Changes in P_r.

To assess P_r, we recorded from BCs in whole-cell voltage clamp, isolated individual presynaptic AN fibers, and delivered pairs of stimuli with various intervals. A representative recording is shown in Fig. 1A, Left, with the endbulb showing typically strong depression at intervals up to 20 ms, reflecting high P_r. From these data, we calculated the paired-pulse ratio (PPR) by normalizing the amplitude of the second EPSC to the first (PPR = EPSC₂/EPSC₁) (Fig. 1B). PPR was below 1 at all interpulse intervals (Δt) (Fig. 1B, open circles). Similar results were found in 28 cells in this study (Fig. 1C) and are consistent with previous recordings from BCs confirmed by cell filling (14).

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Fig. 1.

Noise exposure lowers P_r at the endbulb of Held. AN fibers were stimulated with pairs of pulses of different interpulse intervals, Δt = 3–20 ms. (A) Representative traces recorded from P26 BCs from control and noise-reared animals. In control conditions (Left), EPSCs show depression at all intervals whereas, after noise rearing (Right), EPSCs show no depression, but rather facilitation. (B) Paired-pulse ratio (PPR) for the representative experiments in A. (C) Average PPR for control (n = 17) and noise-reared (n = 22) in animals P21 or older. PPR differed significantly (P < 0.005) at all intervals. (D) PPR for control (n = 28) and noise-reared (n = 26) at Δt = 3 ms as a function of age. Noise rearing commenced at P12. (E) PPR at Δt = 3 ms for control (open circles) and recovery (closed triangles) BCs. (F) Average PPR is slightly elevated over control after recovery (control n = 15; recovery n = 20, P < 0.05 for each interval). (G) Cumulative frequency of EPSC₁ amplitudes in BCs from control (n = 34) and noise-reared animals (n = 22). The amplitudes do not differ significantly (P > 0.5, Kolmogorov–Smirnov test). Averages in C and F and in subsequent figures are represented by the mean ± the SEM.

We tested the impact of activity on P_r by exposing mice to constant noise beginning at postnatal day 12 (P12) and assessing P_r beginning at P18. The example endbulb in Fig. 1 A, Right and B showed no depression, but rather facilitation for interpulse intervals from 3 to 20 ms. This PPR is well above the range observed under normal conditions. Recordings from a number of BCs showed significant increases in PPR after noise rearing. We confirmed the cell identity using neurobiotin in the intracellular solution in some experiments (control n = 12, noise-reared n = 13) (Fig. S1), and cell structure seemed normal (26⇓⇓⇓⇓⇓–32). PPR for Δt = 3 ms was 0.61 ± 0.04 in control (n = 17) whereas, after noise rearing, PPR was 0.93 ± 0.06 (n = 22), which is significantly higher (P = 0.005, Student’s unpaired t test). PPRs at all intervals up to 20 ms were significantly higher (P < 0.05) (Fig. 1 B and C). The increase in PPR was first observed around P22, during which time PPR is relatively stable in control synapses (Fig. 1D). An increase in PPR results from a decrease in P_r so these results suggest that activity plays an important role in setting P_r.

We next tested whether synapses were permanently altered by exposing mice to noise until P26, then allowing recovery in normal sound conditions for at least 2 wk. After restoration of a normal sound environment, the PPR eventually recovered to near normal levels (Fig. 1E) although it remained significantly higher than control at all intervals. For Δt = 3 ms, PPR after recovery was 0.767 ± 0.031 (n = 20), which was significantly higher than control BCs (PPR = 0.606 ± 0.028, n = 15, P < 0.05, t test) (Fig. 1F). Thus, P_r decreased in noise and increased back near normal levels when normal sound conditions were restored, suggesting that changes in P_r are adaptive and depend on sensory experience.

We tested the effects of noise rearing on general auditory function using auditory brainstem responses (ABRs). There were no significant changes in either threshold or amplitudes of the individual ABR components (Fig. S2). We also assessed hair cell synapses onto auditory nerve afferents by counting CtBP2-immunopositive synaptic ribbons. There was no significant change after noise rearing in the number of ribbons per inner hair cell in base, middle, or apical regions of the basilar membrane (Fig. S3). There were also no noticeable changes in the positions of the synaptic ribbons. Thus, the levels of noise we used for these experiments did not cause any visible, permanent hearing loss or damage to the cochlea.

We also assessed how noise rearing affected the first EPSC (EPSC₁) in each pair. There were no significant changes in half-width (control, 0.43 ± 0.01 ms, n = 34; noise-reared, 0.42 ± 0.01 ms, n = 22, P > 0.5) or in decay τ (control, 0.186 ± 0.006 ms, n = 34; noise-reared, 0.174 ± 0.007 ms, n = 22, P > 0.2). These values fit well within the range that we have observed before (14). In addition, the distribution of EPSC₁ amplitudes was not significantly changed by noise rearing [P > 0.5, Kolmogorov–Smirnov (K–S) test] (Fig. 1G). This lack of change in EPSC₁ is surprising because, all else being equal, a decrease in P_r should cause a decrease in EPSC₁ amplitude. Therefore, it appears that other factors compensate for the decrease in P_r.

