Noise-induced damage to ribbon synapses without permanent threshold shifts in neonatal mice

Neuroscience 304 (2015) 368–377

NOISE-INDUCED DAMAGE TO RIBBON SYNAPSES WITHOUT PERMANENT THRESHOLD SHIFTS IN NEONATAL MICE L. SHI, a  X. GUO, a,b  P. SHEN, a  L. LIU, a S. TAO, a X. LI, a Q. SONG, c Z. YU, d S. YIN c* AND J. WANG a,d*

exposure did produce a significant loss of ribbon synapses, particularly in P14d mice, which continued to increase from P4w to P8w. Additionally, a corresponding reduction in the amplitude of compound action potential (CAP) was observed in the noise-exposed groups at P4w and P8w, and the CAP latency was elongated, indicating a change in synaptic function. Ó 2015 The Authors. Published by Elsevier Ltd. on behalf of IBRO. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/).

a

Department of Physiology, Medical College of Southeast University, 87 Dingjiaoqiao Road, Nanjing 210009, China b Children’s Medical Center, The Second Affiliated Hospital of Nanjing Medical University, 262 Zhongshan Road North, Nanjing 210003, China c Department of Otolaryngology, 6th Affiliated Hospital, Jiaotong University, 600 Yishan Road, Shanghai 200233, China d School of Human Communication Disorders, Dalhousie University, 1256 Barrington Street, Halifax, NS B3J1Y6, Canada

Key words: noise, permanent threshold synapse, cochlea, neonatal mice.

Abstract—Recently, ribbon synapses to the hair cells (HCs) in the cochlea have become a novel site of interest in the investigation of noise-induced cochlear lesions in adult rodents (Kujawa and Liberman, 2009; Lin et al., 2011; Liu et al., 2012; Shi et al., 2013). Permanent noise-induced damage to this type of synapse can result in subsequent degeneration of spiral ganglion neurons (SGNs) in the absence of permanent changes to hearing sensitivity. To verify whether noise exposure during an early developmental period produces a similar impact on ribbon synapses, the present study examined the damaging effects of noise exposure in neonatal Kunming mice. The animals received exposure to broadband noise at 105-decibel (dB) sound pressure level (SPL) for 2 h on either postnatal day 10 (P10d) or postnatal day 14 (P14d), and then hearing function (based on the auditory brainstem response (ABR)) and cochlear morphology were evaluated during either postnatal weeks 3–4 (P4w) or postnatal weeks 7–8 (P8w). There were no significant differences in the hearing threshold between noise-exposed and control animals, which suggests that noise did not cause permanent loss of hearing sensitivity. However, noise

shift,

ribbon

INTRODUCTION Noise exposure is the most common cause of acquired sensorineural hearing loss (SNHL) in modern society (NIDCD, 1995; Stucken and Hong, 2014), but the mechanisms underlying this type of hearing loss remain unclear. A majority of studies have focused on mechanical damage and the deleterious effects of noise exposure on outer hair cells (OHCs) as the primary factors involved in this process (Ivory et al., 2014; Stucken and Hong, 2014; Furness, 2015). While the damaging effects of noise on ribbon synapses between spiral ganglion neurons (SGNs) and cochlear hair cells (HCs), particularly inner hair cells (IHCs), have also been assessed by previous studies, (Puel et al., 1997; Pujol and Puel, 1999), this type of damage was largely considered to be repairable and, therefore, not important. More recently, however, quantitative studies have demonstrated the development of permanent noise-induced damage in ribbon synapses, which suggests that the repair of these synapses following such damage is incomplete (Kujawa and Liberman, 2009; Lin et al., 2011; Liu et al., 2012; Shi et al., 2013). For example, in adult rodents (both mice and guinea pigs), brief exposure to relatively low-level noise that does not cause a permanent threshold shift (PTS) in hearing can produce massive damage to the ribbon synapses (Kujawa and Liberman, 2009; Lin et al., 2011; Liu et al., 2012; Shi et al., 2013). Without a PTS, this type of damage would likely go unnoticed by human subjects and would almost certainly be missed during routine audiology evaluations that focus primarily on hearing threshold levels. Therefore, this kind of acoustic trauma might be silent or subclinical. Even though noise-induced damage to the ribbon synapses may be partially repairable, permanent damage to these synapses can result in the

