Sensory processing in the Drosophila antennal lobe increases reliability and separability of ensemble odor representations

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1 27 Nture Pulishing Group Sensory processing in the Drosophil ntennl loe increses reliility nd seprility of ensemle odor representtions Viks Bhndwt 1, Shwn R Olsen 1,2, Nthn W Gouwens 1,2, Michelle L Schlief 1,2 & Rchel I Wilson 1 Here we descrie severl fundmentl principles of olfctory processing in the Drosophil melnogster ntennl loe (the nlog of the verterte olfctory ul), through the systemtic nlysis of input nd output spike trins of seven identified glomeruli. Repeted presenttions of the sme odor elicit more reproducile responses in second-order projection neurons () thn in their presynptic olfctory receptor neurons (). PN responses rise nd ccommodte rpidly, emphsizing odor onset. Furthermore, wek ORN inputs re mplified in the PN lyer ut strong inputs re not. This nonliner trnsformtion rodens PN tuning nd produces more uniform distnces etween odor representtions in PN coding spce. In ddition, portions of the odor response profile of PN re not systemticlly relted to their direct ORN inputs, which proly indictes the presence of lterl connections etween glomeruli. Finlly, we show tht liner discrimintor clssifies odors more ccurtely using PN spike trins thn using n equivlent numer of ORN spike trins. Ech glomerulus in the olfctory system receives synptic input from mny, ll of which express the sme odornt receptor gene. Ech second-order neuron sends dendrite into single glomerulus, so for ech odornt receptor gene there is n identifile ORN type nd corresponding type of second-order neuron. An odornt typiclly ctivtes multiple ORN types, nd so ech odor is represented s popultion code cross different glomerulr processing chnnels 1,2. Wht hppens to olfctory signls s they move through these chnnels? It is techniclly chllenging to ddress this question in vertertes ecuse there re so mny glomeruli. In Drosophil, the prolem is comprtively simpler ecuse the ntennl loe contins only B5 glomeruli. Ech of these glomeruli hs stereotyped position tht is identifile cross flies, nd lmost ll hve een mtched to n identified ORN type 3 6.Fortheseresons,itmyeesiertodiscover the sic principles of erly olfctory processing in this model orgnism. In generl, effective informtion trnsmission requires tht the response evoked y stimulus should e highly relile, nd tht the responses evoked y different stimuli should e distinctive. Therefore, we hve focused on two fundmentl questions. First, how reproducile is the numer of spikes evoked y repeted presenttions of the sme odor? There hs een remrkly little ttention pid to the reproduciility of olfctory responses, nd the smll numer of previous studies on this issue hve een concerned with the precision of spike timing rther thn the reproduciility of spike counts 7,8. Response reproduciility is centrl issue in sensory processing ecuse the signl-to-noise rtio of neurl response limits the rte of informtion trnsmission y tht neuron. The second fundmentl question concerns the distinctiveness of neurl responses to different stimuli. How selective re, nd how does their selectivity compre with tht of second-order olfctory neurons? Three studies pulished more thn 2 yers go in vertertes reched conflicting conclusions on this issue, ut it ws not fesile for these investigtors to directly compre the selectivity of pre- nd postsynptic neurons corresponding to the sme glomerulus More recently, three studies mde this direct comprison in the Drosophil ntennl loe, ut gin the results were conflicting Two of these studies used geneticlly encoded sensors, which my not report spike trins fithfully owing to their limited dynmic rnge 15,16 ; the third study recorded spike trins directly, ut exmined only one glomerulus. Here we im to resolve these issues with systemtic nlysis of the inputs nd outputs of seven glomeruli in the Drosophil ntennl loe (Supplementry Fig. 1 online). Our results show tht there is mjor trnsformtion of olfctory representtions in this region of the rin. The most importnt effects of this trnsformtion re to improve the signl-to-noise rtio of individul spike trins nd to distriute odor representtions more uniformly in neuronl coding spce. RESULTS Odor responses re more relile in thn in The vriility of neuronl response cn e quntified y ssessing the vriility in the numer of spikes evoked y sensory stimulus. In most sensory systems, the spike-count vriility of stimulus-evoked responses increses t ech successive level of processing in sensory 1 Deprtment of Neuroiology, Hrvrd Medicl School, 22 Longwood Avenue, Boston, Msschusetts 2115, USA. 2 These uthors contriuted eqully to this work. Correspondence should e ddressed to R.I.W. (rchel_wilson@hms.hrvrd.edu). Received 16 July; ccepted 16 August; pulished online 7 Octoer 27; doi:1.138/nn VOLUME 1 [ NUMBER 11 [ NOVEMBER 27 NATURE NEUROSCIENCE

2 27 Nture Pulishing Group Figure 1 Odor responses re more relile in thn in. () Odor responses of n ORN nd PN pre- nd postsynptic to the glomerulus (glomerulus VA2, odor is gernyl cette). Ech tick represents spike, nd ech row in rster represents different tril. The gry r indictes 5-ms odor stimulus period. () Men odor responses re lrger in (mgent) thn in (green). Spikes were counted in 5-ms ins nd verged cross five trils with the sme odor, then verged cross ll locks of trils (ll odors nd ll experiments). The gry r indictes the stimulus period; the lck r indictes 1-ms period when verge PN firing rtes re mximl. (c) Stndrd devitions (s.d.) of spike counts in five trils with the sme odor, verged cross ll locks of trils (ll odors nd ll experiments). (d) Coefficient of vrition (s.d./men) of spike counts in five trils with the sme odor, verged cross ll locks of trils. Note tht the coefficient of vrition of PN responses drops gin fter odor offset. This is ecuse some responses contin zero spikes for n epoch following odor offset, so the s.d. in these ins is zero for some responses. (e) The verge s.d. of spike counts is lower for thn for even when men firing rtes re mtched. s.d. vlues were mesured for ll counting windows in ll locks of trils, inned ccording to men firing rte nd verged cross ll counting windows in the sme in. Note tht ecuse the s.d. devition depends sulinerly on the men, the verge coefficient of vrition is lrger thn (the verge s.d.)