Mechanotransduction is required for establishing and maintaining mature inner hair cells and regulating efferent innervation

Abstract

In the adult auditory organ, mechanoelectrical transducer (MET) channels are essential for transducing acoustic stimuli into electrical signals. In the absence of incoming sound, a fraction of the MET channels on top of the sensory hair cells are open, resulting in a sustained depolarizing current. By genetically manipulating the in vivo expression of molecular components of the MET apparatus, we show that during pre-hearing stages the MET current is essential for establishing the electrophysiological properties of mature inner hair cells (IHCs). If the MET current is abolished in adult IHCs, they revert into cells showing electrical and morphological features characteristic of pre-hearing IHCs, including the re-establishment of cholinergic efferent innervation. The MET current is thus critical for the maintenance of the functional properties of adult IHCs, implying a degree of plasticity in the mature auditory system in response to the absence of normal transduction of acoustic signals.

Fig. 1

Current and voltage responses from IHCs of Pcdh15 mutant mice. a, b Potassium currents recorded from IHCs of control Pcdh15^+/av3j (a) and littermate mutant Pcdh15^av3j/av3j mice (b) using 10 mV depolarizing voltage steps from –84 mV to the various test potentials shown by some of the traces. The adult-type currents (I_K,f and I_K,n) were only present in IHCs from control Pcdh15^+/av3j mice (a). IHCs from Pcdh15^av3j/av3j mice retained the currents characteristic of immature cells (b_, I_K and I_K1). The absence of the rapidly activating I_K,f in Pcdh15 ^av3j/av3j IHCs is also evident when comparing the activation time course of the total outward currents on an expanded time scale (see insets). a, b are representative recordings from 10 (3 mice) and 12 (3 mice) IHCs, respectively. c, d Voltage responses elicited by applying depolarizing current injections to control, Pcdh15^+/av3j c and mutant, Pcdh15^av3j/av3j d IHCs from their resting membrane potentials. Depolarizing current injections caused slow APs in the Pcdh15^avj3/av3j mutant IHC. c, d are representative recordings from 8 (3 mice) and 15 (3 mice) IHCs, respectively. Recordings were performed near body temperature

Fig. 2

Disappearance of the MET current in IHCs of Ush1c^fl/fl conditional KO mice. a MET currents recorded in P7 IHCs from control Ush1c^fl/fl and knockout Ush1c^fl/flMyo15-cre^+/- mice by displacing the hair bundles while applying 20 mV voltage steps between −121 mV to + 99 mV. For clarity, only the traces at −121 mV and + 99 mV are shown (dashed lines: holding current; V_Piezo: driver voltage to the fluid jet). b Maximum MET current-voltage curves obtained from P7 (left) and P8 (right) Ush1c^fl/fl and Ush1c^fl/flMyo15-cre^+/- IHCs (3 and 6 mice, respectively). c MET currents in P10 IHCs from both genotypes. Protocol as in a. d Maximum MET currents obtained from 9 Ush1c^fl/fl and 17 Ush1c^fl/flMyo15-cre^+/- (P10-P12) IHCs (4 and 8 mice, respectively). e SEM images of P15 IHC bundle from both genotypes. Scale bars, 1 μm. f TEM images (left) of P25 IHC stereocilia from both genotypes. Scale bars, 400 nm. Right panels show the height and width of the tallest (s1) and second tallest (s2) stereocilia in P25 IHCs from Ush1c^fl/fl and Ush1c^fl/flMyo15-cre^+/- mice. g Depolarizing current used to elicit repetitive APs in Ush1c^fl/fl (P9-P10, 8 mice) and Ush1c^fl/flMyo15-cre^+/- (P9-P11, 7 mice) IHCs. Experimental conditions: body temperature (BT: 34–37 °C); 1.3 mM extracellular Ca²⁺. h Size of the resting MET current (I_MET) recorded from Ush1c^fl/fl IHCs (P9-P11, 2 mice) and Ush1c^fl/flMyo15-cre^+/- (P10-P11, 5 mice) IHCs. Room temperature (RT: 22–24 °C, left bars); 1.3 mM extracellular Ca²⁺. I_MET was interpolated from its current-voltage relation near the V_m of IHCs (−73 mV). Right bars show the RT values corrected for BT using a Q₁₀ of 1.45 (ref. ). i Size of the resting I_MET at −73 mV from Ush1c^fl/fl (P10, 3 mice) and Ush1c^fl/flMyo15-cre^+/- (P10, 3 mice) IHCs using the endolymph-like Ca²⁺ concentration (0.3 mM: see panel j). Left and right bars are as described in h. j I_MET recorded at −81 mV at P10 from Ush1c^fl/fl and Ush1c^fl/flMyo15-cre^+/- IHCs in 0.3 mM Ca²⁺. Protocol as in a. Number of IHCs in g–i is shown above the columns. Average data is shown as mean ± SEM in b, d, g, h or mean ± SD in f

