Supplmntary Information NMDA spiks nhan ation potntial gnration uring snsory input Luy M. Palmr, Aam S. Shai, Jams E. Rv, Harry L. Anrson, Ol Paulsn, Matthw E. Larkum Natur Nurosin: oi:1.138/nn.3646
1 2 3 4 5 μm 2 3 4 APs 1 25 % ΔG/R 25 % ΔG/R Norm. Amplitu AP Frquny (Hz) 1..5-3 -2-1 1 2 3 Dnriti Spra (μm) 15 1 5 2 4 6 Dnriti Spra (μm) f g I h.5 ΔF/F I I 5 μm f g II h II II 5 μm.5 ΔF/F Nois Nois Numr of vnts Numr of vnts 1 25 3xSD (ΔF/F) 1.5 3xSD 1.5 (ΔF/F) Supplmntary Figur 1. Tuft transints ar spatially rstrit, hav a istint imoal istriution, an ar not gra in amplitu. In prinipl, NMDA spiks in multipl ranhs oul an iniation of many NMDA spiks or on vry spatially istriut NMDA vnt. W thrfor masur th spatial xtnt of tuft transints. (a) Twophoton imag of a tuft nrit with spatially rstrit rgions of intrst (ROIs; 5 μm lngth; olour oxs). () Spontanous transints ror in th nriti ROIs shown in (a) an somati APs iniat y ashs. To ompar iffrnt nriti rgions, transints ar rport as ΔG/R. () Ovrlay of th transints in () illustrating th spra of a vnt along a tuft nrit. () Gaussian istriution of th normaliz transint amplitus at iffrnt loations along tuft nrits. () Th spatial spra of th transints within a tuft nrit was not rlat to th somati AP firing frquny. (f-h) Sin NMDA spiks hav a lar thrshol, plotting fluorsn amplitus woul rsult in a imoal istriution whras oost potntials woul hav a gra istriution. W thrfor plott th fluorsn amplitu istriutions from two xampl tuft nrits (I an II). (f) Two-photon imag of two xampl tuft nrits. (g) Exampl fluorsn tras with two transints aov th nois from tuft nrit shown in (f). (h) Histogram of th pak amplitu of fluorsn from th tuft nrit shown in (f) illustrating imoal istriutions. transints wr inlu in th analysis if thy ha an amplitu largr than 3x th stanar viation of th nois (mil, gry ar; right, ash lin). This ritria sparat th rport transints (r) from th fluorsn akgroun nois (lak). Natur Nurosin: oi:1.138/nn.3646
.5 ΔF/F 1 mv Prntag of APs 1 5 no MK81 Count (#) 5 25 Avrag CF 5 1 15 2 Firing Frq uring tuft (Hz) Ca2 + APs.5 ΔF/F 5 ms 5 Hz Ca2 +.5 ΔF/F 125 ms 1 mv Normaliz ount 1. Ca2+ for aftr (5-1 Hz).5-4 -2 2 Timing of tranisnt (T - T ) (ms) Ca2+ AP f.2 ΔF/F 5 ms Supplmntary Figur 2. Tuft transints ar not from APs. Bakpropagating ation potntials (APs) aus influx into th nrits of layr 2/3 pyramial nurons in a istan-pnnt mannr (Svooa t al, 1999; Watrs t al, 23). W tst th possiility that APs aus transints in tuft nrits. (a) Tuft tra (top) an simultanous somati voltag (ottom). Bars highlight somati APs with (grn) an without (gry) assoiat tuft influx. APs trunat. () Only 26 ± 4 % of all somati APs wr assoiat with a transint in ontrol tuft nrits (n = 28). Although thr wr still spontanous APs whn all NMDA hannls wr lok y intrnal MK81, thr wr no tuft transints illustrating th rquirmnt of ativ NMDA hannls (n = 6 nrits). () Histogram of firing frquny ror uring tuft transints. Th avrag firing frquny (6 ± 3 Hz) uring tuft transints is onsiraly lowr than th rport ritial frquny (CF; r arrow) for voking spiks in layr 2/3 pyramial nurons (Larkum t al, 27). Thrfor APs alon oul not vok spiks whih woul aus influx into th tuft nrits. () To invstigat th timing of APs with nriti ativity, imaging frquny was inras to a maximum of 1 Hz. Typial tuft transints ror at 5 Hz an somati APs (lak ashs). Gry ars, timing of transints ompar to APs. Box, magnifiation of transint (top) an simultanous somati voltag (ottom) from th rgion mark