SUPPLEMENTARY INFORMATION

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1 SUPPLEMENTARY INFORMATION oi:1.138/nture NX3673 NX3675 NX285 NX # Session Pro. Lik Response NX3673 NX3675 NX285 NX12527 Touh No touh Supplementry Figure 1 Behviorl performne efore n uring the strt of imging sessions., Behviorl performne of the ttile etetion tsk mesure y (see Methos) uring trining perio. Colors inites the 4 nimls use in the urrent stuy. Imging session egn typilly fter rehe >1.5 (typilly > 75% of overll orret)., Behviorl response uring imging session mesure y perent of trils with lik response in trils with (Touh) n without (No touh) whiskerojet ontt, s etermine from high spee vieogrphy. 1

2 RESEARCH SUPPLEMENTARY INFORMATION men projetion IC #1 IC #2 IC #3 IC #4 1 µm 1.48 s 1.86 s 2.23 s 2.6 s 1% F/F 1 s 2.97 s 3.34 s 3.71 s 4.8 s 15 5% F/F.5 s Events Count Events Durtion (fwhm, se) Supplementry Figure 2 ICA se ROI seletion n enriti C 2+ signls., Exmple of inepenent omponents showing hot spots of sptilly n temporlly more inepenent pixels tht mth unerlying enriti strutures. ROIs were mnully selete se on ICA hot spots n two-photon imges. Overlpping of ROIs ws voie se on the men projetion of ll ICA imges., Motion orreltion n exmple C 2+ signl. Left, Men projetion of 1 two-photon imging frmes from single ehviorl tril efore (upper) n fter (lower) motion orretion using omintion of whole frme imge registrtion n line-y-line registrtion. The motion rtifts ue to niml movement re lrgely remove. Right, extrte fluoresene time series from ientifie ROIs (lele y re lines) n exmple two-photon iming frmes., Exmple tres of enriti C 2+ trnsients uring ehvior showing oth simple (upper row) n omplex (ottom row) kinetis., Distriution of urtion of lium events uring ehviorl sessions. 2

3 SUPPLEMENTARY INFORMATION RESEARCH Tril # % F/F Tril time (s) Supplementry Figure 3 Exmple enriti ROIs showing less relile response outsie whisker smpling epoh. Upper pnels, olor rster plot of C 2+ signl (ΔF/F) from 3 exmple enriti ROIs 3 experiments. Eh row of pixels epits tril. The group of trils ontining whisker-ojet ontt (inite y the re r on the left) re seprte from trils without etetle whisker-ojet ontt (gry r on the left). Trils within eh group re rrnge y temporl orer with erly trils t the ottom. Lower pnels, verge enriti C 2+ signl (men ± s.e.m.) trils ontining whisker ontts (re), n trils with no ontts (lk). 3

4 RESEARCH SUPPLEMENTARY INFORMATION ir puff 1st touh 2% F/F.5 s Ative Air puff Anesth. tive touh ir puff κ, 1 mm -1.1 se Men F/F, pssive stim n = 2 rnhes Men F/F, tive touh 2% F/F Ative touh Air puff wke 1 s ir puff 1st touh e C Amp Air puff C Signl Ampitue ( F/F) f 18% 71% Touh only Puff only Both Neihter C Amp Ative Touh 6% 6% n = 5 nimls, 156 ROIs Supplementry Figure 4 Pssive stimultion uner wke n nesthetize onitions., C 2+ signls (men ± s.e.m.) from n exmple enriti rnh uring ehvior with tive touh with C2 whisker (ligne to touh onset, lk rrow) n uring nesthesi with ir puff stimultion to the sme whisker (ligne to the onset of ir puff, 3 pulses, gry rrows)., Whisker urvture hnge inue y tive touh (top tre), n y ir puff (ottom tre)., Summry of popultion t of verge C 2+ signls from the sme enriti rnhes uring ehvior with tive touh n uring nesthesi with ir puff stimuli. Pssive stimultion uring 4

5 SUPPLEMENTARY INFORMATION RESEARCH nesthesi is ineffetive in evoking C 2+ signls from enrites tht were otherwise tive uring tive touh. -f, Denriti C 2+ signls evoke y ir puff to wke mie n y tive touh uring ehvior., C 2+ signls (men ± s.e.m.) from enriti rnh showing responses to tive touh ut not to ir puff (left), enriti rnh showing responses to ir puff ut not to tive touh (mile), n enriti rnh showing responses to oth ir puff n tive touh. e, Compring C 2+ signls in response to ir puff n tive touh for ll enriti ROIs (n = 156 ROIs, 5 nimls). Denrites often show istint response to ir puff n tive touh. f, The frtion of enriti ROIs responing to tive touh n ir puff uring wke stte. Note tht t were only from imging fiels ontining tive enriti ROIs responing to tive touh, n there were potentil ses where more thn one ROIs were from the sme ell, therefore the totl frtion of responsive enriti ROIs is likely n overestimtion. 5

