© 2010 Nature America, Inc. All rights reserved. R.I.W. ([email protected]). Boston, USA. Massachusetts, 1 logically diverse and are hypothesized to be in important any gain control, in ratios morpho­ are interneurons bulb neuron Olfactory region. brain mammalian interneuron/projection local highest the of neuron projection each for interneurons local ~100 are there characterized. well less much are lobe antennal PNs class ORN one from input direct receive to glomerulus single a into dendrites send (PNs) neurons projection lobe antennal most and glomerulus, lobe antennal same to the axons their project receptor odorant same the express that (ORNs) neurons receptor olfactory All tractability. genetic its and organization functional and anatomical elegant its of how nervous systems process information characterization of local interneurons is a prerequisite to asunderstanding such organisms simpler in exists diversity of kind same the whether clear less is However,it and physiology. Different types are thought to have distinct functions local interneuron types, as defined by morphology, histological markers transmitted into the brain. Every vertebrate brain region contains many contrast, motion and color can be encoded by retinal ganglion cells and incoming signals from photoreceptors, such that information regarding and amacrine cells in the vertebrateneurons retinaare important in neural arecomputation. instrumentalFor example, horizontal in transformingneurons,processes whose areregion.restrictedsingle a to Local inter­ whoseaxons connect discrete neuraltissue, regions of localinter­and Neurons can be divided into two general categories: projection neurons, Drosophila degree of complexity and individual variation in an invertebrate neural circuit, a result that creates challenges for solving the during development, and some individual variability also likely reflected LN–LN Our interactions. results reveal an unexpected variability within each or morphological genetic class. Finally, LN innervation required interaction with olfactory receptor neurons However, the finer-scale connectivity of an individual LN varied considerably across brains, and there was notable physiological stereotyped glomerular innervation patterns. Some of these classes morphological showed distinct properties. physiological properties. physiological Analysis of >1,500 individual LNs revealed principal classes morphological by characterized coarsely first olfactory processing center in the brain. We found LNs to be diverse in their profiles, neurotransmitter connectivity and anatomical and analysis of electrophysiological local interneurons (LNs) in the Local interneurons are essential in information processing by neural circuits. Here we present a genetic, comprehensive Ya-Hui Chou interneurons in the Diversity and wiring variability of olfactory local nature nature Received 28 September 2009; accepted 22 December 2009; published online 7 February 2010; Howard Howard Hughes Medical Institute, Department of Biology, Stanford University, Stanford, California, USA. In the olfactory bulb, the vertebrate analog of the antennal lobe, lobe, antennal the of analog vertebrate the bulb, olfactory the In because circuit neural model attractive an is lobe antennal fly The 3 . In contrast to these input and output neurons, LNs in the fly fly the in LNs neurons, output and input these to contrast In . NEUR 3 . Genetic access is available to many classes of ORNs and and ORNs of classes many to available is access . Genetic connectome. OSCI 1 , 3 , Maria L Spletter EN C E

3 These These authors contributed equally to this work. should Correspondence be addressed to L.L. or ([email protected]) advance online publication online advance Drosophila . Systematic identification and identification Systematic . 1 2 , . 3 , Emre Yaksi Drosophila 2 ,

3 , Jonathan C S Leong 4 , one one , 1 .

kinds of fine of kinds these reflect part in may behavior in variations individual that and differs the fly diagram considerably between individual wiring brains, that suggests LNs of variability wiring The lobe. antennal fly the in imply that LNs play diverse unexpectedly findings roles in processing olfactory Our development. during interactions cell–cell of role the may in reflect variation part This individual properties. physiological in variability considerable LNs, we found of class defined genetically or morphologically a within even Similarly, brains. across varied LN individual an of pattern innervation the that demonstrated also sis the circuit. At a our within analy scale, fine functions potential their classes have distinctive electrophysiological properties that help define to classes define of distinct LNs.morphological used Main LN morphological be can which patterns, innervation glomerular of diversity >1,500 LNs at the single method marker) cell repressible with analysis (mosaic MARCM the with conjunction in the in LNs izing LNs. lobe antennal of understanding into PN by a output requires lobe the deeper antennal is transformed been have described in more recent studies panglomerular not are that LNs although glomeruli, all innervating LN each with morphologies, simple relatively have LNs that suggested available data limited the morphologies LN of variety a described have of odorants similar representations the of decorrelation and synchronization spike Here we took a systematic approach to identifying and character and identifying to approach systematic a took we Here antennal lobe doi:10.1038/nn.248 - Drosophila Drosophila melanogaster scale variations at the cellular level. cellular the at variations scale 1 , Rachel I Wilson Drosophila 4–6 2 Department Department of Neurobiology, Harvard Medical School, . Studies of antennal lobe LNs in larger insects insects larger in LNs lobe antennal of Studies . 1 9 and dye fills, we analyzed the morphology of of morphology the analyzed we fills, dye and - cell cell level. This analysis revealed a remarkable antennal lobe. Using specific Gal4 lines lines Gal4 specific Using lobe. antennal 9 12–18 2 . Understanding how ORN input & Liqun Luo antennal lobe, the Drosophila 7–11 t r a . In comparison, comparison, In .

1 antennal lobe lobe antennal C I e l s  ­ ­ © 2010 Nature America, Inc. All rights reserved. Antibody specificity was validated by loss of stainingacetyltransferase. choline in and MARCMGABA to clones antibodies with staining ble We examined the neurotransmitter type of different have Gal4 lines by dou studies excitatory be can LNs recent some that suggested neurotransmitter, their as GABA release that projecting projecting and ~100 bilaterally projecting LNs for each antennal lobe. bilaterally mostly bound for lower the labeled Therefore, cluster. ventral 10 the in below) (see Line LNs clus lateral below). the in (see LNs ter projecting ipsilaterally 3–9, and mostly 1 are lines by which labeled LNs ~100 were there that estimate we ( analysis clone neuroblast from positions ( fly same lines the by in 5 labeled and LNs 4 3, of number total the of basis the On lobe. anten the nal within were 5 line from below) (see fill biocytin and sis analy MARCM systematic from cells single 578 all of arborizations ( lobe antennal the of out 5–driven processes send line (i) mCD8 because LNs marker are 5 membrane line by labeled cells ~56 All completely. or partly overlap lines other whereas populations, LN nonoverlapping fly largely same the in combinations their ( from as well as Gal4s, vidual indi from driven markers nuclear by labeled nuclei of numbers the counted we lines, Gal4 these by labeled LNs of overlap potential the 10 line by labeled ( LNs most included which lobe, antennal the to ventral cluster separate a and 3–9, and 1 lines by labeled LNs most included which lobe, antennal the to dorsolateral and lateral cluster ( brain the of areas other to project that lobe antennal the near neurons and/or were processes whose to restricted the lines antennal also lobes—several ORNs,labeled and PNs lobe antennal the near were bodies cell whose neurons as defined we LNs—which to addition In Methods). Online ( (see 1–10 LNs lines as lines of Gal4 these to handful refer we a simplicity, For just to lobe of antennal hundreds the from near range a neurons span lines these of patterns expression The LNs. lobe antennal access genetically to lines Gal4 ten Weused Antennal lobe LNs and their neurotransmitter profiles RESULTS 3–10.) 1 and lines for same the 20 bars, Scale marker. a neuropil for specific nc82, antibody monoclonal by staining nuclear Gal4-driven red, mCD8-GFP; Gal4-driven 1 Figure t r a  of mutation null a for homozygous Supplementary Table 1 Supplementary Fig. Fig.

Although Although LNs are considered traditionally to be neurons inhibitory 6–9. lines by labeled were that cells all include to seems 5 Line continuous but large a clusters: two in located are bodies cell LN Drosophila 1 and and

C I Antennal lobe LNs. Expression patterns of the ten Gal4 lines used in this study. Green, study. Green, this in used lines Gal4 ten the of patterns Expression LNs. lobe Antennal Supplementary Fig. 1 Fig. Supplementary Supplementary Fig. 1 Fig. Supplementary e l LNs (labeled by our Gal4 lines) is ~100 ipsilaterally ipsilaterally ~100 is lines) Gal4 our by (labeled LNs s ). These data suggested that some lines label label lines some that ). suggested data These - Supplementary Table Supplementary GFP did not label any neurons that that neurons any label not did GFP ). ). To determine the number and and number the Todetermine ). Glutamic acid Glutamic Fig. Fig. 14,20 Supplementary Fig. Supplementary β 1 -galactosidase or nuclear RFP; blue, neuropil neuropil blue, RFP; nuclear or -galactosidase and ), and (ii) the entire entire the (ii) and ),

