Letter. Autocrine BDNF TrkB signalling within a single dendritic spine

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1 Letter doi:1.138/nture19766 Autorine BDNF TrkB signlling within single dendriti spine Stephen C. Hrwrd 1, Nthn G. Hedrik 1, Chrles E. Hll 1, Pul Prr-Bueno 2, Teres A. Milner 3,4, Enhui Pn 1, Tl Lviv 2, Brr L. Hempsted 3,5, Ryohei Ysud 1,2 & Jmes O. MNmr 1 Brin-derived neurotrophi ftor (BDNF) nd its reeptor TrkB re ruil for mny forms of neuronl plstiity 1 6, inluding struturl long-term potentition (sltp) 7,8, whih is orrelte of n niml s lerning 7,9 12. However, it is unknown whether BDNF relese nd TrkB tivtion our during sltp, nd if so, when nd where. Here, using fluoresene resonne energy trnsfer-sed sensor for TrkB nd two-photon fluoresene lifetime imging mirosopy 13 16, we monitor TrkB tivity in single dendriti spines of CA1 pyrmidl neurons in ultured murine hippompl slies. In response to sltp indution 9,14 16, we find fst (onset < 1 min) nd sustined (>2 min) tivtion of TrkB in the stimulted spine tht depends on NMDAR (N-methyl-d-sprtte reeptor) nd CMKII signlling nd on postsynptilly synthesized BDNF. We onfirm the presene of postsynpti BDNF using eletron mirosopy to lolize endogenous BDNF to dendrites nd spines of hippompl CA1 pyrmidl neurons. Consistent with these findings, we lso show rpid, glutmte-unging-evoked, time-loked BDNF relese from single dendriti spines using BDNF fused to superelipti phluorin We demonstrte tht this postsynpti BDNF TrkB signlling pthwy is neessry for oth struturl nd funtionl LTP 2. Together, these findings revel spine-utonomous, utorine signlling mehnism involving NMDAR CMKIIdependent BDNF relese from stimulted dendriti spines nd susequent TrkB tivtion on these sme spines tht is ruil for struturl nd funtionl plstiity. To ddress the role of BDNF TrkB signlling in sltp, we developed fluoresene resonne energy trnsfer (FRET)-sed sensor for TrkB onsisting of two omponents: (1) TrkB fused to monomeri enhned green fluoresent protein (TrkB egfp), nd (2) n SH2 domin of the TrkB inding prtner phospholipse Cγ1 (PLC-γ1) 21 fused to two opies of monomeri red fluoresent protein-1 (mrfp1 PLC mrfp1; Fig. 1 nd Supplementry Informtion). After TrkB tivtion vi phosphoryltion of Tyr816, the ffinity of mrfp1 PLC mrfp1 for TrkB egfp inreses 21, therey llowing FRET to our etween the fluorophores (Supplementry Informtion). We vlidted the sensor in HeL ells nd ultured ortil neurons y showing it to e sensitive to BDNF, speifi for Tyr816phosphoryltion, nd reversile when imged y two-photon fluoresene lifetime imging mirosopy (2pFLIM) (Extended Dt Fig. 1, Supplementry Informtion). Furthermore, we demonstrted tht the sensor ould funtionlly reple endogenous TrkB in neurons of ultured hippompl slies (Extended Dt Fig. 2, Supplementry Informtion). Using this sensor, we iolistilly trnsfeted ultured rt hippompl slies nd imged CA1 pyrmidl neurons with 2pFLIM. In response to glutmte unging trgeted to single dendriti spine (3 pulses t.5 Hz), spine volume rpidly inresed y ~22% (trnsient phse) efore relxing to n inresed stte of ~9% lsting t lest 6 min (sustined phse; Fig. 1,, Extended Dt Fig. 3) hnges independent of protein synthesis (Extended Dt Fig. 3, d) nd lrgely onsistent with previous desriptions of sltp (Extended Dt Fig. 3, Supplementry Informtion) 9, At the sme time, TrkB rpidly tivted in the stimulted spine, peking t ~1 2 min nd remining elevted for t lest 6 min (Fig. 1, d, e, Extended Dt Figs 3, 4 nd Supplementry Informtion). For the first 3 6 s fter the onset of glutmte unging, this tivtion ws lrgely restrited to the stimulted spine (Fig. 1d f, Supplementry Informtion). However, with time, TrkB tivtion in djent regions slowly inresed, suggesting spreding of TrkB tivtion (Fig. 1d f). The vlidity of the oserved signl ws onfirmed y its dependene on kinse tivity nd Tyr816 phosphoryltion nd independene of sensor onentrtion nd temperture (Extended Dt Figs 5 7, Supplementry Informtion). To explore mehnisms underlying this TrkB tivtion, we sked whether it required NMDAR-medited C 2+ influx 7,9,15,16 nd susequent CMKII tivtion 9,15. Applition of either the NMDAR inhiitor d-2-mino-5-phosphonovlerte (d-; 1 μm) or the CMKII inhiitor (ref. 22; 1 μm) impired TrkB tivtion during the trnsient nd sustined phses of sltp while lso inhiiting spine volume hnge (Fig. 2 d), suggesting tht TrkB is in prt downstrem of oth NMDAR nd CMKII tivtion. Next, we sked whether BDNF ontriutes to this TrkB tivtion. Using the extrellulr BDNF svenger TrkB-Ig (6 8 μg ml 1 ), we found impired TrkB tivtion throughout sltp with similr impirment of spine volume hnge (Fig. 2e h), suggesting ruil role for BDNF in mediting glutmte-unging-indued TrkB tivtion. To exmine the ellulr soure of BDNF underlying this tivtion, we sprsely trnsfeted the sensor with Cre reominse in slies from Bdnf fl/fl mie, therey seletively knoking-out BDNF synthesized in the postsynpti ell, perturtion without detetle effet on sl spine morphology 23,4,41 (Extended Dt Fig. 8, ). This mnipultion ttenuted glutmte-unging-evoked TrkB tivtion nd sltp (Fig. 2i l) while leving CMKII tivtion intt (Extended Dt Fig. 8 f). These results implite utorine BDNF s one mehnism underlying TrkB tivtion during sltp: dditionl mehnisms ould inlude other soures of BDNF (pre-synpti, prrine) or nonneurotrophin TrkB tivtors (suh s zin) 24. The dependene of glutmte-unging-indued TrkB tivtion on postsynptilly synthesized BDNF ontroversilly suggests the existene of BDNF in dendrites or spines 25. To provide more diret evidene, we used eletron mirosopy to exmine BDNF loliztion in previously hrterized mouse line in whih C-terminl hemgglutinin (HA) epitope tg ws dded to the Bdnf oding sequene (Bdnf-HA) 26. Using highly sensitive ntiodies ginst the HA-tg, we found BDNF not only in xons ut lso in dendrites nd spines of CA1 pyrmidl ells of these mie (Fig. 3). 1 Neuroiology Deprtment, Duke University Medil Center, Reserh Drive, Durhm, North Crolin 2771, USA. 2 Mx Plnk Florid Institute for Neurosiene, 1 Mx Plnk Wy, Florid 33458, USA. 3 Feil Fmily Brin nd Mind Reserh Institute, Weill Cornell Mediine, 47 Est 61st St, New York 165, USA. 4 Lortory of Neuroendorinology, The Rokefeller University, 123 York Avenue, New York 165, USA. 5 Deprtment of Mediine, Weill Cornell Mediine, 13 York Avenue, New York 165, USA. Present ddress: Neuroiology Setion, Center for Neurl Ciruits nd Behvior, nd Deprtment of Neurosienes, University of Cliforni, Sn Diego, 95 Gilmn Drive, L Joll, Cliforni 9293, USA. These uthors ontriuted eqully to this work. 6 otoer 216 VOL 538 NATURE Mmilln Pulishers Limited, prt of Springer Nture. All rights reserved.