/(the verge men). system However,intheDrosophil ntennl loe, B4 with the sme receptive field converge onto B4 in ech glomerulus Thus, y pooling cross these inputs, might e le to reduce their response vriility. We therefore compred the reliility of odor-evoked spike counts in nd. We presented n odor stimulus in multiple consecutive trils to the sme cell ( lock of trils; Fig. 1). To quntify the spike-count reliility cross trils, we divided ech set of repeted responses into 5-ms windows tht overlpped y 25 ms. In ech time window, we computed the men nd the stndrd devition of the spike count cross repeted responses y the sme cell to the sme odor. Odors typiclly evoked more vigorous responses in thn in (Fig. 1, nd Supplementry Fig. 2 online). So, lthough the typicl stndrd devition of PN responses is slightly greter thn tht of ORN responses (Fig. 1c), PN responses re less vrile in proportion to the mgnitude of the response (P o 1 15,whethercompring over the entire stimulus period or the 1-ms epoch t the response pek, Mnn-Whitney U-test, n ¼ 779 ORN responses nd 843 PN responses; Fig. 1d). Thus, individul re more relile thn individul, which should tend to mke their responses more informtive. We lso compred the stndrd devitions of ORN nd PN spike counts s function of the men spike count for ech time window. For ll men spike counts, hve lower stndrd devition thn (Fig. 1e). Furthermore, the stndrd devition is not strongly dependent on the men, nd so stronger responses hve lower coefficient of vrition. Becuse PN responses re on verge stronger thn ORN responses (Fig. 1), this lso tends to mke more relile thn. preferentilly trnsmit the rising phse of ORN signls ORN responses typiclly do not pek until 1 3 ms fter odor onset 21. This is proly ecuse spiking is coupled to odornt receptor ctivtion y the genertion of second messengers. However, odors cn trigger rpid ehviorl responses in flies, with totl ltency from stimulus to motor rection of less thn 3 ms 23. This suggests tht neurons in the rin re preferentilly tuned to detect the rising phse of ORN signls, rther thn the response pek. This motivted us to compre the onset kinetics of odor responses in synpticlly connected nd. Spikes per in d ORN PN Men s.d./men Spikes per in s.d. (spikes per in) Compring peri-stimulus time histogrms verged cross ll odor responses in ll cells, we noted tht PN responses rise more rpidly thn ORN responses (Fig. 2). Furthermore, PN responses egin to decy while ORN responses re still growing. This is lso cler in most direct comprisons etween synpticlly connected nd (Figs. 1 nd 2,c). Overll, PN responses pek significntly fster thn ORN responses (P o 1 7,piredt-test, n ¼ 69 odor-glomerulus comintions; Fig. 2d), nd the time to hlf-decy of the response is shorter for thn for their presynptic (P o 1 5, pired t-test; Fig. 2e). Tken together, fster rise nd fster decy men tht more excittory drive to third-order neurons occurs within n erly epoch of the odor response (P o 1 11,piredt-test; Fig. 2f). Therefore, ct s high-pss filters tht preferentilly signl the rising phse of the ORN response. Becuse PN responses ccommodte rpidly, we chose to quntify response mgnitudes in y mesuring the verge firing rtes during n erly epoch of the response ( 1-ms time window eginning 1 ms fter odor onset; Fig. 1). Becuse fruitfly cn respond rpidly fter encountering n odor, this erly epoch should e prticulrly informtive to downstrem neurons. Throughout this study, we lso quntified PN responses in different wy: following other investigtors 21,24, we mesured the verge spike rtes over the entire 5-ms stimulus period. The min conclusions from this study re the sme for oth of these response metrics. nd differ in odor selectivity nd odor preferences Setting side the issues of tril-to-tril reliility nd response kinetics, we exmined the verge response mgnitudes for ech cell type to our odor stimuli (Fig. 3). How does the response profile of ech PN type compre with the response profile of its corresponding? We egn y sking simply whether these responses re linerly correlted. For ech glomerulus we found sttisticlly significnt correltion etween the ORN nd PN response profile (P o.5 for ll seven glomerulr comprisons, Person s correltion; Supplementry Tle 1 online), ut r 2 vlues re only in the rnge of This mens tht liner scling of ORN responses explins only 26 81% of the odor-dependent vrince in PN responses. c e s.d Men (spikes per in) NATURE NEUROSCIENCE VOLUME 1 [ NUMBER 11 [ NOVEMBER

3 27 Nture Pulishing Group Frction of mximum c Spikes per s Time (ms) Time (ms) 25 Time (ms) Two fetures of PN odor responses diminish this liner correltion. First, for ech glomerulus, re less selective thn their presynptic (P o.5, Wilcoxon signed-rnk test; Fig. 3, similr results in Supplementry Fig. 3 online). To test the generlity of this result, we lso compred ORN nd PN selectivity for glomerulus DM4 t three odor concentrtions. As with our stndrd concentrtion (1:1, dilution), weker stimuli (1:1, nd 1:1, dilutions) produce PN response profiles tht were less selective thn the corresponding ORN response profiles (Fig. 4; seelsosupplementry Fig. 4 online). Other investigtors who used identicl stimulus conditions hve shown tht ORN responses re very sprse t the 1:1, dilution, indicting tht this concentrtion is ner the ottom of the dynmic rnge of this system 21,22,24,25. These results show tht rod PN tuning is phenomenon tht is not limited to high odor concentrtions. Another fctor tht diminishes this liner correltion is tht the rnk order of odor preferences differs for nd. For exmple, wheres ethyl utyrte is the 3rd-rnked odor of DL1, it is only rnked 16th mong the odor responses of DL1 (Fig. 3). Some of this difference is due to errors in estimting ech verge response profile on the sis of limited smple of individul experiments. However, smpling error cnnot completely ccount for this result. This cn e shown y piring n individul ORN with corresponding individul PN nd computing the correltion etween their odor rnks, nd then compring the distriution of these correltions with the correltions otined from ORN-ORN or PN-PN pirings. Becuse we were not le to test every odor in every experiment, we ssemled mny simulted response profiles y drwing rndomly from norml distriution defined y the men nd stndrd devition of ech verge response profile (Fig. 5; see lso Supplementry Methods Figure 3 nd differ in their odor selectivity. () Response profiles of synpticlly connected (green) nd (mgent) for seven glomeruli. Brs show verges cross ll experiments (±s.e.m.; see Supplementry Tle 2 for n). Responses re mesured s the men spike rte during the 1-ms epoch when firing rtes re peking (lck r in Fig. 1 d), minus the seline firing rte. Results re similr over the entire 5-ms stimulus period (Supplementry Fig. 3). () The selectivity of ech response profile is quntified s lifetime sprseness 29,48 (see Supplementry Methods; ¼ nonselective, 1 ¼ mximlly selective). nd tht correspond to the sme glomeruli re connected. re consistently less selective thn their corresponding. The highest ORN sprseness vlue is for glomerulus DL1 nd the lowest is for glomerulus VM2. Spikes per s 2 1 d Time (ms) e f Percentge Figure 2 preferentilly trnsmit the rising phse of ORN signls. () Averge pek-normlized peri-stimulus time histogrms (PSTHs), verged cross ll odors nd ll glomeruli (±s.e.m.). Note tht PN responses rise nd decy more rpidly thn ORN responses. Odor stimultion egins t ms nd ends t 5 ms. () An exmple compring the responses of prend postsynptic neurons to the sme odor. PSTHs show the verge response of nd in glomerulus VA2 to gernyl cette (men ± s.e.m., verged cross experiments). Note tht the PN response is roust t time point when the hve just egun to respond, nd the PN response egins decying efore the hve peked. (c) Another exmple of PSTHs for nd in glomerulus DM1 showing responses to ethyl utyrte. The PN response rises fster nd peks erlier, even though in this cse the PN pek is smller. (d) Compred with ORN responses, PN responses hve shorter ltency to rech 9% of the response pek (men ± s.e.m., cross ll locks of trils; see Supplementry Methods). (e) PN responses hve fster decy from pek to hlf-pek. (f) A lrger percentge of the totl spike count occurs in the first 2 ms fter odor onset for PN responses compred with ORN responses. online). The medin correltion etween ORN nd PN rnks ws only.47, which is sustntilly lower thn the correltion etween of the sme type or etween of the sme type (.65 nd.61, respectively; Fig. 5). The simplest explntion for this result is tht the odor preferences of PN re influenced y lterl connections etween glomeruli 26,27. A nonliner trnsformtion function for ech glomerulus So, the output of glomerulus is not simple liner scling of its inputs. Furthermore, ecuse nd differ in their rnked Spikes per s DL1 DM1 DM2 DM3 DM4 VA2 VM2 Benzldehyde Butyric cid 2,3-Butnedione 1-Butnol Cyclohexnone Ethyl utyrte Ethyl cette Gernyl cette Isomyl cette 4-Methyl phenol Methyl slicylte 3-Methylthio-1-propnol Octnl 2-Octnone cis-3-hexen-1-ol Pentyl cette trns-2-hexenl γ-vlerolctone Lifetime sprseness VOLUME 1 [ NUMBER 11 [ NOVEMBER 27 NATURE NEUROSCIENCE

4 27 Nture Pulishing Group Spikes per s :1, 1:1, 1:1, Benzldehyde Butyric cid 2,3-Butnedione 1-utnol cis-3-hexen-1-ol Ethyl utyrte Ethyl cette Gernyl cette Methyl slicylte 2-Octnone Pentyl cette Lifetime sprseness 1 odor preferences, no monotonic function cn descrie the reltionship etween the ORN nd PN response profile for glomerulus. We therefore sked whether there is ny systemtic reltionship etween ORN nd PN responses. For ech glomerulus, we plotted PN responses to ech odor s function of ORN responses to the sme odor (Fig. 6; see lso Supplementry Fig. 5 online). This reveled consistent trnsformtion function for ech glomerulus, leit with some sctter. These functions hve similr shpe for most glomeruli: they initilly slope steeply, mening tht the gin of the trnsformtion function is high for wek inputs. As ORN input levels increse these curves fltten, mening tht the gin of the trnsformtion function decreses. (This is true for ll the glomeruli we tested prt from DL1.) Therefore, these plots show tht inherit much of their tuning from their presynptic, ut the trnsformtion is nonliner. This type of trnsformtion function my e useful ecuse it mkes etter use of the ville response rnge of PN. This is illustrted y projecting the points in ech plot onto oth the x nd y xes (Fig. 6). do not use ll prts of their dynmic rnge with equl frequency in response to our stimuli. However, two odors tht elicit similrly Figure 5 The rnk order of ORN nd PN odor preferences is different. () An exmple illustrting how we computed correltions etween the odor rnks of individul cell response profiles. Here we show the men nd stndrd devition of the ORN nd PN response profiles for glomerulus DM4. (Note the tle is truncted fter six odors.) We drew rndomly from these distriutions to produce representtive simulted profiles for two individul nd two individul (rrows). Next we rnked the odors in ech individul response profile (lue). In this exmple, the correltion coefficient etween the 18 odor rnks of ORN smple 1 nd ORN smple 2 (r s )is.79. Correltion coefficients re lower for PN-PN comprisons (.58 in this exmple). In comprison with ech of these, ORN-PN correltions re much lower (.33,.39,.42 nd.46 in this prticulr exmple). () Histogrms showing the distriution of Spermn s rnk correltion coefficients (r s ), ccumulted cross 2, runs of the simultion procedure for ech glomerulus. Arrowheds indicte the medin of ech distriution. The ORN- PN correltions (gry) do not lie etween the ORN-ORN (green) nd PN-PN (mgent) correltions, indicting tht ORN nd PN odor