From hidden hearing loss to supranormal auditory processing by neurotrophin 3-mediated modulation of inner hair cell synapse density

Abstract

Loss of synapses between spiral ganglion neurons and inner hair cells (IHC synaptopathy) leads to an auditory neuropathy called hidden hearing loss (HHL) characterized by normal auditory thresholds but reduced amplitude of sound-evoked auditory potentials. It has been proposed that synaptopathy and HHL result in poor performance in challenging hearing tasks despite a normal audiogram. However, this has only been tested in animals after exposure to noise or ototoxic drugs, which can cause deficits beyond synaptopathy. Furthermore, the impact of supernumerary synapses on auditory processing has not been evaluated. Here, we studied mice in which IHC synapse counts were increased or decreased by altering neurotrophin 3 (Ntf3) expression in IHC supporting cells. As we previously showed, postnatal Ntf3 knockdown or overexpression reduces or increases, respectively, IHC synapse density and suprathreshold amplitude of sound-evoked auditory potentials without changing cochlear thresholds. We now show that IHC synapse density does not influence the magnitude of the acoustic startle reflex or its prepulse inhibition. In contrast, gap-prepulse inhibition, a behavioral test for auditory temporal processing, is reduced or enhanced according to Ntf3 expression levels. These results indicate that IHC synaptopathy causes temporal processing deficits predicted in HHL. Furthermore, the improvement in temporal acuity achieved by increasing Ntf3 expression and synapse density suggests a therapeutic strategy for improving hearing in noise for individuals with synaptopathy of various etiologies.

Fig 1. Timeline of the experiments.

Experimental timeline showing the ages of mice for tamoxifen treatments, ABR measurements, behavioral assay (ASR, PPI, GPIAS), and sample collections for quantitative RT-PCR and immunostaining (P = postnatal day, wk = weeks). ABR, auditory-brainstem response; ASR, acoustic startle response; DPOAE, distortion product otoacoustic emission; GPIAS, gap-inhibition of the acoustic startle; PPI, prepulse inhibition.

Fig 2. Ntf3 expression in Plp1⁺ cells impacts TrkC signaling in the cochlea, not in the CNS.

mRNA level of Ntf3 and VGF, a gene downstream of TrkC signaling, are reduced in Ntf3-KD cochleas (A) and increased in Ntf3-OE cochleas (B). Furthermore, cochlear of Ntf3 and VGF mRNA levels are correlated (C). In contrast, cortical Ntf3 mRNA level is slightly decreased in Ntf3-KD mice (D) and unchanged in Ntf3-OE mice (E). No changes in VGF mRNA levels are observed in the brains of either Ntf3-KD or Ntf3-OE mice (D, E). n = 6–8, ns = p > 0.05, * p < 0.05, ** p < 0.01, mRNA levels were compared by two-tailed unpaired t test. The data underlying this figure can be found in S1 Data. Error bars represent SEM. CNS, central nervous system.

Fig 3. Ntf3 regulates IHC synapse density.

Representative confocal images of IHC synapses at the 16 kHz cochlear region from Ntf3-KD (A) and Ntf3-OE (E) mice and their respective controls immunolabeled for presynaptic ribbons (CtBP2—red), postsynaptic receptor patches (GluA2—green), and hair cells (Myo7a - blue). Mean counts (± SEM) of ribbons (B, F), GluA2 patches (C, G), and colocalized markers (D, H) in Ntf3 KDs and OEs. n = 5, ns = p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, *** p < 0.0001. Synaptic markers were compared by two-way ANOVA. The data underlying this figure can be found in S1 Data, the raw images were deposited in the Dryad repository (https://doi.org/10.5061/dryad.k6djh9w8v). Error bars represent SEM. IHC, inner hair cell.

Fig 4. Ntf3 knockdown or overexpression influence ABR peak I amplitudes without effecting cochlear thresholds.

DPOAE (A, D) and ABR (B, E) thresholds in Ntf3-KD and Ntf3-OE mice are not different than their controls. In contrast, Ntf3 knockdown reduces ABR P1 amplitudes (C), whereas overexpression leads to increased peak I amplitudes (F). Representative traces of DPOAEs (G) and ABRs (H). n = 15–24. ABR P1 amplitudes were assessed at 80 dB SPL. ns = p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001 by two-way ANOVA. The data underlying this figure can be found in S1 Data. Error bars represent SEM. ABR, auditory-brainstem response; DPOAE, distortion product otoacoustic emission.

Fig 5. Ntf3 knockdown or overexpression have different effects on the input-output function of ABR peaks I–IV.

Mean amplitude vs. level functions for ABR peaks I–IV in Ntf3-KD (A) and Ntf3-OE (B) mice and their respective controls at 16 kHz. Whereas peak I amplitudes are reduced in Ntf3-KD mice, the amplitudes of the other peaks remain normal, indicative of central compensation (A). In contrast, Ntf3 overexpression increases amplitudes of ABR peaks I to IV (B). N = 14–20, ns = p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001 by two-way ANOVA. The data underlying this figure can be found in S1 Data. Error bars represent SEM. ABR, auditory-brainstem response.