RESEARCH SUPPLEMENTARY INFORMATION 2 5 surfe 6 1 15 15 μm 1 2 6 1 e 5 μm 2 5 1 15 2 intermeite ROI

Coef. surfe intermeite eep Supplementry Figure 5 Pir wise orreltion for enriti ROIs imge t ifferent

Left, two-photon imges with tive enriti ROIs lele with she lines.

Mgent, enriti ROIs from the sme ell. Light green enriti not trele to single pil trunk.

rnges (surfe, 15-4 μm, R =.13 ±.14, n = 1 for ense leling, R =.69 ±.

6 RESEARCH SUPPLEMENTARY INFORMATION 2 5 surfe μm e 5 μm intermeite ROI # μm f 8 μm ROI # eep 1 μm g Correltion Coeff ROI # 1 μm multi ells single ells ROI # 1 Corr. Coef. surfe intermeite eep Supplementry Figure 5 Pir wise orreltion for enriti ROIs imge t ifferent epth. -, Sprse leling onition [~2 μm in, ~5 μm in ~12 μm in ]. Left, two-photon imges with tive enriti ROIs lele with she lines. Right, Color mps of orreltion oeffiient mtries for ROIs shown on the left. Mgent, enriti ROIs from the sme ell. Light green enriti not trele to single pil trunk. -f, Dense leling onition [~25 μm in, ~46 μm in e, ~15 μm in f], similrly presente s in -. g, Summry for t shown in -f. Densely lele (multi ells) n sprsely lele (single ells) imging fiels were groupe into 3 fol epth rnges (surfe, 15-4 μm, R =.13 ±.14, n = 1 for ense leling, R =.69 ±.13, n = 15 for sprse leling; intermeite rnhing points, 45-8 μm, R =.84 ±.13, n = 1 for ense leling, R =.7 ±.13, n = 23 for sprse leling; eep primry rnhes, 12-2 μm, R =.85 ±.11, n = 1 for ense leling, R =.69 ±.11, n = 9 for sprse leling). 6

7 SUPPLEMENTARY INFORMATION RESEARCH Tle1: Pire n unpire enriti plteu properties Preprtion (onition) Amplitue (mv) Durtion (ms) Anesth. (unpire tuft) 3.6 ± ± 1.1 Anesth. (pire tuft) 54.2 ± ± 2.6 Slie (unpire tuft) 45.2 ± ±.9 Slie (pire tuft) 66.4 ± ± 5.2 Slie (unpire trunk) 69.2 ± ± 1.1 Slie (pire trunk) 71.9 ± ± 6.5 Anesthetize unpire: 38 events from 6 ells, Anesthetize pire: 69 events from 6 ells, Slie: 78 events from 13 ells. C 2+ signl mplitue (ΔF/F) pire unpire mplitue (mv) 6 C 2+ signl mplitue (ΔF/F) mplitue -urtion single ur = 7.2±4.3 ms totl plteu ur. (ms) C 2+ signl urtion (s) nesthetize wke (Fig 1) 1% ΔF/F 53% ΔF/F 5 mv 5 ms tuft Vm Supplementry Figure 6 Tuft plteu potentil properties., Tle ompring plteu potentil properties ross reoring onitions., For reorings from nesthetize mie, C2+ signl mplitue orsely vrie s funtion of lol tuft event mplitue. Pire events were lrger in mplitue thn unprie events n were ssoite with lrger C2+ signls., C2+ signl mplitue n urtion s funtion of totl urtion of plteu potentils from simultneous in vivo imging n voltge reorings., C 2+ n voltge tres from n nesthetize mouse (re tres) show tht multiple plteu potentils enhne the mplitue n urtion of the C2+ signls. These tres re superimpose y C2+ signls reore from ehving mie (lue; gry lines inite smpling perio; tres from Fig. 1). 7

RESEARCH SUPPLEMENTARY INFORMATION Tuft 3 mv Trunk 9 pa 6 ms 6 e 8 f Hlf-with (ms) Pire / unpire with 5 4 3 2 1 Tuft Trunk.2.4.6 Current injetion (na) Trunk 4. 3.5 3. 2.5 2. 1.5 Amplitue (mv) Pire / unpire with 6 4 2 Tuft Trunk.