*Corresponding authors at: School of Human Communication Disorders, Dalhousie University, 1256 Barrington Street, Halifax, NS B3J1Y6, Canada. Tel: +1-9024945149; fax: +1-902 4945151 (J.Wang) and Dept. of Otolaryngology, 64h Affilicated Hospital, Jiaotong Unversity, Shanghai, 200233, China. Tel: 8621-64834143 (S. Yin). E-mail addresses: [email protected] (L. Shi), guoxiaojing [email protected] (X. Guo), [email protected] (P. Shen), [email protected] (L. Liu), [email protected] (S. Tao), [email protected] (X. Li), [email protected] (Q. Song), [email protected] (Z. Yu), [email protected] (S. Yin), Jian.Wang@ dal.ca (J. Wang).   These authors contributed equally. Abbreviations: ANOVA, analysis of variance; CAP, compound action potential; CaV1.3, voltage-sensitive calcium channels; CtBP2, C-terminal-binding protein 2; dB, decibel; HCs, hair cells; IHCs, inner hair cells; OHCs, outer hair cells; PBS, phosphate-buffered saline; PSD, postsynaptic density; PTS, permanent threshold shift; P4w, postnatal week 4; P8w, postnatal week 8; P10d, postnatal day 10; P14d, postnatal day 14; SEM, standard errors of the mean; SGNs, spiral ganglion neurons.

http://dx.doi.org/10.1016/j.neuroscience.2015.07.066 0306-4522/Ó 2015 The Authors. Published by Elsevier Ltd. on behalf of IBRO. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 368

L. Shi et al. / Neuroscience 304 (2015) 368–377

degenerative death of SGNs that are disconnected from the IHCs (Kujawa and Liberman, 2009; Lin et al., 2011). Furthermore, although noise-induced damage can occur in the absence of hearing loss (as defined by threshold elevations), there may still be functional deficits in cochlear coding. More specifically, this type of massive damage will likely result in a significant reduction in cochlear output to the auditory cortex that can be identified by reductions in the amplitude of the compound action potential (CAP) and, ultimately, impairments in the coding of sound intensity. The synaptic ribbon, which is a characteristic structure of ribbon synapses located mainly in the inner ear and on the retina (Nouvian et al., 2006), is responsible for the fast release of neurotransmitters by HCs (Fuchs et al., 2003; Fuchs, 2005; Sterling and Matthews, 2005; Nouvian et al., 2006; Moser et al., 2006a,b). Therefore, this structure likely plays a critical role during temporal resolution in the cochlea (Nouvian et al., 2006; Moser et al., 2006a,b; Schmitz, 2009). The importance of ribbon synapses in the cochlea has been demonstrated in animal studies involving a mutation in Bassoon, a critical ribbon protein (Buran et al., 2010). In these mutant cochlea, the synapses between SGNs and IHCs are of the conventional type without ribbons, and although the mutated animals show no differences from normal controls in singleunit thresholds, their temporal coding ability is largely reduced (Buran et al., 2010; Jing et al., 2013). Additionally, a recent study from our group revealed that the damage and repair of ribbon synapses resulting from noise exposure is likely accompanied by deterioration in temporal processing (Shi et al., 2013). The clinical implications of noise-induced damage are important. Because it is more likely that an individual will be exposed to noise that does not result in a change in PTS than to a stronger stimulus in an industrial setting, the deterioration in temporal processing that occurs with aging may be due, at least in part, to the ‘‘silent’’ noise-induced damage of ribbon synapses. Moreover, these effects may go unnoticed and accumulate with age. To our knowledge, all previous investigations of noiseinduced damage in cochlear ribbon synapses have been conducted in adult animals. However, accumulating evidence suggests that auditory sensory organs appear to be more sensitive to a variety of ototoxic factors, including noise, during early development (Hall, 2000; Surenthiran et al., 2003; Li and Steyger, 2009; Reeves et al., 2010). The postnatal period in rodents has long been identified as one that is sensitive to acoustic trauma (Lenoir and Pujol, 1980; Saunders and Chen, 1982), but early studies focused primarily on noise damage to OHCs. To date, no studies have investigated the effects of noise on ribbon synapses in the neonatal mammalian cochleae. Because exposure to noise during early development is a highly probable occurrence, particularly for preterm infants exposed to life-support systems (Surenthiran et al., 2003; Lahav, 2014; Laubach et al., 2014; Park et al., 2014), a better understanding of the effects of noise-induced damage on ribbon synapses during early development is important for the formulation of noise safety guidelines. Thus, the present study examined the

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effects of noise-induced silent damage on ribbon synapses in neonatal mice during the early onset of hearing. The present results demonstrated that noise exposure shortly after birth resulted in similar but less marked damage to the ribbon synapses than has been observed in previous studies conducted in adult mice (Kujawa and Liberman, 2009; Lin et al., 2011; Liu et al., 2012; Shi et al., 2013). However, in contrast to adult animals, the loss of ribbon synapses progressed continuously in the young mice for more than 1 month following exposure to noise.

