Dynamic Patterns of Neurotrophin 3 Expression in the

Dynamic Patterns of Neurotrophin3 Expression in the Postnatal Mouse

Inner Ear

MITSURU SUGAWARA,1,2,3 JOSHUA C. MURTIE,1 KONSTANTINA M. STANKOVIC,1,2

M. CHARLES LIBERMAN,2AND GABRIEL CORFAS1*

1Neurobiology Program, Children’s Hospital and Department of Neurology,Harvard Medical School, Boston, Massachusetts 02115

2Department of Otology and Laryngology, Harvard Medical School and Eaton-PeabodyLaboratory, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts 02114-30963Department of Otolaryngology, Head and Neck Surgery, Tohoku University Graduate

School of Medicine, Sendai, Japan 980-8574

ABSTRACTRecent studies indicate that neurotrophin 3 (NT3) may be important for the maintenance

and function of the adult inner ear, but the pattern of postnatal NT3 expression in this organ hasnot been characterized. We used a reporter mouse in which cells expressing NT3 also express�-galactosidase, allowing for their histochemical visualization, to determine the pattern of NT3expression in cochlear and vestibular organs. We analyzed animals from birth (P0) to adult(P135). At P0, NT3 was strongly expressed in supporting cells and hair cells of all vestibular andcochlear sense organs, Reissner’s membrane, saccular membrane, and the dark cells adjacent tocanal organs. With increasing age, staining disappeared in most cell types but remained rela-tively high in inner hair cells (IHCs) and to a lesser extent in IHC supporting cells. In the cochlea,by P0 there is a longitudinal gradient (apex � base) that persists into adulthood. In vestibularmaculae, staining gradients are: striolar � extrastriolar regions and supporting cells � hair cells.By P135, cochlear staining is restricted to IHCs and their supporting cells, with strongerexpression in the apex than the base. By the same age, in the vestibular organs, NT3 expressionis weak and restricted to saccular and utricular supporting cells. These results suggest that NT3might play a long-term role in the maintenance and functioning of the adult auditory andvestibular systems and that supporting cells are the main source of this factor in the adult. J.Comp. Neurol. 501:30–37, 2007. © 2007 Wiley-Liss, Inc.

Indexing terms: NT3; postnatal; cochlea; utricle; saccule; ampulla; spiral ganglion

It is well established that the trophic factor neurotro-phin 3 (NT3) and its receptor, TrkC, are expressed in theinner ear during embryonic development (e.g., Farinas etal., 2001) and are essential for normal development of theinner ear (Ernfors et al., 1995; Fritzsch et al., 1997a,b).Similarly, evidence that NT3 plays important roles in theadult inner ear is beginning to emerge. For example,Gacek and Khetarpal (1998) showed that recovery fromunilateral surgical labyrinthectomy is impaired in micewith reduced NT3 expression but not in mice with reducedbrain-derived neurotrophic factor (BDNF) or NT4. Morerecently, our analysis of transgenic mice in whichneuregulin-erbB receptor signaling is blocked in cochlearsupporting cells in adults suggested that neuregulin-induced NT3 production by supporting cells of the organ ofCorti is critical for long-term survival of spiral ganglionneurons (Stankovic et al., 2004). In these mice, which

express a dominant-negative erbB receptor in inner earsupporting cells, the cochlea develops normally until �3

This article includes Supplementary Material available via the Internetat http://www.interscience.wiley.com/jpages/0021-9967/suppmat.

Grant sponsor: National Institute on Deafness and Other Communica-tion Disorders: Grant numbers: R01 DC004820 (to G.C.), Core Grant P30DC05209 (to M.C.L.), and RO1 DC0188 (to M.C.L.); Grant sponsor: MentalRetardation Developmental Disabilities Research Center, National Insti-tutes of Health; Grant number: P30-HD 018655 (to G.C.); Grant sponsor:Children’s Hospital Otolaryngology Foundation Research Fund (to G.C.).