5 1. 1. 2 4 6 8 1 2 3 Voltge (mv) Voltge (mv) g Supplementry figure 7 Simultneousul ul whole-ell voltge reorings from the istl pil trunk n tuft regions of L5 pyrmil neuron.

8 RESEARCH SUPPLEMENTARY INFORMATION Tuft 3 mv Trunk 9 pa 6 ms 6 e 8 f Hlf-with (ms) Pire / unpire with Tuft Trunk Current injetion (na) Trunk Amplitue (mv) Pire / unpire with Tuft Trunk Current injetion (na) Tuft Voltge (mv) Voltge (mv) g Supplementry figure 7 Simultneousul ul whole-ell voltge reorings from the istl pil trunk n tuft regions of L5 pyrmil neuron., Wie-fiel fluoresent imge of reorings (trunk t 7 um; tuft t 9 um)., Bk-spre of trunk spike into tuft pire with lol urrent injetions rnging from -6 pa (6 ms)., Trunk spikes evoke y 9 pa injetion (2 ms)., Trunk spike n tuft plteu potentil urtions or (e) mplitues versus lol urrent injetion mplitue. f, Inrese in trunk spike or (g) tuft plteu potentil urtions s funtion of epolriztion. 8

SUPPLEMENTARY INFORMATION RESEARCH Supplementry Figure 8 v v 8 iii 6

F/F) iv 5 μm GCMP3 1 iv 6 5 4 3 2 1 n=3 peri somti mitrunk istl

not suffiient to generte tuft C2+ trnsients.

n ute slie initing eletril n optil reoring lotions.

injetion (ottom) long pil tuft enrites (top).

Line-sn two-photon imging of CMP3 s performe t ifferent istnes long the

e, Averge two-photon fluoresene tres reore from the enriti lotions inite

9 SUPPLEMENTARY INFORMATION RESEARCH Supplementry Figure 8 v v 8 iii 6 ΔF/F (%) iv ii 4 5 μm iii 2 i 2% F/F 2 mv ii Vm Iinj i e vi vi v Brnh orer 4.1 s 1.2 na f 2 mv v 2 ms iv iii iii ii ii i 2% F/F 1 ms i g Pek C2+ signl ( F/F) iv 5 μm GCMP3 1 iv n=3 peri somti mitrunk istl Supplementry Figure 8 Plteu potentils n k-propgting tion potentils re not suffiient to generte tuft C2+ trnsients., Mximl z-projetion of n exmple GCMP3(+) rt L5 pyrmil neuron pil tuft in n ute slie initing eletril n optil reoring lotions., GCMP3 signls in response to plteu potentils evoke y nexus urrent injetion (ottom) long pil tuft enrites (top)., Summry of plteu potentil-evoke GCMP3 signls in the istl pil ror of L5 pyrmis (n = 9 ells n 42 rnhes)., Z-stk imge of GCMP3(+) positive L5 pyrmil neuron pthe t the som. Line-sn two-photon imging of CMP3 s performe t ifferent istnes long the pil enrite from the som inite y the ornge rs n numerls. e, Averge two-photon fluoresene tres reore from the enriti lotions inite in () in response to trin of high frequeny somti tion potentils t the som s shown in (f). f, Voltge reoring of trin of high frequeny tion potentils from the som of the neuron shown in -e, evoke y somti urrent injetion. g, Groupe t summrizing the ttenution of C2+ signl reore from pil enrites with inresing istne from som in response to high frequeny somti tion potentils y urrent injetion. W W W. N A T U R E. C O M / N A T U R E 9

RESEARCH SUPPLEMENTARY INFORMATION Supplementry Figure 9 S u p p l e m e n t r y F i g u r e 9 D i s t l s y n p t i i n p u t r e s u e s p l t e u p o t e n t i l k p r o p g t i o n i n L 5 t u f