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morphological diversity. morphological ten from single these images 1,439 from LNs times development. of throughout lines Gal4 developmental labeling MARCM specific systematic at performed born neurons single individual LNs ( LNs individual of patterns innervation by the glomerular therefore started analyzing to.We information channels send and from olfactory information receive may which LN the determines this as pattern, innervation glomerular its is LN an of feature important An network. LN the of principles organizational data identifying of these aim the with analyzed systematically next we morphologies, of diversity the Given patterns innervation glomerular of analysis Statistical innervate. they glomeruli the of a subset to only information deliver ( synaptotagmin selective more a had LNs bilateral selectivity. glomerular notable without zation as a general rule, LNs broadcast transmitter release across their arbori ( processes synaptotagmin general, In terminals presynaptic labels which marker, (HA) hemagglutinin single MARCM of subset large a performed we LNs, individual within distributed are terminals presynaptic how To arbor, examine zone? its restricted a in only or throughout neurotransmitter releases LN each does but lobe, antennal the within occurs LNs of release neurotransmitter All LNs in terminals presynaptic of distribution Subcellular ( patterns arborization contralateral and lateral Fig. 5 in different regions of the antennal lobe ( or arborized glomeruli different skipped individuals region, different Fig. 4 tion and the thickness of their processes ( nearly the entire antennal lobe,arborized they LNs differed in many the density of arboriza although example, For diversity. considerable (oligoglomerular; uous or patchy regions of the antennal lobe ( ( (panglomerular; lobe antennal entire the lobe: antennal the in arborization their of extent the of basis the Fig. 2 Fig. 2e, Fig. We identified five broad categories of ipsilaterally projecting LNs on However, in exception to the above and rule, some oligoglomerular ). Finally, LNs ipsi showed projecting heterogeneous bilaterally ). For LNs that skip a few glomeruli or arborize in a continuous in a continuous or arborize ). a For skip few glomeruli that LNs b µ ), a continuous region of the antennal lobe ( m. (Scale is (Scale m. f and and advance online publication online advance Fig. Fig. Supplementary Fig. Supplementary 2 and Fig. Fig. 2 Fig. - cell labeling with an additional synaptotagmin additional an with labeling cell

3 Supplementary Fig. 4 Supplementary ). The MARCM method can be used to label label to used be can method MARCM The LNs of diversity Morphological LNs. different of roles functional the combinations, emphasizing their or neurotransmitters different use LNs most some GABAergic, are LNs although that indicate results These ( LNs ventral) glutamatergic few (mostly a many and identified dopaminergic types neurotransmitter 2 Fig. Supplementary ( cholinergic nor GABAergic neither were that minority there were a few cells, cholinergic and a larger Fig. 2 or e - ). Within each category, however, there was was however, category, there each ). Within cell LN clones. The LNs showed substantial cell LN clones. The LNs showed substantial Choline acetyltransferase Choline - HA labeled puncta throughout an LN’s an throughout puncta labeled HA ). Most LNs were GABAergic; however, GABAergic; ). were LNs Most

4 ). These LNs therefore seem to to seem therefore LNs These ). Fig. 2 upeetr Tbe 1 Table Supplementary Figure Figure Fig. 2b Fig. 2a

potential diversity in the the in diversity potential Fig. 2 ). These data suggest ). that, data suggest These a nature nature ), all but a few glomeruli ), but all a few glomeruli Supplementary Fig. 3 Fig. Supplementary 2 , , shows representative representative shows b ). Staining for other other for Staining ). c Fig. 2 d and and ) and 1–3 glomeruli Fig. 2 - ( HA distribution distribution HA Supplementary Supplementary NEUR Supplementary Supplementary f Supplementary ). c ), discontin OSCI 21,22 We . and and EN C 2 ). 3 E ­ ­ ­ ­ - .

© 2010 Nature America, Inc. All rights reserved. presents examples from categories of from LNs categories ( examples all that presents glomeruli innervate 2 Figure ( lobe and antennal the in glomeruli 54 of innervation their for whole during fills dye from 93 clones; nature nature of the antennal lobe. Scale bar, 20 fourth and fifth from left) and Some in images structures. glomerular highlighting blue, nc82 staining, a or terminals; N-cadherin for marker presynaptic synaptotagmin-HA, lobe, and a ( few glomeruli We scored 1,532 individual LNs (1,439 from MARCM single MARCM from (1,439 LNs individual 1,532 Wescored Supplementary Tables Supplementary f e d c b a

Diversity of Diversity LN morphology. ( NEUR OSCI EN C E e

). ). ( advance online publication online advance f f (third, fifth and sixth from top left) do not include synaptotagmin-HA staining. Arrowheads, cell bodies; dashed lines, outline

) Representative arborization patterns of bilaterally projecting LNs. mCD8-GFP,Green, projecting of patterns bilaterally arborization ) Representative red, LN labeling processes; 2 2 and µ m. Information about corresponding Gal4 lines in this and subsequent figures can be found in 3 a – ). We used hierarchical clustering e ) Representative arborization patterns of single-cell MARCM clones of ipsilaterally projecting LNs. Each row projecting MARCM of clones of patterns ipsilaterally single-cell arborization ) Representative - cell patch cell - clamp recording) recording) clamp

Fig. 3 Fig. a - ), ), all but a ( few glomeruli cell cell b

glomerular innervation patterns. This analysis revealed several several ­subcluster C tended to by avoid innervated trichoid glomeruli certain revealed analysis This binary ( classes morphological ­distinct patterns. their to innervation according LNs ­glomerular 1,532 the organize to b ), continuous ( ), continuous c Fig. 3 ) ) or ( patchy a ). For example, neurons within d ) regions ) of regions the antennal Supplementary Table 4 t r a c

C I (left and ipsilateral ipsilateral e l s .  © 2010 Nature America, Inc. All rights reserved. is adapted to the average level of afferent input to glomerulus each input afferent of level to average the is adapted innervation inhibitory of density the that idea the with be consistent odor mean and ability found a significant positive correlation between LN innervation prob set data published averaging the responses a of each ORN across type using 110 odor stimuli. type We ORN each of activity lifetime input. ORN We more receive average the that estimated in glomeruli denser be might innervation LN that hypothesis the considered we morphological ( patterns of diversity a included subtypes both that revealed below). (see recordings electrophysiological using detail more in idea this examine We will population. functionally heterogeneous a label others whereas classes, functional few a just to This suggests that some Gal4 lines predominantly label LNs belonging a labeled others group more (for example, heterogeneous lines 1, 2, whereas 3 and 5). 6–8), lines example, (for cells of population logical morpho uniform relatively a labeled lines Gal4 some that revealed ( innervation after their shape cells, ( dumbbell neurons these call we glomeruli; ventral the and dorsal skipped and lobe antennal central the innervated D subcluster pheromones sense that classes ORN PC2. and PC1 in distributions cell of histograms and lines 8 Figure Supplementary See colors. different with marked are lines Gal4 different by labeled LNs types. cell other all from separates distinctly (bracket) LNs of subclass dumbbell the dimension, second the In patterns. innervation glomerular ( glomeruli. trichoid represent symbols Filled odors. 110 across averaged rates Firing n ( glomerulus that to presynaptic ORNs the of rate firing evoked odor- mean the with positively correlated glomerulus a of probability innervation 20 bar, Scale VA1l/m. and VA1d DA1, glomeruli pheromonal in lines Dashed staining. N-cadherin or nc82 blue, staining; mCD8-GFP Green, in side right the on indicated diagram cluster LN the of regions selected three from images ( brains. stained sources of number a after modified is model This posterior. to anterior from lobe antennal the of sections three in outlined are study this for scored we glomeruli 54 The model. ( innervated. not glomeruli blue, innervated; glomeruli Yellow, analysis. clustering the in included were patterns ipsilateral the only LNs, projecting bilaterally 43 the Of LNs. projecting ipsilateral were 1,489 glomeruli. 54 represent columns cells; individual of patterns innervation represent Rows clustering. hierarchical by organized LNs labeled singly 1,532 of patterns innervation ( patterns. innervation 3 Figure t r a  odors. natural of representative are stimuli these that assuming g = 23 glomeruli). ORN data from ref. 25. 25. ref. from data ORN glomeruli). 23 =

) Principal component analysis of LN LN of analysis component Principal ) lseig l GABA all Clustering Clustering all cells labeled by the same Gal4 ( 3,43 C I

Statistical analysis of glomerular glomerular of analysis Statistical Supplementary Fig. 7 Fig. Supplementary and derived from tracing nc82- tracing from derived and e l Fig. 3 Fig. c – s Fig. 3 e r ) Three representative representative Three ) e = 0.63, 0.63, = ). a µ ) Binary glomerular glomerular Binary ) - d m. ( m. for color code of Gal4 Gal4 of code color for evoked ORN firing rate ( rate firing ORN evoked ). Neurons within subcluster E showed patchy + b ess GABA versus ) Antennal lobe lobe Antennal ) f ) The LN LN The ) P < 0.005, 0.005, < ). Because most LNs are GABAergic, GABAergic, are LNs most ). Because c

