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1 oi:1.138/ntur1844 Rptitiv motor lrning inus oorint formtion of lustr nriti spins in vivo Min Fu 1, Xinzhu Yu 1,JuLu 2 &YiZuo 1 Mny lins of vin suggst tht mmory in th mmmlin brin is stor with istint sptiotmporl pttrns 1,2. Dspit rnt progrsss in intifying nuronl popultions involv in mmory oing 3 5, th synps-lvl mhnism is still poorly unrstoo. Computtionl mols n ltrophysiologil t hv shown tht funtionl lustring of synpss long nriti brnhs ls to nonlinr summtion of synpti inputs n grtly xpns th omputing powr of nurl ntwork 6 1. Howvr, whthr nighbouring synpss r involv in noing similr mmory n how tsk-spifi ortil ntworks vlop uring lrning rmin lusiv. Using trnsrnil two-photon mirosopy 11, w follow pil nrits of lyr 5 pyrmil nurons in th motor ortx whil mi prtis novl forlimb skills. Hr w show tht thir of nw nriti spins (postsynpti struturs of most xittory synpss) form uring th quisition phs of lrning mrg in lustrs, n tht most suh lustrs r nighbouring spin pirs. Ths lustr nw spins r mor likly to prsist throughout prolong lrning sssions, n vn long ftr trining stops, thn non-lustr ountrprts. Morovr, formtion of nw spin lustrs rquirs rptition of th sm motor tsk, n th mrgn of sunt nw spin(s) ompnis th strngthning of th first nw spin in th lustr. W lso show tht unr ontrol onitions nw spins ppr to voi xisting stbl spins, rthr thn bing uniformly long nrits. Howvr, sunt nw spins in lustrs ovrom suh sptil onstrint n form in los viinity to nighbouring stbl spins. Our finings suggst tht lustring of nw synpss long nrits is inu by rptitiv tivtion of th ortil iruitry uring lrning, proviing struturl bsis for sptil oing of motor mmory in th mmmlin brin. Spins r nriti protrusions tht ontin ll th ssntil omponnts for postsynpti signlling n r thus goo initor of synpti onntivity 12,13. Th lustr plstiity mol suggsts tht nighbouring spins tn to trnsmit similr informtion to th postsynpti nuron 6,7. To invstigt th formtion n funtionl signifin of spin lustrs uring lrning, w trin thy1-yfp-h mi 14 with s-rhing tsk 15 n follow th ynmis of spins on pil nrits of lyr 5 (L5) pyrmil nurons in th motor ortx ontrltrl to th trin limb uring iffrnt lrning phss. W foun tht % of nw spins tht form uring th quisition phs of lrning (rly trining, ys 1 4) mrg in lustrs; tht is, two or mor nighbouring nw spins without intrsprs xisting spin(s) (Fig. 1, b). Most suh lustrs (61 ss) ompris two ontiguous nw spins, n th othr two lustrs ompris thr. In ontrst, fwr nw spin lustrs mrg in untrin ontrol mi ovr th sm prio ( %, P,.1) or in trin mi uring th onsolition phs of lrning (lt trining, ys 13 16; %, P,.1; Fig. 1b). In ition to lustring of ontiguous nw spins, w obsrv fw ss in whih two or mor nw spins form in los viinity to h othr, but with up to thr xisting spins intrsprs mong thm, s wll s ss in whih nw filopoi lustr with nw spins (Supplmntry Fig. 1). W inorport ths ss in nothr st of nlyss, in whih lustr ws fin s st of nw spins/filopoi form within 5 mm of h othr, rgrlss of th prsn or bsn of xisting spin(s) btwn thm (Supplmntry Informtion). Ths nlyss gin rvl tht signifintly highr prntg of nw spins lustr uring rly trining, ompr with tht in ontrols or uring lt trining (P,.1 for both ss; Supplmntry Fig. 2). Thy lso show tht filopoi only m minor ontribution to nw protrusion lustrs (Supplmntry Fig. 3). Mor intrstingly, mong th nw spins obsrv t th n of th quisition phs (y 