Diversity and wiring variability of olfactory local interneurons in the Drosophila antennal lobe

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1 Diversity n wiring vriility of olftory lol interneurons in the Drosophil ntennl loe Y-Hui Chou 1,3, Mri L Spletter 1,3, Emre Yksi 2,3, Jonthn C S Leong 1, Rhel I Wilson 2 & Liqun Luo 1 21 Nture Ameri, In. All rights reserve. Lol interneurons re essentil in informtion proessing y neurl iruits. Here we present omprehensive geneti, ntomil n eletrophysiologil nlysis of lol interneurons (LNs) in the Drosophil melnogster ntennl loe, the first olftory proessing enter in the rin. We foun LNs to e iverse in their neurotrnsmitter profiles, onnetivity n physiologil properties. Anlysis of >1,5 iniviul LNs revele prinipl morphologil lsses hrterize y orsely stereotype glomerulr innervtion ptterns. Some of these morphologil lsses showe istint physiologil properties. However, the finer-sle onnetivity of n iniviul LN vrie onsierly ross rins, n there ws notle physiologil vriility within eh morphologil or geneti lss. Finlly, LN innervtion require intertion with olftory reeptor neurons uring evelopment, n some iniviul vriility lso likely reflete LN LN intertions. Our results revel n unexpete egree of omplexity n iniviul vrition in n inverterte neurl iruit, result tht retes hllenges for solving the Drosophil onnetome. Neurons n e ivie into two generl tegories: projetion neurons, whose xons onnet isrete regions of neurl tissue, n lol interneurons, whose proesses re restrite to single region. Lol interneurons re importnt in neurl omputtion. For exmple, horizontl n mrine ells in the verterte retin re instrumentl in trnsforming inoming signls from photoreeptors, suh tht informtion regring ontrst, motion n olor n e enoe y retinl gnglion ells n trnsmitte into the rin. Every verterte rin region ontins mny lol interneuron types, s efine y morphology, histologil mrkers n physiology. Different types re thought to hve istint funtions 1. However, it is less ler whether the sme kin of iversity exists in simpler orgnisms suh s Drosophil. Systemti ientifition n hrteriztion of lol interneurons is prerequisite to unerstning how nervous systems proess informtion 2. The fly ntennl loe is n ttrtive moel neurl iruit euse of its elegnt ntomil n funtionl orgniztion n its geneti trtility. All olftory reeptor neurons (ORNs) tht express the sme oornt reeptor projet their xons to the sme ntennl loe glomerulus, n most ntennl loe projetion neurons (PNs) sen enrites into single glomerulus to reeive iret input from one ORN lss 3. Geneti ess is ville to mny lsses of ORNs n PNs 3. In ontrst to these input n output neurons, LNs in the fly ntennl loe re muh less well hrterize. In the olftory ul, the verterte nlog of the ntennl loe, there re ~1 lol interneurons for eh projetion neuron 4, one of the highest lol interneuron/projetion neuron rtios in ny mmmlin rin region. Olftory ul interneurons re morphologilly iverse n re hypothesize to e importnt in gin ontrol, spike synhroniztion n eorreltion of the representtions of similr oornts 4 6. Stuies of ntennl loe LNs in lrger insets hve esrie vriety of LN morphologies In omprison, the limite t ville suggeste tht Drosophil ntennl loe LNs hve reltively simple morphologies, with eh LN innervting ll glomeruli, lthough LNs tht re not pnglomerulr hve een esrie in more reent stuies Unerstning how ORN input is trnsforme into PN output y the ntennl loe requires eeper unerstning of ntennl loe LNs. Here we took systemti pproh to ientifying n hrterizing LNs in the Drosophil ntennl loe. Using speifi Gl4 lines in onjuntion with the MARCM (mosi nlysis with repressile ell mrker) metho 19 n ye fills, we nlyze the morphology of >1,5 LNs t the single-ell level. This nlysis revele remrkle iversity of glomerulr innervtion ptterns, whih n e use to efine istint morphologil lsses of LNs. Min morphologil LN lsses hve istintive eletrophysiologil properties tht help efine their potentil funtions within the iruit. At fine sle, our nlysis lso emonstrte tht the innervtion pttern of n iniviul LN vrie ross rins. Similrly, even within morphologilly or genetilly efine lss of LNs, we foun onsierle vriility in physiologil properties. This iniviul vrition my in prt reflet the role of ell ell intertions uring evelopment. Our finings imply tht LNs ply unexpetely iverse roles in olftory proessing in the fly ntennl loe. The wiring vriility of LNs suggests tht the wiring igrm iffers onsierly etween iniviul fly rins, n tht iniviul vritions in ehvior my in prt reflet these kins of fine-sle vritions t the ellulr level. 1 Howr Hughes Meil Institute, Deprtment of Biology, Stnfor University, Stnfor, Cliforni, USA. 2 Deprtment of Neuroiology, Hrvr Meil Shool, Boston, Msshusetts, USA. 3 These uthors ontriute eqully to this work. Corresponene shoul e resse to L.L. (lluo@stnfor.eu) or R.I.W. (rhel_wilson@hms.hrvr.eu). Reeive 28 Septemer 29; epte 22 Deemer 29; pulishe online 7 Ferury 21; oi:1.138/nn.2489 NATURE NEUROSCIENCE ADVANCE ONLINE PUBLICATION 1

2 21 Nture Ameri, In. All rights reserve. Figure 1 Antennl loe LNs. Expression ptterns of the ten Gl4 lines use in this stuy. Green, Gl4-riven mcd8-gfp; re, Gl4-riven nuler -gltosise or nuler RFP; lue, neuropil stining y monolonl ntioy n82, speifi for neuropil mrker. Sle rs, 2 m. (Sle is the sme for lines 1 n 3 1.) RESULTS Antennl loe LNs n their neurotrnsmitter profiles We use ten Gl4 lines to genetilly ess ntennl loe LNs. The expression ptterns of these lines spn rnge from hunres of neurons ner the ntennl loe to just hnful of LNs (Fig. 1). For simpliity, we refer to these Gl4 lines s lines 1 1 (see Online Methos). In ition to LNs whih we efine s neurons whose ell oies were ner the ntennl loe n whose proesses were restrite to the ntennl loes severl lines lso lele ORNs, PNs n/or neurons ner the ntennl loe tht projet to other res of the rin (Supplementry Fig. 1). LN ell oies re lote in two lusters: lrge ut ontinuous