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1 Vol 45 5 Mrh 9 doi:1.13/nture7615 LETTERS Sleep nd sensorimotor integrtion during erly vol lerning in songird Sylvn S. Shnk 1 & Dniel Mrgolish 1, Behviourl studies widely implite sleep in memory onsolidtion in the lerning of rod rnge of ehviours 1 4. During sleep, rin regions re retivted 5,6, nd speifi ptterns of neurl tivity re replyed 7 1, onsistent with ptterns oserved in previous wking ehviour. Birdsong lerning is prdigmti model system for skill lerning Song development in juvenile zer finhes (Teniopygi guttt) is hrterized y sleepdependent irdin flututions in singing ehviour, with immedite post-sleep deteriortion in song struture followed y reovery lter in the dy 15. In sleeping dult irds, spontneous ursting tivity of forerin premotor neurons in the roust nuleus of the ropllium (RA) rries informtion out dytime singing 16. Here we show tht, in juvenile zer finhes, plyk during the dy of n dult tutor song indued profound nd tutor-song-speifi hnges in ursting tivity of RA neurons during the following night of sleep. The night-time neuronl hnges preeded tutor-song-indued hnges in singing, first oserved the following dy. Interruption of uditory feedk gretly redued sleep ursting nd prevented the tutor-songspeifi neuronl remodelling. Thus, night-time neuronl tivity is shped y the intertion of the song model (sensory templte) nd uditory feedk, with hnges in night-time tivity preeding the onset of prtie ssoited with vol lerning. We hypothesize tht night-time ursting indues dptive hnges in premotor networks during sleep s prt of vol lerning. By this hypothesis, dptive hnges driven y reply of sensory informtion t night nd y evlution of sensory feedk during the dy intert to produe the omplex irdin ptterns seen erly in vol development. To explore the role of sleep in the erly phses of song lerning, we hrterized the properties of single RA neurons in hed-fixed, sleeping juvenile zer finhes during nights efore nd fter the onset of tutor song exposure. Sleep ws defined on the sis of ehviourl nd eletroenephlogrphi riteri (see Supplementry Informtion) 17. During sleep, RA neurons tended to dishrge irregulrly or urst, s seen in distriutions of inter-spike intervls (ISIs; Fig. 1, lk urve) 16. Strting on the night fter the first dy of exposure to the tutor song, there ws shrp inrese in the mount of high-frequeny spiking tivity (Fig. 1, red urve). Aross ll irds, we quntified the effet of tutor song exposure s normlized hnge in the perentge of ISIs #4 ms, whih showed signifint inrese strting on the night fter the first dy of tutor song exposure nd persisted therefter (Fig. 1). A signifint result ws lso otined onsidering firing rtes tht were normlized y linerly sling the ISIs for eh ell (P 5., repeted mesures nlysis of vrine (ANOVA), 5.5; Fig. 1, open irles). This verified tht the inrese in high-frequeny tivity ws not dependent on hnges in men spike rtes fter tutoring, ut ws the result of speifi inrese in high-frequeny tivity inluding ursting. Within eh ird there ws some vrition in the mount of highfrequeny tivity of RA ells on nights fter the onset of song lerning, ut the tendeny towrds shorter ISIs ws pprent in most ells (Fig. 1). Emerging RA ursting tivity, furthermore, ws shped y the speifi tutor song tht ird herd. Nightly men ISI distriutions were lulted for ll RA neurons reorded for eh ird fter tutor song exposure (whih showed little differene from night to night; Fig. ), nd nightly men distriutions were verged together to generte one men urve per ird. For the resulting post-exposure urves, within the high-frequeny rnge (ISIs #4 ms), the shpes s ssessed using Person orreltion oeffiients were more