4), lustr nw spins h signifintly highr survivl rt thn non-lustr ons (tht is, iniviul nw spins flnk by two xisting spins) by trining y 16 (P,.1), s wll s 4 months ftr trining stopp (P,.5; Fig. 1). Togthr, our rsults rvl tht motor lrning inus oorint formtion of lustr synpss, whih prsumbly blong to th sm nuronl iruit n prsist ovr tim to no motor informtion. Prftion of motor skill rquirs rpt prti, usully through multipl trining sssions. W thrfor sought to fin out whthr lustr nw spins obsrv on trining y 4 wr form within th sm trining sssion or ross iffrnt sssions. W img th mi thr tims (on th y bfor trining, n ftr 1 n 4 ys of trining), n foun tht mong nw spin lustrs obsrv on trining y 4, only 2.4% wr ompos of spins tht form togthr btwn trining ys n 1. On th othr hn, 43.9% of lustrs wr ompos of spins form btwn ys 1 n 4, n th rmining 53.7% of lustrs onsist of on spin form btwn ys n 1 (th first nw spin) n nothr spin form btwn ys 1 n 4 (th son nw spin). Thus most nw spin lustrs mrg through rurrnt trining sssions. To trmin how th formtion of th son nw spin in lustr orrlts with funtionl hngs of th first nw spin, w tgoriz first nw spins into thr groups bs on thir survivl n nighbouring spin ition: trnsint nw spins (form on trining y 1 but lost by y 4); prsistnt lustr nw spins (form on trining y 1, surviv until y 4, with n jnt nw spin form btwn ys 1 n 4); n prsistnt non-lustr nw spins (form on trining y 1, surviv until y 4, with no jnt nw spin formtion) (Fig. 1). As spin h siz losly orrlts with synpti strngth, w follow h sizs of first nw spins ovr tim. On trining y 1, w foun tht h sizs of both trnsint n prsistnt nw spins wr signifintly smllr thn thos of xisting stbl spins long th sm nrit (P,.1 for both ss, Supplmntry Fig. 4). By trining y 4, h sizs of prsistnt lustr nw spins inrs signifintly (P,.1; Fig. 1 n Supplmntry Fig. 5), whrs h sizs of prsistnt non-lustr nw spins rmin omprbl to y 1 (P..2; Fig. 1f n Supplmntry Fig. 5b). Bus spin h siz is goo proxy for synpti strngth, ths t suggst tht formtion of th son nw spins 1 Dprtmnt of Molulr, Cll n Dvlopmntl Biology, Univrsity of Cliforni, Snt Cruz, Cliforni 9564, USA. 2 Dprtmnt of Biologil Sins n Jms H. Clrk Cntr, Stnfor Univrsity, Stnfor, Cliforni 9435, USA. MONTH 12 VOL NATURE 1

2 RESEARCH LETTER Dy Dys Imging Nw spins Prsistnt lustr b Prsistnt non-lustr Clustr spins/totl nw spins (%) 15.8% 38.3% Trnsint 45.9% Nighbourhoo nlysis Erly Lt Normliz spin h imtr Clustr nw spins (n=23) 1 Nw spin survivl (%) f Normliz spin h imtr Dy Clustr Non-lustr Dy 4 ult Non-lustr nw spins (n=56) Figur 1 Aquisition of novl motor skill inus formtion of spin lustrs., Rpt imging of th sm nriti brnh uring motor lrning rvls tht son nw spin tht form btwn ys 1 n 4 (r rrowh) is lot nxt to stbiliz nw spin tht h form on y 1 (blu rrowh). Sl br, 1 mm. b, A highr prntg of nw spins form in lustrs ovr 4 ys uring rly trining (n 5 18 mi), ompr with ontrol (n 5 7) n lt trining (n 5 4)., Clustr nw spins obsrv on trining y 4 hv highr survivl rt thn non-lustr ountrprts by th n of th 16- y trining (n 5 6), s wll s 4 months ftr trining stops (n 5 4)., Nw spins form on trining y 1 r lssifi oring to thir ft n nighbouring spin formtion., Spin h sizs of prsistnt lustr nw spins inrs btwn trining ys 1 n 4. f, Spin h sizs of prsistnt non-lustr nw spins show no hng btwn trining ys 1 n 4. Spin h siz is quntifi by th normliz spin h imtr, fin s th rtio of th spin h imtr to th