luster lterl n orsolterl to the ntennl loe, whih inlue most LNs lele y lines 1 n 3 9, n seprte luster ventrl to the ntennl loe, whih inlue most LNs lele y line 1 (Fig. 1 n Supplementry Fig. 1). To etermine the numer n the potentil overlp of LNs lele y these Gl4 lines, we ounte the numers of nulei lele y nuler mrkers riven from iniviul Gl4s, s well s from their omintions in the sme fly (Supplementry Tle 1). These t suggeste tht some lines lel lrgely nonoverlpping LN popultions, wheres other lines overlp prtly or ompletely. Line 5 seems to inlue ll ells tht were lele y lines 6 9. All ~56 ells lele y line 5 re LNs euse (i) line 5 riven memrne mrker mcd8-gfp i not lel ny neurons tht sen proesses out of the ntennl loe (Fig. 1), n (ii) the entire roriztions of ll 578 single ells from systemti MARCM nlysis n ioytin fill (see elow) from line 5 were within the ntennl loe. On the sis of the totl numer of LNs lele y lines 3, 4 n 5 in the sme fly (Supplementry Tle 1) n ell oy positions from neurolst lone nlysis (Supplementry Fig. 1), we estimte tht there were ~1 LNs lele y lines 1 n 3 9, whih re mostly ipsilterlly projeting LNs in the lterl luster (see elow). Line 1 lele mostly ilterlly projeting LNs (see elow) in the ventrl luster. Therefore, the lower oun for Drosophil LNs (lele y our Gl4 lines) is ~1 ipsilterlly projeting n ~1 ilterlly projeting LNs for eh ntennl loe. Although LNs re tritionlly onsiere to e inhiitory neurons tht relese GABA s their neurotrnsmitter, reent stuies hve suggeste tht some LNs n e exittory 14,2 n holinergi 14,17. We exmine the neurotrnsmitter type of ifferent Gl4 lines y oule stining with ntioies to GABA n holine etyltrnsferse. Antioy speifiity ws vlite y loss of stining in MARCM lones homozygous for null muttion of Glutmi i eroxylse-1 or Choline etyltrnsferse (Supplementry Fig. 2). Most LNs were GABAergi; however, there were few holinergi ells, n lrger minority tht were neither GABAergi nor holinergi (Supplementry Tle 1 n Supplementry Fig. 2). Stining for other neurotrnsmitter types ientifie few opminergi n mny (mostly ventrl) glutmtergi LNs (Supplementry Fig. 3). These results inite tht lthough most LNs re GABAergi, some LNs use ifferent neurotrnsmitters or their omintions, emphsizing the potentil iversity in the funtionl roles of ifferent LNs. Morphologil iversity of LNs The MARCM metho n e use to lel single neurons orn t speifi evelopmentl times 21,22. We performe systemti MARCM leling of LNs from these ten Gl4 lines throughout evelopment. Figure 2 shows representtive imges from 1,439 single-ell LN lones. The LNs showe sustntil morphologil iversity. We ientifie five ro tegories of ipsilterlly projeting LNs on the sis of the extent of their roriztion in the ntennl loe: the entire ntennl loe (pnglomerulr; Fig. 2), ll ut few glomeruli (Fig. 2), ontinuous region of the ntennl loe (Fig. 2), isontinuous or pthy regions of the ntennl loe (Fig. 2) n 1 3 glomeruli (oligoglomerulr; Fig. 2e). Within eh tegory, however, there ws onsierle iversity. For exmple, lthough mny LNs rorize nerly the entire ntennl loe, they iffere in the ensity of roriztion n the thikness of their proesses (Fig. 2, n Supplementry Fig. 4). For LNs tht skip few glomeruli or rorize in ontinuous region, ifferent iniviuls skippe ifferent glomeruli or rorize in ifferent regions of the ntennl loe (Fig. 2, n Supplementry Fig. 5). Finlly, ilterlly projeting LNs showe heterogeneous ipsilterl n ontrlterl roriztion ptterns (Fig. 2f). Suellulr istriution of presynpti terminls in LNs All neurotrnsmitter relese of LNs ours within the ntennl loe, ut oes eh LN releses neurotrnsmitter throughout its ror, or only in restrite zone? To exmine how presynpti terminls re istriute within iniviul LNs, we performe lrge suset of MARCM single-ell leling with n itionl synptotgminhemgglutinin (HA) mrker, whih lels presynpti terminls 23. In generl, synptotgmin-ha lele punt throughout n LN s proesses (Fig. 2 n Supplementry Fig. 4). These t suggest tht, s generl rule, LNs rost trnsmitter relese ross their roriztion without notle glomerulr seletivity. However, in exeption to the ove rule, some oligoglomerulr n ilterl LNs h more seletive synptotgmin-ha istriution (Fig. 2e,f n Supplementry Fig. 4). These LNs therefore seem to eliver informtion to only suset of the glomeruli they innervte. Sttistil nlysis of glomerulr innervtion ptterns Given the iversity of morphologies, we next nlyze these t systemtilly with the im of ientifying orgniztionl priniples of the LN network. An importnt feture of n LN is its glomerulr innervtion pttern, s this etermines whih olftory hnnels the LN my reeive informtion from n sen informtion to. We therefore strte y nlyzing the glomerulr innervtion ptterns of iniviul LNs (Fig. 3). 2 ADVANCE ONLINE PUBLICATION NATURE NEUROSCIENCE

3 We sore 1,532 iniviul LNs (1,439 from MARCM single-ell lones; 93 from ye fills uring whole-ell pth-lmp reoring) for their innervtion of 54 glomeruli in the ntennl loe (Fig. 3 n Supplementry Tles 2 n 3). We use hierrhil lustering to orgnize the 1,532 LNs oring to their inry ipsilterl glomerulr innervtion ptterns. This nlysis revele severl istint morphologil lsses (Fig. 3). For exmple, neurons within suluster C tene to voi ertin glomeruli innervte y trihoi 21 Nture Ameri, In. All rights reserve. e f Figure 2 Diversity of LN morphology. ( e) Representtive roriztion ptterns of single-ell MARCM lones of ipsilterlly projeting LNs. Eh row presents exmples from tegories of LNs tht innervte ll glomeruli (), ll ut few glomeruli (), ontinuous () or pthy () regions of the ntennl loe, n few glomeruli (e). (f) Representtive roriztion ptterns of ilterlly projeting LNs. Green, mcd8-gfp, leling LN proesses; re, synptotgmin-ha, mrker for presynpti terminls; lue, n82 or N-herin stining, highlighting glomerulr strutures. Some imges in (left n fourth n fifth from left) n f (thir, fifth n sixth from top left) o not inlue synptotgmin-ha stining. Arrowhes, ell oies; she lines, outline of the ntennl loe. Sle r, 2 m. Informtion out orresponing Gl4 lines in this n susequent figures n e foun in Supplementry Tle 4. NATURE NEUROSCIENCE ADVANCE ONLINE PUBLICATION 3