2 PLC RESEARCH Letter Δ Binding frtion GFP GFP Ativted TrkB Y515 Y816 P FRET RFP RFP Before 1 3 min 6 1 min min 16 2 min Time (s) Δ Binding frtion Pek Stim spine Adj spine Dendrite ns Δ Volume Sustined d Stim spine e f Adj spine Dendrite Figure 1 sltp indues rpid, persistent, nd lrgely spine speifi TrkB tivtion., Sensor design., 2pFLIM imges of TrkB tivtion verged ross indited time points. Arrowhed represents point of unging. Wrmer olours indite shorter lifetimes nd higher TrkB tivity. Imge size is μm., Time ourse of volume hnge for the stimulted spine. n = 5 ells/54 spines. d, e, Time ourse (d) nd quntifition (e) of pek ( min) nd sustined (1 2 min) tivtion for experiments in Stim spine Adj spine Dendrite Δ Binding frtion Spine 3 s 1 2 min 11 2 min 5 1 Distne (μm) mesured s the hnge in sensor inding frtion in stimulted spines, djent spines nd dendrites. Right pnel in d shows mgnified time ourse. n = 5/54 for stimulted spines nd dendriti shfts, nd 5/59 for djent spines (ells/spines). f, Sptil profile of TrkB tivtion hnge in inding frtion of the dendrite plotted s funtion of the distne from the stimulted spine. n = 48/52 (ells/stimulted spines). Dt re men ± s.e.m. P <.5, nlysis of vrine (ANOVA) with Dunnet s test. The loliztion of BDNF to dendriti spines, together with the rpid kinetis nd initil spine restrition of glutmte-ungingindued TrkB tivtion, suggested n eqully rpid relese of BDNF from dendriti spines during the trnsient phse of sltp. To ssess this possiility, we used iolistis to trnsfet CA1 pyrmidl ells with full-length BDNF ontining the ph-sensitive fluorophore superelipti phluorin (SEP) fused to its C terminus, thus llowing visuliztion of exoytosed BDNF 18,19 (Extended Dt Fig. 9, Δ Binding frtion Rt Δ Binding frtion Pek Sustined Trnsient Sustined d Rt e Δ Binding frtion Δ Binding frtion Rt HIgG TrkB-Ig f g h Δ Binding frtion Δ Binding frtion Rt HIgG TrkB-Ig i j k l Bdnf fl/fl Pek Sustined Trnsient Sustined HIgG Figure 2 TrkB tivtion during sltp depends on NMDAR CMKII signlling nd postsynpti BDNF.,, Time ourse () nd quntifition () of pek nd sustined TrkB tivtion in stimulted spines in the presene of phrmologil inhiitors., ontrol; denotes n NMDAR inhiitor; denotes CMKII inhiitor. n = 19/19 ontrol, 6/1, nd 7/16 (ells/spines)., d, Time ourse () nd quntifition (d) of trnsient (1 2 min) nd sustined (1 2 min) spine volume hnge for experiments in nd TrkB-Ig HIgG TrkB-Ig Pek Sustined Trnsient Sustined Bdnf.8 4 fl/fl HIgG TrkB-Ig HIgG TrkB-Ig. e h, Similr experiments to d ut with different phrmologil onditions. HIgG, humn IgG; TrkB-Ig, n extrellulr svenger of BDNF. n = 16/18 ontrol, 6/11 HIgG, nd 8/14 TrkB-Ig (ells/spines). i l, Similr experiments to d ut in Bdnf fl/fl hippompl slies trnsfeted with the TrkB sensor with or without Cre-reominse (Cre or, respetively). n = 7/15 Cre nd 9/17 (ells/spines). Dt re men ± s.e.m. P <.5, ANOVA with Dunnet s test (, d), Tukey s test (f, h) or two-tiled t-test (j, l). 1 NATURE VOL otoer Mmilln Pulishers Limited, prt of Springer Nture. All rights reserved.

3 Letter RESEARCH 1 nm Dendrite (Bdnf-HA) d Spine (Bdnf-HA) Dendrite Gli 1 nm 2 Terminl Gli 5 Unknown 1 WT (psr) WT (dsr) Bdnf-HA (psr) Bdnf-HA (dsr) 1 Spine 3 15 Dendrite 4 Axon f WT (psr) WT (dsr) Bdnf-HA (psr) Bdnf-HA (dsr) Numer of oservtions (linded quntifition) 5 Neuron e Numer of oservtions (linded quntifition) 2 nm Figure 3 Endogenous BDNF lolizes to xons, dendrites nd dendriti spines., Immunoperoxidse lelling of HA in hippompl res CA1 (left) nd CA3 (right) from Bdnf-HA nd wild-type (WT) mie, visulized y light mirosopy. dsr, distl strtum rditum; PCL, prinipl ell lyer; psr, proximl strtum rditum; SLM, strtum lunosum-moleulre; SO, strtum oriens. d, Immunoperoxidse lelling of HA in CA1 pyrmidl neuron xon terminls (), dendrites (), nd dendriti spines (d) of Bdnf-HA mie, visulized y eletron mirosopy. e, f, Quntifition of oserved immunoperoxidse lelling of HA in vrious ellulr types (e) nd suellulr omprtments (f) in proximl nd distl strtum rditum in hippompl slies from wildtype nd Bdnf-HA mie. n = 3 nimls eh. Supplementry Informtion). In response to glutmte unging, we oserved n inrese in SEP fluoresene lrgely restrited to the stimulted spine (Fig. 4,, Supplementry Video 1), with two ΔF/F 11th 15th pulses th 3th pulses Spine Time (s) Dendrite Bdnffl/fl 4 Cre 3 BDNF SEP 8 Cre Time (s) h Cre Cre Pek Sustined Figure 4 Glutmte unging indues rpid relese of postsynpti BDNF., Two-photon imges of glutmte-unging-evoked hnges in BDNF SEP fluoresene in dendriti spines of CA1 hippompl neurons. Eh row represents the unging-triggered verge of the BDNF SEP signl in response to individul unging pulses for the designted time window. Imge size is μm., Averged time ourse of BDNF SEP fluoresene hnge in spines nd djent dendriti shfts in response to glutmte unging (timing of glutmte pulses indited y lk rs (top)). Inset shows the hnge in mcherry (mch) fluoresene (red) in response to glutmte unging, inditive of spine volume hnge (sltp). n = 26/187 (ells/spines)., -triggered verge of the inrese in BDNF SEP fluoresene with glutmte unging. TeTx, neurons trnsfeted with tetnus toxin, n inhiitor of exoytosis; POMC, neurons trnsfeted with the POMC peptide, n inhiitor of tivity-dependent Bdnffl/fl g Time (s) f 448 ms ΔF/F d.4 1 e TeTx POMC +NBQX 1.6 ΔF/F 1st 5th pulses ΔR/R BDNF SEP mch TeTx POMC +NBQX NBQX SR Slu PCL SO 5 μm Time (s) Cre dsr SLM distint kineti profiles. The first ws trnsient, spike-like inrese time-loked to eh unging pulse (Fig. 4 ), perhps orrelting with tivity-indued BDNF relese. The seond ws slow inrese in fluoresene ommening with the strt of unging nd peking t its end, perhps due to extrellulr umultion of relesed BDNF SEP (Fig. 4, ). After termintion of unging, SEP fluoresene deyed k to seline over the ourse of 1 min (Extended Dt Fig. 9e). Severl lines of evidene suggested tht these trnsient, spike-like inreses in SEP fluoresene were in ft due to BDNF relese from the spine. First, the oserved fluoresene signl depended on ph, exoytosis, nd BDNF sorting mhinery27 (Fig. 4, d, Extended Dt Fig. 9d, Supplementry Informtion). Seond, expression of BDNF SEP resued struturl plstiity in the setting of postsynpti BDNF knokout, thus suggesting tht it ould funtionlly reple endogenous BDNF in neurons (Fig. 4e, f, Supplementry Informtion). Third, the oserved BDNF SEP signl ws independent of the presene of endogenous postsynpti BDNF (Fig. 4g, h). Fourth, the kineti profile of the signl prlleled the time ourse of TrkB tivtion, s one would expet for BDNF relese (Extended Dt Fig. 4). Colletively, these results suggest tht the oserved inrese in SEP signl proly reports glutmte-dependent exoytosis nd relese of BDNF from stimulted spines. To explore mehnisms underlying glutmte-indued BDNF relese, we inhiited NMDARs (with ) or AMPARs (with NBQX) individully nd together s well s inhiiting CMKII with (Fig. 4, d). We found the SEP signl to e lrgely loked (), unffeted (NBQX), ompletely loked ( plus NBQX), nd prtilly loked () y these perturtions (Fig. 4, d). These findings suggest tht BDNF relese from spines is lrgely NMDAR CMKII dependent, onsistent with our results for TrkB tivtion. The onverging evidene impliting utorine BDNF TrkB signlling in sltp led us to sk whether this pthwy ws lso involved Cre+ CA3 WT CA3 Bdnf-HA ΔF/F Terminl (Bdnf-HA) CA1 WT CA1 Bdnf-HA SO PCL psr ΔF/F BDNF relese. n = 31/218 ontrol, 6/82 TeTx, 2/29 POMC, 3/5, 2/46 + NBQX, 4/4 NBQX, nd 7/88 (ells/spines). d, Pek of the unging-triggered verged inrese of BDNF SEP fluoresene in. e, Time ourse of glutmte-unging-indued spine volume hnge for Bdnffl/fl hippompl slies trnsfeted with egfp (Cre ), egfp plus Cre (Cre+), or egfp, Cre nd BDNF SEP. n = 9/13 Cre, 6/11 Cre+ nd 8/13 Cre+ plus BDNF SEP (ells/spines). f, Trnsient (1 2 min) nd sustined (1 4 min) spine volume hnge for experiments in e. g, h, Similr experiments to nd d ut in Bdnffl/fl hippompl slies in the sene or presene of Cre. n = 1/15 Cre nd 15/132 Cre+ (ells/spines). Dt re men ± s.e.m. See Extended Dt Fig. 9h for dt in d represented s medin ± interqurtile intervl. P <.5, Kruskl Wllis test with Dunn s test (d) or n ANOVA with Tukey s test (f). 6 o t o e r V O L N A T U RE Mmilln Pulishers Limited, prt of Springer Nture. All rights reserved.

