Conductive Hearing Loss Has Long-Lasting Structural and Molecular Effects on Presynaptic and Postsynaptic Structures of Auditory Nerve Synapses in the Cochlear Nucleus

Abstract

Sound deprivation by conductive hearing loss increases hearing thresholds, but little is known about the response of the auditory brainstem during and after conductive hearing loss. Here, we show in young adult rats that 10 d of monaural conductive hearing loss (i.e., earplugging) leads to hearing deficits that persist after sound levels are restored. Hearing thresholds in response to clicks and frequencies higher than 8 kHz remain increased after a 10 d recovery period. Neural output from the cochlear nucleus measured at 10 dB above threshold is reduced and followed by an overcompensation at the level of the lateral lemniscus. We assessed whether structural and molecular substrates at auditory nerve (endbulb of Held) synapses in the cochlear nucleus could explain these long-lasting changes in hearing processing. During earplugging, vGluT1 expression in the presynaptic terminal decreased and synaptic vesicles were smaller. Together, there was an increase in postsynaptic density (PSD) thickness and an upregulation of GluA3 AMPA receptor subunits on bushy cells. After earplug removal and a 10 d recovery period, the density of synaptic vesicles increased, vesicles were also larger, and the PSD of endbulb synapses was larger and thicker. The upregulation of the GluA3 AMPAR subunit observed during earplugging was maintained after the recovery period. This suggests that GluA3 plays a role in plasticity in the cochlear nucleus. Our study demonstrates that sound deprivation has long-lasting alterations on structural and molecular presynaptic and postsynaptic components at the level of the first auditory nerve synapse in the auditory brainstem.

Significance statement: Despite being the second most prevalent form of hearing loss, conductive hearing loss and its effects on central synapses have received relatively little attention. Here, we show that 10 d of monaural conductive hearing loss leads to an increase in hearing thresholds, to an increased central gain upstream of the cochlear nucleus at the level of the lateral lemniscus, and to long-lasting presynaptic and postsynaptic structural and molecular effects at the endbulb of the Held synapse. Knowledge of the structural and molecular changes associated with decreased sensory experience, along with their potential reversibility, is important for the treatment of hearing deficits, such as hyperacusis and chronic otitis media with effusion, which is prevalent in young children with language acquisition or educational disabilities.

Keywords: 3D reconstructions; AMPA receptors; postembedding immunogold labeling; postsynaptic density; synaptic vesicles; vGluT1.

Figure 1.

Scheme of the innervation of auditory nerve synapses on bushy cells and ABR recordings. A, Scheme of the innervation of the auditory nerve on bushy cells of the anteroventral cochlear nucleus. AN, Auditory nerve; BC, bushy cell; Ch, cochlea. B, Diagram illustrating the experimental procedure. Animals were divided in two groups: shams and earplugged. Earplugged animals were recorded before earplugging (at P30), during earplugging (at P40, i.e., 10 d after earplugging), and after earplug removal (at P50). Shams were recorded at P30, P40, and P50 as age-matched controls without the earplug. C, Representative ABR waveforms from the same animal before earplugging (gray line), during earplugging (red line), and 10 d after earplug removal (green line) in the ipsilateral side. Waveform patterns between waves I and IV were similar before earplugging (EP) and after earplug removal. D, Scatterplots of the ABR thresholds to clicks for the two animal groups (shams and earplugged) at the different ages tested (P30, P40, and P50). Means are displayed as error bars (±SEM). Earplug significantly increased ABR thresholds during earplugging (post hoc comparisons, Holm–Sidak method, within P40: earplugged vs sham, t = 17.80, p < 0.001). Earplugged animals did not recover to control levels 10 d after earplug removal (post hoc comparisons, Holm–Sidak method, within P50: earplugged vs sham, t = 3.42, p < 0.001). Asterisks indicate significance between earplugged animals and shams within the same age. E₁, E₂, Average population ABR thresholds to the different frequencies tested (4, 16, 24, and 32 kHz) in shams (E₁) and earplugged animals (E₂). Earplugged animals significantly increased ABR thresholds for each frequency during earplugging (post hoc comparisons, Holm–Sidak method, within P40: earplugged vs sham; 4 kHz: t = 13.5, p < 0.001; 8 kHz: t = 14.14, p < 0.001; 16 kHz: t = 11.36, p < 0.001; 24 kHz: t = 13.07, p < 0.001; 32 kHz: t = 13.52, p < 0.001). Earplugged animals did not recover their hearing thresholds after earplug removal to frequencies higher than 16 kHz (post hoc comparisons, Holm–Sidak method, within P50: earplugged vs sham; 16 kHz: t = 3.25, p < 0.01; 24 kHz: t = 4.38, p < 0.001; 32 kHz: t = 5.54, p < 0.001) but did recover their hearing thresholds in response to lower frequencies (post hoc comparisons, Holm–Sidak method, within P50: earplugged vs sham; 4 kHz: t = 0.84, p = 0.41; 8 kHz: t = 1.04, p = 0.3₁). Asterisk indicates significance between earplugged animals and shams within the same age. F₁–F₃, Scatterplots of the ABR wave I (F₁), II (F₂), and IV (F₃) amplitudes measured at 10 dB SPL above threshold in the two animal groups for the three age levels. Earplugging had no significant effect on ABR amplitudes (2-way repeated-measures ANOVAs showed no significant interactions between age and animal group). Wave I amplitudes significantly decreased between P30 and P40 (post hoc comparisons, Holm–Sidak method, t = 4.45, p < 0.001), but this decrease can be attributed to the effect of age occurring in both animal groups independently of earplugging. G, Relative central gain in earplugged animals. Wave ratios of the earplug groups (II/I and IV/I) were normalized by the wave ratios of the age-matched sham groups as described in Materials and Methods.

