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. 2018 Oct 1;9:4015. doi: 10.1038/s41467-018-06307-w

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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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