Editor's evaluation: The Cl--channel TMEM16A is involved in the generation of cochlear Ca2+ waves and promotes the refinement of auditory brainstem networks in mice

Abstract

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Before hearing onset (postnatal day 12 in mice), inner hair cells (IHCs) spontaneously fire action potentials, thereby driving pre-sensory activity in the ascending auditory pathway. The rate of IHC action potential bursts is modulated by inner supporting cells (ISCs) of Kölliker’s organ through the activity of the Ca2+-activated Cl--channel TMEM16A (ANO1). Here, we show that conditional deletion of Ano1 (Tmem16a) in mice disrupts Ca2+ waves within Kölliker’s organ, reduces the burst-firing activity and the frequency selectivity of auditory brainstem neurons in the medial nucleus of the trapezoid body (MNTB), and also impairs the functional refinement of MNTB projections to the lateral superior olive. These results reveal the importance of the activity of Kölliker’s organ for the refinement of central auditory connectivity. In addition, our study suggests the involvement of TMEM16A in the propagation of Ca2+ waves, which may also apply to other tissues expressing TMEM16A. Editor's evaluation This study addresses the extremely interesting question of how spontaneous activity in the cochlea prior to hearing onset impacts the development of auditory circuits in the brainstem. The study has many strengths, including the use of complementary in vitro and in vivo recording techniques to characterize both peripheral and central defects resulting from conditional deletion of the gene for the chloride channel TMEM16A. The reviewers identified some concerns over the interpretation of the data, but all of these concerns were addressed in the subsequent revisions. https://doi.org/10.7554/eLife.72251.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Before hearing onset around postnatal day 12 (P12) in mice (Müller et al., 2019; Shnerson and Pujol, 1981; Sonntag et al., 2009), the afferent auditory system exhibits cochlea-driven spontaneous activity (Jones et al., 2007; Lippe, 1994). Inner hair cells (IHCs) fire bursts of action potentials (Kros et al., 1998), which drive afferent transmission to spiral ganglion neurons (SGNs) (Beutner and Moser, 2001; Glowatzki and Fuchs, 2002) and thus trigger bursting discharges through ascending auditory pathways (Babola et al., 2018; Tritsch and Bergles, 2010; Tritsch et al., 2007). Similar to developing motor and visual systems (Hanson and Landmesser, 2004; Katz and Shatz, 1996), patterned activity of auditory neurons was proposed to promote activity-dependent refinement of auditory circuits before hearing onset (Clause et al., 2014; Clause et al., 2017). In the developing inner ear, non-sensory inner supporting cells (ISCs) form a transient epithelial structure known as Kölliker’s organ (Hinojosa, 1977; Hou et al., 2019). ATP released from ISCs through connexin hemichannels (Mazzarda et al., 2020) activates purinergic receptors in a paracellular manner, leading to cell volume decrease of ISCs and cochlear Ca2+ transients (Babola et al., 2018; Tritsch and Bergles, 2010; Tritsch et al., 2007). It was proposed that the Ca2+-activated Cl--channel TMEM16A, which is expressed in ISCs, might be the pacemaker for spontaneous cochlear activity (Yi et al., 2013). Indeed, spontaneous osmotic cell shrinkage was shown to be mediated by TMEM16A-dependent Cl- efflux, which forces K+ efflux from ISCs and thus the transient depolarization of IHCs (Yi et al., 2013). Thereby, bursting activity of nearby IHCs, which will later respond to similar sound frequencies, becomes synchronized (Eckrich et al., 2018; Harrus et al., 2018; Wang et al., 2015), establishing a possible scenario for tonotopic map refinement in central auditory structures. Using Ano1 conditional knockout mice, we show that TMEM16A not only modulates