors 0270-6474/17/371117-22$15.00/0 glomerularlayer(GL)-projectingdSACs(GCL/EPL/GL-dSACs,
1118•J.Neurosci.,February1,2017•37(5):1117–1138 Burtonetal.•DeepShort-AxonCellsInhibitTuftedCells
respectively)(Eyreetal.,2008).GL-dSACaxonsarborizeacross For chromogenic Neurobiotin labeling, acute slices containing
multipleglomeruliandthusmayregulatesensory-evokedMOB Neurobiotin-filledcellswerefixedinparaformaldehyde(4%inPB)for
activitysignificantly(Eyreetal.,2008).Moreover,GL-dSACsare (cid:3)24hat4C,quenchedwithH2O2/methanol(1%/10%inPB)for1hat
concentrated in the internal plexiform layer (IPL), a region of roomtemperature,washedwithPB,permeabilizedwithTritonX-100
(2%inPB)for1hatroomtemperature,andthenincubatedovernightat
denseinnervationbyETCandTCaxoncollaterals(Macrideset
4CinVectastainABCcomplexsolution(VectorLaboratories;1%Re-
al.,1985;LiuandShipley,1994),glutamatergicandGABAergic
agentA,1%ReagentBinPBwith1%TritonX-100).Sliceswerethen
centrifugalfeedback(Boydetal.,2012;Markopoulosetal.,2012;
washedextensivelywithPBbeforeincubationwithDAB(Sigma-Aldrich;
Nunez-Parraetal.,2013),andneuromodulatoryinput(Macrides
0.5mg/mlwith0.01%H O inPB)for10minatroomtemperature.
2 2
etal.,1981;ShipleyandAdamek,1984),andarethuspoisedto Imaging.ImmunofluorescentimageswerecollectedoninvertedZeiss
coordinate widespread MOB activity patterns across distinct LSM510METADuoscanand880confocalmicroscopesusing40–100(cid:4)
brainstates. oil-immersionobjectivesinmaximumintensityprojectionsof0.5(cid:3)m-
Here, we identify nicotinic acetylcholine receptor (nAChR) stepz-stacksat2048(cid:4)2048resolution.Chrna2-Cre-labeleddSACabun-
subunit(cid:2)2(chrna2)asthefirstselectivemarkerofGL-dSACs. dance was calculated by counting Chrna2-Cre-labeled dSACs within
Using Chrna2-Cre mice, we demonstrate that GL-dSACs inte- manuallydrawnIPLandGCLbordersacrossstitched10(cid:4)2(cid:3)m-step
z-stacktile-scansoftheentireMOB.Threesagittalsectionswereimaged
grate highly convergent feedforward sensory input from ETCs
peranimalandusedtoevaluatemediolateralbiasandcalculateatotalcell
(and possibly TCs) of several glomeruli and inhibit the apical
densityandabundanceperanimal.Dorsoventralbiaswasevaluatedby
dendritictuftsofETCsandTCsselectivelyacrossmultipleglom-
comparingtotalcelldensitieswithindorsalandventralhalvesofeachof
eruli.Strikingly,GL-dSACsneitherreceiveMCinputnorprovide the three MOB tile scans per animal. GABA R(cid:2)1-expressing dSAC
outputontoMCs.GL-dSACactivityisalsoregulatedbydirect abundance was calculated by counting GABAA R(cid:2)1-positive dSACs
A
centrifugalcholinergicinput.Intotal,theuniquelydivergentyet withinmanuallydrawnIPLandGCLbordersacross5–6random-field
selective connectivity of GL-dSACs supports a novel interglo- 40(cid:4)0.5(cid:3)m-stepz-stacksperanimal,calculatingtotalIPL-andGCL-
merular inhibitory circuit between ETCs and TCs that can be specificcelldensitiesperanimalandthenaveragingacrossanimals,sim-
modulatedbytop-downcholinergicinput. ilartopreviousmethods(Eyreetal.,2009).ForDAB-stainedtissueand
whole-brain imaging, images were collected on an Olympus BX51WI
microscopeusing10–20(cid:4)airobjectivesanda100(cid:4)oil-immersion
MaterialsandMethods objective.
Animalsandvirusinjections.Allexperimentswerecompletedincompli- Slicepreparation.P18–P28miceofbothsexeswereanesthetizedwith
ancewiththeguidelinesestablishedbytheInstitutionalAnimalCareand isofluraneanddecapitatedintoice-coldoxygenateddissectionsolution
Use Committee of Carnegie Mellon University and the University of containing the following (in mM): 125 NaCl, 25 glucose, 2.5 KCl, 25
Pittsburgh.Chrna2-Cremice(lineOE29)wereobtainedfromGENSAT NaHCO ,1.25NaH PO ,3MgCl ,and1CaCl .Brainswereisolatedand
3 2 4 2 2
(Gongetal.,2007)andusedasheterozygousF1–2hybridcrosses(ona acutehorizontalorsagittalslices(310(cid:3)mthick)werepreparedusinga
mixedFVB/N,Crl:CD1[ICR],andC57BL/6background)withC57BL/6 vibratome(5000mz-2;CampdenorVT1200S;Leica).Slicesrecovered
and Ai3 mice (Madisen et al., 2010). Approximately one-third of for30minin(cid:2)37°CoxygenatedRinger’ssolutionthatwasidenticalto
Chrna2-Cre mice exhibited markedly denser Cre-dependent labeling thedissectionsolutionexceptwithlowerMg2(cid:5)concentrations(1mM
throughoutthebrainandwerenotusedinthisstudy.ForCre-dependent MgCl2)andhigherCa2(cid:5)concentrations(2mMCaCl2).Sliceswerethen
ChR2:mCherryexpression,(cid:2)1(cid:3)lofAAV1-EF1a-DIO-hChR2(H134R): storedatroomtemperatureuntilrecording.