rnks re not drwn from the sme underlying men distriution. Figure 4 nd differ in their odor selectivity even t low stimulus intensities. () Response profiles for DM4 nd to pnel of 11 odors t three concentrtions. Brs show verges cross ll experiments (±s.e.m.; see Supplementry Tle 3 for n). Becuse the response pek tends to occur lter for more dilute stimuli, we mesured responses s the men spike rte during the entire 5-ms stimulus period, minus the seline firing rte (s in Supplementry Fig. 3). () The selectivity of ech response profile for the three odor dilutions. nd tht correspond to the sme dilution re connected. Note tht DM4 re consistently less selective thn DM4 t ll three concentrtions. (Selectivity t the 1:1, dilution is slightly different from the selectivity vlue plotted in Supplementry Figure 3 for this glomerulus ecuse here we used only suset of our 18 test odors.) wek ctivity in n ORN cn elicit different levels of ctivity in postsynptic PN, ecuse ech glomerulr trnsformtion function shows high gin t low ORN input levels. This tends to distriute the responses to these stimuli more uniformly throughout the response rnge of ech PN. This type of sensory trnsformtion hs een termed histogrm equliztion 28 ecuse it produces fltter histogrm of response intensities. To exmine whether this is the cse cross the entire popultion of cells in our dt set, we plotted the distriution of response intensities for nd, ccumulted cross ll odors nd ll glomeruli (Fig. 6). The ORN response histogrm shows lrge pek t low response intensities, nd long, flt til covering the rest of the ORN dynmic rnge. The PN response histogrm, y contrst, is much fltter, indicting tht ll ville response intensities re used with more uniform frequency. In this sense, encode our odor stimuli more efficiently thn do. Although there is cler overll reltionship etween ORN nd PN responses for ech glomerulus, it is lso importnt to note tht these functions do not predict PN odor responses completely. Becuse nd differ in their rnked odor preferences, no monotonic function will ccount for ll these dt. Men rte ± s.d. (spikes per s) ORN 4.3 ± ± ± ± ± ± 21.8 Frction etc PN 28.7 ± ± ± ± ± ± 5.1 ORN smple (Smple rte Smple rnk) ORN smple PN smple Spermn's rnk correltion coefficient (r s ) PN smple NATURE NEUROSCIENCE VOLUME 1 [ NUMBER 11 [ NOVEMBER

5 27 Nture Pulishing Group PN response (spikes per s) Frction DL1 DM1 DM2 DM Odor representtions in multiglomerulr coding spce Third-order neurons receive convergent input from multiple PN types It is therefore importnt to exmine odor representtions in multiglomerulr coding spce. In the simplest cse, histogrm equliztion in one dimension should lso produce more uniform distriution of odors in multiple dimensions. To visulize odor representtions in seven-dimensionl spce, we reduced the dimensionlity of this spce y performing principl components nlysis. The first two principl components define the two-dimensionl projection tht mximizes the vrince of the dt. In this projection, most odors re still clustered ner the origin of the ORN spce, with only few odors locted fr from this cluster (Fig. 7). In the equivlent PN spce, odors fill the ville coding spce more uniformly (Fig. 7). Thus, s result of the ORN-to-PN trnsformtion, odor representtions re distriuted more efficiently in multiglomerulr coding spce. In concrete terms, the ensemle ptterns of spiking ctivity elicited y ny two odors ecome more different. We quntified this y mesuring Eucliden distnces etween odors in seven-dimensionl spce for ll possile pirwise comintions of odors ([18 choose 2] ¼ 153 pirs). During the erly epoch of odor responses, distnces re significntly lrger in PN spce thn in ORN spce (P o.1, pired t-test, n ¼ 153). Moreover, the distriution of distnces is nrrower for thn for over the entire stimulus period (note the differing interqurtile rnges in Fig. 7c,d). This mens tht odors re distriuted more uniformly in PN coding spce. Some odor distnces decrese, ut others increse. Is the seprtion of odors in multidimensionl spce lrger or smller thn we would predict, sed solely on the independent odor seprtion in ech one-dimensionl coding chnnel? The nswer depends on the degree of correltion etween the different glomeruli. Lterl connections shpe PN odor responses 26,27 (Fig. 5); if these connections increse correltions etween different PN types, this would decrese inter-odor distnces in multidimensionl spce. To ddress this issue, we constructed simulted dt set tht preserves the distriution of response mgnitudes for ech glomerulr cell type, ut reks ny dependencies etween odor responses in different DM4 VA2 VM2 All glomeruli 2 1 ORN response (spikes per s) Frction Response mgnitude (spikes per s) Response mgnitude (spikes per s) Figure 6 PN odor responses re prtly explined y highly nonliner trnsformtion of their direct ORN inputs. () For ech glomerulus, the verge PN response to n odor is plotted ginst the verge ORN response to tht odor (lck symols, ± s.e.m.). Curves re exponentil fits (y ¼ y + Ae kx ). Green nd mgent symols re projections of the dt onto the x nd y xes, showing tht odor responses generlly occupy the dynmic rnge of PN more evenly thn they occupy tht of n ORN. Responses re mesured s the men spike rte during the 1-ms epoch when firing rtes re peking (with no seline sutrction), ut results re similr if responses re mesured s the men spike rte during the entire 5-ms stimulus period (Supplementry Fig. 5). () Histogrms of ORN nd PN response mgnitudes. Ech histogrm is ccumulted cross ll 126 response mgnitudes (¼ 7 glomeruli 18 odors). The PN histogrm is fltter thn the ORN histogrm, indicting tht use their dynmic rnge more efficiently. 