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1 doi:.38/nture896 c Resting stte Resting stte Resting stte Supplementry Figure Illustrtion of pttern clssifiction nd its effects on neuronl representtions. Dynmic ctivity ptterns evoked y different stimuli re represented y trjectories in coding spce tht strt from common resting stte into different directions. Stle output ptterns re represented y locl minim in the lndscpe. The deflection of trjectories towrds minim illustrtes the clssifiction of inputs into discrete nd defined output ctivity ptterns., Trjectories representing similr stimuli (smll ngulr seprtion) re deflected into the sme locl minimum if their vrince occurs long dimension where minim re rod. Ptterns re therefore not seprted, s oserved for mitrl cell ctivity ptterns evoked y rnge of different odor concentrtions., Trjectories representing other similr stimuli ecome seprted into two locl minim, consistent with the rupt trnsition etween mitrl cell ctivity ptterns evoked y morphing series of similr odors. c, Trjectories re seprted into more thn two minim if stimuli re morphed over lrger distnce in stimulus spce (lrger ngulr seprtion), consistent with multiple pttern trnsitions during morphing of dissimilr odors.

2 doi:.38/nture896 MC mrker 3 M 4 M 5 M 6 M M nk 5 M 55 F/F (%) MCs F/F (%) c f MC MC MC 3 MC 4 MC 5 3 Popultion verge Min TDC signlmx d Men response (normlized). Lys Concentrtion (x M) e Reference pttern nk 5 56 ms 5 ms 768 ms,4 ms,8 ms,536 ms,79 ms.. g h PC.. PC.8.3 PC PC Supplementry Figure Concentrtion-dependence of mitrl cell odor responses: second exmple., Mitrl cell (MC) mrker expression (left) nd rw, time-verged clcium signls evoked y different concentrtions of, lnk, nd the similr control odor. Bottom: Rw clcium signls of mitrl cells mrked y rrows s function of time (gry r indictes stimultion period). Scle r: 5 µm., Temporlly deconvolved clcium signls (TDC signls) s function of time, evoked y the sme stimuli in five individul mitrl cells (different mitrl cells thn in ). Gry r indictes odor ppliction. Color scle normlized for ech mitrl cell. c, TDC signls verged over the popultion of ll recorded

3 doi:.38/nture896 mitrl cells (n = 8 mitrl cells from 7 OBs). d, Time-verged popultion TDC signl (,9 ms) s function of concentrtion of (lck) nd Lys (gry; see Fig. ), normlized to the mximum of the popultion response verged over ll stimuli. Note tht the TDC signl reflects the chnge in firing rte, rther thn the solute firing rte. e, etween ctivity ptterns evoked y different stimuli (different concentrtions of, lnk, nd -5 M; gry r indictes stimultion period) t different times (n = 8 mitrl cells from 7 OBs). Ech pnel depicts the Person correltion etween reference pttern (indicted y cross) nd ctivity ptterns evoked y ll stimuli in ll time ins. Pnels in top row show correltions to reference ptterns evoked y different stimuli t n erly time point (56 ms fter response onset). Pnels in ottom row show correltions to reference ptterns t lte time point (48 ms). f, Time series of correltion mtrices depicting the pirwise similrity etween ctivity ptterns evoked y the sme stimuli in successive time ins. The men correltion etween ctivity ptterns evoked y ( -6 M to -3 M) nd decresed significntly during the odor response (P <., regression nlysis). The men correltions (± s.d.) during n erly (56 5 ms) nd lte (,48,34 ms) time window were.77 ±.7 nd.4 ±.4, respectively. s etween ctivity ptterns evoked y different concentrtions of ( -6 M to -3 M), in contrst, did not significntly decrese (P >.5, regression nlysis). Men correltions (± s.d.) during the erly nd lte time windows were.77 ±.9 nd.65 ±., respectively. g, Representtions of odor-evoked ctivity ptterns s function of time in principl component spce. Trjectories show the evolution of ctivity ptterns etween,48 ms efore response onset nd 4,96 ms fter response onset. Time is indicted y incresing size of plot symols (intervl: 56 ms). Arrowheds mrk onset of odor response nd point in direction of time. h, Mitrl cell ctivity ptterns in principl component spce, time-verged during the stedystte (,536,34 ms). 3