mCherry(UniversityofPennsylvaniaVectorCore)wasinjectedunilat- Electrophysiology.Sliceswerecontinuouslysuperfusedwith34°Coxy-
erallyintotheMOBorforebrainatpostnatalday2(P2)–P3(Cheetham genatedRinger’ssolution.Cellswerevisualizedusinginfrareddifferen-
etal.,2015).ForCre-dependentEGFPexpression,(cid:2)1(cid:3)lofAAV1-CAG- tialinterferencecontrastvideomicroscopy.Current-clamprecordings
flex-GFP(UniversityofNorthCarolinaVectorCore)wasinjecteduni- weremadeusingelectrodes(4–9M(cid:6))filledwiththefollowing(inmM):
laterallyintotheadultMOB. 120K-gluconate,2KCl,10HEPES,10Na-phosphocreatine,4Mg-ATP,
Immunohistochemistry.Micewereanesthetizedwithintraperitonealin- 0.3Na GTP,0.2EGTA,and0.025AF594,alongwith0.2%Neurobiotin
3
jectionof0.4mlofketamine(20mg/ml)/xylazine(3.3mg/ml)andthen (Vector Labs). Voltage-clamp recordings were made using electrodes
transcardiallyperfusedwith1%NaClin0.1Mphosphatebuffer(PB)fol- filledwiththefollowing(inmM):140Cs-gluconate,10QX-314,2KCl,10
lowedbyparaformaldehyde(4%inPB).Brainswereextracted,postfixedin HEPES, 10 Na-phosphocreatine, 4 Mg-ATP, 0.3 Na GTP, and 0.025
3
paraformaldehydeovernight,sunkinsucrose(30%w/vinPB),andthen AF594,alongwith0.2%Neurobiotin.Liquidjunctionpotentialswere
cryogenicallyslicedinto25-(cid:3)m-thicksagittalsectionswithaslidingmi- 12–14mV(K(cid:5)-internal)and11mV(Cs(cid:5)-internal)andwerenotcor-
crotome (Leica, SM2000R). Sections were incubated with Triton X-100 rectedforexceptwherenoted.Datawerelow-passfilteredat4kHzand
(0.1%inPB)andnormaldonkeyserum(NDS;2%inPB)for1hatroom digitizedat10kHzusingaMultiClamp700Aamplifier(MolecularDe-
temperature,washedwithPB,andthenincubatedfor1hatroomtempera- vices)andanITC-18acquisitionboard(Instrutech)controlledbycus-
ture(unlessotherwisenoted)withcombinationsofthefollowingprimary tomsoftwarewritteninIGORPro(WaveMetrics).OSNfiberbundles
antibodies(inPBwith2%NDSand0.05%Tween20):goatanti-GFP(Ab- projectingintosingleglomeruliwerestimulated(100-(cid:3)s-longconstant
cam,ab6673;1:10000),rabbitanti-GFP(Invitrogen,A-11122;1:1000),rab- current pulses) using an extracellular monopolar glass electrode. For
bitanti-GABA R(cid:2)1(AlomoneLabs,AGA-001;1:1000),rabbitanti-Na 1.6 optogeneticstimulation,sliceswereilluminatedbya75Wxenonarc
A V
(Abcam,ab83764;1:2000;overnightincubationat4C),sheepanti-tyrosine lamppassedthroughaYFPfiltersetand60(cid:4)water-immersionobjective
hydroxylase(TH)(EMDMillipore,AB1542;1:1000),mouseanti-PV(clone centeredontherecordedcell(GL-dSACs)ortheGL(allothercells).
PARV-19; Sigma, P3088; 1:1000; overnight incubation at 4C), rat anti- NBQX(TocrisBioscienceandSigma-Aldrich),AP5(TocrisBioscience),
m2AChR(cloneM2–2-B3;EMDMillipore,MAB367;1:500),goatanti-SST GBZ(TocrisBioscience),DMPP(AlomoneLabs),andMec(TocrisBio-
(SantaCruzBiotechnology,sc-7819;1:500;overnightincubationat4C), scienceandAlomoneLabs)wereaddedtothebathorfocallyappliedas
goatanti-OMP(Wako,544–10001;1:5000;overnightincubationat4C), indicated. Cell morphologies were reconstructed under a 100 (cid:4) oil-
andgoatanti-CR(SantaCruzBiotechnology,sc-11644;1:1500).Sections immersionobjectiveandanalyzedwithNeurolucidasoftware(MBFBio-
werethenwashedinPBandincubatedfor1hatroomtemperaturewith science). Reconstructed GL-dSAC dendrites and axons are drawn in
combinationsofthefollowingsecondaryantibodiesraisedindonkey(1:600 blackandred,respectively.GraylinesinreconstructionsdenoteGL/EPL,
inPBwith2%NDSand0.05%Tween20):anti-goatAF488,anti-rabbit EPL/MCL,MCL/IPL,andIPL/GCLborders.
AF488,anti-rabbitAF594,anti-sheepAF594,anti-mouseAF594,anti-goat Data analysis. Synaptic events were detected and analyzed in
AF594,anti-ratAF594(Invitrogen,A-11055,A-21206,A-21207,A-11016, Axograph.Synapticeventsweredetectedwitha1msminimumevent
A-21203,A-11058,A-21209,respectively). separationtime.sEPSCsweredetectedusinga4.5-ms-longdoubleexpo-
Burtonetal.•DeepShort-AxonCellsInhibitTuftedCells J.Neurosci.,February1,2017•37(5):1117–1138•1119
nentialtemplatewith0.5msbaseline,0.4msrisetimeconstant,and2ms GCL-locateddSACsand(cid:2)50%ofIPL/sGCL-locateddSACsto
decaytimeconstant.sIPSCsweredetectedusinga21-ms-longdouble estimate(cid:2)13,500totaldSACsintheadultratMOB(Eyreetal.,
exponentialtemplatewith1msbaseline,0.6msrisetimeconstant,and8 2009).Therefore,todeterminewhatfractionoftotaldSACsthat
msdecaytimeconstant.Alleventsweredetectedwithathresholdampli- Chrna2-Cremicelabel,wequantifiedthenumberandcolocal-
tudeof3(cid:4)SDofthebaselinenoise.Eventamplitudes,20–80%rise izationofChrna2-Cre-labeledandGABA R(cid:2)1-labeleddSACsin
times,anddecayconstantswerecalculatedusingAxograph.Extracellular A
adultChrna2-Cre/Ai3mice.Usingpreviousvolumetricmeasures
stimulation-evokedsynapticlatenciesweredefinedastheintervaloftime
(Parrish-Aungst et al., 2007), GABA R(cid:2)1 was moderately to
from extracellular stimulation to the onset of the synaptic response, A
strongly expressed in 2706.0 (cid:7) 405.4 IPL-located dSACs,
whichwasdefinedasthetimeatwhichthesynapticresponsereached5%
ofitspeakamplitude.Photostimulation-evokedsynapticlatencieswere 7558.9(cid:7)962.4GCL-locateddSACs(bothsGCLanddeepGCL),
defined as the median photostimulation-evoked GL-dSAC first-spike and10,231.1(cid:7)909.2totaldSACsperMOB(193cellscounted,
latency (see Fig. 11H) subtracted from the absolute synaptic latency n (cid:8) 3 mice) (Fig. 1D). Consistent with the negligible to weak
(calculatedasabove).Synapticinputdurationsweredefinedastheinter- expressionofGABA R(cid:2)1in(cid:2)50%ofIPL/sGCL-locateddSACs
A
valoftimefromtheonsetofthesynapticresponsetothetimeatwhich (Eyreetal.,2009)andtherestrictedlocalizationofChrna2-Cre-
thesynapticresponsedecayedto5%ofitspeakamplitude.Forpaired labeled dSACs to the IPL/sGCL, only 53.7% of Chrna2-Cre-
GL-dSAC/MCrecordings,chargetransferredwascalculatedbyintegrat- labeled dSACs (29/54 cells) exhibited detectable GABA R(cid:2)1
ingcurrenttracesfromtheendofthestimulusartifactto1sor50msafter A
expression(Fig.1D).AssumingthatChrna2-Cre-labeleddSACs
OSN stimulation for MCs and GL-dSACs, respectively. For all other
comprisetheentirepopulationofGABA R(cid:2)1-negativedSACs,
recordings, charge transferred was calculated by integrating current A
wethereforeestimatethattheadultmouseMOBcontains(cid:2)3500
tracesfromtheonsetofthesynapticresponsetothetimeatwhichthe
synapticresponsedecayedto5%ofitspeakamplitude.Stimulusartifacts IPL-located dSACs, (cid:2)8000 GCL-located dSACs, and (cid:2)11,500
areblankedforvisualclarityofsynapticresponses.Intrinsicbiophysical totaldSACs,ofwhich(cid:2)51%locatedintheIPLand(cid:2)12%lo-
propertieswerecalculatedasdescribedpreviously(BurtonandUrban, catedintheGCLarelabeledselectivelyinChrna2-Cremice.