*Correspondence to: Gabriel Corfas, Division of Neuroscience, Children’sHospital, 300 Longwood Ave., Boston, MA 02115.E-mail: [email protected]

Received 17 March 2006; Revised 31 August 2006; Accepted 29 Septem-ber 2006

DOI 10.1002/cne.21227Published online in Wiley InterScience (www.interscience.wiley.com).

THE JOURNAL OF COMPARATIVE NEUROLOGY 501:30–37 (2007)

© 2007 WILEY-LISS, INC.

weeks of age, when type I spiral ganglion neurons begin todegenerate. Immediately preceding this degeneration,there is a specific and significant reduction in the levels ofNT3 mRNA in the cochlea. Based on these results, and theobservation that NT3 is expressed by inner hair cell (IHC)supporting cells, we proposed that NT3 plays an impor-tant role in promoting the long-term survival of type Ispiral ganglion neurons.

Information on the pattern of NT3 expression in thepostnatal ear is limited. Some studies reported that NT3is expressed by IHCs of the adult cochlea (Farinas et al.,2001; Pirvola et al., 1994), whereas other studies havedemonstrated that this neurotrophin is expressed by IHCsand their supporting cells (Stankovic et al., 2004). Todefine the pattern of expression of NT3 in the postnatalinner ear, we studied mice in which the Escherichia colilacZ gene is integrated into the NT3 locus (Farinas et al.,1994). In this mouse strain, cells that normally expressNT3 also express �-galactosidase, allowing for their visu-alization and identification by histochemical staining(Fritzsch et al., 1997a). We analyzed cochlear and vestib-ular organs from P0 to P135, to determine the pattern ofNT3 expression during the morphological and functionalmaturation in the early postnatal period and in the ma-ture organ. We found that NT3 is expressed in a dynamicpattern, with the levels of expression and the areas ex-pressing this gene decreasing with age. In the adult innerear, NT3 expression was restricted to inner hair cells andtheir supporting cells in the cochlea and largely to sup-porting cells in the vestibular epithelia. This findingstrengthens the notion that supporting cells are necessaryfor the long-term maintenance and function of the adultauditory and vestibular systems.

MATERIALS AND METHODS

Animals

NT3-lacZ mice (courtesy of Dr. Reichardt, University ofCalifornia-San Francisco) were generated by targeted re-placement of the NT3-coding exon with a construct con-taining a lacZ gene cDNA and the PKCneo marker (Fari-nas et al., 1994). Mice were maintained at the animalfacility at Children’s Hospital, and all procedures werecarried out following protocols approved by the Children’sHospital Animal Care and Use Committee. At least twoanimals (four ears) were evaluated at every age except forP135, which included only one animal. Age-matched, wild-type littermates were used as controls.

Physiological testing

Because the NT3-lacZ mice have only one normal NT3gene, we assessed cochlear function by measuring audi-tory brainstem responses (ABRs) and distortion productotoacoustic emissions (DPOAEs) according to standardtechniques (Kujawa and Liberman, 2006). Threshold sen-sitivity was no different in NT-lacZ hemizygotes and wild-type controls.

LacZ (�-Gal) staining

Mice were anesthetized with 2.5% Avertin (0.2 ml/10 gbody weight) and fixed by intracardial perfusion with2% paraformaldehyde/0.2% glutaraldehyde in 0.1 Mphosphate-buffered saline (PBS, pH 7.4). The temporalbones were dissected, and the cochleae were perfused by

flushing the fixative solution through the oval window.Tissues were then washed three times for 30 minutes atroom temperature and incubated in staining solution (5mM K3Fe(CN)6, 5 mM K4Fe(CN)6 � 3 H2O, 2 mM MgCl2 �6 H2O, 0.01% Na-deoxycholate, 0.02% NP-40, 1 mg/mlX-gal) at 37°C in the dark for 1 or 4 (P0), 5 (P5 and P10),or 6 hours (P15-P135). Tissues were rinsed with PBS for3–5 minutes, postfixed overnight with the same fixingsolution as above, washed with PBS, and decalcified in 4%EDTA. Tissues were then embedded in Araldite by usinga rapid dehydration protocol to minimize washout of re-action product. No differences in the intensity or patternof lacZ staining were observed before and after dehydra-tion. Care was taken to treat all cochleae identically:staining was done in large batches, with ears from allpostnatal ages included in a single staining run. Araldite-embedded materials were sectioned (at 20 �m) on a His-torange (LKB Instruments). Ears of wild-type mice wereprocessed in parallel for control. At least three wild typeswere processed at each age evaluated (up to and includingP20).