Fig. 3

IHCs from Ush1c^fl/flMyo15-cre^+/- conditional knockout mice fail to mature. a, b Potassium currents recorded from P15 (upper panels) and P29 (lower panels) IHCs from control Ush1c^fl/fl (a) and Ush1c^fl/flMyo15-cre^+/- (b) mice using 10 mV depolarizing and hyperpolarizing voltage steps from −64 mV. Note that the K⁺ currents characteristic of adult IHCs (I_K,f and I_K,n) were only present in IHCs from control Ush1c^fl/fl mice (a). IHCs from Ush1c^fl/flMyo15-cre^+/- mice retained the K⁺ currents characteristic of pre-hearing stages (I_K and I_K1). c, d Voltage responses elicited by applying hyperpolarizing and depolarizing current injections from the respective membrane potentials in P15 IHCs of control Ush1c^fl/fl (c) and Ush1c^fl/flMyo15-cre^+/- (d) mice. Note that APs are still present in post-hearing Ush1c^fl/flMyo15-cre^+/- IHCs. For more details see Supplementary Table 2. e, f Mean ABR thresholds ( ±SD ) for clicks and pure tone stimulation from 3 kHz to 24 kHz obtained from control Ush1c^fl/fl and littermate Ush1c^fl/flMyo15-cre^+/- mice at P16 (e) and P37-45 (f). Ush1c^fl/flMyo15-cre^+/- mice are almost completely deaf already at P16. Open symbols represent data from single mice. Closed symbols represent averages (mean ± SEM)

Fig. 4

IHCs from Myo7a^fl/flMyo15-cre^+/- conditional knockout mice acquire their mature phenotype. a, b MET currents from IHCs of control Myo7a^fl/fl (left, P10) and knockout Myo7a^fl/flMyo15-cre^+/- (right, P9) mice. The experimental protocol is as in Fig. 2. c Cochlear whole mount preparations from P10 Myo7a^fl/fl (control, upper panel) and Myo7a^fl/flMyo15-cre^+/- (lower panel) mice, immunostained for Myo7a (green) and for F-actin with phalloidin (red). Myo7a is detected in both OHCs and IHCs in the Myo7a^fl/fl mouse, but not in the Myo7a^fl/flMyo15-cre^+/- mouse (but see also Supplementary Fig. 4 for one example of normal hair cell Myo7a labeling in a P10 Myo7a^fl/flMyo15-cre^+/- mouse). Scale bars, 5 µm. d IHC voltage response recorded during the superfusion of 0.3 mM Ca²⁺ alone and together with the MET channel blocker DHS (0.2 mM) in a Myo7a^fl/flMyo15-cre^+/- mouse. The MET depolarizing current substantially contributes to the IHC membrane potential. e Images showing relative fluo-4 fluorescence changes (ΔF/F₀) before (left) during (middle) and after (right) application of an extracellular solution containing 0.3 mM Ca²⁺. Images were obtained as maximal back projection (200 frames, 6.6 s). Scale bar, 15 µm. f Representative ΔF/F₀ traces from two IHCs (from images in panel e) during bath application of 0.3 mM Ca²⁺. Traces are computed as pixel averages of regions of interest centered on IHCs. Ca²⁺ spikes are evident in IHCs; however, the relative long fluorescence decay time constant of the Ca²⁺ signal, prevents the isolation of single APs during high-frequency bursting. g, h Current responses in post-hearing IHCs (P15) from control (g) and Myo7a^fl/flMyo15-cre^+/- (h) mice. Note that both I_K,f and I_K,n are present in IHCs from both genotypes. Voltage protocol as in Fig. 3a. i Voltage responses recorded from P15 IHCs of control Myo7a^fl/fl (top panel) and Myo7a^fl/flMyo15-cre^+/- (lower panel) P15 IHCs (protocol as in Fig. 3c). Panel (g, j: Myo7a^fl/fl) and (b, j: Myo7a^fl/flMyo15-cre^+/-) are representative recordings from 9 IHCs (4 mice) and 8 IHCs (4 mice), respectively. j Mean ABR thresholds ( ± SD) for clicks and frequency-specific pure tone stimulation from 3 Hz to 24 kHz obtained from control Myo7a^fl/fl and littermate Myo7a^fl/flMyo15-cre^+/- mice at P15