similr in irds hering the sme tutor song thn in irds hering different tutor songs (see Supplementry Informtion). This grouping of ISI distriution shpes y tutor song n e visulized y ompring the verge ISI distriutions for individul irds on nights efore tutor song exposure to those on nights fter tutor song exposure (Fig., lk nd oloured lines, respetively). The differenes etween groups n e visulized y ompring glol verge ISI distriutions one for eh group of irds hering given tutor song (Fig. ). One ird ws exposed to tutor song, prototypil postexposure ISI distriution shpe ws quikly otined nd then mintined. To quntify this, we ompred (using Person orreltions) the nightly ISI distriutions (#4 ms) for eh ird efore nd fter song exposure to the orresponding glol men urve (Fig. ), exluding dt from the ird eing nlysed from the glol men distriutions. Before tutor song exposure, oth the within- nd etween-group omprisons (Fig. d, lk nd grey dots, respetively) hd lrge vriility nd were not signifintly different from eh other on ny night (P 5.5 to P 5.73). By the first night fter tutor song exposure, ISI distriutions hd lredy ssumed their post-exposure shpe, showing inresed Person orreltions with muh lower vriility (Fig. d, red dots), wheres the ross-group orreltions did not inrese (Fig. d, pink dots). These differenes were sttistilly signifint strting from the first night (P 5., two-tiled t-test, 5.5), nd ontinued to show signifint differene throughout the post-exposure period (P,.5 for out of 11 post-exposure nights; see lso Supplementry Fig. ), emphsizing tht stle hnges in ISI distriutions were rpidly hieved nd then mintined in the dys fter tutor song exposure. Zer finhes egin singing s erly s 5 dys of ge 1, so our,4- dy-old irds hd extensive sensorimotor experiene efore neurl reordings were performed. To explore the influene of singing nd uditory feedk on the struture of RA neuronl ursting, we performed two dditionl experiments. We prevented singing y surgilly muting two irds ( third ird, M, sng in spite of the surgery), nd rised seond group of four irds in ontinuous 1 db white noise environments to suppress uditory feedk. 1 Deprtment of Psyhology, Deprtment of Orgnisml Biology nd Antomy, University of Chigo, Chigo, Illinois 6637, USA. 9 Mmilln Pulishers Limited. All rights reserved 73

2 Correltion LETTERS NATURE Vol 45 5 Mrh Song 1 Song Song 3..1 Post-exposure Pre-exposure Bursting tivity (reltive to seline) log[] Night reltive to trining onset log[] Song trining onset 6* * * Song trining onset Cell numer All seven irds ued tutor song plyk; for irds in the white noise environments, this lso riefly eliminted the msking noise. A noisenelltion tehnique llowed us to qulittively ssess the mount of singing in white noise onditions (Fig. 3e). The sene of uditory feedk hd lrge effets on RA sleep ursting. Even efore the onset of tutor song exposure, experimentl irds showed notly redued levels of ursting tivity ompred to irds with intt uditory feedk. ISI distriutions showed suppressed short-durtion ISIs for ll six feedk-deprived irds (tht is, exluding M; 14 nights, 65 ells; Fig. 3, lower lk line) 7* * 6 3 Figure 1 High-frequeny ursting in RA., ISI distriutions for ll ells for eh night from ird S6 were verged; eh urve (6 s.e.m.) shows the glol verge of ll nights efore tutor song exposure (lk; n 5 19 ells, 3 nights, PHD 43 47) nd fter exposure (red; n 5 59 ells, 1 nights, PHD 49 6)., The tutor song triggered n inrese in high-frequeny ursting (13 irds, n 5 49 ells, min per ell, 5.6 ells per ird per night). The numer of irds ontriuting to eh point is shown ove eh point. *P,.5 y sign test ompring men pre- nd post-exposure vlues within irds, 5.5. Filled irles: untrnsformed dt (6 s.e.m.); open irles: spike-rte normlized dt ( s.e.m.; see Methods nd Supplementry Informtion)., ISI distriutions for ll 44 onseutively reorded ells from ird S9. White lines indite dytime d * * * * * * * * Night reltive to trining onset 9 Mmilln Pulishers Limited. All rights reserved e ms Figure Tutor songs shpe RA ursting., ISI distriutions verged ross ll post-exposure nights, one per ird, y tutor song. Blk lines, preexposure; oloured lines, post-exposure. Song 1, n 5 3 irds, 59 ells (only representtive 3 out of 7 irds re shown to redue lutter, see Supplementry Fig. 4 for ll irds); song, n 5 3 irds, 5 ells; song 3, n 5 3 irds, 3 ells., Glol men (6 s.e.m.) ISI distriutions fter tutor song exposure; eh urve is the verge for eh tutor song group. For song 1, two irds with the lrgest high frequeny peks were removed from the verge so tht urves hve omprle pek heights. The rown line is the single song 4 ird., Green lines re verges for eh of the six post-exposure nights in one ird; the lk line is the song glol men (from ). d, ISI distriutions hnge on exposure to tutor song. Shown re Person orreltion oeffiients ompring nightly urves for given ird with glol post-exposure mens (see text). Nightly mens (6 s.d.) ompring irds hering the sme (pre- nd post-exposure, lk nd red dots, respetively) or different (grey nd pink dots, respetively) tutor songs re plotted. e, Tutor song spetrogrphs. ompred to irds with norml feedk (n 5 13 irds, 3 nights, 163 ells; Fig. 3, upper lk line). Considering ISIs #4 ms, these differenes were signifint (P 5.3, two-tiled t-test on rsin-trnsformed dt, 5.5). The sene of feedk lso disrupted the hnges in RA indued y sensory exposure. In feedk-deprived irds, tutor song exposure did not indue n inrese in ursting tivity (ompring ISIs #4 ms efore to those fter exposure, P 5.45, two-tiled t-test on rsin-trnsformed dt, 5.5, n 5 6 irds, 31 nights, 151 ells). Furthermore, on the nights fter tutor song exposure, highfrequeny ursting ws gretly suppressed in feedk-deprived irds (n 5 6 irds, 1 nights, 9 ells; Fig. 3, lower red line) ompred with norml irds (Fig. 3, upper red line; P 5.4, two-tiled t-test on rsin-trnsformed dt, 5.5). Susequent reovery of sensorimotor feedk relesed RA neurons to rpidly reover to norml ptterns. After exposure to song 1, ut efore esstion of the msking noise, the four irds in the white noise environment showed suppressed high-frequeny tivity ompred to

3 NATURE Vol 45 5 Mrh 9 LETTERS e Intt irds No singing/uditory feedk 1 1 Intt irds Postexposure WN off WN on Pre-exposure Mirophone reording 4 d.1.4. log[] Cell numer irds with norml ess to their feedk (ompring ISIs #4 ms, P 5.4, two-tiled t-test on rsin-trnsformed dt, 5.5, n 5 1 nights, 56 ells), with similr ISI distriutions to those oserved efore tutor song exposure (n 5 nights, 44 ells; Fig. 3, lk urve). The white noise ws turned off for portion of the fourth dy of tutor song exposure (turned off for the first three hours, n 5 irds; turned off for the lst five hours, n 5 irds) nd then turned off ompletely strting on the fifth dy. In two irds tht sng roustly during white noise exposure (one relesed from white noise t the strt of the dy, the other t the end of the dy), during sleep on the night fter the fourth dy of exposure, the expeted tutor-song-speifi ISI distriutions were oserved in the tivity of RA neurons for the first time. A third roust singer lso showed the expeted shift, ut only on the following night (Fig. 3d). Singing ws suppressed in the fourth ird (white noise (WN)4) throughout the period of white noise exposure nd showed roust inrese only y the fourth dy fter white noise ws turned off. Conomitntly, sleep ursting of RA neurons lso Cll