jnt nriti shft imtr. P,.5, P,.1. Error brs, s..m. Dy.5.5 ompnis synpti potntition t first nw spins uring motor lrning. It is onsistnt with prvious rports tht long-trm potntition t singl spin n filitt formtion n potntition of nighbouring spins 16,17. Diffrnt sts of synpss hv bn shown to b involv in iffrnt motor tsks 15. W thrfor trin th sm mi squntilly with two motor skills (ross-trining) to trmin if spins inu by iffrnt motor tsks lustr. strt with th rhing tsk on y 1 n thn swith to th pllini-hnling tsk, whih lso rquirs forlimb oorintion, on ys 2 4 (Fig. 2 n Supplmntry Tbl 1). W foun tht % nw spins form uring th pllini-hnling tsk btwn ys 1 n 4, mong whih % ourr in lustrs (Fig. 2b ). Both th spin formtion rt n th prntg of lustr nw spins wr omprbl to thos in mi ontinuously trin with th rhing tsk (rh-only) (P..5 in both ss), n wr signifintly highr thn thos in ontrol mi ovr th sm prio of tim (P,.1 in both ss, Fig. 2, ). Thus, th pllini-hnling tsk itslf n inu lustr spin formtion. Howvr, only % of pllini-hnling-inu nw spins lustr with rhinginu nw spins in ross-trining. This ontrsts with th outom of rh-only trining ( %, P,.1, Fig. 2), suggsting tht nw spins inu by iffrnt tsks hv low inin of lustring with h othr. To hrtriz furthr th tsk-spifi ntur of lustr spin formtion, w hous nimls in motor nrih nvironmnt with ily hng of motor hllngs (Fig. 2; s Mthos). lso robustly nhn spinognsis: % nw spins form btwn ys 1 n 4, omprbl to th prntgs unr rh-only n ross-trining onitions (P..1 for both ss). Howvr, only % of ths nw spins ppr in lustrs, prntg omprbl to ontrols (P..2, Fig. 2) but signifintly lowr thn tht unr rh-only or ross-trining onitions (P,.1 for both ss). Togthr ths t init tht, whrs novlty in lrning stimults spinognsis, rptitiv tivtion of th sm ortil iruit is ruil in lustr spin formtion (Supplmntry Fig. 6). Th phnomnon of lrning-inu, oorint spinognsis l us to invstigt furthr th sptil istribution of nw spins. W first xmin th istn btwn nw spin (n) n its nrst xisting spin (s) (D n s, Fig. 3) in ontrol mi. W thn simult D n s istribution unr th null hypothsis tht nw spins form uniformly n inpnntly long th nrit (s Mthos). Compr with simultion rsults, th min of msur vlus of D n s ws signifintly lrgr (Fig. 3b), n th umultiv probbility istribution of msur vlus of D n s ws shift towrs longr istns (Fig. 3). Ths rsults suggst tht nw spins r not rnomly isprs long nriti sgmnts, n thir pprnt voin of xisting stbl spins unr ontrol onitions is onsistnt with th i tht nighbouring spins shr n ompt for lol rsours To trmin if motor lrning ltrs th sptil istribution of nw spins, w xmin vlus of D n s in mi trin with th rhing tsk. W foun tht th istn btwn nw spin form on trining y 1 (n 1 ) n th nrst xisting spin (D n1 s ) ws omprbl for trin n ontrol mi (P..7). W lssifi nw spins form btwn trining ys 1 n 4 (n 2 ) into two tgoris: lustr n 2 (tht is, thos tht form nxt to stbiliz first nw spin; Fig. 3) n nonlustr n 2 (thos tht i not form nxt to stbiliz first nw spin). W foun tht lustr n 2 wr signifintly losr to thir nrst stbl spins (n 1 or stbl spin xisting sin y ; Fig. 3) (D n2 s, lustr ) thn wr n 1 (D n1 s )(P,.5). In ontrst, th istn btwn non-lustr n 2 n its nrst stbl spin (D n2 s, non-lustr )ws omprbl to D n1 s (P..9, Fig. 3). In ition, whn n n 1 form btwn two jnt stbl spins, th istn btwn th two stbl spins (D s n1 s ) ws omprbl for ontrol n trin mi (P..7; Supplmntry Fig. 7). Howvr, th istn btwn