4 RESEARCH Letter EPSC hnge Trk F616A Veh WT Veh 5 TrkF616A Veh Before Before 3 45 min 1 pa 1 ms 3 45 min EPSC hnge min Trk F616A WT Veh Veh Trk F616A Veh d 4 2 Trnsient Trk F616A Veh 1 5 Sustined Trk F616A Veh e EPSC hnge Bdnf fl/fl Before Before 4 6 min 4 6 min 5 pa 1 ms f EPSC hnge 4 6 min g Bdnf fl/fl + BDNF h Trnsient Bdnf fl/fl + BDNF 1 5 Sustined Bdnf fl/fl + BDNF Figure 5 Funtionl nd struturl LTP depends on postsynpti BDNF TrkB signlling.,, Time ourse () nd quntifition (; 3 45 min) of exittory postsynpti urrent (EPSC) hnge reorded in CA1 pyrmidl ells of hippompl slies from Trk F616A nd wildtype mie, efore nd fter LTP indution in the presene of vehile or. Representtive tres of Trk F616A slies with vehile or re shown ove the grphs. n = 11 Trk F616A vehile, 1 Trk F616A, 11 wild-type vehile, nd 13 wild-type (ells)., d, Time ourse () nd quntifition (d) of trnsient nd sustined glutmte-unging-indued spine volume hnge for Trk F616A hippompl slies in the sene or presene of vehile or. n = 2/2 ontrol, 1/13 vehile, nd 16/2 (ells/spines). e h, Similr experiments to nd ut from Bdnf fl/fl mie infeted with or without Cre. Representtive tres re shown ove the grphs. n = 2 Cre nd 19 (ells). g, h, Time ourse (g) nd quntifition (h) of trnsient nd sustined glutmte-unging-indued spine volume hnge for Bdnf fl/fl hippompl slies trnsfeted with egfp or egfp plus Cre. For plus BDNF, Cre-positive ells were treted with BDNF for 1 min efore glutmte unging. n = 13/14 Cre, 22/32, nd 6/7 plus BDNF (ells/spines). Dt re men ± s.e.m. P <.5, two-tiled t-test (, f) or ANOVA with Tukey s test (d, h). in funtionl LTP (fltp) t the CA3 CA1 synpse. To ddress this question, we indued fltp y piring low-frequeny Shffer ollterl xon stimultion with depolriztion of single CA1 pyrmidl ells through whole-ell pth lmping. First, we exmined the role of TrkB y using knok-in mie ontining point muttion in the TrkB kinse domin (F616A; Trk F616A, lso known s Ntrk2 F616A ), rendering the mutnt TrkB uniquely suseptile to inhiition y the smll moleule (ref. 28). inhiited oth sltp (in ultured slies) nd fltp (in ute slies) in slies isolted from Trk F616A ut not wildtype mie, reveling requirement for TrkB kinse (Fig. 5 d). In ddition, svenging extrellulr BDNF with TrkB-Ig (2 μg ml 1 ) impired oth sltp (in ultured slies) nd fltp (in ute slies) (Extended Dt Fig. 1 d), impliting BDNF s one mehnism underlying TrkB tivtion in this ontext. To determine whether utorine BDNF TrkB signlling in prtiulr ontriuted to these forms of plstiity, we knoked out BDNF in smll popultion of CA1 pyrmidl ells using either in utero infetion of deno-ssoited virus enoding synpsin-cre in Bdnf fl/fl mie for fltp in ute slies or iolisti trnsfetion of Cre in orgnotypi slies prepred from Bdnf fl/fl mie for sltp. The knokout of postsynpti BDNF impired oth fltp nd sltp, the ltter of whih ws resued y th-pplied BDNF (2 ng ml 1 for 1 min) (Fig. 5e h). Colletively, these results revel requirement of ell-utonomous, postsynpti BDNF relese nd susequent tivtion of postsynpti TrkB for oth struturl nd funtionl synpti plstiity (Extended Dt Fig. 1e, Supplementry Informtion). Overll, we hve desried n utorine signlling system within single spine hieved y rpid BDNF relese from the stimulted spine nd susequent TrkB tivtion on the sme spine tht, potentilly in oopertion with other soures of BDNF nd tivtors of TrkB, is essentil for oth struturl nd funtionl plstiity. Online Content Methods, long with ny dditionl Extended Dt disply items nd Soure Dt, re ville in the online version of the pper; referenes unique to these setions pper only in the online pper. reeived 5 April; epted 15 August 216. Pulished online 28 Septemer Lohof, A. M., Ip, N. Y. & Poo, M. M. Potentition of developing neuromusulr synpses y the neurotrophins NT-3 nd BDNF. Nture 363, (1993). 2. Kng, H., Welher, A. A., Shelton, D. & Shumn, E. M. Neurotrophins nd time: different roles for TrkB signling in hippompl long-term potentition. Neuron 19, (1997). 3. Minihiello, L. et l. Essentil role for TrkB reeptors in hippompus-medited lerning. Neuron 24, (1999). 4. Figurov, A., Pozzo-Miller, L. D., Olfsson, P., Wng, T. & Lu, B. Regultion of synpti responses to high-frequeny stimultion nd LTP y neurotrophins in the hippompus. Nture 381, (1996). 5. Korte, M. et l. Hippompl long-term potentition is impired in mie lking rin-derived neurotrophi ftor. Pro. Ntl Ad. Si. USA 92, (1995). 6. Kovlhuk, Y., Hnse, E., Kfitz, K. W. & Konnerth, A. Postsynpti indution of BDNF-medited long-term potentition. Siene 295, (22). 7. Tnk, J. et l. Protein synthesis nd neurotrophin-dependent struturl plstiity of single dendriti spines. Siene 319, (28). 12 NATURE VOL otoer Mmilln Pulishers Limited, prt of Springer Nture. All rights reserved.