EXPERIMENTAL PROCEDURES Subjects and experimental outline Pregnant Kunming mice were obtained from Qinglongshan Animal Farm (Nanjing, Jiangsu, China), which is a qualified provider of laboratory animals, and 36 neonatal mice were recruited from four litters for the present study. The neonatal mice were divided randomly into three groups of equal size (n = 12) with an equal number of males and females. Experimental Group one was exposed to noise on postnatal day 10 (P10d), Experimental Group two was exposed to noise on postnatal day 14 (P14d), and the control group was not exposed to noise. Within each group, the animals were divided into two subgroups (n = 6 each) based on the performance of the end tests: either postnatal week 4 (P4w) or postnatal week 8 (P8w). The six subgroups were labeled such that the timing of both the noise exposure and end tests is indicated, as follows: Ctrl4w, Ctrl8w, Exp10d4w, Exp10d8w, Exp14d4w, and Exp14d8w. For example, Exp14d4w indicates that the mice in this group were exposed to noise on P14d and examined at 4 weeks of age. The animals in the experimental groups were exposed to broadband noise at a 105-decibel (dB) sound pressure level (SPL) for 2 h on either P10d or P14d, whereas the animals in the control group underwent sham exposure (environmental change) on P14d. All mice were returned to their mothers after the exposure session. The end tests included auditory brainstem response (ABR) and CAP assessments for functional evaluation and morphological examination of the ribbon synapses. All animal procedures were approved by the University Committee for Laboratory Animals of Southeast University, China (Permit number: SYXK 2011-0009). Noise exposure The experimental subjects were exposed to the broadband noise while awake and unrestrained in a cage. The floor of the cage was 60 cm below the horns of one low-frequency woofer and one high-frequency tweeter, and electrical Gaussian noise was delivered through the two loudspeakers after power amplification. The acoustic spectrum of the sound was distributed mainly below 20 kHz, as described previously (Liu et al., 2012), and the frequency range for sound density 10 dB below the peak was between 3 and 14 kHz. The noise level was monitored using a 0.25-inch microphone linked to a sound level meter (microphone: 2520, sound level meter: 824, Larson Davis; Depew, NY, USA).

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Physiological tests Prior to the ABR and CAP recordings, the animals were anesthetized using pentobarbital (80 mg/kg, intraperitoneally), and their body temperature was maintained at 37.5–38 °C on a thermostatic heating pad. Three subdermal needle electrodes were used to record the ABRs: the non-inverting electrode was inserted at the vertex in the middle point between the two eyes, and the reference and grounding electrodes were on the two earlobes. To record the CAPs, a silver ball electrode that was led to the non-inverting channel of the pre-amplifier was placed on the round window membrane of the ear via a small hole that penetrated the bulla inferior and posterior to the external ear canal. TDT hardware and software (TDT: Tucker-Davis Technologies; Alachua, FL, USA) were used for the stimulus generation and bio-signal acquisition. The acoustic stimuli used were as follows: (1) 10-ms tone bursts with cos2 gating and a 0.5-ms rise/fall time and (2) equal-level paired clicks lasting 80 ls with inter-click intervals (ICIs) varying from 2 to 20 ms. The stimuli were played through a broadband speaker (MF1; TDT) that was placed 10 cm in front of the heads of the animals. The evoked responses were amplified 20-fold and then filtered between 100 and 3000 Hz using a preamplifier (RA16PA; TDT), which digitized the signal at a sampling rate of 25 kHz. The responses were averaged 1000 times for the ABR and 100 times for the CAP. The ABR thresholds were measured across 2–48 kHz with tone bursts at a rate of 21.1/s. At each frequency, the test was performed in a down sequence starting at 90dB SPL and decreasing in 5-dB steps until the ABR response disappeared; the threshold was defined as the lowest level at which a repeatable wave III response could be obtained. The CAP was recorded in response to paired clicks with an overall repetition rate of 11.1/s. The clicks were presented at four supra-threshold levels (50-, 60-, 70-, and 80-dB peak equivalent [peSPL]), and the amplitude of the CAP to the second click (CAP2) was measured as a function of the ICI at each of the four levels to determine the response change to time stress. Morphology The primary morphological measure used in the present study was the number of ribbons; this assessment was performed successfully in 6–10 cochleae per group. Following the endpoint functional tests, the cochleae were immediately harvested and transferred to phosphate-buffered saline (PBS) kept at 4 °C. They were perfused rapidly three times with 4% paraformaldehyde in PBS buffer and then underwent a brief 1 h postfixation at 4 °C and then decalcification in 10% EDTA at room temperature for 6–10 h. Next, each cochlea was transferred to PBS, and the bone over the middle earfacing portion of the cochlear spiral was removed using fine forceps. Following the removal of the tectorial membrane, the cochlea was permeabilized with 0.01% Triton X-100 in PBS for 30 min, incubated for 30 min in 5% goat serum in PBS, incubated in primary antibody