Immunohistochemistry

Mice were anesthetized with 2.5% Avertin (0.2 ml/10 gbody weight) and fixed by intracardial perfusion with 4%paraformaldehyde in 0.1 M PBS (pH 7.4). The temporalbones were dissected, and the cochleae were perfused byflushing the fixative solution through the oval window andpostfixed for 2 hours. Temporal bones were decalcified in4% EDTA for 3 days at 4°C, cryoprotected in 30% sucroseovernight at 4°C, embedded in OCT, and sectioned at 15�m. Sections were washed in PBS and blocked for 30minutes in 25% normal goat serum, 0.4% Triton-X, 100mM L-lysine, 1% bovine serum albumin, and 0.05% so-dium azide. Mouse anti-�-galactosidase (1:500, Promega,Madison, WI) and rabbit anti-calretinin (1:500, Chemicon,Temecula, CA) in 3% bovine serum albumin were incu-bated on sections overnight at 4°C. Sections were washedin PBS and blocked with 5% normal donkey serum for 20minutes followed by incubation with donkey anti-mouseAlexa-610 (1:250, Molecular Probes, Eugene, OR) and don-key anti-rabbit Oregon Green (1:250, Molecular Probes)for 1 hour at room temperature.

Imaging

All images of LacZ-stained tissues were obtained withdigital cameras (Orca, Hamamatsu, or Spot). Images wereprocessed by gamma correction and unsharp mask algo-rithm (amount: 70%; radius: 9px; threshold: 1 level) byusing Adobe Photoshop (version 6.0). Images of immuno-fluorescence were obtained with an LSM 510 Zeiss laserscanning confocal microscope with a 40� objective.

RESULTS

Expression gradients in inner-ear wholemounts

Temporal bones of NT3-LacZ mice of different postnatalages from P0 to P135 were processed by �-galactosidasehistochemistry and embedded in plastic. The staining pat-tern was first analyzed in whole mounts (Figs. 1, 2), whichallow visualization of the general pattern of expression aswell as of gradients over space and time. As depicted inFigure 1, when temporal bones of P0 mice were developed

The Journal of Comparative Neurology. DOI 10.1002/cne

31NT3 EXPRESSION IN THE POSTNATAL INNER EAR

for the LacZ reaction for 1 hour, the �-galactosidase stain-ing was most intense in the cochlea, where it presented inan apex � base gradient similar to that observed duringembryogenesis (Farinas et al., 2001). With this incubationtime, the vestibular sensory epithelia exhibited very lightstaining. When tissues from older animals were subjectedto similar treatment, staining was very light (not shown).Therefore, we extended the incubation time to increasethe sensitivity of the staining.

As shown in Figure 2, longer incubations times showedthat �-galactosidase is expressed in the postnatal murineinner ear in a dynamic pattern, with the levels of expres-sion steadily decreasing from apex to base and with in-creasing postnatal age, even beyond the age at which theepithelia are otherwise mature, both structurally andfunctionally (about P21 for the cochlear portion; Kujawaand Liberman, 2006). Control ears (without the lacZ geneinsert) never showed consistent positive reaction productin any structures of the inner ear. At P0, the saccular andutricular maculae were also clearly and darkly stained,the former appearing as a dark U-shaped band and thelatter as an ovoid spot. The saccular membrane wasstrongly stained, and the label continued into Reissner’smembrane of the cochlear duct, especially in the basalturn. The crista from each of the semicircular canals pre-sented a more complex staining pattern, because each ofthese sensory organs consists of two centrally located lacZ-positive hair-cell patches (one at each end of the ampul-lary ridge) and two peripherally located lacZ-positivepatches of dark cells, the cells responsible for ion trans-port in the vestibular portion of the inner ear (see below).