stbiliz n 1 n th jnt stbl spin, btwn whih lustr n 2 form (D n1 n2 s ), ws signifintly smllr thn th istn btwn two jnt stbl spins, btwn whih non-lustr n 2 form (D s n2 s ; P,.1; Supplmntry Fig. 7). Ths rsults suggst tht lrning-inu lustr nw spins n ovrom th sptil onstrint of xisting spins n b pk into tightr nriti sp. Rnt stuis hv shown tht nriti spins r ynmi in th living brin, n tht rrrngmnt of ortil onntions through novo growth n loss of spins provis struturl substrt for xprin-pnnt plstiity Built upon ths works, our stuy rvls novl sptil rul of spinognsis uring motor lrning. W foun tht lrning-inu nw spins tn to form in smll lustrs (mostly pirs). Th orrltion btwn th mrgn of th son 2 NATURE VOL MONTH 12

3 RESEARCH Dys b Dy Imging Spin formtion btwn ys 1 n 4 (%) Clustr/totl spin formtion btwn ys 1 n 4 (%) Dys 1 4 nw spins lustr with ys 1 nw spins (%) 1 5 Figur 2 Clustr nw spins form ovr multipl trining sssions of th sm, but not iffrnt, motor tsks., Timlins of rh-only, rosstrining n motor nrihmnt xprimnts. b, Rpt imging of th sm nriti brnh rvl tht two nighbouring nw spins (rrowhs) form btwn ys 1 n 4 uring ross-trining. Sl br, 1 mm., Highr prntgs of nw spins form btwn ys 1 n 4 in rh-only, rosstrining n motor nrihmnt, ompr with ontrols., Highr prntgs of nw spins form in lustrs btwn ys 1 n 4 in rh-only n rosstrining, ompr with ontrols., A highr prntg of nw spins tht form btwn ys 1 n 4 lustr with nw spins tht h form btwn ys n 1 in th rh-only onition, ompr with ontrols. Numbr of mi xmin: six ontrol, nin rh-only, fiv ross-trining n six motor nrihmnt. P,.1, P,.1. Error brs, s..m. b D n s D n s (μm) Cumultiv probbility Simultion Msurmnt D n s (μm) 1.5 D n1 s D n2 s D n2 s Distn (μm) 1..5 (53) (147) (144) (23) Figur 3 Th sptil istribution of nw spins long nrits., Shmti illustrting th msurmnt of D n s. b, Th min of msur vlus of D n s (r irl) is signifintly lrgr thn tht of simult vlus of D n s (box plot of rsults from 1, simultions, with whiskrs rprsnting th minimum n th mximum) in ontrol mi. Th simultion is bs on th null hypothsis tht nw spins r inpnntly n uniformly long linr nrit., Cumultiv probbility istribution of msur D n s is shift towrs longr istns thn th simult D n s in ontrol mi. or. D n1 s D n1 s D n2 s D n2 s (non-lustr) (lustr), Shmti illustrting th msurmnt of D n2 s, lustr. Th nrst spin to lustr n 2 oul b ithr prsistnt first nw spin (n 1 ) or stbl spin xisting sin y, pning on rltiv n 2 lotion., D n1 s in ontrol mi is omprbl to tht of trin mi. In trin mi, D n2 s, lustr is signifintly smllr thn D n1 s, whrs D n2 s, non-lustr is omprbl to D n1 s. Th numbr of spins nlys in h onition is init on h olumn. P,.5. Error brs, s..m. MONTH 12 VOL NATURE 3

4 RESEARCH LETTER nw spin n th strngthning of th first nw spin lso suggsts thir potntil prtiiption in th sm nuronl iruit. Ths finings support th lustr plstiity mol, whih postults tht synpss lot los togthr long th sm nriti brnh r mor likly to b llot for th sm informtion thn synpss isprs throughout th nriti rbor 7. In, in th mous uitory ortx, lthough spins tun for iffrnt frqunis r highly intrsprs, 26% of nighbouring spins xhibit similr fftiv frqunis, muh mor frquntly thn ntiipt from rnom istribution (1%) 26. Thrfor, lthough nurons tn to mximiz thir ovrll onntions 27, lustr plstiity nsurs strngthning of iruit-spifi onntions n nbls sptil oing for tsk-rlt informtion. Prvious ltron mirosopy stuis hv rvl tht nighbouring spins n