10 RESEARCH SUPPLEMENTARY INFORMATION Supplementry Figure 9 S u p p l e m e n t r y F i g u r e 9 D i s t l s y n p t i i n p u t r e s u e s p l t e u p o t e n t i l k p r o p g t i o n i n L 5 t u f t e n r i t e s i n u t e r i n s l i e s., M x i m l t w o - p h o t o n z - p r o j e t i o n o f L 5 p y r m i l n e u r o n t u f t p t h e n e r t h e n e x u s i l l u s t r t i n g t w o - p h o t o n l i n e s n l o t i o n s ( n u m e r e g r e e n r s )., V o l t g e t r e s r e o r e t t h e n e x u s ( t o p l e f t ) n s s o i t e l o l O G B - 6 F s i g n l s r e o r e t t h e t h r e e r n h e s o f i n t e r e s t i n i t e i n ( ) ( t o p r i g h t n o t t o m ) i n r e s p o n s e t o n e x u s u r r e n t i n j e t i o n l o n e ( 1. n A, j u s t o v e t h r e s h o l f o r p l t e u p o t e n t i l g e n e r t i o n ; l k t r e s ), s y n p t i s t i m u l t i o n l o n e ( 1 p u l s e s o f. 2 m s t 1 H z e l i v e r e v i i p o l r s t i m u l t i n g e l e t r o e p l e ~ 5-1 µ m f r o m t h e e n r i t e s o f i n t e r e s t ; g r e y t r e s ) n p i r i n g t h e s e t w o p r i g m s ( u r r e n t i n j e t i o n e l y e y 2 5 m s r e l t i v e t o t h e s y n p t i s t i m u l t i o n ; r e t r e s )., S u m m r y o f t u f t r n h O G B - 6 F s i g n l s i n 2 3 r n h e s s s e s s e i n 9 n e u r o n s w h e r e i s t l s y n p t i s t i m u l t i o n w s p i r e w i t h n e x u s u r r e n t i n j e t i o n ( p i r i n g s i g n i f i n t l y g r e t e r t h n e i t h e r p l t e u o r s y n p t i s t i m u l t i o n l o n e : p <. 1, o n e - w y r e p e t e m e s u r e s A N O V A )., Z - s t k o f L 5 n e u r o n t u f t p t h e t t h e n e x u s w i t h t w o - p h o t o n g l u t m t e u n g i n g n i m g i n g l o t i o n h i g h l i g h t e y t h e y e l l o w o x. e, V o l t g e r e o r e t t h e n e x u s ( l e f t ) n l o l r n h O G B - 6 F s i g n l s ( r i g h t ) i n r e s p o n s e t o u r r e n t i n j e t i o n t t h e n e x u s ( 1. 4 n A, j u s t o v e t h r e s h o l ; l k t r e s ), p r o l o n g e s u t h r e s h o l u n g i n g t t h e r n h o f i n t e r e s t ( 3 p o i n t s, y l e 3 t i m e s, 1. m s i s i ; g r e y t r e s ), n p i r i n g ( u n g i n g l e i n g u r r e n t i n j e t i o n y 2 m s ; r e t r e s ). f, S u m m r y o f t u f t r n h O G B - 6 F s i g n l s f r o m 1 r n h e s i n 6 n e u r o n s w h e r e n e x u s u r r e n t i n j e t i o n w s p i r e w i t h p r o l o n g e, s u t h r e s h o l u n g i n g ( p i r i n g s i g n i f i n t l y g r e t e r t h n e i t h e r p l t e u o r u n g i n g l o n e : p <. 1, o n e - w y r e p e t e m e s u r e s A N O V A ). g, P i r i n g s i g n i f i n t l y i n r e s e p l t e u p o t e n t i l r e ( p <. 1, p i r e t - t e s t ) n h, f u l l w i t h t h l f m x i m l m p l i t u e ( p =. 6, p i r e t - t e s t ). 1 W W W. N A T U R E. C O M / N A T U R E

11 SUPPLEMENTARY INFORMATION RESEARCH Control Musimol M1 Musimol ontrol 15 F/F Tril time (s) 5 n=16 rnhes *** 2 n = 5 rnhes * 2 n = 4 nimls ΔF/F mplitue Control Mus M1 ΔF/F mplitue Mus Con. Mus M1 Performne (-prime) Control Mus M1 Reov Mus Cont. Supplementry Figure 1 Effets of M1 silening on ir puff evoke responses n ehviorl performne., Color rster plot of C 2+ signls from ir puff trils uner wke stte uring ontrol session (left), uring session with musimol injete to M1 (mile), n with musimol injete to the ontrol site (right)., Summrize t ompring C 2+ signls (verge from ll trils for eh enriti ROI) from enriti rnhes imge uring ontrol sessions n sessions with musimol injete to M1., Summrize t ompring C 2+ signls from enriti rnhes imge uring sessions with musimol injete to M1, n to the ontrol site., Behviorl performne (-prime) uner ifferent onitions in onseutive sessions. Note tht tsk performne ws strongly suppresse y M1 intivtion, n i not reover to ontrol level in the following sessions, suggesting n unlerning proess ourre uring M1 intivtion. 11

RESEARCH SUPPLEMENTARY INFORMATION 3 Numer of liks 25 2 15 1 R =.26, P >.5 5 P(lik C) =.93 P(lik) =.76 5 5 1 15 2 25 1 C 2+ signl (men F/F) 1 se 3 % F/F P (lik C).9.8.7.6.5.5.6.7.8.9 1 P (lik) P (lik C) =.