2014,2015)usinga20mV/msthresholdfordetectingactionpotentials. To better resolve the morphologies of Chrna2-Cre-labeled
Tocalculatepowerspectraldensitiesfromspontaneousfiringepochs, dSACs,wefilledindividualEYFP-expressingdSACswithNeuro-
binaryspiketrainswereconvertedtosinusoidalspikephasesandana- biotin via targeted whole-cell recordings in acute slices from
lyzedusingWelch’smethod(500mswindows,50%windowoverlap).
Chrna2-Cre/Ai3mice(Fig.3A,B).Posthocreconstructionofa
Values are reported as mean (cid:7) SD. Error bars and shading denote
largesubset(Fig.3C,D)revealedthatChrna2-Cre-labeleddSACs
mean(cid:7)SEM.Normallyandnon-normallydistributeddata(Anderson–
extend4.7(cid:7)1.5(n(cid:8)24)sparselyspinyandbeadeddendritesup
Darlingtest)wereanalyzedusingparametricandnon-parametrictests,
to(cid:2)200(cid:3)mthroughtheIPLparalleltotheMCLandfrequently
respectively,asindicated.
projectasinglethinputativeaxonsuperficiallytoarborizeacross
Results theGL(Fig.3E,Table1).Importantly,weneverobservedapu-
TargetingGL-dSACsgenetically tativeaxonarborizeacrosstheEPLorGCLorenterthelateral
GL-dSACsareconcentratedintheMOBIPLandsuperficialGCL olfactorytract,thoughwenotethattheprocessesofsomecells
(sGCL) (Fig. 1A) and are composed of horizontal cells and a weretruncatedproximaltothesomaandcouldnotbetraced.In
subset of stellate Golgi cells (Eyre et al., 2008). To study GL- addition,processesenteringtheGLcouldonlybetracedashort
dSACssystematically,wethereforesearchedforcandidatemark- distanceduetotheirthin,tortuousprofileandthehighcellden-
ersthatareexpressedinIPL/sGCL-locatedcellsselectively.Insitu sityoftheGL.Chrna2-Cre-labeleddSACmorphologiesnever-
hybridization of chrna2 labels sparse IPL/sGCL-located cells theless closely match the GL-dSAC morphologies recovered
withintheMOB(Ishiietal.,2005).Consistentwiththisprevious previously from serial thin section reconstruction (Eyre et al.,
report, localized AAV injection in the adult MOB of transge- 2008).Moreover,highdensitiesofaxoninitialsegmentchannel
nic Chrna2-Cre mice (Fig. 1B) yielded highly selective Cre- Na 1.6(LorinczandNusser,2008)withintheproximalregions
V
dependent EGFP expression in multipolar neurons within the of the superficial processes of Chrna2-Cre-labeled dSACs (Fig.
IPL/sGCL (Fig. 1C). Moreover, putative axons in the GL ex- 1F)confirmedthatChrna2-Cre-labeleddSACsprojecttheirax-
tended up to (cid:2)1 mm away from the nearest EGFP-expressing ons superficially into the GL. Chrna2-Cre-labeled somata like-
soma(Fig.1C),matchingtheextensiveaxonalarborizationsob- wisepoorlycolocalizedwithnonselectivemarkersofEPL-and
servedpreviouslyforGL-dSACs(Eyreetal.,2008). GCL-dSACs(Fig.4)andChrna2-Cre-labeledaxonalboutonsin
Compared with adult AAV injection, constitutive Chrna2- theGLpoorlycolocalizedwithsuperficialshort-axoncellmarker
Cre/Ai3reportercrosses(Fig.1B)yieldedlessselectivelabeling, TH(Fig.1E).
withasmallfractionofM/TCs,GCs,andnon-MOBcellsexpress- Collectively,ourresultsthusshowthatChrna2-Cremicelabel
ing EYFP (Fig. 2A–G; see Materials and Methods). However, GL-dSACsselectivelyintheMOB,establishingthefirstselective
EYFP expression was still heavily concentrated in IPL/sGCL- marker of any dSAC subclass. Further, across 158 untargeted
locateddSACsthroughouttheMOB(Fig.2A,B,G).Quantifica- recordings,NusserandcolleaguespreviouslyobservedthatGL-
tion of labeled cell densities yielded 282.2 (cid:7) 92.6 IPL-located dSACs constitute (cid:2)59% and (cid:2)18% of IPL- and GCL-located
dSACs/mm3(316cellscounted,n(cid:8)4mice)and147.4(cid:7)63.6 dSACs, respectively (Eyre et al., 2008), fractions closely corre-
sGCL-located dSACs/mm3 (174 cells counted, n (cid:8) 4 mice) sponding to the (cid:2)51% and (cid:2)12% of IPL- and GCL-located
acrosstheentireMOBvolume,withnodorsoventralormedio- dSACslabeledselectivelyinChrna2-Cremice.Ourresultsthus
lateralbias(Fig.2H–J).Applyingpreviousvolumetricmeasures further suggest that Chrna2-Cre mice label most, if not all,
(Parrish-Aungst et al., 2007), we therefore estimate that adult GL-dSACs.