2 glomeruli. We chieved this y independently shuffling the odor lels on ech glomerulr response profile. We then mesured interodor distnces in seven-dimensionl spce for ll possile pirwise comintions of odors. When we repeted this simultion mny times, the rnge of distnces we otined ws indistinguishle from the distnces we mesured in our rel dt set (Fig. 7c,d). This mens the seprtion etween ensemle odor representtions is roughly wht we would predict, sed solely on histogrm equliztion in ech glomerulus individully. Correltions etween cell types nd odors We hve seen tht use ll prts of their dynmic rnge with pproximtely equl frequency, nd in this sense encode odors more efficiently thn do 28. However, the term efficient coding hs lso een pplied to the ide tht the responses of different neurons should e mximlly independent from ech other 33.Wemesuredthe independence of different glomerulr coding chnnels y computing the percentge of the vrince in the ensemle odor responses tht is cptured y ech of the seven principl components of the sevendimensionl ORN or PN coding spce. If ll seven cell types were completely correlted, then the first principl component would ccount for 1% of the vrince in the dt. In other words, ll the dt would lie long single line in multidimensionl spce. Conversely, if ll cell types were perfectly decorrelted, nd if the dt were drwn from multidimensionl Gussin distriution, then ech principl component would ccount for n equl mount of the totl vrince (1% C 7 ¼ B14%). (Even in this cse, we would need very lrge odor set to discern this perfect decorreltion.) The principl components of our ORN dt set fll etween these hypotheticl extremes (Fig. 8). In prt, this reflects the limited size of our odor set nd the non-gussin distriution of the ORN response histogrms (Fig. 6). We demonstrted this y independently nd rndomly shuffling the odor lels on ech of the seven ORN response profiles nd re-computing the principl components of this simulted dt set. These simultions lwys produced first principl component tht ccounted for disproportiontely lrge shre of the vrince (usully 3 4%; Fig. 8). However, the principl components of the rel (non-shuffled) dt set re even more skewed, with the first principl component ccounting for 54% of the vrince. This 1478 VOLUME 1 [ NUMBER 11 [ NOVEMBER 27 NATURE NEUROSCIENCE

6 27 Nture Pulishing Group Principl component 1 (spikes per s) c Inter-odor distnces (spikes per s) Principl component 2 (spikes per s) Third qurtile 2 Medin First qurtile Dt Simultion mens tht in different glomeruli hve odor preferences tht re more highly correlted thn we would expect, sed solely on the distriution of response mgnitudes in ech glomerulr chnnel. Similrly, out hlf the vrince in the PN dt is cptured y the first principl component (51%; Fig. 8). This is minly due to the limited size of our odor set nd the non-gussin distriution of the PN response histogrms, s shuffling the odor lels on ech PN response profile lwys produced skewed distriution of principl component contriutions (Fig. 8). Becuse rel PN dt produced distriution tht ws even more skewed thn the simulted dt, (like ) re more correlted thn we would expect, sed solely on the distriution of response mgnitudes for ech PN type. In summry, sensory processing in the Drosophil ntennl loe does not chnge the degree of independence etween different glomerulr coding chnnels. The conclusions of this nlysis re similr regrdless of whether we mesure spike rtes round the response pek (Fig. 8) or over the entire stimulus period (Supplementry Fig. 6 online). PN responses re more linerly seprle thn ORN responses Incresed PN reliility nd more uniform odor distnces in PN coding spce should men tht odors re more discriminle on the sis of PN spike trins thn on the sis of n equivlent numer of ORN spike trins. We tested this prediction y mesuring the ility of n lgorithm to identify the odor stimulus on the sis of the ensemle neurl response elicited y tht odor. Becuse our dt come from single (not multiple) unit ORN nd PN recordings, we simulted multi-unit responses y ssemling dt from different glomerulr clsses. Ech simulted dt set consisted of 9 multi-unit responses (18 odors with 5 spike trins per odor per cell). We performed liner discriminnt nlysis to identify the liner comintions of input vriles tht est seprted ll 18 odor response clusters from ech other. To evlute the qulity of these discrimintions, we withheld 1 multi-unit odor response from the dt set, trined the lgorithm with the remining 89, nd predicted the odor corresponding to the one withheld response. The predicted odor ws then compred 1 d Figure 7 Odors re distriuted more uniformly in ensemle PN coding spce thn in ensemle ORN coding spce. () Averge odor responses from seven ORN types projected onto the spce defined y the first two principl components. Ech point represents different odor. () Sme s for PN dt (with the sme color conventions), showing more uniform seprtion etween odor representtions. (c) The difference etween ensemle ORN responses to different odors is quntified s the Eucliden distnce etween odor representtions in seven-dimensionl spce. Distnces re computed for ll 153 pirwise comintions of the 18 odor stimuli, nd the medin nd interqurtile rnge of this distriution re plotted here for ech time point. The interqurtile rnge is wide ecuse some odors re well seprted in ORN spce, ut mny re poorly seprted. Blue nds indicte the rnge of results otined y shuffling odor lels on ech glomerulr response profile (see Supplementry Methods). The gry r indictes the 5-ms stimulus period nd the lck r indictes the 1-ms period when firing rtes were mesured for nd. (d) Smesc for PN responses. At the pek of the response (lck r), distnces re significntly lrger in PN spce compred with ORN spce. PN responses then quickly ccommodte (Fig. 2), nd so inter-odor distnces shrink. However, the interqurtile rnge of distnces remins smller thn in ORN spce. This indictes more uniform distriution of distnces. As in c, shuffling odor lels on ech glomerulr response profile produces rnge of results (lue nds) tht resemles the rel dt. with the ctul odor. We repeted this nlysis with mny independently ssemled multi-unit responses t ech time point in the odor response. Before odor onset, the prediction success rte hovers ner chnce (Fig. 9). (The success rte is slightly ove chnce ecuse different cells hve different spontneous