12 RESEARCH SUPPLEMENTARY INFORMATION 3 Numer of liks R =.26, P >.5 5 P(lik C) =.93 P(lik) = C 2+ signl (men F/F) 1 se 3 % F/F P (lik C) P (lik) P (lik C) =.95 P(lik) =.74 Supplementry Figure 11 Denriti C 2+ signls n pereptul etetion reporte y liking., Color rster plot showing C 2+ signls from one exmple enriti rnhe. Lik times were mrke y mgent tiks., Numer of liks for eh tril were plotte ginst men C 2+ signls. In trils showing lrge C 2+ signls, no signifint tril y tril orreltion etween C 2+ signls n liking ehvior (R =.26, P>.5, for the urrent ROI; R =.11 ±.163 for 57 ROIs). Note tht trils with strong C 2+ signls (> 1 stnr evition, re symols), show high proility of lik response [P(lik C) =.93]., Popultion t of proility of lik response in trils with lrge C 2+ signls (> 1 stnr evition; P(lik C)) is signifintly higher thn proility of lik response over ll trils [P(lik)]. 12

13 SUPPLEMENTARY INFORMATION RESEARCH Denriti nonliner proessing in ojet loliztion INPUT: ontt strength n whisker lotion OUTPUT: orreltion epenent, plteu riven ursting i) 1 mm ii) iii) iv) v) ~2 2 Fe whisker lotion (lyer 1, vm1) whisker ontt (thlmi, lol ortil) firing rte firing rte whisker ngle ontt fore lotion oinient ontt wiespre C 2+ signls plteu potentils urst firing output whisker position ~ ontt strength (Δ urvture) i) 2 mm ii) iii) iv) v) 2 ~-5 Fe whisker lotion (lyer 1, vm1) whisker ontt (thlmi, lol ortil) firing rte firing rte whisker ngle ontt fore lotion nonoinient ontt No C2+ signls APs only single spiking output whisker position ~ ontt strength (Δ urvture) Supplementry Figure 12 Propose role of enriti nonlinerity in ehviorl relevnt omputtion., Conition of oinient input (lue). i) shemti of whisking profile for ojet ontt t 1 mm n whisker ngles roun 2. ii) informtion representing whisker lotion oul rrive through long-rnge ortio-ortil lyer 1 inputs, mong others, with virssl motor ortex s one potentil soure 3,23. These inputs re shown here to hve preferre whisking feture tht proues mximum firing rte roun 2. Whisker ontt sensory input is elivere vi thlmoortil n lol rrel ortil inputs from lyers 2/3 n 4 3 n is shown here to inrese in mplitue with inrese ontt fores. Ativity in these inputs oul lso posses some whisking relte moultion. iii) whisker lotion epenent input is elivere to the enriti tuft regions of lyer 5 pyrmil neurons 3,23 while whisker ontt sensory input is elivere to the perisomti enriti regions 3. iv) oinient perisomti n lyer 1 inputs onvert APs into multiple lrge mplitue, long urtion regenertive plteu potentils in the istl enriti region 4-7. This nonliner form of enriti integrtion uses lrge wiespre C 2+ influx throughout the pil tuft region n urst of high frequeny tion potentil output from the involve lyer 5 neurons 21. v) the plteu potentil inue urst firing will enhne the slope of the stimulus-response reltionship t the preferre whisking feture (2 ) of the ominnt lyer 1 inputs., Nonoinient input (re). The pnel lyout is the sme s. Here whisker ontt is in ifferent position (2 mm or ~-5 ) n there is oringly less lyer 1 input rriving uring the ontt. With reue mounts of lyer 1 input, plteu potentils re not generte n the neuron remins in low frequeny single spiking moe. Without the lrge enriti nonlinerity the slope of the stimulus-response urve is reue. 13

Supplementary Figure 1

Supplementary Figure 1 Supplementry Figure (nesthetized) (wke) Normlized mplitude.5 Pek width (ms).6.4.2 4 2 2 x 3 Wveform slope Normlized mplitude.5 Pek width (ms).6.4.2 x 3 3 2 Wveform slope c (nesthetized) d (wke) Normlized