Sharpened cochlear tuning in a mouse with a genetically modified tectorial membrane

Abstract

Frequency tuning in the cochlea is determined by the passive mechanical properties of the basilar membrane and active feedback from the outer hair cells, sensory-effector cells that detect and amplify sound-induced basilar membrane motions. The sensory hair bundles of the outer hair cells are imbedded in the tectorial membrane, a sheet of extracellular matrix that overlies the cochlea's sensory epithelium. The tectorial membrane contains radially organized collagen fibrils that are imbedded in an unusual striated-sheet matrix formed by two glycoproteins, alpha-tectorin (Tecta) and beta-tectorin (Tectb). In Tectb(-/-) mice the structure of the striated-sheet matrix is disrupted. Although these mice have a low-frequency hearing loss, basilar-membrane and neural tuning are both significantly enhanced in the high-frequency regions of the cochlea, with little loss in sensitivity. These findings can be attributed to a reduction in the acting mass of the tectorial membrane and reveal a new function for this structure in controlling interactions along the cochlea.

Figure 1

Targeted deletion of exons 1–4 of Tectb. (a) Structure of the 5′ region of Tectb, the targeting vector and the targeted Tectb locus. Dark bars = genomic DNA; exons = open boxes; grey bar = homologous DNA in targeting vector; thin grey line = vector DNA. Lightly shaded boxes = external probes A and B; the sizes of the hybridising restriction fragments are shown. (Neo) Neomycin resistance gene; (TK) HSV thymidine kinase cassette; (S) SacI; (EV), EcoRV; (Xh), XhoI; (Ac), Acc65I. (b) Southern blots of SacI or EcoRV digested genomic DNA probed with external probes. (1) Wild type control: probe A hybridises to a 1.6 kb SacI band, probe B hybridises to a 9.8 kb EcoRV band. (2, 3) Homologous recombinants: probes A and B hybridise to wild type bands, in addition probe A hybridises to a 3.4 kb SacI band and probe B hybridises to a 6.2 kb EcoRV band. (c) RT-PCR of total RNA from Tectb^+/+ (+/+), Tectb^+/− (+/–) and Tectb^−/− (−/−) mice with primers for an 816 bp product spanning exons 1 to 8 of the Tectb mRNA (Tectb 5′), a 557 bp product spanning exons 7 to 10 of Tectb RNA (Tectb 3′), and a 615 bp product specific for Tecta (Tecta). C1, no reverse transcriptase control; C2, no cDNA control. (d) Western blots of P57 tectorial membranes and P2 cochlear epithelia from Tectb^+/+ (+/+) and Tectb^−/− mutant (−/−) mice probed with anti chick β-tectorin. Arrow indicates the 45 kDa Tectb band.

Figure 2

Tectorial membrane morphology and composition in mature Tectb^+/− and Tectb^−/− mutant mice. (a–f) Cryosections from the basal coils of Tectb^+/− (a,c,e) and Tectb^−/− mutant (b,d,f) mice stained with antisera specific for Tectb (a,b), Tecta (c,d) and otogelin (e,f). The tectorial membranes from Tectb^−/− mutant mice stain strongly for Tecta and otogelin but are negative for Tectb. (g–j) Toluidin blue stained sections from the apical (g,h) and basal (i,j) coils of the cochleae of adult Tectb^+/− (g,i) and Tectb^−/− (h,j) mice. Insets in i and j show a region of the lower surface of the tectorial membrane that lies just above the inner hair cells. Hensen’s stripe is visible in i (arrowhead in inset). The tectorial membranes in Tectb^−/− mice have a less dense structure compared to those of heterozygotes. (k–n) Wholemount preparations of Alcian blue stained tectorial membranes from Tectb^+/− (k,m) and Tectb^−/− (l,n) mice photographed with Nomarski interference contrast optics. (k,l) Basal region, HS = Hensen’s stripe. (m,n) Apical region, MB = marginal band. Bar in f = 100 μm and applies to a–f, bar in j = 50 μm and applies to g–j, bar in n = 20 μm and applies to k– n.

