ARTICLES. Lateral presynaptic inhibition mediates gain control in an olfactory circuit. Shawn R. Olsen 1 & Rachel I. Wilson 1

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1 doi:1.138/nture6864 ARTICLES Lterl presynpti inhiition medites gin ontrol in n olftory iruit Shwn R. Olsen 1 & Rhel I. Wilson 1 Olftory signls re trnsdued y lrge fmily of odornt reeptor proteins, eh of whih orresponds to unique glomerulus in the first olftory rely of the rin. Crosstlk etween glomeruli hs een proposed to e importnt in olftory proessing, ut it is not ler how these intertions shpe the odour responses of seond-order neurons. In the Drosophil ntennl loe ( region nlogous to the verterte olftory ul), we seletively removed most interglomerulr input to genetilly identified seond-order olftory neurons. Here we show tht this rodens the odour tuning of these neurons, implying tht interglomerulr inhiition domintes over interglomerulr exittion. The strength of this inhiitory signl sles with totl feedforwrd input to the entire ntennl loe, nd hs similr tuning in different glomeruli. A sustntil portion of this interglomerulr inhiition ts t presynpti lous, nd our results imply tht this is medited y oth ionotropi nd metotropi reeptors on the sme nerve terminl. A sensory stimulus generlly triggers tivity in multiple neurl proessing hnnels, eh of whih rries informtion out some feture of tht stimulus. The onept of proessing hnnel hs prtiulrly ler ntomil sis in the first rely of the olftory system, whih is typilly divided into glomerulr omprtments. Eh glomerulus reeives input from mny first-order olftory reeptor neurons (ORNs), ll of whih express the sme odornt reeptor. Eh seond-order neuron reeives diret ORN input from single glomerulus, nd thus ll the first- nd seond-order neurons orresponding to glomerulus onstitute disrete proessing hnnel. An odornt typilly triggers tivity in multiple glomeruli, nd lol interneurons tht interonnet glomeruli provide sustrte for rosstlk etween hnnels. The Drosophil ntennl loe is fvoured model for investigting olftory proessing euse it ontins only,5 glomeruli 1, eh of whih orresponds to n identified type of ORN nd n identified type of postsynpti projetion neuron (PN) 2 5. Severl reent studies of the Drosophil ntennl loe hve produed divergent views regrding the reltive importne of interglomerulr onnetions. One model proposes tht PN odour responses re lmost ompletely determined y feedforwrd exittion 6,7. This model sries little importne to rosstlk etween glomerulr proessing hnnels. An lterntive model proposes tht interglomerulr onnetions mke n importnt ontriution to shping PN odour responses However, this hs not een demonstrted y showing hnge in PN odour responses when lterl inputs to tht PN re removed. (We use the word lterl s synonym for interglomerulr.) In priniple, severl fetures of olftory proessing in the Drosophil ntennl loe ould reflet either intr- or interglomerulr events. For exmple, most PNs re more rodly tuned to odours thn their presynpti ORNs 8,1. This ould reflet purely intrglomerulr nonliner proess, suh s short-term synpti depression t ORN PN onnetions. Alterntively, it ould e due to the ft tht lterl exittory onnetions exist etween glomeruli 7,11,12. It is lso unler whether inhiitory epohs in PN odour responses 8 1 reflet inter- or intrglomerulr events. Mny interneurons tht re immuno-positive for GABA (-minoutyri id) form onnetions etween glomeruli 9,13,14, ut severl reent studies hve filed to oserve ny interglomerulr inhiition 7,11,12. These studies removed ll the diret ORN inputs to n identified PN, nd sked whether lterl input ould e evoked in tht PN y olftory stimultion of other ORN types. In ll ses, lterl inputs to PNs were exittory. This rises the possiility tht interglomerulr inhiition might not exist, nd tht inhiitory PN odour responses might merely reflet intrglomerulr feedk This is n importnt issue euse intr- nd interglomerulr inhiition hve different onsequenes for how olftory representtions re trnsformed in this iruit. In this study, we ddress three questions. Do interglomerulr intertions mke sustntil ontriution to PN odour responses? Do these intertions inlude lterl inhiition? If so, how does this our, nd why hs it een diffiult to oserve? Removing lterl input to projetion neurons We egn y investigting wht hppens to PN odour responses when most lterl input to tht PN is removed. We took dvntge of the ft tht the fly hs two olftory orgns. Aout 9% of ORNs re ontined in the ntenne, wheres 1% re in the mxillry plp (Fig. 1). Plp ORNs express odornt reeptors not expressed in the ntenne, nd projet to plp glomeruli tht re distint from glomeruli trgeted y ntennl ORNs 3,4. Antennl nd plp glomeruli re interonneted y lol interneurons. Aute removl of the ntenne elimintes 9% of the input to the ntennl loe, nd therefore most exittory drive to lol interneurons (Fig. 1). If lterl onnetions re minly exittory 7,11,12, then removing the ntenne should derese the odour responses of plp PNs. We performed this experiment in two different plp glomeruli (VM7 nd VC1). Surprisingly, removing the ntenne inresed most of the odour responses of these PNs (Fig. 1, d nd Supplementry Figs 2 nd 3). No odour responses were deresed. This implies tht most of our odours normlly evoke lterl inhiitory input to these glomeruli, nd tht this outweighs the effet of lterl exittory input. We exmined the input output funtion of eh glomerulus y plotting the strength of eh PN odour response ginst the strength of the ognte ORN response to the sme odour. These input output funtions were nonliner (Fig. 1d), whih is typil for most glomeruli 1. When we removed most lterl input to VM7 nd VC1 PNs, 1 Deprtment of Neuroiology, Hrvrd Medil Shool, 22 Longwood Avenue, Boston, Msshusetts 2115, USA. 28 Nture Pulishing Group 1