firing rtes, nd spontneous firing rtes sometimes drift during experiments; thus, spontneous firing rtes were slightly predictive of the odor ecuse successive trils with n odor were presented consecutively rther thn interleved). After odor stimulus onset, success rtes rise rpidly. As expected, including more glomerulr clsses in the dt set produced higher success rtes Percentge of vrince 5 25 Dt Simultion Principl component Principl component Figure 8 Correltions etween different glomeruli re similr for nd. () Principl components nlysis (PCA) ws pplied to the 18 7 ORN response mtrix. The mgnitude of the vrince ccounted for y ech principl component (green circles) is mesure of the correltions etween different ORN types. Blue nds indicte the rnge of results otined y shuffling odor lels on ech glomerulr response profile (see Supplementry Methods). Compring the dt nd the simultion shows tht re less independent in their odor responses thn we would expect, sed solely on the distriution of response mgnitudes within ech glomerulr coding chnnel. () Sme s for the 18 7 PN response mtrix. Correltions etween PN types re similr to correltions etween ORN types. NATURE NEUROSCIENCE VOLUME 1 [ NUMBER 11 [ NOVEMBER

7 Averge success rte 1..5 Averge success rte 1..5 mens the verge PN pool inputs from 1 4 (depending on whether ech ORN contcts ll in glomerulus). Becuse pooling N ORN inputs should decrese the vriility of the pooled verge y ON, we would expect the coefficient of vrition to improve y O1 to O4. The effect we descrie is on the low end of this rnge, suggesting tht ech ORN contcts only single PN, or tht receive dditionl noise from other neuronl sources. 27 Nture Pulishing Group Numer of glomeruli Figure 9 A liner discrimintor cn clssify odors more ccurtely with responses from multiple thn with responses from the sme numer of. () Odor clssifiction success rte from liner discrimintor nlysis with dt sets tht include cells from three glomerulr clsses. All possile comintions of three glomeruli were smpled. Points re the men ± s.e.m., verged cross 2 runs of the clssifiction procedure. The dotted line represents chnce performnce. () Success rte is higher for PN dt thn for ORN dt, regrdless of how mny glomerulr clsses re included in the dt set. Points re the men ± s.e.m., verged over the 1-ms window shown in, nd then verged cross 2 runs of the clssifiction procedure. Dshed green nd mgent lines plot the clssifiction success rte during the seline period efore odor onset; this is n rtifct of vrying spontneous ctivity rtes (see text), nd ORN nd PN performnce is similr. Dotted lck lines indicte perfect nd chnce performnce. (Fig. 9). Becuse success rtes using PN dt plteu t 1% for some clssifictions tht use dt from more thn three glomeruli, this procedure underestimtes the difference etween ORN nd PN responses. Nevertheless, success rtes were significntly higher for PN dt thn for ORN dt for ll conditions in Figure 9 (P o.5, Mnn-Whitney U-tests, n ¼ 2 runs of the clssifiction procedure for nd for ech condition). This demonstrtes tht liner discrimintor cn clssify odors more ccurtely with responses from severl thn with responses from the sme numer of. DISCUSSION An improved signl-to-noise rtio Studies in other systems hve implied tht the vriility of stimulusevoked spike counts lmost lwys increses t successively higher levels of sensory processing 34. For exmple, the visul responses of higher corticl neurons re often very noisy 17, in contrst to the reliility of retinl gnglion cells 35. A direct comprison of the responses evoked y identicl stimuli in the retin, thlmus nd visul cortex hs confirmed tht spike-count reliility decreses t ech successively higher level of the visul strem 18. This is despite the fct tht simple cell in primry visul cortex pools signls from B3 thlmic neurons 36, which should improve its reliility. Similrly, direct comprison of spike trins t successive levels of n insect uditory circuit hs found tht noise increses t successively higher levels 19.Ourresultsshow different trend: spike counts in individul re more consistent thn spike counts in individul. This is prtly ecuse tend to fire more vigorously thn their presynptic in response to the sme stimulus, nd stronger responses re more relile for oth nd. This my imply n incresingly deterministic control of spike timing t high firing rtes owing to intrinsic refrctoriness 37. However, even t the sme firing rtes, PN responses re more relile thn ORN responses. This my reflect the enefits of pooling: ech PN is postsynptic to mny, nd ll these respond in similr wy to odors 21,22,38. If noise is uncorrelted cross, then pooling these inputs should improve the reliility of PN responses. On lnce, the improvement in reliility is smller thn one might predict. Ech glomerulus corresponds to B4 nd B4 ; this. High-pss filtering of olfctory signls Our results show tht cn e extremely sensitive to smll differences etween wek ORN inputs. Even smll increse in ORN spike rte ove the seline cn produce roust response in postsynptic. As result, PN responses rise rpidly even when ORN responses uild slowly. This is prticulrly useful ecuse the onset kinetics of re intrinsiclly limited y the speed of the signl trnsduction cscdes tht link odornt receptor ctivtion to spike initition. PN responses then rpidly decline while ORN spike rtes continue to rise. This mens tht ct s high-pss filters, trnsmitting the rising phse of ORN responses preferentilly over the tonic component of ORN responses. This rpid ccommodtion might e due to ny of severl mechnisms, including short-term synptic depression t the ORN-to-PN synpse. Tken together, fster rise nd fster decy should shrpen the estimte of odor rrivl time y downstrem neurons. For fly in flight, this should trnslte to n improved estimte of odor plume loction. Notly, Drosophil cn turn in flight less thn 3 ms fter encountering n odor plume 23. A similr phenomenon opertes in the visul system: sluggish photoreceptor responses trigger speedy depolriztions in downstrem neurons 39 nd ultimtely rpid