No singing/uditory feedk Song trining White noise onset turned off Noise-nelled signl Song syllles Intt irds Figure 3 RA sleep tivity in sene of uditory feedk (white noise or muted)., ISI distriutions efore tutor song exposure, verged ross two muted irds nd four irds rised in white noise (lower lk line), nd verged (for omprison) ross 13 irds with norml feedk (upper lk line)., Birds exposed to white noise nd those tht were muted fil to show tutor-song-speifi ISI distriutions fter song exposure. Lower red line: men post-exposure distriution from muted irds nd those exposed to white noise (5 out of 6 irds herd song 1). Upper red line: men distriution from intt song 1 irds (sme s in Fig. )., Bursting returns when feedk is restored. Birds exposed to white noise (n 5 4) oth preexposure (lk line) nd post-exposure ut in white noise (lower red line) show suppression of ursting; ursting reovers fter white noise (WN) is turned off (upper red line). Compre with distriution for intt song 1 irds (middle red line). d, Profound suppression of ursting for ll ells in ird exposed to white noise. Bursting ppers the seond night fter withdrwl of white noise. White lines represent dytime. e, Reording of singing in the presene of 1 db white noise without nd with noise nelltion (left nd right, respetively). Right, old yellow lines mrk voliztions. 1 s 9 Mmilln Pulishers Limited. All rights reserved remined suppressed for those three dditionl nights, nd then on the night fter tht first dy of singing RA neurons expressed ursting with n ISI distriution pproprite for song 1 (r ,. within-group on the two nights post-singing onset versus r ,.46 for the three nights efore singing onset). The men ISI urve for ll four irds (n 5 17 nights, 4 ells), representing only the dt olleted fter white noise ws turned off (n 5 3 irds) or fter white noise ws turned off nd singing hd ommened (for WN4), ws typil of irds exposed to song 1 with norml feedk (Fig. 3, upper red urve). Anlysis of the fine struture of vol development further demonstrted the tight temporl orreltion etween song development nd night-time ursting. We exmined the entropy vrine mesure of song omplexity tht shows n upwrd developmentl trend trking song lerning 15 for ll song segments eh ird sng eh dy. By this mesure, songs gined struture in slttory mnner on the seond dy of tutor song exposure (P 5.6, repeted mesures ANOVA, 5.5; Fig. 4) 19. The inrese in entropy vrine on the seond dy of trining ws not present with the first voliztions of tht dy. Insted, the inrese seen on the seond dy ws hieved ross singing in the erly prt of the dy. On dys efore tutor song exposure nd on the first dy of exposure, there ws no signifint inrese in entropy vrine ross morning singing (Fig. 4,, see Supplementry Informtion for sttistil nlysis). However, strting on the seond dy of exposure nd on the dys therefter, irds showed ler inrese in entropy vrine ross the erly prt of the dy (Fig. 4, ), similr to wht hs een reported previously 15,, nd this ws signifint (see Supplementry Informtion). Beuse irds volized on the first dy of song tutoring efore nd fter they ued tutor song plyk, however, this ould onfound the implitions of hnges in night-time RA ursting if the irds modified their songs on the first dy ut only fter tutor song exposure, nd this ws not refleted in men hnges for tht dy. Anlysis of singing t the end of the dy, nlysis of the most omplex songs nd nlysis of singing immeditely fter tutoring, however, ll filed to support this lterntive explntion (Supplementry Informtion). In irdsong lerning, memories of onspeifi songs re quired erly in development nd t s referent to guide susequent uditory-feedk-dependent sensorimotor lerning 1 14,1 