Brief hearing loss disrupts binaural integration during two early critical periods of auditory cortex development

Transcription

1 Received 3 Apr 23 Accepted 3 Sep 23 Pulished 3 Sep 23 Brief hering loss disrupts inurl integrtion during two erly criticl periods of uditory cortex development Dniel B. Polley,2,3, John H. Thompson 2 & Wei Guo 2,3 DOI:.38/ncomms3547 Erly inurl experience cn reclirte centrl uditory circuits tht support sptil hering. However, it is not known how inurl integrtion mtures shortly fter hering onset or whether vrious developmentl stges re differentilly impcted y disruptions of norml inurl experience. Here we induce rief, reversile unilterl conductive hering loss (CHL) t severl experimentlly determined milestones in mouse primry uditory cortex (A) development nd chrcterize its effects B week fter norml hering is restored. We find tht CHL shpes A inurl selectivity during two erly criticl periods. CHL efore P6 disrupts the norml coregistrtion of interurl frequency tuning, wheres CHL on P6, ut not efore or fter, disrupts interurl level difference sensitivity contined in long-ltency spikes. These dt highlight n evolving plsticity in the developing uditory cortex tht my relte to the etiology of mlyudi, inurl hering impirment ssocited with outs of otitis medi during humn infncy. Deprtment of Otology nd Lryngology, Hrvrd Medicl School, Boston Msschussetts 24, USA. 2 Eton-Peody Lortories, Msschusetts Eye nd Er Infirmry, Boston Msschussetts 24, USA. 3 Center for Computtionl Neuroscience nd Neurl Technology, Boston University, Boston Msschussetts 225, USA. Correspondence nd requests for mterils should e ddressed to D.B.P. (emil: dniel_polley@meei.hrvrd.edu). NATURE COMMUNICATIONS 4:2547 DOI:.38/ncomms & 23 Mcmilln Pulishers Limited. All rights reserved.

2 NATURE COMMUNICATIONS DOI:.38/ncomms3547 Perturtions of norml sensory experience during criticl periods of corticl development cn hve specific nd lsting effects on the orgniztion of synpses, receptive fields, topogrphic mps nd sensory-guided ehviours (for recent reviews see refs,2). Often, criticl period regultion of prticulr sensory representtion coincides with stge in normtive development when its ssocited neurl circuitry is most lile 3. In the primry visul cortex, the effects of the eyelid suture re gretest severl weeks fter eye opening, when ipsilterl eye responses egin to rpidly mture 4 nd the preferred stimulus orienttion for inputs to ech eye ecome incresingly well mtched 5. In the primry uditory cortex (A) of ltricil rodents, the effect of hering loss on intrcorticl inhiition is gretest when inhiitory synpses re ctively mturing 6. Tonotopic mp plsticity ssocited with pure tone rering is limited to 3-dy window eginning t the onset of hering, when tone-evoked response thresholds drop most precipitously 7, synptic excittion nd inhiition re still uncorrelted 8 nd dendritic spines on lyer 4 thlmorecipient neurons re rpidly mturing 9. By contrst, the effect of frequency-modulted sweep rering on uditory directionl tuning is gretest weeks lter, when frequency-modulted sounds first egin to elicit high firing rtes. Temporrily disrupting hering in one er of young nimls cn negtively impct inurl selectivity in centrl uditory nuclei 6, much like the effects of monoculr deprivtion on the development of coordinted inoculr tuning in the visul cortex 2. Unlike tonotopic mp plsticity in the uditory cortex or oculr dominnce plsticity in the visul cortex, the precise timing nd orgniztion of criticl periods governing the experience-dependent mintennce of inurl integrtion in the developing uditory cortex re unknown. Moreover, it is not known how or whether these chnges re ligned to the norml mturtionl time course for corticl inurl tuning. Thus, one of the ims for these experiments ws to introduce temporry hering loss t precise time points suggested from dy-y-dy nlysis of normtive inurl development in A. Asymmetric conductive hering loss (CHL) is quite common in humn infnts with otitis medi 7. Affected individuls re t greter risk to experience constelltion of rin-sed inurl hering impirments in lter life, collectively known s mlyudi (nmed fter its visul nlogue, mlyopi (for review see ref. 8)). However, developmentl studies of temporry CHL in niml models generlly involve recording immeditely following extended periods of severe CHL rther thn simulting the more enduring effects of the trnsient, moderte-intensity CHL tht cn ccompny otitis medi 7. Therefore, second gol of these studies ws to determine whether mimicking nturlly occurring CHL t empiriclly defined developmentl milestones is ssocited with specific neuronl deficits in inurl integrtion. To ddress these ims, we trcked the dy-y-dy mturtion of A sound representtions over the first week of hering in mouse pups. We noted rupt shifts in monurl response threshold nd preferred frequency tuning etween P nd P4 followed y more grdul mturtion of ipsilterl interurl level difference (ILD) sensitivity contined in long-ltency spikes tht ws mximlly pronounced t P5 P6. To test the hypothesis tht the stepwise time course of normtive mturtion would correspond to distinct points in development when ech feture ws specificlly vulnerle to the effects of degrded inurl experience, we introduced rief, reversile monurl CHL t P2, P6 or P2. We found tht disrupting inurl experience t P2 interfered with the norml coregistrtion of frequency-receptive fields etween the contrlterl nd ipsilterl ers, wheres the sme mnipultion t P6 primrily ffected corticl ILD sensitivity. These findings highlight sequence of rief developmentl windows occurring shortly fter hering onset tht regulte the specific nd lsting influence of inurl experience on the corticl representtion of sound fetures underlying sptil hering. Results Defining the onset of hering. The mturtion of corticl sound representtions ws chrcterized on dy-y-dy sis for week fter the onset of hering. The nlysis ws sed on recordings from,262 A units in 35 CBA/CJ mice. The onset of hering ws opertionlly defined s P, s it ws the erliest ge when contiguous monurl frequency response re (FRA) could e delineted from the nesthetized cortex using irorne tone ursts r9 db sound pressure level (SPL) (leit, in only 3/ 64 recordings sites from one of the three P pups tested). This does not set n solute lower ound on the onset of hering in ll mice, s mesurements in different strins or with different functionl mrkers of hering could provide different results. However, P or P is the ge t which the er cnl first opens in severl mouse strins, Preyer s reflex is first evident nd soundevoked ctivity is first oserved in the uditory nerve nd inferior colliculus, mking it resonle estimte 9 2. Development of monurl sound representtions. Monurl FRAs exhiit series of profound chnges during the first week of hering (Fig. ). At the onset of hering, contrlterl nd ipsilterl est frequencies (BFs) were predominntly found etween nd 22 khz, frequencies tht fll within the most sensitive region of the CBA mouse udiogrm 22. Beginning t P4, contrlterl nd ipsilterl BFs were oserved t progressively more eccentric sound frequencies until they reched n dult-like distriution y P6 or P7 (Fig. ). Contrlterl nd ipsilterl minimum response thresholds lso decresed significntly during the initil dys of hering, reching pproximtely dult-like levels y P5 (one-wy nlysis of vrinces (ANOVAs), F42.43, Po 6 for oth contrlterl (n ¼ 964) nd ipsilterl (n ¼ 684) response mesurements, Fig. c). The rpid mturtion of corticl response properties in the first dys fter hering onset could reflect neurodevelopmentl chnges in the centrl uditory system nd/or the uditory periphery. To estimte postntl chnges in cochler sensitivity, we mesured the outer hir cell function vi distortion product otocoustic emissions (DPOAEs), iocoustic signl tht is sed upon sound-evoked chnges in hir cell receptor potentils tht cn e mesured s sound pressure chnges in the er cnl 23. In so doing, it ws evident tht oth the chnge in A response thresholds nd the incresing rnge of BF tuning etween P nd P4 occurred in prllel with commensurte chnges in cochler sensitivity (ANOVA, min effect for DPOAE threshold chnges y ge nd ge frequency interction term n ¼ 56 ers, F ¼ nd 3.52, respectively, Po 6 for oth comprisons; Fig. d). The contriution of peripherl mturtion to developmentl chnges in A threshold nd frequency sensitivity cn e ccounted for if one ssumes tht peripherl mturtion is symmetricl; nmely, whtever developmentl processes re unfolding in the left er re lso unfolding in the right er. In this scenrio, the interul difference in vrious response fetures would remin constnt, despite ongoing cochler mturtion. Alterntively, peripherl development could ccompny chnges in CNS circuits tuned to interurl response fetures. This could result in developmentl chnges in oth monurl nd interurl response properties. 2 NATURE COMMUNICATIONS 4:2547 DOI:.38/ncomms & 23 Mcmilln Pulishers Limited. All rights reserved.