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© 2010 Nature America, Inc. All rights reserved. during the first 100 during ms the of odor response. All four proper these of fired spikes of the percentage we the odor response, of measured also course time the To response. quantify odor maximum and response respect. this in diverse particularly were 9 line ( profiles response different LNs very label having could line a 8 Gal4 cases, LNs. as were line In other properties, tional func their in stereotyped relatively were 7 line in recorded we LNs ( manner similar a in odors to odors responded across than cells ( cell across a within more varied typically notably and were diverse responses odor LN odors. by modulated always was successfully filled and scored the innervation patterns of 93 LNs. with biocytin using the patch pipette for visualization aftercell to recording.a panel of diverse odorsWe (see Online Methods) and filled each cell from GFP subsets of LNs (lines 5–9) and made whole ties? To address this, we selected five Gal4 lines that label relatively small (glomerularinnervation patterns) correlate with physiological proper categories morphological and lines) (Gal4 categories genetic LN Do properties physiological of stereotypy and Diversity expression. Gal4 and timing distinct groups of cells defined by a combination of morphology, birth windows. time limited in Together,born were these analyses indicate that LNs and are composed of genetically lines Gal4 labeled few a were LNs only by of types morphological all) not (although some types cell those ( for further expression and Gal4 and patterns timing birth innervation investigated identifiable with classes LN of nature nature ( line Gal4 on dependent significantly were ties Supplementary Fig. 5 Fig. Supplementary In all these LNs, we measured spontaneous firing rate, mean odor odor mean rate, firing spontaneous measured we LNs, these all In All these LNs fired action potentials, spontaneous and their number a minimal we identified clustering, Finally, of on basis the c b a Line Line Line Line Line Meth Meth Eth Eth Geran Geran Heptanoic acid Heptanoic acid NEUR Pe Pe - Eth Eth 2-Heptanone 2-Heptanone Eth Eth yl cinnamate yl cinnamate labeled somata in each line. We recorded the responses of each 9 8 7 6 5 Butyr Butyr ntyl acetate ntyl acetate yl salicylate yl salicylate Cada Cada yl b yl b yl acetat yl acetat yl acetat yl acetat Spontaneous (spik 16.8 OSCI fir Fig. 4a Fig. utyrate utyrate ic acid ic acid 7.8 5.2 4.5 4.0 ve ve ing rate r r es ine ine ± ± ± ± ± 2.1 0.6 0.7 0.4 1.7 e e e e s EN –1 0 0 ) , 52 glomer b C and and odor response ). Some LNs recorded in the same Gal4 line line Gal4 same the in recorded LNs Some ). E (spik

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± ± ± ± ± uli inner 0.5 0.5 1.1 1.2 1.0 5.5 1.7 s –1 9 ) v odor response ated (spik –0.3 –3.1 2.5 8.7 7.7 1.0 1.0 Mean es ± ± ± ± ± s s 0.6 1.4 3.5 0.7 1.2 - 0 0 s cell patch 36 glomer –1 Fig. 4 Fig. ) Pe Fig. 4 Fig. rcentage ofspik a Line Line in first100ms ). For example, the the example, For ). Fig. Fig. 4 of response uli inner 0.5 0.5 - 17 43 23 21 14 clamp recordings 7 9 ). We that found c ± ± ± ± ± ). This implies implies ). This b

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s s spiking spiking ­ ­ ­ phological variation was variation phological the number each of glomeruli LN innervates mor for axis principal the Because class. morphological their with correlated were properties physiological LN whether Next, we asked classes morphological between differences Physiological lines. these by labeled are that cells the in overlap partial the and line Gal4 each within variability the of LNs, in spite between differences physiological reflect expression Gal4 in differences that rate. firing spontaneous the from a change as expressed are rates firing Odor-evoked lines. Gal4 between comparisons pair-wise ( differences significant ANOVA, (one-way LN recorded the of line Gal4 the on dependent significantly were ( diverse. were 9 LNs line Overall, odors. all by excited was and glomeruli of subset a smaller innervated second the whereas odors, all by inhibited mainly was and 54) of (52 glomeruli all almost innervated these of first fly. the ( reaches odor before ms 100 about of a delay is there period; stimulus odor nominal the box, Gray inhibition. odor-evoked strong and excitation odor-evoked weak rates, firing spontaneous high having 7 in line of typical are LNs ( 4 Figure innervation patterns in innervation 1,489 projecting LNs ipsilaterally ( distinct 847 of finding by our implied is alternative first The viduals? from many diverse LN each types patterns with stereotypic in all indi or flies, individual different across LNs same the of variability from Does the diversity we observed in LN morphology and physiology arise variability fine and stereotypy coarse 6 LNs: Line inhibition. of pulse excluded from this transient be selectively would evidently glomeruli pheromone some so, then If onset. odor at inhibition GABAergic of ( LNs other did than 100 the ms response in of the spikes first their of percentage ( excitation cells These recordings. electrophysiological in filled we cells of 10% and ( 6 line by labeled those notably 1–7, lines from for all LNs accounted ~15% of in our data set variations) slight (with pheromones for selective be to thought avoided also ( DL3 frequently and VA1d glomerulus avoided selectively ( LNs other in than cells panglomerular in weaker significantly were rate firing rate ( firing odors of their presence the in increased modestly others whereas onset, burst odor brief at a after sometimes completely, down shut panglo­ cells many in merular spiking ( LNs spontaneous odor, an other of than presence the rates In firing signifi spontaneous had LNs higher cantly panglomerular average, On from recorded. cells the we of which 28% comprised class This glomeruli. of largest the number innervate cells these because particular in LNs merular (Pearson’s responses odor its of strength average the with anticorrelated weakly) (although significantly was by an LN innervated glomeruli of number The glomeruli. fewer vate many odor lower innervate had that glomeruli LNs Unexpectedly, axis. this with correlated ( c a Fig. 3 Fig. ) Functional properties of lines 5–9 (mean (mean 5–9 lines of properties ) Functional These 7 LNs. line two of responses spiking similar the showing ) Rasters A second main class of LNs that we recorded from were LNs that that LNs were from recorded we that LNs of class main second A panglo­ of physiology the examine to us motivated finding This

differed from other LNs in having especially transient bursts of of bursts transient especially having in LNs other from differed Fig. 3 Fig. g

), we began by asking whether any response properties were were properties response any whether asking by began ), we P Functional stereotypy and diversity among genetic classes. classes. genetic among diversity and stereotypy Functional < 0.0001). For each property, property, each For < 0.0001). Fig. Fig. 5 Fig. Fig. Fig. Fig. c ). These glomeruli are innervated by trichoid ORNs ORNs trichoid by innervated are glomeruli These ). 5 5 c c a , d ). Thus, these LNs could create a transient pulse pulse transient a create could LNs these Thus, ). ). b ). Overall, this morphological class fired a fired class higher ). morphological this Overall, ) Dissimilar responses of two line 9 LNs. The The 9 LNs. line two of responses ) Dissimilar P < 0.05) between some but not all of the ten ten the of all not but some between < 0.05) Fig. Fig. - evoked firing rates than LNs that inner that LNs than rates firing evoked 5 b ). Overall, odor ). Overall, post hoc post 2 4 ± r . This innervation pattern pattern innervation This . s.e.m.). All these properties properties these All s.e.m.). = −0.2, −0.2, = Tukey’s tests yielded Tukey’s yielded tests - Fig. 3 Fig. evoked changes in changes evoked t r a P < 0.05). < a ; see below), below), see ; Fig. 3 C I Fig. Fig. e l a

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© 2010 Nature America, Inc. All rights reserved. Innervation density of Innervation line 6 neurons in DA1 and VA1d was glomeruli DA1.VA1dDM1, and for densities innervation similar showed LNs DA1 or VA1d than panglomerular selected whereas randomly density innervated), (occasionally innervation higher significantly a has We missed. or often innervated) (always DM1 that always found innervated either were that glomeruli selected for LNs 6 line of innervation density the measured also we morphology, LN’s a of measure 9). of (7 LNs 6 line biocytin the of most Indeed, omitted. often most VA1d was glomeruli, of group latter this Among missed. often were glomeruli of subset a whereas ( prediction the by vary should standard glomerulus error, a innervating samples of number the distribution, probability binomial a Assuming innervated. being of probability 94.4% a have would glomerulus every then random, were completely pattern innervation the If on average. = s.e.m. 0.22) ( glomeruli 51.1 innervated LN 6 line Each random. from ( LNs all across than population 6 the line within variable were less also rates firing tion, spontaneous ( as a whole LN population the ( population 6 line the within variable less were odor maximum and Mean LNs. all of properties response odor the than cells across varied less were significantly cells domly distributed. For example, the odor response properties of line 6 not stereotyped, the rigidly properties of these LNs were far from ran stereotyped. highly was 6 LNs line of physiology odor more ( cells by Other inhibition. odor, followed every almost of onset the at burst transient a showed diverse functional properties ( flies. across identical be than the cells per of number fly, LNs cannot line 6–labeled individual ( terns pat innervation glomerular distinct 76 observed we 6, line for cells per lobe; LNs antennal (seven population small a labeled that 6) (line line Gal4 a flies. across stereotyped be completely therefore cannot patterns LN arborization lobe antennal an ( within (~100) individual LNs projecting ipsilaterally of number total estimated the exceeding LNs. pheromone-avoiding three of responses histogram, time peristimulus (mean LNs other for than LNs these for transient more was course time response odor Right: rasters.) in shown period 1-s the during spikes total by divided was this onset; odor nominal after ms 100–200 period the during counted were fly, the spikes reaches actually odor before ms 100 about ( LNs other all to compared as response odor the of ms 100 first the during spikes their of ( LNs pheromone-avoiding ( t ( ( LNs glomerular 5 Figure t r a  ( LNs panglomerular of that than lower significantly n reconstructed successfully were that LNs other all to compared as -tests) P

= 67). ( = 67). < 0.01) and weaker mean and maximum odor responses ( responses odor maximum and mean weaker and < 0.01) Because Because our binary scores for patterns innervation are only a coarse far also were LNs 6 line of patterns innervation glomerular The Despite the clear evidence that individual LNs labeled by line 6 were had also LNs 6 line that showed recordings Electrophysiological of sampling dense by question this address further to Wesought Fig. 6 Fig.