4 21 Nture Ameri, In. All rights reserve. Figure 3 Sttistil nlysis of glomerulr innervtion ptterns. () Binry glomerulr innervtion ptterns of 1,532 singly lele LNs orgnize y hierrhil lustering. Rows represent innervtion ptterns of iniviul ells; olumns represent 54 glomeruli. 1,489 were ipsilterl projeting LNs. Of the 43 ilterlly projeting LNs, only the ipsilterl ptterns were inlue in the lustering nlysis. Yellow, glomeruli innervte; lue, glomeruli not innervte. () Antennl loe moel. The 54 glomeruli we sore for this stuy re outline in three setions of the ntennl loe from nterior to posterior. This moel is moifie fter numer of soures 3,43 n erive from tring n82- stine rins. ( e) Three representtive imges from three selete regions of the LN luster igrm inite on the right sie in. Green, mcd8-gfp stining; lue, n82 or N-herin stining. Dshe lines in mrk pheromonl glomeruli DA1, VA1 n VA1l/m. Sle r, 2 m. (f) The LN innervtion proility of glomerulus orrelte positively with the men oorevoke firing rte of the ORNs presynpti to tht glomerulus (r =.63, P <.5, n = 23 glomeruli). ORN t from ref. 25. Firing rtes verge ross 11 oors. Fille symols represent trihoi glomeruli. (g) Prinipl omponent nlysis of LN glomerulr innervtion ptterns. In the seon imension, the umell sulss of LNs (rket) istintly seprtes from ll other ell types. LNs lele y ifferent Gl4 lines re mrke with ifferent olors. See Supplementry Figure 8 for olor oe of Gl4 lines n histogrms of ell istriutions in PC1 n PC2. ORN lsses tht sense pheromones 24 (Fig. 3). Neurons within suluster D innervte the entrl ntennl loe n skippe the orsl n ventrl glomeruli; we ll these neurons umell ells, fter their shpe (Fig. 3). Neurons within suluster E showe pthy innervtion (Fig. 3e). Clustering ll ells lele y the sme Gl4 (Supplementry Fig. 6) revele tht some Gl4 lines lele reltively uniform morphologil popultion of ells (for exmple, lines 6 8), wheres others lele more heterogeneous group (for exmple, lines 1, 2, 3 n 5). This suggests tht some Gl4 lines preominntly lel LNs elonging to just few funtionl lsses, wheres others lel funtionlly heterogeneous popultion. We will exmine this ie in more etil using eletrophysiologil reorings (see elow). Clustering ll GABA + versus GABA ells from our olletion revele tht oth sutypes inlue iversity of morphologil ptterns (Supplementry Fig. 7). Beuse most LNs re GABAergi, we onsiere the hypothesis tht LN innervtion might e enser in glomeruli tht reeive more ORN input. We estimte the verge lifetime tivity of eh ORN type using pulishe t set 25 y verging the responses of eh ORN type ross 11 oor stimuli. We foun signifint positive orreltion etween LN innervtion proility n men oor-evoke ORN firing rte (Fig. 3f). This woul e onsistent with the ie tht the ensity of inhiitory innervtion is pte to the verge level of fferent input to eh glomerulus 26, ssuming tht these stimuli re representtive of nturl oors. Single-ell innervtion ptterns Innervte Glomerulr ientity Not innervte e e f LN innervtion proility DM6 DM5 VM2 Anterior VM5v VA2 DA2 VA6 VA7m VA3 DA3 DA4m DA4l VA7l DL3 VA1m VA5 DA1 VA1l VA1 DA1 VA1 VA1lm In ition to hierrhil lustering, we use prinipl omponent nlysis to ientify emergent reltionships in the glomerulr innervtion t set without pplying ny ritrry threshol of signifine. The first n seon prinipl omponents (PC1 n PC2; Fig. 3g n Supplementry Fig. 8) for the entire set of innervtion ptterns ounte for 4% n 1% of the vrine in the t, respetively, ut 13 itionl prinipl omponents were neee to ount for 25% more. The high numer of imensions neessry to ount for vrine inites tht innervtion ptterns re not well esrie s the liner omintion of ny smll numer of si glomerulr reltionships. PC1 ws essentilly proxy for the numer of glomeruli innervte (orreltion oeffiient.9993). Thus, efining feture of n LN s innervtion pttern is the numer of glomeruli it onnets to. LNs were imolly istriute on PC2, with istintive suluster ontining minly umell ells (Fig. 3), onfirming tht the umell ells onstitute istintive morphologil lss. Next, we onsiere the LNs tht rorize ilterlly. We ompre ipsilterl innervtion ptterns (Supplementry Fig. 9) with orresponing ontrlterl innervtion ptterns (Supplementry Fig. 9). We foun tht mny ilterl LNs h symmetri glomerulr innervtion ptterns n overll, ipsi- n ontrlterl ptterns were signifintly orrelte (Person s orreltion oeffiient r =.46.5 (s.e.m.), P <.1, n = 38; Supplementry Fig. 9). In generl, the exeptions were LNs with nerly pnglomerulr ipsilterl innervtion. DM2 VM5 5 VC3l VM3 Mile DC2 VM4 D DC1 VC2 VA4 Men oor-evoke hnge in ORN firing rte (spikes s 1 ) g Seon prinipl omponent DL4 DC3 VL1 DL2v DL2 VL2 DM1 DM4 VM7 VC3m VM1 Posterior DM3 DC4 1 VP1 VM6 VP2 V DL5 DP1m VC1 VP3 DL1 DP1l VL2p First prinipl omponent 4 ADVANCE ONLINE PUBLICATION NATURE NEUROSCIENCE

5 21 Nture Ameri, In. All rights reserve. 2-Heptnone Heptnoi i Methyl sliylte Pentyl ette Gernyl ette Ethyl innmte Ethyl utyrte Butyri i Cverine Ethyl ette 2-Heptnone Heptnoi i Methyl sliylte Pentyl ette Gernyl ette Ethyl innmte Ethyl utyrte Butyri i Cverine Ethyl ette Spontneous firing rte (spikes s 1 ) Line 7 Line s.5 1. s Line 9 Line 9 52 glomeruli innervte 36 glomeruli innervte.5 1. s.5 1. s Mximum oor response (spikes s 1 ) Men oor response (spikes s 1 ) Perentge of spikes in first 1 ms of response Line 5 4. ± ± ± ± 1 Line 6 Line 7 Line 8 Line ± ± ± ± ± ± ± ± ± ± ± ± ± 2 23 ± 2 43 ± 4 17 ± 3 Finlly, on the sis of lustering, we ientifie miniml numer of LN lsses with ientifile innervtion ptterns n further investigte irth timing n Gl4 expression for those ell types (Supplementry Fig. 5 n Supplementry Tle 2). We foun tht some (lthough not ll) morphologil types of LNs were lele y only few Gl4 lines n were orn in limite time winows. Together, these