ISC volume but also drives the amplification of localized Ca2+ transients to propagating Ca2+ waves within the cochlea. Prior to hearing onset, knockout mice show reduced burst firing of neurons in the medial nucleus of the trapezoid body (MNTB), downstream of SGNs and neurons of the cochlear nucleus. Moreover, the frequency selectivity of individual MNTB neurons is diminished shortly after hearing onset (P14) pointing toward reduced refinement of auditory connections. Indeed, neurons from the lateral superior olive (LSO) received twice as many functional MNTB afferents in knockout mice compared to wildtype littermates. Taken together, these results suggest that the Ca2+-activated Cl--channel TMEM16A plays a significant role in the propagation of Ca2+ waves and contributes to the refinement of auditory brainstem circuitries prior to hearing onset. Results TMEM16A is required for the generation of cochlear Ca2+ waves The Ca2+ activated Cl--channel TMEM16A is expressed in ISCs of Kölliker’s organ (for a schematic representation of a part of the organ of Corti, see Figure 1A; for a differential interference contrast [DIC] image, see Figure 1B; Wang et al., 2015; Yi et al., 2013) and is activated by an ATP-induced increase in Ca2+ concentration (Wang et al., 2015; Yi et al., 2013). The opening of TMEM16A triggers Cl- efflux, followed by K+ efflux and cell shrinkage. The ensuing rise of extracellular K+ drives electrical activity of immature IHCs (Wang et al., 2015). To assess the role of TMEM16A in the developing cochlea and the impact of TMEM16A-dependent cochlear signaling on the development of auditory brainstem nuclei, we disrupted Ano1 in the inner ear. This was achieved by mating our floxed Ano1 line (Ano1fl/fl) (Heinze et al., 2014) with a line expressing Cre-recombinase under the control of the Pax2 promoter (Ohyama and Groves, 2004), which is active in the otic placode (Lawoko-Kerali et al., 2002). This line is subsequently referred to as cKO mice. Ano1 deletion was confirmed by immunohistochemistry (Figure 1—figure supplement 1A) and Western blot analysis (Figure 1—figure supplement 1B). Importantly, organs of Corti of cKO mice showed no obvious morphological defects. The development of the tectorial membrane and the morphology of the hair cells appeared normal before hearing onset (P6), at hearing onset (P12), or in the weeks thereafter (3 weeks and 6 weeks after birth) (Figure 1—figure supplement 1C and D), indicating that TMEM16A and TMEM16A-dependent activity of Kölliker’s organ is not essential for the morphological development of the organ of Corti. Figure 1 with 2 supplements see all Download asset Open asset 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, Kölliker’s organ; OHC, outer hair cells; SGN, spiral ganglion neurons; SM, scala media; ST, scala tympani; SV, scala vestibuli; TM, tectorial membrane. (B) Left: example differential interference contrast (DIC) image from the area of the cochlea turn imaged in (C–F). Right: the schematic drawing highlights the location of IHCs (blue) and ISCs (yellow) of KÖ. HC, Hensen cells; DC, Deiter’s cells; PC, pillar cells. (C, D) DIC time-lapse imaging at P7 reveals spontaneous volume changes of ISCs in a wildtype mouse indicated by changes in light intensity, which are almost absent in the cKO littermate. The arrow in (D) indicates erythrocytes moving in a blood vessel. Scale bar 50 μm. (E, F) Ca2+ imaging at P6 reveals spontaneous Ca2+ waves traveling across ISCs of KÖ in a wildtype mouse (E) that are reduced to small local Ca2+ transients in the cKO littermate (F). Scale bar 50 μm. (G, H) Quantification of areas and frequencies of spontaneous ISC volume changes. A time-lapse series of 1200 images with one image per second was analyzed. Values represent mean ± SEM (P5–7; n = 7 WT, n = 9 cKO; two-tailed unpaired Student’s t-test: area: p=0.0000056, frequency: p=0.0042). (I, J) Quantification