4. In n influentil ount of this proess, the quired memory of song ws envisioned to t s sensory templte, diretly mthing uditory feedk rising from singing with the sensory representtion of the memorised song guiding hnges in vol output 13,5. We found tht the effets of tutor song exposure were immedite, profound nd distriuted expressed s rpid, fundmentl nd long-lsting hnges in song-speifi high-frequeny spiking tivity in forerin nuleus one synpse from the motor nuleus innervting the syrinx. The isoltion rering our irds experiened efore tutor song exposure is likely to hve enhned the mgnitude of these effets eyond wht would otherwise e seen in normlly rised irds 6. Nevertheless, the timing nd song-type speifiity of the hnges re onsistent with the tion of song (sensory) memory influening the funtionl orgniztion of motor iruit 7, lthough usl link hs yet to e estlished. The rpidity of hnges in RA sleep tivity nd its susequent stility olsters the ide tht the sensory representtion ws quikly estlished, opening gte or enling dormnt iruit, nd tht song development proeeded with the sensory representtion expressed t night guiding hnges in dytime premotor ptterns. This motivtes new hypothesis: sensory memories n t indiretly (offline) on sensorimotor performne y mens of spontneous tivity. We speulte tht during sleep, hnges in the RA network ( onsolidtion ) reflet updting of sensory memories. Night-time hnges would estlish new onfigurtion in the RA network. The following morning, plsti responses relesed y singing susequent to nighttime reonfigurtion might first drive the network wy from stility efore it reovers stility with dditionl itertions; this ould explin 75

4 LETTERS NATURE Vol 45 5 Mrh 9 Entropy vrine (perentge hnge from seline) 6 4 First night of sleep First dy of song trining whih reeives inputs from the desending motor pthwy nd from the ortio-sl gngli pthwy. Spontneous tivity guides erly development of the nervous system. Here, high-frequeny urst-mode firing, ommon feture of verterte forerin neurons, is strutured y sleep-medited lerning mehnisms, emerging strikingly lte in development s the orgnism experienes pproprite environmentl ues. It remins to e seen whether ursting sleep tivity represents the tivtion of sensory tres in other forms of skill lerning, nd perhps more generlly. Strutured spontneous dishrge is oserved rodly in the forerin during sleep, nd hs een ssoited with rell 9,3. Entropy vrine Entropy vrine (reltive to dily verge) Dy reltive to trining onset Dy reltive to trining onset First night of sleep 6:3 1:3 14:3 1:3 Time of dy Dy 1 Dy 1 Dy Dy 5 Figure 4 Entropy vrine hnges fter night of sleep., Men dily entropy vrine for ird voliztions. Dt for eh ird were normlized y dividing within-dy mens y the men of the four nights preeding exposure., Entropy vrine of ll sounds produed y one ird ross 15 dys, smoothed y 4 dt points within eh dy. The entropy vrine inrese ross the seond dy of trining is hieved ross morning singing., Entropy vrine dily trends, verged ross ll (nonmnipulted) irds in two-hour ins (6 s.e.m.) for the dy efore trining onset (lk), the first dy of trining (lue), the seond dy of trining (red) nd the verge of the seond to the fifth dy of trining (green). the non-monotoni hnges in singing oserved eh morning during development 15. A lrger mgnitude of non-monotoni singing ehviour whih is positively orrelted with the eventul degree of song opying 15 would result from stronger or more urte sensory influenes on premotor networks. Beuse the differenes etween sensory (night time) nd motor (dytime) network onfigurtions derese during development (improved sensorimotor integrtion), the mgnitude of the irdin singing ptterns would derese, s