Figure 2.

Monaural conductive hearing loss decreases vGluT1 expression within the cytoplasm of auditory nerve endings, and vGluT1 expression increases after earplug removal. A, Top row, Examples of auditory nerve endings on bushy cells immunolabeled with vGluT1 (10-nm-diameter gold particles) at low magnification. White boxed areas are magnified on the bottom row. Arrowheads note the edge of the PSD. AN, Auditory nerve; BC, bushy cell. Scale bars: top row, 250 nm; bottom row, 200 nm. B, Summary of the mean area of auditory nerve endings and number and density of gold particles for vGluT1 per auditory nerve ending. Areas of endbulb profiles were the same between the earplugged group and the age-matched P40 shams (p > 0.05, Mann–Whitney U test). vGluT1 gold labeling was significantly decreased in the earplugged animals compared with age-matched shams (number: P40 sham, 197.6 ± 30.57; P40 EP, 54.4 ± 7.91; ****p < 0.0001; density: P40 sham, 54.51 ± 7.10; P40 EP, 15.68 ± 1.7; ****p < 0.0001, Mann–Whitney U test). Density and gold labeling for vGluT1 after earplug removal was higher than age-matched shams (number: P50 sham, 183.6 ± 27.45; P50 EP-removal, 486.2 ± 61.75; ***p < 0.003; density: P50 sham, 57.95 ± 5.93; EP-removal, 150 ± 23.4; ****p < 0.001, Mann–Whitney U test). Number and density of vGluT1 labeling was the same in P40 and P50 shams (p > 0.05, Mann–Whitney U test). C, Average number of vGluT1 gold particles per SV in sham and earplugged animal groups. The number of gold particles per SV was significantly decreased in the earplugged animals compared with age-matched shams (****p < 0.0001, Mann–Whitney U test). No difference was found in the number of vGluT1 gold particles per SV after earplugged removal compared with age-matched shams (p = 0.20, Mann–Whitney U test). The difference between earplugged animals and age-matched controls was not found statistically significant (p > 0.05, Mann–Whitney U test test).

Figure 3.

Changes in SV density, size, and volume in response to monaural conductive hearing loss. A, Electron micrographs of auditory nerve synapses on bushy cells of sham (P40), earplugged (P40), and postearplug-removal (P50) groups. White boxed areas are shown at higher magnification on the bottom row. Arrowheads note the edge of the PSD. AN, Auditory nerve; BC, bushy cell. Scale bars: top row, 250 nm; bottom row, 200 nm. B, Plot of the density of SVs per square micrometer of auditory nerve endings. SV density was similar in age-matched shams and earplugged animals (p = 0.07, Mann–Whitney U test) but was significantly increased in the earplug-removal group compared with age-matched shams (p < 0.0001, Mann–Whitney U test). Error bars are ± SEM. C, The mean SV size was significantly lower in the earplugged animals compared with age-matched shams (***p < 0.0001, Mann–Whitney U test), but it was significantly higher in the earplug-removal group compared with age-matched shams (***p < 0.0001, Mann–Whitney U test). Error bars are ± SEM. The mean SV volume was significantly lower in the earplugged animals compared with age-matched shams (***p < 0.0001, Mann–Whitney U test), but it was significantly higher in the earplug-removal group compared with age-matched shams (***p < 0.0001, Mann–Whitney U test). Error bars are ±SEM.

Figure 4.

Monaural conductive hearing loss enlarges the postsynaptic membrane of auditory nerve synapses on bushy cells. A, Drawings of an auditory nerve (AN) synapse on a bushy cell (BC) showing how the thickness of the PSD was calculated. B, Representative PSD diagrams of the sham, earplug, and postearplug-removal groups that were obtained from the 3D reconstructions of serial ultrathin sections. Scale bar, 50 nm. C, Plot showing a significant increase in the mean PSD area of the synapse of the auditory nerve on bushy cells after earplug removal (sham P40, 0.036 ± 0.003 μm²; earplugged P40, 0.045 ± 0.005 μm²; **p = 0.02, Mann–Whitney U test; sham P50, 0.037 ± 0.004 μm²; earplug-removal P50, 0.058 ± 0.005 μm²; p = 0.0006, Mann–Whitney U test). Error bars are ±SEM. D, Plot showing a significant increase in the average PSD thickness during earplug and after 10 d of earplug removal (sham P40, 26.21 ± 0.88 nm; earplugged P40, 41.72 ± 1.72 nm; ****p < 0.0001, Mann–Whitney U test; sham P50, 25.47 ± 1.56 nm; earplug-removal P50, 43.28 ± 1.793 nm; ****p < 0.0001, Mann–Whitney U test). Error bars are ±SEM.