4 doi:.38/nture896.8 Liner Concentrtion series Liner Qudrtic c Morphing series Liner Qudrtic TDC signl (normlized) Qudrtic Sigmoid Lys Sigmoid -,4 ms,4 -,48 sec % 8% 35% 37% 35% 44% 56% 45% 3% 7% 3% 38% 49% 5% 54 % 4% / / -,4 ms,4 -,48 sec 5 % 3 % 8 % 5 % 6 % 59 % 76 % 88 % 9 % % 7 % 6 % 7 % 67 % Sigmoid 63 % 63 % Supplementry Figure 3 Clssifiction of mitrl cell responses to concentrtion nd morphing series into ctegories of liner, qudrtic nd sigmoid fits. In order to chrcterize responses of individul mitrl cells to series of different concentrtions or mixture rtios we used n pproch similr to the one descried y Leutge et l. nd Khn et l.. Responses of ech mitrl cell were fit y liner, qudrtic nd sigmoid functions. If the response of mitrl cell to the stimulus series shows sudden trnsition, it is expected tht responses would e est fit y sigmoid function. Best fits y liner function would suggest grdul trnsitions; est fits y qudrtic function would suggest other trnsitions or mximum responses to intermedite stimuli. Responses for which none of the fits ws significnt (P >.5) were excluded from the nlysis. See Methods for detils., Exmples of responses to morphing series (/) tht were est fit y liner, qudrtic nd sigmoid functions. TDC signls were normlized to the mximum of the popultion verge., Percentge of mitrl cell responses to different concentrtions of Lys (n = 4 mitrl cells from 4 OBs; top) nd (n = 8 mitrl cells from 7 OBs; ottom) tht were est fit y liner (green), qudrtic (red) nd sigmoid (lue) functions. The percentge of mitrl cells for which t lest one fit ws significnt (P <.5) is given in the lower right of ech plot. Anlyses were performed seprtely for responses verged etween,4 ms (left) nd etween,4,48 ms (right) fter response onset. c, Percentge of mitrl cell responses to morphing series of / (n = 56 mitrl cells in 9 OBs; top) nd / (n = 4 mitrl cells in 7 OBs; ottom) tht were est fit y liner (green), qudrtic (red) nd sigmoid (lue) functions. 4

5 doi:.38/nture896 Lys Lys Reference pttern nk 5. Supplementry Figure 4 etween ctivity ptterns evoked y concentrtion series of Lys t different time points. etween ctivity ptterns evoked y different concentrtions of Lys, lnk, nd ( -5 M) t different times (n = 4 mitrl cells from 4 OBs). Ech pnel depicts the Person correltion etween reference pttern (indicted y cross) nd ctivity ptterns evoked y ll stimuli in ll time ins. Pnels in top row show correltions to reference ptterns evoked y different stimuli t n erly time point (56 ms fter response onset, gry r indictes stimultion period). Pnels in ottom row show correltions to reference ptterns t lte time point (,48 ms). 5

6 doi:.38/nture896 : Glomeruli 8 99: 9: 7:3 5:5 3:7 :9 :99 : F/F (normlized) F/F (normlized) 4.5 Men s.d. Pre-odor ms 56 ms 5 ms 768 ms,4 ms,8 ms,536 ms,79 ms. c Pre-odor. Supplementry Figure 5 morphing did not cuse rupt trnsitions etween glomerulr ctivtion ptterns. Ptterns of glomerulr ctivtion were mesured y imging of clcium signls from fferent xon terminls in glomeruli of the lterl OB 3 (Methods)., Responses of ll glomeruli to ll stimuli in the morphing series (/) s function of time (n = 8 glomeruli from 5 fish). Clcium signls were normlized to the mximum of the popultion verge. Responses follow stereotyped time course nd do not chnge ruptly within the morphing series. Right: men clcium signl (lck) nd s.d. (gry; cross glomeruli) s function of time., Person correltion etween glomerulr ctivtion ptterns evoked y the morphing series (/) s function of time. The first correltion mtrix (left) shows correltions efore stimulus onset. s re high nd constnt throughout the odor response. No rupt trnsitions etween glomerulr ctivtion ptterns re oserved. c, s etween glomerulr ctivtion ptterns evoked y morphing one odor () into dissimilr odor (; n = 59 glomeruli in 5 fish). Pttern trnsitions re grdul nd do not chnge much during the odor response. 6