is tully oserved 15. The plsti hnges tht we hve desried re relesed y the onfluene of two sensory signls: the model nd feedk. We further speulte this ould our t the level of RA itself, 76 METHODS SUMMARY Birds were red in-house in sound-isoltion oxes, nd were femle-rised to isolte them from dult mle song eginning on or efore post-hth-dy (PHD) 15. Mles (n 5 1) were isolted in individul sound-isoltion oxes etween PHD 3 nd PHD 35, nd reeived preprtory surgery etween PHD 37 nd PHD 4. Song ws reorded ontinuously strting on or efore the first dy postsurgery until t lest PHD 9, nd neurl reordings egn two to three dys lter. Birds were permitted to self-eliit song plyk y pulling string in the ge fter nights of neuronl reordings. Bursting inrese. We quntified the inrese in high-frequeny neuronl tivity s normlized hnge in the perentge of the ISI distriution #4 ms, whih ptures short intervls ssoited with ursting in these juvenile irds nd the differene in ursting efore nd fter song exposure (Fig. 1). Nightly vlues were lulted y first tking the men ISI distriution ross ells for given night for given ird. The proportion of the men distriution #4 ms ws found nd nightly vlues were normlized within irds y the men from the four nights preeding tutor song exposure onset (Fig. 1). We lso performed the sme lultion ut first pplying liner trnsformtion of ISIs so tht the men spike rte ws the sme for ll ells efore nd fter tutor song exposure, finding similr inrese in ursting fter song exposure (Fig. 1, open irles). Correltion oeffiients. For sttistil nlysis of Person orreltion oeffiients, Fisher s r-to-z trnsformtion ws performed on the orreltion vlues. Where the trnsformed dt re plotted (Fig. d), the r vlues hve symmetril stndrd devitions euse the trnsformtion yields vlues etween 1 nd 1. Full Methods nd ny ssoited referenes re ville in the online version of the pper t Reeived 11 June; epted 31 Otoer. Pulished online 14 Deemer. 9 Mmilln Pulishers Limited. All rights reserved 1. Krni, A., Tnne, D., Ruenstein, B. S., Askensy, J. J. & Sgi, D. Dependene on REM sleep of overnight improvement of pereptul skill. Siene 65, (1994).. Fenn, K. M., Nusum, H. C. & Mrgolish, D. Consolidtion during sleep of pereptul lerning of spoken lnguge. Nture 45, (3). 3. Wgner, U., Gis, S., Hider, H., Verleger, R. & Born, J. Sleep inspires insight. Nture 47, (4). 4. Wlker, M. P. & Stikgold, R. Sleep, memory, nd plstiity. Annu. Rev. Psyhol. 57, (6). 5. Peigneux, P. et l. Are sptil memories strengthened in the humn hippompus during slow wve sleep? Neuron 44, (4). 6. Rsh, B., Buhel, C., Gis, S. & Born, J. Odor ues during slow-wve sleep prompt delrtive memory onsolidtion. Siene 315, (7). 7. Ji, D. & Wilson, M. A. Coordinted memory reply in the visul ortex nd hippompus during sleep. Nture Neurosi. 1, 1 17 (7).. Qin, Y. L., MNughton, B. L., Skggs, W. E. & Brnes, C. A. Memory reproessing in ortioortil nd hippompoortil neuronl ensemles. Phil. Trns. R. So. Lond. B 35, (1997). 9. Ndsdy, Z., Hirse, H., Czurko, A., Csisvri, J. & Buzski, G. Reply nd time ompression of reurring spike sequenes in the hippompus. J. Neurosi. 19, (1999). 1. Hennevin, E., Huetz, C. & Edeline, J. M. Neurl representtions during sleep: from sensory proessing to memory tres. Neuroiol. Lern. Mem. 7, (7). 11. Mrler, P. & Tmur, M. Culturlly trnsmitted ptterns of vol ehvior in sprrows. Siene 146, (1964). 1. Mrler, P. A omprtive pproh to vol lerning: song development in whiterowned sprrows. J. Comp. Physiol. Psyhol (197). 13. Konishi, M. The role of uditory feedk in the ontrol of voliztion in the white-rowned sprrow. Z. Tierpsyhol., (1965). 14. Zeigler, H. P. & Mrler, P. R. (eds) Behviorl Neuroiology of Birdsong (Ann. NY Ad. Si., 4).