5 Letter RESEARCH 8. Li, K.-O. O. et l. TrkB phosphoryltion y Cdk5 is required for tivitydependent struturl plstiity nd sptil memory. Nt. Neurosi. 15, (212). 9. Mtsuzki, M., Honkur, N., Ellis-Dvies, G. C. & Ksi, H. Struturl sis of long-term potentition in single dendriti spines. Nture 429, (24). 1. Okmoto, K., Ngi, T., Miywki, A. & Hyshi, Y. Rpid nd persistent modultion of tin dynmis regultes postsynpti reorgniztion underlying idiretionl plstiity. Nt. Neurosi. 7, (24). 11. Kim, I. H. et l. Disruption of Arp2/3 results in symmetri struturl plstiity of dendriti spines nd progressive synpti nd ehviorl normlities. J. Neurosi. 33, (213). 12. Kim, I. H., Wng, H., Soderling, S. H. & Ysud, R. Loss of Cd42 leds to defets in synpti plstiity nd remote memory rell. elife 3, e2839 (214). 13. Ysud, R. Imging sptiotemporl dynmis of neuronl signling using fluoresene resonne energy trnsfer nd fluoresene lifetime imging mirosopy. Curr. Opin. Neuroiol. 16, (26). 14. Hrvey, C. D., Ysud, R., Zhong, H. & Svood, K. The spred of Rs tivity triggered y tivtion of single dendriti spine. Siene 321, (28). 15. Lee, S.-J. R. J., Esoedo-Lozoy, Y., Sztmri, E. M. & Ysud, R. Ativtion of CMKII in single dendriti spines during long-term potentition. Nture 458, (29). 16. Murkoshi, H., Wng, H. & Ysud, R. Lol, persistent tivtion of Rho GTPses during plstiity of single dendriti spines. Nture 472, 1 14 (211). 17. Miesenök, G., De Angelis, D. A. & Rothmn, J. E. Visulizing seretion nd synpti trnsmission with ph-sensitive green fluoresent proteins. Nture 394, (1998). 18. Mtsud, N. et l. Differentil tivity-dependent seretion of rin-derived neurotrophi ftor from xon nd dendrite. J. Neurosi. 29, (29). 19. Den, C. et l. Synptotgmin-IV modultes synpti funtion nd long-term potentition y regulting BDNF relese. Nt. Neurosi. 12, (29). 2. Hedrik, N. G. et l. Rho GTPse omplementtion underlies BDNF-dependent homo- nd heterosynpti plstiity. Nture nture19784 (216). 21. Middlems, D. S., Meisenhelder, J. & Hunter, T. Identifition of TrkB utophosphoryltion sites nd evidene tht phospholipse C-γ1 is sustrte of the TrkB reeptor. J. Biol. Chem. 269, (1994). 22. Vest, R. S., Dvies, K. D., O Lery, H., Port, J. D. & Byer, K. U. Dul mehnism of nturl CMKII inhiitor. Mol. Biol. Cell 18, (27). 23. Lu, W. et l. Suunit omposition of synpti AMPA reeptors reveled y single-ell geneti pproh. Neuron 62, (29). 24. Hung, Y. Z., Pn, E., Xiong, Z.-Q. Q. & MNmr, J. O. Zin-medited trnstivtion of TrkB potentites the hippompl mossy fier-ca3 pyrmid synpse. Neuron 57, (28). 25. Dieni, S. et l. BDNF nd its pro-peptide re stored in presynpti dense ore vesiles in rin neurons. J. Cell Biol. 196, (212). 26. Yng, J. et l. Neuronl relese of probdnf. Nt. Neurosi. 12, (29). 27. Lou, H. et l. Sorting nd tivity-dependent seretion of BDNF require intertion of speifi motif with the sorting reeptor roxypeptidse E. Neuron 45, (25). 28. Chen, X. et l. A hemil-geneti pproh to studying neurotrophin signling. Neuron 46, (25). 4. He, X.-P. et l. Conditionl deletion of TrkB ut not BDNF prevents epileptogenesis in the kindling model. Neuron 43, (24). 41. Luikrt, B. W. et l. TrkB hs ell-utonomous role in the estlishment of hippompl Shffer ollterl synpses. J Neurosi. 25, (25). Supplementry Informtion is ville in the online version of the pper. Aknowledgements We thnk A. West nd Y. Hung for ritil disussion. This work ws supported y grnts from the Ntionl Institutes of Helth (F31NS78847 (S.C.H.), R1NS6841 (R.Y.), DP1NS96787 (R.Y.), R1NS5621 (J.O.M.), R1MH847 (R.Y.), R1DA8259 (T.A.M.), R1HL98351 (T.A.M.), P1HL96571 (T.A.M.), nd RO1NS3687 (B.L.H.)), the Wkemn Fellowship (S.C.H.), nd Humn Frontier Siene Progrm (T.L.). Author Contriutions S.C.H., N.G.H., R.Y. nd J.O.M. designed experiments; S.C.H. nd N.G.H. olleted nd nlysed imging dt with ssistne from C.E.H.; P.P.-B. nd E.P. olleted nd nlysed pth lmp dt; T.A.M. olleted eletron mirosopi imges nd nlysed them with B.L.H.; T.L. performed in utero virl injetions; S.C.H., N.G.H., R.Y. nd J.O.M. nlysed remining dt nd wrote the pper. All uthors disussed results nd omments on this mnusript. Author Informtion Reprints nd permissions informtion is ville t The uthors delre no ompeting finnil interests. Reders re welome to omment on the online version of the pper. Correspondene nd requests for mterils should e ddressed to R.Y (Ryohei. Ysud@mpfi.org). Reviewer Informtion Nture thnks B. Bingol, H. Zhng nd the other nonymous reviewer(s) for their ontriution to the peer review of this work. 6 otoer 216 VOL 538 NATURE Mmilln Pulishers Limited, prt of Springer Nture. All rights reserved.