7 doi:.38/nture896 MC mrker : 99: 9: 7:3 5:5 3:7 :9 :99 : 5 F/F (%).5 MCs F/F (%) 8 Responding MCs (%) 6 4 c Responding MCs (%) d MC mrker : 99: 9: 7:3 5:5 3:7 :9 :99 : 5 MCs F/F (%) F/F (%).5 75 e MC MC MC 3 MC 4 TDC signl Mx Min f Popultion verge TDC signl Mx Min Supplementry Figure 6 Mitrl cell responses to morphed odors: dditionl dt., Mitrl cell (MC) mrker expression (left) nd rw, time-verged clcium signls evoked y mixtures of nd. Bottom: Rw clcium signls of mitrl cells mrked y rrows s function of time. Scle r: 5 µm., Frction of mitrl cells responding to different odors s function of time (TDC signl > s.d. of spontneous fluctutions) for morphing series /. c, Sme for morphing series /. d, Rw, time-verged clcium signls evoked y the morphing series /. Bottom: Rw clcium signls of mitrl cells mrked y rrows s function of time. Scle r: 5 µm. e, Temporlly deconvolved clcium signls (TDC signls) s function of time, evoked y the sme stimuli in four individul mitrl cells (different mitrl cells thn in d). Gry r indictes odor ppliction. f, TDC signls evoked y morphing series / verged over the popultion of ll recorded mitrl cells (n = 4 mitrl cells from 7 fish). 7

doi:.38/nture896 coefficient.8.6.4.. / coefficient.8.6.4.. /.4,4,4,48 Time (ms).

(cluster centers) : :99 :9 3:7 5:5 7:3 9: 99: : d Mx = 4.

(lue) nd etween ptterns from seprte clusters (red) s function of time.

Insets outline clusters of correltion coefficients on correltion mtrices t,8 ms (Figs.

, Eucliden distnces etween mitrl cell ctivity ptterns evoked y the morphing series from

8 doi:.38/nture896 coefficient / coefficient /.4,4,4,48 Time (ms).4,4,4,48 Time (ms) 56 ms 5 ms 768 ms,4 ms,8 ms Eucliden distnce (.u.) Mx Mx = 8.6 Mx =. Mx =.3 Mx =. Mx = 9.3 Mx = 8. c Proximity score (pure components) : :99 :9 3:7 5:5 7:3 9: 99: : Proximity score (cluster centers) : :99 :9 3:7 5:5 7:3 9: 99: : d Mx = ms 5 ms 768 ms,4 ms,8 ms Mx =.6 Mx = 5. Mx =.9 Mx = 9. Mx = 6.3 Eucliden distnce (.u.) Mx Supplementry Figure 7 Additionl nlyses of trnsitions etween mitrl cell ctivity ptterns evoked y odor morphing., Person correltion coefficients etween ptterns within the sme high-correltion cluster (lue) nd etween ptterns from seprte clusters (red) s function of time. Thin lines represent correltions etween individul pirs of ptterns; thick lines re verges. Insets outline clusters of correltion coefficients on correltion mtrices t,8 ms (Figs. c nd 3). Left: morphing of into ; right: morphing of into., Eucliden distnces etween mitrl cell ctivity ptterns evoked y the morphing series from to s function of time (n = 56 mitrl cells in 9 fish). Eucliden distnce ws mesured in the 56-dimensionl spce in which ech dimension represents the TDC signl of one mitrl cell. Color is scled individully for ech mtrix; mxim re indicted elow. c, Evolution of ctivity ptterns evoked y the morphing series from to towrds two discrete sttes, visulized in metric tht represents the sttes y 8