Chrna2-Cremicelabel(i.e.,driveCre-dependentexpressionin)
1797.9(cid:7)589.6IPL-locateddSACs,939.1(cid:7)404.8sGCL-located GL-dSACsfireintrinsicallyatthetafrequencies
dSACs,and2736.9(cid:7)765.8totaldSACsperMOB. Cell-attachedrecordingsfromGL-dSACsinacuteslices(Fig.5A)
ThetotalnumberofdSACsintheadultmouseMOBisnot revealedspontaneousfiringat(cid:2)6Hzthatwashighlyregularand
currently known, though Nusser and colleagues have used the stable,yieldingrobustspectralpeaksat5–20Hz(Fig.5B,C)and
specific but nonselective expression of GABA R(cid:2)1 in all deep lowinterspikeinterval(ISI)coefficientofvariation(CV )values
A ISI
1120•J.Neurosci.,February1,2017•37(5):1117–1138 Burtonetal.•DeepShort-AxonCellsInhibitTuftedCells
Figure1. Chrna2-CremicelabelGL-dSACsselectively.A,MOBcircuitry.Glutamatergiccellsareshowninblack.GABAergiccellstargetingglutamatergiccellsareshowninred.GABAergiccells
putativelytargetingotherGABAergiccellsareshowninblue.B,ExperimentalstrategytodriveCre-dependentexpressionofEYFP,EGFP,orChR2:mCherryunderthecontrolofthechrna2promoter.
C,Cre-dependentexpressionofEGFP2weeksafterAAVinjectionintotheadultdorsalMOB.Scalebar,500(cid:3)m(inset:50(cid:3)m).A,Anterior;D:dorsal.D,HalfoftheChrna2-Cre-labeledGL-dSACs
(46.3%;25/54cells,n(cid:8)2mice)exhibitweakGABAR(cid:2)1expression(arrowheads),whereastheremainingcellsexhibitnoornegligibleGABAR(cid:2)1expression(arrow).Scalebar,50(cid:3)m.
A A
E,Chrna2-Cre-labeledaxonalboutonscolocalizepoorlywithTH.Scalebar,50(cid:3)m.F,Na1.6localizestoproximalregionsofsuperficiallyprojectingprocessesofChrna2-Cre-labeleddSACs
V
(arrowhead).Scalebar,20(cid:3)m.
(Table 2). Application of NBQX (10 (cid:3)M), AP5 (50 (cid:3)M), and enablingexaminationofGL-dSACexcitabilitythroughsomatic
gabazine(GBZ;10(cid:3)M)hadnoeffectonspontaneousfiringrates stepcurrentinjections.ThemajorityofGL-dSACsrespondedto
(Fig. 5B,C) or regularity (Table 2), demonstrating that highly depolarizingcurrentwithhighlyregularfiring(Fig.5G,K)and
regular firing is an intrinsic property of GL-dSACs. Consistent relatively uniform excitability (Fig. 5L, Table 2). Surprisingly,
withthisfinding,whole-cellrecordingsfromalargepopulation whereasallGL-dSACsexhibitedregularfiringatrest(Fig.5F,I),
ofGL-dSACs(n(cid:8)33)likewiserevealedhighlyregularandstable 23.1%(6/26cells)respondedtoweakdepolarizingcurrent((cid:10)50
firingat(cid:2)8Hz(Fig.5D–F,H,I,Table2). pA)fromhyperpolarizedV withprominent3–4Hzburstfiring
m
Spontaneousfiringwasabolishedbymoderatehyperpolariza- (Fig.5J),whereasstrongercurrents((cid:3)50pA)evokedhighlyreg-
tion from a mean membrane potential (V ) measured during ularfiring(Fig.5K).Likewise,hyperpolarizingcurrentsevoked
m
ISIs(V )of(cid:9)55.0(cid:7)3.0mVto(cid:9)61.6(cid:7)2.7mV(n(cid:8)20), reboundbursting(Fig.5J)in39.1%(9/23cells)ofGL-dSACs.
m,ISI
Burtonetal.•DeepShort-AxonCellsInhibitTuftedCells J.Neurosci.,February1,2017•37(5):1117–1138•1121
Figure2. CharacterizationofthetransgenicChrna2-Cremouseline.A–F,SagittalsectionfromarepresentativeChrna2-Cre/Ai3mouse.WithintheMOB,EYFPexpressionislocalizedtodSACsin
theIPLandtosparseM/TCsandGCs(B).OutsideoftheMOB,EYFPexpressionislargelylocalizedtosparseneuronsintheaccessoryolfactorybulb(C),septalnucleus(D),andmultiplebasalforebrain
regions,includingtheventralpallidum,bednucleusstriaterminalis,andsubstantiainnominata(E,F).Scalebar,500(cid:3)m(insets:200(cid:3)m).G,Chrna2-Cre/Ai3micelabeledsignificantlymoredSACs
thanothercelltypesintheMOB(429.7(cid:7)120.2,n(cid:8)4,vs134.3(cid:7)78.5,n(cid:8)4,vs24.2(cid:7)22.5,n(cid:8)4,vs40.0(cid:7)46.3,n(cid:8)4,cells/mm3,dSACsvsTCsvsMCsvsGCs;p(cid:8)2.2(cid:4)10(cid:9)5,one-way
ANOVA;dSACsvsTCs,p(cid:8)7.1(cid:4)10(cid:9)4,dSACsvsMCs,p(cid:8)3.6(cid:4)10(cid:9)5,dSACsvsGCs,p(cid:8)5.4(cid:4)10(cid:9)5,posthocTukey–Kramer).ColorscorrespondtodifferentChrna2-Cre/Ai3mice.
H,Chrna2-Cre/Ai3micelabeleddSACswithintheIPLandsGCL,withastrongtrendtowardgreaterlabeleddSACdensityintheIPL(p(cid:8)0.08,pairedtwo-sidedttest).I,J,Chrna2-Cre/Ai3-labeled
dSACswereequallydistributedalongdorsoventral(I;p(cid:8)0.34,pairedtwo-sidedttest)andmediolateral(J;p(cid:8)0.87;two-sidedttestoflinearregressionslopes)axesoftheMOB.
Collectively,thisstate-dependentburstingcloselymatchesrecent and morphological analyses. First, direct comparison of 18
characterizationofratGolgicells(Pressleretal.,2013)andsug- physiologicalproperties(Table3)and13morphologicalproper-
gests that GL-dSACs may comprise distinct functional and/or ties (Table 4) across the two subpopulations revealed overall
morphologicalsubtypes(e.g.,regularfiringhorizontalcellsand minordifferences,withburstingcellsexhibitingsmallerafterhy-
burstingGolgicells). perpolarizations,moreregularspontaneousfiring,greatertotal
To evaluate the heterogeneity of GL-dSACs, we subdivided dendritic length, and more dendritic branch points. Second,
GL-dSACs into regular firing or bursting cells based on their principalcomponentanalysisacrossthe18physiologicalproper-
aboveresponsetodepolarizingandhyperpolarizingstepcurrent ties(Fig.5M–O)and13morphologicalproperties(Fig.3F–H)
injections and performed a series of multivariate physiological revealednoobviousclusteringofregularfiringandburstingcells.