6 RESEARCH Letter Methods Regents. Humn reominnt BDNF nd humn reominnt β-ngf were purhsed from Millipore, K252 nd d-2-mino-5-phosphonovlerte (d-) nd 2,3-dihydroxy-6-nitro-7-sulfmoyl-enzo[f]quinoxline-2,3-dione (NBQX) were from Toris, humn-igg ws from Sigm, nd 1 -nphthylmethyl-4-mino-1-tert-utyl-3-(p-methylphenyl)pyrzolo[3,4-d] pyrimidine () ws from Snt Cruz nd Shnghi Institute of Mteri Medi, Chinese Ademy of Sienes. TrkB-Ig ws gift from Regeneron nd the tt- peptide (YGRKKRRQRRRKRPPKLGQIGRSKRVVIEDDR) ws synthesized y GenSript. Plsmids. TrkB egfp ws prepred y inserting the oding sequene of mouse TrkB (otined from previously desried plsmid 29 ) into pegfp-n1 (Clonteh) ontining the A26K monomeri muttion in egfp nd the CAG promoter 3. The linker etween TrkB nd egfp is TGRH. mrfp1 PLC mrfp1 ws prepred y inserting the oding sequene for the C-terminl SH2 domin of humn PLC-γ1 ( ; otined from full-length, humn PLC-γ1 purhsed from Origene) into tndem-mrfp1 plsmid ontining the CAG promoter. The linkers etween the mrfp1s nd PLC-γ1 ( ) re RSRAQASNS for the N terminus nd GSG for the C terminus. TrkB Y816F egfp ws prepred y introduing point muttion using the Site-Direted Mutgenesis Kit (Strtgene). Tndem mcherry (mch mch) ws generted s previously desried 16. HA BDNF Flg ws gift from A. West. The oding sequene for SEP (otined from SEP-GluA1; ref. 31) ws inorported onto the 3 end of HA BDNF Flg to generte HA BDNF Flg-SEP. HA BDNF Flg mrfp1 ws generted in similr fshion. A plsmid ontining mch-ires-tetx ws gift from M. Ehlers. POMC-mCh ws generted y mplifying the POMC peptide (MWCLESSQCQDLTTESNLLACIRACRLDL) 27 using overhng PCR with C-terminl linker (GGGGGGGGGGGGGGG GGGGGGGGGMADQLTEEWHRGTAGPGS). This mplion ws then inserted into the tndem mch plsmid y repling the oding sequene of the first mch. Animls. All niml proedures were pproved y the Duke University Shool of Mediine Animl Cre nd Use Committee, Mx Plnk Florid Institute for Neurosiene, nd Weill Cornell Medil College Institutionl Animl Cre nd Use Committees nd were onduted in ordne with the NIH Guide for the Cre nd Use of Lortory Animls. We used oth mle nd femle rts nd mie. Rts nd C57/B6 mie were otined from Chrles River, Trk F616A mutnt mie were provided y D. Ginty 28, Bdnf fl/fl nd Trk fl/fl mie were provided y L. Prd 32, nd Bdnf-HA mie were generted s previously desried 26. The genotype of eh niml used ws verified efore nd fter prepring slies using PCR of genomi DNA isolted from til DNA efore nd slie smples fter. Preprtion of HeL ells. HeL ells were otined from the Duke University Cell Culture Fility. These ells hd een uthentited using short-tndem repet profiling nd evluted for myoplsm ontmintion. Cells were ultured nd mintined s previously desried 16. Cells were trnsfeted with Lipofetmine 2 using the mnufturer s protool (Invitrogen). Conentrtions used were.5 μl ml 1 Lipofetmine nd 1 μg ml 1 totl DNA (1:1 rtio of TrkB egfp to mrfp1 PLC mrfp1 DNA). Then, h lter, ulture medi ws repled with HEPES-uffered ACSF for imging (HACSF; 2 mm HEPES, 13 mm NCl, 2 mm NHCO 3, 25 mm d-gluose, 2.5 mm KCl nd 1.25 mm NH 2 PO 4 ; djusted to ph 7.4 nd 31 mosm). After 3-min equilirtion period, trnsfeted ells were imged using 2pFLIM s desried elow. Cell stimultion ws performed y diretly dding BDNF or vehile to the HACSF thing the ells. Preprtion of mixed ortil ultures. Mixed ortil ultures were prepred s desried previously 33 nd trnsfeted with Lipofetmine 2 using modified protool. For trnsfetion of neurons in 3.5 m dishes, 1 μl Lipofetmine ws mixed with 1 μg of plsmid DNA (1 μg per onstrut trnsfeted) in 1 μl of ulture medi for 2 min. Culture medi ws removed from the 3.5 m dish until only 1 ml remined. The Lipofetmine/DNA solution ws dded to the neurons for 45 min. At this point, ll the medi ws removed nd repled with 2 ml onditioned ulture medi. After h, ulture medi ws repled with HACSF. To stimulte ells, we dded BDNF or NGF diretly to the HACSF thing the ells. 3 min fter stimultion, we dded K252 to the HACSF. Preprtion of orgnotypi hippompl slies. Cultured hippompl slies were prepred from post-ntl dy 5 7 rts or mie, s previously desried 34, in ordne with the niml re nd use guidelines of Duke University Medil Center. After 5 12 dys in ulture, CA1 pyrmidl neurons were trnsfeted with iolisti gene trnsfer using gold eds (12 mg; Biord) oted with plsmids ontining 2 4 μg of totl DNA (TrkB sensor: 15 μg TrkB egfp nd 15 μg mrfp1 PLC mrfp1; TrkB sensor plus mch: 5 μg TrkB egfp, 5 μg mrfp1 PLC mrfp1, nd 2 μg mch mch; TrkB sensor plus mch nd Cre: 5 μg TrkB GFP, 5 μg mrfp1 PLC mrfp1, 5 μg tdtom-cre, nd 15 μg mch mch; BDNF SEP plus mch: 2 μg BDNF SEP nd 1 μg mch mch; BDNF SEP plus TeTX: 2 μg BDNF SEP nd 1 μg mch-ires-tetx; BDNF SEP plus POMC: 2 μg BDNF SEP nd 1 μg POMC mch; egfp: 2 μg egfp; nd egfp plus Cre: 1 μg egfp plus 1 μg tdtom-cre). Neurons expressing the TrkB sensor were imged h fter trnsfetion. Neurons expressing the TrkB sensor with mch or mch plus Cre were imged 5 7 dys fter trnsfetion. The ddition of the mch proved ritil in limiting the TrkB sensor expression therey llowing neurons to survive longer with the sensor present. Neurons expressing only egfp were imged 1 7 dys fter trnsfetion. Neurons expressing egfp plus Cre were imged 5 9 dys fter trnsfetion. 2pFLIM. FRET imging using ustom-uilt two-photon fluoresene lifetime imging mirosope ws performed s previously desried 13,35. Two-photon imging ws performed using Ti-spphire lser (MiTi, Spetrphysis) tuned to wvelength of 92 nm, llowing simultneous exittion of egfp, mrfp1 nd mch. All smples were imged using <2 mw lser power mesured t the ojetive. Fluoresene emission ws olleted using n immersion ojetive (6, numeril perture.9, Olympus), divided with dihroi mirror (565 nm), nd deteted with two seprte photoeletron multiplier tues (PMTs) pled downstrem of two wvelength filters (Chrom, HQ51-2p to selet for green nd HQ62/9-2p to selet for red). The green hnnel ws fitted with PMT hving low trnsfer time spred (H7422-4p; Hmmtsu) to llow for fluoresene lifetime imging, while the red hnnel ws fitted with wide-perture PMT (R3896; Hmmtsu). Photon ounting for fluoresene lifetime imging ws performed using time-orrelted single photon ounting ord (SPC-15; Beker nd Hikl) ontrolled with ustom softwre 13, while the red hnnel signl ws quired using seprte dt quisition ord (PCI-611) ontrolled with Snimge softwre 36. Two-photon glutmte unging. A seond Ti-spphire lser tuned t wvelength of 72 nm ws used to unge 4-methoxy-7-nitroindolinyl-ged-lglutmte (MNI-ged glutmte) in extrellulr solution with trin of 4 6 ms, 4 5 mw pulses (3 times t.5 Hz) ner spine of interest. Experiments were performed in Mg 2+ free rtifiil ererl spinl fluid (ACSF; 127 mm NCl, 2.5 mm KCl, 4 mm CCl 2, 25 mm NHCO 3, 1.25 mm NH 2 PO 4 nd 25 mm gluose) ontining 1 μm tetrodotoxin (TTX) nd 4 mm MNI-ged l-glutmte erted with 95% O 2 nd 5% CO 2. Experiments were performed t C (room temperture) or 3 32 C using heting lok holding the ACSF ontiner. Temperture mesurements were mde from ACSF within the perfusion hmer holding the slie. 2pFLIM dt nlyses. To mesure the frtion of donor ound to eptor, we fit fluoresene lifetime urve summing ll pixels over whole imge with doule exponentil funtion onvolved with the Gussin pulse response funtion: Ft () = F [ P Htt (,, τ, τ ) + P Htt (,, τ, τ )] D D G AD AD G in whih τ AD is the fluoresene lifetime of donor ound with eptor, P D nd P AD re the frtion of free donor nd donor ound with eptor, respetively, nd H(t) is fluoresene lifetime urve with single exponentil funtion onvolved with the Gussin pulse response funtion: 1 τ Htt (,, τ, τ ) = 2 exp G 2 D G 2τ D 2 t τ τ t G 2 D( t t) erf τ D 2 ττ DG in whih τ D is the fluoresene lifetime of the free donor, τ G is the width of the Gussin pulse response funtion, F is the pek fluoresene efore onvolution nd t is the time offset, nd erf is the error funtion. We fixed τ D to the fluoresene lifetime otined from free megfp (2.6 ns). To generte the fluoresene lifetime imge, we lulted the men photon rrivl time, t, in eh pixel s: t = tftdt () / Ftdt () then, the men photon rrivl time is relted to the men fluoresene lifetime, τ, y n offset rrivl time, t, whih is otined y fitting the whole imge: τ = t t For smll regions-of-interest (ROIs) in n imge (spines or dendrites), we lulted the inding frtion (P AD ) s: P = τ ( τ τ )( τ τ ) ( τ + τ τ ) 1 (3) AD D D D AD 1 D AD BDNF SEP nd BDNF mrfp1 imging. BDNF SEP imging ws performed y interleving 8 Hz two-photon imging with two-photon glutmte unging (3 pulses t.5 Hz). Multiple (1 3) spines were imged on eh neuron. Chnge in BDNF SEP fluoresene ws mesured s ΔF/F fter sutrting kground fluoresene. -triggered verges were lulted s the verge inrese in SEP fluoresene fter eh individul unging pulse. Red fluoresene inrese ws smoothed using 16-frme window. 216 Mmilln Pulishers Limited, prt of Springer Nture. All rights reserved.