(1:200, mouse anti-C-terminal-binding protein 2 [CtBP2] IgG1, cat. # 612044; BD Biosciences, San Jose, CA, USA) overnight at 4 °C, and then incubated in secondary antibodies (1:1000, goat anti-mouse IgG1, A21124; Invitrogen, Shanghai, China) for 2 h at room temperature. All of the antibodies were diluted in 5% goat serum in PBS. Following the immunostaining procedure, the basilar membrane of each cochlea was dissected into four pieces, mounted onto microscope slides, and coverslipped. To reduce the variability and increase the reliability of the results, control and experimental samples from the same time points were processed together under identical conditions. Confocal images were acquired using a confocal laser-scanning microscope (510 META; Zeiss, Shanghai, China) with 100 oil-immersion objectives and the image stacks were ported to image-processing software (Lsmix and ImageJ). The laser excitation power and microscope emission and detection settings were identical for all observations. Across the entire basilar membrane, the CtBP2-immunoreactive puncta were counted from a total of 11 regions in terms of the percentage distance from the apex. In each region, counting was performed in all of the IHCs and OHCs that were identified within 2–3 microscopic fields, which typically produced a total of 9–11 IHCs and 25–35 OHCs. The total numbers of CtBP2-stained puncta were divided by the total number of IHC and OHC nuclei to obtain the average number of ribbons for each HC. Statistical analysis All data are expressed as mean ± standard errors of the mean (SEM). Post hoc multiple comparisons were conducted following an analysis of variance (ANOVA). P < 0.05 was considered to indicate statistical significance.

RESULTS The ABR thresholds of the three groups from the two study time points are compared in Fig. 1; all curves largely overlapped. For the sake of simplicity, hearing sensitivity was summarized as the averaged threshold across the frequency evaluated, and the overall effect of the noise exposure was assessed by a one-way ANOVA for each end test time point. There were no significant differences in the frequency-averaged thresholds among the three groups at any of the time points (data not shown). Representative images of the ribbons immunostained for CtBP2 are shown in Fig. 2. Compared with the control group, the P14d group exhibited a reduction in ribbon counts in the IHC region and possibly in the OHC region following noise exposure. In both noise-exposed groups, there were fewer IHC ribbons in the P8w subgroups, which were assessed approximately 6 weeks after noise exposure, than in the P4w subgroups, suggesting a continuation of ribbon damage after P4w. To quantitatively evaluate the changes in the ribbons, ribbon counts were expressed as the ribbon number for each IHC (ribbon#/IHC) at 11 different spots across the

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Fig. 1. ABR thresholds-frequency curves. Animals exposed to noise at both P10d and P14d did not show any significant ABR threshold differences from the control groups as measured at the two ages (P4w and P8w). See Experimental procedures section for grouping information.

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cochlea to form a ribbon-count cochleogram (Fig. 3A). The cochleograms of the ribbons overlapped largely between the two control groups (Ctrl4w and Ctrl8w), suggesting that the number of ribbons remained stable after P4w in the control subjects. Similar to previous reports (Liu et al., 2012; Shi et al., 2013), the ribbon#/IHC was highest in the middle-frequency region in both the control and noise-damaged cochleae (Fig. 3A). Compared with the control group, the ribbon#/IHC cochleograms in the noise-exposed groups exhibited a downward shift due to the ribbon loss that was generally parallel across the entire cochlea. Additionally, noise exposure at P14d produced a greater ribbon loss than that at P10d, and there was a greater degree of ribbon loss on P8w compared with P4w in both noise-exposed groups (Fig. 3B). The noise-induced changes in ribbon count are summarized as the cochlear averages of the

Fig. 2. Representative images of cochlear ribbons showing the noise-induced changes in ribbon numbers. Ctrl: control sample at age of P8w. Exp14d4w: the sample observed at P4w from an animal exposed to noise at P14d. Exp14d8w: the sample observed at P8w from animal exposed to noise at P14d.

Fig. 3. Comparison of ribbon counts across groups. A: ribbon count cochleograms, B: averaged ribbon#/IHC for the whole cochlea. ***p < 0.001 against control, ###p < 0.001 between the two time points (P4w and P8w) of observation within animals exposed to noise at the same times (P10d or P14d), &&&p < 0.001 between the two groups of noise-exposed animals (P10d and P14d) within animals tested at two different ages (P4w or P8w).