2 ARTICLES NATURE the nonlinerity persisted (Fig. 1d) nd these PNs eme even more rodly tuned (Fig. 1e). This rgues tht rod tuning of PNs 8,1 results minly from purely intrglomerulr mehnisms. These ould inlude short-term synpti depression t ORN PN onnetions nd/or n intrinsi eiling on PN firing rtes. Lterl exittion should tend to roden PN tuning even more, ut lterl inhiition evidently ounterts this. One lue to the signifine of lterl inputs is tht, in the intt ntennl loe iruit, PN responses nnot e predited purely on the sis of feedforwrd exittory inputs 1. Two odours n eliit similr responses in n ORN ut divergent responses in postsynpti PN. For exmple, pentyl ette nd 4-methyl phenol evoke similr tivity in VM7 ORNs ut not in VM7 PNs (Fig. 1), implying tht these odours reruit different lterl inputs to this glomerulus. After ntenne were removed, these odours evoked similr responses in VM7 PNs (Fig. 1). Overll, removing the ntenn inresed the orreltion etween the rnked odour preferenes of ORNs nd their ognte PNs (Spermn s r inresed from.82 to.88 for VM7 nd from.89 to.94 for VC1; P,.2 for eh omprison, Mnn Whitney U-test). Lterl inhiition suppresses ORN input Severl reent studies hve filed to oserve lterl inhiition in the Drosophil ntennl loe 7,11,12. These studies silened ll diret ORN Spikes s 1 Antenn Mxillry plp 2 Ethyl utyrte Sever ntennl nerves Pentyl ette Antenn Mxillry plp 4-Methyl phenol ORNs Glomeruli PNs inputs to PN nd foused on lterl input to tht PN evoked y stimulting ORNs presynpti to other glomeruli. We resoned tht some lterl inhiition might trget ORN xon terminls; if so, this would only e oserved when the diret ORN inputs to PN re tive. GABAergi inhiition t ORN xon terminls in the olftory ul hs een desried previously 15 21, nd synpses from GABAergi interneurons onto ORN xon terminls hve een found in n inset ntennl loe 22. To test the ide tht some lterl inhiition is presynpti, we sked how lterl input to glomerulus VM7 depends on the tivity of VM7 ORNs. Eh VM7 ORN fires spontneously t,1 spikes s 21, nd onsequently these PNs re omrded y spontneous spike-driven exittory postsynpti potentils (EPSPs; Supplementry Fig. 4). When we silened VM7 ORNs y removing the mxillry plps, lrge spontneous EPSPs disppered in VM7 PNs (Fig. 2). In this experimentl onfigurtion, stimulting the ntenne with odornts depolrized VM7 PNs (Fig. 2), whih is onsistent with previous reports tht lterl input is exittory when diret ORN inputs re silent 7,11,12. Next, we sked whether preserving spontneous tivity in VM7 ORNs would llow us to oserve lterl inhiition. To prevent odour-evoked tivity in VM7 ORNs, we overed the mxillry plps Antenn Plp removed Antenn Plp Memrne potentil (mv) Ethyl utyrte Ethyl utyrte Spikes s 1 d PN response (spikes s 1 ) ORN response (spikes s 1 ) 1 ORN response (spikes s 1 ) e 1 Lifetime sprseness VM7 VC1 Figure 1 Removing lterl input disinhiits projetion neurons., Drosophil olftory orgns., Experimentl onfigurtion., Spiking responses of ORNs (green) nd PNs (lue nd mgent) for the plp glomerulus VM7 (n for eh response). PNs re shown with (mgent) nd without (lue) lterl input from ntennl glomeruli. The lk rs represent the 5-mse odour-stimulus period. d, Input output funtions for plp glomeruli VM7 (left) nd VC1 (right). Eh point represents the verge PN response to n odour versus the response of the ognte ORNs. PNs re shown with ntenne intt (mgent) or removed (lue). Responses to 18 out of 2 odours re signifintly disinhiited in VM7; 13 out of 2 re signifintly disinhiited in VC1 (P,.5, t-tests). e, Odour seletivity of ORNs (green) nd ognte PNs with ntenne intt (mgent) or removed (lue). Lifetime sprseness is for n unseletive ell nd 1 for mximlly seletive ell. All within-glomerulus omprisons re signifint (P,.2, Mnn Whitney U-test), exept ORNs versus ntenne-intt PNs for VC1. Dt re shown s men 6 s.e.m. 2 d 2 VM7 PN disinhiition (spikes s 1 ) Ethyl utyrte 4 mv 28 Nture Pulishing Group 1 Lterl input to VM7 (mv s) e 1 Lterl input to VM7 (mv s) Pentyl ette 1,5 Totl ntennl ORN tivity (spikes s 1 ) f 2 VC1 PN disinhiition (spikes s 1 ) 4-Methyl phenol 1 Lterl input to VM7 (mv s) Figure 2 Lterl inhiition suppresses spontneous EPSCs nd sles with totl ORN input., Left: experimentl onfigurtion. Right: reording from PN in glomerulus VM7. Olftory stimultion evokes depolriztion. Spontneous EPSPs re sent., Left: plps re shielded from odours. Right: reording from PN in glomerulus VM7. Olftory stimultion suppresses spontneous EPSPs., Averge VM7 PN responses s in (green) or (lk); n PNs for eh ondition. When ORNs re shielded (lk), inhiition domintes. When ORNs re sent (green), exittion domintes throughout the stimulus period. (Off-inhiition proly reflets lterl postsynpti inhiition, see Supplementry Fig. 5.) Ple nds re s.e.m. d, Lterl input to VM7 (s in ) is orrelted with the disinhiition in VM7 PNs fter ntennl removl (see Fig. 1). Eh point represents different odour. Lterl input is mesured s the time-integrted hnge in memrne potentil. e, Lterl input to VM7 is orrelted with totl ntennl ORN spiking tivity evoked y eh odour. f, Lterl input to VM7 is orrelted with the disinhiition in VC1 PNs fter ntennl removl.