7 Letter RESEARCH For visulizing BDNF mrfp1 loliztion in CA1 pyrmidl neurons, imges were otined using Lei SP5 lser snning onfol mirosope (Lei). Spine volume nlysis. During 2pFLIM nd BDNF SEP imging (Figs 2, 3), spine volume ws reported using the red fluoresent intensity from mrfp1 or mch. For two-photon imging without FLIM (Fig. 4), green fluoresent intensity from egfp ws used. In ll experiments, spine volume ws mesured s the integrted fluoresent intensity fter sutrting kground (F). Spine volume hnge ws lulted y F/F, in whih F is the verge spine volume efore stimultion. Additionlly, to ompre sl spine size/morphology etween vrious onditions, mximl spine (F mx(spine) ) nd dendrite (F mx(dendrite) ) fluoresent intensities were mesured nd the F mx(spine) /F mx(dendrite) rtio ws lulted fter sutrting kground fluoresene. In utero virl injetion for single-ell BDNF knokout. E14.5/15.5 timed-pregnnt Bdnf fl/fl mie were deeply nesthetized using n isoflurne oxygen mixture. The uterine horns were exposed nd pproximtely 1 2 μl of AAV solution mix (ontining AAV1.CAG.EGFP, AAV1.CAG.Flex.tdTomto nd AAV1.hSyn.Cre, ll from U Penn vetor ore) ws injeted through pulled-glss pillry tue into the right lterl ventrile of eh emryo. To hieve suffiient lelling of egfp CA1 neurons longside sprse expression of BDNF knokout tdtomto neurons, egfp nd Flex-tdTomto viruses were used t onentrtion of ~1 12 virl genome opies per μl, nd Cre ws diluted (~1-fold) in PBS t dilution determined to hieve sprse lelling density of Cre-positive CA1 neurons. Funtionl LTP. LTP experiments in Fig. 5 nd Extended Dt Fig. 1 were performed in Mx Plnk Florid Institute (MPFI) nd Duke University, respetively. Mie (wild type, Trk F616A, or Bdnf fl/fl ge dys) were sedted y isoflurne inhltion, nd the rin ws removed nd disseted in hilled utting solution (124 mm holine hloride, 2.5 mm KCl, 26 mm NHCO 3, 3.3 mm MgCl 2, 1.2 mm NH 2 PO 4, 1 mm d-gluose nd.5 mm CCl 2 : MPFI or 11 mm surose, 6 mm NCl, 3 mm KCl, 1.25 mm NH 2 PO 4, 28 mm NHCO 3,.5 mm CCl 2, 7. mm MgCl 2, nd 5 mm d-gluose. The solutions were sturted with 95% O 2 plus 5% CO 2, ph 7.4) 37. Coronl slies (25 μm: MPFI) or trnsverse hippompl slies (4 μm: Duke) were prepred nd mintined in oxygented ACSF (MPFI/Duke: 127/124 mm NCl, 2.5/1.75 mm KCl, 1/11 mm d-gluose, 26/25 mm NHCO 3, 1.25/ mm NH 2 PO 4, /1.25 mm KH 2 PO 4, 1.3/2. MgCl 2 nd 2.4/2. CCl 2,) in sumerged hmer t C for t lest 1 h efore use. Eletrophysiologil reordings were performed in ACSF (plus pirotoxin t MPFI). CA1 pyrmidl neurons in ute hippompl slies from wild-type nd Trk F616A mie were visulized using olique illumintion or differentil interferene ontrst (DIC). For Bdnf fl/fl experiments, Cre-negtive (egfp-expressing) nd Cre-positive (tdtomto-expressing) neurons were identified nd trgeted with fluoresene mirosopy. Pth pipettes (3 6 MΩ) were filled with n internl solution (13 mm K gluonte, 1 mm, N phosphoretine 4 mm MgCl 2,4 mm NATP,.3 mm MgGTP, 3 mm l-sori id nd 1 mm HEPES, ph 7.4, nd 31 mosm t MPFI or K-gluonte 14 mm, HEPES 1 mm, EGTA 1 mm, NCl 4 mm, Mg 2 ATP 4 mm, nd Mg 2 GTP.3 mm, ph 7.25, nd 29 mosm t Duke). Series resistnes (1 4 MΩ) nd input resistnes (1 3 MΩ) were monitored throughout the experiment using negtive voltge steps. The memrne potentil ws held t 7 mv. Experiments were performed t room temperture (~21 C) nd slies were perfused with oxygented ACSF. For Trk F616A /wild-type experiments, 1NMMP1 or vehile ws dded to the ACSF efore stimultion. For TrkB-Ig experiments, slies were inuted in 2 μg ml 1 TrkB-Ig or ontrol humn IgG for t lest 2 h efore the experiments. EPSCs were evoked y extrellulr stimultion of Shffer ollterls using onentri ipolr stimulting eletrode (World Preision Instruments) t rte of.3 Hz. LTP ws indued y piring 2-Hz stimultion with postsynpti depolriztion to mv for 15 s (MPFI) or 75 s (Duke). EPSC potentition ws ssessed for 3 45 min (for Trk F616A experiments), 4 6 min (for Bdnf fl/fl experiments) or 2 3 min (for TrkB-Ig experiments) fter stimultion. Immunopreipittion. HeL ells were trnsfeted with the TrkB sensor (TrkB egfp nd mrfp1 PLC mrfp1) using Lipofetmine 2 s desried ove. Then, h fter trnsfetion, the medi thing the ells ws exhnged for HEPES uffered ACSF for iohemistry (15 mm NCl, 3 mm KCl, 1 mm HEPES ph 7.35, 2 mm gluose, nd 31 mosm). After 3-min equilirtion period, ells were stimulted with 1 ng ml 1 BDNF for 1 min. Following stimultion, ells were wshed in ie-old PBS (Gio), nd then lysed in modified RIPA uffer (5 mm Tris-HCl ph 7.4, 15 mm NCl, 1% NP-4,.25% sodium deoxyholte, 1 mm EDTA, 1 mm PMSF, 1 mm N 3 VO 4, nd protese inhiitors) for 1 min on ie. The superntnt ws olleted fter 1 min entrifugtion t 16,g t 4 C. At this point, smll volume of the superntnt ws dded to SDS-smple uffer nd sved s the ell lyste smple. The remining superntnt ws pre-lered using protein G Sephrose eds (25 μl, Rohe) for 3 min t 4 C. After pre-lering, the superntnt ws inuted with 2 μg mouse monolonl nti-phosphotyrosine (BD Trnsdution Ls) t 4 C overnight. The immunoomplexes were preipitted with protein G Sephrose eds (5 μl) for 3 h t 4 C nd then nlysed with western lotting. Antiodies used in western lotting inluded TrkB (Millipore), GFP (Am), tin (Sigm), nd ptrkb(y515) (Sigm). Eletron mirosopi immunohistohemistry. Mle dult (~2 3 months old) Bdnf-HA knok-in mie 26 nd ged mthed wild-type C57/BL mie were used. The sme investigtor (T.A.M.) perfused ll mie (Bdnf-HA nd wild type) to mintin onsisteny etween groups. Mie (3 per group) were deeply nesthetized with sodium pentoritl (15 mg kg 1, i.p.) nd perfused sequentilly through the sending ort with: (1) ~5 ml sline (.9%) ontining 2% heprin, nd (2) 3 ml of 3.75% rolein nd 2% prformldehyde in.1 M phosphte uffer (PB; ph 7.4) 38. Following removl from the skull, the rin ws post-fixed for in 2% rolein nd 2% prformldehyde in PB 3 min. Brins were then setioned (4 μm thik) on Virtome nd stored t 2 C in ryoprotetnt until use. For eh niml, two dorsl hippompl setions were proessed for immunoeletron mirosopy (immunoem) experiments using previously desried methods 38. Before immunohistohemil proessing, setions were rinsed in PB, nd experimentl groups were oded with hole-punhes so tht tissue ould e run in single ruiles, ensuring identil exposure to ll regents. Before proessing for immunolelling, setions were treted with 1% sodium orohydride for 3 min to remove free ldehyde sites. Setions then were rinsed in PB followed y rinse in.1 M Tris-sline (TS; ph 7.6) nd then 3 min inution in.5% BSA in TS. Setions then were inuted in primry rit nti-ha (1:1,; Sigm) in.25% Triton-X 1 nd.1% BSA in TS for 1 dy t room temperture nd 4 dys t 4 C. Setions then were inuted in donkey nti-rit iotinylted IgG (1:4; Jkson Immunoreserh Lortories) for 3 min followed y 3 min inution in vidin-iotin omplex (ABC; Vetstin Elite Kit, Vetor Lortories) in TS (1:1 dilution). Setions were developed in 3,3 -diminoenzidine (Sigm-Aldrih) nd H 2 O 2 in TS. All ntiody inutions were performed in.1% BSA/TS nd seprted y wshes in TS. Setions were post-fixed in 2% osmium tetroxide for 1 h, dehydrted, nd flt emedded in Emed-812 (EMS) etween two sheets of Alr plsti. Brin setions ontining the CA1 nd dentte gyrus were seleted from the plsti emedded setions, glued onto Epon loks nd trimmed to 1 mm-wide trpezoids. Ultr-thin setions (7 nm thikness) through the tissue-plsti interfe were ut with dimond knife (EMS) on Lei EM UC6 ultrtome, nd setions were olleted on 4-mesh, thin-r opper grids (EMS). Grids were then ounterstined with urnyl ette nd Reynold s led itrte. Ultrstruturl nlysis. An investigtor linded to niml ondition performed the dt olletion nd nlysis. One setion from eh of Bdnf-HA nd wild-type nimls ws nlysed (n = 3 eh group). The thin setions were exmined nd photogrphed on Teni Biotwin trnsmission eletron mirosope (FEI). Cell profiles were identified y defined morphologil riteri 39. Dendriti profiles generlly were postsynpti to xon terminls nd ontined regulr mirotuule rrys. Dendriti spines lso were usully postsynpti to xon terminl profiles nd sometimes ontined spine pprtus. Axon terminls ontined smll synpti vesiles nd osionl dense-ore vesiles. Unmyelinted xons were profiles smller thn.15 μm tht ontined few smll synpti vesiles nd lked synpti juntion in the plne of setion. Glil profiles were distinguished y the presene of glil filments (stroyti profiles), y the presene of mirotuules nd/or their tendeny to onform irregulrly to the oundries of surrounding profiles. Unknown profiles were those tht ontined immunoperoxidse retion produt ut ould not e definitively pled in one of the ove tegories. From eh lok, 4 grid squres (eh squre ws μm 2 ) eh from the CA1 ner strtum rditum (nsr in Fig. 3; tht is, djent to the pyrmidl ell lyer) nd distl strtum rditum (dsr in Fig. 3; tht is, 5 15 μm wy from the pyrmidl ell lyer) were rndomly smpled for nlysis. Thus, 12,1 μm 2 ws smpled for eh re in eh lok. Grid squres were seleted plsti-tissue interfe to ensure even ntiody tissue penetrtion 38. Immunoperoxidse lelling for HA ws evident s hrteristi, eletron-dense DAB retion produt preipitte. All peroxidse lelled profiles from eh squre were photogrphed nd tegorized. Animl odes were not roken until ll 6 loks were nlysed. Sttistil nlysis. Smple sizes for ll experiments were hosen sed on signl-to-noise rtios identified in pilot experiments. Vrines of ll dt sets were estimted nd ompred using Brtlett s or Levene s test efore further sttistil nlysis. Rndomiztion of nimls nd/or slies ws not needed. To evlute distriution ptterns of TrkB sensor tivity, spine volume hnge, nd BDNF SEP signl, pek responses for eh dt set (the sme points used for sttistil omprisons) were sujeted to Shpiro Wilk test for normlity. TrkB sensor tivity dhered to the null hypothesis (norml distriution) while spine volume hnge nd BDNF SEP signl did not. Beuse TrkB sensor tivity hd norml distriution, prmetri sttistis were used: pired nd unpired two-tiled t-test, ANOVA, nd repeted-mesures ANOVA with pproprite post-ho nlysis, s indited in the figure legends nd supplementry note. For t-tests, homosedstiity etween groups ws evluted 216 Mmilln Pulishers Limited, prt of Springer Nture. All rights reserved.