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ribbon#/IHC in Fig. 3B. Because the values from the two control subgroups (Ctrl4w and Ctrl8w) did not differ significantly, they were pooled (Ctrl4/8w; 14.535 ± 0.0834 ribbons/IHC). The values for the two P10d subgroups were 13.280 ± 0.0303 at P4w (Exp10d4w) and 10.895 ± 0.0493 at P8w (Exp10d8w), which corresponded to reductions of 8.6% and 25%, respectively, compared with the control group. The ribbon#/IHC values of the two P14d subgroups were lower than those of the P10d subgroups at P8w: 12.734 ± 0.0720 (Exp14d8w) and 10.052 ± 0.0606 (Exp14d8w), corresponding to reductions of 12.4% and 30.8%, respectively, compared with the control group. A two-way ANOVA conducted using noise exposure (control, ExP10d, and ExP14d) and the times of observation (control, P4w, and P8w) as factors revealed main significant effects for both factors (noise exposure: F2 = 819.376, p < 0.001 and observation time: F2 = 601.129, p < 0.001). Tukey’s post hoc pairwise comparisons for noise exposure revealed that the ribbon counts pooled over the two observation times were significantly lower in the two noise-exposed groups than in the control group: for Exp10d, the difference from the mean of the control was 3.142/IHC (q = 42.439, p < 0.001) and for Exp14d, the difference from the mean of the control was 2.447/IHC (q = 54.496, p < 0.001). Additionally, the count of the Exp14d group was significantly lower than that of the Exp10d group with the difference between the means being 0.695 (q = 12.057, p < 0.001). All of the values of the noise-exposed groups at the time points of the end tests (P4w and P8w) were significantly lower than those of the control group (as indicated by * in Fig. 3B), and in each noise-exposed group the ribbon/IHC count was significantly lower at P8w than at P4w (as indicated by # in Fig. 3B). Moreover, the ribbon counts were significantly lower in the Exp14d than Exp10d groups at each end test time point (as indicated by & in Fig. 3B). Exposure to noise also appeared to result in a loss of ribbons in the OHCs, which are innervated by type II SGNs. The ribbon#/OHC was 2.878 ± 0.171 in the control group at P8w (Ctrl8w), which was considerably higher than those in the two experimental subgroups receiving noise exposure at P14d (Exp14d4w: 1.643 ± 0.0792 and Exp14d8w: 1.924 ± 0.0159). A one-way ANOVA revealed a significant effect of noise treatment (F2 = 35.109; p < 0.001), and Tukey’s post hoc pairwise comparisons showed significant differences between the control and noise-exposed animals (p < 0.001). However, there was no significant difference in ribbon#/OHC between the Exp14d4w and Exp14d8w groups, suggesting that unlike the IHC ribbons, the ribbon loss in OHCs did not progress after P4w. Similar to the reduction in ribbon counts, there was also a reduction in CAP amplitude in the noise-exposed groups in the present study, which assessed CAP in response to paired clicks for the purpose of examining temporal processing. Because the CAP was evaluated across four sound levels, the amplitude/sound level function was established against the CAP evoked by the

first click (CAP1; Fig. 4). Because the values from the two control subgroups (Ctrl4w and Ctrl8w) did not differ significantly and for the sake of simplicity, these values were pooled (Ctrl4/8w). The amplitude/sound level curves of the two subgroups exposed to noise at P10d generally overlapped with that of the control, while the amplitude/sound level curves of the two subgroups exposed to noise at P14d were largely downshifted, particularly in the Exp14d8w subgroup. A two-way ANOVA using groups and sound levels as factors revealed a significant effect of noise exposure on CAP1 amplitude. A Tukey’s post hoc comparison of the groups showed that the CAP1 amplitudes of the Exp14d4w and Exp14d8w groups were significantly lower than that of the control (q = 4.773, p = 0.007 and q = 14.519, p < 0.001, respectively). There were no significant differences between the control group and the two P10d subgroups. A post hoc pairwise comparison for each sound level showed that the reduction in the CAP1 amplitude in the Exp14d4w subgroup was only significant at the highest sound level, but that this reduction was significant at all sound levels in the Exp14d8w subgroup (as indicated by the number of asterisks in Fig. 4). Changes in CAP1 latency as a function of sound level are summarized in Fig. 5; again, the data from the two control subgroups were pooled (Ctrl4/8w). In general, the CAP1 latency was longer in the noise-exposed groups than the control group; more specifically, there was a longer latency in the P14d group, and this manifested to a greater degree at P8w than at P4w. Two-way ANOVA using group and sound levels as factors revealed a significant effect of noise exposure on CAP1 latency, and Tukey’s post hoc comparison showed that the CAP1 latencies in the Exp10d8w and Exp14d8w subgroups were significantly longer than that of the control group (q = 8.025, p < 0.001 and q = 16.499, p < 0.001 respectively). There were no significant differences between the control group and the other two subgroups. Significant differences identified by post hoc pairwise comparisons conducted

Fig. 4. Comparison of click-evoked CAP amplitude across groups. The control obtained at P4w and P8w are pooled into Ctr4w/8w. *, ** and *** were for p < 0.05, 0.01 and 0.001 (post hoc tests against the control after a two-way ANOVA against group and sound level).