form synpss with th sm xon 28 3 (s Supplmntry Fig. 8,, ). Positioning multipl synpss btwn pir of nurons in los proximity llows nonlinr summtion of synpti strngth, n potntilly inrss th ynmi rng of synpti trnsmission wll byon wht n b hiv by rnom positioning of th sm numbr of synpss. Altrntivly, lustr nw spins my synps with istint (but prsumbly funtionlly rlt) prsynpti prtnrs (Supplmntry Fig. 8b, ). In this s, thy oul potntilly intgrt inputs from iffrnt nurons nonlinrly n inrs th iruit s omputtionl powr. Distinguishing btwn ths two possibilitis woul probbly rquir iruit ronstrution by ltron mirosopy following in vivo imging to rvl th intitis of prsynpti prtnrs of nwly form spins. Profiling spin formtion uring novl xprins, our t rvl ritil rol of rptitiv tivtion of th sm nuronl iruit. Th ft tht th son nw spin in lustr n ovrom th sptil onstrint impos by xisting spins suggsts tht rptitiv tivtion of nuronl iruit my moify or rllot lol rsours for spinognsis. Suh rsours my b prmissiv or instrutiv molulr us t th pr- or postsynpti sit, or th vilbility of suitbl prtnrs (for xmpl, xonl boutons). Unrstning th ntur n rgultion of suh rsours my hol th ky to luiting th llulr mhnisms of lustr spin formtion. It will b onuiv to th vlopmnt of tools to lbl n mnipult spifi synpti popultions, n ultimtly to th isstion of th usl rltionship btwn synpti ynmis n lrning. METHODS SUMMARY YFP-H lin mi 14 xprssing yllow fluorsnt protin (YFP) in smll subst of ortil nurons wr us in ll th xprimnts. Mi of both sxs wr trin with iffrnt motor-skill tsks or hous in motor-nrih nvironmnt, strting t 1 month of g (s Mthos). Th prours for trnsrnil twophoton imging n quntifition of spin ynmis hv bn srib prviously 11,15. ImgJ ws us to msur spin h siz, s wll s intr-spin istns. Simultion ws prform with ustom-writtn os in Mtlb (MthWorks) n sttistil nlyss wr prform using GrphP Prism 5 (GrphP Softwr) (s Mthos). All t wr prsnt s mn 6 s..m. P vlus wr lult using th Mnn Whitny U-tst for inpnnt smpls, n th Wiloxon sign-rnk tst for pir smpls. Full Mthos n ny ssoit rfrns r vilbl in th onlin vrsion of th ppr t Riv 22 Sptmbr 11; pt 9 Jnury 12. Publish onlin 19 Fbrury Silv, A. J., Zhou, Y., Rogrson, T., Shob, J. & Blji, J. Molulr n llulr pprohs to mmory llotion in nurl iruits. Sin 326, (9). 2. Aimon, J. B., Wils, J. & Gg, F. H. Potntil rol for ult nurognsis in th noing of tim in nw mmoris. Ntur Nurosi. 9, (6). 3. Hn, J. H. t l. Sltiv rsur of fr mmory. Sin 323, (9). 4. Komiym, T. t l. Lrning-rlt fin-sl spifiity img in motor ortx iruits of bhving mi. Ntur 464, (1). 5. K, N., Tixir, C. M., Wng, A. H. & Frnkln, P. W. Prfrntil inorportion of ult-gnrt grnul lls into sptil mmory ntworks in th ntt gyrus. Ntur Nurosi. 1, (7). 6. Lrkum, M. E. & Nvin, T. Synpti lustring by nriti signlling mhnisms. Curr. Opin. Nurobiol. 18, (8). 7. Govinrjn, A., Kllhr, R. J. & Tongw, S. A lustr plstiity mol of longtrm mmory ngrms. Ntur Rv. Nurosi. 7, (6). 8. Poirzi, P. & Ml, B. W. Impt of tiv nrits n struturl plstiity on th mmory pity of nurl tissu. Nuron 29, (1). 9. Sjostrom, P. J. & Hussr, M. A ooprtiv swith trmins th sign of synpti plstiity in istl nrits of noortil pyrmil nurons. Nuron 51, (6). 1. Losonzy, A. & Mg, J. C. Intgrtiv proprtis of ril obliqu nrits in hippompl CA1 pyrmil nurons. Nuron 5, (6). 11. Zuo, Y., Lin, A., Chng, P. & Gn, W. B. Dvlopmnt of long-trm nriti spin stbility in ivrs rgions of rbrl ortx. Nuron 46, (5). 