C I Functional differences between morphological classes. ( classes. morphological between differences Functional b ) Odor responses of three pan-glomerular LNs. ( LNs. pan-glomerular three of responses ) Odor - specific tuning. Thus, neither the morph the neither Thus, tuning. specific Fig. 6 Fig. a e l - filled LNs that selectively avoided VA1d( avoided selectively that LNs filled ). Because there were many more innervation patterns patterns innervation more many were there Because ). ± n Supplementary Table 1 Table Supplementary n 2%. The actual distribution clearly deviates from this = 26) had significantly higher spontaneous activity activity spontaneous higher significantly had = 26) s = 84, = 84, f ). Most glomeruli were almost always almost were glomeruli Most ). P n < 0.01, < 0.01, = 9) fired a significantly higher percentage percentage higher a significantly fired = 9) Fig. 6d Figs. 6b n = 92, P t , -test). (Because there is a delay of a delay is there (Because -test). e < 5 × 10 × 5 < ) showed sustained excitation and excitation ) showed sustained – ± e P s.e.m. across cells). ( cells). across s.e.m. and ). Among 131 labeled single single labeled 131 Among ). < 5 × 10 −6 Supplementary Table 1 Supplementary 5c , F ). Some cells ( - test). −5 n - evoked firing rates rates firing evoked = 16) than across across than 16) = , F - Fig. 6 Fig. tests). In addi tests). o c Fig. 5 Fig. P ) Left: ) Left: logy nor the the nor logy

< 0.05, < 0.05, innervated, innervated, d g Fig. 6b n ) Odor ) Odor a ). These These ). d = 131, 131, = ) ) Pan- ) were were )

, c ). ) ­ ­ ­ method to visualize a pair of sister patchy cells with two different different two genetic with a cells patchy sister designed of we pair fly, a same visualize to the method in LNs different of patterns actions among LNs. To examine the relationships between these innervation patterns might be established through cell–cell inter across variable that flies. highly individual clear are different is LNs patchy it of LNs, patterns projecting innervation the ipsilaterally small a ~100 and the line LN of each by fraction labeled cells of fraction represent small a cells only patchy these that Given glomeruli. of sets identical patchy 161 ( of cells patterns innervation glomerular the of comparison ( innervation patchy distinc of a showed pattern that tive 5 and 3 1, lines from LNs of subset small a in exemplified is patterns innervation glomerular of variability The variability for mechanism a potential LNs: Patchy experience. sensory and plasticity developmental as such factors finer their whereas preprogrammed, genetically are cells these of properties coarse the that idea the with from the entire distribution of LN properties. This picture is consistent brains. Yet the properties of these LNs are clearly not drawn randomly in the same cells across must different variations represent variability this of some and class, a within variability marked showed LNs LNs. of classes defined genetically and morphologically other most to ble avoided. completely being not when innervation, less much had group other the whereas always was group One innervation. LN 6 line to regard with two these of glomeruli of groups the distinction data strengthen Meth d c b a Meth Eth Eth Geran Geran Heptanoic acid Heptanoic acid Pe Pe Eth The The patchy nature of these cells’ innervation patterns suggested that applica likely principle a indicated 6 line of analysis summary, In Eth 2-Heptanone Eth 2-Heptanone Eth yl cinnamate yl cinnamate Butyr Butyr ntyl acetate yl salicylate ntyl acetate Cada yl salicylate Cada yl b yl b yl acetat yl acetat yl acetat yl acetat Fig. 7 Fig. utyrate utyrate ic acid ic acid ve ve Percentage of r r advance online publication online advance ine ine spikes in first 100 ms e e e e a 0 0

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10 20 30 Firing rate 0 spikes s–1 10 15 0 5 Line Line 8 0.5 0.5 Spontaneous ** 9 * 1.0 s Firing rate 1.0 (spikes s–1) s odor-e 10 20 30 0 0 0 0 Mean vo ** - ke scale properties may also reflect properties scale Figs. 2d 2d Figs. Line Line 0.5 0.5 d 0.5 odor-e 7 6 Maximum

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© 2010 Nature America, Inc. All rights reserved. sister patchy LNs may tile the antennal lobe the way ganglion cells cells ganglion way the lobe antennal the tile may LNs patchy sister had been dilated much more ( signals their after only overlap of degrees similar showed patchy cells whereas dilation, of amount small a just with overlap of deal great a Non Methods). Online (see degrees varying to signals of the two cells after the signals had been systematically dilated qualitative difference by quantifying the degree of overlap between the ( scales finer much at intermixed types 10 Fig. in (outlined glomerulus single a ting each other to carve out their innervation domains, sometimes by split patchy sister of avoided cells that processes it brains, sected is evident dis of thousands among cells patchy of pairs five only low,yielding colors ( glomeruli. different from measurements separate lines Dotted 5). 3 and 1, lines from selected randomly LNs (pan) (panglomerular LNs (Tukey’s * lower test, significantly was (LN6) 6 LNs line VA1d by and DA1 of Innervation volume. / glomerulus glomerulus in length dendritic = total density Innervation values. minimum and maximum the to extend whiskers the and quartiles bottom and top the of limits the to encloses box the average, the denotes line Center glomeruli. three all innervated that class each of LNs chosen randomly ten VA1d from and DA1 ( glomeruli. pheromonal are DM5 except all VA1d; and DL3 VA1l, VA1m, the DM5, to are DA1 of right identities glomerular The distribution. theoretical the than frequently less significantly innervated were glomeruli other distribution; theoretical the above significantly innervated, always almost were glomeruli many frequency, experimental the In frequencies. innervation of distribution binomial of envelope The line). black (center innervated randomly were glomeruli if innervation of frequency theoretical ( ( fixation d before 10 for removed been had both or palps maxillary antennae, which in flies from were cells Some cluster. a single form patterns innervation 6 binary line in Cells 6 Figure nature nature retina vertebrate the tile b – e

) Odor response of four line 6 LNs. Shaded regions of plots denote odor stimulus period (500 ms). ( ms). (500 period stimulus odor denote plots of regions Shaded 6 LNs. line four of response ) Odor Single-cell innervation patterns a ). By contrast, differentially labeled sister cells of other LN LN other of cells sister labeled differentially contrast, By ). Fig. 7 b

NEUR – Variability and stereotypy of line 6 LNs. ( 6 LNs. line of stereotypy and Variability e are indicated. Glomeruli innervated by trichoid ORNs (pheromonal glomeruli) are highlighted in orange. orange. in highlighted are glomeruli) (pheromonal ORNs trichoid by innervated Glomeruli indicated. are b Inner OSCI ). Although the efficiency of this method was extremely va ted EN Fig. 8e Fig. C Glomer E

2 7 , f advance online publication online advance and sensory neurons tile the the tile neurons sensory and ), but these treatments did not affect the number or the variability of glomerular innervation ( innervation glomerular of variability the or number the affect not did treatments these but ), ular identity Fig. 7 Not inne i ). These observations suggest that rv Fig. 7d Fig. Fig. 7f Fig. ated , – e a h and and ) Hierarchical clustering of innervation patterns, as in in as patterns, innervation of clustering ) Hierarchical - ). We confirmed the the ). Weconfirmed patchy cells showed showed cells patchy Supplementary Supplementary d b e c f Drosophila