1122•J.Neurosci.,February1,2017•37(5):1117–1138 Burtonetal.•DeepShort-AxonCellsInhibitTuftedCells
Figure3. MorphologicalpropertiesofGL-dSACs.A,B,EYFP-expressingdSACsinChrna2-Cre/Ai3micewerefilledwithAF594andNeurobiotinviawhole-cellrecordings.Insetsshowaxon
projectingthroughtheEPLandintotheGL.Scalebar,20(cid:3)m(A);100(cid:3)m(B).C,ReconstructedmorphologyofthecellshowninAandB.ScaleisthesameasinB.D,Additionalreconstructions,
sortedbyfiringpattern.GraynumbersmarkGL-dSACsinwhichthefiringpatternwasundetermined;blacknumbersmarkregularlyfiringGL-dSACs;greennumbersmarkburstingGL-dSACs.
E,DendriticShollanalysis.Inset,Meanangulardistributionofreconstructeddendriticandaxonalprocesslengthsshowinghorizontaldendriticextensions(black)andsuperficialaxonalprojections
(red)(n(cid:8)24).F,Projectionofregularfiring(black)andbursting(green)GL-dSACsandGL-dSACswithundeterminedfiringpatterns(gray)ontothefirsttwoprincipalcomponents(PC1andPC2)
identifiedfromprincipalcomponentanalysisofGL-dSACmorphologicalproperties.NoobviousclusteringofGL-dSACsbyfiringpatternemerged.G,VarianceinGL-dSACmorphologicalproperties
explainedbyeachprincipalcomponent.H,DecompositionofPC1andPC2intotheirconstituentpropertycoefficients.I,UnbiasedagglomerativehierarchicalclusteringofGL-dSACsbytheEuclidean
distancesamongthez-scoredmorphologicalpropertieslistedinH(usingWard’smethod).Applicationofthegapstatisticmethodyieldedonecluster.
Burtonetal.•DeepShort-AxonCellsInhibitTuftedCells J.Neurosci.,February1,2017•37(5):1117–1138•1123
Table1.MorphologicalGL-dSACproperties temporalpatterningofspecificaspectsofMOBactivityattheta
Soma frequencies(seeDiscussion).
Area((cid:3)m2) 227.7(cid:7)46.0(196.2–257.2)(24) Feedforward excitatory input to GL-dSACs may originate
Majordiameter((cid:3)m) 21.2(cid:7)3.0(19.1–22.7)(24) from ETCs, TCs, and/or MCs. However, at least three lines of
Minordiameter((cid:3)m) 14.5(cid:7)1.6(13.3–15.0)(24) evidencesuggestthatETCsprovidetheprimaryfeedforwardex-
Dendrite citatoryinputtoGL-dSACs.First,thesomataanddendritesof
Totallength((cid:3)m) 1150.3(cid:7)349.1(876.4–1431.0)(24)
GL-dSACsoverlapwithETCaxonsandTCaxoncollateralsex-
Totalvolume((cid:3)m3) 699.7(cid:7)342.1(465.4–849.7)(24)
tensively within the IPL/sGCL (Macrides et al., 1985; Liu and
Spines 6.0(cid:7)6.3(2.0–6.5)(24)
Branchpoints 9.0(cid:7)3.6(7.0–11.0)(24) Shipley, 1994). This overlap provides an anatomical basis for
LengthinMCL(%) 8.7(cid:7)9.9(0.5–11.7)(24) ETC/TC-mediated input to GL-dSACs while strongly arguing
LengthinIPL(%) 80.4(cid:7)17.5(70.8–94.9)(24) againstinputfromMCaxoncollaterals,whichextendthrough-
LengthinGCL(%) 10.9(cid:7)14.6(1.2–16.9)(24) outthedeepGCL(Macridesetal.,1985).Second,theshortla-
MaximumShollradius((cid:3)m) 161.3(cid:7)37.9(140.0–180.0)(24) tency(Fig.6H)andduration(Fig.6I)offeedforwardexcitatory
Fractalindex 1.07(cid:7)0.03(1.05–1.09)(24) inputtoGL-dSACsmarkedlyprecedesthefirst-spikelatencyof
Somatodendritic MCsunderidenticalexperimentalconditions(BurtonandUr-
Convexhullvolume((cid:4)105(cid:3)m3) 3.5(cid:7)2.3(1.9–3.9)(24) ban,2014,2015).Third,theshortduration(Fig.6I)andburst-
Valuesareshownasmean(cid:7)SD(Q (cid:9)Q )(n). likenature(Fig.6E–G,R–U)offeedforwardexcitatoryinputto
1 3
GL-dSACsmirrorstheETC-mediatedexcitatoryinputtotype2
Finally, unbiased agglomerative hierarchical clustering of GL- periglomerularcells(PGCs)(Hayaretal.,2004;Shaoetal.,2009;
dSACsbyeitherthe18physiologicalproperties(Fig.5P)or13 Najac et al., 2015), but contrasts strongly with the long-lasting
morphological properties (Fig. 3I) and application of the gap M/TC-mediatedexcitationobservedingranulecells(GCs)(Bur-
statisticmethodconsistentlyyieldedasingleclusterofcells.Itis tonandUrban,2015;Geramitaetal.,2016).