12. Sgl, M. Dnriti spins n long-trm plstiity. Ntur Rv. Nurosi. 6, (5). 13. Hrms, K. J. & Dunvsky, A. Dnriti spin plstiity: looking byon vlopmnt. Brin Rs. 1184, (7). 14. Fng, G. t l. Imging nuronl substs in trnsgni mi xprssing multipl sptrl vrints of GFP. Nuron 28, (). 15. Xu, T. t l. Rpi formtion n sltiv stbiliztion of synpss for nuring motor mmoris. Ntur 462, (9). 16. Hrvy, C. D. & Svobo, K. Lolly ynmi synpti lrning ruls in pyrmil nuron nrits. Ntur 45, (7). 17. DRoo, M., Klusr, P. & Mullr, D. LTP promots sltiv long-trmstbiliztion n lustring of nriti spins. PLoS Biol. 6, 219 (8). 18. Gry, N. W., Wimr, R. M., Buru, I. & Svobo, K. Rpi ristribution of synpti PSD-95 in th noortx in vivo. PLoS Biol. 4, 37 (6). 19. Hrvy, C. D., Ysu, R., Zhong, H. & Svobo, K. Th spr of Rs tivity triggr by tivtion of singl nriti spin. Sin 321, (8).. Ros, J., Jin, S. X. & Crig, A. M. Htrosynpti molulr ynmis: lolly inu propgting synpti umultion of CM kins II. Nuron 61, (9). 21. Tsuril, S. t l. Lol shring s prominnt trminnt of synpti mtrix molulr ynmis. PLoS Biol. 4, 271 (6). 22. Yu, X. & Zuo, Y. Spin plstiity in th motor ortx. Curr. Opin. Nurobiol. 21, (11). 23. Chn, J. L. & Nivi, E. Nuronl struturl rmoling: is it ll bout ss? Curr. Opin. Nurobiol., (1). 24. Fu, M. & Zuo, Y. Exprin-pnnt struturl plstiity in th ortx. Trns Nurosi. 34, (11). 25. Holtmt, A. & Svobo, K. Exprin-pnnt struturl synpti plstiity in th mmmlin brin. Ntur Rv. Nurosi. 1, (9). 26. Chn, X., Lishnr, U., Rohfort, N. L., Nlkn, I. & Konnrth, A. Funtionl mpping of singl spins in ortil nurons in vivo. Ntur 475, (11). 27. Yust, R. Dnriti spins n istribut iruits. Nuron 71, (11). 28. Knott, G. W., Holtmt, A., Wilbrht, L., Wlkr, E. & Svobo, K. Spin growth prs synps formtion in th ult noortx in vivo. Ntur Nurosi. 9, (6). 29. Fil, J. C., Allwrt, B. & Hrris, K. M. Dnriti spins o not split uring hippompl LTP or mturtion. Ntur Nurosi. 5, (2). 3. Toni, N., Buhs, P. A., Nikonnko, I., Bron, C. R. & Mullr, D. LTP promots formtion of multipl spin synpss btwn singl xon trminl n nrit. Ntur 42, (1999). Supplmntry Informtion is link to th onlin vrsion of th ppr t Aknowlgmnts W thnk D. Stts, D. Gri, L. Hink, T. Jons, S. Song, W. Thompson n G. Wng for ommnts on this mnusript. W thnk A. Prlik n T. Xu for thnil support. This work ws support by grnts from th DANA Fountion n th Ntionl Instituts of Mntl Hlth to Y.Z. Author Contributions M.F. n X.Y. i th in vivo imging n m th figurs. M.F. prform bhviourl trining n ll spin nlyss, n m figurs for rptitiv imging. J.L. n M.F. prform Mtlb simultion n sttistil nlyss. J.L., M.F. n X.Y. prtiipt in isussion bout th ppr. Y.Z. initit n sign th projt, n wrot th mnusript. Author Informtion Rprints n prmissions informtion is vilbl t Th uthors lr no ompting finnil intrsts. Rrs r wlom to ommnt on th onlin vrsion of this rtil t Corrsponn n rqusts for mtrils shoul b rss to Y.Z. (yizuo@us.u). 4 NATURE VOL MONTH 12

5 RESEARCH METHODS Exprimntl nimls. Thy1-YFP-H lin mi 14 wr obtin from th Jkson Lbortory. Mi wr group-hous n br in th Univrsity of Cliforni, Snt Cruz, niml fility, with ll xprimnts prform in orn with pprov niml protools. Motor skill trining n motor nrihmnt. Both th mous singl-s rhing tsk n pllini-hnling tsk protools hv bn prviously srib 15. Motornrih mi wr rr in groups of 8 12 in lrg gs (9 m 3 25 m 3 15 m) ontining vrious toys, suh s rops, lrs, hins, hnging msh/brs t., ll of whih rquir substntil motor oorintion. Th ntur of toys