nlyses inite tht LNs re ompose of genetilly istint groups of ells efine y omintion of morphology, irth timing n Gl4 expression. Diversity n stereotypy of physiologil properties Do LN geneti tegories (Gl4 lines) n morphologil tegories (glomerulr innervtion ptterns) orrelte with physiologil properties? To ress this, we selete five Gl4 lines tht lel reltively smll susets of LNs (lines 5 9) n me whole-ell pth-lmp reorings from GFP-lele somt in eh line. We reore the responses of eh ell to pnel of iverse oors (see Online Methos) n fille eh ell with ioytin using the pth pipette for visuliztion fter reoring. We suessfully fille n sore the innervtion ptterns of 93 LNs. All these LNs fire spontneous tion potentils, n their spiking ws lwys moulte y oors. LN oor responses were notly iverse n typilly vrie more ross ells thn ross oors within ell (Fig. 4,). Some LNs reore in the sme Gl4 line respone to oors in similr mnner (Fig. 4). For exmple, the LNs we reore in line 7 were reltively stereotype in their funtionl properties, s were line 8 LNs. In other ses, Gl4 line oul lel LNs hving very ifferent response profiles (Fig. 4). Line 5 n line 9 were prtiulrly iverse in this respet. In ll these LNs, we mesure spontneous firing rte, men oor response n mximum oor response. To quntify the time ourse of the oor response, we lso mesure the perentge of spikes fire uring the first 1 ms of the oor response. All four of these properties were signifintly epenent on Gl4 line (Fig. 4). This implies Figure 4 Funtionl stereotypy n iversity mong geneti lsses. () Rsters showing the similr spiking responses of two line 7 LNs. These LNs re typil of line 7 in hving high spontneous firing rtes, wek oor-evoke exittion n strong oor-evoke inhiition. Gry ox, the nominl oor stimulus perio; there is ely of out 1 ms efore oor rehes the fly. () Dissimilr responses of two line 9 LNs. The first of these innervte lmost ll glomeruli (52 of 54) n ws minly inhiite y ll oors, wheres the seon innervte smller suset of glomeruli n ws exite y ll oors. Overll, line 9 LNs were iverse. () Funtionl properties of lines 5 9 (men s.e.m.). All these properties were signifintly epenent on the Gl4 line of the reore LN (one-wy ANOVA, P <.1). For eh property, post ho Tukey s tests yiele signifint ifferenes (P <.5) etween some ut not ll of the ten pir-wise omprisons etween Gl4 lines. Oor-evoke firing rtes re expresse s hnge from the spontneous firing rte. tht ifferenes in Gl4 expression reflet physiologil ifferenes etween LNs, in spite of the vriility within eh Gl4 line n the prtil overlp in the ells tht re lele y these lines. Physiologil ifferenes etween morphologil lsses Next, we ske whether LN physiologil properties were orrelte with their morphologil lss. Beuse the prinipl xis for morphologil vrition ws the numer of glomeruli eh LN innervtes (Fig. 3g), we egn y sking whether ny response properties were orrelte with this xis. Unexpetely, LNs tht innervte mny glomeruli h lower oor-evoke firing rtes thn LNs tht innervte fewer glomeruli. The numer of glomeruli innervte y n LN ws signifintly (lthough wekly) ntiorrelte with the verge strength of its oor responses (Person s r =.2, P <.5). This fining motivte us to exmine the physiology of pnglomerulr LNs in prtiulr euse these ells innervte the lrgest numer of glomeruli. This lss omprise 28% of the ells from whih we reore. On verge, pnglomerulr LNs h signifintly higher spontneous firing rtes thn other LNs (Fig. 5). In the presene of n oor, spontneous spiking in mny pnglomerulr ells shut own ompletely, sometimes fter rief urst t oor onset, wheres others moestly inrese their firing rte in the presene of oors (Fig. 5). Overll, oor-evoke hnges in firing rte were signifintly weker in pnglomerulr ells thn in other LNs (Fig. 5). A seon min lss of LNs tht we reore from were LNs tht seletively voie glomerulus VA1 n frequently lso voie DL3 (Fig. 3). These glomeruli re innervte y trihoi ORNs thought to e seletive for pheromones 24. This innervtion pttern (with slight vritions) ounte for ~15% of ll LNs in our t set from lines 1 7, notly those lele y line 6 (Fig. 3; see elow), n 1% of ells we fille in eletrophysiologil reorings. These ells iffere from other LNs in hving espeilly trnsient ursts of exittion (Fig. 5,). Overll, this morphologil lss fire higher perentge of their spikes in the first 1 ms of the response thn i other LNs (Fig. 5). Thus, these LNs oul rete trnsient pulse of GABAergi inhiition t oor onset. If so, then some pheromone glomeruli woul eviently e seletively exlue from this trnsient pulse of inhiition. Line 6 LNs: orse stereotypy n fine vriility Does the iversity we oserve in LN morphology n physiology rise from vriility of the sme LNs ross ifferent iniviul flies, or from mny iverse LN types eh with stereotypi ptterns in ll iniviuls? The first lterntive is implie y our fining of 847 istint innervtion ptterns in 1,489 ipsilterlly projeting LNs (Fig. 3), fr NATURE NEUROSCIENCE ADVANCE ONLINE PUBLICATION 5

6 21 Nture Ameri, In. All rights reserve. Figure 5 Funtionl ifferenes etween morphologil lsses. () Pnglomerulr LNs (n = 26) h signifintly higher spontneous tivity (P <.1) n weker men n mximum oor responses (P <.5, t-tests) s ompre to ll other LNs tht were suessfully reonstrute (n = 67). () Oor responses of three pn-glomerulr LNs. () Left: pheromone-voiing LNs (n = 9) fire signifintly higher perentge of their spikes uring the first 1 ms of the oor response s ompre to ll other LNs (n = 84, P <.1, t-test). (Beuse there is ely of out 1 ms efore oor tully rehes the fly, spikes were ounte uring the perio 1 2 ms fter nominl oor onset; this ws ivie y totl spikes uring the 1-s perio shown in rsters.) Right: oor response time ourse ws more trnsient for these LNs thn for other LNs (men peristimulus time histogrm, s.e.m. ross ells). () Oor responses of three pheromone-voiing LNs. exeeing the estimte totl numer of ipsilterlly projeting LNs (~1) within n iniviul ntennl