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Fig. 5. Comparison of click-evoked CAP peak latency across groups. * ** , and *** were for p < 0.05, 0.01 and 0.001 (post hoc tests against the control after a two-way ANOVA against group and sound level). Fig. 7. Comparison of CAP2 peak latency at 50-dB peSPL and 20ms ICI. ***p < 0.001 as compared to the control; ###p < 0.001 between Exp14d4w and Exp14d8w.

for each sound level are indicated by the number of asterisks for each data point in Fig. 5. To further analyze the impact of noise-induced damage in the ribbon synapses on temporal processing, the amplitudes and latencies of the CAPs evoked by CAP2 were measured as a function of the ICI, which varied from 2 to 20 ms. Fig. 6A illustrates the changes in CAP2 amplitude as a function of the ICIs from each group. Because the two CAPs partially overlapped when the ICI was 64 ms, the waveform obtained at any ICI 64 ms was subtracted from the waveform obtained during the 20-ms ICI prior to the measurement of CAP2. This method of subtraction was also applied to the CAP2 latency measurement reported later. The most striking finding was an overall reduction in CAP2 amplitude across the different ICIs, which was greater in the animals exposed to noise at P14d and similar to the results for CAP1. To determine whether CAP2 dropped off more rapidly as the ICI decreased in the noiseexposed groups, the data shown in Fig. 6A were normalized in Fig. 6B by setting the largest CAP amplitude value obtained at the 20-ms ICI to 100%. This demonstrated a decrease in CAP2 percentage with decreasing ICI, but there were no significant differences among the groups.

The CAP2 latency was also compared among groups. Because the ICI did not impact latency, and a relatively larger cross-group difference was observed at the lower sound levels, only the CAP2 latencies for the 20-ms ICI at the 50-dB peSPL were compared (Fig. 7). Once again, the data from the two control subgroups were pooled (Ctrl4/8w) because they did not differ significantly. In general, exposure to noise on either P10d or P14d caused an increase in the CAP2 latencies. More specifically, a one-way ANOVA revealed a significant overall difference among the groups (F4 = 8.526; p < 0.001), and Tukey’s post hoc tests showed that the latency significantly increased only in animals that received noise exposure on P14d (***for comparisons between the Exp14d and Ctrl4/8w groups), particularly in the Exp14d8w subgroup (###for the comparison between the Exp14d8w and Exp14d8w groups in Fig. 7).

DISCUSSION The present study assessed the impact of noise exposure during a critical period of postnatal development, in which the peripheral auditory system is immature, on the

Fig. 6. Comparison of CAP2 amplitude-ICI functions across groups. (A) The CAP2 amplitude in microvolts, and (B) normalized from A using the largest CAP2 (at ICI = 20 ms) as 100%.

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hearing sensitivity of mice. Mice are born with no hearing function (Ehret, 1976, 1985a; Walters and Zuo, 2013), because their external ear has not yet formed and their middle ear is filled with a jelly-like material (Mikaelian and Ruben, 1965; Mikaelian, 1979). On the other hand, the cochleae of mice at birth are formed with an immature organ of Corti in which OHCs are differentiable from IHCs. During the same time period, SGNs can be identified but maintain an immature morphology, and their basic synaptic structure with HCs does not fully develop until P8d when the external ear starts to open (Sobkowicz et al., 1982; von Kriegstein and Schmitz, 2003; Safieddine et al., 2012; Johnson et al., 2013). The IHC/SGN synapses develop from the basal to the apical turn and from the modiolar side to the pillar side of the IHCs (Sobkowicz et al., 1986). Ultimately, mice begin to exhibit the ability to hear at approximately P10–P12d even though the ribbon synapses have yet to fully mature. After this point, hearing function develops very rapidly, and cochlear function is almost fully mature by P14d (Mikaelian and Ruben, 1965; Ehret, 1976, 1985a; Mikaelian, 1979). In the present study, the time points chosen for noise exposure were closely related to the above timeline of hearing development in mice. P10d was chosen for the earlier noise exposure, because at this age, cochlear microphonics and the CAP can be recorded from almost all individuals (Mikaelian and Ruben, 1965; Mikaelian, 1979; Ehret, 1985b), which suggests that this age represents hearing onset. P14d was chosen for the later noise exposure, because cochlear potentials begin to exhibit similar characteristics as those recorded from adult subjects, which suggests functional maturation of the cochlea at this age (Ehret, 1985b). The type of noise exposure employed in the present study presumably did not cause a PTS in hearing, as there were no significant threshold differences among the control and noise-exposed groups at either P4w or P8w (Fig. 1). Furthermore, exposure to the same noise at P14d, when the cochlea is closer to maturity, produced larger morphological and functional impacts. Compared with previous studies in adult mice (Kujawa and Liberman, 2009), the present study observed a lesser degree of noise-induced loss of ribbons in young mice following comparable levels of noise exposure. However, in contrast to the findings in adult mice (Kujawa and Liberman, 2009), the loss of ribbon synapses following postnatal exposure to noise did not stabilize in young mice 2 weeks (P4w) after exposure (at P14d). Rather, the loss of ribbons continued to increase from P4w to P8w in conjunction with a reduction in the CAP amplitude. This deficit in temporal processing was evidenced by an elongation of the CAP latency, which also continued to deteriorate from P4w to P8w. Additionally, there was a reduction in the number of ribbons in the OHCs following noise exposure. Using noise levels comparable to those of the present study, a previous study investigating the effects of noise exposure in adult CBA/CaJ mice found an initial 60% decrease in the number of ribbon synapses as well as damage to the postsynaptic terminals (Kujawa and