In the cochlear portion of the inner ear, the whole mountimages of Figure 2 clearly show a general decrease instaining intensity with increasing age. Also visible at P10(and later) was the tunnel of Corti, the light strip thatseparates the spiraling cochlear label into inner and outerbands, corresponding to the inner and outer hair cell ar-eas, respectively (see below). By P15, even in these low-power views, it is clear that the staining in the inner haircell area was more intense than that in the outer hair cellarea.

Fig. 1. Left: NT3-LacZ expression (blue reaction product) in wholemounts of the inner ear at postnatal day 0 (P0). The tissue wasdeveloped for the LacZ reaction for 1 hour. The staining was mostintense in the cochlea, where it presented in an apex-to-base gradient.Tissues of wild-type (WT) animals at all postnatal ages (see P0, rightpanel) did not produce any LacZ staining, even when developed for 4hours. Scale bar � 0.5 mm in right panel (also applies to left panel).

Fig. 2. NT3-LacZ expression (blue reaction product) in wholemounts of the inner ear at different postnatal ages. Key structures areidentified in the image of the ear at postnatal day 0 (P0). Cochlearapex and base are indicated. Vestibular organs are labeled, including

the cristae of the three semicircular canals (posterior [P], lateral [L],and superior [S]) and the maculae of the utricle and sacccule. Thepostnatal age, which ranged from P0 to P135, is shown. Scale bar �0.5 mm in lower right panel (applies to all).


32 SUGAWARA ET AL.

In the vestibular organs of the inner ear, the wholemount images of Figure 2 also show a decrease in thestaining intensity of all the hair cell organs. At P20 andbeyond, the expression levels appeared highest in thesaccule and lowest in the semicircular canal cristae; how-ever, even at the oldest age investigated (P135), there wasstill detectable staining in all hair cell organs of the ves-tibular system.

Tissue localization in sectioned material

To investigate the tissue localization of NT3 expressionwith higher resolution, i.e., to identify the cells expressingthe �-galactosidase, the plastic-embedded whole mountswere sectioned and analyzed at higher magnification. Rep-resentative images of the cochlear duct, the utricular andsaccular epithelia, and the epithelia of the posterior am-pulla are shown in Figures 4, 5, and 6, respectively. Im-portantly, to ensure that the blue reaction product trulyidentified �-galactosidase expressing, cells we also stainedsections of the apical region of the P15 cochlea with anti-�-galactosidase antibodies. Figure 3 shows that�-galactosidase antibodies labeled all hair cells and sup-porting cells, the same cells that were labeled by the�-galactosidase reaction (Fig. 4). Furthermore, the rela-tive intensity of the immunostaining of the different celltype was similar to that of the histochemistry (IHC �OHCs � supporting cells). Thus, these results indicatethat the blue-labeled cells represent NT3-expressing cellsand that the intensity of the labeling reaction providessemiquantitative information about the levels of NT3 ex-pression by the different cells.

Cochlea. In the cochlear portion of the inner ear, de-velopment of hair cells proceeds from basal to apical turns.Thus, at any given age, apical regions appear more imma-ture than basal regions. The images of the entire cochlearduct (left panels of Fig. 4) across all ages and cochlearregions indicate that strong NT3-lacZ expression is re-stricted to the cells of the organ of Corti, with weakerexpression seen in the cells of Reissner’s membrane. Ex-

pression was not detected at any age in the spiral liga-ment, the spiral limbus, or the stria vascularis, or in anyneural structures in the spiral ganglion or modiolus.(These neural structures do not appear in the images ofFig. 4.)

