Repeated Moderate Sound Exposure Causes Accumulated Trauma to Cochlear Ribbon Synapses in Mice

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NEUROSCIENCE 1

RESEARCH ARTICLE Y. Luo et al. / Neuroscience xxx (2020) xxx–xxx

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Repeated Moderate Sound Exposure Causes Accumulated Trauma to Cochlear Ribbon Synapses in Mice

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Yangtuo Luo, a Tengfei Qu, b Qingling Song, b Yue Qi, b Shukui Yu, b Shusheng Gong, b Ke Liu b* and Xuejun Jiang a*

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Department of Otolaryngology, The First Affiliated Hospital of China Medical University, Shenyang 110001, China

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Department of Otolaryngology Head and Neck Surgery, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China

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Abstract—Repeated induction of a temporary threshold shift (TTS) may result in a permanent threshold shift (PTS) and is thought to be associated with early onset of age-related hearing loss (ARHL). The possibility that a PTS might be induced by administration of repeated TTS-inducing noise exposures (NEs) over a short period during early adulthood has not been formally investigated. We aimed to investigate possible cumulative acoustic overstimulation effects that permanently shift the auditory threshold. Young adult C57BL/6J mice were exposed twice to moderate white noise in an experimental design that minimized the effects of aging. The first exposure resulted in a reversible noise-induced hearing loss (NIHL) measured as recoverable alterations in auditory brainstem response (ABR) thresholds, waveform amplitudes, and numbers of ribbon synapses. The second NE with the same parameters caused persistent threshold shifts, wave I amplitude reductions, wave IV/I ratio enhancements, and synaptic losses, even though recovery time sufficient for a TTS had been provided. The pattern of PTS resembled NIHL since the observed impairments tonotopically followed the power spectrum of the noise insult, rather than ARHL, which distributes at higher frequencies. No significant changes were observed in the control group as the mice aged. To conclude, our results demonstrate a cumulative effect of repetitive TTS-inducing NE on hearing function and synaptic plasticity that does not cause premature ARHL, thereby providing insight into the pathophysiological mechanisms underlying NIHL and ARHL. Ó 2020 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: repeated noise exposure, noise-induced hearing loss, age-related hearing loss, threshold shift, ribbon synapse.

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INTRODUCTION

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Sensorineural hearing loss is a common disease that has become the fourth largest source of disability in the population, averaging across all ages (The WHO, 2018). Noise and aging are the most prevalent triggers (Liberman, 2017; Kujawa and Liberman, 2019). With the industrialization and urbanization of modern society, people are more likely to be exposed to noise from daily life, occupational sources, and recreational sources. This type of noise is characterized by a wide frequency distribution

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and an intensity that is moderate or slightly greater. Depending on the intensity and duration of the exposure, noise can cause mechanical damage to the cochlea of the inner ear. This can result in two types of attenuation of hearing acuity, either a temporary threshold shift (TTS) or a permanent threshold shift (PTS) (Kujawa and Liberman, 2009; Ryan et al., 2016; Le et al., 2017). Accumulating evidence indicates that exposure to modest sound levels can cause TTS without hair cell loss but with significant loss of the ribbon synapses between cochlear inner hair cells (IHCs) and type-I spiral ganglion neurons (Kujawa and Liberman, 2009; Furman et al., 2013). Recent studies on guinea pigs have revealed significant recovery of synapse counts following a massive initial loss induced by noise exposure (NE) that did not lead to PTS (Liu et al., 2012; Shi et al., 2013). The reduction in ribbon synapses is reversible, which may contribute to hearing recovery in mice after NE (Wang et al., 2015). Previous research findings in our laboratory in C57BL/6J mice indicated a relationship between hearing impairment and the plasticity of the ribbon synapses. After 2 h of exposure to 110 decibel (dB) and 100 dB white noise, we observed reversible threshold shifts in the auditory brainstem responses (ABRs) (Shi et al., 2015b; Wang et al.,