We first considered the quantal size (Q) because it can change homeostatically with activity (1). We recorded miniature EPSCs (mEPSCs) from BCs of control and noise-reared mice. The representative cells in Fig. 2A had similar mEPSC frequency (control = 6.4 events per s; noise-reared = 5.3 events per s), similar average mEPSC amplitude (control, 80 pA; noise-reared, 96 pA), and similar distributions of mEPSC amplitude (Fig. 2B). On average, noise-reared BCs were not significantly different from control in average mEPSC amplitude (control, 89 ± 5 pA, n = 19; noise-reared, 96 ± 6 pA, n = 12, P = 0.30, K–S test) (Fig. 2C) or frequency (control, 3.5 ± 0.32/s, n = 19; noise-reared, 4.8 ± 0.75/s, n = 12, P = 0.37, K–S test) (Fig. 2D). Thus, AN synapses did not show obvious homeostatic changes in Q with activity, indicating that another factor must compensate for the decrease in P_r, most likely the number of release sites, N.

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Fig. 2.

Noise rearing has no effect on mEPSCs. (A) Representative traces showing mEPSCs measured in BCs from control (Top) and noise-reared (Bottom) animals. (B) Cumulative histogram of mEPSC amplitude for the representative cells in A. (C) Cumulative histogram of average mEPSC amplitudes measured in BCs from control (n = 19) and noise-reared (n = 12) animals. The distributions do not differ significantly (P = 0.3, K–S test). (D) Cumulative histogram of mEPSC frequency in BCs from control (n = 19) and noise-reared (n = 12) animals. The distributions of frequency do not differ significantly (P = 0.37). (E) Representative EPSC trains recorded in the presence of 1 mM kynurenate from a control and noise-reared BC in response to stimulation of a single presynaptic AN fiber (45 pulses, 100 Hz). (F) Derivation of N and P_r from trains data in E, using the integration method. EPSC amplitudes are integrated, and a fit is extrapolated back to the y axis. Arrowheads mark the estimated value of N. P_r is calculated by dividing the first EPSC by N. (G) Average P_r and N measured using the integration method. Noise-reared endbulbs have significantly lower P_r (0.30 ± 0.01, P < 0.005) and larger N (10.1 ± 0.1 nA, P < 0.05) compared with control (P_r = 0.45 ± 0.04, N = 5.9 ± 1.2 nA).

To verify quantitative changes in P_r and N, we used the integration method (20, 33⇓–35). Single AN inputs were stimulated with a long train (45 pulses, 100 Hz) (Fig. 2E) in the presence of 1 mM kynurenate to prevent AMPA receptor saturation and desensitization. The peak amplitudes of the evoked EPSCs were integrated (markers in Fig. 2F), and the steady-state rate of replenishment was estimated by a straight-line fit to the last 20 pulses of the cumulative EPSC (lines in Fig. 2F). The y intercept of the fit is proportional to N (arrows in Fig. 2F), which significantly increased after noise rearing (12 control endbulbs, 5.9 ± 1.2 nA vs. 9 noise-reared, 10.1 ± 1.0 nA, P < 0.05) (Fig. 2G, black bars). P_r is calculated by dividing the first EPSC in the train by N. P_r decreased significantly after noise rearing (control, 0.45 ± 0.04 vs. noise-reared, 0.30 ± 0.01, P < 0.005) (Fig. 2G, white bars). These data reinforce the conclusions based on PPR and EPSC₁ data that noise rearing leads to a decrease in P_r and an increase in N.

Anatomical Changes.

We considered whether there were changes in synaptic structure consistent with an increase in N. AN synapses are primarily axosomatic (36⇓–38). To quantify such changes, we immunolabeled endbulbs using an antibody against vesicular glutamate transporter-1 (VGLUT-1), which shows strong punctate staining around the BC soma (Fig. 3 A and B) (30, 38⇓–40). These puncta represent areas of the endbulb containing releasable vesicles, and thus the areas containing release sites. We quantified the average cross-sectional area of puncta in the optical section taken at the midline of each cell. The sizes of puncta around BCs from noise-reared animals were larger than those from controls (control, 1.71 ± 0.18 µm², n = 10; noise-reared, 2.37 ± 0.25 µm², n = 15; P < 0.05, t test) (Fig. 3C). This increase in synaptic area is consistent with an increase in the number of release sites.

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Fig. 3.

