Changes in Properties of Auditory Nerve Synapses following Conductive Hearing Loss

Abstract

Auditory activity plays an important role in the development of the auditory system. Decreased activity can result from conductive hearing loss (CHL) associated with otitis media, which may lead to long-term perceptual deficits. The effects of CHL have been mainly studied at later stages of the auditory pathway, but early stages remain less examined. However, changes in early stages could be important because they would affect how information about sounds is conveyed to higher-order areas for further processing and localization. We examined the effects of CHL at auditory nerve synapses onto bushy cells in the mouse anteroventral cochlear nucleus following occlusion of the ear canal. These synapses, called endbulbs of Held, normally show strong depression in voltage-clamp recordings in brain slices. After 1 week of CHL, endbulbs showed even greater depression, reflecting higher release probability. We observed no differences in quantal size between control and occluded mice. We confirmed these observations using mean-variance analysis and the integration method, which also revealed that the number of release sites decreased after occlusion. Consistent with this, synaptic puncta immunopositive for VGLUT1 decreased in area after occlusion. The level of depression and number of release sites both showed recovery after returning to normal conditions. Finally, bushy cells fired fewer action potentials in response to evoked synaptic activity after occlusion, likely because of increased depression and decreased input resistance. These effects appear to reflect a homeostatic, adaptive response of auditory nerve synapses to reduced activity. These effects may have important implications for perceptual changes following CHL.

Significance statement: Normal hearing is important to everyday life, but abnormal auditory experience during development can lead to processing disorders. For example, otitis media reduces sound to the ear, which can cause long-lasting deficits in language skills and verbal production, but the location of the problem is unknown. Here, we show that occluding the ear causes synapses at the very first stage of the auditory pathway to modify their properties, by decreasing in size and increasing the likelihood of releasing neurotransmitter. This causes synapses to deplete faster, which reduces fidelity at central targets of the auditory nerve, which could affect perception. Temporary hearing loss could cause similar changes at later stages of the auditory pathway, which could contribute to disorders in behavior.

Keywords: conductive hearing loss; endbulbs of Held; release probability; release sites; synaptic transmission.

Figure 1.

Assessing hearing thresholds using ABRs before, during occlusion, and following recovery from occlusion. A, Representative traces from a P20 control mouse (left), a P19 occluded mouse (middle), and a P27 recovered mouse (right). Traces were evoked by click stimuli and were averages of 1024 trails. Thresholds are indicated by arrows. B, Average ABR thresholds for control (5 ears), bilateral occlusion (8 ears), and recovery (7 ears). After occlusion, thresholds were significantly elevated by 48.0 ± 1.6 dB SPL (p < 0.001). After recovery, thresholds were not significantly different compared with control (p > 0.1).

Figure 2.

P_r at endbulbs increased after occlusion. A, Representative EPSC traces recorded from a BC in a P20 control mouse (left) and in a P23 occluded mouse (right). Single auditory nerve fibers were stimulated in pairs with interpulse intervals from 3 to 20 ms. B, Quantification of depression using PPR (EPSC₂/EPSC₁) for control (open circles) and occluded (closed circles) cells in A. C, Average PPR for control (13 cells) and occlusion (22 cells) from mice aged P19–P30. Occlusion induced greater depression at all intervals. *p < 0.05. **p < 0.01. Markers are shown as mean ± SEM. Some errors bars are obscured by markers. D, EPSC₁ amplitude distributions. There was no significant difference between control (28 cells) and occlusion (45 cells) (NS, p > 0.5, Kolmogorov–Smirnov test). E, PPR at 3 ms interval for control (10 cells) and occluded (29 cells) endbulbs as a function of age from P15 to P29. F, PPR as a function of age, averaged over ≥2 days from control and occluded mice. PPRs after occlusion were significantly smaller starting at P21 (P15–P18: p = 0.3; P19–P20: p = 0.15; P21–P22: p = 0.004; P23–P24: p = 0.01).

Figure 3.

Q remained the same after occlusion. A, Representative spontaneous mEPSCs from a control mouse (left) and an occluded mouse (right). Cumulative frequency plots of average mEPSC frequency (B) and amplitude (C) from mice under control (13 cells) and occlusion (12 cells) condition. Occlusion had no significant effect on mEPSC amplitude (p > 0.1, Kolmogorov–Smirnov test) or frequency (p > 0.5, Kolmogorov–Smirnov test) of mEPSC. D, Cumulative frequency plots of 100 events total per cell under control (6 cells) and occlusion (8 cells) conditions. No significant difference was observed between control and occluded BCs (p > 0.1, Kolmogorov–Smirnov test).