Liberman, 2009). However, the damage observed in that study was much more severe than that seen in the present study, likely due to different mouse strains used, variations in the noise exposure procedures, and potentially different synaptic sensitivities to noise between adult and young mice as a result of age differences. This notion is supported by the fact that both the ribbon loss and CAP deterioration in the present study were greater in animals that received noise exposure on P14d versus P10d. Nonetheless, the exact underlying reasons for the discrepant findings between these two studies are not entirely clear in part, because the mechanism by which noise exposure damages the presynaptic ribbon has yet to be fully elucidated. Many studies have investigated the mechanisms that potentially underlie noise-induced damage in postsynaptic terminals, and it is generally accepted that the damage is mediated by excitotoxicity resulting from the over-release of glutamate from IHCs in response to noise stimulation (Pujol et al., 1990, 1993; Puel et al., 1997; Pujol and Puel, 1999; Hakuba et al., 2000; D’Sa et al., 2007; Moser et al., 2013). In contrast, to the best of our knowledge, the mechanisms underlying the disruption of presynaptic ribbons following noise exposure have yet to be fully characterized, even though the loss of ribbons and their partial repair after damage have been well documented by recent reports from our group (Liu et al., 2012; Shi et al., 2013) and others (Kujawa and Liberman, 2009; Lin et al., 2011). Furthermore, little is known about the general mechanisms of ribbon formation in normal HCs, which assemble from the protein units of ribeye. This structure may mediate the processes of disassembly and reassembly in response to acoustic stimulation, which has been identified in the photoreceptor cells of the retina as a mechanism underlying sensorial adaptation (Spiwoks-Becker et al., 2008). However, this has not been verified in cochlear HCs, and no further research on this mechanism is available. Several lines of evidence indicate that calcium and calcium ion channels also potentially play roles in the disruption of ribbons following acoustic overstimulation. First, the IHC ribbon is anchored to the active zone, which is very close to voltage-sensitive calcium channels CaV1.3 (Nouvian et al., 2006; Uthaiah and Hudspeth, 2010). Second, the inflow of calcium through the CaV1.3 channel is triggered by depolarization graded according to the strength of the acoustic stimulation (Fettiplace, 1992). Third, although it plays an important role in HC transduction and transmission, calcium is also a major factor in the damage sustained by cellular structures induced by a variety of factors (Smaili et al., 2009; Albrecht et al., 2010; Szydlowska and Tymianski, 2010; Orrenius et al., 2011; Zhivotovsky and Orrenius, 2011; Zundorf and Reiser, 2011). Finally, the potential roles of calcium and the CaV1.3 channel in the disruption of ribbons following noise exposure is supported by recent findings showing that the CaV1.3 channels regulate the size of ribbons and that the genetic disruption or acute blockage of this channel increases ribbon size in HCs (Sheets et al., 2012). Taken together, these data suggest that the modulation of calcium levels by the CaV1.3

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channel is likely involved in sound-induced changes to ribbon structure. Future studies can verify the role of this ion channel during noise-induced damage and whether the selective blockage of CaV1.3 reduces ribbon loss due to acoustic overstimulation. Nonetheless, speculation regarding the role of CaV1.3 in noise-induced ribbon damage appears to be in agreement with the findings of the present study, which demonstrated a greater degree of ribbon loss in mice that received noise exposure at P14d compared with P10d. Based on limited developmental data, CaV1.3 channels appear to be detectable by approximately P6d in C57 mice but are not mature until P14d (Zampini et al., 2010; Wong et al., 2014). A plausible explanation for the lesser degree of ribbon loss following noise exposure on P10d is that the immature status of the CaV1.3 channels might have limited the acoustic overstimulation-induced calcium overload in IHCs. The most striking finding of the present study was the greater reduction in ribbon synapses at P8w than at P4w. However, it remains unclear whether the ribbon damage ceased after P8w, and further study is needed to clarify this issue. However, the reliability of the present data was confirmed by repeating the ribbon counts and verifying the changes using different samples and different independent testers. Furthermore, the reliability of the present results is also supported by the consistency between the morphological and functional data, such that a greater degree of ribbon loss (and presumably greater synapse disruption) was typically accompanied by a greater reduction in CAP amplitude and a greater elongation of the CAP latency. To the best of our knowledge, no existing studies have reported long-term worsening of noise-induced cochlear lesions. Rather, in all reported cases, noise-induced hearing loss and morphological changes in the cochlea exhibit a greater recovery within 1 week of the cessation of noise exposure. Because the control data in the present study did not reveal changes in either the ribbon counts or CAP characteristics from P4w to P8w, the deteriorations in morphology and function in the noise-exposed groups were likely not due to differences in cochlear structure or function between the two time points. Thus, at this time, an explanation for this result cannot be provided, but the immaturity of the cochlear ribbon synapses between P10d and P14d may have been responsible for the long-term effects observed. During early development, more than one ribbon is connected to each active zone, but between P10d and P14d, the number of ribbons decreases such that eventually only one active zone will be connected to one ribbon (Zampini et al., 2010; Wong et al., 2014). Noise exposure in this window of time may alter the ribbons and render them vulnerable to later damage by unknown factors. Similar to findings from studies in adult animals, the functional effects of noise-induced damage in neonatal mice include a reduction in CAP amplitude (Liu et al., 2012; Shi et al., 2013). This reduction is likely due to the loss of ribbon synapses and, subsequently, a decrease in available auditory nerve fibers that send