3 NATURE COMMUNICATIONS DOI:.38/ncomms3547 ARTICLE Contrlterl Ipsilterl c Threshold (db SPL) e Asolute BF dif. (octves) 8 db SPL Frequency (khz) P P4 P7 Spike count Mx P P P2 P3 P4 P5 P6 P7 Adult Postntl ge P P2 P3 P4 P5 P6 P7 Adult Postntl ge Best frequency (khz) d Cochler threshold (db SPL) f Threshold dif. (I - C, db) > Contrlterl stimulus Ipsilterl stimulus P P P2 P3 P4 P5 P6 P7 Adult Postntl ge f2 frequency (khz) P P P2 P3 P4 P5 P6 P7 Adult P P2 P3 P4 P5 P6 P7 Adult Postntl ge Figure Rpid mturtion of monurl response properties in A is ssocited with concurrent chnges in cochler sensitivity. () Representtive contrlterl nd ipsilterl FRAs recorded from A on P, P4 nd P7. (,c) Rpid expnsion of BF () nd drop in threshold (c) of contrlterl (lck) nd ipsilterl (red) FRAs mesured from, units in 35 mice. (d) Minimum sound levels sufficient to elicit mesurle DPOAE t five test frequencies. N ¼ 56 ers, vlues represent men±s.e.m. DPOAEs re sed on outer hir cell receptor currents tht cn e mesured s sound level chnges in the er cnl. (e,f) Neither the solute difference etween contrlterl nd ipsilterl BFs (e) nor the contrlterl is in minimum response threshold (f) were gretly chnged over the first week of hering. C, contrlterl; I, ipsilterl; dif., difference. Lines represent men vlues t ech ge. Ech dt point is one recording site. A slight rndom jitter is imposed on ll dt points to visully distinguish identicl vlues in the sctterplots. To ddress these possiilities, we nlysed developmentl chnges in the interurl reltionship etween vrious response fetures for units with contrlterl nd ipsilterl receptive fields (7% of ll recording sites). We found tht interurl BFs devited y B.25 octves throughout the first week of hering (one-wy ANOVA, n ¼ 77, F ¼.54, P ¼.5; Fig. e). This suggested tht the rpid increse in the rnge of preferred frequency tuning noted in Fig. cn e primrily ttriuted to postntl chnges in cochler development, s hs een suggested previously 24,25. The interurl threshold difference incresed slightly ut significntly cross the ges tested (one-wy ANOVA, n ¼ 77, F ¼ 8.49, Po 6 ; Fig. f). Although this supports previous description of incresing contrlterl sensitivity is cross n erly period of hering in the ferret cortex 26, the increse in the interurl threshold difference is fr less thn the chnge in overll response threshold (Fig. c). Collectively, these findings suggest tht mny chnges in monurl threshold nd frequency tuning cn e scried to cochler development. Despite its provennce, the fct remins tht the sensitivity nd frequency rnge of sound-evoked spiking chnges drmticlly etween P nd P4 in A. The timing of this rupt chnge in corticl spiking ptterns my predict the timing of criticl period window for experience-dependent influences on coordinted interurl frequency tuning. Monurl deprivtion disrupts receptive field lnce. To test this hypothesis, we developed new pproch to temporrily degrde uditory inputs to one er during rief windows of postntl development. This ws chieved y injecting thermoreversile poloxmer hydrogel into the middle er cvity, which simulted temporry out of symmetric CHL tht cn ccompny otitis medi in humn infnts (Fig. 2). Poloxmers offer the dvntge of remining liquid when injected in the middle er t cool temperture, then rpidly trnsition to gel s they wrm to ody temperture nd, finlly, spontneously NATURE COMMUNICATIONS 4:2547 DOI:.38/ncomms & 23 Mcmilln Pulishers Limited. All rights reserved.

