THE JOURNAL OF COMPARATIVE NEUROLOGY 501:30–37 (2007) Dynamic Patterns of Neurotrophin 3 Expression in the Postnatal Mouse Inner Ear MITSURU SUGAWARA,1,2,3 JOSHUA C. MURTIE,1 KONSTANTINA M. STANKOVIC,1,2 M. CHARLES LIBERMAN,2 AND 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-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts 02114-3096 3Department of Otolaryngology, Head and Neck Surgery, Tohoku University Graduate School of Medicine, Sendai, Japan 980-8574 ABSTRACT Recent 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 has not 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 NT3 expression 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 and cochlear sense organs, Reissner’s membrane, saccular membrane, and the dark cells adjacent to canal 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 Ͼ base) that persists into adulthood. In vestibular maculae, staining gradients are: striolar Ͼ extrastriolar regions and supporting cells Ͼ hair cells. By P135, cochlear staining is restricted to IHCs and their supporting cells, with stronger expression in the apex than the base. By the same age, in the vestibular organs, NT3 expression is weak and restricted to saccular and utricular supporting cells. These results suggest that NT3 might play a long-term role in the maintenance and functioning of the adult auditory and vestibular 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- express a dominant-negative erbB receptor in inner ear phin 3 (NT3) and its receptor, TrkC, are expressed in the supporting cells, the cochlea develops normally until ϳ3 inner ear during embryonic development (e.g., Farinas et al., 2001) and are essential for normal development of the inner ear (Ernfors et al., 1995; Fritzsch et al., 1997a,b). This article includes Supplementary Material available via the Internet Similarly, evidence that NT3 plays important roles in the at http://www.interscience.wiley.com/jpages/0021-9967/suppmat. adult inner ear is beginning to emerge. For example, Grant sponsor: National Institute on Deafness and Other Communica- Gacek and Khetarpal (1998) showed that recovery from tion Disorders: Grant numbers: R01 DC004820 (to G.C.), Core Grant P30 unilateral surgical labyrinthectomy is impaired in mice DC05209 (to M.C.L.), and RO1 DC0188 (to M.C.L.); Grant sponsor: Mental Retardation Developmental Disabilities Research Center, National Insti- with reduced NT3 expression but not in mice with reduced tutes of Health; Grant number: P30-HD 018655 (to G.C.); Grant sponsor: brain-derived neurotrophic factor (BDNF) or NT4. More Children’s Hospital Otolaryngology Foundation Research Fund (to G.C.). recently, our analysis of transgenic mice in which *Correspondence to: Gabriel Corfas, Division of Neuroscience, Children’s neuregulin-erbB receptor signaling is blocked in cochlear Hospital, 300 Longwood Ave., Boston, MA 02115. E-mail: [email protected] supporting cells in adults suggested that neuregulin- Received 17 March 2006; Revised 31 August 2006; Accepted 29 Septem- induced NT3 production by supporting cells of the organ of ber 2006 Corti is critical for long-term survival of spiral ganglion DOI 10.1002/cne.21227 neurons (Stankovic et al., 2004). In these mice, which Published online in Wiley InterScience (www.interscience.wiley.com). © 2007 WILEY-LISS, INC. The Journal of Comparative Neurology. DOI 10.1002/cne NT3 EXPRESSION IN THE POSTNATAL INNER EAR 31 weeks of age, when type I spiral ganglion neurons begin to flushing the fixative solution through the oval window. degenerate. Immediately preceding this degeneration, Tissues were then washed three times for 30 minutes at there is a specific and significant reduction in the levels of room temperature and incubated in staining solution (5 ⅐ ⅐ NT3 mRNA in the cochlea. Based on these results, and the mM K3Fe(CN)6,5mMK4Fe(CN)6 3H2O, 2 mM MgCl2 observation that NT3 is expressed by inner hair cell (IHC) 6H2O, 0.01% Na-deoxycholate, 0.02% NP-40, 1 mg/ml supporting cells, we proposed that NT3 plays an impor- X-gal) at 37°C in the dark for 1 or 4 (P0), 5 (P5 and P10), tant role in promoting the long-term survival of type I or 6 hours (P15-P135). Tissues were rinsed with PBS for spiral ganglion neurons. 