possiblethataninsufficientnumberofneuronswererecordedto Tofurtherevaluatethesourceoffeedforwardexcitatoryinput
resolve multiple clusters statistically across 13–18 properties. toGL-dSACs,weperformedpairedrecordingsofGL-dSACsand
However, repeating the agglomerative hierarchical clustering nearby MCs (Fig. 9A–D) while stimulating OSNs at intensities
analysisusingthefirsttwotofourprincipalcomponentsderived perithresholdforgeneratinganall-or-nonelong-lastingdepolar-
fromeitherthe18physiologicalpropertiesor13morphological ization(LLD)intheMC(Carlsonetal.,2000;GireandSchoppa,
properties, as performed previously to differentiate between 2009).TheMCLLDismediatedbytheregenerativeactivationof
GCL-,EPL-,andGL-dSACs(Eyreetal.,2008),likewiseyieldeda allrecurrentlycoupledETCswithinaglomerulus(DeSaintJanet
single cluster of cells (data not shown). These results thus al., 2009; Gire and Schoppa, 2009). If GL-dSACs receive excit-
stronglyarguethatGL-dSACscompriseasinglefunctionaland atoryinputfromMCs,thenLLDgenerationshouldbeanimpor-
morphologicalclass,withregularfiringandburstingGL-dSACs tant predictor of excitatory input to GL-dSACs. Strikingly,
andhorizontalandGolgicellmorphologiesrepresentingdistinct however, GL-dSACs received robust excitatory input on both
endsofacontinuum. LLDsuccessandLLDfailuretrials(Fig.9E,F).Togetherwiththe
threelinesofevidencedescribedabove,thisresultstronglyargues
GL-dSACsreceivehighlyconvergent thatETCs(andpossiblyTCs)providetheprimaryfeedforward
ETC-mediatedexcitation excitatoryinputtoGL-dSACs,andmoreoverthatGL-dSACsare
Under baseline conditions, GL-dSACs are continuously bom- excitedevenwhenOSNinputtotheMOBfailstoactivateprin-
bardedwithspontaneousexcitatoryandinhibitorypostsynaptic cipalMCs.
currents (sEPSCs and sIPSCs, respectively) (Figs. 6B–D, 7A,B, ETCaxonscanextendlongdistanceswithintheIPL(seeDis-
Tables2,3),identifyingtheIPLasaregionofhighsynapticac- cussion)(Macridesetal.,1985;LiuandShipley,1994;Belluscioet
tivity.Physiologicalactivationofasinglenearbyglomerulusvia al.,2002;Lodovichietal.,2003).ItisthuspossiblethatGL-dSACs
extracellularstimulationofOSNfiberbundles(Fig.6A),analo- integrateETC-mediatedexcitatoryinputfrombothnearbyand
gous to the presentation of a low concentration odor in vivo distant glomeruli. To examine the convergence of feedforward
(Mainlandetal.,2014),evokedrobust,short-latencyexcitatory excitatoryinputtoGL-dSACs,weactivatedsingleglomerulihun-
inputtoGL-dSACsthatwasgradedacrossstimulationintensities dredsofmicrometersawaywhilerecordingexcitatoryinputto
(Fig.6E–H).Thisrobustexcitatoryinputwasobservedregardless GL-dSACs(Fig.9A).InpairedrecordingsfromGL-dSACsand
ofwhethertheGL-dSACaxonenteredtheGLorwastruncatedin nearbyMCs,activationofdistantglomeruli(Fig.9B)evokedno
theEPLduringslicepreparation(Fig.6R–U),excludingthepos- inputtoMCs(Fig.9E),confirmingthatextracellularstimulation
sibility of direct OSN input to GL-dSAC axons within the GL. does not activate OSN axons in passage. In contrast to MCs,
Excitatory input to GL-dSACs after glomerular activation was GL-dSACsreceivedrobustexcitatoryinput(Fig.9E),evenwhen
alsoreliablyfollowedbyfeedforwardinhibition(Fig.7C–F). activatingsingleglomeruli(cid:3)700(cid:3)manteriortotherecordedcell
Theexcitatoryinputevokedbyglomerularactivationreliably (Fig.9G–I).Equivalentresultswerelikewiseobservedwhenac-
triggered one or two short-latency action potentials in GL- tivating single glomeruli posterior to the recorded cell (Fig.
dSACs, followed by a consistent (cid:2)100 ms pause in firing (Fig. 9J,K).Collectively,ourresultsthusshowthatGL-dSACsinte-
6J–Q). This temporal response (rather than a change in firing gratehighlyconvergentexcitatoryinputfromseveralglomeruli,
rate)waslikewiseobservedacrosstrainsofOSNstimulationat5 suggestingbroadodortuninginvivo.
and10Hz(Fig.8),frequenciesmimickingphysiologicalboutsof
sniffing(Wachowiak,2011).Glomerularactivationthusactsto CentrifugalcholinergicfibersinnervateGL-dSACs
reset the phase of spontaneous firing in GL-dSACs. The high Cholinergic signaling critically regulates olfaction (Devore and
precision and temporal nature of this response to glomerular Linster, 2012; D’Souza and Vijayaraghavan, 2014; Linster and
activation,togetherwithhighlyregulartheta-frequencysponta- Fontanini,2014;LinsterandCleland,2016).Understandinghow
neous firing, suggests that GL-dSACs may be involved in the cholinergicsignalinginfluencessensoryprocessingintheMOB
1124•J.Neurosci.,February1,2017•37(5):1117–1138 Burtonetal.•DeepShort-AxonCellsInhibitTuftedCells
Figure4. NeurochemicalpropertiesofGL-dSACs.A,SmallsubsetofChrna2-Cre-labeledGL-dSACs(9.3%;4/43cells,n(cid:8)2mice)expressparvalbumin(PV).ArrowheadmarksaPV-expressingcell.
B,C,Chrna2-Cre-labeledGL-dSACsdonotexpresscalretinin(CR)(B;0.0%;0/25cells,n(cid:8)2mice)orsomatostatin(SST)(C;0.0%;0/61cells,n(cid:8)3mice).D,Chrna2-Cre-labeleddSACsexhibit
negligiblemuscarinicAChRM2(m2AChR)expression.
hasprovendifficult,however,becausemultipleneurontypesex- neurontype,implicatingGL-dSACsasanimportantcomponent
press cholinergic receptors but exhibit no or weak response to ofthecholinergicregulationofolfaction.
endogenous ACh release (Liu et al., 2015; Smith et al., 2015).
Expression of chrna2 suggests that GL-dSACs may express GL-dSACsinnervatePGCs
nAChRs and respond to endogenous ACh release, consistent ToexaminethesynapticoutputofGL-dSACs,weinjectedAAV
withthedensecholinergicinnervationoftheIPL(Macridesetal., into the MOB to drive Cre-dependent channelrhodopsin
1981;ShipleyandAdamek,1984). (ChR2):mCherryexpressioninGL-dSACs(Fig.11A;seeMateri-
We used a pharmacological approach to test for functional alsandMethods).Photostimulationwithbrief(10ms)bluelight
nAChRexpressioninGL-dSACs.Focalapplicationofnicotinicag- pulsesreliablyevokedonetofouractionpotentialsinGL-dSACs
onistdimethylphenylpiperazinium(DMPP;100(cid:3)M)(Fig.10A)in- inthepresenceofglutamatergicantagonists(Fig.11B–H),con-
creasedthespontaneousfiringrateofGL-dSACsdramatically(Fig. firmingthedirectinfluenceofChR2onGL-dSACs.