of area and frequency of spontaneous Ca2+ events. A time-lapse series of 400 images with one image per second was analyzed. Values represent mean ± SEM (P5–7; n = 14 WT, n = 16 cKO; two-tailed unpaired Student’s t-test: area: p=0.00032; frequency: p=0.0027). Figure 1—source data 1 Source data for Figure 1. https://cdn.elifesciences.org/articles/72251/elife-72251-fig1-data1-v2.xlsx Download elife-72251-fig1-data1-v2.xlsx To investigate the volume changes and Ca2+ waves that spontaneously appear in the ISCs of Kölliker’s organ (Anselmi et al., 2008; Tritsch and Bergles, 2010; Tritsch et al., 2007), acutely isolated cochleae from P5–7 wildtype and cKO littermates were used. While wildtype cochleae showed notable volume changes of groups of ISCs (Figure 1C, G and H; n = 7; mean event area ± SEM = 7021 ± 1128 μm2; mean event frequency ± SEM = 0.0171 ± 0.0056 Hz), volume changes were almost absent in cochleae acutely isolated from 5- to 7-day-old cKO mice (Figure 1D, G and H; n = 9; mean event area ± SEM = 40 ± 30 µm2, p=0.0000056; mean event frequency ± SEM = 0.0003 ± 0.0002 Hz, p=0.0042), in agreement with previously published results (Wang et al., 2015). In wildtype cochleae, these events propagated in waves along the tonotopic axis of the cochlea also affecting phalangeal cells that surround IHCs (Video 1). To visualize changes in intracellular Ca2+ concentrations, cochlear explants were loaded with the Ca2+ indicator dye Fura-2 AM. Wildtype mice showed Ca2+ transients that traveled in waves along the tonotopic axis of Kölliker’s organ (Figure 1E, I and J; n = 14; mean event frequency ± SEM = 0.0282 ± 0.0050 Hz; mean event area ± SEM = 3839 ± 794 µm2). In cochleae from cKO littermates, Ca2+ transients were rare and restricted to small areas (Figure 1F, I and J, Video 2; n = 16; mean event frequency ± SEM = 0.0123 ± 0.0020 Hz; p=0.0027; mean event area ± SEM = 656 ± 214 µm2; p=0.00032). Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg 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 cochlea turn. In contrast, volume changes are almost absent in the cochlea isolated from a cKO littermate (the bottom of the video shows erythrocytes moving in a blood vessel). Images were processed using a custom-written ImageJ macro and the ImageJ software. Each frame was subtracted from an average of five preceding frames to highlight the changes in light scattering caused by the changes in cell volume. The video (seven images per second) shows the top view of an area from an isolated cochlea turn. KÖ, Kölliker's organ; IHC, inner hair cells. Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg 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, Ca2+ waves are reduced to local Ca2+ events in the cochlea of a cKO littermate. Images were processed using a custom-written ImageJ macro and the ImageJ software. Each frame was subtracted from an average of five preceding frames to highlight the changes in Ca2+ concentration. The video (seven images per second) shows the top view of an area from an isolated cochlea turn. KÖ, Kölliker’s organ; IHC, inner hair cells. Spontaneous Ca2+ waves are elicited by ATP-induced ATP release from ISCs (Mazzarda et al., 2020), a mechanism possibly involving P2Y1, P2Y2, and P2Y4 receptors (Babola et al., 2018; Piazza et al., 2007), Cx26 and Cx30 heteromeric hemichannels (Anselmi et al., 2008; Mazzarda et al., 2020; Schütz et al., 2010), and TMEM16A (Wang et al., 2015). To assess what role TMEM16A might play in ATP-mediated Ca2+ signals, we applied the nonselective P2 receptor antagonist suramin (150 µM) (Burnstock, 2014; von Kügelgen and Wetter, 2000). In wildtype mice, suramin application reduced Ca2+ waves to uncoordinated, locally restricted Ca2+ transients (n = 6; mean event area before suramin application ± SEM = 4476 ± 1007 µm2 and after suramin application ± SEM = 1172 ± 389 µm2; p=0.0324 paired two-tailed