with a r ar (lft). R markrs on transint, ata points (3 ata points on th rising phas of th transint). () Histogram of th timing of transints ror at 5-1 Hz ompar to th timing of somati APs. Timing was trmin as th iffrn in tim (ms) twn th first ata point on th rising phas of th tuft transint an th tim at 1 % of th AP thrshol for th first AP. On avrag, th onst of th tuft transint was 16 ± 6 ms for th somati AP (n=17). (f) Ovrlay of spontanous transints from a singl tuft nrit illustrating thir strotypi wavform (n=9). Th amplitu of transints ror in all nrits wr within on stanar viation of th man. Natur Nurosin: oi:1.138/nn.3646
Extrallular Stimulation 5 mv 5 ms soma 2n Puls Intgral (ms mv) 12 6 4 6 8 1 Stimulus Intnsity (ma) 1 μm 5 mv 5 ms soma 2n Puls Intgral (ms mv) 12 6 4 6 8 1 Stimulus Intnsity (ma) Supplmntary Figur 3. NMDA spiks our in tuft nrits of layr 2/3 pyramial nurons in vitro. NMDA spiks hav n shown in th asal nrits of layr 2/3 pyramial nurons ut not in th tuft nrit. (a) Exprimntal paraigm. Somati rorings wr ma from layr 2/3 pyramial nurons fill with Alxa Fluor 594 (5 μm) to ai th plamnt of an xtrallular stimulation piptt in los proximity to a tuft nrit. () Squntially inrasing th intnsity of pair pulss (2x 1 ms pulss at 5 Hz) appli to th tuft nrit shown in (a) rsult in a supralinar voltag rspons (top; lak) whih was lok y ath appliation of (1 μm; ottom; grn). Inst, ovrlay of somati voltag uring supralinar stimulation uring ontrol (lak) an (grn). () Intgral of th somati voltag uring squntial inras in stimulus intnsity for th nuron shown in (a) an (). Blok of NMDA hannls y signifiantly ras th intgral of th somati voltag uring a NMDA spik y on avrag 69 ± 1 % (2n puls; n = 3; p <.5; Data not shown). Data fitt with linar rgrssion. R arrows iniat suprathrshol rspons. Natur Nurosin: oi:1.138/nn.3646
In vitro a Unag 2 mv 5 ms soma soma Normaliz Intgral (post-unag /pr-unag) 1..5 In vivo g Amplitu (proportion of max) 1..8.6.4 Unag Spik EPSP Prntag of maximum stimulus strngth (%) 2 mv 5 ms Unag 1 mv Pr-2P Post 2P 5 ms Loal NMDA lok pr-2p.2 Max 6 8 1 1..8.6.4.2 6 8 Max 1 h post-2p 2n Puls Intgral (ms mv) f 6 3 Normaliz Intgral (/post-unag) i Pr-2P Unag 4 8 Stim. Strngth (ma) Post-2P 1..5 tmk81 Amplitu (ΔF/F) 3 2 1.2 ΔF/F Pr-2P Post-2P Supplmntary Figur 4. Two-photon unaging of t-mk81 loks NMDA spiks in vitro an in vivo. (a) Somati voltag rsponss to squntially inrasing intnsity of xtrallular stimulation (2x 1 ms pulss at 5 Hz) for (lak; top) an aftr (r; ottom) twophoton ativation (71-73 nm) of a ag NMDA hannl agonist (t-mk81) at th stimulat ranh in vitro. Arrow iniats a suprathrshol rspons. () Ovrlay of NMDA spik for (lak) an aftr (r) unaging t-mk81 for th nrit in (a). () Intgral of th voltag rspons to inrasing stimulus strngth for th xampl shown in (a) an () for (lak) an aftr (r) two-photon unaging. Data fitt with linar rgrssion. () Normaliz intgral aftr two-photon unaging uring a NMDA spik (r soli; n = 5), EPSP (r mpty; n = 11) an ontrol (lasr xposur in nurons without t-mk81; light r; n = 8). () Exampl of xtrallularly stimulat potntials from a ontrol nuron (no t-mk81) for (lak) an aftr (r) two-photon xposur (73 nm for ~3 min) an uring ath appliation of (1 μm; grn). (f) Normaliz intgral uring ath appliation of aftr two-photon xposur in nurons fill with t-mk81 (mpty ar; n = 3) an in ontrol (soli ar; n = 5). (g) Amplitu of rsponss to loal xtrallular stimulation (2x 1ms pulss at 5 Hz) normaliz to th maximum vok rspons for (lak; lft) an aftr (r; right) two-photon ativation of th ag NMDA hannl agonist t-mk81 in vivo (n = 6 nrits from 3 nurons). Unaging t-mk81 aolishs th supralinar rspons to inrasing xtrallular stimulation intnsity. (h) Hr, w show that xposur to twophoton xitation alon ha no masural avrs ffts on th nriti morphology or amplitu of transints. Dnriti morphology for (top) an aftr (ottom) twophoton lasr xposur (69 nm, ~ 3 min). Sal ar, 2 μm. (i) transints for (lak; top) an aftr (r; ottom) xposur to two-photon light (69 nm for ~3 min) from a ontrol layr 2/3 pyramial nuron fill with OGB1 an Alxa Fluor 594 (an not t-mk81). Inst, thr was no iffrn in th pak amplitus of transints for (lak) an aftr (r) xposur to two-photon xitation in ontrol layr 2/3 pyramial nuron nrits. iniats p <.5. Error ars rprsnt S.E.M. Natur Nurosin: oi:1.138/nn.3646
5 μm Ca2 + -6 mv a Puff.1 ΔF/F Trun 5 mv Evok (%) Evok APs (#) 2 1 2 1 Supplmntary Figur 5. Hinpaw airpuff rlialy voks nriti an somati rsponss whih ar pnnt on NMDA rptors. Dspit th iffrnt mos of hinpaw stimulation, rif ltrial (Figur 2) an air puff stimulation (4 psi) vok similar nriti an somati rsponss - approximatly on ation potntial (AP) pr stimulation an a nriti rspons in approximatly 2 % of stimulations. (a) Dnriti fluorsn (top) an somati voltag (ottom) uring airpuff stimulation of th hinpaw. Inst; two-photon imag of th imag tuft nrit. () Prntag of hinpaw stimulation trials whih rsult in a masural transint uring ontrol (lak; n=16) an NMDA lok y ortial appliation of (r; n = 15). () Numr of APs vok y hinpaw stimulation uring ontrol (lak; n = 5) an NMDA lok y ortial appliation of (r; n = 15). Error ars rprsnt S.E.M. Natur Nurosin: oi:1.138/nn.3646
Expos OGB1 t-mk81 1 μm Naiv Norm Frq aftr unaging (post unag/pr unag) 1..5 ontrol Naiv Expos OGB1 t-mk81 1 μm APs APs.2 ΔF/F % Isolat % Isolat pr-uv pr-uv 5 % 2P % 15 % 2P % Supplmntary Figur 6. Unaging of t-mk81 loally loks NMDA hannls. Hr, w show that two-photon unaging of t-mk81 loally lok NMDA hannls y omparing th frquny of transints (a & ) an th ourrn of isolat transints (-) aftr susqunt unaging at a nighoring ranh. (a) Nurons wr fill with th ag NMDA hannl lokr t-mk81. transints wr ror in oth naiv nrits (no prior two-photon unaging anywhr in th nuron; grn; n = 8 nrits) an xpos nrits (nighoring nrits ha loal NMDA hannls lok y unaging t-mk81; orang; n = 16 nrits). Insts, ontrol (pr unaging) transints; sal,.2 ΔF/F,. Th loal lok of NMDA hannls y two-photon unaging of t-mk81 i not afft th frquny of spontanous ontrol transints in nighoring tuft nrits (naiv,.6 ±.2 Hz; xpos,.6 ±.3 Hz). () Th frquny of spontanous transints aftr loal NMDA hannl lok (normaliz to th ontrol frquny) in naiv nrits (grn) ompar to xpos nrits (orang). Not, lok of NMDA hannls os not afft th fftivnss of unaging t-mk81 in nighoring tuft nrits. () Ronstrution of a L2/3 pyramial nuron fill with t-mk81. () & (), Dnriti tra an simultanous somati voltag from two iffrnt tuft nrits from th nuron shown in (). Both nrits hav transints in th asn of somati APs (iniat y olor ots). APs ar iniat y ashs an ar trunat. Both nrits ha transints (i not orrlat with a somati AP) for (olor) ut not aftr (r) ativation of t-mk81 with two-photon lasr. Error ars rprsnt S.E.M. Natur Nurosin: oi:1.138/nn.3646