5 NATURE Vol 45 5 Mrh 9 LETTERS 15. Derégnuourt, S., Mitr, P. P., Feher, O., Pytte, C. & Thernihovski, O. How sleep ffets the developmentl lerning of ird song. Nture 433, (5). 16. Dve, A. S. & Mrgolish, D. Song reply during sleep nd omputtionl rules for sensorimotor vol lerning. Siene 9, 1 16 (). 17. Low, P. S., Shnk, S. S., Sejnowski, T. J. & Mrgolish, D. Mmmlin-like fetures of sleep struture in zer finhes. Pro. Ntl Ad. Si. USA 15, (). 1. Roper, A. & Znn, R. The onset of song lerning nd song tutor seletion in fledgling zer finhes. Ethology 11, (6). 19. Thernihovski, O., Mitr, P. P., Lints, T. & Notteohm, F. Dynmis of the vol imittion proess: how zer finh lerns its song. Siene 91, (1).. Crndll, S. R., Adm, M., Kinnishtzke, A. K. & Nik, T. A. HVC neurl sleep tivity inreses with development nd prllels nightly hnges in song ehvior. J. Neurophysiol. 9, 3 4 (7). 1. Mrler, P. & Peters, S. Sprrows lern dult song nd more from memory. Siene 13, 7 7 (191).. Hultsh, H. & Todt, D. Memoriztion nd reprodution of songs in nightingles (Lusini megrhynhos): evidene for pkge formtion. J. Comp. Physiol. A 165, (199). 3. Funiki, Y. & Konishi, M. Long memory in song lerning y zer finhes. J. Neurosi. 3, (3). 4. Leonrdo, A. & Konishi, M. Derystlliztion of dult irdsong y perturtion of uditory feedk. Nture 399, (1999). 5. Konishi, M. in Pereption nd Experiene (eds Wlk, R. D. & Pik, H. L. J.) (Plenum, 197). 6. Adret, P. & Mrgolish, D. Metoli nd neurl tivity in the song system nuleus roustus rhistritlis: effet of ge nd gender. J. Comp. Neurol. 454, (). 7. Nik, T. A. & Konishi, M. Neurl song preferene during vol lerning in the zer finh depends on ge nd stte. J. Neuroiol. 6, 31 4 (5).. Evrts, E. V., Bentl, E., Bihri, B. & Huttenloher, P. R. Spontneous dishrge of single neurons during sleep nd wking. Siene 135, 76 7 (196). 9. Pstlkov, E., Itskov, V., Amrsinghm, A. & Buzsáki, G. Internlly generted ell ssemly sequenes in the rt hippompus. Siene 31, (). 3. Gelrd-Sgiv, H., Mukmel, R., Hrel, M., Mlh, R. & Fried, I. Internlly generted retivtion of single neurons in humn hippompus during free rell. Siene 3, (). Supplementry Informtion is linked to the online version of the pper t Aknowledgements We thnk M. Fukushim for extensive disussions, nd J.-M. Rmirez, H. C. Nusum, S. M. Shermn nd M. Konishi for omments on the mnusript. A. S. Dve nd M.F. designed nd implemented the white noise reording/nelltion environment. Author Contriutions S.S.S. performed the experimentl work. Author Informtion Reprints nd permissions informtion is ville t Correspondene nd requests for mterils should e ddressed to D.M. (dn@igird.uhigo.edu). 9 Mmilln Pulishers Limited. All rights reserved 77

6 doi:1.13/nture7615 METHODS Birds were mintined on reverse light shedule (light, 6:3 p.m. 1:3.m.; light:drk, 16 h: h) to permit experiments during dytime hours. Around PHD 3, sujets reeived single surgery, inluding implnttion of hedrestrint pin nd durl EEG eletrodes, nd, for some irds, muting. Neurl reordings egn two to three dys fter the surgery, in the sme oxes tht housed the irds. Birds were wrpped in loth nd the hed ws immoilized, onnetors were tthed to EEG eletrode leds, nd high-impedne eletrode ws lowered into the rin ove RA. The ox ws then losed for the durtion of the eight-hour night. Birds were illuminted with infrred light, nd EEG signls nd video were reorded for the durtion of the night while singleell reordings in RA were otined. Birds experiened norml levels of rpid eye movement (REM) nd non-rem sleep s tht whih hs een reported in songirds previously 17,31, nd rief