Student’s t-test). Suramin application had no effect on Ca2+ transients in cKO mice (n = 5; mean event area before suramin application ± SEM = 466 ± 172 µm2 and after suramin application ± SEM = 379 ± 103 µm2; p=0.4842 paired two-tailed Student’s t-test) (Figure 1—figure supplement 2). This supports the notion that TMEM16A is important for the propagation of spontaneous activity between ISCs of Kölliker’s organ, probably via P2 receptors. TMEM16A-dependent cochlear activity modulates the burst-firing pattern of MNTB neurons By increasing the K+ concentration, TMEM16A-dependent activity of Kölliker’s organ leads to the generation of Ca2+ action potentials in IHCs. This is followed by Ca2+-dependent exocytosis of glutamate at the IHC synapse, which drives burst firing of action potentials in SGNs (Wang et al., 2015). The bursting activity is then relayed to central auditory neurons (Babola et al., 2018; Tritsch and Bergles, 2010; Figure 2A shows a schematic representation of the auditory brainstem) and is believed to be important for the proper development of synaptic contacts and tonotopic maps (Clause et al., 2014; Clause et al., 2017). Figure 2 with 1 supplement see all Download asset Open asset 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; LSO, lateral superior olive; SGN, spiral ganglion neurons. (B–E) Patterns of spontaneous discharge activity from individual MNTB neurons recorded from mice before hearing onset (P8) in wildtype and cKO littermates. Dotplot graphs show respective interspike interval (ISI) distributions for 100 s of spontaneous discharge activity. On top of each dotplot raster is a 10 s period of original spike trains. Note that the wildtype MNTB neuron shows prominent burst firing, which is either strongly reduced (D) or absent in cKO mice (C, E). (F) Quantification of spike bursting patterns by calculating the coefficient of variation of ISIs yields significant differences between wildtype (n = 14) and cKO units (n = 15) (Mann–Whitney rank-sum test: p=0.006); also shown are boxplots indicating medians and 25% and 75% quartiles. (G) The mean cumulative distribution of ISIs reveals the significant shift toward larger values in cKO mice (wildtype n = 14 and cKO n = 15; Kolmogorov–Smirnov test: p=0.0008, D = 0.19), with the median ISI increasing from 26.9 ms in wildtype to 76.3 ms in cKO mice. (H) The overlaid log-binned histogram compares the distribution of ISIs between wildtype and cKO mice. Values represent mean ± SEM (n = 14 wildtype, n = 15 cKO [P8]). For statistical analysis, the chi-square test was used (see Supplementary file 1a for p-values). (I) Iontophoretic injection with Fluorogold verifies recording site from in vivo juxtacellular voltage recordings from MNTB neurons in a prehearing wildtype mouse (P8). Scale bar 200 μm. Figure 2—source data 1 Source data for Figure 2A–G. https://cdn.elifesciences.org/articles/72251/elife-72251-fig2-data1-v2.xlsx Download elife-72251-fig2-data1-v2.xlsx Figure 2—source data 2 Source data for Figure 2H. https://cdn.elifesciences.org/articles/72251/elife-72251-fig2-data2-v2.xlsx Download elife-72251-fig2-data2-v2.xlsx To investigate the effects of Ano1 knockout on the burst-firing patterns in auditory brainstem neurons, juxtacellular single-unit recordings from MNTB neurons were obtained from in vivo prehearing mice (P8). Similar to SGNs (Tritsch et al., 2010; Jing et al., 2013), MNTB neurons exhibit spontaneous bursts of spikes that could last several seconds (Figure 2—figure supplement 1A; Sonntag et al., 2009). These long bursts are made up of a series of short ‘mini-bursts consisting of several spikes and occurring at intervals of approximately 100 ms (Figure 2—figure supplement 1B and C). Bursting activity was quantified by the coefficient of variation (CV) of interspike intervals (ISIs), whereby values below 1 correspond to random firing and higher values indicate more patterned