Examination of the organ of Corti (right panels of Fig. 4)showed NT3-lacZ signal in all structures of the nascentsensory epithelium at P0; however, even at this earlystage, the darkest signal was in the region of the hair cellsand their immediately adjacent supporting cells (seedashed circles in P0, fourth column). By P10–P15, thelabel was more clearly restricted to hair cells and support-ing cells (see P15, third column), i.e., Deiters’ cells (sup-porting the outer hair cells), pillar cells (surrounding thetunnel of Corti), and the inner border and inner phalan-geal cells (surrounding inner hair cells). By P15, a radialgradient (inner hair cell area darker than outer hair cellarea) was clearly established. In this regard, the pillar-cellstaining (both inner and outer pillars) appeared to trackwith that of the outer hair cell area, not the inner hair cellarea. At the oldest age examined, label appeared to berestricted to the inner hair cell area. In the basal turn,label appeared only in the inner hair cells themselves andnot in their support cells.

Saccule and utricle. The low-power cross-sections ofthe saccule and utricle (Fig. 5, left panels) showed thatNT3-lacZ staining is restricted at all ages beyond P0 to thesensory epithelia, i.e., the macular regions containing haircells, and the saccular membrane. At P0, there was somestaining of cells in the marginal zone, visible in the imagefrom the utricle. No staining at any age was visible in theutricular membrane, nor in the mesenchymal cells under-lying the epithelia or in neural structures including Scar-pa’s ganglion.

The sensory epithelia of the saccule and utricle displayregional variation in their cellular, synaptic, and neuralarchitecture (reviewed by Desai et al., 2005). One impor-tant organizational landmark in these hair cell organs isthe striola, a swathe of hair cells running along the middleof the epithelium, which marks the dividing line betweengroups of hair cells with opposing hair-bundle polarities.At all ages, NT3 expression was higher in striolar thanextrastriolar regions in both the saccule and the utricle(Fig. 5, left panels). In the mouse inner ear, the saccularstriola is more curvilinear than the utricular striolar (asseen clearly in the whole mounts of Fig. 2); thus thesaccular striola typically appears as two dark lacZ-positive bands in the sections, whereas the utricular st-riolar appears as only one band.

Examination at higher magnification (Fig. 5, right pan-els) showed that, at the earliest ages (P0–P5), the stainingintensity within the sensory epithelium was comparablein hair cells and their supporting cells. With increasingage, the signal became weaker and, in contrast to thecochlea, became increasingly limited to the supportingcells. By P30, hair cell staining was very weak.

Crista ampullaris. Ampullae are expanded regions ineach semicircular canal that contain the sense organs forrotational motion, the crista ampullaris. Each ridge-likecrista is covered with patches of sensory cells and flankingregions of dark cells surrounding the ridge, which playimportant roles in the regulation of ionic composition ofendolymph. As seen in the low-power views of Figure 6(left panels), NT3 expression was seen in hair cells andsupporting cells at the center of the sensory cell patch and

Fig. 3. Immunostaining for �-galactosidase (�-Gal). Sectionsthrough the apical region of the P15 cochlea were stained with anti-bodies against �-galactosidase (red) and the inner hair cell markercalretinin (green). The �-galactosidase staining is present in all haircells and supporting cells (SCs) but absent from the tectorial mem-brane. As is the case for the LacZ staining (Fig. 4), the intensity of theimmunostaining in the inner hair cell (IHC) is stronger than that inOHCs and supporting cells. Scale bar � 25 �m in upper right panel(applies to all). DIC: differential interference contrast.



in ampullary dark cells. Expression in both cell types wasstrong at P0 and decreased with age thereafter (Fig. 6).

Based on hair cell density, the sensory cell patches inthe ampulla are divided into three regions of approxi-mately equal area (Lindeman, 1969): the innermost cen-tral zone, the outermost peripheral zone, and the transi-tional intermediate zone. Similar to the striolar region ofthe utricle and saccule, type I hair cells outnumber type IIhair cells in the central zone of the crista with roughlyequal numbers of type I and type II hair cells in theperipheral zone (Desai et al., 2005). As seen in the low-power images in Figure 6, NT3 expression was observed

only in the central zone. As seen in the saccule and utricle,NT3 expression was robust in both supporting cells andhair cells at P0 (Fig. 6, right panels). After P0, NT3 ex-pression levels decreased and became restricted to sup-porting cells.