loe (Supplementry Tle 1). LN roriztion ptterns therefore nnot e ompletely stereotype ross flies. We sought to further ress this question y ense smpling of Gl4 line (line 6) tht lele smll popultion (seven LNs per ntennl loe; Supplementry Tle 1). Among 131 lele single ells for line 6, we oserve 76 istint glomerulr innervtion ptterns (Fig. 6). Beuse there were mny more innervtion ptterns thn the numer of ells per fly, iniviul line 6 lele LNs nnot e ientil ross flies. Eletrophysiologil reorings showe tht line 6 LNs lso h iverse funtionl properties (Figs. 6 e n 5). Some ells (Fig. 6,) showe trnsient urst t the onset of lmost every oor, followe y inhiition. Other ells (Fig. 6,e) showe sustine exittion n more oor-speifi tuning. Thus, neither the morphology nor the physiology of line 6 LNs ws highly stereotype. Despite the ler eviene tht iniviul LNs lele y line 6 were not rigily stereotype, the properties of these LNs were fr from rnomly istriute. For exmple, the oor response properties of line 6 ells were signifintly less vrie ross ells thn the oor response properties of ll LNs. Men n mximum oor-evoke firing rtes were less vrile within the line 6 popultion (n = 16) thn ross the LN popultion s whole (n = 92, P < 5 1 5, F-tests). In ition, spontneous firing rtes were lso less vrile within the line 6 popultion thn ross ll LNs (P < 5 1 6, F-test). The glomerulr innervtion ptterns of line 6 LNs were lso fr from rnom. Eh line 6 LN innervte 51.1 glomeruli (n = 131, s.e.m. =.22) on verge. If the innervtion pttern were ompletely rnom, then every glomerulus woul hve 94.4% proility of eing innervte. Assuming inomil proility istriution, the numer of smples innervting glomerulus shoul vry y the stnr error, 2%. The tul istriution lerly evites from this preition (Fig. 6f). Most glomeruli were lmost lwys innervte, wheres suset of glomeruli were often misse. Among this ltter group of glomeruli, VA1 ws most often omitte. Inee, most of the ioytin-fille LNs tht seletively voie VA1 (Fig. 5) were line 6 LNs (7 of 9). Beuse our inry sores for innervtion ptterns re only orse mesure of LN s morphology, we lso mesure the innervtion ensity of line 6 LNs for selete glomeruli tht were either lwys innervte or often misse. We foun tht DM1 (lwys innervte) hs signifintly higher innervtion ensity thn VA1 or DA1 (osionlly innervte), wheres rnomly selete pnglomerulr LNs showe similr innervtion ensities for DM1, VA1 n DA1. Innervtion ensity of line 6 neurons in DA1 n VA1 glomeruli ws signifintly lower thn tht of pnglomerulr LNs (Fig. 6g). These 2-Heptnone Heptnoi i Methyl sliylte Pentyl ette Gernyl ette Ethyl innmte Ethyl utyrte Butyri i Cverine Ethyl ette Perentge of spikes in first 1 ms of response 2-Heptnone Heptnoi i Methyl sliylte Pentyl ette Gernyl ette Ethyl innmte Ethyl utyrte Butyri i Cverine Ethyl ette Firing rte spikes s 1 Line s 3 2 ** * Firing rte (spikes s 1 ) Line 7 Mximum oor-evoke.5 1s Pnglomerulr LNs All other LNs Line s.5 1. s LNs voiing pheromone glomeruli All other LNs Line 8 Line 6 Line s.5 1. s.5 1. s t strengthen the istintion of these two groups of glomeruli with regr to line 6 LN innervtion. One group ws lwys innervte, wheres the other group h muh less innervtion, when not eing ompletely voie. In summry, nlysis of line 6 inite priniple likely pplile to most other morphologilly n genetilly efine lsses of LNs. LNs showe mrke vriility within lss, n some of this vriility must represent vritions in the sme ells ross ifferent rins. Yet the properties of these LNs re lerly not rwn rnomly from the entire istriution of LN properties. This piture is onsistent with the ie tht the orse properties of these ells re genetilly preprogrmme, wheres their finer-sle properties my lso reflet ftors suh s evelopmentl plstiity n sensory experiene. Pthy LNs: potentil mehnism for vriility The vriility of glomerulr innervtion ptterns is exemplifie in smll suset of LNs from lines 1, 3 n 5 tht showe istintive pttern of pthy innervtion (Figs. 2 n 3e). Remrkly, omprison of the glomerulr innervtion ptterns of 161 pthy ells (Fig. 7) inite tht none of these single ells innervte ientil sets of glomeruli. Given tht these pthy ells represent only smll frtion of ells lele y eh LN line n smll frtion of the ~1 ipsilterlly projeting LNs, it is ler tht the innervtion ptterns of pthy LNs re highly vrile ross ifferent iniviul flies. The pthy nture of these ells innervtion ptterns suggeste tht these innervtion ptterns might e estlishe through ell ell intertions mong LNs. To exmine the reltionships etween innervtion ptterns of ifferent LNs in the sme fly, we esigne geneti metho to visulize pir of sister pthy ells with two ifferent ** Spontneous Men oor-evoke * 6 ADVANCE ONLINE PUBLICATION NATURE NEUROSCIENCE

7 Glomerulr ientity 21 Nture Ameri, In. All rights reserve. Single-ell innervtion ptterns Innervte Not innervte e f 2-Heptnone Heptnoi i Methyl sliylte Pentyl ette Gernyl ette Ethyl innmte Ethyl utyrte Butyri i Cverine Ethyl ette 2-Heptnone Heptnoi i Methyl sliylte Pentyl ette Gernyl ette Ethyl innmte Ethyl utyrte Butyri i Cverine Ethyl ette Perentge of smples innervte DM1 Experimentl Theoretil Glomeruli DA1 VA1 Length/volume Pn LN6 Pn * * LN6 Pn LN6 DM1 DA1 VA1 Figure 6 Vriility n stereotypy of line 6 LNs. () Hierrhil lustering of innervtion ptterns, s in Figure 3 ut only for line 6 LNs (n = 131). Cells in e re inite. Glomeruli innervte y trihoi ORNs (pheromonl glomeruli) re highlighte in ornge. k-mens lustering verifie tht line 6 inry innervtion ptterns form single luster. Some ells were from flies in whih ntenne, mxillry plps or oth h een remove for 1 efore fixtion (Fig. 8e,f), ut these tretments i not ffet the numer or the vriility of glomerulr innervtion (Supplementry Fig. 11). ( e) Oor response of four line 6 LNs. She regions of plots enote oor stimulus perio (5 ms). (f) Comprison of experimentl (lue ots) n theoretil frequeny of innervtion if glomeruli were rnomly innervte (enter lk line). The envelope