10B–D,F).Thisincreaseinratewasdrivenbyanon-desensitizing Previous ultrastructural examination of biocytin-filled GL-
inward current, which was abolished by nicotinic antagonist dSACaxonterminalsidentifiedsynapticcontactsontoputative
mecamylamine(Mec;20(cid:3)M)(Fig.10E,G).Importantly,blockade PGCs(Eyreetal.,2008).Consistentwiththisresult,alargesubset
ofglutamatergicandGABAergictransmissionhadnoeffectonthe ofPGCs,whichwereidentifiedbytheirsmallsomataandden-
responsetoDMPP(Fig.10F,G),confirmingthatDMPPactsdi- dritictuft(Fig.11I,J),exhibitedrobust,short-latencypostsyn-
rectlyonfunctionalnAChRsexpressedbyGL-dSACs. apticcurrentsafterbrief(10ms)lightpulses(Fig.11K–M).These
TotestwhetherGL-dSACsrespondtoendogenousAChre- postsynapticcurrentswereunaffectedbyglutamatergicantago-
lease, we recorded postsynaptic currents in GL-dSACs while nistsbutwereabolishedbysubsequentapplicationofGBZ(Fig.
stimulatingthenearbyIPLinthepresenceofglutamatergicand 11K–N), consistent with monosynaptic release of GABA from
GABAergicantagonists(Fig.10H).IPLstimulationevokedro- GL-dSACsontoPGCs.Interestingly,thepostsynapticcurrentsin
bust EPSCs in all GL-dSACs tested (Fig. 10I–L). These EPSCs PGCsreversedatarelativelydepolarizedpotential((cid:9)44mV;Fig.
wereabolishedbybriefapplicationofMec(Fig.10K)andexhib- 11N)despitetherecordingelectrodecontainingalow-Cl(cid:9)inter-
itedkineticsconsistentwiththeexpressionof(cid:2)2(cid:4)4-containing nalsolution(seeMaterialsandMethods).Thisdepolarizedrever-
nAChRs(Fig.10L)(FiglandCohen,2000).Ofgreatinterest,the salpotentialisconsistentwithmultiplepreviousreports(Smith
observednicotinicEPSCsare,toourknowledge,thelargestre- and Jahr, 2002; Parsa et al., 2015), however, and suggests that
sponse to endogenous ACh release observed among any MOB GABAreleasefromGL-dSACsmayacttoincreasePGCfiring,as
Burtonetal.•DeepShort-AxonCellsInhibitTuftedCells J.Neurosci.,February1,2017•37(5):1117–1138•1125
Figure5. FunctionalpropertiesofGL-dSACs.A–C,EYFP-expressingGL-dSACsfiretonicallyincell-attachedrecordingsindependentlyofsynapticactivity(5.7(cid:7)5.2vs6.5(cid:7)6.3Hz,beforevsafter
NBQX/AP5/GBZapplication;n(cid:8)10;p(cid:8)0.10,two-tailedpairedttest).RedpointsdenotecellshowninAandB.Inset,Meanpowerofcell-attachedspiketimes(n(cid:8)10).D,DistributionofGL-dSAC
spontaneousfiringratesrecordedinwhole-cellmode(8.3(cid:7)5.6Hz;n(cid:8)33).Inset:meanpowerofspiketimes(n(cid:8)33).E–G,Whole-cellrecordingsfromarepresentativeGL-dSACshowingregular
firingatrest(F)andatahyperpolarizedV inresponsetoadepolarizingstepcurrent(G).Singleactionpotentialexpandedatright.H–J,ExampleGL-dSACexhibitingburstfiringwhendepolarized
m
orhyperpolarizedfromahyperpolarizedV .Singleburstisexpandedatright.K,GL-dSACsexhibithighlyregularfiringinresponsetostrong((cid:3)50pA)depolarizingstepcurrents(without
m
NBQX/AP5/GBZ,n(cid:8)20;withNBQX/AP5/GBZ,n(cid:8)6).L,GL-dSACsexhibitrelativelyuniformexcitabilityinresponsetostepcurrentinjections(withoutNBQX/AP5/GBZ,n(cid:8)20;withNBQX/AP5/GBZ,
n(cid:8)6).M,Projectionofregularfiring(black)andbursting(green)GL-dSACsontothefirsttwoprincipalcomponents(PC1andPC2)identifiedfromprincipalcomponentanalysisofGL-dSAC
functionalproperties.NoobviousclusteringofGL-dSACsbyfiringpatternemerged.N,VarianceinGL-dSACfunctionalpropertiesexplainedbyeachprincipalcomponent.O,DecompositionofPC1
andPC2intotheirconstituentpropertycoefficients.P,UnbiasedagglomerativehierarchicalclusteringofGL-dSACsbytheEuclideandistancesamongthez-scoredfunctionalpropertieslistedinO
(usingWard’smethod).Applicationofthegapstatisticmethodyieldedonecluster.