4 NATURE COMMUNICATIONS DOI:.38/ncomms khz ABR threshold (db SPL) d 32 khz ABR threshold (db SPL) Poloxmer 47 Injected er Control er Time from CHL onset (dys) Time from CHL onset (dys) Time from CHL onset (dys) dissolve through hydrolysis severl dys lter. Intrtympnic poloxmer injections circumvent some of the chllenges ssocited with using er plugs in very young nimls, such s permnent stenosis of the er cnl nd degrdtion of the tympnic memrne 27. It lso provides n lterntive to surgiclly ligting the er cnl, which is disdvntged y the fct tht norml hering thresholds cn only e restored y n invsive surgery on the ligted er, therey necessitting tht the neurophysiology experiment tke plce within minutes or hours fter hering is restored. By trcking dy-to-dy chnges in uditory rinstem response (ABR) in mice injected with poloxmer t P6 nd P7 (n ¼ 4) or ge-mtched control mice (n ¼ 9), we determined tht c 6 khz ABR threshold (db SPL) e ABR thresh. t A recording (db SPL) A unit recordings ABR recordings Injected er Uninjected er P2 P6 P2 Age t the onset of CHL Figure 2 Intrtympnic poloxmer injections crete short-term nd fully reversile mild CHL. () Reversile CHL ws creted y injecting solution of Poloxmer 47, thermoreversile hydrogel, into the middle er spce of young mice. Injections were mde on two consecutive dys into the left er. Unit recordings were mde in the right A, contrlterl to the injected er. ( d) Minimum response threshold for ABR wve elicited with 8 (), 6 (c) or 32 khz (d) tone ursts delivered to the left er. Thresholds re plotted reltive to dy of the first poloxmer injection on P6 (solid lines, n ¼ 4) or from ge-mtched control mice (dshed lines, n ¼ 9). (e) ABR threshold ws mesured t 6 khz from left injected er (lck) nd the right uninjected er (grey) in poloxmer-injected mice. Mesurements were tken immeditely efore A recordings, 4 dys fter n initil poloxmer injection t P2 (n ¼ 5), P6 (n ¼ 4) or P2 (n ¼ 5). All vlues represent men±s.e.m. Asterisks indicte significnt differences etween poloxmer nd control threshold vlues sed on ANOVA post-hoc pirwise comprisons (Po.5). poloxmer injections induced significnt elevtion of ABR wve threshold for 5 dys t 8 khz (Fig. 2), 6 dys t 6 khz (Fig. 2c) nd 7 3 dys t 32 khz (Fig. 2d). We lso confirmed tht the ABR threshold shift ws fully resolved efore our corticl neurophysiology experiments 4 5 dys fter the initil poloxmer injection (tht is B week fter norml hering ws restored, Fig. 2e). To compre the effects of temporry unilterl hering loss on the coordinted development of interurl frequency tuning, recordings were mde from A in the right hemisphere of mice 2 weeks fter poloxmer injection into the left middle er cvity on P2 (CHL2), P6 (CHL6) or P2 (CHL2), nd lso from mice tht underwent shm procedure t one of these ges. Unless otherwise indicted, descriptive sttistics re provided s men±s.e.m. P-vlues for ll sttisticl tests reported elow re sed on n ANOVA followed up with pirwise contrsts tht were djusted for multiple comprisons with the Bonferroni method. In shm-operted juvenile mice, s ws previously descried in nive dult mice, contrlterl FRA thresholds were 5 db lower on verge thn ipsilterl FRA thresholds (2.±.94 versus 25.9±.25 db, Fig. f nd Fig. 3,). A doule dissocition ws oserved for tone sensitivity in CHL2 mice, such tht thresholds for the developmentlly deprived contrlterl er were significntly elevted (26.4±.77 db, ANOVA post-hoc comprison, n ¼ 22, Po.5), wheres sensitivity to the non-deprived ipsilterl er ws significntly enhnced reltive to shms (2.±.79 db, ANOVA post-hoc comprison, n ¼ 22, Po.5; Fig. 3,). CHL initited t P6 ws ssocited with significnt elevtion in the contrlterl deprived er threshold compred with shm (25.28±.9 db, ANOVA post-hoc comprison, n ¼ 24, Po.5) ut no chnge in ipsilterl threshold (24.76±.96 db, ANOVA post-hoc comprison, n ¼ 24, P4.5). Contrlterl nd ipsilterl FRA thresholds from CHL2 recordings were not different thn shm controls (22.46±.83 nd 27.46±.84 db for contrlterl nd ipsilterl, respectively, ANOVA post-hoc comprison, n ¼ 95, P4.9 for oth). The spontneous firing rte ws lso elevted when CHL ws initited t P2 compred with shm, ut not t ny other ge (8.64±.85 versus 2.7±.8 spikes per s, ANOVA post-hoc comprison, n ¼ 35, Po 6, Fig. 3c). In ddition to idirectionl djustment in sensitivity, we lso oserved tht contrlterl nd ipsilterl receptive fields, which were precisely coregistered for frequency preference even t the onset of hering (Fig. e), hd slipped out of lignment when CHL ws initited t P2 (Fig. 3). Quntittive nlysis confirmed tht the percentge of overlpping points in contrlterl nd ipsilterl FRAs ws significntly reduced (53±% versus 44±%; ANOVA post-hoc comprison, n ¼ 22, Po5 5, Fig. 3d) nd tht BFs were pproximtely twice s fr prt in CHL2 recordings (.59±.5 versus.27±.3 octves; ANOVA post-hoc comprison, n ¼ 22, Po 6 ), ut were not different thn shm recordings in CHL6 or CHL2 conditions (ANOVA post-hoc comprisons, n ¼ 24 nd n ¼ 95, respectively, P4.24 for ll comprisons, Fig. 3e). The increse in solute BF difference in CHL2 recordings ws ttriuted to systemtic high-frequency shift in contrlterl compred with ipsilterl BF, s hs lso een reported shortly fter mild sensorineurl hering loss in primtes 28 (.33±.6 octves, pired Student s t-test, n ¼ 22, Po.5, Fig. 3f). As finl comprison of interurl dominnce, the verge rtio of firing rtes for ll points within the union of the contrlterl nd ipsilterl FRAs were compred, where the degree of positivity is commensurte with the degree of contrlterl is in evoked firing rte (right-most column in Fig. 3). The contrlterl is mesured from ll three deprivtion ges ws reduced compred with shms (CHL2,.6±.2; CHL6,.28±.; CHL2, 4 NATURE COMMUNICATIONS 4:2547 DOI:.38/ncomms & 23 Mcmilln Pulishers Limited. All rights reserved.

5 NATURE COMMUNICATIONS DOI:.38/ncomms3547 ARTICLE 8 db SPL 8 db SPL Contr Contr/deprived 4 Freq (khz) 45 Ipsi Ipsi/non-deprived Overly Overly I>C C=I C>I Threshold (db SPL) Contrlterl Ipsilterl CHL2 CHL6 CHL2 Shm c Spontneous rte (sp s ) 2 Spike count 4 d Receptive field overlp (%) e Asolute BF dif. (octves) CHL2 CHL6 CHL2 Shm f BF dif. (contr ipsi, octves) CHL2 CHL6 CHL2 Shm g.5 CHL2 CHL6 CHL2 Shm. CHL2 CHL6 CHL2 Shm.4 Aurl dominnce.3.2. CHL2 CHL6 CHL2 Shm Figure 3 An erly criticl period for the disruptive effect of trnsient unilterl hering loss on interurl receptive field lignment. () Representtive contrlterl nd ipsilterl FRAs recorded from A of mice tht received shm procedure (top) or poloxmer injection to the contrlterl er on P2 (ottom). All recordings re mde 4 5 dys following the initil poloxmer injection or shm procedure. For purposes of direct comprison, the monurl FRA colour scle is mpped to the sme rnge of firing rtes rther thn normlizing individully. Pnels to the right represent the rtio of firing rtes for ech point within the union of the contrlterl nd ipsilterl FRAs. () Contrlterl (lck) nd ipsilterl (red) FRA thresholds. (c) Spontneous firing rte mesured during the -ms period preceding tone onset. (d) The percentge of tone comintions contined in oth contrlterl nd ipsilterl FRAs (the intersection) reltive to ll tone comintions contined in the contrlterl nd ipsilterl FRAs comined (the union). (e) Asolute difference in BF mesured from the contrlterl nd ipsilterl FRAs. (f) Incresed BF disprity in CHL2 group reflects systemtic high-frequency shift in contrlterl versus ipsilterl BFs. Asterisk indictes significnt difference sed on pired Student s t-test (Po.5). (g) Men rtio of contrlterl versus ipsilterl firing rtes from tone comintions within the union of oth FRAs. More positive vlues indicte greter contrlterl dominnce. All vlues represent men±s.e.m. dif., difference. Smple size: CHL2 (n ¼ 63 (totl units)/35 (units with ilterl FRAs) from 6 mice), CHL6 (n ¼ 59/27 units from 5 mice), CHL2 (n ¼ 49/27 units from 5 mice) nd shm-operted controls (n ¼ 42/77 units from 5 mice). Asterisks nd horizontl lines indicte significnt differences reltive to the shm group or ssocited pir, respectively, ccording to ANOVA post-hoc pirwise sttistics djusted for multiple comprisons (Po.5). NATURE COMMUNICATIONS 4:2547 DOI:.38/ncomms & 23 Mcmilln Pulishers Limited. All rights reserved.