ws hng on ily bsis. mi wr hous in stnr mous gs, with up to fiv mi pr g. Surgil prour for in vivo trnsrnil imging. Th prour for trnsrnil two-photon imging hs bn srib prviously 11,31. Trin mi wr img immitly ftr h trining sssion. Dt quntifition. All nlyss of spin ynmis wr on using ImgJ softwr, blin for xprimntl onitions. Quntifition ritri of nriti spins hv bn srib prviously 15. All nriti protrusions wr trk mnully in thr-imnsionl stks to nsur th onsistny of protrusion intifition ross imging sssions, spit possibl tissu movmnt or rottion. Th numbr n lotion of nriti protrusions (fin s protrusion lngth lrgr thn on-thir of nriti shft imtr) wr intifi in h viw. Filopoi wr intifi s long, thin struturs with th rtio of h imtr to nk imtr bing lss thn 1.2 n th rtio of lngth to nk imtr bing grtr thn 3. Th rmining protrusions wr lssifi s spins. Formtion n limintion of spins n filopoi wr trmin by ompring imgs ollt t two iffrnt tim points. Spins or filopoi wr onsir intil btwn th two imgs if thy wr within.7 mm of thir xpt positions, bs on thir sptil rltionship to jnt lnmrks n/or thir positions rltiv to immitly jnt spins. A stbl spin ws fin s spin tht ws prsnt in both imgs. A nw spin ws spin tht ppr in subsqunt img but ws bsnt from th initil img. Prntgs of form n limint spins (or nriti protrusions) wr normliz to th numbr of spins (or nriti protrusions) in th initil img. Spin imtr nlyss hv bn prviously srib 11. Bus imging n niml onitions vri ovr tim, th rtio of th spin h imtr to th jnt nriti shft imtr ws us s th normliz spin h imtr. Msurmnt of spin h intnsity, s srib prviously 32, ws lso prform to onfirm ths spin siz rsults. Brifly, w trmin th signl intnsity (fin s th sum intnsity of ll pixls omposing th spin in th bst fol pln) n subtrt th bkgroun intnsity (fin s th sum intnsity of rgion ompos of th sm numbr of pixls s th spin but with no YFP-lbll strutur). Th iffrn ws thn ivi by th mn intnsity of th jnt nriti shft (fin similrly s th iffrn btwn th mn signl intnsity of th shft n th mn bkgroun intnsity) to orrt for vrying imging onitions. Th finl vlu ws trm intgrt spin brightnss. All istn msurmnts wr on in ImgJ. To simult spin formtion, w first obtin th rltiv lotion of stbl spins by msuring intr-spin istns long tr nrits in svn ontrol nimls, n ontnt nriti sgmnts from h niml into singl synthti nrit. W thn us ustom-writtn Mtlb os to simult th ition of nw spins. As w obsrv, two spins n xtn from th sm linr lotion long th nriti sgmnt n point towrs iffrnt irtions, givn th ylinril shp of nrits. In our nlysis n simultion, w m th simplifying pproximtion tht th nriti sgmnt is on-imnsionl rthr thn tub. Thrfor, zro intr-spin istn in our nlysis rprsnts two spins ovrlpping in linr position but tully lot t iffrnt sits roun th irumfrn of th nriti sgmnt. In h roun of simultion, th sm numbrs of nw spins s obsrv in xprimnts wr gnrt inpnntly n uniformly long synthti nrits. Th istn btwn h nw spin n its nrststblspin(d n s ) ws lult. Th simultion ws rpt 1, tims n th rsultnt t wr pool to omput th simult smpl min n th umultiv probbility urv. All t wr prsnt s mn 6 stnr rror of mn (s..m.). P vlus wr lult using th Mnn Whitny U-tst for inpnnt smpls, n th Wiloxon sign-rnk tst for pir smpls. 31. Grutznlr, J., Ksthuri, N. & Gn, W. B. Long-trm nriti spin stbility in th ult ortx. Ntur 4, (2). 32. Hofr, S. B., Mrsi-Flogl, T. D., Bonhoffr, T. & Hubnr, M. Exprin lvs lsting struturl tr in ortil iruits. Ntur 457, (9).