1126•J.Neurosci.,February1,2017•37(5):1117–1138 Burtonetal.•DeepShort-AxonCellsInhibitTuftedCells
Table2.FunctionalGL-dSACproperties
(cid:5)NBQX/AP5/GBZ
Cell-attachedrecordings
Spontaneousfiringrate(Hz) 5.7(cid:7)5.2(0.24–8.5)(10) 6.5(cid:7)6.3(0.0–11.1)(10)
SpontaneousfiringCV 0.46(cid:7)0.45(0.13–0.70)(10) 0.21(cid:7)0.22(0.08–0.20)(10)
ISI
Whole-cellrecordings
Spontaneousfiringrate(Hz) 8.3(cid:7)5.6(3.9–12.2)(33) 8.2(cid:7)4.1(6.3–11.5)(6)
SpontaneousfiringCV 0.19(cid:7)0.21(0.08–0.21)(32) 0.09(cid:7)0.07(0.04–0.11)(6)
ISI
V (mV) (cid:9)54.5(cid:7)2.9((cid:9)56.0to(cid:9)52.3)(33) (cid:9)54.4(cid:7)4.4((cid:9)57.2to(cid:9)51.1)(6)
m,ISI
sEPSCproperties(Cs-internal)
Frequency(Hz) 10.0(cid:7)5.4(6.3–12.1)(16) —
Amplitude(pA) 32.2(cid:7)8.2(27.1–34.5)(16) —
20–80%risetime(ms) 0.42(cid:7)0.05(0.39–0.47)(16) —
Decayconstant(ms) 2.8(cid:7)1.0(2.3–3.0)(16) —
sIPSCproperties(Cs-internal)
Frequency(Hz) 12.8(cid:7)5.2(8.7–16.9)(16) —
Amplitude(pA) 38.6(cid:7)8.0(32.4–45.4)(16) —
20–80%risetime(ms) 0.82(cid:7)0.11(0.74–0.90)(16) —
Decayconstant(ms) 7.7(cid:7)1.3(6.9–8.1)(16) —
Inputresistance(M(cid:6)) 551.6(cid:7)193.8(410.4–702.5)(18) 429.0(cid:7)110.8(355.4–478.3)(6)
Membranetimeconstant(ms) 25.8(cid:7)7.2(21.4–30.6)(18) 22.0(cid:7)5.0(18.6–23.8)(6)
Membranecapacitance(pF) 48.6(cid:7)10.2(43.1–54.4)(18) 52.0(cid:7)7.8(47.1–55.2)(6)
Sagamplitude(mV) 10.0(cid:7)5.5(5.9–12.9)(17) 7.7(cid:7)6.4(4.4–9.7)(6)
Actionpotentialproperties
Threshold(mV) (cid:9)45.0(cid:7)4.0((cid:9)48.1to(cid:9)42.8)(20) (cid:9)43.6(cid:7)7.8((cid:9)49.1to(cid:9)37.5)(6)
Amplitude(mV) 73.6(cid:7)5.3(71.7–78.2)(20) 71.2(cid:7)2.7(69.7–72.5)(6)
Width(ms) 0.67(cid:7)0.14(0.60–0.80)(20) 0.60(cid:7)0.09(0.53–0.68)(6)
Risingslope(mV/ms) 253.0(cid:7)41.8(218.3–277.7)(20) 247.2(cid:7)38.6(236.5–260.8)(6)
Fallingslope(mV/ms) (cid:9)121.2(cid:7)31.0((cid:9)144.3to(cid:9)98.0)(20) (cid:9)132.2(cid:7)19.0((cid:9)141.3to(cid:9)123.8)(6)
Afterhyperpolarization(mV) 17.5(cid:7)6.1(13.2–21.8)(20) 15.3(cid:7)5.0(11.2–19.2)(6)
Firingratecurrentgain(Hz/pA) 0.59(cid:7)0.27(0.40–0.68)(20) 0.53(cid:7)0.16(0.46–0.61)(6)
Valuesareshownasmean(cid:7)SD(Q (cid:9)Q )(n).
1 3
Table3.FunctionaldifferencesbetweenregularfiringandburstingGL-dSACs
Regularfiring Bursting
Spontaneousfiringrate(Hz) 6.2(cid:7)4.9(2.9–8.3)(14) 10.6(cid:7)6.0(8.5–12.9)(6)
SpontaneousfiringCV 0.24(cid:7)0.19(0.13–0.27)(13) 0.07(cid:7)0.04(0.06–0.09)(6)#
ISI
V (mV) (cid:9)55.5(cid:7)2.9((cid:9)57.6to(cid:9)53.3)(14) (cid:9)53.9(cid:7)3.2((cid:9)57.5to(cid:9)51.4)(6)
m,ISI
sEPSCproperties(K-internal)
Frequency(Hz) 11.9(cid:7)9.8(4.8–20.6)(14) 5.8(cid:7)4.6(2.7–10.7)(6)
Amplitude(pA) 32.5(cid:7)7.0(25.9–40.1)(14) 30.5(cid:7)5.0(26.0–34.8)(6)
20–80%risetime(ms) 0.46(cid:7)0.04(0.42–0.50)(14) 0.42(cid:7)0.05(0.40–0.42)(6)
Decayconstant(ms) 2.2(cid:7)0.4(2.0–2.5)(14) 1.9(cid:7)0.2(1.7–2.0)(6)
Inputresistance(M(cid:6)) 605.6(cid:7)163.5(441.9–747.3)(12) 443.7(cid:7)219.0(324.5–411.8)(6)
Membranetimeconstant(ms) 28.3(cid:7)6.0(23.9–30.9)(12) 20.7(cid:7)7.1(16.6–24.8)(6)
Membranecapacitance(pf) 48.5(cid:7)10.7(43.2–52.2)(12) 48.8(cid:7)10.0(38.8–57.6)(6)
Sagamplitude(mV) 8.5(cid:7)4.9(4.6–11.1)(11) 12.7(cid:7)5.8(6.3–16.0)(6)
Actionpotentialproperties
Threshold(mV) (cid:9)44.9(cid:7)4.3((cid:9)48.8to(cid:9)43.8)(14) (cid:9)45.3(cid:7)3.6((cid:9)47.8to(cid:9)42.7)(6)
Amplitude(mV) 73.2(cid:7)5.3(71.5–78.1)(14) 74.6(cid:7)5.8(73.2–79.0)(6)
Width(ms) 0.68(cid:7)0.16(0.60–0.80)(14) 0.65(cid:7)0.08(0.60–0.70)(6)
Risingslope(mV/ms) 253.9(cid:7)48.4(214.4–282.8)(14) 251.0(cid:7)23.1(230.0–270.0)(6)
Fallingslope(mV/ms) (cid:9)121.9(cid:7)32.9((cid:9)152.5to(cid:9)97.8)(14) (cid:9)119.8(cid:7)29.1((cid:9)131.9to(cid:9)98.8)(6)
Afterhyperpolarization(mV) 20.1(cid:7)4.6(17.8–24)(14) 11.3(cid:7)4.2(9.4–13.7)(6)*
Firingratecurrentgain(Hz/pA) 0.55(cid:7)0.28(0.35–0.65)(14) 0.67(cid:7)0.25(0.48–0.90)(6)
*p(cid:10)0.05,two-tailedunpairedttestwithBonferronicorrectionformultiplecomparisons;#p(cid:10)0.05,two-sidedWilcoxonrank-sumtestwithBonferronicorrectionformultiplecomparisons.Valuesareshownasmean(cid:7)SD
(Q (cid:9)Q )(n).
1 3
demonstratedrecentlywithexogenousGABAapplication(Parsa Shaoetal.,2009;Nagayamaetal.,2014;Najacetal.,2015).To
etal.,2015). investigatewhetherGL-dSACstargettype1and/or2PGCs,we
Approximately one-third of PGCs (i.e., type 1 PGCs) re- first examined the overlap between GL-dSAC axonal bo-
ceive direct sensory-evoked input from OSNs and colocalize utons and OMP-positive glomerular compartments. EYFP-
with OMP-positive glomerular compartments, whereas the expressingGL-dSACboutonsinnervatedOMP-negativecom-
remaining PGCs (i.e., type 2 PGCs) colocalize with OMP- partments profusely while strikingly avoiding OMP-positive
negativeglomerularcompartmentsandreceiveindirectfeed- compartments (Fig. 12A), strongly arguing that GL-dSACs
forwardsensory-evokedinputfromETCs(Hayaretal.,2004; targettype2PGCs.