9 doi:.38/nture896 vlues of + nd -. Two sttes were defined either s the ptterns evoked y the two pure components (left) or s the cluster centers, i.e. the centroids (verges) of the groups tht ecme seprted (: 9: versus 7:3 : /). Ptterns were reduced to one dimension y extrcting the first principl component. At ech time point, principl component coefficients were scled y sutrcting the men coefficient of the two sttes nd normlizing y hlf their difference, thus ssigning + nd - to the two sttes. The resulting proximity score cn vry etween positive nd negtive infinity. Vlues ner + nd - indicte tht ptterns re similr to one or the other stte, respectively. When sttes re defined s ptterns evoked y pure odors (left), proximity scores of the pure components re + nd - y definition. Both proximity scores show tht, fter response onset, ctivity ptterns converge onto discrete sttes tht re tightly ssocited with one of the pure components. d, Eucliden distnces etween mitrl cell ctivity ptterns evoked y the morphing series from to (n = 4 mitrl cells in 7 fish). 9

10 doi:.38/nture896 r r r 3 r : correltion within cluster r : correltion within cluster r 3 : correltion cross clusters. r r r : men correltion within centrl cluster r : correltion etween ptterns in centrl cluster nd ptterns in mrginl clusters / 56 ms 5 768,4,8 Fish ( n = 8 MCs) Fish ( n = MCs) Fish 3 ( n = 5 MCs) Fish 4 ( n = MCs) Fish 5 ( n = 7 MCs) Fish 6 ( n = MCs) Fish 7 ( n = 4 MCs) Fish 8 ( n = MCs) Fish 9 ( n = 4 MCs) r =.56 ±.3 r =.58 ±.5 r 3 =.3 ±.5 P < -4 r =.7 ±.4 r =.7 ±.7 r 3 =.66 ±. P =.3 r =.7 ±. r =.78 ±. r 3 =.3 ±.9 P < -6 r =.39 ±.7 r =.38 ±. r 3 =.4 ±.3 P =.67 r =.84 ±.7 r =.7 ±.9 r 3 =.79 ±.7 P =.7 r =.63 ±. r =.76 ±.8 r 3 =.73 ±.4 P =.8 r =.7 ±.9 r =.68 ±.8 r 3 =.43 ±.4 P <.5 r =.84 ±.6 r =.76 ±.9 r 3 =.79 ±.8 P =.46 r =.6 ±. r =.86 ±.4 r 3 =.49 ±.3 P < -5 / 56 ms 5 768,4,8 Fish ( n = 3 MCs) Fish ( n = 6 MCs) Fish 3 ( n = 7 MCs) Fish 4 ( n = 8 MCs) Fish 5 ( n = 5 MCs) Fish 6 ( n = MCs) Fish 7 ( n = 3 MCs) r =.57 ±. r =.36 ±. P <.5 r =.65 ±. r =.3 ±. P < -4 r =.65 ±. r =.37 ±.4 P =.3 r =.66 ±.3 r =.5 ±.7 P <.5 r =.46 ±. r =.3 ±.8 P =.6 r =.63 ±. r =.4 ±.7 P < -3 r =.75 ±. r =.8 ±.76 P <.5 Supplementry Figure 8 Anlysis of response ptterns in individul fish., s etween ctivity ptterns evoked y morphing series / in ech individul fish (n = 9). Numer of mitrl cells (MCs) recorded in ech fish is indicted. Inset t top defines two clusters of high correltion coefficients (lue) nd correltion coefficients etween clusters (red) on the correltion mtrix of the full dt set (Fig. c,,8 ms). r, r nd r 3 show men correltions (± s.d.) within nd cross these clusters in ech fish. P-vlues quntify the sttisticl significnce of comprisons etween the men correltion within high-correltion clusters (clusters nd ; lue res) nd the men correltion cross clusters (red re). P-vlues in old indicte tht the difference etween men correltions is sttisticlly significnt (P <.5) in given individul (Mnn-Whitney U-test). Underlined vlues indicte tht sttisticl significnce (P <.5) is mintined fter Bonferroni correction for multiple comprisons cross ll fish., s etween ctivity ptterns evoked y morphing series / in ech individul fish (n = 7). r gives the men correltion (± s.d.) within the centrl cluster (lue), r gives the men correltion etween the centrl (9/ /9) nd mrginl (:, 99:, :99 nd :) stimuli in the series (red). Corresponding regions in the correltion mtrix (see Fig. 3,,8 ms) re outlined. P-vlues give sttisticl significnce for comprisons etween r nd r ; conventions s in. In nd, note tht sttisticlly significnt differences re oserved in susets of fish despite the reltively low numer of neurons recorded in ech individul. This oservtion is consistent with the finding tht trnsitions etween ptterns re medited y smll ensemles of neurons. Moreover, note tht rupt pttern trnsitions, if pprent, occur t similr positions within the morphing series in different fish.