Percentage of Meth Meth Eth Eth Geran Geran Heptanoic acid Heptanoic acid

samples innervated 100 P Pe Eth Eth 40 60 80 2-Heptanon 2-Heptanon Eth Eth entyl acetate yl cinnamate yl cinnamate Butyr Butyr yl salicylate ntyl acetate yl salicylate 0 Cada Cada yl b yl b yl acetate yl acetate yl acetate yl acetate utyrate utyrate ic acid ic acid ve ve ­ ­ DM r

r Theoretical Exper ine ine e e 1 development of LNs has not previously been described. been previously not has LNs of development glomeruli distinguishable of in formation the results nition into the antennal lobe and subsequent ORN axon–PN dendrite recog axon invasion ORN lobe. antennal adult the in found are they where to those similar to positions dendrites by axons targeting pioneering of arrival the before formation pattern initiate dendrites PN circuit, lobe antennal the of construction the During LNs. of pattern zation Finally, we tested the contributions of ORNs to the glomerular arbori patterns innervation LN of maintenance and Development patchy morphologies. LN establishing in role a play interactions LN–LN that suggest findings These lobe. antennal entire an tile could LNs patchy eight (mean 1.6% wall body b d imental Glomer 2 8 . We also estimated that each patchy LN occupied 13.1 13.1 occupied LN patchy each that . Weestimated also uli ± s.d., g ) Box plot quantification of innervation density of DM1, DM1, of density innervation of quantification plot ) Box n DA = 8) of the antennal lobe volume, implying that that implying volume, lobe = antennal the 8) of VA 1 1d f ) Comparison of experimental (blue dots) and and dots) (blue experimental of ) Comparison Figure 3 Figure g ±

2% is the standard error assuming a assuming error standard the is 2% Length/volume 0.15 0.00 0.05 0.10 e c a but only for line 6 LNs ( 6 LNs line for only but k -means clustering verified that that verified clustering -means

DM Pan P < 0.05) than that of control control of that than < 0.05) Supplementary Fig. 11 Fig. Supplementary D 1 LN6

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© 2010 Nature America, Inc. All rights reserved. ORNs ( by synaptotagmin LN processes and the total number of presynaptic terminals as marked volume glomerular the to contribute lower( significantly was glomeruli ( ORN axons. We found that LN processes still innervated maxillary palp after removal of the maxillary palps to allow complete ( experiment previous the in the processes line of 6 LNs (which are a subset line of 5 LNs examined We examined completed. is wiring lobe antennal after well is which adults, in palps maxillary the removed bilaterally we processes, LN axons. ORN of presence the requires LNs ( processes were devoid present mostly still ORN of in axons glomeruli to compared nearby the ( ORNs antennal of innervated are targets that glomeruli still processes LN ORNs. palp maxillary of targets normal are that glomeruli the in present not were processes palps (~1 loss in bilateral of maxillary 250; mCD8 ORN input of are ORNs devoid palp maxillary of targets normal are that glomeruli but axons, ORN antennal of targets normal are that glomeruli innervate would nae intact antennae the leaving while palps maxillary of loss occasional in results naling, sig Hedgehog for essential a component Smoothened, of loss clonal and proliferation, disc antennal for required is signaling Hedgehog ( axons ORN required ment clustering into two clusters. and non-patchy classes was verified by cells. A quantitative distinction between patchy Colored lines indicate individual pairs of sister shifted) and non-patchy sister cells (left-shifted). indicate the mean for patchy sister cells (right- dilation kernel (see Online Methods). Dashed lines overlap index as a function of edge length of the and DC2 ( boundaries of glomeruli VA1l/m ( processes from two sister cells. Dashed lines, antennal lobe sections, showing nonoverlapping projections from anterior ( lobe. ( staining. ( N-cadherin (blue), GFP (green) and CD2 (red) ( patchy cells ( instead of CD2. ( cells, this cell will express mCD8-GFP (green) FLP-out event occurs in one of the two daughter derived from this GMC. However, if an additional (red) should be expressed in both daughter cells of Gal80 in the ganglion mother cell (GMC), CD2 mediated mitotic recombination causes the loss (ref. 40) serves as a reporter of Gal4. After FLP- different colors. which allows two sister cells to be labeled by patterns. ( Figure 3 of 161 patchy cell innervation patterns, as in Figure 7 t r a  ity of glomerular innervation patterns of line 6 LNs between wild f Fig. Fig. 8c

– h To test whether ORN axons are required for the maintenance of of maintenance the for required are axons ORN whether test To develop pupal during arborization LN whether tested first We ) labeled by MARCM–FLP-out, shown with d , - e a Fig. 8 , GFP into this genetic background and examining flies with with flies examining and background genetic this into GFP C I d , Variability of patchy LNs. ( . No two cells had identical innervation c e g b ). This experiment suggests that glomerular innervation of of innervation glomerular that suggests ).experiment This , ). Scale bars, 20 , ) Schematic of MARCM–FLP-out, f h Fig. 8 ) Projection of the entire antennal c ) High magnification of 5- e l – f e Fig. 8 Fig. , middle and bottom panels). We also compared variabil ) and two sister control cells UAS-FRT-CD2-FRT-mCD8GFP c 3 – e 0 s h ). The volume of one of the two glomeruli quantified . Under this condition, ORN .axons ORN from anten the Under condition, this - ) Examples of two sister HA puncta were unaffected by the adult removal of b , top panels; panels; top , d , Fig.8 g µ ) and middle ( m. ( Fig. Fig. 8a d Supplementary Table1 Supplementary f i ) and DL1 , top panel), likely because ORN axons ORN ,panel),because toplikely ) Sister cell a 3 0 ) Clustering . By introducing line 5 Gal4–driven 5 line .Gal4–driven By introducing Fig. 8 Fig. µ k – -means m d 3 ). Wethat facts the of use made e 1

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S>CD2 v some combination of (i) geneticfrom specification, result (ii) likely cell LNs of patterns innervation glomerular final The established? LNs of diversity morphological the is How Drosophila lobe antennal the of function and development the for findings these of implications we the Below discuss LNs. of subclasses defined fine genetically in of degree variability scale unexpected an also but stereotypy coarse revealed responses odor and patterns innervation glomerular of of analysis our distribution Furthermore, and properties. response odor and densities terminals, presynaptic structures, innervation dendritic glomerular fine types, patterns, neurotransmitter their in diverse were LNs insect. any in antennal previously of described that diversity beyond LNs a lobe revealed study our Methods), even Online entirely (see not was LNs of sampling our that caveat access. the easy Despite with cells large favors which filling, dye with involved published yet LNs insect of analysis comprehensive most the of that than greater Drosophila single systematic a present we Here under information. to processes circuit essential neural is a how stand properties interneuron local of Knowledge DISCUSSION tenance their of glomerular innervation in adulthood. LN innervation during development but are not required for the main for essential axonsare ORN that together,indicate experiments these Takenadult. in axons ORN of presence the on dependent not is LNs patterns ( innervation unique or innervated glomeruli of number the either in flies and those with ORNs removed. We found no significant differences ated y + > CD8GFP FLP f c ase Supplementary Fig.Supplementary 11 advance online publication online advance brain as a model for neural circuit analysis. circuit neural for model a as brain . The scale of our analysis is an order of magnitude magnitude of order an is analysis our of scale The . 8 UA . Our genetic strategy also avoided sampling biases biases sampling avoided also strategy genetic Our . Single cell S>CD2 UA S>CD8GFP - cell analysis of >1,500 antennal lobe LNs in in LNs lobe antennal >1,500 of analysis cell y + > CD8GFP d g

circuit, and for the complexity of the the of complexity the for and circuit, Overlap i ), suggesting that variability of line 6 ), of suggesting that variability 0.2 0.4 0.6 0.8 1.0 0 0 Dilation (edgelength,pix

nature nature e h Pa Non-patch - NEUR cell interactions tc hy LNpairs OSCI y LNpairs els) EN 5 0 C E ­ ­ -

© 2010 Nature America, Inc. All rights reserved. terminals within glomeruli that were always innervated were higher higher were innervated always were that glomeruli within terminals glomeruli. Furthermore, the densities and of arborization presynaptic pheromonal certain against biased were patterns these patterns, tion finer level, even line 6 though LNs showed innerva many glomerular lar ( defined stage a val during born likewise were and 9 and 2 1, lines from exclusively derive cells Dumbbell 5. and 3 1, lines from exclusively ( in mid the window time a defined well during were born ( neuroblast distinct a from bodies cell located had ventral to the LNs antennal lobe, were labeled by projecting line 10, and derive bilaterally most expres example, gene For sion. specific and timing birth lineage, neuroblast include These PNs. lobe antennal as such neurons, central of types other ify largely by to similar specified mechanisms genetic those used to spec Methods). Online (see conditions rearing and age rule sex, of cannot contributions also additional We out diversification. LN of aspects different ifying spec in differentially used are mechanisms these that Our suggests study plasticity. and activity neuronal (iii) and development during nature nature than the those within densities that were only innervated occasionally Supplementary Supplementary Fig. 5 We envisage the distinctions between major LN categories being being categories LN major between distinctions the Weenvisage e c a VC2 VA7l 2 AT 0 MP 2 AT 2 MP 2 AT 0 MP 2 AT 2 MP NEUR Supplementary Fig. 5 Fig. Supplementary OSCI Uncut EN C and E

b advance online publication online advance Supplementary Supplementary Table 2 Supplementary Fig. 1 Fig. Supplementary MP cut and and Supplementary Table2 Supplementary AT +MPcut ) ) and were derived ). All patchy LNs LNs patchy All ). - larval stage stage larval