activity (Jones et al., 2007). In wildtype mice, MNTB neurons showed the typical bursting activity (Figure 2B and F; n = 14, median CV [25%, 75% quartiles] = 3.2 [2.65, 3.56]) (Sonntag et al., 2009), while in cKO littermates MNTB firing patterns had significantly smaller CVs (Figure 2C–F; n = 15, median CV [25%, 75% quartiles] = 1.7 [1.2, 2.9]; p=0.006, Mann–Whitney test). Intermediate ISIs, representing the interval between mini-bursts, were severely diminished in all MNTB neurons recorded from cKO mice. A presentation of the firing pattern as mean cumulative distribution of ISIs (Figure 2G; Kolmogorov–Smirnov test: p=0.0008, D = 0.19) and in an overlaid log-binned histogram (Figure 2H; chi-square test, p-values are shown in Supplementary file 1a) illustrates a shift toward longer ISIs in cKO mice. The additional detailed analysis of burst-firing patterns in MNTB neurons revealed further significant differences between wildtype and cKO littermates for (i) the number of bursts per 100 s (median [25%, 75% quartiles]: WT = 3.9 [3.3, 5.8]; cKO = 0.6 [0, 3.8]; p=0.009, Mann–Whitney test), (ii) the number of spikes per burst (median [25%, 75% quartiles]: WT = 45 [21, 89]; cKO = 19 [14, 31]; p=0.00001, Mann–Whitney test), and (iii) the duration of bursts (median [25%, 75% quartiles]: WT = 1.6 [0.9, 3.3]; cKO = 0.7 [0.3, 1.8]; p=0.000002, Mann–Whitney test) (Figure 3A–C). MNTB neurons recorded from cKO mice showed a large variation of action potential firing rates, but still the mean firing rate did not differ from wildtype cells (Figure 3D; mean firing rate ± SEM: WT = 6.2 ± 0.8 Hz, n = 14; cKO = 5.8 ± 1.1 Hz, n = 15; p=0.73, Student’s t-test). Also, firing rates within bursts did not differ between TMEM16A cKO and WT mice (data not shown; WT: 26 [19; 36.8] AP/s, n = 85; cKO: 32 [16.4; 48.8] AP/s, n = 60; p=0.34, Mann–Whitney rank-sum test). Figure 3 with 1 supplement see all Download asset Open asset 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 (C) compared to wildtype. Values represent median with 25%, 75% quartiles (n = 14 WT; n = 15 cKO [P8]; Mann–Whitney rank-sum test: number of bursts per 100 s: p=0.009; number of spikes per burst: p=0.00001; duration of burst: p=0.000002). (D) The overall discharge rates did not differ between wildtype (n = 14) and cKO (n = 15) (two-tailed unpaired Student’s t-test: p=0.73). Figure 3—source data 1 Source data for Figure 3. https://cdn.elifesciences.org/articles/72251/elife-72251-fig3-data1-v2.xlsx Download elife-72251-fig3-data1-v2.xlsx Since TMEM16A is neither expressed in SGNs nor in CN, MNTB, and LSO neurons in wildtype mice and expression in the brainstem was limited to vascular smooth muscle cells (Figure 3—figure supplement 1), we primarily attribute the severely altered burst-firing activity to impaired Kölliker’s organ activity in cKO mice. Frequency selectivity of MNTB neurons is reduced in cKO mice To test whether the changes in burst-firing patterns of cKO MNTB neurons have consequences on neuronal function after hearing onset, auditory brainstem responses (ABRs) were measured at P13–14. cKO mice had similar ABR thresholds in response to stimulation with clicks or tone bursts at 6, 12, and 24 kHz as wildtypes (Figure 4A and B, Supplementary file 1b; n = 6 WT; n = 7 cKO). In both genotypes, ABRs to click stimuli of various intensities (40–100 dB) mainly consisted of three waves (labeled I–III), which were comparable in latency and amplitude (Figure 4—figure supplement 1, Supplementary file 1c and d). These data indicate that cKO mice have a normal sensitivity to sound stimulation and normal temporal precision of the spiking response to sound onset in the lower auditory pathway. Figure 4 with 1 supplement see all Download asset Open asset 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 stimuli did not differ between wildtype (n = 7) and cKO (n = 6) at P13–14. For values, see Supplementary