11 doi:.38/nture896 Mitrl cells (rnked y covrince) 56 TDC signl (centered) ms 56 ms 5 ms 768 ms,4 ms,8 ms Mitrl cells (rnked) 8 5 Pre-odor TDC Signl (centered) 5 Supplementry Figure 9 Trnsitions etween ctivity ptterns medited y mitrl cell susets., Response mtrices of mitrl cells stimulted y the morphing series from to (n = 56 from 9 fish) t fixed time point (768 ms), sorted y covrince with different templtes. Systemticlly chnging the trnsition point in the templte did not result in equivlent shifts in the ligned trnsition pprent in the sorted mitrl cell responses (top nd ottom of mtrix). The trnsition etween the 9: nd 7:3 / mixtures is therefore slient feture in the dt nd not chnce result. Similr oservtions were mde for the morphing series from to., Top: centered responses of eight mitrl cells, selected for highest covrince with the templte t 768 ms, s function of time. Below, response profiles of individul mitrl cells (gry) nd their verge (red) shown s line plots. Left: efore response onset. Steep trnsitions emerge from response profiles tht initilly showed grdul trnsitions.

12 doi:.38/nture896 Lys Lys 56 ms 5 ms 768 ms,4 ms,8 ms,536 ms,79 ms Suset. Suset Suset Suset PC.. PC... PC... PC 56 ms 5 ms 768 ms,4 ms,8 ms Suset Shuffled Suset.. Suset. Suset.... PC..8.4 PC...4 PC.. PC c 56 ms 5 ms 768 ms,4 ms,8 ms Suset Suset. Shuffled Suset Suset PC.. PC... PC.. PC Supplementry Figure Anlysis of pttern trnsitions in rndomly reduced dt sets., Top: correltion mtrices of ctivity ptterns evoked y different concentrtions of Lys nd the control odor cross two rndom susets of mitrl cells, ech contining

13 doi:.38/nture896 n = 7 out of the 4 mitrl cells (4 fish). Bottom: ctivity ptterns cross the two rndom susets of mitrl cells in principl component spce s function of time. Results from ech suset re similr to ech other nd to the full dt set (Fig. d, e)., Sme nlysis for ctivity ptterns evoked y the morphing series from to cross two rndom susets of mitrl cells (n = 78 mitrl cells ech out of 56 mitrl cells in totl). Line plots on the right (lck lines) show sections through correltion mtrices t,8 ms for ll rndomly selected mitrl cell susets. The position of the section is indicted y the lck line; utocorreltions were replced y interpolted vlues. Note consistent trnsitions etween 9: nd 7:3 / mixtures. Gry lines show sections through correltion mtrices fter shuffling of cell identity (see Supplementry Fig. ). c, Sme nlysis for ctivity ptterns cross rndom susets of mitrl cells evoked y the morphing series from to (n = 7 mitrl cells in ech suset out of 4 mitrl cells in totl). Sections through correltion mtrices show two pttern trnsitions etween the 99: nd 9: nd etween the :9 nd :99 mixtures of / in ll cses. 3