). At a At ). d f Total process 3 ­ ­ ­ ­ ­ No. of Syt-HA puncta length (µm) Volume (µm ) VC2 VA7l 400 600 800 200 12 16 20 40 60 percentage (%) percentage (%) 100 0 4 8 VA Un-inner Inner 0 0 100 20 40 60 80 widely in widely their odor LN of classes. correlates Overall, we found that LNs functional varied akr ae huh t hv dsic functions to assess opportunity an unusual represents lobe antennal distinct have to molecular thought and are morphology markers by defined classes interneuron Local implications Functional are LNs lobe. antennal entire patchy the individual of u patterns innervation glomerular specific the that likely seems it perspective, functional a morphological From types. LN some of patterns in innervation role a final play establishing might repulsion homotypic that suggest data These three the tile dendrites complex these that idea the with consistent is analysis quantitative avoid each common Our other even glomeruli. when they innervated achieving for to tended diversity. cells patchy this sister Weof processes that found mechanism developmental a suggests analysis clone two Our patterns. innervation glomerular distinct 161 showed noteworthy example was patchy LNs: the 161 single cells we examined most The deterministic. less and variable more much were patterns 20 40 60 80 0 7l/VC2 absent 0 n Compared Compared to ORNs and PNs, however, innervation LN glomerular important as long as a group of similar types of patchy LNs cover LNs patchy of types similar of group a as long as important v 30 GH146 V WT ated A7l * 2 AT 2 MP

–M v * ated 41

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2 AT 0 MP A7l/VC2 V 28 VC2 A7l/VC2 WT Line –M 2 AT 2 MP

–A–M 35 5 2 AT 0 MP - evoked firing evoked rates firing and their dynamics. temporal - certain types of LNs. of types certain of specificity innervation glomerular specify mechanisms targeting glomerular other and PNs of specificity ing that lineage and translate birth timing to wir by 6.line mechanisms that One may imagine Scale bars, 20 20 bars, Scale PNs. GH146-negative by innervated are that glomeruli designate Arrows processes. PN by innervated still was but flies 0-MP in processes 5 line by innervated not was circle) (dotted VA7l glomerulus target ORN MP The indicated. as mCD8GFP Gal4-driven and nc82 by labeled carrying flies (bottom) 0-MP and (top) normal from 100 bars, Scale arrowheads). induced eyFLP- occasionally, Bottom: arrowheads). (MP, palps AT, (2 maxillary two and arrows) ( ORNs. on depends arborization LN of 8 Figure VA7l and VC2. Blue, nc82; green, mCD8-GFP; mCD8-GFP; green, nc82; Blue, VC2. VA7l and ATs, and MPs innervated both still 6 LNs line of or MPs of removal adult After (bottom). VC2 and VA7l (top) of innervation 6 LN line show ATs. (uncut) and MPs both samples of Control or MPs of removal adult after clones single-cell ( (bottom). VC2 and VA7l (top) glomeruli target MP of innervation ORN of absence or GH146-Gal4 by innervation glomerular reduced. Error bars, s.e.m. bars, Error reduced. significantly was VC2 VA7l and in puncta synaptotagmin-HA of number the nor length process neither but removed, were processes ORN when lower significant was VA7l volume The ATbrains. and MP or (–A–M) (–M) cut cut MP (WT), control in puncta Syt-HA of number the and length process LN volume, glomerular of ( (Syt)-HA. synaptotagmin red, a e dimensional volume of the antennal lobe. lobe. antennal the of volume dimensional ) Top: normal adult fly head with two antennae antennae two with head fly ) Top:adult normal ) Representative single sections of line 6 LN line of sections single ) Representative

Line5-Gal4 smo Development but not maintenance maintenance not but Development clones eliminate both MPs (open (open MPs both eliminate clones PN processes in the presence presence the in processes PN µ m. ( m. ( d b ) Quantification of ) Quantification ) or ) or 3 2 Line5-Gal4 can also be used to to used be also can GH146-Gal4 t r a 1 . The The . µ f ) Quantification ) Quantification m. ( m. b Drosophila , C I in in vivo LN and and LN c

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© 2010 Nature America, Inc. All rights reserved. the function of neural circuits. However, our results imply that the the that imply results our However, circuits. neural of understanding function for the prerequisite sufficient) not certainly (although brain. insect simple relatively the in even exception, an than upon detailed examination, such lack of is stereotypy the norm rather in targets the mushroom body their postsynaptic non suggests study in body the mushroom stereotypy less show horn, lateral the in patterns arborization terminal stere otyped notably showed which PNs, lobe antennal of axons the even is connectivity a variable general feature of the fly brain. Interestingly, the in variability of demonstration unequivocal and convenient a vides functional the PNs. and ORNs LNs, in of connectivity variability into translate should variability Such of glomerular innervation patterns of individual LNs was unexpected. notion this of in support example notable a provided PNs lobe antennal individual of patterns jection stere and neurons identifiable individually with species, same the of bers mem different across stereotyped more much are vertebrates systems nervous to compared that, believed widely is It the of Complexity diversity. this underlying principles developmental to we and establish have begun the in diversity in types cell system” nervous of central the notion the endanger to perhaps as diverse so be to seem cells that the remark “inhibitory prompting varied, remarkably are also cortex mammalian the of Notably,markers. lar interneurons LN’s physiology on the basis solely of its morphology or a few molecu cells. such Thus, ~7 we are only unlikely to be were able to accurately there predict every aspect of that an considering especially geneous, LNs had odor varied responses. Similarly, line 6 LNs were also hetero­ diversity. For example, and both panglomerular pheromone substantial contained detail finer in examined we class LN each that properties and or morphological genetic classifications, we also found PNs postsynaptic in amplified strongly be can pheromones ­pheromones by elicited rates firing ORN weak relatively the to adaptation an be may This inhibition. this from excluded evidently are glomeruli one peak rates firing ms PN when 100 responses, initial odor PN the of inhibiting in role special a onset. have odor may at LNs These bursts transient unusually fired glomeruli ­selective odors. to response in lobe antennal entire the disinhibit to tend would LNs panglomerular of inhibition GABAergic, mainly are they Because excited. weakly only bulb in olfactory odor the of detection mammalian the regulate sensitivity to thought is Tonic inhibition lobe. antennal the throughout bition inhi tonic broadcasting in important particularly be may they that activity, suggesting spontaneous of ular rates LNs high had unusually ferences or in markers. genetic morphology For example, panglomer activity. PN of pattern temporal the modulate to way could be a dynamics particular LNs of with modulation The selective t r a 1 a of single diagram the of wiring reconstruction ­complete 0 e n others and We pro lobe antennal the of organization glomerular the Although functional some between correlation coarse a found we Although pheromone certain avoid that LNs example, another take To dif with correlated diversity physiological some level, On a coarse

Drosophila 33,34 o typed connection patterns. Indeed, previous analysis of pro of analysis previous Indeed, patterns. connection typed C I . Panglomerular LNs either were inhibited by odors or were were or odors by inhibited were either LNs . Panglomerular e l 2 4 and may help explain why weak ORN responses to to responses ORN weak why explain help may and ‘connectome’, it remains to be determined whether whether determined be to remains it ‘connectome’, Drosophila s 4 2 - believe that a wiring diagram is a necessary necessary a is diagram wiring a that believe Drosophila stereotypic connections between PN axons and and axons PN between connections stereotypic brain rivals that of the mammalian brain, brain, mammalian the of that rivals brain 3 7 . Here we have shown that interneuron interneuron .that Here we have shown brain 39,40 23,39,40 . Thus, the degree of variability . variability Thus, of the degree . Indeed, a recent physiology 4 1 3 . It is that, possible 5 . Some pherom Some . 3 8 , invertebrate invertebrate , Drosophila - 3 avoiding 6 . ­ ­ ­ ­ ­ ­ ­ ­ ­ -

analyses. We thank T. Clandinin, G. Jefferis and the members of Luo and Wilson for Consulting Statistics Service technical of help statistical with Department M.L.S. is for grateful the J.help of Brooks in data collection. We thank the Stanford (GETDB), and Developmental Studies Hybridoma Bank for other reagents. Drosophila Genetic Resource Center, Gal4 Enhancer Trap Database Insertion for rabbit anti (Stanford University) for mouse anti Medical Institute) for providing LCCH3unpublished (line 7) Gal4; K. Wehner line 1 of identification and line 6; J. Simpson Farm,(Janelia Howard Hughes for respectively providing and screening Gal4 unpublished lines, which lead to We thank U. Heberlein (University California,of San Francisco) and E. Marin Acknowledgmen is availableNote: information on the Supplementary online the in at http://www.nature.com/natureneuroscience/. paper the of ­version available are references associated any and Methods M brain. own our standing Drosophila the solving brain, fly the of connectivity the map to assumed ously previ than challenging more prove may it Although connectomes. their of variability the in brains vertebrate and insect between all larity for simi diagram fundamental a identify findings wiring our time, same the At general brains. a yield not will brain 8. 4. 3. 2. 1. reprintsandpermissions/. http://npg.nature.com/ at online available is information permissions and Reprints Published online at http://www.nature.com/natureneuroscience/. The authors declare no competing financial interests. COM analysis. L.L. and R.I.W. the project and supervised wrote the paper. E.Y. the performed physiological experiments. J.C.S.L. statistical with helped Y. AUTH Medical Institute. Stanford University. L.L. receives investigator fromsupport the Howard Hughes Fellowship. J.C.S.L. is bysupported the Medical Scientist Training Program at award. E.Y. is bysupported a Human Frontiers Science Program Long Term (to R.I.W). M.L.S. is bysupported a US National Research predoctoral Service Foundation research fellowship, and a Beckman Young Investigator award (R01 US National Institutes Health of to grants L.L. (R01 for laboratories helpful comments on the manuscript. This work was bysupported 10. 9. 7. 6. 5. -

ethods H.C. and M.L.S. the performed anatomical and developmental experiments.