6 NATURE COMMUNICATIONS DOI:.38/ncomms3547.3±.2; Shm,.44±.2; ANOVA post-hoc comprisons, n ¼ 22, 24 nd 95, respectively, Po5 5 for ll comprisons), yet the decrese ws significntly greter in CHL2 recordings compred with either CHL6 or CHL2 (ANOVA post-hoc comprisons, n ¼ 262, Po5 5 for oth, Fig. 3g), mtching n erlier description tht A url dominnce plsticity effects susequent to unilterl CHL re oserved into dulthood ut re most pronounced when symmetric hering loss is initited shortly fter hering onset 5. Other thn this grded chnge in contrlterl response is, most spects of interurl frequency tuning were only modified y CHL initited t P2, the ge in normtive development when response thresholds nd preferred frequency tuning were poised to chnge most ruptly. Development of ILD selectivity. The position of sound source long the horizon is encoded centrlly y neurons sensitive to differences in the loudness or timing of sounds rriving t ech er. Therefore, the development of inurl integrtion must predominntly reflect mturtion of centrl inurl circuits. In mmmls such s rts nd mice, which hve smll hed circumferences, high-frequency hering rnges nd virtully nonexistent medil superior olivry nucleus, ILD contriutes fr more ctionle informtion for sound locliztion thn interurl time differences 29,3. In response to rief dichotic sounds tht vry in ILD or sptil loction, most corticl units exhiit roust responses to contrlterl stimuli nd suppressed responses to ipsilterl stimuli Thus, most uditory cortex units, whether recorded in dulthood or shortly fter hering onset hve een descried s EI, ctegoricl lel denoting net excittory (E) input from the contrlterl er nd net inhiitory (I) input from the ipsilterl er 35,36. Severl studies hve demonstrted tht the corticl representtion of inurl sound level is considerly more nunced thn the ctegoricl lel EI would suggest 34. In fct, spikes evoked y dichotic sounds my contin informtion out multiple inurl level comintions, nd the representtionl slience of disprte stimulus fetures cn e wx nd wne on distinct time scles within the overll stimulus-evoked response period To further explore the possiility tht spikes ssocited with contrlterl versus ipsilterl ILD comintions my occur in distinct periods within the overll stimulus-evoked response, nd the mturtion of this dichotomous ILD sensitivity my e regulted on distinct developmentl time scles, we dopted n unised rute force serch lgorithm to identify nd seprtely nlyse the post-stimulus time periods ssocited with the strongest differentil sensitivity to contrlterl versus ipsilterl ILD comintions. At the onset of hering, short-ltency spikes were strongly tuned for contrlterl ILDs in mnner consistent with the EI designtion previously descried in infnt nd dult A units (Fig. 4). Moreover, t the onset of hering, even the optiml temporl windowing filed to cpture ny selectivity for ipsilterl ILDs (Fig. 4). Recordings mde from mice just few dys older reveled similr short-ltency response period contining contrlterl ILD sensitivity (Fig. 4c), ut in contrst to recordings mde t P cler sensitivity for ipsilterl ILDs ws present t longer ltencies (Fig. 4d). Looking cross the developmentl ges studied here, the optiml period for contrlterl ILD sensitivity occurred significntly erlier within the overll response period thn the optiml ipsilterl ILD period (mixed-design ANOVA, n ¼ 6, F4,66, Po 6 for oth onset nd offset min effects; Fig. 4e). ILD sensitivity chnged significntly over the erly period of hering, lthough in different wys nd t different rtes within the erly- nd lte-occurring response windows. At the onset of hering, A inurl tuning ws essentilly purely EI. Contrlterl ILD sensitivity ws reltively constnt cross ge, lthough ILD slopes flttened somewht etween P7 nd dulthood, resulting in sutle ut significnt reduction in the short-ltency contrlterl ILD slope cross development (onewy ANOVA, n ¼ 6, F ¼ 56.25, Po 6 ; lck symols in Fig. 4f). Conversely, negtively sloped ipsilterl ILD sensitivity ws rrely oserved t the onset of hering (Fig. 4, ottom row, nd Fig. 4f, red symols). However, negtive slope vlues indicting seprle ipsilterl ILD sensitivity in long-ltency spikes emerged severl dys lter, ecme mximlly defined t P5 nd P6 nd returned to n intermedite, lthough still clerly seprle, stte in dulthood (one-wy ANOVA, n ¼ 6, F ¼ 47., Po 6 ; Fig. 4f). Trnsient hering loss wekens corticl ILD sensitivity. The normtive course of ILD mturtion in A suggested tht contrlterl ILD sensitivity ws roughly dult-like t the onset of hering, wheres the weker, temporlly delyed response to ipsilterl ILDs ws effectively sent t the onset of hering nd continued to chnge significntly through P7. Working off the premise tht the developmentl windows for experience-dependent reorgniztion would coincide with the period of gretest chnge in normtive development, we hypothesized tht contrlterl ILD sensitivity would e only wekly ffected y CHL. By contrst, the effect of CHL on ipsilterl ILD sensitivity would e oth greter in mgnitude nd developmentlly delyed compred with oth contrlterl ILD nd interurl FRA chnges (Fig. 3). The exmple recordings shown in Fig. 5 c confirm tht trnsient unilterl CHL disrupted distinct spects of inurl integrtion depending upon whether it egn t P2 or P6. Compred with shm recordings (Fig. 5), CHL t P2 ws ssocited with slightly reduced contrlterl ILD slope vlues (.8±. versus.±., ANOVA post-hoc comprison, n ¼ 292, Po 6 ) ut comprle sensitivity to ipsilterl ILDs (.3±. versus.3±., ANOVA post-hoc comprison, n ¼ 292, P ¼.; Fig. 5,d). The opposite pttern ws noted in CHL6 recordings; nmely, contrlterl ILD sensitivity ws similr to controls (.±., ANOVA post-hoc comprison, n ¼ 37, P ¼.), yet ipsilterl ILD sensitivity ws olished (.±., ANOVA post-hoc comprison, n ¼ 37, Po 6 ;Figs.5c,d).Thesesmetrendswerenotedinthe est ILD, defined s the ILD comintion tht yielded the highest verge firing rte. In CHL2 recordings, the preferred ILD within the contrlterl period ws shifted closer to zero thn shms (3±.77 versus 7.8±.46 db, ANOVA post-hoc comprison, n ¼ 292, Po 5 5 ), yet the est ipsilterl ILD ws equivlent ( 6.37±.92 versus 4.78±.3 db, ANOVA post-hoc comprison, n ¼ 292, P ¼.; Fig. 5e). Conversely, inititing hering loss t P6 hd no effect on the preferred ILD in the contrlterl period (6.79±.36 db, ANOVA post-hoc comprison, n ¼ 37, P ¼.), yet the est ILDs from the ipsilterl response window were significntly more contrlterlly ised thn in shm recordings (4.23±.2 db, ANOVA post-hoc comprison, n ¼ 37, Po 6 ;Fig.5e).Inititing trnsient CHL t P2 hd no effect on the ILD slope or est ILD vlues derived from either of the response windows when compred with shm controls (ANOVA post-hoc comprisons, n ¼ 26, P4.6 for ll comprisons; Fig. 5d,e). Systemtic differences in the timing of the contrlterl (23 3 ms poststimulus onset cross ll groups) or ipsilterl ILD windows (5 56 ms post-stimulus onset cross ll groups) were not oserved. 6 NATURE COMMUNICATIONS 4:2547 DOI:.38/ncomms & 23 Mcmilln Pulishers Limited. All rights reserved.