14 doi:.38/nture896 Pre-odor correltion Mixture rtio -,79 ms -,536 ms -,8 ms -,4 ms -768 ms -. Shuffled odor responses 56 ms 5 ms 768 ms,4 ms,8 ms s.d Supplementry Figure etween spontneous nd shuffled ctivity ptterns., s etween ctivity ptterns efore stimulus onset (morphing series /; n = 56 mitrl cells in 9 OBs)., s etween ctivity ptterns evoked y morphing series / (n = 56 mitrl cells in 9 OBs) fter shuffling of cell identity. Numer elow ech mtrix shows the s.d. of correltion coefficients (utocorreltions excluded). 4

15 doi:.38/nture896. Leutge, J.K., et l. Progressive trnsformtion of hippocmpl neuronl representtions in "morphed" environments. Neuron 48, (5).. Khn, A.G., Thtti, M. & Bhll, U.S. representtions in the rt olfctory ul chnge smoothly with morphing stimuli. Neuron 57, (8). 3. Friedrich, R.W. & Korsching, S.I. Comintoril nd chemotopic odornt coding in the zerfish olfctory ul visulized y opticl imging. Neuron 8, (997). 4. Murphy, G.J., Glickfeld, L.L., sen, Z. & Iscson, J.S. Sensory neuron signling to the rin: properties of trnsmitter relese from olfctory nerve terminls. J. Neurosci. 4, (4). 5. Bozz, T., McGnn, J.P., Momerts, P. & Wchowik, M. In vivo imging of neuronl ctivity y trgeted expression of geneticlly encoded proe in the mouse. Neuron 4, 9- (4). 6. Higshijim, S., Msino, M.A., Mndel, G. & Fetcho, J.R. Imging neuronl ctivity during zerfish ehvior with geneticlly encoded clcium indictor. J. Neurophysiol. 9, (3). 7. Miywki, A., et l. Fluorescent indictors for C + sed on green fluorescent proteins nd clmodulin. Nture 388, (997). 8. Li, J., et l. Erly development of functionl sptil mps in the zerfish olfctory ul. J. Neurosci. 5, (5). 9. Mthieson, W.B. & Mler, L. Morphologicl nd electrophysiologicl properties of novel in vitro preprtion: the electrosenspry lterl line loe rin slice. J. Comp. Physiol. A 63, (988).. Yksi, E. & Friedrich, R.W. Reconstruction of firing rte chnges cross neuronl popultions y temporlly deconvolved C + imging. Nt. Methods 3, (6).. Yksi, E., Judkewitz, B. & Friedrich, R.W. Topologicl Reorgniztion of Representtions in the Olfctory Bul. PLoS Biol. 5, e78 (7).. Crr, W.E.S. The moleculr nture of chemicl stimuli in the qutic environment. in Sensory iology of qutic nimls (ed. J. Atem, R.R. Fy, A.N. Popper & W.N. Tvolg) 3-7 (Springer, New York, 988). 3. Michel, W.C. & Luomudrov, L.M. Specificity nd sensitivity of the olfctory orgn of the zerfish, Dnio rerio. J. Comp. Physiol. A 77, 9-99 (995). 4. Denk, W., Strickler, J.H. & We, W.W. Two-photon lser scnning fluorescence microscopy. Science 48, (99). 5. Yksi, E., von Sint Pul, F., Niessing, J., Bundschuh, S.T. & Friedrich, R.W. Trnsformtion of odor representtions in trget res of the olfctory ul. Nt. Neurosci., (9). 6. Pologruto, T.A., Stini, B.L. & Svood, K. ScnImge: flexile softwre for operting lser scnning microscopes. BioMed. Eng. OnLine, 3 (3). 5

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION doi:.38/nture8499 doi:.38/nture8499 5 6 5 4.5 Firing rte (Hz) -67-65 -66-6 -58 V m (mv) -7-67 -68-66 -64 c Thet power (mv ) -73-69 -7-7 -7.5.8 3....9.9.4.6.6. 9 8 9 8 9 8 9 8 9 8 Supplementry Figure Firing