Seki, Y. & Kanzaki, R. Comprehensive morphological identification and GABA and identification morphological Comprehensive R. Kanzaki, & Y. Seki, in Bulb. Olfactory C.A. Greer, & W.R. Chen, G.M., Shepherd, Vosshall, L.B. & Stocker, R.F. Molecular architecture of smell and taste in approaches new brain: tiny a in circuits neural Cracking R.I. Wilson, & S.R. Olsen, H. Markram, ot, . Sn XJ & asn C Mrhlg ad pta dsrbto o bee of distribution spatial and Morphology C. Masson, & X.J. Sun, C., Fonta, temporal and synchronization for mechanisms Distinct G. Laurent, & K. MacLeod, Christensen, T.A., Waldrop, B.R., Harrow, I.D. & Hildebrand, J.G. Local interneurons Wachowiak, M. & Shipley, M.T. Coding and synaptic processing of sensory information bulb olfactory of function and Origin F.T.P.M.,A. Merkle, Lledo, Alvarez-Buylla, & immunocytochemistry of antennal lobe local interneurons in A Physiol. Comp. J. Neurosci. Rev. Annu. (2008). for understanding the neural circuitry of Neurosci. nenl oe nenuos epnie o odours. (1993). to responsive interneurons lobe antennal assemblies. neural odor-encoding of patterning Neurol. moth the of glomeruli olfactory the in processing information and (2006). bulb. olfactory the of layer glomerular the in diversity. interneuron 2004). Brain the of Organization - P DC008174), a Pew Scholar award, a McKnight Scholar award, a Sloan O E TI R R CON

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© 2010 Nature America, Inc. All rights reserved. a pH of 7.3.a pH of The internal patch to 270–275 mOsm). The saline was bubbled with 95% O2/5% CO glucose, 26 NaHCO 5 described previously as Physiology. chicken Cy3 (1:300, JIRL). goat anti anti anti goat (MP), Cy5 ImmunoResearch Laboratories (JIRL) and used at 1:500 include: Zenon mouse A.DiAntonio). fromantibodiesMolecular Secondary ProbesJackson or (MP) Sigma), rabbit anti–vesicular glutamate transporter (DVGLUT; 1:500; gift from ChAT4B1,(1:100,anti rabbit acetyltransferase DSHB),choline anti– chicken Biomedicals), anti anti mouse DSHB), 8, no. anti mouse anti mouse anti or with the aid of Imaris. the sum of areas from each 1 each confocal section collected at 1 N by (marked lobe antennal the of outline the trace to used was software LSM cells,patchy software. To determine the volume the of antennal lobe occupied by individual 5.0Excel PrismGraphpadin and analysisperformedstatistical (Bitplane)and drite length and synaptotagmin r confocal. Images were processed using LSM software (Zeiss) and Image J ( mounted in SlowFade Gold (Molecular Probes) and imaged on a Zeiss Meta were dissected, fixed and stained as described before shock time windows for all cells are listed in induced LN clones rarely in stages our pupal early late experimentsand middle andat thusshocks Heat weredevelopment. pupal not early used further. Heat heat samples and h) 2–24 from (ranging time of lengths various for collected crossed,eggs formed according to established protocols M all developmental windows were equally sampled. single cells is not proportional to the number of LNs in a given Gal4 line, regardand to Gal4 lines and heat not reflect the fraction of these cells in a given fly because of our sampling bias with experiments and at room temperature (22–25 °C) for physiology experiments. 2–3 females using performed aftereclosion.d 10–20electrophysiologybetween sectedAll were experiments dis werecontrolsuntreated their as well as flies removed,werethese palpsso subset of line 6 single ments were performed with male flies, and line 9, which used exclusivelyused female males.flies 0–14 A d old except for lines 2, 3 and 10, where a subset of experi over the course 2of years, so experimental conditions varied. MARCM analysis Single-cell collections. 10 is OK107 (ref. (ref. H24 is (ref. 3 line neurons: brain other in or LNs in line from J. Simpson. Other lines haveunpublished beenan describedis (LCCH3) before7 Line forscreens. unpublished their various expressionour of basis (NP6277) and line 5 (NP3056) were ordered from the NP Gal4 library 2 Line Heberlein.U. from library Gal4 unpublished an of screen anatomical G ONLINE METHODS nature nature buffer,HEPESaspartate, 10sium Mg 4 s al4 lines. al4 b N AR Primary antibodies used included rat anti necessarily not does collection our in cells single of frequency relative The w - - - - tris(hydroxymethyl)methyl 46); line 8 is Nan e rat Cy5 rat (JIRL),antigoat HA (1:1,000, Abcam), rabbit anti– (1:1,000,Abcam),rabbit HA GFP (1:1,000; Aves Labs), rabbit anti rabbit Labs), Aves (1:1,000; GFP CM b . n - i ,immunostaining and image processing. rabbit Cy5 (JIRL), donkey anti h NEUR . Line 1 (HB4 1 Line - g - Whole rCD2 (1:100, OX34, Serotec), rat anti–N rat OX34,Serotec),(1:100, rCD2 c2 13, eeomna Suis yrdm Bn (DSHB)), Bank Hybridoma Studies Developmental (1:30, nc82 o - shocked at time points of interest during embryonic, larval and larval embryonic, during interest of points time at shocked v - / cadherin) or patchy cell processes (labeled by GFP or CD2) in CD2) or GFP by (labeled processes cellpatchy or cadherin) i OSCI j / 21). ). Further measurements, including glomerular volume, den - cell patch 3 , 1 NaH - - - mouse Cy3 (JIRL), goat anti goat (JIRL), Cy3 mouse cell clones were collected after antennae and/or maxillary EN Gal4 (ref. Our 1,532 single cells from ten Gal4 lines were collected 1 3 . External saline contained (in mM) 103 NaCl,103 .mM)containedKCl, Externalsaline(in 3 - C 93) and line 6 (HB8 6 line and 93) E - HA (1:100, 12CA5, gift from K. Wehner), rabbit rabbit Wehner),K. from gift 12CA5, (1:100, HA - 2 - - µ β PO - clamp recordings from LN somata were performed shock windows: the MARCM number of - rat Cy3 rat (JIRL),antigoat - m section. Cell counts were performed manually - d - aatsds (:,0, ba) mue anti– mouse Abcam), (1:1,000, galactosidase pipette solution contained (in mM) 140 potas HA density ( - 47); line 9 is krasavietz 4 - old. Flies were reared at 25 °C for MARCM for °C 25 at reared were Flies old. , 1.5 CaCl 2 - - µ aminoethane m intervals. The volume was determined as - β - ATP,Na0.5 chicken FITC (JIRL) and donkey anti - 1 galactosidase (1:1,000, CAPPEL/MB(1:1,000, galactosidase 9 Supplementary Table 2 - . Briefly, MARCM 2 - mCD8a (1:100, Invitrogen), chicken and 4 MgCl GFP (1:1,000, Molecular Probes), Molecular (1:1,000, GFP Figs. 6 - 145) were identified from an an from identified were 145) - sulfonic acid, 8 trehalose,10 8 acid,sulfonic 2 and 2 MARCM analysis wasper - 3 . After staining, brains were rat Alexa 488 (MP), goat goat (MP), 488 Alexa rat - GTP,EGTA,1 KCl1 and - - cadherin (1:40, DN (1:40, cadherin 8 - rabbit Alexa(MP),488 Gal4 (ref. 2 ), were made in Imaris 45); line 4 is GH298 GH298 is 4 line 45); (osmolarity adjusted - ready stocks were - GABA (1:200, GABA 2 . Adult brains 48); and line and reached 4 - 4 labeled on the h t - t - 510 p EX : / - ­ ­ ­ ­ ­ - /