Fig. 5

The functional MET current is required for retaining mature IHC characteristics. a, b MET currents recorded from mature IHCs of control Myo7a^fl/fl (a, P15) and Myo7a^fl/flMyo15-cre^+/- (b, P14 and P16) mice. Protocol as in Fig. 2a. c Peak-to-peak MET current-voltage curves obtained from six P15 Myo7a^fl/fl (2 mice), four P14 Myo7a^fl/flMyo15-cre^+/- and six P16 Myo7a^fl/flMyo15-cre^+/- IHCs (3 mice). d Scanning electron microscopy images showing the stereociliary bundle of apical-coil IHC from P30 control Myo7a^fl/fl and Myo7a^fl/flMyo15-cre^+/- mice. s1 and s2 represent the tallest and the second tallest stereocilia, respectively. The arrows in the middle panel highlights that tip links, which are required to gate the MET current, are present in Myo7a^fl/flMyo15-cre^+/- IHCs. Scale bars, 1 μm. e Transmission electron microscopy images (left) showing the stereocilia s1 and s2 (see panel d) of apical-coil IHCs from P37 control Myo7a^fl/fl and Myo7a^fl/flMyo15-cre^+/- mice. Scale bars, 400 nm. The right panels show that the height and width of s1 and s2 stereocilia in IHCs are comparable between the two genotypes (Myo7a^fl/fl s1: height 4.42 ± 0.48 μm, width 0.36 ± 0.07 μm, n = 6 IHCs, s2: height 1.58 ± 0.10 μm, width 0.32 ± 0.04 μm, n = 7, s1/s2: 2.77 ± 0.36, n = 6; Myo7a^fl/flMyo15-cre^+/- s1: 4.44 ± 0.20 μm, P < 0.8915, 0.38 ± 0.03 μm, P < 0.4441, n = 7, s2: 1.61 ± 0.18 μm, P < 0.7849, 0.33 ± 0.06 μm, P < 0.8959, n = 7, s1/s2: 2.80 ± 0.35, P < 0.9059, n = 6). f–h Potassium currents recorded from IHCs of young adult (f, P19 and P21), adult (g, P59) and aged (h, P210 and P218) control Myo7a^fl/fl (left panels) and Myo7a^fl/flMyo15-cre^+/- (right panels) mice. Protocols are as in Fig. 3a. i Size of I_K,f, I_K,n, membrane capacitance (C_m) and resting membrane potential (V_m) as a function of postnatal age. Number of IHC investigated is shown above or below the symbols (for more details see Supplementary Table 3). Statistical tests are performed with two-way ANOVA with Bonferroni correction. Average data is shown as mean ± SEM (c, i) or mean ± SD (e)