file 1b. (C, D) Representative frequency response areas of MNTB neurons (P14) recorded juxtacellularly in a wildtype mouse (C) (characteristic frequencies [CF]: 24 kHz, threshold 0.1 dB SPL, Q10/20/30 = 6.4/4.3/4.2), and in a cKO littermate (D) (CF: 18.4 kHz, threshold = 3 dB SPL, Q10/20/30 = 3.7/3.2/2.8). Note that the response area is broader and that the CF thresholds are increased in cKO. (E) Frequency selectivity of MNTB neurons was reduced in cKO mice as indicated by lower Q-factors (shown are medians and 25%, 75% quartiles [n = 25 WT; n = 32 cKO, P14]; Mann–Whitney rank-sum test: Q10: p=0.03, Q20: p=0.008, Q30: p=0.002). (F) Sound thresholds of individual MNTB neurons are elevated in cKO mice compared to wildtype (p=0.006). (G) Average rate-level functions at CF show decreased action potential firing in cKO at SPLs above 10 dB (two-way ANOVA: effect of strain p<0.001, effect of intensity p<0.001). (H) Maximum firing rates during acoustic stimulation are significantly decreased in cKO mice compared to wildtype littermates (two-tailed unpaired Student’s t-test: p=0.015). Figure 4—source data 1 Source data for Figure 4A and B. https://cdn.elifesciences.org/articles/72251/elife-72251-fig4-data1-v2.xlsx Download elife-72251-fig4-data1-v2.xlsx Figure 4—source data 2 Source data for Figure 4E–H. https://cdn.elifesciences.org/articles/72251/elife-72251-fig4-data2-v2.xlsx Download elife-72251-fig4-data2-v2.xlsx Next, we assessed whether the disruption of TMEM16A-dependent cochlear activity affects the frequency tuning properties of MNTB neurons. Therefore, the frequency response areas (FRAs) of single MNTB neurons were acquired in four cKO and four wildtype littermates using in vivo electrophysiology and tone burst stimulation. Juxtacellular recordings were performed at P14, that is, shortly after the onset of hearing to avoid possible compensatory effects of acoustically driven activity on neuron responsiveness (Werthat et al., 2008; Bogart et al., 2011). The characteristic frequencies (CFs) of the recorded MNTB neurons, that is, the frequency value at which the neuron is excited with the lowest intensity , ranged between 5.3 and 30.5 kHz and did not differ between the two groups (mean CF ± SEM: WT = 15.4 ± 1.1 kHz [n = 25]; cKO = 16.2 ± 1.3 kHz [n = 32]; p=0.51, Student’s t-test). MNTB neurons recorded from wildtype mice showed the typical V-shaped FRAs with acoustically driven excitation sharply narrowing toward lower intensities (Figure 4C). The filter characteristics of the FRAs were quantified by the Qn-value, a measure of the unit’s sharpness of tuning, which is calculated as the ratio of CF to bandwidth at 10, 20, and 30 dB above threshold (e.g., Q10 = CF/BW10 with BW10 = bandwidth at 10 dB above threshold). For wildtype mice, the median [25%, 75% quartiles] was Q10 = 5.5 [4.7, 9.2], Q20 = 4.6 [3.8, 6.8], and Q30 = 3.6 [3.4, 5] (Figure 4E). Neurons recorded from the cKO littermates (n = 32) had significantly broader excitatory response areas, that is, lower frequency selectivity as indicated by significantly lower Q-factors at all three above-threshold levels (Figure 4D and E; median [25%, 75% quartiles]: Q10 = 4.7 [2.8, 6.3], p=0.03; Q20 = 3.1 [2.5, 4.9], p=0.008; Q30 = 2.7 [2.0, 3.7], p=0.002, Mann–Whitney test). Additionally, MNTB neurons in cKO mice had elevated thresholds in comparison to the wildtype littermates (median threshold [25%, 75% quartiles]: cKO: 6.3 [0.8, 29.6] dB SPL; WT = 0.6 [0.0, 4.6] dB SPL; p=0.006, Mann–Whitney test) (Figure 4F). Overall CF threshold levels tended to show a larger variability in knockout compared to wildtype mice (cKO: –7.3 dB SPL to 48.3 dB SPL; WT: –8.4 dB SPL to 18.7 dB SPL). Furthermore, the sound-evoked firing properties of the MNTB neurons in cKO mice were also affected. The rate-level functions at CF showed significantly lower firing rates for sound intensities at and above 10 dB SPL of