ehviorl responses to visul stimuli. We note tht Drosophil PN responses differ from the responses of locust, which typiclly show more complex temporl ptterning 29,4,41. Locust lso show higher verge level of mintined ctivity throughout the odor response (reltive to the response pek) nd often show excittory responses to odor offset 41. By contrst, Drosophil ccommodte rpidly nd typiclly do not urst fter stimulus offset (ut for some exceptions see Supplementry Fig. 2f). A nonliner trnsformtion increses coding efficiency An importnt finding from this study is tht lthough inherit sustntil portion of their odor tuning from their presynptic, this reltionship is nonliner. This nonlinerity disproportiontely mplifies smll differences etween wek ORN inputs. By contrst, smll differences etween strong ORN inputs re not mplified to the sme degree. Most ORN odor responses cluster in the wek end of the dynmic rnge of the ORN. As result of this nonliner trnsformtion, use their dynmic rnge more uniformly thn do. If ll portions of the dynmic rnge of neuron re used with equl frequency, the crrying cpcity of tht informtion chnnel is mximized ecuse the entropy of the neuron s response is mximized. This tends to protect signls from contmintion y noise dded t lter stges in the processing chnnel 42. This hs long een recognized s useful computtion in sensory processing 28. If roder tuning curves re useful, why hs evolution not simply produced rodly tuned? ORN responses re directly linked to the wy odornt receptor proteins interct with odor molecules; therefore, rodening ORN tuning might require chnging the iophysics of odornt receptors in wys tht re unfvorle for other resons. Brod PN tuning my seem counterintuitive: we tend to think of higher-order neurons s eing more selective thn their presynptic inputs. These expecttions re founded in prt on the prdigm of 148 VOLUME 1 [ NUMBER 11 [ NOVEMBER 27 NATURE NEUROSCIENCE

8 27 Nture Pulishing Group visul processing, in which successive lyers of higher-order neurons re incresingly specilized to represent complex conjunctions of visul fetures. However, there is lso huge expnsion in the numer of higher-order neurons devoted to representing complex visul fetures compred with the numer of retinl gnglion cells. Thus the dimensionlity of higher visul representtions is incresingly lrge, so these rin regions cn fford to code informtion s ensemles of nrrowly tuned neurons. By contrst, in erly olfctory processing, the dimensionlity of the second-order representtion is the sme s the dimensionlity of the first-order representtion. Therefore, totl coding spce cnnot increse (unless time is used s nother coding dimension 43 ). In truly efficient coding scheme, neurons should efficiently encode nturl stimulus distriutions, not ritrry stimulus distriutions 28. Although our odor set is chemiclly diverse nd reltively lrge, it my not e representtive of the odors wild fly would encounter. In the future, it would e interesting to lern whether the principles of olfctory processing we descrie here lso pply to more nturlistic distriution of odor stimuli. Another cvet is tht we hve not smpled ll ntennl loe glomeruli. However, ecuse most of the glomeruli in our dt set showed similr nonliner trnsformtion function, our conclusions proly generlize to mny glomeruli outside our dt set. An interesting specil cse is the glomerulus DA1, which ws not included in our dt set. projecting to this glomerulus respond wekly ut selectively to Drosophil pheromone. Their postsynptic re lso selective for this odor, ut respond much more roustly 44.Thus, this processing chnnel shows mplifiction without chnge in response selectivity. Ensemle odor responses Extending our nlysis from one glomerulr chnnel to multiglomerulr ensemles, we found tht odors re more uniformly seprted in ensemle PN coding spce thn in ensemle ORN coding spce. This seprtion is pproximtely wht we would expect, sed on the incresed seprtion etween odors within ech glomerulr coding chnnel. We demonstrted this using simultion tht mde the odor preferences of ech glomerulus independent from ech other, while preserving the chrcteristic response mgnitudes of ech cell type. We lso found tht sensory processing in the ntennl loe does not sustntilly lter the independence of different glomerulr coding chnnels. The degree of correltion etween different PN types is similr to the degree of correltion etween different ORN types. Notly, hve more correlted odor preferences thn we would expect sed solely on their tuning redth nd the size of our odor set. We lso otined the sme result y re-nlyzing lrge pulished dt set 24 comprising 24 Drosophil ORN types nd 11 odors (result not shown). This result my e due to the common evolutionry origin of different Drosophil odornt receptors in gene dupliction events 45.In ddition, some odors re intrinsiclly more voltile thn others, which will tend to produce similrities in the odor preferences of different glomeruli. Like, re lso more correlted thn we would expect, sed on their tuning redth nd the size of our odor set. This my reflect correltions tht re inherited from. The role of lterl connections etween glomeruli Glomeruli in the Drosophil ntennl loe re connected y GABAergic interneurons 2,46 nd cholinergic interneurons 26,27.Whtistheroleof these connections in the trnsformtions we hve descried? We hve noted tht the rnk order of PN odor preferences is different from the order of ORN odor preferences. We show tht this difference is too lrge to e explined y the uncertinty in our estimtes of ech verge odor response profile (Fig. 5). Therefore, some of this difference is proly cused y lterl interglomerulr connections, ecuse lterl inputs would hve n odor tuning tht reflects the odor preferences of tht re presynptic to other glomeruli. In principle, either inhiitory or excittory lterl connections could cuse this phenomenon. The computtionl significnce of this phenomenon is not cler, s it does not seem to decorrelte the responses of in different glomeruli (Fig. 8). It is esy to see how lterl