Figure 3

Fine structure of the tectorial membrane and hair bundles. (a,b) Transmission electron micrographs of apical-coil tectorial membranes from adult Tectb^+/− (a) and Tectb^−/− (b) mice. In Tectb^+/− mice (a), the tectorial membranes show the typical pattern of striated-sheet matrix (SM) and collagen fibrils (COL). In Tectb^−/− mice (b), the tectorial membranes lack striated sheet matrix, and have irregular 8 nm diameter filaments (arrowheads) and straight filaments (arrow) between the collagen fibrils. (c–f) Scanning electron micrographs showing the apical surface of the organ of Corti in the apical (c,d) and basal (e,f) cochlear regions of Tectb^+/− (c,e) and Tectb^−/− mice (d,f). In panel c, the arrow indicates the single row of inner hair cell hair bundles, and the arrowheads indicate the three rows of outer hair cell hair bundles. (g– h) Scanning electron micrographs revealing the presence of OHC hair-bundle imprints (arrows) in the lower surface of the tectorial membranes of Tectb^+/− (g) and Tectb^−/− (h) mice. Bar in b = 200 nm and applies to a–b, bar in f = 5 μm and applies to c–f, bar in h = 2 μm and applies to g–h.

Figure 4

Electrical and mechanical recordings from the cochleae of wild type (red) and Tectb^−/− (blue) mice. (a) Set up for recording from the mouse cochlea. The round window is accessed via a caudal opening in the ventrolateral aspect of the bulla and the sound system probe tip is within 1 mm of the tympanum. Cochlear cross-section showing the organ of Corti, the tectorial (TM) and basilar (BM) membranes, the location of the recording pipette, and the laser diode beam used for measuring basilar membrane vibrations. (b) Magnitude and form of OHC extracellular receptor potentials in response to a 10 kHz tone as a function of level. Each record is an average of 10 presentations. (c) Iso-response displacement frequency tuning curves (mean ± s.d., n = 8 for each genotype) response criterion 0.2 nm, 53 kHz location). Arrow, low-frequency resonance. Black symbols: difference between wild type and Tectb^−/− tuning curves. Dotted line, 6 dB.octave⁻¹. (d) The phases of basilar membrane (BM) responses (mean ± s.d., n = 4 for each genotype) to 70 dB SPL tones as a function of frequency. Vertical dashed lines indicate frequencies of the low-frequency resonance (43 kHz) and the characteristic frequency (CF) (53 kHz). (e) Phase difference (in degrees, mean ± s.d., n = 4 for each genotype) between basilar membrane displacements at 45 and 75 dB SPL. Zero crossing slopes: wild type 33.28° kHz⁻¹, Tectb^−/− mice 61.75° kHz⁻¹.

Figure 5

Neural and acoustical recordings from the cochleae of wild type (red) and Tectb^−/− (blue) mice. (a) Simultaneous masking tuning curves (n = 30, mean ± s.d) of compound action potentials in response to 10, 20, 35 and 54 kHz probe tones. The levels and frequencies of the probe tone are indicated by stars. Dotted lines indicate SPLs of 30 and 100 dB. (b) The means (n = 10) of masking tuning curves for a 20 kHz probe tone for individual mice from each of 3 different F1 hybrid families (C37:CBA/Ca f6 × C57/BL6 f5; C41: CBA/Ca f6 × 129SvEv; C42: C57/BL6 f7 × 129SvEv). (c) Neural masking tuning curves (n = 30 for each genotype) for 20 kHz (upper) and 35 kHz (lower) probe tones. Masker frequency is expressed as a ratio of the probe tone frequency, stars indicate the notches of insensitivity. (d) Notch frequency in octaves below the tuning curve tip-frequency as a function of probe-tone frequency. (e) Mean ± s.d. (n = 10) of the CAP detection threshold as a function of tone frequency. Grey shading indicates +s.d. of wild type and –s.d. of Tectb^−/− data. (f) Mean ± s.d. (n =10) of the magnitude of 2f1–f2 DPOAEs as functions of f2 frequency. Levels of f2/f1 tones in dB SPL are shown in the upper left of each panel.