5 μm 1 % ΔG/R amplitu (% ΔG/R) 2 1 ~ 38 μm 3x SD nois -4-2 2 4 Dnriti spra (μm) OGB1 1 μm D1 D1 D2 D2 5 μm Multipl.2 ΔF/F.2 ΔF/F Singl f g Av amplitu Av somati voltag (ΔF/F) 2.5 2. 1.5 1..5 Multipl Singl (mv) 15 1 5 Multipl Singl Supplmntary Figur 7. transints in th prsn of th Na + hannl lokr ar similar to ontrol - thy ar spatially rstrit an our in oth singl an multipl ranhs. In thory, akpropagating APs from th ll oy an inva th tuft nrit an influn nriti ltrognsis. W thrfor tst whthr tuft transints wr influn y somati ativity y aing th Na + hannl lokr to th path piptt. (a) Two-photon imag illustrating a tuft ranh with 5 μm rgions of intrst. Inst, r fluorsn. () Spontanous transints from th nrit shown in (a). Not th iffrnt spatial spra of th two transints. To ompar iffrnt nriti rgions, transints ar rport as ΔG/R. () Avrag transint spatial spra along nriti tuft ranhs (5 μm rgions of intrst; n = 4 transints). Data fitt with Gaussian fit; ash lin. Gry lin; thrshol for vnts (> 3x stanar viation of th nois). () Ronstrution of layr 2/3 pyramial nuron fill with an two-photon imag of two tuft nrits. () Spontanous transints whih ourr in only on (singl) or oth (multipl) of th nrits shown in (). (f) Avrag pak amplitus of th transints whih ourr in singl (light grn) an multipl (ark grn) ranhs (n = 58 transints in 41 ranhs). (g) Avrag somati voltag uring transints whih ourr in singl (light grn) an multipl (ark grn) ranhs (n = 9 ranhs) orrt for th inras in input rsistan uring (s Fig. 3 an S9). Error ars rprsnt S.E.M. Natur Nurosin: oi:1.138/nn.3646
L1 tmk81 OGB1 L2/3 2P 1 μm Estim Estim Unag Estim Amp (ΔF/F).5 ΔF/F.5 ΔF/F 2. 1. Unag With (ms) Amp (ΔF/F) Amp (ΔF/F) 1.5 1..5 4 6 1 Estim strngth Spont Ctrl Unag f.2 ΔF/F g.3.8 Pr-2P 1. Post-2P.5.2.1.4 i OGB1 Ryanoin 1 μm j HS vok spontanous.2 ΔF/F Ryan k Amplitu (ΔF/F) 1.5 1..5 h Spont Frq (Hz) Ryan l Frquny (Hz).1 MK81 Ryan Supplmntary Figur 8. Extrallular stimulation voks larg transints in tuft nrits whih is pnnt on NMDA an not voltag-snsitiv hannls nor intrnal stors. Ation potntial (AP) initiation was lok y inluing in th path piptt whih ompltly or partially loks Na +, Ih, K + an hannls (Prkins an Wong, 1995; Talot an Sayr, 1996). (a) Ronstrution of a layr 2/3 pyramial nuron illustrating th xprimntal sign. Nurons wr fill with an th ag NMDA hannl lokr t-mk81, an an xtrallular stimulating piptt was pla in los proximity to a ranh of intrst. () Ovrlay (spatially shift for isplay purposs) of transints in rspons to inrasing stimulus intnsity from th tuft nrit ox in (a). () Avrag transint amplitu uring foal xtrallular stimulation of inrasing intnsity (lak, n = 7) an uring spontanous ativity (lu; n = 7). Linar rgrssion for suthrshol rsponss is shown y gry lin. () Ovrlay (spatially shift for isplay purposs) of transints in rspons to inrasing stimulus intnsity aftr (ottom) lok of NMDA rptors y twophoton (69 nm) unaging of t-mk81 from th ox tuft nrit in (a). () Th vok transint amplitu to th sam suprathrshol stimulation strngth was signifiantly largr for (lak) than aftr (r) lok of NMDA rptors y two-photon (69 nm) unaging of t-mk81. (f) Two-photon lasr alon osn t afft ativity. Hr, w show that xposur to two-photon xitation alon ha no masural avrs fft on th amplitu or with of transints in nurons fill with. transints for (lak) an aftr (r) xposur to two-photon light (69 nm for ~3 min) from a layr 2/3 pyramial nuron fill with (an not t-mk81). (g) Pak amplitus (lft) an with (right) of transints for (lak) an aftr (r) xposur to two-photon xitation from ontrol layr 2/3 pyramial nurons (n = 9). (h) Avrag frquny of spontanous tuft transints uring alon (lu; n=4 nrits) an an MK81 (r; n=11 nrits) in th path piptt. (i) In aition