3–5 minutes, postfixed overnight with the same fixing Information on the pattern of NT3 expression in the solution as above, washed with PBS, and decalcified in 4% postnatal ear is limited. Some studies reported that NT3 EDTA. Tissues were then embedded in Araldite by using is expressed by IHCs of the adult cochlea (Farinas et al., a rapid dehydration protocol to minimize washout of re- 2001; Pirvola et al., 1994), whereas other studies have action product. No differences in the intensity or pattern demonstrated that this neurotrophin is expressed by IHCs of lacZ staining were observed before and after dehydra- and their supporting cells (Stankovic et al., 2004). To tion. Care was taken to treat all cochleae identically: define the pattern of expression of NT3 in the postnatal staining was done in large batches, with ears from all inner ear, we studied mice in which the Escherichia coli postnatal ages included in a single staining run. Araldite- lacZ gene is integrated into the NT3 locus (Farinas et al., embedded materials were sectioned (at 20 ␮m) on a His- 1994). In this mouse strain, cells that normally express torange (LKB Instruments). Ears of wild-type mice were NT3 also express ␤-galactosidase, allowing for their visu- processed in parallel for control. At least three wild types alization and identification by histochemical staining were processed at each age evaluated (up to and including (Fritzsch et al., 1997a). We analyzed cochlear and vestib- P20). ular organs from P0 to P135, to determine the pattern of NT3 expression during the morphological and functional Immunohistochemistry maturation in the early postnatal period and in the ma- Mice were anesthetized with 2.5% Avertin (0.2 ml/10 g ture organ. We found that NT3 is expressed in a dynamic body weight) and fixed by intracardial perfusion with 4% pattern, with the levels of expression and the areas ex- paraformaldehyde in 0.1 M PBS (pH 7.4). The temporal pressing this gene decreasing with age. In the adult inner bones were dissected, and the cochleae were perfused by ear, NT3 expression was restricted to inner hair cells and flushing the fixative solution through the oval window and their supporting cells in the cochlea and largely to sup- postfixed for 2 hours. Temporal bones were decalcified in porting cells in the vestibular epithelia. This finding 4% EDTA for 3 days at 4°C, cryoprotected in 30% sucrose strengthens the notion that supporting cells are necessary overnight at 4°C, embedded in OCT, and sectioned at 15 for the long-term maintenance and function of the adult ␮m. Sections were washed in PBS and blocked for 30 auditory and vestibular systems. minutes in 25% normal goat serum, 0.4% Triton-X, 100 mM L-lysine, 1% bovine serum albumin, and 0.05% so- dium azide. Mouse anti-␤-galactosidase (1:500, Promega, MATERIALS AND METHODS Madison, WI) and rabbit anti-calretinin (1:500, Chemicon, Animals Temecula, CA) in 3% bovine serum albumin were incu- bated on sections overnight at 4°C. Sections were washed NT3-lacZ mice (courtesy of Dr. Reichardt, University of in PBS and blocked with 5% normal donkey serum for 20 California-San Francisco) were generated by targeted re- minutes followed by incubation with donkey anti-mouse placement of the NT3-coding exon with a construct con- Alexa-610 (1:250, Molecular Probes, Eugene, OR) and don- taining a lacZ gene cDNA and the PKCneo marker (Fari- key anti-rabbit Oregon Green (1:250, Molecular Probes) nas et al., 1994). Mice were maintained at the animal for 1 hour at room temperature. facility at Children’s Hospital, and all procedures were carried out following protocols approved by the Children’s Imaging Hospital Animal Care and Use Committee. At least two All images of LacZ-stained tissues were obtained with animals (four ears) were evaluated at every age except for digital cameras (Orca, Hamamatsu, or Spot). Images were P135, which included only one animal. Age-matched, wild- processed by gamma correction and unsharp mask algo- type littermates were used as controls. rithm (amount: 70%; radius: 9px; threshold: 1 level) by Physiological testing using Adobe Photoshop (version 6.0). Images of immuno- fluorescence were obtained with an LSM 510 Zeiss laser Because the NT3-lacZ mice have only one normal NT3 scanning confocal microscope with a 40ϫ objective. gene, we assessed cochlear function by measuring audi- tory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs) according to standard RESULTS techniques (Kujawa and Liberman, 2006). Threshold sen- sitivity was no different in NT-lacZ hemizygotes and wild- Expression gradients in inner-ear whole type controls.