tone-burst stimulation in comparison to wildtype littermates (effect of strain p<0.001, effect of intensity p<0.001, interaction strain × intensity p=0.006; two-way ANOVA) (Figure 4G). The maximal firing rate of individual neurons in response to any CF/intensity combination was markedly diminished in cKO mice (mean FR ± SEM: WT = 263.6 ± 12.4 action potentials/s, n = 25; cKO = 223.9 ± 11.1 action potentials/s, n = 32; p=0.015, t-test) (Figure 4H). Apparently, MNTB neurons in cKO mice achieve rates that are typically observed in wildtype littermates. Taken together, these data demonstrate that frequency selectivity and sensitivity to acoustic stimulation in single MNTB neurons are impaired upon disruption of Ano1 in the cochlea. The developmental refinement of functional connections of the MNTB-LSO pathway is impaired in cKO mice Despite the above-described differences in the pattern of spontaneous and sound-evoked activity in MNTB neurons between wildtype and cKO mice, the gross morphology of the MNTB and the LSO appeared normal (mean MNTB area ± SEM: WT = 63 × 103 ± 4.6 µm2 [n = 10]; KO = 55 × 103 ± 3.4 µm2 [n = 10]; p=0.0831; mean LSO area ± SEM: WT = 117 × 103 ± 6.8 µm2 [n = 10]; KO = 110 × 103 ± 3.9 µm2 [n = 10]; p=0.4325). Since spontaneous burst activity of auditory neurons might promote the targeting and refinement of their projections as suggested for the developing visual system (Torborg et al., 2005), we assessed the synaptic and topographic refinement of MNTB and LSO neurons. MNTB neurons were activated via photolysis of caged glutamate. A 405 nm continuous diode laser was used for illumination. Laser flashes were delivered through a light guide of 20 µm diameter, which produced circular spots of 20 µm diameter in the focal plane. Laser pulses of 10 ms duration were delivered with 6 s delay time between uncaging sites. The number of functional inputs on individual LSO neurons was assessed by whole-cell current-clamp recordings. In a pilot experiment, the distance was measured at which glutamate uncaging produces action potentials in MNTB neurons. The mean distance (± SEM) was 19.2 ± 0.8 µm mediolaterally and 20.0 ± 1.3 µm dorsoventrally, indicating that glutamate uncaging was locally restricted to a surface area of 20 × 20 µm2 and that light scattering that might influence the amount of uncaged glutamate in the tissue was negligible (Figure 5—figure supplement 1A–C). Notably, the size of MNTB input areas in cKO mice was doubled compared to wildtype (Figure 5A–C; mean cross-sectional input area ± SEM: WT = 10% ± 1% [n = 10] and cKO: 20% ± 3% [n = 10] of the respective MNTB cross-sectional areas; p=0.0017, Student’s t-test). Moreover, the input width, defined as the maximal distance of responsive uncaging sites along the mediolateral axis, was twice as large (Figure 5A, B and D; mean input width ± SEM: WT = 18% ± 3% of the MNTB’s mediolateral length; cKO = 36% ± 5% of the MNTB’s mediolateral length; p=0.0073, Student’s t-test). The large increase in MNTB input width in cKO mice reveals that LSO neurons located in the medial high-frequency region of the nucleus not only received input from neurons of the high-frequency (medial) region of the MNTB, but also from neurons in the mid-nuclear region tuned to lower frequencies. For additional examples comparing the size and width of MNTB input areas between wildtype and cKO mice, see Figure 5—figure supplement 1D and E. These data strongly point toward an impairment of the tonotopic refinement of MNTB-to-LSO projections in cKO mice. Figure 5 with 1 supplement see all Download asset Open asset 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 sites). The location of responsive (colored squares) and unresponsive (open squares) uncaging sites is indicated. Scale bar 40 µm. (B) Uncaging of glutamate close to presynaptic MNTB neurons gives rise to synaptic responses of various peak amplitudes (left),