3 NATURE ARTICLES with plsti shield. We inferred tht the shield did not prevent spontneous tivity in VM7 ORNs euse we oserved norml spontneous EPSPs in VM7 PNs (Fig. 2). The shield ws lerly n effetive rrier to odours euse it loked the norml strong exittory response to ethyl utyrte in VM7 PNs (Supplementry Fig. 4). Insted, when the plps were shielded, stimulting the ntenne with odornts suppressed spontneous EPSPs in VM7 PNs (Fig. 2, ). This ws ompnied y hyperpolriztion of the memrne potentil, whih would reflet (t lest in prt) the removl of ongoing depolriztion produed y EPSP omrdment. For ll 2 odours in our pnel, lterl input depolrized VM7 PNs when their ognte ORNs were sent (s in Fig. 2) nd hyperpolrized these PNs when their ORNs were spontneously tive (s in Fig. 2). This rgues tht sustntil omponent of lterl inhiition ts t presynpti lous. This does not men tht ll lterl inhiition is presynpti; indeed there is evidene for n dditionl postsynpti omponent (Supplementry Fig. 5). Inhiition sles with totl ORN input When plp ORNs were shielded, different odours evoked different mounts of inhiition in glomerulus VM7 (Fig. 2, lk tres). We hypothesized tht this odour tuning ould explin why ntennl removl disinhiits some VM7 PN odour responses more thn others (Fig. 1, d). To test this, we mesured the mount of inhiition Olftory stimultion Eletril stimultion Reord d Eletril GABA stimultion iontophoresis Reord EPSC mplitude (% of seline) e f EPSC mplitude (% of seline) 2 pa pa 1 5 Odour Odour No ntgonists PCT + PCT Figure 3 Lterl GABAergi suppression of ORN PN synpses., Experimentl onfigurtion., Eletril stimultion of the ntennl nerve (rrowheds) evokes EPSCs in PN (verge of 2 trils). Olftory stimultion (5 ms) inhiits EPSCs. Odour lso evokes trnsient inwrd urrent refleting lterl postsynpti exittion; this is resistnt to GABA ntgonists (Supplementry Fig. 6)., Inhiition is loked y the GABA B ntgonist () together with the GABA A ntgonist pirotoxin (ontrol, n 5 12; PCT, n 5 5;, n 5 5; 1 PCT, n 5 5). All pirwise omprisons re signifintly different exept ontrol versus PCT (P,.5, t-tests). d, Experimentl onfigurtion, sustituting GABA iontophoresis for olftory stimultion. e, Asin for GABA iontophoresis (sterisk). GABA lso evokes n outwrd urrent. f, Asin for GABA iontophoresis (ontrol, n 5 13; PCT, n 5 5;, n 5 6; 1 PCT, n 5 7). All pirwise omprisons etween onditions re signifintly different exept ontrol versus PCT (P,.5, t-tests). Dt re shown s men 6 s.e.m. 28 Nture Pulishing Group evoked y eh odour in the shielded-plps experiment, nd ompred this to the hnge in PN spiking responses to tht odour fter ntennl removl. These two mesures were well orrelted (Fig. 2d, n 5 2 odours, Person s r , P,.1), whih rgues tht these two experimentl prdigms mesure the sme underlying phenomenon. The odour tuning of lterl input must reflet the onnetivity of the lol interneurons tht medite this inhiition. Mny individul GABAergi interneurons innervte ll glomeruli 9,12,13, suggesting tht lterl inhiitory input to eh glomerulus might reflet pooled input from ll ORNs. If so, then the size of lterl input to glomerulus should orrelte with the totl ORN tivity evoked y tht odour. We estimted totl ORN tivity y summing the spiking responses of eh ntennl ORN type 23, nd found tht this mesure predited the strength of lterl inhiition evoked y eh odour in the shielded-plps experiment (Fig. 2e, n 5 14 odours, Person s r , P,.5). If lterl inhiition to ll glomeruli sles with totl ORN tivity, then lterl inhiitory input to eh glomerulus would show the sme odour tuning. We hve lredy shown tht the odour tuning of lterl input to VM7 is good preditor of whih odour responses were most disinhiited in VM7 PNs fter ntennl removl (Fig. 2d). As expeted, it lso prtilly predited whih odour responses were most disinhiited in different plp glomerulus, VC1 (Fig. 2f, n 5 2 odours, Person s r , P,.1). However, this orreltion ws weker thn the orreltion with disinhiition in VM7. This leves open the possiility tht there my e some differenes in the odour tuning of lterl inhiitory input to different glomeruli (see Disussion). GABA medites presynpti inhiition Our results suggest tht muh of the lterl inhiition in this iruit ts y suppressing ORN PN synpti trnsmission. To test this, we monitored ORN PN synpti strength in one glomerulus while reruiting lterl input to tht glomerulus (Fig. 3). We reorded from n identified PN while eletrilly stimulting the ipsilterl ntennl nerve to evoke exittory postsynpti urrents (EPSCs). Next, we used n odour to stimulte ORNs in the remining intt ntenn (nd the mxillry plps). Beuse most glomeruli reeive ilterl ORN input 2, olftory stimultion of the ontrlterl ntenn drives tivity in ipsilterl glomeruli. Finlly, we prevented odours from reruiting diret ORN input to the reorded PN y mutting the odornt reeptor gene normlly expressed y its ORNs. As predited, olftory stimultion of the ontrlterl ntenn inhiited EPSCs evoked y ipsilterl nerve stimultion (Fig. 3, ). We ould mimi this inhiition y iontophoresing GABA into the ntennl loe neuropil (Fig. 3d f). A GABA B reeptor ntgonist loked the lte phse of this inhiition, ut hd only modest effet on the erly phse (Fig. 3, f). Adding GABA A ntgonist to the GABA B ntgonist loked the residul erly portion of the inhiition (Fig. 3, f). The GABA A ntgonist lone hd no effet (Fig. 3, f). Tken together, these results suggest tht oth GABA A nd GABA B reeptors re present on the sme ORN xon terminls, nd tht either GABA A or GABA B reeptors lone re suffiient to medite sustntil inhiition of EPSCs just fter GABA relese. The lte phse of inhiition evidently involves only GABA B reeptors. Presynpti inhiition is generlly ssoited with hnge in the wy in whih synpse responds to pired eletril pulses 24.We found tht oth the GABA A nd the GABA B omponents of EPSC inhiition re ssoited with n inrese in the pired-pulse rtio (Supplementry Fig. 7). This implies tht the independent tions of oth GABA A nd GABA B reeptors re t lest prtilly presynpti. As further test of this model, we genetilly olished GABA B signlling seletively in presynpti ORNs. We used n ORN-speifi promoter 25 to drive expression of pertussis toxin 26 seletive inhiitor of some types of G proteins. In these flies, GABA still inhiited ORN PN EPSCs, ut now this inhiition hd riefer durtion thn 3