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signals to the central auditory system. Previously, our group reported that a reduction in ribbon counts parallels the loss of postsynaptic terminals, which was verified by postsynaptic density (PSD) staining (Shi et al., 2013), even though the number of ribbons lost was slightly greater than the loss of PSD. Therefore, ribbon counts may be considered a good index of the survival of afferent innervations to IHCs. However, two discrepancies between the ribbon loss and the changes in CAP amplitude in the present study were identified. First, there was no significant reduction in CAP amplitude in animals exposed to noise on P10d. Second, in animals exposed to noise on P14d, the reduction in CAP amplitude was greater than what could be expected based on the number of ribbons lost. Compared with the control group, the CAP amplitude in the Exp14d8w group was reduced to 41.7%, while the ribbon count in the Exp14d8w group was reduced to approximately 70%. The reasons behind these discrepancies are unclear, but uncontrolled variations in the CAP recording procedure are one likely contributing factor. To evaluate the impact of noise-induced damage in the ribbon synapses on temporal processing, the present study measured the CAP response to paired clicks with a focus on the manner in which the CAP evoked by the second click in the pair (CAP2) changed as a function of ICI (Figs. 6 and 7). In this test, the cochlear ability to handle the quick change in signal produced by a shortening of the ICI was examined. To avoid confounds from the overall reduction in CAP amplitude, normalized % CAP–ICI curves were used to evaluate potential changes in temporal processing abilities (Fig. 7). In a previous study from our group using adult guinea pigs, a temporal processing deficit was demonstrated by an increasing reduction in the CAP amplitude percentage as the ICI decreased (Shi et al., 2013). In that study, the larger than normal reduction in the percentage of CAP2 due to changes in the ICI suggests that the auditory nerve responded poorly to the second click when the ICI was short. The present study did not observe a similarly large decrease in % CAP2–ICI function in the noise-exposed groups (Fig. 6), but a temporal processing deficit was revealed by the elongated CAP latency following noise exposure. Theoretically, the change in CAP evoked by the change in the latency of the clicks may be due to two reasons: a latency change in single auditory nerve fibers or changes in the population of fibers dominating the response (e.g., a greater loss of high-frequency nerve fibers). Although the second reason could not be rejected entirely, it was considered to be less likely in the present study due to the parallel losses of ribbon synapses across the whole cochlea and the larger increase in latency at P8w. Similar to the changes in ribbon loss and CAP amplitude, elongation of the latency was also greater in animals exposed to noise on P14d. Further investigation is needed to explore the meaning of the latency and % CAP amplitude–ICI function tests in terms of temporal processing and the mechanisms underlying the noiseinduced changes. Nonetheless, the elongation of the

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response latency likely reflected the slow neurotransmitter release from ribbon synapses and possibly a slower reaction of the postsynaptic membrane. The impact of noise exposure during early development has received an increasing amount of attention in recent years. A number of studies have suggested that hearing loss during early development (even if temporary) can greatly influence the development of the central auditory system (Xu et al., 2007; Hatano et al., 2012; Butler and Lomber, 2013; Polley et al., 2013). Interestingly, exposure to noise on P14d in rats, which was a time point used in the present study, has a huge impact on startle responses and frequency coding differing from the effects of noise exposure in adult animals (Rybalko et al., 2015; Suta et al., 2015). The present study extended the concept of hearing loss by showing that exposure to noise at an early age can induce cochlear damage without a PTS in hearing, and that this damage may be quantitatively different from the damage induced by noise at later ages. However, one issue that remains unclear is whether the parallel ribbon loss occurred across the entire cochlea. This is puzzling considering the fact that cochlear development follows a tonotopic gradient (Manley and Jones, 2011; Mann and Kelley, 2011), but at this point, this issue cannot be explained adequately. However, in addition to the maturity of the Organ of Corti, other factors may play a role in the degree of damage that occurs in the ribbon synapse.

CONCLUSION The present study documented noise-induced damage to the ribbon synapses during an important developmental period of the peripheral auditory organs as well as the functional impacts of this lesion. Although an exact match between the timelines of cochlear development for mice and humans is impossible, P10–P14d roughly corresponds to 28–42 weeks of human gestation. Thus, the present data may further the understanding of the potential effects of noise-induced damage in human fetuses and/or neonates, which are exposed to noise from life-support systems.

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(Accepted 24 July 2015) (Available online 30 July 2015)