7 NATURE COMMUNICATIONS DOI:.38/ncomms3547 ARTICLE No. of spikes Ipsilterl db SPL Spikes/s e Post-stimulus time (ms) 5 2 Time (ms) Contrlterl db SPL 2 ILD Mx No. of spikes Slope = ILD (db) P contr period Slope =.5 Entire response Contr ILD period Ipsi ILD period 5 P ipsi period P/2 P3/4 P5/6 P7 Adult Postntl ge 2 f ILD slope (.u.) c P5 contr period Slope = Contr ILD period P5 ipsi period 2 Slope = Ipsi ILD period P/2 P3/4 P5/6 P7 Adult Postntl ge Figure 4 Mturtion of ILD sensitivity during the first week of hering. ( d) Top rows: post-stimulus time histogrms of spikes recorded from n A units t P (,) or P5 (c,d) following -ms dichotic white noise urst (horizontl r). Histogrm colouring depicts timing of spikes tht showed the strongest differentil sensitivity to contrlterl ILDs (lck) nd ipsilterl ILDs (red). Middle rows: ule plots present firing rte s function of ILD cross five men inurl levels sed on the spike windowing shown ove. Het mp is scled to the normlized firing rte within ech time window, wheres circle dimeter is normlized to the mximum firing rte cross oth time windows. Bottom rows: fint lines represent firing rte chnges cross ILD t ech of five inurl levels etween threshold nd 2 db4threshold. Thick nd dshed lines represent the men ILD-rte function nd its liner fit, respectively. Numericl slope of the fit line (the ILD slope vlue) is provided. (e) Men±s.e.m. onset (lower vlue) nd offset (higher vlue) of the entire stimulus-evoked response period (light gry), optiml contrlterl (drk grey) nd ipsilterl (red) ILD periods. (f) ILD slope vlues cross ll ges (n ¼ 39 units from 6 P/P2 mice, n ¼ 48 form 7 P3/4 mice, n ¼ 8 from 6 P5/6 mice, n ¼ 96 from 3 P7 mice nd n ¼ 47 from 9 dult mice). Positive nd negtive slope vlues indicte preference for contrlterl nd ipsilterl ILDs, respectively, for spikes contined within the optiml contrlterl (lck) or ipsilterl (red) ILD response periods. Lines represent men vlues t ech ge. Ech dt point is one recording site. A slight rndom jitter is imposed on ll dt points to visully distinguish identicl vlues in the sctterplots. d To ddress the possiility tht ny stimulus feture encoded y long-ltency spikes might e regulted y developmentlly delyed criticl period rther thn just ILD per se, we revisited the developmentl regultion of url dominnce plsticity (Fig. 3g), only this time we seprtely performed the nlysis on spikes occurring in the erly, mid nd finl portion of the tone-evoked response period. Although contrlterl is decresed in lter epochs of the tone-evoked response, the chnge ws consistent for units recorded in ll groups, such tht the sme developmentl regultion of frequency tuning ws oserved in oth erly nd lte response periods (Fig. 6). This suggests tht long-ltency spikes representing ipsilterl ILDs re specificlly vulnerle to the effects of CHL t lter ges. Discussion A inurl response properties were firly mture t the onset of hering. The preferred frequency for ech er ws closely ligned (Fig. e) nd the contrlterl is in response threshold ws similr to dults (Fig. f). A mrked elortion of BF rnge nd steep decline in response thresholds were noted etween P nd P4 (Fig.,c), ut these chnges could e ccounted for y ongoing cochler mturtion (Fig. d). We lso noted roust EI-like ILD sensitivity in short-ltency spikes t the onset of hering tht ws qulittively similr to recordings in older mice (Fig. 4,c), in keeping with previous reports of well-defined ILD sensitivity shortly fter the onset of hering in cts nd ts 35,36. The precocious mturtion of inurl sensitivity is likely to rise from the sustntil refinement of rinstem inurl circuits tht is known to occur efore the onset of hering However, y utilizing rute force serch lgorithm tht scnned the poststimulus response period for spike epochs tht were optimlly sensitive to oth contrlterl nd ipsilterl ILDs, we noted the delyed mturtion of long-ltency response period contining fewer spikes tht were nevertheless clerly ssocited with ipsilterl ILDs. Ipsilterl ILD sensitivity ws sent t the NATURE COMMUNICATIONS 4:2547 DOI:.38/ncomms & 23 Mcmilln Pulishers Limited. All rights reserved.

8 NATURE COMMUNICATIONS DOI:.38/ncomms3547 No. of spikes 5 25 Shm contr period Shm ipsi period CHL 2 contr period CHL 2 ipsi period Time (ms) Ipsilterl db SPL ILD Spikes/s c No. of spikes Time (ms) Contrlterl db SPL CHL 6 contr period No. of spikes Mx CHL 6 ipsi period 2 35 d ILD slope (.u.) Contr ILD period Slope =. 2 Slope =. Slope =.8 2 Slope = ILD (db) Ipsi ILD period Ipsilterl db SPL e CHL 2 CHL 6 CHL 2 Shm Spikes/s Contrlterl db SPL 35 Slope = ILD (db) Slope = Best ILD (db) 5 5 CHL 2 CHL 6 CHL 2 Shm Figure 5 Temporry CHL disrupts short- nd long-ltency ILD sensitivity t distinct ges during the first week of hering. ( c), PSTHs (top row), inurl interction mtrices (middle row) nd ILD-rte functions (ottom row) derived from the optiml contrlterl nd ipsilterl ILD response periods (lck nd red, respectively) from representtive A recording sites in shm nd poloxmer-injected mice. All plotting conventions re identicl to those used in Fig. 4. Note the doule dissocition etween disruptions of short-ltency contrlterl ILD sensitivity in the CHL2 unit () nd long-ltency ipsilterl ILD sensitivity in the CHL6 unit (c) compred with shm (). (d) Slope of the liner fits pplied to ILD-rte functions derived from the erly contrlterl (lck) nd lte ipsilterl (red) ILD response windows (n ¼ 27 shm units from 5 mice, 65 CHL2 units from 6 mice, 43 CHL6 units from 5 mice nd 7 CHL2 units from 5 mice). (e) ILD ssocited with the highest firing rte (tht is, the est ILD) sed on the contrlterl (lck) versus ipsilterl (red) ILD response windows. All vlues represent men±s.e.m. Asterisks indicte significnt difference reltive to the shm group with n ANOVA fter correcting for multiple comprisons (Po.5). onset of hering (Fig. 4) ut cme online rpidly, reching its pek y P5 P6 (Fig. 4d,f). One hypothesis is tht the delyed mturtion of ipsilterl ILD sensitivity in long-ltency spikes might e trced to the development of intrcorticl inhiition, which is thought to feture more protrcted development thn excittion 8,46 (lso see ref. 47). Auditory deprivtion hs een shown to interrupt the progressive reduction in inhiitory response durtion tht 8 NATURE COMMUNICATIONS 4:2547 DOI:.38/ncomms & 23 Mcmilln Pulishers Limited. All rights reserved.