of 2% is the stnr error ssuming inomil istriution of innervtion frequenies. In the experimentl frequeny, mny glomeruli were lmost lwys innervte, signifintly ove the theoretil istriution; other glomeruli were innervte signifintly less frequently thn the theoretil istriution. The glomerulr ientities to the right of DA1 re DM5, VA1m, VA1l, DL3 n VA1; ll exept DM5 re pheromonl glomeruli. (g) Box plot quntifition of innervtion ensity of DM1, DA1 n VA1 from ten rnomly hosen LNs of eh lss tht innervte ll three glomeruli. Center line enotes the verge, the ox enloses to the limits of the top n ottom qurtiles n the whiskers exten to the mximum n minimum vlues. Innervtion ensity = totl enriti length in glomerulus / glomerulus volume. Innervtion of DA1 n VA1 y line 6 LNs (LN6) ws signifintly lower (Tukey s test, *P <.5) thn tht of ontrol LNs (pnglomerulr (pn) LNs rnomly selete from lines 1, 3 n 5). Dotte lines seprte mesurements from ifferent glomeruli. g e olors (Fig. 7). Although the effiieny of this metho ws extremely low, yieling only five pirs of pthy ells mong thousns of issete rins, it is evient tht proesses of sister pthy ells voie eh other to rve out their innervtion omins, sometimes y splitting single glomerulus (outline in Fig. 7,e n Supplementry Fig. 1). By ontrst, ifferentilly lele sister ells of other LN types intermixe t muh finer sles (Fig. 7f h). We onfirme the qulittive ifferene y quntifying the egree of overlp etween the signls of the two ells fter the signls h een systemtilly ilte to vrying egrees (see Online Methos). Non-pthy ells showe gret el of overlp with just smll mount of iltion, wheres pthy ells showe similr egrees of overlp only fter their signls h een ilte muh more (Fig. 7i). These oservtions suggest tht sister pthy LNs my tile the ntennl loe the wy gnglion ells tile the verterte retin 27 n sensory neurons tile the Drosophil oy wll 28. We lso estimte tht eh pthy LN oupie % (men s.., n = 8) of the ntennl loe volume, implying tht eight pthy LNs oul tile n entire ntennl loe. These finings suggest tht LN LN intertions ply role in estlishing pthy LN morphologies. Development n mintenne of LN innervtion ptterns Finlly, we teste the ontriutions of ORNs to the glomerulr roriztion pttern of LNs. During the onstrution of the ntennl loe iruit, PN enrites initite pttern formtion efore the rrivl of pioneering xons y trgeting enrites to positions similr to those where they re foun in the ult ntennl loe. ORN xon invsion into the ntennl loe n susequent ORN xon PN enrite reognition results in the formtion of istinguishle glomeruli 29,3. The evelopment of LNs hs not previously een esrie. NATURE NEUROSCIENCE ADVANCE ONLINE PUBLICATION 7

8 21 Nture Ameri, In. All rights reserve. Figure 7 Vriility of pthy LNs. () Clustering of 161 pthy ell innervtion ptterns, s in Figure 3. No two ells h ientil innervtion ptterns. () Shemti of MARCM FLP-out, whih llows two sister ells to e lele y ifferent olors. UAS-FRT-CD2-FRT-mCD8GFP (ref. 4) serves s reporter of Gl4. After FLPmeite mitoti reomintion uses the loss of Gl8 in the gnglion mother ell (GMC), CD2 (re) shoul e expresse in oth ughter ells erive from this GMC. However, if n itionl FLP-out event ours in one of the two ughter ells, this ell will express mcd8-gfp (green) inste of CD2. ( h) Exmples of two sister pthy ells ( e) n two sister ontrol ells (f h) lele y MARCM FLP-out, shown with N-herin (lue), GFP (green) n CD2 (re) stining. (,f) Projetion of the entire ntennl loe. (,e,g,h) High mgnifition of 5- m projetions from nterior (,g) n mile (e,h) ntennl loe setions, showing nonoverlpping proesses from two sister ells. Dshe lines, ounries of glomeruli VA1l/m () n DL1 n DC2 (e). Sle rs, 2 m. (i) Sister ell overlp inex s funtion of ege length of the iltion kernel (see Online Methos). Dshe lines inite the men for pthy sister ells (rightshifte) n non-pthy sister ells (left-shifte). Colore lines inite iniviul pirs of sister ells. A quntittive istintion etween pthy n non-pthy lsses ws verifie y k-mens lustering into two lusters. We first teste whether LN roriztion uring pupl evelopment require ORN xons (Fig. 8 ). We me use of the fts tht Hegehog signling is require for ntennl is prolifertion, n lonl loss of Smoothene, omponent essentil for Hegehog signling, results in osionl loss of mxillry plps while leving the ntenne intt 3. Uner this onition, ORN xons from the ntenne woul innervte glomeruli tht re norml trgets of ntennl ORN xons, ut glomeruli tht re norml trgets of mxillry plp ORNs re evoi of ORN input 3. By introuing line 5 Gl4 riven mcd8-gfp into this geneti kgroun n exmining flies with ilterl loss of mxillry plps (~1 in 25; Fig. 8), we foun tht LN proesses were not present in the glomeruli tht re norml trgets of mxillry plp ORNs. LN proesses still innervte the nery glomeruli tht re trgets of ntennl ORNs (Fig. 8, ottom pnels, ompre to Fig. 8, top pnels; Fig. 8). By ontrst, PN enriti proesses were mostly still present in glomeruli evoi of ORN xons (Fig. 8,). This experiment suggests tht glomerulr innervtion of LNs requires the presene of ORN xons. To test whether ORN xons re require for the mintenne of LN proesses, we ilterlly remove the mxillry plps in ults, whih is well fter ntennl loe wiring is omplete. We exmine the proesses of line 6 LNs (whih re suset of line 5 LNs exmine in the previous experiment (Supplementry Tle 1)) t lest 1 fter removl of the mxillry plps to llow omplete egenertion of ORN xons. We foun tht LN proesses still innervte mxillry plp glomeruli (Fig. 8e). The volume of one of the two glomeruli quntifie ws signifintly lower (Fig. 8f, top pnel), likely euse ORN xons ontriute to the glomerulr volume 31. However, the totl length of LN proesses n the totl numer of presynpti terminls s mrke y synptotgmin-ha punt were unffete y the ult removl of ORNs (Fig. 8f, mile n ottom pnels). We lso ompre vriility of glomerulr innervtion ptterns of line 6 LNs etween wil-type Single-ell innervtion ptterns