4 ARTICLES NATURE Eletril GABA stimultion iontophoresis Reord EPSC mplitude (% of seline) EPSC mplitude (% of seline) Pertussis toxin in ORNs Norml ORNs No ntgonists 1 2 Pertussis toxin in ORNs No ntgonists PCT + PCT 1 2 Figure 4 Geneti evidene tht GABA B reeptors inhiit ORN PN synpses t presynpti lous., Experimentl onfigurtion., When pertussis toxin is speifilly expressed in ORNs, GABAergi inhiition of EPSCs (solid line, n 5 11) is more trnsient thn in ontrol flies (dotted line, n 5 13, reprodued from Fig. 3f)., Pertussis toxin expression in ORNs renders the GABAergi inhiition of EPSCs ompletely insensitive to, nd ompletely sensitive to PCT (lk tre reprodued from ;, n 5 5; PCT, n 5 6; 1 PCT, n 5 5). Compre to Fig. 3f. Asterisk represents GABA iontophoresis. Dt re shown s men 6 s.e.m. ORN PN, ntenne removed PN, ntenne intt Antenne removed 1 mv Control PCT +PCT 1 Control Antenne intt PCT + PCT 8 Spikes s 1 Figure 5 GABA reeptor ntgonists mimi removl of lterl input to projetion neuron., Rsters show spiking responses to pentyl ette (grey) in VM7 ORN nd VM7 PN. With ntenne intt, oth ntgonists re required to mimi the effet of ntennl removl on PNs. (Some offinhiition in PNs persists fter ntennl removl or in the presene of ntgonists; this proly reflets the off-inhiition in VM7 ORNs.), Averge spike rtes during odour stimulus period, minus seline spike rtes (n PNs for eh ondition)., Averge memrne potentil responses to pentyl ette in VM7 PNs (n projetion neurons for eh). Dt re shown s men 6 s.e.m. in wild-type flies (Fig. 4, ). Unlike in wild-type flies, this inhiition ws ompletely resistnt to the GABA B ntgonist nd ompletely loked y the GABA A ntgonist (ompre Fig. 4 to Fig. 3f). As negtive ontrol, we onfirmed tht this phenotype requires oth the ORN-speifi promoter nd the toxin trnsgene (Supplementry Fig. 8). This demonstrtes tht GABA B reeptors inhiit ORN PN synpses t purely presynpti lous. If tivtion of either GABA A or GABA B reeptors is suffiient to medite strong lterl inhiition, then lokde of oth reeptors should e required to mimi the removl of lterl input. To test this, we gin reorded from plp PNs in glomerulus VM7. Normlly, the odour pentyl ette wekly exites VM7 ORNs nd inhiits VM7 PNs. When most lterl input is removed (y removing the ntenne), this odour strongly exites these PNs (Fig. 1). We ould not mimi this disinhiition y pplying either GABA A or GABA B reeptor ntgonist lone. However, the two ntgonists together produed strong disinhiition tht resemled the effet of removing the ntenne (Fig. 5). Disussion Mny previous studies hve shown tht odours n inhiit spiking in olftory ul mitrl ells nd ntennl loe PNs (see refs for erly exmples), ut in priniple this inhiition ould e purely intrglomerulr Experiments in vitro hve reveled severl types of interglomerulr iruits 33 35, ut some of these iruits re evidently not reruited y olftory stimuli 15. Here we hve diretly demonstrted n importnt role for inhiitory intertions etween olftory glomeruli in vivo. Our results rgue tht sustntil omponent of interglomerulr inhiition ours t presynpti lous. Previous studies in other speies hve shown tht GABA n inhiit relese from ORN xon terminls Our results imply tht in Drosophil this is medited y oth GABA A nd GABA B reeptors. This rrngement is unusul ut not unique; for exmple, there re severl instnes of GABA A nd GABA B inhiition t the sme presynpti site in other neurl iruits Ionotropi nd metotropi reeptors t with different kinetis, nd so this rrngement might ensure tht inhiition spns rod time window. Although oth reeptor types were o-tive during most of the odour response, we notied tht GABA A reeptors were required for rief erly phse of inhiition fter odour onset 4 28 Nture Pulishing Group (Fig. 5) wheres GABA B reeptors were required for the long, lte phse (Figs 3 nd 5). We hve shown tht lterl inhiitory input to glomerulus roughly sles with totl ORN tivity. This would imply tht the odour tuning of GABAergi input to eh glomerulus is pproximtely similr. However, the effets of lterl input my nevertheless e somewht glomerulus-speifi. Even if the odour tuning of GABA relese were identil in ll glomeruli, the effiy of presynpti inhiition will vry with presynpti memrne potentil 4,41.As result, the sme inhiitory signl should e more effetive in some glomeruli thn in others. Also, in some glomeruli, lterl inhiition might e outweighed y lterl exittion. This would explin why other studies hve found tht some PNs n e exited y odours tht do not exite their ognte ORNs 8,1,12,42. We propose tht this form of gin ontrol represents flexile lne etween sensitivity nd effiieny. When totl ORN input is wek, lterl inhiition is miniml, nd ORN PN synpses re strong. When n odornt reruits vigorous ORN input to mny glomeruli, GABAergi interneurons inhiit ORN neurotrnsmitter relese. This should prevent stimulus from sturting the dynmi rnge of mny types of PNs simultneously. Beuse this mehnism suppresses responses tht re strong nd redundnt, it my tend to derese ross-orreltions etween the output of different glomeruli, nd thus promote more effiient neurl ode for odours. METHODS SUMMARY In vivo whole-ell pth-lmp reordings from PNs nd extrellulr reordings from ORNs were performed essentilly s desried previously 8 11,42. Reordings in Fig. 3 were mde from VM2 PNs in Or43 1 mutnt flies. Antgonists (5 mm nd 5 mm pirotoxin, PCT) were dded to the sline, whih perfused the rin. Pertussis toxin ws expressed under ontrol of the Or83- GAL4 driver. All dt re shown s men vlues, verged ross experiments, 6 s.e.m. (exept for rw eletrophysiologil tres nd rsters). The odour stimulus period (shown s lk r in Figures) ws 5 ms, exept in Fig. 3,. Full Methods nd ny ssoited referenes re ville in the online version of the pper t Reeived 22 Septemer 27; epted 25 Ferury 28. Pulished online 16 Mrh Lissue, P. P. et l. Three-dimensionl reonstrution of the ntennl loe in Drosophil melnogster. J. Comp. Neurol. 45, (1999).

