The Cl--channel TMEM16A is involved in the generation of cochlear Ca2+ waves and promotes the refinement of auditory brainstem networks in mice
- Neuroscience Department, Max Delbrück Center for Molecular Medicine, Germany
- Faculty of Biology, Chemistry, Pharmacy, Freie Universität Berlin, Germany
- Institute of Human Genetics, University Hospital Jena, Germany
- Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Germany
- Institute of Biology, Faculty of Life Sciences, University of Leipzig, Germany
Figures
Figure 1 with 2 supplements
TMEM16A is required for the generation of spontaneous volume changes and Ca2+ waves in Kölliker’s organ.
(A) Schematic representation of the organ of Corti at birth (left) and after hearing onset (right). BM, basilar membrane; IHC, inner hair cells; IS, inner sulcus; ISC, inner supporting cells; KÖ, …
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Figure 1—source data 1
Source data for Figure 1.
- https://cdn.elifesciences.org/articles/72251/elife-72251-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Comparison of morphology and TMEM16A expression patterns in the developing organ of Corti between wildtype and cKO mice.
(A) Immunohistochemical stainings for TMEM16A of cryosections from the organ of Corti in P9 wildtype (left) and cKO (right) mice. TMEM16A is broadly expressed in plasma membranes of inner supporting …
Figure 1—figure supplement 2
Absence of P2 receptor activation in cKO mice.
The purinergic receptor blocker suramin reduces Ca2+ waves to uncoordinated Ca2+ transients in wildtype (black) but does not have an effect on Ca2+ transients in cKO littermates (red) (mean values ± …
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Figure 1—figure supplement 2—source data 1
Source data for Figure 1—figure supplement 2.
- https://cdn.elifesciences.org/articles/72251/elife-72251-fig1-figsupp2-data1-v2.xlsx
Figure 2 with 1 supplement
Disruption of Kölliker’s organ activity changes prehearing burst firing of medial nucleus of the trapezoid body (MNTB) neurons in vivo.
(A) Simplified model of auditory connections in the brainstem. The pathways relevant for this experiment are marked in green. Inhibitory pathways are indicated by dotted arrows. CN, cochlear nucleus;…
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Figure 2—source data 1
Source data for Figure 2A–G.
- https://cdn.elifesciences.org/articles/72251/elife-72251-fig2-data1-v2.xlsx
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Figure 2—source data 2
Source data for Figure 2H.
- https://cdn.elifesciences.org/articles/72251/elife-72251-fig2-data2-v2.xlsx
Figure 2—figure supplement 1
Spontaneous activity of an exemplary P8 medial nucleus of the trapezoid body (MNTB) wildtype neuron in vivo.
(A) A representative 20 s voltage trace showing one prominent 3.5 s burst. (B) Fine structure of the burst shown in (A) reveals mini-bursts separated by 50–200 ms silent periods. (C) The mini-burst …
Figure 3 with 1 supplement
Lack of TMEM16A in cochlear inner supporting cells (ISCs) changes bursts but not the overall firing rate in medial nucleus of the trapezoid body (MNTB) auditory brainstem neurons in vivo.
(A–C) Spontaneous discharge patterns of MNTB neurons recorded from cKO mice show a reduced number of bursts per 100 s (A), a reduced number of spikes per burst (B), and a reduced duration of bursts …
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Figure 3—source data 1
Source data for Figure 3.
- https://cdn.elifesciences.org/articles/72251/elife-72251-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
TMEM16A is not expressed in spiral ganglion (SG), cochlear nucleus (CN), medial nucleus of the trapezoid body (MNTB), and lateral superior olive (LSO) neurons before hearing onset in wildtype mice.
(A) At P2, TMEM16A is expressed in inner supporting cells (ISCs) of Kölliker’s organ (KÖ) and phalangeal cells (PCs) but not in SG neurons (dashed line). IHC, inner hair cells . Scale bar 50 μm. (B) …
Figure 4 with 1 supplement
Wildtype and cKO mice show similar auditory brainstem response (ABR) thresholds, but differences in frequency selectivity, response threshold, and maximal firing rate in neurons of the medial nucleus of the trapezoid body (MNTB).
(A) Grand averages of ABR waveforms to 80 dB click stimulation recorded from cKO (red) and wildtype (black) at P13–14. (B) ABR thresholds in response to 6, 12, or 24 kHz tone bursts and click …
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Figure 4—source data 1
Source data for Figure 4A and B.
- https://cdn.elifesciences.org/articles/72251/elife-72251-fig4-data1-v2.xlsx
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Figure 4—source data 2
Source data for Figure 4E–H.
- https://cdn.elifesciences.org/articles/72251/elife-72251-fig4-data2-v2.xlsx
Figure 4—figure supplement 1
Wildtype and cKO mice have similar auditory brainstem responses (ABRs).
Shown are latencies (A) and amplitudes (B–D) of the first three ABR peaks (wave I–III) in response to click stimuli presented at different intensities (40–100 dB) (n = 6 WT; n = 7 cKO; P13–14).