8 RESEARCH Letter using the F-test. If vrine ws unequl, Welh s orreted t-test ws performed. For ANOVA, homosedstiity ws evluted with Brtlett s test. For multiple omprisons of sensor tivity, dt were sujeted to ANOVA or repeted-mesures ANOVA followed y post-ho test to determine sttistil signifine. In ses where eh ondition ws ompred to ll other onditions in the experiment, the Tukey Krmer method ws employed. In ses where eh ondition ws ompred to single ontrol, Dunnet s test ws used. Sine spine volume hnge hd non-norml distriution, dt were log-trnsformed to resolve skewness nd then nlysed with prmetri sttistis (the sme tests desried ove), s indited in the figure legends. For the BDNF SEP signl, log-trnsformtion of the dt did not resolve the skewness. As suh, non-prmetri sttistis were used Wiloxon rnk-sum test nd Kruskl Wllis test with followed y Dunn s test. Dt were only exluded if ovious signs of poor ellulr helth (dendriti leing, spine ollpse, et.) were pprent. 29. Hung, Y. Z. & MNmr, J. O. Mutul regultion of Sr fmily kinses nd the neurotrophin reeptor TrkB. J. Biol. Chem. 285, (21). 3. Zhris, D. A., Violin, J. D., Newton, A. C. & Tsien, R. Y. Prtitioning of lipid-modified monomeri GFPs into memrne mirodomins of live ells. Siene 296, (22). 31. Ptterson, M. A., Sztmri, E. M. & Ysud, R. AMPA reeptors re exoytosed in stimulted spines nd djent dendrites in Rs-ERK-dependent mnner during long-term potentition. Pro. Ntl Ad. Si. USA 17, (21). 32. He, X.-P. P. et l. Conditionl deletion of TrkB ut not BDNF prevents epileptogenesis in the kindling model. Neuron 43, (24). 33. Xiong, Z. Q. & MNmr, J. O. Fleeting tivtion of ionotropi glutmte reeptors sensitizes ortil neurons to omplement ttk. Neuron 36, (22). 34. Stoppini, L., Buhs, P. A. & Muller, D. A simple method for orgnotypi ultures of nervous tissue. J. Neurosi. Methods 37, (1991). 35. Murkoshi, H., Lee, S.-J. J. & Ysud, R. Highly sensitive nd quntittive FRET-FLIM imging in single dendriti spines using improved non-rditive YFP. Brin Cell Biol. 36, (28). 36. Pologruto, T. A., Stini, B. L. & Svood, K. SnImge: flexile softwre for operting lser snning mirosopes. Biomed. Eng. Online 2, 13 (23). 37. Pn, E. et l. Vesiulr zin promotes presynpti nd inhiits postsynpti long-term potentition of mossy fier-ca3 synpse. Neuron 71, (211). 38. Milner, T. A., Wters, E. M., Roinson, D. C. & Piere, J. P. in Neurodegenertion, Methods nd Proedures (eds Mnfredi, G. & Kwmt, H.) (Spring, 211). 39. Peters, A., Ply, S. L. & Wester, H. D. The Fine Struture of the Nervous System 3rd edn (Oxford Univ. Press, 1991). 216 Mmilln Pulishers Limited, prt of Springer Nture. All rights reserved.

9 Letter RESEARCH BDNF HeL Cells Before BDNF IP: ptyr IB: TrkB IB: GFP TrkB 2.15 Cell Lystes IB: TrkB IB: GFP IB: Atin TrkB(Y816F) ns 2.75 e.8.4 Before BDNF or Vehile d Mixed Cortil Cultures min BDNF Vehile Y816F BDNF 1 min 5 min 15 min 3 min K ns f BDNF or NGF K252 g min Post- K BDNF NGF Extended Dt Figure 1 Design nd development of FRET-sed sensor for TrkB tivtion., Top, western lot nlysis of ell extrts from HeL ells stimulted with either BDNF or vehile. Extrts were immunopreipitted with n ntiody for phosphorylted tyrosine residues (ptyr) nd then proed with ntiodies for TrkB nd GFP. Bottom, immunolot (IB) of BDNF nd vehile stimulted ell extrts efore immunopreipittion (IP) using ntiodies for TrkB, GFP nd tin. For soure dt, see Supplementry Fig. 1., FLIM imges of TrkB nd TrkB Y816F tivtion quired efore nd 2 6 min fter BDNF stimultion (verged multiple imges tken over 5 min). Wrmer olours indite shorter lifetimes nd higher TrkB tivity., Time ourse of TrkB nd TrkB Y816F tivtion mesured s the hnge in inding frtion of TrkB egfp or TrkB Y816F egfp ound to mrfp1 PLC mrfp1 efore nd fter BDNF or vehile stimultion. n = 22/8 TrkB plus BDNF, 9/4 TrkB plus vehile, nd 11/4 TrkB Y816F plus BDNF (ells/experiments). d, TrkB tivtion (verged over 6 1 min) for experiments in. e, FLIM imges of TrkB tivtion in neuron in mixed ortil dissoited ulture efore nd fter BDNF stimultion followed y K252 pplition t 3 min. f, Time ourse of TrkB tivtion mesured s desried in efore nd fter BDNF or NGF stimultion followed y K252 pplition. n = 8 BDNF nd 4 NGF (neurons). g, TrkB tivtion (verged over 1 3 min nd 3 5 min following K252 pplition) for experiments in f. Dt re men ± s.e.m. P <.5 s determined y two-tiled unpired smples t-test (g) or n nlysis of vrine (ANOVA) followed y Tukey s method to orret for multiple omprisons. (d). P <.5 s determined y two-tiled pired smples t-test. 216 Mmilln Pulishers Limited, prt of Springer Nture. All rights reserved.