Anatomical changes at light and ultrastructure levels. (A and B) Endbulbs immunolabeled for VGLUT-1 in control (A) and noise-reared (B) animals. BC somata are surrounded by multiple VGLUT-1–positive puncta. (Scale bar: A and B, 20 µm.) (C) Puncta area for endbulbs from control and noise-reared mice. Horizontal lines mark average areas of puncta around individual BCs, and bars are overall averages across all cells (10 control, 15 noise-reared). The average area of puncta around noise-reared BCs is significantly greater than around control BCs (P < 0.05). (D and E) Representative profiles of endbulbs from control (D) and noise-reared (E) animals. Each profile has numerous synaptic vesicles, curved and asymmetric postsynaptic densities (asterisks). (Scale bar: D and E, 1 µm.) (F–H) Endbulbs in noise-reared mice had significantly more postsynaptic densities per profile (P < 0.05, Fisher exact test; F), larger profile perimeter and cross-sectional area (P < 0.001; G) and larger form factors (P < 0.001; H).

To gain a better understanding of the ultrastructural changes underlying the larger area in VGLUT-1 puncta, we examined electron micrographs. AN synapses from control animals resembled those described previously for the CBA/CaJ strain (30). Each section through an endbulb (a “profile”) displayed clusters of mitochondria, numerous large round synaptic vesicles, and curved, asymmetric postsynaptic densities (Fig. 3 D and E). Extended extracellular spaces, mitochondrion adherens complexes, and puncta adherentia were sometimes observed in profiles. Glial sheaths surrounded the synaptic terminals. AN profiles from noise-reared animals seemed generally similar to control.

We quantified differences between quiet- and noise-reared mice (n = 22 profiles from control mice, and n = 33 profiles from noise-reared mice). Noise-reared mice had more postsynaptic densities (PSDs) per profile (P < 0.05, Fisher exact test) (Fig. 3F), larger profile cross-sectional areas (control, 1.23 ± 0.14 µm² vs. noise-reared, 2.59 ± 0.32 µm², P < 0.001, t test) (Fig. 3G), and larger form factors (area/perimeter, control, 0.19 ± 0.01 µm vs. noise-reared, 0.27 ± 0.02 µm, t test, P < 0.001) (Fig. 3H). Thus, noise-reared endbulbs show clear changes in structure that are consistent with an increase in release site density.

Postsynaptic Effects.

Sound-driven activity can influence postsynaptic excitability in other parts of the auditory pathway, such as the medial nucleus of the trapezoid body (41). We examined how noise rearing might influence the intrinsic properties of BCs in the AVCN. Representative responses to depolarizing current injection for control and noise-reared BCs are shown in Fig. 4. Spikes maintained the undershooting amplitude characteristic of BCs (Fig. 4A). There were small, but significant, shifts in peak of AP (control, −18.10 ± 2.06 mV, n = 30, vs. noise-reared, −22.61 ± 1.38 mV, n = 32, P = 0.03, t test) and threshold voltage (control, −42.49 ± 0.79 mV vs. noise-reared, −45.3 ± 0.77 mV, P < 0.005, t test) (Fig. 4B). Spikes in BCs from noise-reared animals had shorter half-width than control BCs (control, 0.76 ± 0.08 ms, n = 15 vs. noise-reared, 0.54 ± 0.05 ms, n = 26, P = 0.03). We saw no significant difference in the resting membrane potential (control, −56 ± 1 mV; noise-reared, −57 ± 0.5 mV, P > 0.05) between control and noise-reared BCs. We also saw no significant change in the input resistance (Fig. S4). The decrease in spike threshold, together with the speeding of the action potential and the decreased synaptic depression, might enhance the likelihood of BCs to respond more reliably during high rates of synaptic activity.

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Fig. 4.

Effects of noise rearing on spike generation and fidelity of BC spiking. (A) Representative current-clamp recordings in response to current pulses of 0.6 nA in control and noise-reared BCs. (B) Average measurements of 30 BCs from control and 32 BCs from noise-reared mice, showing significant decreases in action potential peak voltage (Top, P = 0.03), threshold voltage (Middle, P < 0.005), and spike half-width (Bottom, P < 0.05). (C) Representative traces showing responses to fiber stimulation at 100 (Top) and 333 Hz (Bottom) in BCs from control (Left) and noise-reared (Right) mice. (D) Cumulative frequency histogram of spike probability (P_spike) over the second half of the stimulation train (pulses 11–20), for experiments similar to C. P_spike increased significantly after noise rearing (P < 0.05, asterisks) for both 100 (i) and 333 (ii) Hz.

We tested these possibilities directly by doing fiber stimulation in current-clamp recordings. We activated AN fibers using trains of stimuli at different frequencies (100, 200, and 333 Hz). In control conditions, BCs fired reliably early in the trains of stimuli but failed to fire at later pulses, especially for the 333 Hz stimulation rate (Fig. 4C, Left), presumably because of synaptic depression. However, in noise-reared BCs, spiking was much more reliable for those later pulses (Fig. 4C, Right). When we quantified spike probability for pulses 11‒20, it was much higher for noise-reared BCs at all frequencies tested (K–S test, P < 0.05) (Fig. 4D). We also quantified spike latency and jitter, but neither was affected significantly by noise rearing (P > 0.1) (Fig. S4). Thus, spike reliability increased greatly after noise rearing.