9 NATURE COMMUNICATIONS DOI:.38/ncomms3547 ARTICLE Lst third Second third First third Entire response 8 Level (db) Contr/deprived er Ipsi/non-deprived er Overly Aurl dominnce Frequency (khz) ADI =.2 ADI =.3 ADI =.28 ADI =. Spike count I>C C=I C>I Mx ADI (.u.).5 Shm CHL2 CHL6 CHL2 Figure 6 Delyed criticl period for sound fetures coded y long-ltency spikes is specific to ILD sensitivity. () A FRAs derived from stimuli delivered seprtely to the contrlterl/deprived (left column) nd ipsilterl/non-deprived (middle column) ers in CHL6 mouse. Aurl dominnce, quntified s the rtio of contrlterl/ipsilterl firing rtes for ll tone comintions contined within the union of the two receptive fields is provided in the right column, where more positive url dominnce index (ADI) reflects contrlterl dominnce in the men of ll rtio vlues. The overll url dominnce cptured cross the entire post-stimulus spiking response period (top row) cn e ttriuted to strong contrlterl is in the first nd middle third of the response period (second nd third rows, respectively) nd more lnced contrlterl nd ipsilterl response strength in the finl portion of ech response period (ottom row). () When url dominnce is nlysed for ll recording sites with ilterl FRAs (n ¼ 35 CHL2 units from 6 mice, 27 CHL6 units from 5 mice, 27 CHL2 units from 5 mice nd 77 shm units from 5 mice), it is evident tht lthough contrlterl dominnce my lessen in the finl portion of the tone-evoked response period, it does so eqully for shm controls nd ll CHL groups. Therefore, the ge-dependent regultion of url dominnce effects is similr cross ll portions of the monurl tone-evoked spiking response, unlike the dichotomous effect of CHL on ILD sensitivity contined in erly- versus lte-response periods. Vlues represent men±s.e.m. I, ipsilterl; C, contrlterl. Horizontl lines indicte significnt difference etween two groups fter correcting for multiple comprisons (ANOVA post-hoc pirwise comprisons, Po.5). normlly occurs in the erly postntl period 46,48,49. Thus, longltency responses to wek, delyed inputs (such s ipsilterl ILDs) my emerge only when intrcorticl inhiition hs contrcted to its mture durtion, ut not when inhiition is normlly prolonged, s would occur t hering onset 5 or following hering loss 6. The vlidity of this hypothesis remins to e tested with in vivo whole-cell recording, which offers the dvntge of relting ILD spike tuning to the underlying interctions etween synptic excittion nd inhiition 5. Looking eyond underlying mechnisms, multiplexed code for ILD sensitivity could gretly increse the ndwidth for representing inurl sound properties. With this strtegy, units could dynmiclly encode multiple comintions of ILD nd men inurl level (rther thn single fixed property) ccording to when spikes occur during the post-stimulus period, s hs een suggested for oth spectrl 38,52 nd inurl locliztion cues Although mny spects of inurl sound representtions were dult-like t the onset of hering, the mintennce of these response properties ws strongly disrupted y erly experience with imlnced inurl cues. Trnsient hering loss in the contrlterl er ws ssocited with elevted thresholds (Fig. 3), enhnced sensitivity to the non-deprived er (Fig. 3), elevted spontneous firing rtes (Fig. 3c), decresed precision of interurl FRA coregistrtion (Fig. 3d f) nd reduced contrlterl dominnce in tone-evoked response rtes (Fig. 3g). Other thn the elevtion of contrlterl response threshold t P6 nd comprtively wek shifts in url dominnce t oth P6 nd P2, reorgniztion of interurl frequency tuning ws only oserved when CHL ws initited t P2. By contrst, unilterl deprivtion t P2 hd comprtively wek effects on ILD tuning tht were limited to short-ltency spikes encoding contrlterl ILDs (Fig. 5). However, long-ltency sensitivity to ipsilterl ILDs ws strongly disrupted when CHL ws introduced t P6, ut not t P2 or P2, the sme dy ipsilterl ILD selectivity reched its mximl level in normtive development (Fig. 5c,e). A previous study from our group descried drmtic chnge in contrlterl nd ipsilterl frequency tuning ccompnied y ctegoricl remodelling of ILD sensitivity following reversile er cnl ligtion in rts 5. The findings reported here re similr in kind, lthough comprtively sutle in degree. We ttriute this difference to the fct tht CHL ssocited with poloxmer injection ws less severe thn er cnl ligtion (n verge of 5 2 db here versus 3 db in the previous study), the period of deprivtion ws fr shorter (B7 dys here versus 6 dys in the previous study), nd the post-chl recovery time efore neurophysiologicl recordings ws longer (B7 dys here versus severl hours in the previous study). The outcome of our originl study motivted us to more precisely proe the developmentl regultion of enduring plsticity effects stemming from mild CHL tht resemled out of otitis medi in humn infnts. We would rgue tht these two studies provide resonle upper nd lower ound on the mgnitude of corticl plsticity effects ssocited with reversile unilterl CHL in erly life, lthough the precise timing of developmentl vulnerility to NATURE COMMUNICATIONS 4:2547 DOI:.38/ncomms & 23 Mcmilln Pulishers Limited. All rights reserved.