DISCUSSION

By using NT3-LacZ mice, we show that NT3 is ex-pressed in the postnatal inner ear in dynamic patternsand that these patterns continue to change with age wellbeyond the periods in which the inner ear structures be-

Fig. 4. NT3-LacZ expression in plastic sections through the co-chlear duct (left columns) and the organ of Corti (right columns) showthe postnatal changes in expression level and locus. Each row repre-sents a different postnatal age, as indicated. The “apex” views aretaken from the middle of the second turn; the “base” views are taken

from the middle of the first turn. These two regions correspond to bestfrequencies of roughly 4 and 25 kHz, respectively. IHC, inner hair cell;OHC, outer hair cell; SC, supporting cell; IP, inner pillar cell; OP,outer pillar cell; RM, Reissner’s membrane. Scale bar � 100 �m forleft two columns; 25 �m for right two columns.


34 SUGAWARA ET AL.

come functionally and morphologically mature. Therefore,it is possible that NT3 plays a long-term role in the main-tenance and functioning of the adult auditory and vestib-ular systems.

Previous studies have used either in situ hybridizationor an NT3-lacZ reporter to study the expression patternsof NT3 in embryonic and fetal cochleae from either rat ormouse (Pirvola et al., 1992, 1994; Fritzsch et al., 1999;Qun et al., 1999; Farinas et al., 2001). All studies agreethat NT3 expression can be seen in hair cells and/or theirsupporting cells throughout late embryonic development.However, these earlier studies include only fragmentary

observations in postnatal ears, and none provides datafrom an age-graded series of postnatal animals. Thus thepresent report complements the existing literature. The insitu study in rats suggests that an apical-basal gradient inNT3 expression levels may appear in the P7 ear, althoughno supporting images are presented. Our data clearlyshow an apical-basal gradient in NT3 expression in allears at ages beyond P0. The postnatal gradient in expres-sion of this neurotrophic factor could be a contributor toneural presbycusis, i.e., the observation that age-relateddegeneration of cochlear neurons in the absence of signif-icant hair cell loss occurs in a base-apex gradient, with a

Fig. 5. NT3-LacZ expression in plastic sections through the utric-ular and saccular hair cell epithelia at low power (left columns) andhigh power (right columns) show the postnatal changes in expressionlevel and locus. Postnatal ages are as indicated. For each organ, thesection chosen for photographic documentation was that for which the

number of hair cells was maximal, roughly corresponding to themiddle section through the maculae. HC, hair cell; SC, supportingcell. Scale bar � 100 �m for left two columns; 25 �m for right twocolumns.



prominent steady decline in the numbers of surviving cellsin the basal turn (Otte et al., 1978).

This gradient of NT3 expression may also explain re-sults from our previous studies using transgenic miceexpressing a dominant-negative form of the receptorerbB4 in supporting cells of the inner ear (Stankovic et al.,2004). This dominant-negative receptor was designed toabolish glial response to neuregulin, a factor that caninduce the expression of glial signals that have retrogradeeffects on their associated neurons (Chen et al., 2003;Prevot et al., 2003). In the cochlea, this retrograde signalappears to be NT3, which contributes to the long-termsurvival of adult spiral ganglion neurons (Stankovic et al.,

2004). When DN-erbB4 was expressed in the inner ear bysupporting cells, mice with one copy of the transgene hada reduction in high-frequency hearing, whereas mice withtwo copies had hearing loss extending to all frequencyregions. These results suggest that a relatively low level ofexpression of DN-erbB4 could induce a significant reduc-tion in NT3 expression in the base, where expression ofthis neurotrophin is normally the lowest, thus resulting inthe frequency-specific hearing loss.