Figure 5 with 1 supplement
Medial nucleus of the trapezoid body-lateral superior olive (MNTB-LSO) input maps are enlarged upon disruption of TMEM16A.
(A) Exemplary MNTB input maps from wildtype (P9) and cKO mice (P10) as revealed by whole-cell current-clamp recordings (dotted line outlines the MNTB area, grid points indicate glutamate uncaging …
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Figure 5—source data 1
Source data for Figure 5A-D.
- https://cdn.elifesciences.org/articles/72251/elife-72251-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Spatial resolution of glutamate uncaging and additional examples of medial nucleus of the trapezoid body (MNTB) input maps recorded from wildtype and cKO mice.
(A) Schematic representation of the MNTB from a wildtype mouse recorded at P8 (dotted line represents the MNTB area, grid points show glutamate uncaging sites, black cross indicates the location of …
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Figure 5—figure supplement 1—source data 1
Source data for Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/72251/elife-72251-fig5-figsupp1-data1-v2.xlsx
Author response image 1
(A-C) Example traces from the WT LSO cell for which the MNTB input map is shown in Figure S6D on the right.
The upper part of each trace shows the response of the LSO cell to glutamate uncaging in the MNTB. The timepoints of glutamate uncaging are shown in the lower part. (A) 1.5 min recording time. (B) …
Videos
Video 1
TMEM16 A is required for the generation of spontaneous volume changes in Kölliker’s organ (KÖ).
Time-lapse imaging (one image per second) reveals spontaneous volume changes of inner supporting cells of in a wildtype mouse cochlea (P7), which propagate in a wave-like manner up and down the …
Video 2
TMEM16A is required for the propagation of cochlear Ca2+ waves.
Time-lapse imaging (one image per second) reveals spontaneous Ca2+ signals in the inner supporting cells that propagate up and down the cochlear turn in a wildtype mouse cochlea (P7). In contrast, …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (mouse) | Ano1(Tmem16a) | GenBank | MGI:2142149 | |
| Strain, strain background (Mus musculus) | Ano1fl/fl | Published in Heinze et al., 2014 | ||
| Strain, strain background (M. musculus) | Ano2 KO | Published in Billig et al., 2011 | ||
| Strain, strain background (M. musculus) | Pax2Cre | Published in Ohyama and Groves, 2004 | ||
| Strain, strain background (M. musculus) | C57BL6/J | Jackson | 000664 | |
| Antibody | Anti-TMEM16A polyclonal rabbit | Published in Heinze et al., 2014 | IF (1:500) | |
| Antibody | Anti-TMEM16B polyclonal rabbit | Published in Billig et al., 2011 | IF (1:100)WB (1:1000) | |
| Antibody | Anti-CD31monoclonal rat | BioLegend | 102,502 | IF (1:500) |
| Antibody | Alexa Fluor 488, polyclonal goat anti-rabbit | Molecular Probes | A11008 | IF (1:1000) |
| Antibody | Cy3, polyclonal donkey anti-rabbit | Jackson ImmunoResearch | 711-165-152 | IF (1:10,000) |
| Antibody | DyLight 488polyclonal goat anti-rat | Bethyl Laboratories | A110-305D2 | IF (1:1000) |
| Antibody | HRP polyclonal goat anti-guinea pig | Merck | AP108P | WB (1:1000) |
| Chemical compound, drug | Fura-2 AM | Thermo Fisher | F1221 | Ca2+ dye |
| Chemical compound, drug | MNI-caged glutamate trifluoroacetate | Femtonics | Caged glutamate | |
| Chemical compound, drug | DAPI stain | Invitrogen | D1306 | (1 µg/ml) |
| Chemical compound, drug | Fluorogold hydroxystilbamidin-bis-(methansulfonat) | Sigma-Aldrich | 39286 | Fluorescent tracer |
| Chemical compound, drug | KetavetKetamine hydrochloride | Pfizer | 0.1 mg/g body weight | |
| Chemical compound, drug | RompunXylazine hydrochloride | Bayer | 0.5 µg/g body weight | |
| Software, algorithm | SigmaPlot 12.5 | Systat Software Inc | Statistical analysis | |
| Software, algorithm | GraphPad Prism | GraphPad Software Inc | Statistical analysis |
Additional files
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Supplementary file 1
Distribution of interspike intervals and quantification of auditory brainstem response.
(a) Comparison of the distribution of interspike intervals (ISIs) between wildtype and cKO mice. (b) Quantification of auditory brainstem response (ABR) thresholds (mean ± SEM) in response to stimulation with tone bursts at 6, 12, and 24 kHz, or click stimulation. (c) Quantification of peak amplitudes (mean ± SEM) of the first three ABR waves (I–III) in response to click stimuli of various intensities (40–100 dB). (d) Quantification of latencies (mean ± SEM) of the first three ABR waves (I–III) in response to click stimuli of various intensities (40–100 dB).
- https://cdn.elifesciences.org/articles/72251/elife-72251-supp1-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/72251/elife-72251-transrepform1-v2.pdf