10 NATURE COMMUNICATIONS DOI:.38/ncomms3547 CHL will likely depend upon the species, severity nd durtion of hering loss. Sptil hering rises from three redundnt coustic cues: ILD, interurl time differences nd monurl spectrl cues (lthough the slience of ech cue will vry etween species ccording to fctors such s hed size, hering rnge nd outer er shpe). When one cue is devlued, dult humns nd ferrets lern to dptively reweight informtion from other cues or remp the correspondence etween the ltered cues nd sptil loctions. For instnce, with severl consecutive dys of prctice, dult ferrets or humns outfitted with monurl erplug cn lern to shift their sensitivity to other cues nd eventully recover norml zimuthl locliztion ccurcy 53,54. Juvenile ferrets rered with n intermittent unilterl erplug develop istle neurl nd ehviourl representtion of sound loction tht dptively switches etween emphsizing spectrl cues through the unplugged er or dichotic cues during periods when oth ers re unostructed 55. Similrly, dult humn sujects lern over severl week period to ccurtely loclize sound sources long the elevtion xis when the spectrl locliztion cues nturlly provided y the geometry of their own outer ers re exchnged for new set of spectrl cues from foreign ers 56. Wheres the dult nervous system is le to flexily nd dptively reweight inurl cues ccording to the listening demnds imposed y the prticulr environment, chronic experience with consistently impoverished locliztion cues in erly life cn hve lsting nd negtive impct on inurl hering. Mmmls rered with monurl er plugs exhiit persistent inurl hering deficits when tested lter in life, fter their hering is udiometriclly norml 57,58. Similrly, humns orn with unilterl er cnl mlformtions tht re surgiclly repired in lter life exhiit sunorml ility to mke use of inurl cues, where the degree of ehviourl deficit is positively correlted with their ge t the time of corrective surgery 59,6. Persistent inurl hering deficits re fr more common in instnces where CHL ccompnies otitis medi in erly childhood. According to peditric udiology studies nd the ltest US census dt, B2% of children (or 2.6 million children in the United Sttes lone) will experience t lest one out of otitis medi severe enough to cuse rief, mild CHL (425 dbhl) efore reching 5 yers of ge 7,8. These children re t fr greter risk to develop mlyudi in lter life thn children who hve otitis medi tht is not severe enough to introduce CHL 8. Individuls tht fll under the mlyudi spectrum exhiit reduced inurl relese from msking 6,62, normlly poor sound locliztion ccurcy 63 nd deficits in complex spectrotemporl processing 64 tht persist for yers fter the peripherl hering loss hs een resolved. Prdoxiclly, plsticity of inurl circuits in the uditory cortex my e oth the cuse of irregulr inurl sound encoding during development s well s its most likely source of remedition in lter life. Sustntil ehviourl nd neurl improvements in discriminting sutle differences in sound locliztion cues hve een reported fter severl dys of perceptul trining in rodents nd humns 65,66. The lifelong plsticity of the dult uditory cortex is thought to e essentil for the ility of mmmls to grdully dpt to disordered inurl cues s phrmcologicl inctivtion of the uditory cortex prevents the lerning-induced recovery of ehviourl sound locliztion ccurcy following unilterl er plugging 67. Future work will e required to ddress the permnence of the chnges descried here nd to estlish whether pplied neuroplsticity protocols in the form of uditory trining or neuromodultory piring strtegies 68 could e used to remedite suoptiml inurl sensitivity in nimls, nd eventully humns, recovering from impoverished uditory experience in erly life. Methods Animl preprtion. All procedures were pproved y the Msschusetts Eye nd Er Infirmry Animl Cre nd Use Committee nd followed the guidelines estlished y the Ntionl Institutes of Helth for the cre nd use of lortory nimls. A totl of 78 CBA/CJ mice of either sex were used for ll experiments. The morning tht new litter of pups ws first oserved ws designted s postntl dy P. Mice were nesthetized with ketmine nd xylzine for ll experiments involving physiologicl mesurements (induction with 8 mg kg ketmine nd 6 mg kg xylzine; 45 mg kg mintennce doses of ketmine, s needed). Mice were nesthetized with isoflurne (5% induction,.5 2.5% mintennce in oxygen) for cses where mnipultions or inspections of the er were performed without physiologicl mesurements. For ll procedures, mice were plced top homeothermic lnket system with skin temperture mintined t 36.5 C nd the hed immoilized with modified hed-holder. All experiments were performed inside sound-ttenuting chmer. Acoustic ssemlies consisted of two miniture dynmic erphones used s sound sources nd n electret condenser microphone coupled to proe tue to mesure sound pressure ner the erdrum. For terminl recording procedures, the externl er ws severed t the tympnic ring. The tip of ech sound delivery tue ws positioned ginst the tympnic ring nd clirted efore ech experiment. In P mice, cre ws tken to seprte the metus from the tympnic memrne to crete direct pth for the coustic stimulus. A lumen hd formed in the externl metus y P2; thus, this step ws unnecessry. Middle er poloxmer hydrogel injection. A slit ws mde in the left trgus to llow etter visuliztion of the tympnic memrne. A smll hole ws mde in the prs flccid to permit the outflow of excess solution. Borosilicte glss cpillry tuing ws pulled to fine, tpering point (B5 mm) nd the lunt end fixed to syringe infusion set. The syringe contined 2% (w/w) solution of poloxmer 47 (Spectrum Chemicls) nd lue dye (FD&C Blue ) dissolved in doule deionized wter. The solution ws prepred immeditely efore use nd mintined t B5 C up to the point of injection. The injection pipette ws secured within threedimensionl micropositioner nd the pipette tip dvnced through the posterior qudrnt of the prs tens. Approximtely 5 ml of poloxmer solution ws injected under visul inspection through n operting microscope until lue fluid hd filled the middle-er cvity. Excess solution ws quickly removed with n sorent point efore it trnsitioned to gel. Injections were mde on two successive dys eginning on P2, P6 or P2. For the shm procedure, the mouse ws nesthetized t one of the ges listed ove nd the trgus ws snipped. The middle er ws left intct owing to concerns tht tympnic memrne puncture, on its own, ws form of CHL s well s portl for middle er infection. Otocoustic emission mesurement. The proe-tue microphone ws clirted in smll coupler with /8 condenser microphone. Stimuli were generted digitlly, nd sound pressure ws mplified nd digitlly smpled t 5 ms. Two tones with different crrier frequencies were delivered through the erphones. The frequencies were kept t constnt rtio (f 2 /f ¼.2), nd the f 2 primry ws presented t db elow the f level. The resultnt DPOAE ws mesured t the 2f f 2 frequency. Ech er cnl SPL mesurement ws otined from.6 s of spectrl nd wveform verging. DPOAEs were mesured t five proe frequencies (f 2 ¼ 8 32 khz in.5 octve intervls) nd 3 levels (f 2 ¼ 2 8 db SPL in 5 db increments). The coustic noise floor ws mesured t frequencies strddling the DPOAE frequency in the spectrum verge. DPOAE threshold ws defined s the lowest of t lest two contiguous f 2 levels for which the DPOAE mplitude ws t lest 3 db greter thn the noise floor. ABR mesurement. Suderml needle electrodes (Grss) were inserted into the ventrl spect of ech pinn nd ground electrode t the se of the til. ABRs were ndpss filtered (.3 3 khz), mplified, (Grss) nd smpled y the A/D ord t khz. ABR ws mesured with tone ursts (5 ms durtion,.5 ms onset/offset gtes, 3 Hz repetition rte) t 8, 6 nd 32 khz (chronic experiments) or 6 khz (cute experiments). Stimulus level ws vried from 2 8 db SPL, initilly in 5-dB steps ut incresing to -db steps fter criterion response mplitude ws reched (.55 mv pek-to-pek). Stimuli were presented in opposite-polrity pirs to ttenute the contriution of microphonic potentils to the ABR wveforms. ABRs were verged from 52 stimulus pirs (or 256 in the event the criterion response mplitude hd een reched). Wve threshold ws defined for ech stimulus type sed on visul inspection of the stcked ABR wveforms. Threshold ws defined s the lowest sound level tht could relily produce stimulus-evoked pek tht followed the progressive trend for decresing mplitude nd incresing ltency cross the full rnge of sound levels. Corticl unit recordings. A crniotomy ws mde over the right uditory cortex nd the rin surfce ws covered with high viscosity silicone oil. The dur mter ws left intct. Recordings were mde from the middle lyers of A with 4-shnk 6-chnnel silicon proe (77 mm 2 contct re, 5 mm contct seprtion, 4 contcts per shnk, 25 mm seprtion etween shnks; NeuroNexus Technologies). A ws distinguished from other corticl fields on the sis of crnil lndmrks nd low-to-high, cudl-to-rostrl tonotopic orgniztion 69,7. One to NATURE COMMUNICATIONS 4:2547 DOI:.38/ncomms & 23 Mcmilln Pulishers Limited. All rights reserved.