10 RESEARCH Letter 4 2 TrkB fl/fl Cre Neg Cre Pos Cre Pos + TrkB-EGFP Sustined Cre Neg Cre Pos Cre Pos + TrkB-EGFP Extended Dt Figure 2 Resue of sltp with TrkB egfp following postsynpti TrkB knokout.,, Time ourse () nd quntifition () of glutmte-unging-indued spine volume hnge for Trk fl/fl hippompl slies trnsfeted with egfp (Cre Neg), egfp plus Cre (Cre Pos), nd mch, TrkB egfp nd Cre (Cre Pos + TrkB egfp). n = 7/2 Cre Neg, 9/24 Cre Pos, nd 5/11 Cre Pos + TrkB egfp (ells/spines). Dt re men ± s.e.m. P <.5 s determined y n ANOVA followed y Tukey s method to orret for multiple omprisons. 216 Mmilln Pulishers Limited, prt of Springer Nture. All rights reserved.

11 Letter RESEARCH d TrkB sensor GFP Anisomyin Extended Dt Figure 3 Chrteriztion of prolonged TrkB tivtion nd spine volume hnge during single spine sltp., Prolonged time ourse of spine volume hnge fter two-photon glutmte unging in rt hippompl slies trnsfeted with the TrkB sensor or egfp. n = 5/54 for TrkB sensor (9/1 for experiments longer thn 2 min) nd 8/8 for egfp (ells/spines)., Prolonged time ourse of TrkB tivtion in stimulted spines, the se of the spine nek, djent Trnsient Aniso Sustined Aniso Stim Spine Spine Bse Adj Spine Dendrite spines, nd the dendriti shft djent to the stimulted spine. n = 5 ells with 54 stimulted spine, spine se, nd dendrite plus 59 djent spine., d, Time ourse () nd quntifition (d) of the trnsient (verged over 1 2 min) nd sustined (verged over 2 4 min) phses of glutmte unging-indued spine volume hnge in rt hippompl slies in the sene nd presene of nisomyin (25 μm). n = 12/14 ontrol nd 5/5 nisomyin (ells/spines). Dt re men ± s.e.m. 216 Mmilln Pulishers Limited, prt of Springer Nture. All rights reserved.

12 RESEARCH Letter Normlized Chnge TrkB Ativity Spine Volume Normlized Chnge BDNF Signl TrkB Ativity Spine Volume Extended Dt Figure 4 Comprison of temporl dynmis of BDNF relese, TrkB tivtion, nd spine volume hnge during single spine sltp., Time ourse of normlized hnges in TrkB tivity nd spine Time(s) volume hnge (perentge of mximl tivity nd volume hnge)., Mgnified view of normlized hnges of BDNF relese, TrkB tivtion, nd spine volume during nd 1 min fter the unging epoh. 216 Mmilln Pulishers Limited, prt of Springer Nture. All rights reserved.

13 Letter RESEARCH d Rt K252 Rt Y816F Pek K Rt K e TrkB F616A f g h TrkB F616A Pek Sustined Pek Sustined Sustined i j k l Rt Pek Sustined Pek Sustined.8 Y816F Y816F Pek K252 Y816F K252 Y816F Sustined K252 Y816F Extended Dt Figure 5 Determintion of the speifiity of glutmte unging evoked TrkB tivtion., Time ourse of TrkB tivtion following glutmte unging efore nd t lest 3 min fter K252 pplition to the perfusion th. n = 41/45 nd 4/9 K252 (ells/ spines)., Pek (verged over 1 2 min) nd sustined (verged over 1 2 min) TrkB tivtion for experiments in., Time ourse of spine volume hnge for experiments in. d, Trnsient nd sustined spine volume hnge for experiments in. e h, Similr experiments to d ut in Trk F616A hippompl slies trnsfeted with the TrkB F616A sensor efore nd t lest 3 min fter pplition (2 μm). n = 4/5 ontrol nd 3/6 (ells/spines). i l, Similr experiments to d ut with the TrkB nd TrkB Y816F sensors. n = 9/1 ontrol nd 7/11 Y816F (ells/spines). Dt re men ± s.e.m. P <.5 s determined y twotiled unpired smples t-test. 216 Mmilln Pulishers Limited, prt of Springer Nture. All rights reserved.

14 RESEARCH Letter Stim spine (RT) Stim spine (3-32ºC) Dendrite (RT) Dendrite (3-32ºC) Extended Dt Figure 6 Effet of temperture on the sptiotemporl dynmis of TrkB tivtion. Time ourse of TrkB tivtion t room temperture (RT; C) nd 3 32 C in the stimulted nd dendrite. n = 19/2 nd 23/25 t C nd 3 32 C, respetively (spines/ells). Dt re men ± s.e.m. 216 Mmilln Pulishers Limited, prt of Springer Nture. All rights reserved.

15 Letter RESEARCH Binding Frtion (Stim Spine) Bsl Binding Frtion R 2 = Conentrtion (μm) d Volume (%) Binding Frtion (Dendrite) R 2 =.4.5 R 2 = R 2 = Conentrtion (μm) Conentrtion (μm) Extended Dt Figure 7 Effets of sensor expression levels on hnges reported y the sensor. d, Effet of TrkB egfp onentrtion s mesured in individul neurons on orresponding hnge in inding frtion of the stimulted spine (), hnge in spine volume (), inding Conentrtion (μm) frtion efore unging (sl inding frtion) (), nd hnge in inding frtion of the dendrite (d). n = 25/28 (ells/spines). Dt re men vlues nd were fit to liner regression model with orresponding oeffiients of determintion (R 2 ) provided for eh. 216 Mmilln Pulishers Limited, prt of Springer Nture. All rights reserved.

16 RESEARCH Letter Fmx spine /Fmx dendrite Cre Neg Cre Pos Cre Neg Cre Pos Δ Lifetime (ns) Bdnf fl/fl Cre Neg Cre Pos d Δ Lifetime (ns) Pek Cre Neg Cre Pos e Bdnf fl/fl 5 Extended Dt Figure 8 Bsl spine size nd CMKII tivtion in the presene nd sene of post- synpti BDNF.,, Quntifition () nd representtive two-photon imges (f) of sl spine size/morphology in Bdnf fl/fl slies trnsfeted with egfp or egfp plus Cre (Cre Neg or Pos). n = 14/5 Cre Neg nd 29/117 Cre Pos (ells/spines). Sle r, 1 μm., d, Time ourse () nd quntifition (verged over 45 s) (d) of CMKII Cre Neg Cre Pos 1 f Trnsient Cre Neg Cre Pos tivtion in Bdnf fl/fl slies trnsfeted with the CMKII sensor or CMKII plus Cre. n = 7/13 Cre Neg nd 7/15 for Cre Pos (ells/spines). e, f, Time ourse nd quntifition of the trnsient phse of spine volume hnge for experiments in. Dt re men ± s.e.m. P <.5 s determined y two-tiled unpired smples t-test. 216 Mmilln Pulishers Limited, prt of Springer Nture. All rights reserved.

17 Letter RESEARCH d HA Pro HA Pro BDNF-mRFP BDNF BDNF megfp FLAG SEP FLAG RFP e Merge C 2+ NMDAR BDNF Quenhed SEP De-quenhed SEP F/F ph 6.5 ph 8. 1 µm 2 µm F Volume BDNF-SEP mch ell fill f ΔF Red /F Red TeTx POMC NBQX + NBQX g ΔF Red /F Red h 6 F/F Extended Dt Figure 9 Design nd vlidtion of BDNF SEP., Shemti of BDNF SEP nd BDNF mrfp1. Pro, mino ids of humn BDNF; BDNF, mino ids of humn BDNF orresponding to the mture hin., Mehnisti model linking hnges in SEP fluoresene with BDNF relese., Chnge in BDNF SEP fluoresene following glutmte unging under ontrol, idi (ph 6.5), nd si (ph 8.) onditions. d, Confol imges of CA1 pyrmidl neuron trnsfeted with egfp nd BDNF mrfp1. Arrowheds indite dendriti spines. e, Prolonged time ourse of BDNF SEP fluoresene 1 TeTx POMC +NBQX NBQX TeTx hnge (left) nd spine volume hnge (right) in response to glutmte unging. n = 11/2 (ells/spines). f, g, Time ourse (f) nd quntifition (g) of spine volume hnge for experiments in Fig. 4, d. n = 31/218 ontrol, 6/82 TeTx, 2/29 POMC, 3/5, 2/46 + NBQX, 4/4 NBQX, nd 7/88 (ells/spines). h, Dt from Fig. 4d presented s medin ± interqurtile rnge. Dt re men ± s.e.m. unless otherwise indited. P <.5 s determined y n ANOVA followed y Dunnet s method to orret for multiple omprisons. P <.5 s determined y Kruskl Wllis test followed y Dunn s test. POMC +NBQX NBQX 216 Mmilln Pulishers Limited, prt of Springer Nture. All rights reserved.