Innervte GAL8 Glomerulr ientity FLPse GAL4 UAS>CD2 y + >CD8GFP Neurolst Not innervte e f g h FLPse UAS>CD2 y + >CD8GFP Gnglion mother ell UAS>CD8GFP UAS>CD2 y + >CD8GFP Single ell i Overlp Non-pthy LN pirs Pthy LN pirs 5 Diltion (ege length, pixels) flies n those with ORNs remove. We foun no signifint ifferenes in either the numer of glomeruli innervte or unique innervtion ptterns (Supplementry Fig. 11), suggesting tht vriility of line 6 LNs is not epenent on the presene of ORN xons in ult. Tken together, these experiments inite tht ORN xons re essentil for LN innervtion uring evelopment ut re not require for the mintenne of their glomerulr innervtion in ulthoo. DISCUSSION Knowlege of lol interneuron properties is essentil to unerstn how neurl iruit proesses informtion. Here we present systemti single-ell nlysis of >1,5 ntennl loe LNs in Drosophil. The sle of our nlysis is n orer of mgnitue greter thn tht of the most omprehensive nlysis of inset LNs yet pulishe 8. Our geneti strtegy lso voie smpling ises involve with ye filling, whih fvors lrge ells with esy ess. Despite the vet tht our smpling of LNs ws not entirely even (see Online Methos), our stuy revele iversity of ntennl loe LNs eyon tht esrie previously in ny inset. LNs were iverse in their neurotrnsmitter types, glomerulr innervtion ptterns, fine enriti strutures, ensities n istriution of presynpti terminls, n oor response properties. Furthermore, our nlysis of glomerulr innervtion ptterns n oor responses revele orse stereotypy ut lso n unexpete egree of finesle vriility in genetilly efine sulsses of LNs. Below we isuss the implitions of these finings for the evelopment n funtion of the ntennl loe iruit, n for the omplexity of the Drosophil rin s moel for neurl iruit nlysis. How is the morphologil iversity of LNs estlishe? The finl glomerulr innervtion ptterns of LNs likely result from some omintion of (i) geneti speifition, (ii) ell-ell intertions 8 ADVANCE ONLINE PUBLICATION NATURE NEUROSCIENCE

9 21 Nture Ameri, In. All rights reserve. 2 AT 2 MP 2 AT MP 2 AT 2 MP 2 AT MP e VC2 VA7l Unut MP ut AT + MP ut uring evelopment n (iii) neuronl tivity n plstiity. Our stuy suggests tht these mehnisms re use ifferentilly in speifying ifferent spets of LN iversifition. We lso nnot rule out itionl ontriutions of sex, ge n rering onitions (see Online Methos). We envisge the istintions etween mjor LN tegories eing speifie lrgely y geneti mehnisms similr to those use to speify other types of entrl neurons, suh s ntennl loe PNs. These inlue neurolst linege, irth timing n speifi gene expression. For exmple, most ilterlly projeting LNs h ell oies lote ventrl to the ntennl loe, were lele y line 1, n erive from istint neurolst (Supplementry Fig. 1). All pthy LNs were orn uring well efine time winow in the mi-lrvl stge (Supplementry Fig. 5 n Supplementry Tle 2) n were erive exlusively from lines 1, 3 n 5. Dumell ells erive exlusively from lines 1, 2 n 9 n were likewise orn uring efine lrvl stge (Supplementry Fig. 5 n Supplementry Tle 2). At finer level, even though line 6 LNs showe mny glomerulr innervtion ptterns, these ptterns were ise ginst ertin pheromonl glomeruli. Furthermore, the ensities of roriztion n presynpti terminls within glomeruli tht were lwys innervte were higher thn the ensities within those tht were only osionlly innervte f VA7l perentge (%) VC2 perentge (%) Volume (µm 3 ) Totl proess length (µm) No. of Syt-HA punt Innervte VA7l/VC2 Un-innervte VA7l/VC2 VA7l/VC2 sent AT 2 MP 41 GH146 * * WT M A M VA7l 2 AT MP AT 2 MP 2 AT MP Line 5 WT M A M VC2 Figure 8 Development ut not mintenne of LN roriztion epens on ORNs. () Top: norml ult fly he with two ntenne (2 AT, rrows) n two mxillry plps (MP, rrowhes). Bottom: osionlly, eyflpinue smo lones eliminte oth MPs (open rrowhes). Sle rs, 1 m. (,) Brins from norml (top) n -MP (ottom) flies rrying Line5-Gl4 () or GH146-Gl4 () lele y n82 n Gl4-riven mcd8gfp s inite. The MP ORN trget VA7l glomerulus (otte irle) ws not innervte y line 5 proesses in -MP flies ut ws still innervte y PN proesses. Arrows esignte glomeruli tht re innervte y GH146-negtive PNs. Sle rs, 2 m. () Quntifition of glomerulr innervtion y Line5-Gl4 LN n GH146-Gl4 PN proesses in the presene or sene of ORN innervtion of MP trget glomeruli VA7l (top) n VC2 (ottom). (e) Representtive single setions of line 6 LN single-ell lones fter ult removl of MPs or of oth MPs n ATs. Control smples (unut) show line 6 LN innervtion of VA7l (top) n VC2 (ottom). After ult removl of MPs or of oth MPs n ATs, line 6 LNs still innervte VA7l n VC2. Blue, n82; green, mcd8-gfp; re, synptotgmin (Syt)-HA. (f) Quntifition of glomerulr volume, LN proess length n the numer of Syt-HA punt in ontrol (WT), MP ut ( M) or MP n AT ut ( A M) rins. The VA7l volume ws signifint lower when ORN proesses were remove, ut neither proess length nor the numer of synptotgmin-ha punt in VA7l n VC2 ws signifintly reue. Error rs, s.e.m. y line 6. One my imgine tht mehnisms tht trnslte linege n irth timing to wiring speifiity of PNs n other glomerulr trgeting mehnisms 32 n lso e use to speify glomerulr innervtion speifiity of ertin types of LNs. Compre to ORNs n PNs, however, LN glomerulr innervtion ptterns were muh more vrile n less eterministi. The most noteworthy exmple ws pthy LNs: the 161 single ells we exmine showe 161 istint glomerulr innervtion ptterns. Our two-ell lone nlysis suggests evelopmentl mehnism for hieving this iversity. We foun tht proesses of sister pthy ells tene to voi eh other even when they innervte ommon glomeruli. Our quntittive nlysis is onsistent with the ie tht these omplex enrites tile the three-imensionl volume of the ntennl loe. These t suggest tht homotypi repulsion might ply role in estlishing finl innervtion ptterns of some LN morphologil types. From funtionl perspetive, it seems likely tht the speifi glomerulr innervtion ptterns of iniviul pthy LNs re unimportnt s long s group of similr types of pthy LNs over the entire ntennl loe. Funtionl implitions Lol interneuron lsses efine y morphology n moleulr mrkers re thought to hve istint funtions 1. The Drosophil ntennl loe represents n unusul opportunity to ssess in vivo the funtionl orreltes of LN lsses. Overll, we foun tht LNs vrie wiely in their oor-evoke firing rtes n their temporl ynmis. NATURE NEUROSCIENCE ADVANCE ONLINE PUBLICATION 9