Figure 4.

Quantification of P_r, N, and Q. A, C, Example experiments showing effects of different Ca_e on EPSC amplitude over the course of the experiment from a control mouse (P22, A) and an occluded mouse (P23, C). Lines above the points indicate the data that are used for analysis. B, D, Markers indicate variance versus EPSC amplitude for each Ca_e concentration from experiments in A and C. Lines are fits to the equation σ² = Qμ − (μ²/N), with N = 81 and Q = 108 in B, and N = 70 and Q = 90 in D. From these, P_r at 1.5 Ca_e is calculated to be 0.39 in B, and 0.64 in D. E–G, Cumulative frequency plot of N (E), P_r (F), and Q (G) from control mice (9 cells, black) and occluded mice (16 cells, red). After occlusion, N significantly decreased and P_r significantly increased (p < 0.05). Q showed no significant changes (p > 0.1). H, J, Representative long trains recordings in the presence of 1 mm kynurenate from a control mouse (H) and an occluded mouse (J) in response to stimulation of a single AN fiber (100 Hz, 45 pulses). I, K, EPSC amplitude is integrated, and a fit is extrapolated back from the last 20 cumulative EPSCs to the y-axis to estimate N (straight line). P_r is calculated by dividing the first EPSC by N. L, M, Quantification of N (L) and P_r (M) using the integration method from control mice (11 cells) and occluded mice (10 cells). Occluded endbulbs showed significantly smaller N (*p < 0.05) and higher P_r (*p < 0.05).

Figure 5.

Recovery of P_r and N after returning to normal sound conditions. A, Experimental timeline for the 4 experimental groups shown in B and C. One group of mice (Ctrl_early) were raised under normal sound conditions, and recordings were performed around P24. The second group (Occ) were occluded at P21 until recording at P24. The third group (Rec) were ligated at P21, unligated at P24, and recorded after P30. The fourth group (Ctrl_late) were raised under normal sound conditions and recorded after P30. Quantification of N (B) and P_r (C) using the integration method from Ctrl_early group (10 cells), Occ group (11 cells), Ctrl_late group (13 cells), and Rec group (15 cells), similar to Figure 4. D, Measurement of PPR at Δt = 3 ms for treatment groups in A. These measurements were performed in the absence of kynurenate and were therefore done on separate cells from those shown in B and C. Error bars indicate mean ± SEM. Markers indicate individual BCs. *p < 0.05.

Figure 6.

Endbulb structure changed after occlusion. A, Single optical sections of a control (P31, left) mouse and an occluded (P31, right) mouse immunolabeled for VGLUT1 (green). Scale bar, 10 μm. B, Outlines of puncta for a control (left) and an occluded (right) mouse shown in A. C, Number of puncta surrounding BCs for control mice (10 cells) and occluded mice (10 cells). The number of puncta after occlusion was similar compared with control mice (p > 0.1). Error bars indicate mean ± SEM. Circles indicate number of puncta for individual BCs. D, Average area of puncta surrounding BCs for control mice (10 cells) and occluded mice (10 cells). Puncta significantly shrank after occlusion (p < 0.05). Error bars indicate mean ± SEM. Line marks indicate average area of puncta for individual BCs. *p < 0.05.

Figure 7.

BC excitability decreased after occlusion. A, Representative current-clamp traces recorded from a P20 control (left) mouse and a P23 occluded (right) mouse in response to current injection. B, Spike thresholds for control (8 cells) and occluded (24 cells) mice. Open circles represent raw data. Line markers indicate average results for control and occlusion. There were no significant changes in threshold (p > 0.5). C, Average plots of voltage versus injected current from control (8 cells) and occluded (24 cells) mice. D, Input resistances (R_in) at −60 mV for control and occluded mice. R_in significantly decreased after occlusion (p < 0.05). Line markers indicate mean ± SEM. Open circles represent individual data points. E, F, Example traces of current-clamp recordings of control BCs (left) and occluded BCs (right) in response to fiber stimulation at 100 Hz (E) and 200 Hz (F). Strip chart plots of firing probability for both 100 Hz (G) and 200 Hz (H). Firing probability was calculated during the last 10 stimuli during the trains. Spike fidelity decreased significantly after occlusion (*p < 0.05).