11 NATURE COMMUNICATIONS DOI:.38/ncomms3547 ARTICLE four seprte penetrtions were mde per mouse. Rw signls were digitized t 32 it nd ndpss filtered t 3 5, Hz. High SNR multi-unit spikes were detected using n dptive threshold set to s.d. ove the men of 5 s running verge (OpenEx, Tucker-Dvis Technologies). For ech penetrtion, FRAs were derived from tone pips (5 ms durtion, 4 ms rised cosine rmps, khz in.5 octve steps, cross mximum rnge of 9 db SPL, interleved nd rndomized for ech er, repeted twice) delivered independently to ech er (7 ms stimulus onset synchrony). ILD sensitivity ws mesured from the unit responses to 45 dichotic white noise ursts of vrying contrlterl nd ipsilterl level ( ms durtion, 5 ms rised cosine rmps, 5 repetitions). Contrlterl nd ipsilterl sound levels rnged from the minimum response threshold to 2 db ove threshold (5 db steps) with ILDs spnning 2 to 2 db (5 db steps). To estimte the level of coustic ttenution provided y the hed, we performed extrcellulr unit recordings from the left nd right centrl nucleus of the inferior colliculus using the sme methods for nesthesi, sound delivery nd neurophysiologicl recordings descried ove. Before recording, the right stpes ws removed nd the cochler fluids were drined, therey inducing complete unilterl hering loss. Sound delivery tues were then positioned ginst ech tympnic ring (Fig. 7 d). Tones nd white noise ursts were presented to the intct or destroyed er. We were unle to elicit response from tone ursts presented to the cochlectomized er t ny frequency up to 8 db SPL, therey ssuring tht tonl receptive fields were fithful to the stimulted er (Fig. 7e). Noise ursts delivered to the cochlectomized er could drive 69% of the recorded units with thresholds tht were elevted y 6.6±.7 db (n ¼ 45, men±s.e.m.) ove the sme unit s sensitivity to stimultion of the intct cochle (Fig. 7f). Our nlysis of noiseevoked responses ws restricted to stimuli with ILDs of±2 db nd men inurl levels etween threshold to 2 db ove threshold (tht is, for unit with men inurl threshold t 6 db SPL, we presented noise ursts to the contrlterl nd ipsilterl ers rnging from 5 to 9 db SPL s illustrted in Fig. 4). With this pproch, the most intense sound presented would e only 3 db ove the response threshold, which is less thn hlf of the men threshold for ctivting the contrlterl cochle vi cross-tlk. Therefore, the neurl responses presented here cn e ttriuted to the er tht ws directly stimulted. Anlysis of corticl unit recordings. The FRA ws first smoothed y 3 3 point medin filter, then the spike counts were summed cross sound frequencies or sound levels to crete spike-frequency or spike-level functions, ech of which were susequently smoothed gin with five-point medin filter. The spikefrequency function ws inverted (to crete V-shpe) nd the tip set t the minimum threshold, defined s the inflection point on the spike-level function. FRA BF ws defined s the frequency ssocited with the gretest numer of spikes within the tone-evoked response period. The url dominnce index ws defined in sites with ilterl FRAs s the men rtio of contrlterl versus ipsilterl firing rtes seprtely defined for ech frequency-level comintion contined within the union of the contrlterl nd ipsilterl FRAs. Receptive field overlp ws defined s the numer of frequency intensity points contined within the intersection of the contrlterl nd ipsilterl FRAs divided y the numer of points contined within their union. The developmentl nlysis ws sed on,262 unit recordings in 35 mice, of which, units were responsive to ipsilterl or contrlterl sound (P, n ¼ 3/3 (recording sites/nimls); P, n ¼ 55/3; P2, n ¼ 7/3; P3, n ¼ 42/3; P4, n ¼ 96/4; P5, n ¼ 7/3; P6, n ¼ 9/3; P7, n ¼ 62/ 4; dult (8 4 weeks) n ¼ 375/9). The effects of hering loss were sed on 62 units in 2 mice (Shm, n ¼ 42/5; CHL2, n ¼ 63/6, CHL6, n ¼ 59/5 nd CHL2, n ¼ 48/5). For dichotic noise urst stimuli, nlysis ws restricted to recording sites tht yielded FRA for either the contrlterl or ipsilterl er, were significntly driven y dichotic white noise ursts (unpired Student s t-test of pre- versus poststimulus spike rtes for ll stimulus comintions, Po.) nd hd minimum response threshold for verge inurl sound levels r75 db SPL (n ¼ 6 nd 552 recordings sites for the developmentl study nd plsticity study, respectively). Minimum threshold ws defined t ech recording site s the lowest men inurl level for which the firing rte of t lest one ILD comintion ws more thn 3.5 s.d. ove its spontneous firing rte. The timing of the overll noise-evoked c d Inferior colliculus left right Sound level (db SPL) Tone frequency (khz) Tone ursts Sound level (db SPL) Post-stim. time (ms) White noise ursts Firing rte (sp s ) Sound level (db SPL) e Tone urst response threshold (db SPL) f Brodnd noise urst threshold (db SPL) Not responsive Not responsive Right IC recordings, n = 6 Left IC recordings, n = 3 Stimulus position Right IC recordings, n = 32 Left IC recordings, n = 3 Stimulus position Figure 7 FRA nd ILD mesurements re not contminted y the contrlterl trnsfer of coustic energy cross the hed. ( d) Extrcellulr unit recordings were mde from the right (,) nd left (c,d) centrl nucleus of the inferior colliculus fter the right cochle hd een destroyed. Sound delivery tues were positioned ginst the tympnic ring of the left nd right ers, per norml. Exmple FRAs (second column), spike rstergrms (third column) nd rte-level functions (fourth column) for individul recordings corresponding to the electrode nd stimulus configurtion depicted in the first column. Rstergrms nd rte-level functions re derived from ms white noise ursts. (e,f) minimum thresholds for responses evoked y tones (e) nd noise ursts (f) re displyed for sounds delivered to the intct (left) nd cochlectomized (right) ers. Unit responses for white noise stimuli presented to the cochlectomized er could e elicited in 69% of the recordings ut the threshold shift ws fr greter thn the mximum ILD used in Figs 4 nd 5 (n ¼ 45 units, 6.6±.7 db, men±s.e.m.). NATURE COMMUNICATIONS 4:2547 DOI:.38/ncomms & 23 Mcmilln Pulishers Limited. All rights reserved.