to synapti input, larg vnts in th apial nrits of layr 2/3 nurons hav also n shown in vitro to u to rlas from ryanoin-snsitiv intrallular stors (Larkum t al, 23). W thrfor tst whthr th larg vnts w masur in vivo wr ryanoin snsitiv. Ronstrution of layr 2/3 pyramial nuron fill with ryanoin (1 μm) an (1 mm). (j) Spontanous (lak) an vok (hinlim stimulation; r) transints ror from th nuron shown in (i). Th loation of th imag tuft nrit is iniat y a r irl in (i). (k) Avrag pak amplitu an (l) frquny of th transints in nurons fill with alon (lu; n = 465 transints from 8 nrits) an oth an ryanoin (fuhsia; n = 117 transints from 42 nrits). p <.5. Error ars rprsnt S.E.M. Natur Nurosin: oi:1.138/nn.3646
-8 mv -8 mv 2 mv 2 ms Proportion of ontrol 1.5 1..5 25 2 5 mv 15 5 ms 1 5.5 1. Vm soma Stimulus Strngth Thrshol Vm rst f 2n Puls Amp (mv) Input Rsistan 1 5 h i j k l Synaps 2 ms 2 mv Vm soma Proportion of ontrol 1..5 Thrshol Vm rst Input Rsistan 1 5 (MΩ) (MΩ) g 2n Puls Amp (mv) 2n Puls Amp 2n Puls Amp 3 2 1 1..5 12 6 Supplmntary Figur 9. inrass th amplitu of th somati voltag uring NMDA spiks in tuft nrits of layr 2/3 pyramial nurons in vitro an in silio. Intrallular appliation of loks Na + hannls as wll as partially loks, Ih an K + hannls (Prkins an Wong, 1995; Talot an Sayr, 1996). Th fft of on NMDA spiks was tst using somati rorings from layr 2/3 pyramial nurons whih wr initially path with ontrol intrallular solution an thn rpath with intrallular solution ontaining. Nurons wr fill with Alxa Fluor 594 (5 μm) to ai th plamnt of an xtrallular stimulation piptt in los proximity to a tuft nrit. (a) Somati voltag rspons to somati urrnt stp injtions (1 pa stps) for (lak) an aftr (lu). Not th lak of ation potntials in th prsn of. () Squntially inrasing th intnsity of pair pulss (2x 1 ms pulss at 5 Hz) to th tuft nrit rsult in a supralinar voltag rspons oth for (lak) an aftr (lu). A suthrshol an suprathrshol rspons is shown for ah onition. () Amplitu of th somati voltag uring squntial inras in stimulus intnsity for th xampl shown in (). () signifiantly inras th amplitu of th voltag rspons y approximatly two-fol (2n puls; n = 5; p <.5). () Thr was no signifiant influn of on th stimulus intnsity (thrshol) rquir to vok a spik (n = 5) nor th rsting mmran potntial (n = 6). (f) Howvr, signifiantly inras th input rsistan y on avrag 48 ± 15 % (n = 6). (g) Bath appliation of th NMDA hannl agonist (5 μm; ottom; grn) signifiantly ras th voltag rspons. (h) Th ffts of intrallular on synapti input (loation; orang ot) was mol y ompltly or partially loking Na +, Ih, K + an onutans (s supplmntal mthos). (i) Suthrshol an suprathrshol somati rsponss to inrasing NMDA/AMPA input in ontrol (lak) an (lu) simulations at th synaps shown in (h). Not, th omputr simulations ar omparal to th xprimntal ata in (). In th prsn of, th omputational mol shows no fft on thrshol or rsting mmran potntial (j), an inras in th input rsistan (k) an an inras in th amplitu of th son puls of th pair puls stimulation whih was ras uring ath appliation of in ontrol (l). Ths omputr simulation rsults ar omparal to th in vitro ata shown in (a) - (g) an th inras in voltag uring an ntirly xplain y th ras in input rsistan. iniats p<.5. Error ars rprsnt S.E.M. Natur Nurosin: oi:1.138/nn.3646