Fig. 6

The resting MET current drives the maintenance of mature IHC K⁺ currents in cochlear cultures. a Potassium outward currents recorded from a P21 IHC of an acutely dissected gerbil cochlea. Recordings were performed using a perilymph-like solution (1.3 mM extracellular Ca²⁺; 5.8 mM K⁺). Currents were elicited using 10 mV depolarizing voltage steps from −84 mV. Note the fast activating I_K,f characteristic of adult IHCs (arrow). b, c Currents recorded from IHCs maintained in cochlear culture for 1 day (b) or 6 days (c). The culture medium bathing the cochlea (see Methods) contained 1.3 mM Ca²⁺ and 4.2 mM K⁺. d, e Currents recorded from IHCs maintained in culture for 2 day (d) or 7 days (e). Culture medium contained 1.3 mM Ca²⁺ and 40 mM K⁺. f Size of I_K,f recorded from IHCs of acutely isolated gerbil cochleae (gray circle, n = 6, P21-P28, 3 gerbils) or maintained in culture. Culture conditions were: (1) normal medium (black circles, number of IHCs from left to right: 7,3,4,10,2,6,7; from 9 cochlear cultures; gerbil age at the day of culturing: P18-P24); 2) medium supplemented to 40 mM K⁺ (blue circles, number of IHCs: 6,6,5,7, from 4 cochlear cultures, P18-P24). g, h Currents recorded from IHCs maintained in culture for 1 day (g) or 6 days (h) with a medium containing 40 µM Ca²⁺_. i, j, l Currents from IHCs maintained in cochlear culture for 1 day (i), 4 days (j) or 8 days (l) with a medium containing 40 µM Ca²⁺ and 50 μM of the MET channel blocker d-tubocurarine. The inward rectifier K⁺ current (I_K1) l was obtained using 10 mV hyperpolarizing voltage steps from −64 mV. k Size of I_K,f recorded from IHCs of acutely dissected cochleae (gray circle: same as in panel f), maintained in culture with a normal medium (black circles: as in panel f), a medium containing 40 µM Ca²⁺ (red circles: number of IHCs: 5,10,9,5,3,5,8, from 9 cochlear cultures, P18-P24) and a medium containing 40 µM Ca²⁺ and 50 μM d-tubocurarine (orange circles: 4,6,6,4, from 4 cochlear cultures, P18-P24). Average data is shown as mean ± SEM

Fig. 7

IHC exocytosis is unaffected in adult Myo7a^fl/flMyo15-cre^+/- mice but efferent input is altered. a−d Calcium current (I_Ca) and corresponding changes in membrane capacitance (ΔC_m) recorded from IHCs of P16-P17 (a, b) and P48-P49 (c, d) control Myo7a^fl/fl (black) and knockout Myo7a^fl/flMyo15-cre^+/- mice (red). Recordings were obtained in response to 50 ms voltage steps (10 mV increments) from −81 mV and using 1.3 mM extracellular Ca²⁺ and at body temperature. For clarity, only maximal responses at −11 mV are shown. Panels b and d shows average peak I_Ca (bottom) and ΔC_m (top) curve from control Myo7a^fl/fl and Myo7a^fl/flMyo15-cre^+/- IHCs. e, f Membrane currents recorded from adult IHCs of control Myo7a^fl/fl (e) and Myo7a^fl/flMyo15-cre^+/- (f) mice before (left panels) and during superfusion of ACh (right panels). ACh elicited an instantaneous current only in the Myo7a^fl/flMyo15-cre^+/- IHC. g Steady-state slope conductance of the ACh-sensitive current (g_{ACh-sensitive}) at different age ranges in control Myo7a^fl/fl (black symbols) and Myo7a^fl/fl Myo15-cre^+/- (red symbols) IHCs. The isolated g_{ACh-sensitive} was obtained by subtracting the control current (left panels in e, f) from that in the presence of 100 μM ACh (right panels). Number of IHCs is shown near the symbols (2 Myo7a^fl/fl mice at P22 and P33-P49; 3 Myo7a^fl/flMyo15-cre^+/- mice at P22; 5 Myo7a^fl/flMyo15-cre^+/-mice at P34-P48). h–k Voltage-clamp recordings obtained from adult IHCs of control Myo7a^fl/fl (h, P36) and Myo7a^fl/flMyo15-cre^+/- (i, j, P36; k P39) mice during the superfusion of 40 mM extracellular K⁺. Synaptic currents were only evoked in Myo7a^fl/flMyo15-cre^+/- IHCs. Panel j shows an expanded time scale of the blue area shown in panel (i). Panel k shows the effect of 1 μM strychnine on the ACh-induced synaptic currents. l, m TEM showing the IHC synaptic region from P37 control Myo7a^fl/fl (l) and Myo7a^fl/flMyo15-cre^+/- (m) mice. IHCs from Myo7a^fl/fl mice showed the characteristic synaptic organization of mature cells with efferent terminals forming axo-dendritic contact with the afferent fibers, as seen by the presence of active zones (l, black arrows). Scale bars, 1 μm. In Myo7a^fl/flMyo15-cre^+/- mice efferent fibers make direct axo-somatic contact with the IHC (m), which is characteristic of pre-hearing IHCs. Average data is shown as mean ± SEM