Figure 5.

Monaural conductive hearing loss increases GluA3 AMPAR subunits at the synapse of the auditory nerve on bushy cells. A, Examples of high-magnification electron micrographs of auditory nerve synapses on bushy cells that were immunogold labeled (10-nm-diameter gold particles) for GluA2/3, GluA2, and GluA4 from sham and earplugged animals at P40. AN, Auditory nerve; BC, bushy cell. Scale bar, 50 nm. B, Distribution of GluA2/3, GluA2, and GluA4 gold particles per PSD of the synapse of the auditory nerve on bushy cells from sham and earplugged (EP) animals. GluA2/3 labeled/total #PSD: sham, 125/155; EP, 125/144; GluA2 labeled/total #PSD: sham, 48/112; EP, 46/106; GluA4 labeled/total #PSD: sham, 44/112; EP, 32/112. C, Plots of the mean number and density of gold particles showing that GluA2/3 gold labeling was increased in earplugged (EP) animals (number: sham, 3.21 ± 0.23; EP, 3.80 ± 0.21; p = 0.0132, Mann–Whitney U test; density: sham, 12.52 ± 0.66; EP, 10.51 ± 0.68; p = 0.0122, Mann–Whitney U test). Gold labeling for GluA2 and GluA4 was similar between sham and EP animals (GluA2 number: sham, 0.61 ± 0.08; EP, 0.68 ± 0.11; p = 0.8389, Mann–Whitney U test; density: sham, 2.05 ± 0.26; EP, 2.29 ± 0.35; p = 0.9374, Mann–Whitney U test; GluA4 number: sham, 0.64 ± 0.13; EP, 0.42 ± 0.07; p = 0.1690, Mann–Whitney U test; density: sham, 2.1 ± 0.40; EP, 1.47 ± 0.26; p = 0.1603, Mann–Whitney U test). Error bars are ±SEM. #GP, Number of gold particles per PSD.

Figure 6.

Redistribution of GluA2 and GluA3 AMPAR subunits at the synapse of the auditory nerve on bushy cells after earplug removal. A, Examples of high-magnification electron micrographs of auditory nerve synapses on bushy cells that were immunogold labeled (10-nm-diameter gold particles) for GluA2/3, GluA2, and GluA4 from sham and earplug-removal groups at P50. AN, Auditory nerve; BC, bushy cells. Scale bar, 50 nm. B, Distribution of gold particles for GluA2/3, GluA2, and GluA4 per PSD of auditory nerve synapses on bushy cells from sham and earplug-removal animals (GluA2/3 labeled/total #PSD: sham, 89/145; earplug-removal, 178/218; GluA2 labeled/total #PSD: sham, 61/142; earplug-removal, 52/199; GluA4 labeled/total #PSD: sham, 47/139; earplug-removal, 63/207). C, Plots of the mean number and density of GluA2/3, GluA2, and GluA4 gold particles at the synapse of the auditory nerve on bushy cells from earplug-removal animals and age-matched shams. There was a significant increase in GluA2/3 gold particles (sham, 2.60 ± 0.28; earplug-removal, 3.33 ± 0.22; p = 0.0002, Mann–Whitney U test) and a significant decrease in GluA2 gold particles (sham, 0.68 ± 0.08; earplug-removal, 0.37 ± 0.05; p = 0.0008, Mann–Whitney U test) in the earplug-removal group compared with the sham group. The gold labeling for GluA4 was similar between groups (sham, 0.43 ± 0.05; earplug-removal, 1.28 ± 0.06; p = 0.5455, Mann–Whitney U test). Error bars are ±SEM. The density of gold particles for GluA2/3 was increased after earplug removal (sham, 7.40 ± 0.75; earplug-removal, 10.24 ± 0.46; p < 0.0001, Mann–Whitney U test), whereas there was a significant decrease in GluA2 density (sham, 2.26 ± 0.25; earplug-removal, 1.12 ± 0.16; p = 0.0002, Mann–Whitney U test). The density of GluA4 gold particles was similar between groups (sham, 1.47 ± 0.20; earplug-removal, 1.28 ± 0.16; p = 0.4619, Mann–Whitney U test). Error bars are ±SEM. #GP, Number of gold particles per PSD.

Figure 7.

Diagram summary of the data obtained in this study. After 10 d of earplugging, there was a decrease in vGluT1 labeling at auditory nerve (AN) endings and in the size of SVs. Postsynaptically, the PSDs of the AN on bushy cell (BC) somata were thicker and contained increased GluA3 AMPAR subunits. Ten days after earplug removal, there was an increase in the density of SVs in the AN; SVs were also larger in size. On BCs, the PSD apposed to the AN were larger and thicker, and they contained fewer GluA2 but more GluA3 subunits.