Tuft Soma Basal Layr 2/3 pyramial nuron Currnt Into Soma Bakgroun Top-own input Vm soma Conutan (ns) Soma Basal Tuft Axon Hotzon m 1 2 2 1 2 gpassiv.9295.12942.16197.89375.16197 Ca LVA.343.187.187 Ca HVA.992.555.111 SKv3 1.395.3915.3915 SK E2.6615.18.18 K Tst.812 K Pst.223 Nap Et2.172 Na Ta 2.4.213.213 Ih 8.6957E - 6 8.6957E - 6.285642.243 Im.675.675 Currnt Into Dnrit Dnrit 1 mv 2 ms f Input Rsistan 5 (MΩ) 7 Rsting Vm (mv) Soma In Vitro Mol In Vitro Mol g h -4 mv -6 mv -8 mv NMDA soma 2 mv 2 ms gmax ( -t/7 - -t/3 ) 1+.3 -.8 v Soma Dnrit AMPA gmax -t/2 5 ms 5 ms max inrasing synapti onutan 2 mv 6 mv 2 ms 2 mv nrit Supplmntary Figur 1. Dtails of th layr 2/3 omputational mol. (a) A ronstrut L2/3 pyramial nuron from th xprimntal part of this stuy was us for simulations in NEURON. () Top-own synapti inputs (r) wr pla with uniform proaility istriution aross th tuft nrits an a furthr ynapti inputs wr istriut with uniform proaility aross th ntir nuron to simulat akgroun synapti input (lu). Inst, akgroun mmran potntial. R lin: 17.3 mv from rst. Sal, 1 mv; 15 ms. () Tal of onutan valus us in th simulat nuron. All valus ar in units of ns. () A ronstrut L2/3 pyramial nuron. () Computr simulations of th voltag rspons at th soma (grn) an nrit (lu) uring urrnt injtion () into th soma (lft) an nrit (right) for th mol nuron shown in (). (f) Comparison of vitro xprimnts (n = 6) an th mol nuron for input rsistan an rsting mmran potntial. Error ars ar stanar viation. (g) (lft) Nonlinar NMDA onutan us in th mol. Not that th onutan is a funtion of th loal mmran potntial. Plots ar shown at 4, 6, an 8 mv. (Right) Linar AMPA onutan us in th mol. Not that th AMPA onutan is voltag inpnnt. (h) Simulat voltag rspons to pair puls stimulation (5 Hz) with inrasing intnsity with (r) an without (lak; ) NMDA onutan ror at th soma (lft) an at th sit of stimulation (tuft nrit; right). Inst, maximum voltag rspons at th soma as synapti onutan inrass. Th non linarity in th ontrol onition stalishs th vnt as an NMDA spik, as sri in prvious work (Shillr t al., 2, Shillr an Shillr, 21, Spruston an Kath, 24, Rhos, 26, Major t al., 28, Larkum t al., 29, Polsky t al., 29, L, 212). Natur Nurosin: oi:1.138/nn.3646
NMDA onutan rmov from tuft ranhs (r): Zro ranhs On ranh Fiv ranhs Entir tuft soma 6 inputs 1 mv 2 ms zro on fiv all NMDA onutans rmov Numr of synapss rquir for ation potntial gnration 12 8 NMDA/AMPA onutan only AMPA onutan 4 2 4 6 8 1 Numr of ranhs without NMDA onutan Supplmntary Figur 11. Bloking istriut NMDA onutans in th majority of th tuft affts AP gnration. Th xprimntal ata show that loally loking NMDA hannls in a singl tuft ranh os not afft somati ation potntials (APs). To furthr invstigat this, w us omputr simulations in th NEURON simulation platform to manipulat th xtnt of th tuft xprining NMDA lok an masur th rsulting fft on nuronal output. Top-own synapti inputs wr ranomly istriut aross th tuft nrits an a furthr ynapti inputs wr istriut with uniform proaility aross th ntir nuron to simulat akgroun synapti input (s Fig. S1). (a) A mol layr 2/3 pyramial nuron ha NMDA onutans lok in varying numrs of tuft ranhs. Synapti input was ranomly istriut for ah trial an ranhs shown in r ha oth akgroun an top-own NMDA onutans lok. () Mmran potntial tras in rspons to 6 synapti inputs ranomly istriut onto th tuft nrit in nurons whr NMDA onutans wr rmov from no (gry), on (rown), fiv (fuhsia) an all (grn) tuft ranhs. During this simulation, NMDA lok in fiv tuft nrits is suffiint to sustantially hang nuronal output. () Th total numr of synapss n to gnrat an AP ompar to th numr of ranhs with NMDA onutans rmov. Color ots rfr to ata shown in (). Natur Nurosin: oi:1.138/nn.3646