5 NATURE ARTICLES 2. Stoker, R. F., Lienhrd, M. C., Borst, A. & Fishh, K. F. Neuronl rhiteture of the ntennl loe in Drosophil melnogster. Cell Tissue Res. 262, 9 34 (199). 3. Couto, A., Alenius, M. & Dikson, B. J. Moleulr, ntomil, nd funtionl orgniztion of the Drosophil olftory system. Curr. Biol. 15, (25). 4. Fishilevih, E. & Vosshll, L. B. Geneti nd funtionl sudivision of the Drosophil ntennl loe. Curr. Biol. 15, (25). 5. Mrin, E. C., Jefferis, G. S., Komiym, T., Zhu, H. & Luo, L. Representtion of the glomerulr olftory mp in the Drosophil rin. Cell 19, (22). 6. Wng, J. W., Wong, A. M., Flores, J., Vosshll, L. B. & Axel, R. Two-photon lium imging revels n odor-evoked mp of tivity in the fly rin. Cell 112, (23). 7. Root, C. M., Semmelhk, J. L., Wong, A. M., Flores, J. & Wng, J. W. Propgtion of olftory informtion in Drosophil. Pro. Ntl Ad. Si. USA 14, (27). 8. Wilson, R. I., Turner, G. C. & Lurent, G. Trnsformtion of olftory representtions in the Drosophil ntennl loe. Siene 33, (24). 9. Wilson, R. I. & Lurent, G. Role of GABAergi inhiition in shping odor-evoked sptiotemporl ptterns in the Drosophil ntennl loe. J. Neurosi. 25, (25). 1. Bhndwt, V., Olsen, S. R., Gouwens, N. W., Shlief, M. L. & Wilson, R. I. Sensory proessing in the Drosophil ntennl loe inreses reliility nd seprility of ensemle odor representtions. Nture Neurosi. 1, (27). 11. Olsen, S. R., Bhndwt, V. & Wilson, R. I. Exittory intertions etween olftory proessing hnnels in the Drosophil ntennl loe. Neuron 54, (27). 12. Shng, Y., Clridge-Chng, A., Sjulson, L., Pypert, M. & Miesenok, G. Exittory lol iruits nd their implitions for olftory proessing in the fly ntennl loe. Cell 128, (27). 13. Stoker, R. F., Heimek, G., Gendre, N. & de Belle, J. S. Neurolst ltion in Drosophil P[GAL4] lines revels origins of olftory interneurons. J. Neuroiol. 32, (1997). 14. Ng, M. et l. Trnsmission of olftory informtion etween three popultions of neurons in the ntennl loe of the fly. Neuron 36, (22). 15. MGnn, J. P. et l. Odornt representtions re modulted y intr- ut not interglomerulr presynpti inhiition of olftory sensory neurons. Neuron 48, (25). 16. Murphy, G. J., Dry, D. P. & Isson, J. S. Intrglomerulr inhiition: signling mehnisms of n olftory miroiruit. Nture Neurosi. 8, (25). 17. Vuini, D., Cohen, L. B. & Kosmidis, E. K. Interglomerulr enter-surround inhiition shpes odornt-evoked input to the mouse olftory ul in vivo. J. Neurophysiol. 95, (26). 18. Nikell, W. T., Behehni, M. M. & Shipley, M. T. Evidene for GABA B -medited inhiition of trnsmission from the olftory nerve to mitrl ells in the rt olftory ul. Brin Res. Bull. 35, (1994). 19. Whowik, M. & Cohen, L. B. Presynpti inhiition of primry olftory fferents medited y different mehnisms in loster nd turtle. J. Neurosi. 19, (1999). 2. Aronidou-Anderjsk, V., Zhou, F. M., Priest, C. A., Ennis, M. & Shipley, M. T. Toni nd synptilly evoked presynpti inhiition of sensory input to the rt olftory ul vi GABA B heteroreeptors. J. Neurophysiol. 84, (2). 21. Whowik, M. et l. Inhiition of olftory reeptor neuron input to olftory ul glomeruli medited y suppression of presynpti lium influx. J. Neurophysiol. 94, (25). 22. Distler, P. G. & Boekh, J. Synpti onnetions etween identified neuron types in the ntennl loe glomeruli of the okroh, Periplnet merin: II. Lol multiglomerulr interneurons. J. Comp. Neurol. 383, (1997). 23. Hllem, E. A. & Crlson, J. R. Coding of odors y reeptor repertoire. Cell 125, (26). 24. Zuker, R. S. & Regehr, W. G. Short-term synpti plstiity. Annu. Rev. Physiol. 64, (22). 25. Lrsson, M. C. et l. Or83 enodes rodly expressed odornt reeptor essentil for Drosophil olftion. Neuron 43, (24). 26. Ferris, J., Ge, H., Liu, L. & Romn, G. G(o) signling is required for Drosophil ssoitive lerning. Nture Neurosi. 9, (26). 27. Shiuy, T., Ai, N. & Tkgi, S. F. Response types of single ells in the olftory ul. Pro. Jpn. Ad. 38, (1962). 28. Mrides, F. & Chorover, S. L. Olftory ul units: tivity orrelted with inhltion yles nd odor qulity. Siene 175, (1972). 29. Mthews, D. F. Response ptterns of single units in the olftory ul of the rt to odor. Brin Res. 47, (1972). 3. Tne, T., Iino, M. & Tkgi, S. F. Disrimintion of odors in olftory ul, pyriform-mygdloid res, nd oritofrontl ortex of the monkey. J. Neurophysiol. 38, (1975). 31. Meredith, M. & Moulton, D. G. Ptterned response to odor in single neurones of goldfish olftory ul: influene of odor qulity nd other stimulus prmeters. J. Gen. Physiol. 71, (1978). 32. Chput, M. & Holley, A. Single unit responses of olftory ul neurones to odour presenttion in wke rits. J. Physiol. (Pris) 76, (198). 