10 21 Nture Ameri, In. All rights reserve. The seletive moultion of LNs with prtiulr ynmis oul e wy to moulte the temporl pttern of PN tivity. On orse level, some physiologil iversity orrelte with ifferenes in morphology or geneti mrkers. For exmple, pnglomerulr LNs h unusully high rtes of spontneous tivity, suggesting tht they my e prtiulrly importnt in rosting toni inhiition throughout the ntennl loe. Toni inhiition is thought to regulte the sensitivity of oor etetion in the mmmlin olftory ul 33,34. Pnglomerulr LNs either were inhiite y oors or were only wekly exite. Beuse they re minly GABAergi, inhiition of pnglomerulr LNs woul ten to isinhiit the entire ntennl loe in response to oors. To tke nother exmple, LNs tht voi ertin pheromoneseletive glomeruli fire unusully trnsient ursts t oor onset. These LNs my hve speil role in inhiiting the initil 1 ms of PN oor responses, when PN firing rtes pek 35. Some pheromone glomeruli re eviently exlue from this inhiition. This my e n pttion to the reltively wek ORN firing rtes eliite y pheromones 24 n my help explin why wek ORN responses to pheromones n e strongly mplifie in postsynpti PNs 36. Although we foun orse orreltion etween some funtionl properties n morphologil or geneti lssifitions, we lso foun tht eh LN lss we exmine in finer etil ontine sustntil iversity. For exmple, oth pnglomerulr n pheromone-voiing LNs h vrie oor responses. Similrly, line 6 LNs were lso heterogeneous, espeilly onsiering tht there were only ~7 suh ells. Thus, we re unlikely to e le to urtely preit every spet of n LN s physiology on the sis solely of its morphology or few moleulr mrkers. Notly, interneurons of the mmmlin ortex re lso remrkly vrie, prompting the remrk tht inhiitory ells seem to e so iverse s perhps to ennger the notion of ell types in the entrl nervous system 37. Here we hve shown tht interneuron iversity in the Drosophil rin rivls tht of the mmmlin rin, n we hve egun to estlish evelopmentl priniples unerlying this iversity. Complexity of the Drosophil rin It is wiely elieve tht, ompre to vertertes 38, inverterte nervous systems re muh more stereotype ross ifferent memers of the sme speies, with iniviully ientifile neurons n stereotype onnetion ptterns. Inee, previous nlysis of projetion ptterns of iniviul ntennl loe PNs provie notle exmple in support of this notion 39,4. Thus, the egree of vriility of glomerulr innervtion ptterns of iniviul LNs ws unexpete. Suh vriility shoul trnslte into vriility in the funtionl onnetivity of LNs, ORNs n PNs. Although the glomerulr orgniztion of the ntennl loe provies onvenient n unequivol emonstrtion of vriility in the Drosophil onnetome, it remins to e etermine whether vrile onnetivity is generl feture of the fly rin. Interestingly, even the xons of ntennl loe PNs, whih showe notly stereotype terminl roriztion ptterns in the lterl horn, show less stereotypy in the mushroom oy 23,39,4. Inee, reent physiology stuy suggests non-stereotypi onnetions etween PN xons n their postsynpti trgets in the mushroom oy 41. It is possile tht, upon etile exmintion, suh lk of stereotypy is the norm rther thn n exeption, even in the reltively simple inset rin. We n others 42 elieve tht wiring igrm is neessry (lthough ertinly not suffiient) prerequisite for unerstning the funtion of neurl iruits. However, our results imply tht the omplete reonstrution of the wiring igrm of single Drosophil rin will not yiel generl wiring igrm for ll Drosophil rins. At the sme time, our finings ientify funmentl similrity etween inset n verterte rins in the vriility of their onnetomes. Although it my prove more hllenging thn previously ssume to mp the onnetivity of the fly rin, solving the Drosophil onnetome my turn out to e more relevnt to unerstning our own rin. METHODS Methos n ny ssoite referenes re ville in the online version of the pper t Note: Supplementry informtion is ville on the Nture Neurosiene wesite. ACKNOWLEDGMENTS We thnk U. Heerlein (University of Cliforni, Sn Frniso) n E. Mrin for respetively proviing n sreening unpulishe Gl4 lines, whih le to ientifition of line 1 n line 6; J. Simpson (Jneli Frm, Howr Hughes Meil Institute) for proviing unpulishe LCCH3 (line 7) Gl4; K. Wehner (Stnfor University) for mouse nti-ha; A. DiAntonio (Wshington University) for rit nti-dvglut; n the Bloomington Stok Center, Kyoto Stok Center, Drosophil Geneti Resoure Center, Gl4 Enhner Trp Insertion Dtse (GETDB), n Developmentl Stuies Hyriom Bnk for other regents. M.L.S. is grteful for the help of J. Brooks in t olletion. We thnk the Stnfor Deprtment of Sttistis Consulting Servie for tehnil help with sttistil nlyses. We thnk T. Clninin, G. Jefferis n memers of the Luo n Wilson lortories for helpful omments on the mnusript. This work ws supporte y US Ntionl Institutes of Helth grnts to L.L. (R1-DC5982) n R.I.W. (R1-DC8174), Pew Sholr wr, MKnight Sholr wr, Slon Fountion reserh fellowship, n Bekmn Young Investigtor wr (to R.I.W). M.L.S. is supporte y US Ntionl Reserh Servie preotorl wr. E.Y. is supporte y Humn Frontiers Siene Progrm Long Term Fellowship. J.C.S.L. is supporte y the Meil Sientist Trining Progrm t Stnfor University. L.L. reeives investigtor support from the Howr Hughes Meil Institute. AUTHOR CONTRIBUTIONS Y.-H.C. n M.L.S. performe the ntomil n evelopmentl experiments. E.Y. performe the physiologil experiments. J.C.S.L. helpe with sttistil nlysis. L.L. n R.I.W. supervise the projet n wrote the pper. COMPETING INTERESTS STATEMENT The uthors elre no ompeting finnil interests. 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