periods of wkefulness. RA ws identified stereotxilly nd y the redily identifile tivity ptterns of RA neurons. After nights in the reording pprtus (rnge 1 6 nights), when suffiient pre-exposure dt hd een otined ( nights of suessful reordings; rnge 1 5 nights), irds were llowed to self-eliit plyk of song model y pulling string, until PHD 9. Birds were limited to ten renditions of the tutor song during eh morning nd fternoon trining session. Neuronl reordings were olleted over 5 16 nights (exept one ird for whih we only sueeded in olleting dt for nights, 1 nd 1 reltive to song exposure). Beuse lerly identifile repeted syllles do not egin to emerge until severl dys fter tutor song exposure onset 15, rther thn try to luster song elements in proto-syllles, we exmined the entropy vrine of ll song elements produed to ssess dily trends efore nd fter the onset of tutor song exposure. Dt olletion nd nlysis used Sound Anlysis Pro, Mtl, nd in-house softwre. Surgil muting. For some irds, muting proedure 3 ws performed t the time of the surgery. A smll opening ws mde into the interlviulr ir s. A smll fenestr ws mde in the trhe just rostrl to the syrinx nd in the ronhi immeditely udl to the syrinx within the interlviulr ir s. The externl opening ws sutured losed. This proedure proly elimintes the ility to generte pressure ross the syrinx. Birds M1 nd M3 were suessfully muted ut ird M ontinued to volize immeditely fter the surgery (presumly, the vol trt holes seled). Both muted irds eventully reovered the ility to sing. On dys 9 nd 11 post-surgery, M1 nd M3 egn mking very sprse, soft voliztions omposed of simple hrmoni stks of vrious length. Over the following few weeks, irds developed norml plsti song nd oth eventully hieved finl opy tht showed lerning in the rnge of the non-devolized irds. White noise nelltion. White noise ws plyed ontinuously, during the sujetive dy, strting on the dy efore the first night of neurl reordings. To ssess whether irds were singing, we used n tive noise nelltion tehnique 33. In rief, proe uditory signls were plyed nd reorded to estimte the trnsfer funtion of the reording hmer. Using the known input to the speker nd the trnsfer funtion, the white noise signl rodst from the speker ws sutrted from the overll signl rehing the mirophone, llowing us to oserve song (leit with low fidelity). On the sis of this tehnique we oserved tht irds WN1, WN nd WN3 sng frequently while exposed to ontinuous white noise (nd they lso sng immeditely fter the white noise ws turned off). Bird WN4 filed to sing in white noise nd lso only reovered singing four dys fter white noise exposure ws terminted. Song similrity mesures. The similrity of ird s song to its tutor song ws omputed s desried previously 15,19,34 using Sound Anlysis Pro softwre. In rief, mens nd vrines of ousti fetures, priniplly durtion, pith, Weiner entropy nd men frequeny, were lulted for eh syllle produed y eh ird. Similrity etween given song nd the tutor song ird herd ws omputed y quntittive omprison of this rry of ousti fetures. 31. Rttenorg, N. C. et l. Migrtory sleeplessness in the white-rowned sprrow (Zonotrihi leuophrys gmelii). PLoS Biol., E1 (4). 3. Pytte, C. L. & Suthers, R. A. A ird s own song ontriutes to onspeifi song pereption. Neuroreport 1, (1999). 33. Leonrdo, A. Experimentl test of the irdsong error-orretion model. Pro. Ntl Ad. Si. USA 11, (4). 34. Thernihovski, O., Notteohm, F., Ho, C. E., Pesrn, B. & Mitr, P. P. A proedure for n utomted mesurement of song similrity. Anim. Behv. 59, (). 9 Mmilln Pulishers Limited. All rights reserved