33. Isson, J. S. & Strowridge, B. W. Olftory reiprol synpses: dendriti signling in the CNS. Neuron 2, (1998). 34. Urn, N. N. & Skmnn, B. Reiprol intrglomerulr exittion nd intr- nd interglomerulr lterl inhiition etween mouse olftory ul mitrl ells. J. Physiol. (Lond.) 542, (22). 35. Aungst, J. L. et l. Centre-surround inhiition mong olftory ul glomeruli. Nture 426, (23). 36. Sturt, G. J. & Redmn, S. J. The role of GABA A nd GABA B reeptors in presynpti inhiition of I EPSPs in t spinl motoneurones. J. Physiol. (Lond.) 447, (1992). 37. Mtthews, G., Ayou, G. S. & Heidelerger, R. Presynpti inhiition y GABA is medited vi two distint GABA reeptors with novel phrmology. J. Neurosi. 14, (1994). 38. Fisher, Y. & Prns, I. Differentil tivtion of two distint mehnisms for presynpti inhiition y single inhiitory xon. J. Neurophysiol. 76, (1996). 39. Fisher, Y. & Prns, I. Ativtion of GABA B reeptors t individul relese outons of the ryfish opener neuromusulr juntion produes presynpti inhiition. J. Neurophysiol. 75, (1996). 4. Ben, B. P. Neurotrnsmitter inhiition of neuronl lium urrents y hnges in hnnel voltge dependene. Nture 34, (1989). 41. Foldy, C., Neu, A., Jones, M. V. & Soltesz, I. Presynpti, tivity-dependent modultion of nninoid type 1 reeptor-medited inhiition of GABA relese. J. Neurosi. 26, (26). 42. Shlief, M. L. & Wilson, R. I. Olftory proessing nd ehvior downstrem from highly seletive reeptor neurons. Nture Neurosi. 1, (27). Supplementry Informtion is linked to the online version of the pper t Aknowledgements We thnk K. Ito, L. Luo, G. Romn, D. P. Smith, L. M. Stevens nd L. B. Vosshll for gifts of fly stoks. We thnk G. Lurent, A. W. Liu nd memers of the Wilson lortory for onverstions. This work ws funded y grnt from the NIDCD, the Pew, MKnight, Slon, nd Bekmn Foundtions (to R.I.W.). S.R.O. ws prtilly supported y NSF fellowship. Author Contriutions S.R.O. performed the experiments nd nlysed the dt. S.R.O. nd R.I.W. designed the experiments nd wrote the pper. Author Informtion Reprints nd permissions informtion is ville t Correspondene nd requests for mterils should e ddressed to R.I.W. (rhel_wilson@hms.hrvrd.edu). 28 Nture Pulishing Group 5

6 doi:1.138/nture6864 METHODS Fly stoks. Flies were rered t 2 21 uc on onventionl ornmel gr. All experiments were performed on dult femle flies 2 5 dys post-elosion. Fly stoks were provided s follows: Or43 1 (D. Smith); NP513-GAL4, NP3481- GAL4 nd NP5221-GAL4 (K. Ito nd L. Luo); Or46-GAL4 nd Or83-GAL4 (L. Vosshll); UAS-DTl II nd UAS-DTl III (L. Stevens); UAS-PTX (G. Romn); nd UAS-CD8GFP I (Bloomington Stok Center). ORN reordings. Extrellulr reordings of ORN spiking were performed s desried previously 1. To urtely sort spikes reorded from VC1 ORNs, we killed the seond ORN type (VA7l) housed in the sme sensillum. This ws done y expressing diphtheri toxin light hin in the VA7l ORNs (genotype: either Or46-GAL4/UAS-DTl II (n 5 4) or Or46-GAL4/1; UAS-DTl III /1 (n 5 4)). The genotype for VM7 ORN reordings ws NP3481-Gl4 UAS-CD8GFP, whih is the sme genotype s tht used for the VM7 PN reordings. VM7 ORNs fire spontneously t 1 spikes s 21, nd VC1 ORNs fire spontneously t 3 spikes s 21. Projetion neuron reordings. In vivo whole-ell reordings from PNs were performed s previously desried 1. For the urrent-lmp reordings in Figs 1, 2 nd 5 nd in Supplementry Figs 2 5, the omposition of the internl pth-pipette solution ws (in mm): potssium sprtte 14, HEPES 1, MgATP 4, N 3 GTP.5, EGTA 1, KCl 1, nd ioytin hydrzide 13 (ph 7.3, osmolrity djusted to,265 mosm). For the voltge-lmp reordings in Figs 3 nd 4 nd Supplementry Figs 6 8, the omposition of the internl ws (in mm): esium sprtte 14, HEPES 1, MgATP 4, N 3 GTP.5, EGTA 1, KCl 1, ioytin hydrzide 13, nd QX (ph 7.3, osmolrity djusted to,265 mosm). The omposition of the externl sline solution in ll reordings ws (in mm): NCl 13, KCl 3, N-tris(hydroxymethyl) methyl-2-minoethnesulfoni id 5, trehlose 8, gluose 1, NHCO 3 26, NH 2 PO 4 1, CCl 2 1.5, nd MgCl 2 4. Osmolrity ws djusted to mosm. The sline ws uled with 95% O 2 /5% CO 2 nd rehed finl ph of 7.3. Sline perfused the rin ontinuously t 2 ml min 21. Reordings were otined with n A-M Systems Model 24 mplifier (1 MV hedstge), were low-pss filtered t 5 khz, nd digitized t 1 khz. Dt were quired in Igor Pro. In most experiments, we used n enhner trp line to lel speifi PNs with green fluoresent protein (GFP) for trgeted reording. VM7 PNs were lelled y NP3481-Gl4 UAS- CD8GFP (Figs 1, 2 nd 5, nd Supplementry Figs 2, 4 nd 5). VC1 PNs were lelled y NP5221-Gl4 UAS-CD8GFP (Fig. 1 nd Supplementry Fig. 3). VM2 PNs were lelled y NP513-Gl4 UAS-CD8GFP (Fig. 3, nd Supplementry Fig. 6 nd the odour ondition in Supplementry Fig. 