of these morphological classes. Thus, a systematic comparison of all morpho­ of classes. Thus, comparison a systematic morphological these of to a due low abundance or potentially GFP) of low levels may express or they either for technical reasons (for example, their somata may be unusually small, LNs, of classes morphological particular these any of from to record able not we were attempts, these Despite LNs). oligoglomerular and patchy, dumbbell by labeled this line (including types to hit some the of morphological unusual attempt an in 5 line oversampled deliberately we example, For LNs. these of is tribution not representative necessarily of the actual distribution underlying statistical tests is smaller than the number listed here. We emphasize ments,that which isthis why the dis number experimentsof cited in connection with some In some of these cells, we did not (8).complete 11 9,the full line (5),set of 8 physiological8, line measure(11), 21 7, line (17), 18 6, line reconstructed), (51 biocytin fills were successfully reconstructed) was as follows: line 5, 55 recorded described previously visualized was at 10 kHz. One neuron was recorded per brain and the morphology of each cell current clamp mode (10 rate sealconductance andthereby to return thecell to itsnative spontaneous firing amount of hyperpolarizing current was injected constantly to compensate for measuredthe in cell microscope with a ×40 water epifluorescenceunderBX51WI signal Olympus GFP thean illuminationin of the osmolarity was adjusted to ~265 mOsm. LN somata were targeted by means hydrazide. biocytin 13 internalthe solution adjustedwas of pHtoand The7.3 of the assumption of that allglomeruli have an equal chance being innervated. of were centered by subtracting column means but were not rescaled. componentprincipalcustomizedusing performedanalysis wassoftware. Data ing were judged significant. clustering that correlated with those revealed by iterative sum squares of or by using the GAP statistic. Clusters identified by hierarchical Wedetermined clustering. Hierarchicalcomparisoniterativeverifiedbyclusteringtowas bootstrap analysis available through the pvclust package with unbiased approximately sou visualized as dendrograms and heat maps using JavaTreeView ( plete linkage and a Euclidean distance measure in either program. Results were ht Cluster( entered the nc82 channel. A glomerulus was considered innervated if any greenz 1 of planes individual examining by manually collectedwere analyses ing Statistical analysis of glomerular innervation patterns. in IgorPro and in MATLAB. rate relative to the spontaneous firing rate of that cell. Analyses were performed over a 1 cell is expressed as a single time histogram for that combination odor of and cell. Where the odor response a of of 40–60 s, and those trials were then averaged to generated a peristimulus time windows that overlapped by 25 ms. Each odor in was presented histograms six times time at intervals degrees varying to types ORN of variety wide a activate to and ORNs same the of studies other in used odors with overlap maximize to stimuli, our of diversity chemical the maximize to odors ten of fly.panel the reachingWea before chose air in tenfold diluted was headspace ously physiological population. LN The the of diversity the on possible. bound lower a not only therefore is was describe we diversity categories physiological and logical - stacks. We identified 54 antennal glomeruli ( m The total number LNsof recorded in each Gal4 line (and the number whose Theoretical probabilities for LN6 innervation were calculated on the basis the on calculated were innervation LN6 for probabilities Theoretical Innervation data were also imported into Matlab R2007a (Mathworks), where Hierarchical clustering of innervation data was performed using freely available Odors (Sigma) were delivered with a custom olfactometer, as described previ rceforge.net 4 ) or R ( 9 3 . Recordings were acquired with an A 5 . odorsAllwere diluted 1:100(vol/vol) oil,paraffinin odorthevial and - s period beginning at odor onset and are expressed as a change in firing http://bonsai.ims.u http:/ post hoc post / - ) or native R graphics facilities. Coherent clusters (those with with (those clustersCoherentfacilities. graphics R native or ) attached mode before whole - /www.r or N 3 Figure 5 using a Zeiss LSM 510 META confocal microscope. - with biocytin with cadherin - - M project.org P - Ω values >90% or >95%) were identified in R using R in identified were >95%) or >90% values k

- - 5c for each iteration by plotting the within the plotting by iteration each for immersion objective. Spontaneous firing rates were headstage), low tokyo.ac.jp/~mdeh sie ie wr bne i 50 in binned were times spike , - averaged firing rate, these rates were measured - dense region of a glomerulus. / ) software. Data were clustered using com - streptavidin and nc82 histochemistry as histochemistry nc82 and streptavidin - M Systems Model 2400 amplifier in - - cell break Fig. 3 pass filtered at 2 kHz and digitized 5 0 .To peristimulustheconstruct oon/software/cluster b ) in the nc82 or N - in. After break k Scoring data in cluster - means–based cluster doi:10.1038/nn.2489 - n colored LN process iterations http://jtreeview. k - - means–based ms counting counting ms - /software. - in, a small cadherin - ≥ groups 5,000. - µ M ­ ­ ­ ­ ­ ­

© 2010 Nature America, Inc. All rights reserved. opment, opment, experiments. Perturbation plotted the overlap index as a function of dilation kernel size. using the Euclidean norm and where computed as follows: vectorssize.image For varying of kernels percentile set to 0. Such thresholded images were then dilated using cube isolate the innervation patterns from background, to withstacks image cellall sister to pixelsapplied then was threshold intensity belowlobe.An nal the 97.5th first excluded from the analysis cell bodies and brain regions outside the anten we idea, this Tocomparable.test more become interest of volume the within signals green and red overall the of strength the should larger much were est inter of volume the if Only signal. green just or red just containconceivably could lobe antennal of volumesame patchycells,the sisterfor whereas signal, green and red both contain should size small of lobe antennal the of volume non innervation by glomerulus, in presented are probabilities theoretical where uted, the variation in observations can be calculated as s.e.m. = [ Assumingthat the innervation probabilities collection of is binomially distrib / 54) / set data the in glomeruli innervated of number (total = innervated of set given a for Thus, doi:10.1038/nn.2489 Wefollowingtheusedintuition toquantify overlapthe betweenpatchy and - patchy sister cells. For non p is the probability of innervation and smo 3 mutant clones were induced by eyFLP in flies expressing line 5 line expressing in flies by eyFLP induced were clones mutant OI , ( g r   n ) innervation patterns, chance a given glomerulus is glomerulus given a chance patterns, innervation = ± To eliminate both maxillary palps in early devel in early palps To maxillary both eliminate s.e.m. as the upper and lower bounds. y x ∑ - , , patchy sister cells colored red and green, a given z x r   , ( θ is the angle between g r   z y ) , ⋅ Figure r  and ⋅ x g n , ( is the number of samples. These g 

z y 6 , an index of overlap OI was overlapOI of index an, ) , f as the average percentage average the as = cos( r  q and ) p (1 − g  . Finally, we p - ) / shaped n ] 1/2 n ­ ­ ­ ­ , .

47. 46. 45. 44. stanford.edu in LNs all of D using Imaris. Syt as well as glomeruli individual within glomeruli were traced to obtain volumetric measurements. LN projection length immun Dumont no. a 5 forceps. using eclosion afterFlies were d aged 0–2 between both 10 and or 20 palpsd before maxillary antennae, removeddissection and and between 24 and 72 h after egg or LN for analyzed were ORNs. MP by targeted brains glomeruli in their innervation PN and dissected were antennae both GH146 or 48. 50. 49. ata access.ata To remove ORN input in adults, MARCM

Kim, J. Kim, in ablation Neuroblast J.S. Belle, de & N. Gendre, G., Heimbeck, R.F.,Stocker, locomotor suppress bodies Mushroom M. Heisenberg, & R. Ernst, J.R., Martin, S. Hayashi, Dubnau, J. Dubnau, Hallem, E.A., Ho, M.G. & Carlson, J.R. The molecular basis of odor coding in the in coding odor of basis molecular The J.R. Carlson, & M.G. Ho, E.A., Hallem, in propagation Signal R.I. Wilson, & N.W. Gouwens, 443–456 (1997). 443–456 Drosophila in activity traps. enhancer Gal4 of collection a of locations memory. 424 Drosophila Neurosci. J. o , 81–84 (2003). 81–84 , histochemistry. Confocal images were loaded into Imaris, where individual - et al. et Gal4–driven mCD8 Gal4–driven Curr. Biol. Curr. All the raw data used for clustering,forused rawdataconfocalincluding thethe stacks All / P[GAL4] lines reveals origins of olfactory interneurons. olfactory of origins reveals lines P[GAL4] antenna. Drosophila melanogaster Drosophila . Supplementary Tables2 Supplementary et al. et

et al. et 29 A TRPV family ion channel required for hearing in hearing for required channel ion family TRPV A , 6239–6249 (2009). 6239–6249 , The staufen/pumilio pathway is involved in involved is pathway staufen/pumilio The GETDB, a database compiling expression patterns and molecular and patterns expression compiling database a GETDB,

13 Cell , 286–296 (2003). 286–296 ,

117 - laying to generate single , 965–979 (2004). 965–979 , - GFP. Adults missing both MPs but retaining retaining but MPs both GFP. missing Adults . Learn. Mem. Learn. and 3 - - ready line 6 flies were heat A uca ee lo quantified also were puncta HA , are accessible at accessible are ,

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