The presence of NT3 in adult IHC supporting cells, inaddition to IHCs, suggests that supporting cells providetrophic support to sensory neurons in the spiral ganglion.These supporting cells closely ensheath the unmyelinatedperipheral terminals of neurons underneath IHCs andOHCs, and they express multiple glial markers (Anniko etal., 1986; Furness and Lehre, 1997; Vega et al., 1999; Rioet al., 2002). Thus, they may well play roles similar tothose of glial cells, which have been implicated in thepromotion of neuronal survival. The importance of sup-porting cells in regulating long-term neuronal survivaland neuronal sprouting in the organ of Corti was sug-gested by the above-mentioned study in which blockade ofthe neuregulin signaling pathway in cochlear supportingcells led to a hearing loss due to postnatal degeneration ofcochlear neurons (Stankovic et al., 2004). The importanceof supporting cells was further supported by our study oflong-term effects of ototoxic drugs, which showed that, inareas of hair cell loss, neuronal survival was enhancedwhen supporting cells remained intact, even years afterthe insult (Sugawara et al., 2005). Interestingly, the long-term (� 5-year) neuronal survival was particularly strongin the apical turn, where, according to the present study,the levels of NT3 expression remain highest well intoadulthood.

The functional significance of NT3 in cells of the Reiss-ner’s membrane, which separates two markedly differentcochlear fluids, potassium (K�)-rich endolymph and so-dium (Na�)-rich perilymph, is unclear. However, previousworkers have also noted the fetal expression of BDNF bycells of the Reissner’s membrane (Farinas et al., 2001).Cells of Reissner’s membrane express high levels of manyion transporters (Yoshihara and Igarashi, 1987; Stankovicet al., 1997; King et al., 1998; Yeh et al., 1998), and theyparticipate in absorption of Na� from endolymph (Lee andMarcus, 2003). Similarly, NT3 is expressed by cells in thelimiting membrane of the saccule and in dark cells of theampulla, which have roles in regulating vestibular ionicenvironment (Wangemann, 1995; Stankovic et al., 1997).It is not known whether cells of Reissner’s and limitingmembranes express TrkC, but it is interesting to note thatTrk signaling is involved in regulation of the expressionand activities of ion channels (reviewed by Huang andReichardt, 2003). For example, Trk receptor signalingleads to modulation of Na� and Ca� currents, and activa-tion of several members of the TRP family of cation chan-nels (reviewed by Huang and Reichardt, 2003). Therefore,the possibility that NT3 acts in an autocrine manner tofacilitate ionic regulation through activation of TrkC inboth the auditory and vestibular systems needs to beconsidered.

Unlike the adult cochlea, in which IHCs and supportingcells express NT3, the adult vestibular end organs expressNT3 only in supporting cells. Our data suggest that NT3in vestibular supporting cells provides trophic support forperipheral terminals of neurons that synapse on type I

Fig. 6. NT3-LacZ expression in plastic sections through the am-pulla of the posterior canal at low power (left column) and high power(right column). Postnatal ages are as indicated. Sections are throughthe middle of one of the pair of hair-cell-rich “central zones, whichform mirroring patches on either end of the ampullary crest. HCs,hair cells; SCs, supporting cells. Scale bar � 100 �m for left twocolumns; 25 �m for right two columns.


36 SUGAWARA ET AL.

and type II hair cells. A possible role for NT3 in vestibularfunction in adults was hinted at in a study showing thatrecovery from unilateral surgical labyrinthectomy takesmuch longer in NT3 heterozygous mice compared withwild-type mice and mice with reduced expression of BDNFor NT4 (Gacek and Khetarpal, 1998). This suggested thatNT3 is the early trophic regulator of vestibular compen-sation. In light of our current and earlier findings (Stank-ovic and Corfas, 2003), we suggest that NT-3 involved investibular compensation may also be derived from thevestibular periphery.

It is clear that NT3 plays a significant role in the devel-oping auditory and vestibular systems. The current studydemonstrating nonmonotonic changes in NT3 expressionin the adult auditory and vestibular systems suggests thatNT3 may have long-lasting roles in maintenance and func-tioning of these systems. Furthermore, the specific expres-sion patterns suggest that this long-term role for NT3 maybe necessary not only for the most accepted roles for neu-rotrophins, e.g., the preservation of synaptic contacts andneuronal survival, but possibly also for the maintenance ofionic balance in the inner ear through regulation of theexpression and activity of ion channels.

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Dynamic Patterns of Neurotrophin 3 Expression in the