7d). VM2 PNs were reorded in flies in whih the odornt reeptor expressed y VM2 ORNs ws mutted (NP513-Gl4 UAS-CD8GFP; Or43 1, see shemti in Fig. 3). In some experiments for eh genotype, we filled the reorded ell with ioytin nd verified the PN identity with ioytin histohemistry s desried previously 8. In the GABA iontophoresis experiments in Fig. 3d f (lso Supplementry Figs 6d f nd 7), we reorded from PNs in the nterodorsl ell luster 5 lelled y the enhner trp line GH146 (GH146-Gl4 UAS-CD8GFP). For the experiments in whih pertussis toxin ws seletively expressed in ORNs (Fig. 4, lso Supplementry Fig. 8), we reorded from PNs in the nterodorsl ell luster in the genotype Or83-Gl4; UAS-PTX/1. In voltge-lmp reordings, the ommnd potentil ws either 285 mv (Fig. 3 ) or 265 mv (Figs 3d f nd 4). Mnipultion of peripherl orgns. Antennl input ws olished in some experiments y severing the ntennl nerves with fine foreps just efore reording. The ntennl nerve ws gently roken y pplying fores perpendiulr to the long xis of the nerve, leving stump of nerve tthed to the ntennl loe. In other experiments, the mxillry plps were removed with foreps just efore reording. For oth ntennl nd plp mputtion, the ell odies of the ffeted ORNs re removed, ut the proximl portions of their xons remin intt nd ontinue to innervte the ntennl loe. Amputtion removes ORN somt nd thus lrge spontneous spike-driven EPSCs (,5 2 pa) dispper lmost entirely. Axon terminls re still intt, s evidened y the persistene of miniture EPSCs (,1 pa). In some experiments, the mxillry plps were shielded from diret odour stimultion y overing them in 5 minute epoxy (Devon). The epoxy ws llowed to dry for,2 min efore the reording ws strted. We onfirmed tht this sustne is reltively non-toxi to ORNs y overing the ntenne in epoxy, llowing it to dry, peeling the epoxy wy, nd verifying tht odours were still le to eliit norml field-potentil response in the ntenne. Olftory stimultion. Odours were diluted in prffin oil t rtio of 1:1 v/v. Odour dilutions in prffin oil were prepred fresh every 5 dys. Our odour pnel onsisted of enzldehyde, utyri id, 1-utnol, ylohexnone, ethyl utyrte, ethyl ette, ethyl-3-hydroxyutryte, fenhone, gernyl ette, 2-heptnone, linlool, 4-methyl ylohexnol, methyl sliylte, 3-methylthio- 1-propnol, 4-methyl phenol, otnl, 1-oten-3-ol, pentyl ette, trns-2- hexenl nd prffin oil (solvent ontrol). Odour soure detils re posted t Odours were delivered with ustom-uilt olftometer s desried previously 1. Odour stimuli were pplied for 5 ms, exept in Fig. 3 in whih the odour ws pplied for 1 s. For the experiments in Figs 1, 2 nd 5, onseutive odour presenttions were sped s prt. Diret ORN xon stimultion. In Figs 3 nd 4 nd in Supplementry Figs 6 8, we eletrilly stimulted ORN xons. The ipsilterl ntennl nerve ws severed nd inserted into stimulting sution eletrode. Single spikes were evoked in ORN xons with short urrent pulse (5 ms) using stimulus isoltor (A.M.P.I.). In eh sweep we delivered trin of 23 nerve stimuli t 4 Hz. During the trin, the size of the evoked EPSCs deresed over the first 4 8 stimuli owing to short-term depression, ut then rehed stedy vlue. We timed our GABA iontophoresis nd odour stimultion to our during this stedy-stte period. For the pired-pulse experiments in Supplementry Fig. 7, we delivered two eletril stimuli to the nerve with n inter-pulse intervl of 25 ms. This intervl produes depression t the ORN PN synpse under ontrol onditions nd llowed us to oserve inreses in the pired-pulse rtio when we deresed relese proility. GABA iontophoresis. In the GABA iontophoresis experiments, highresistne (,8 VM) shrp glss pipette ws filled with solution of 25 mm GABA in wter, nd the ph ws djusted to 4.3 y dding HCl. GABA ws ejeted into the ntennl loe neuropil with rief (3 2 ms) positive urrent pulse using n iontophoresis unit (World Preision Instruments). A onstnt negtive king urrent ws pplied to retin GABA in the iontophoresis pipette etween ejetion events. To ensure tht similr mount of GABA ws relesed into the neuropil in ll experiments, nd tht the time-ourse of GABA relese ws s onsistent s possile, we djusted the lotion of the iontophoresis pipette, the level of negtive king urrent nd the durtion of the ejetion urrent t the eginning of eh experiment. We did not proeed with n experiment until the following riteri were met: EPSC suppression ws,5% t 15 ms fter iontophoresis, mximl EPSC suppression ws,85% t 4 ms fter iontophoresis, nd GABA evoked n outwrd urrent in the reorded PN whih lsted for,1 1.5 s. Antgonists. Antgonists were prepred s onentrted stok solutions nd mesured volume of stok ws dded to the sline perfuste to hieve