Fig 6. Ntf3 expression levels do not influence the latencies of the ABR waveform peaks.

Plots of peak latency recorded at 16 kHz against sound stimulus level show that latencies of ABR peaks I–V are not altered by Ntf3-KD (A) and Ntf3-OE (B). n = 14–20, ns = p > 0.05 by two-way ANOVA. The data underlying this figure can be found in S1 Data. Error bars represent SEM. ABR, auditory-brainstem response.

Fig 7. IHC synapse density does not influence the acoustic startle response or prepulse inhibition.

Schematics of the protocols for prepulse stimulus (A1), startle stimulus (B1), and PPI stimuli (C1). (A1) The noise prepulse stimulus is a narrowband noise (4 kHz width around variable center frequencies, 65 dB SPL, 50 ms duration). (B1) The startle stimulus is a broadband noise (120 dB SPL, 20 ms duration). (C1) PPI consists of a noise prepulse and a startle stimulus that starts 50 ms after the prepulse. (A2, A3) Reactivity to prepulse is not significantly different between mutant and control mice. (B2, B3) Loud sound (120 dB SPL) elicits startle responses with amplitudes that were similar in mutant mice and their control littermates. (C2, C3) The degree of prepulse inhibition of the startle response by a prepulse was determined using the formula $PPI=1-\frac{\mathrm{s}\mathrm{t}\mathrm{a}\mathrm{r}\mathrm{t}\mathrm{l}\mathrm{e}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{p}\mathrm{o}\mathrm{n}\mathrm{s}\mathrm{e}\mathrm{w}\mathrm{i}\mathrm{t}\mathrm{h}\mathrm{p}\mathrm{r}\mathrm{e}\mathrm{p}\mathrm{u}\mathrm{l}\mathrm{s}\mathrm{e}}{startleresponsewithoutprepulse}$. On average, the prepulse inhibit the startle response by 40%. There is no significant difference between control and mutant mice. N = 20–24 mice/group for response to prepulse (A2, A3); n = 18–20 mice/group for startle response (B2, B3); n = 11–17 mice/group for PPI (C2, C3). ns = p > 0.05 by two-tailed unpaired t tests (B2, B3) or two-way ANOVA (C2, C3). The data underlying this figure can be found in S1 Data. Mean ± SEM are shown. IHC, inner hair cell; PPI, prepulse inhibition.

Fig 8. Ntf3 expression levels influence gap detection thresholds in broadband background noise.

(A) Schematic depiction of NO-GAP trials (left) and GAP trials (right). NO GAP trials consisted of a startle sound (120 dB SPL, 20 ms duration) presented in continuous noise background (broadband noise, BBN, 65 dB SPL). In contrast, in the GAP trials, a silent gap in the background noise of variable length (0–50 ms) was presented ending 50 ms before the startle stimulus (S). (B, C) ASR amplitudes for the NO-GAP trials were similar in Ntf3 mutant mice and their control littermates. (D, E) Show the level of gap inhibition vs. gap length and for Ntf3 KD and OE mice, respectively. The inhibition of the startle reflex increases as the gap duration increases. (F, G) Show gap detection thresholds. Gap detection threshold is increased in Ntf3-KD mice (H) and decreased in Ntf3-OE mice (I) compared to their littermate controls. (H, I) Show level of Rd’ vs. gap length for Ntf3 KD and OE mice, respectively. n = 7–20 mice/group, *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001 by two-tailed unpaired t test (B, C, H, and I) or two-way ANOVA (D, E, F, and G). The data underlying this figure can be found in S1 Data. Mean ± SEM are shown. ASR, acoustic startle response; BBN, broadband background noise.

Fig 9. Ntf3 expression levels influence gap inhibition in NBN.

(A) A schematic view of No gap trials (left) and gap trials (right). No gap trials consist of a startle sound (120 dB SPL, 20 ms duration) presented in continuous noise background (narrowband noise, NBN, 4 kHz width around variable center frequencies, 65 dB SPL). Gap-prepulse inhibition of the acoustic startle (GPIAS) was tested in gap trials with the same background noise with a 50-ms gap included as a prepulse followed 1 ms later by the startle-eliciting stimulus. (B, C) Responses to startle stimulus in continuous NBN background are unaffected in the Ntf3-KD or Ntf3-OE mice. (D, E) GPIAS in narrowband noises were significantly weakened in Ntf3-KD mice and strengthened in Ntf3-OE mice in a two-way ANOVA. Sidak multiple comparison tests revealed a significant reduction at frequency of background noise band 10–14, 14–18, and 22–26 kHz in Ntf3-KD mice and no frequency-specific changes in Ntf3-OE mice. n = 10–14 mice/group, *p < 0.05, **p < 0.01 by two-way ANOVA. The data underlying this figure can be found in S1 Data. Mean ± SEM are shown. GPIAS, gap-inhibition of the acoustic startle; NBN, narrowband background noise.