connections could cuse sctter round ech glomerulr trnsformtion function. However, lterl connections my lso ply n importnt prt in determining the underlying shpe of these trnsformtion functions. For exmple, ll of these functions hve y intercept ove (Fig. 6). This reflects the tendency of to respond wekly to n odor even when their presynptic re not responding t ll. Lterl excittory connections re strong enough to trigger these responses 26,27. Moreover, lterl inhiition could ct on ORN xon terminls to govern the proility of neurotrnsmitter relese, nd therey contriute to nonliner reltionship etween prend postsynptic ctivity. At the nlogous synpse in the olfctory ul, ORN-to-mitrl cell synpses show strong frequency-dependent short-term plsticity tht is modulted y presynptic inhiition through GABAergic locl neurons 47. Thus, we should not ssume tht the systemtic reltionships in Figure 6 re intrinsic to ech glomerulus. More mechnistic experiments will e required to disentngle the role of intr- versus interglomerulr mechnisms in shping these trnsformtion functions. Odor discrimintion We hve shown tht liner discrimintor cn identify odors more ccurtely on the sis of PN spike trins thn on the sis of n equivlent numer of ORN spike trins. This is proly due to oth the incresed distnces etween odors in PN spce nd the improved signl-to-noise rtio mong. It is importnt to point out tht liner discriminnt nlysis is not ment to emulte iologiclly plusile downstrem neuron, nd tht rel third-order neurons will e suject to more constrints thn our lgorithm is. Also, this is not n optiml decoder, nd so its performnce my not reflect the totl mount of informtion in the responses it decodes. Finlly, the totl mount of informtion in the entire PN ensemle cnnot, of course, exceed the totl mount of informtion in the entire ORN ensemle. Wht we hve shown here is tht the informtion in limited suset of the PN ensemle is more useful to liner decoder thn the informtion in n equivlent numer of. This highlights the potentil functionl consequence of incresed PN reliility, comined with incresed inter-odor distnces in PN coding spce. In conclusion, we hve descried two fundmentl tsks tht re ccomplished y the first stge of the olfctory processing strem. On neuron-to-neuron sis, our comprisons show tht signl reproduciility is incresed nd distinctions etween the responses to different stimuli re enhnced. The detils of odor processing in the verterte olfctory ul might e different, especilly ecuse the numer of glomeruli in vertertes is much lrger. Nevertheless, most orgnisms shre common olfctory processing rchitecture, which suggests tht some of the sic principles we hve demonstrted in flies my lso pply to vertertes. METHODS Fly stocks. Flies were rered t room temperture on conventionl cornmel gr. All experiments were crried out on dult femle flies 2 7 d fter eclosion. Supplementry Tle 4 online lists genotypes for ll experiments. See Supplementry Methods for stock origins. NATURE NEUROSCIENCE VOLUME 1 [ NUMBER 11 [ NOVEMBER

9 27 Nture Pulishing Group ORN recordings. Flies were immoilized in the trimmed end of plstic pipette tip under 5 ir ojective mounted on n Olympus BX51WI microscope. A reference electrode filled with sline ws inserted into the eye, nd shrp sline-filled glss cpillry (tip dimeter o1 mm) ws inserted into sensillum. Recordings were otined with n A-M Systems Model 24 mplifier, low-pss filtered t 2 khz nd digitized t 1 khz. ORN spikes were detected using routines in IgorPro (Wvemetrics). See Supplementry Methods for detils. PN recordings. Whole-cell recordings from PN somt were crried out in vivo s previously descried 46. One neuron ws recorded per rin nd the morphology of ech cell ws visulized post hoc with iocytin-streptvidin nd nc82 histochemistry s descried previously 46, except tht in the secondry incution we used 1:25 got nti-mouse AlexFluor633 nd 1:1, streptvidin AlexFluor568 (Moleculr Proes). See Supplementry Methods for detils. Olfctory stimultion. We chose pnel of 18 odors to mximize the chemicl diversity of our stimuli, nd to mximize overlp with odors used in other studies of the sme 22,24. For ll experiments (except in Fig. 4), odors were diluted 1:1 v/v in prffin oil (J.T. Bker, VWR no. JTS894), except 3-methylthio-1-propnol, which ws diluted 1:1 v/v in wter, nd 4-methyl phenol, which ws diluted 1:1 w/v in wter. In Figure 4, odors were diluted 1:1, 1:1,, or 1:1, in prffin oil. See Supplementry Methods for detils. Dt nlysis. See Supplementry Methods for dt nlysis detils. Note: Supplementry informtion is ville on the Nture Neuroscience wesite. ACKNOWLEDGMENTS We thnk K. Ito, L. Luo, L.B. Vosshll nd L.M. Stevens for gifts of fly stocks. We enefited from helpful converstions with S.A. Bccus, V. Jyrmn, H. Kzm, A.W. Liu, J.H.R. Munsell, O. Mzor, M. Meister, R.C. Reid, H. Sompolinsky, G.C. Turner nd Y. Zhou. This work ws funded y grnt from the US Ntionl Institutes of Helth (1R1DC8174-1), Pew Scholr Awrd, McKnight Scholr Awrd, Smith Fmily Foundtion New Investigtors Awrd nd n Armenise-Hrvrd Junior Fculty Awrd (to R.I.W.). S.R.O. is supported y US Ntionl Science Foundtion Predoctorl Fellowship. N.W.G. is supported y Howrd Hughes Medicl Institute Predoctorl Fellowship. M.L.S. is supported y Ntionl Institutes of Helth Postdoctorl Fellowship (1F32DC8741-1A1). Pulished online t Reprints nd permissions informtion is ville online t reprintsndpermissions 1. Brgmnn, C.I. Comprtive chemosenstion from receptors to ecology. Nture 444, (26). 2. Momerts, P. Genes nd lignds for odornt, vomeronsl nd tste receptors. Nt. Rev. Neurosci. 5, (24). 3. Lissue, P.P. et l. Three-dimensionl reconstruction of the ntennl loe in Drosophil melnogster. J. Comp. Neurol. 45, (1999). 4. Hllem, E.A. & Crlson, J.R. The odor coding system of Drosophil. Trends Genet. 2, (24). 5. Couto, A., Alenius, M. & Dickson, B.J. 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Sprse coding nd decorreltion in primry visul cortex during nturl vision. Science 287, (2) VOLUME 1 [ NUMBER 11 [ NOVEMBER 27 NATURE NEUROSCIENCE