the finl drug onentrtions. Pirotoxin (Sigm) ws used t 5 mm, (Toris) ws used t 5 mm, nd CdCl 2 (Sigm) ws used t 1 25 mm. Peri-stimulus time histogrms. For the experiments in Figs 1 nd 5 nd in Supplementry Figs 2 nd 3, spike times were extrted from rw ORN nd PN reordings using routines in Igor Pro. Eh ell ws tested with multiple odours, nd eh odour ws presented six times. The response to the first presenttion ws not inluded in our nlysis. Eh of the five remining trils ws onverted into peri-stimulus time histogrm (PSTH) y ounting the numer of spikes in 5-ms ins tht overlpped y 25 ms. These single-tril PSTHs were verged together to generte PSTH desriing the response to n odour in given experiment. Multiple ells orresponding to eh glomerulr lss nd eh ell type (ORN or PN) were tested with given odour in multiple experiments. Averge PSTHs in Fig. 1 nd Supplementry Figs 2 nd 3 represent the men 6 s.e.m. omputed ross experiments. Input output funtions. For the nlysis of odour responses in Fig. 1d, we omputed the verge spike rte over the 5-ms odour durtion nd sutrted the seline firing rte verged over the 5-ms window efore odour onset. Eh point in the input output funtion represents the men PN response to n odour plotted ginst the men response of the ognte ORNs to the sme odour. Fits re single exponentil funtions. Lifetime sprseness. The seletivity of neuron s odour response profile (Fig. 1e) ws quntified s lifetime sprseness: P 2 1 N S~ 1 B 1{1=N 1{ j~1 r j=n P N j~1 r2 j =N A where N 5 the numer of odours nd r j is the nlogue response intensity of the neuron to odour j minus seline firing rte. Anlogue response intensity ws the men spike rte (verged ross five sweeps) during the entire 5-ms odour stimulus period. Any vlues of r j, were set to zero efore omputing lifetime sprseness (this ws the only nlysis in this study in whih negtive responses were zeroed). Responses to prffin oil (solvent ontrol) were not onsidered in the sprseness nlysis. We omputed the lifetime sprseness for eh individul ell in our study for whih we tested t lest 12 of the odours in our set. We used the Mnn Whitney U-test to ssess the signifine of sprseness differenes. Correltions etween rnked odour preferenes. To ompute orreltions etween ORN nd PN odour preferenes, we omputed Spermn s rnk orreltion oeffiient (r) on the odour response vetors of these neurons. This is 28 Nture Pulishing Group

7 doi:1.138/nture6864 nonprmetri mesure tht quntifies how well ny monotoni funtion ould desrie the reltionship etween ORN nd PN responses. We hose this rther thn liner (Person s) orreltion oeffiient euse the intrinsi input output funtion of these glomeruli is highly nonliner (Fig. 1d). For eh glomerulus, we omputed Spermn s r on eh pirwise omintion of individully reorded ORNs nd PNs for whih we tested t lest 12 of the sme odours on oth ells (for glomerulus VM7, n 5 27 omprisons for ORNs ompred to PNs with ntenne nd n 5 38 for ORNs ompred to PNs without ntenne; for glomerulus VC1, n 5 19 for ORNs ompred to PNs with ntenne nd n 5 25 for ORNs ompred to PNs without ntenne). We report the men of these pirwise orreltions. We ssessed the signifine of differenes in Spermn s r with the Mnn Whitney U-test. Quntifying hnges in memrne potentil. In Figs 2 nd 5, we show memrne potentil responses verged ross multiple experiments. To generte these we first verged 4 6 onseutive sweeps within single experiment for eh odour nd low-pss filtered this t 13 Hz to remove ny spikes. Next we verged these responses ross experiments, with 6 s.e.m. shown in pstel. To quntify the mgnitude of lterl input in Fig. 2d f, we integrted the hnge in memrne potentil (versus seline) over 2-s intervl eginning t odour onset. We hose this intervl euse shorter intervls did not pture s muh odour dependene in the mgnitude of lterl input. In Fig. 2d, f, PN disinhiition ws omputed y verging PN firing rtes over the 2 s fter odour onset, nd then verging ross experiments (4 12 PNs per odour), efore omputing the differene in the PN responses with ntenne versus without ntenne. In Fig. 2e, totl ORN tivity for eh odour ws omputed y summing ll the response rtes in ref. 23. There re only 14 dt points in this pnel euse only 14 of our odours were inluded in ref. 23. EPSC mplitudes. We mesured EPSC mplitudes in Figs 3 nd 4 nd in Supplementry Figs 6 8 y first verging together lok of 8 2 sweeps from the sme experiment. From these verged tres, we omputed the EPSC mplitude s the verge over.9-ms window entred on the pek. Before verging ross experiments, we normlized the trin of evoked EPSCs to the seline EPSCs evoked just efore either odour or GABA onset. 28 Nture Pulishing Group