*Corresponding authors. Addresses: Department of Otolaryngology Head and Neck Surgery, Beijing Friendship Hospital, Capital Medical University, No. 95 Yong’an Road, Xicheng District, Beijing 100050, China (K. Liu). Department of Otolaryngology, The First Affiliated Hospital of China Medical University, No. 155, Nanjing Street, Heping District, Shenyang 110001, China (X. Jiang). E-mail addresses: [email protected] (K. Liu), entxuejunjiang@ outlook.com (X. Jiang). Abbreviations: ABR, auditory brainstem response; ANOVA, analysis of variance; ARHL, age-related hearing loss; CtBP2, C-terminal-binding protein 2; DAPI, 40 ,6-diamidino-2-phenylindole; dB, decibel; DPOAE, distortion product otoacoustic emission; GluR2/3, glutamate receptor 2/3; IHC, inner hair cell; NE, noise exposure; NIHL, noise-induced hearing loss; OHC, outer hair cell; PBS, phosphate-buffered saline; PTS, permanent threshold shift; SEM, standard error of the mean; SPL, sound pressure level; TTS, temporary threshold shift. https://doi.org/10.1016/j.neuroscience.2019.12.049 0306-4522/Ó 2020 IBRO. Published by Elsevier Ltd. All rights reserved. 1

Please cite this article in press as: Luo Y et al. Repeated Moderate Sound Exposure Causes Accumulated Trauma to Cochlear Ribbon Synapses in Mice. Neuroscience (2020), https://doi.org/10.1016/j. neuroscience.2019.12.049

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2015). In the daily life of people, repeated exposure to noise of moderate intensity is the most common situation that may be problematic for hearing conservation. In the clinic, patients with noise-induced hearing loss (NIHL) are found to have been exposed to repeated noise after recovery of hearing following an initial NE and often exhibit significant hearing loss and attenuation of further recovery. Previous studies have indicated that NE that only induces a TTS may induce a PTS if repeated, as occurs in occupational or recreational settings (Lonsbury-Martin et al., 1987; Wang and Ren, 2012). Age-related hearing loss (ARHL), or presbycusis, is a gradual and irreversible age-dependent decline in auditory function. It has been well characterized and consists of a progressive hearing loss mainly at high frequencies, with smaller shifts at low frequencies (Huang and Tang, 2010; Kujawa and Liberman, 2019, 2015). Whether independently or synergistically, aging and NE have long been associated with hearing impairment (Albera et al., 2010; Liberman, 2017; Kujawa and Liberman, 2019). Recent studies have shown that the ribbon synapses are the elements of the cochlea most vulnerable to NE (Kujawa and Liberman, 2009) and that in ARHL, these synaptic connections disappear before the hair cells do (Sergeyenko et al., 2013). Under some conditions, ‘‘common pathogenic pathways” are considered to contribute to both NIHL and ARHL (Huang and Tang, 2010; Alvarado et al., 2019; Kujawa and Liberman, 2019); the rate and nature of ARHL are thought to interact with lifetime doses of sub traumatic NE. A handful of existing studies on repeated exposures to short-duration, loud sound over a long time period (Mannstrom et al., 2015; Alvarado et al., 2019), or to repetitive ‘‘benign” noise (Wang and Ren, 2012), to the point of inducing PTS, are all suggestive of an accelerated onset of ARHL due to NE. Evaluating NIHL and ARHL separately is challenging when studying the mechanism by which repeated TTS from moderate NE leads to PTS (Kujawa and Liberman, 2019). Therefore, in spite of all the accumulated data on NE and aging, a lack of consensus remains on whether repeated TTS-inducing NE alone can cause PTS in early adulthood and well before the onset of ARHL. Moreover, complaints of early hearing loss are common in patients under 50 who have been exposed to repeated noise from occupational and/or recreational sources. Their hearing impairment manifests differently from typical ARHL. Therefore, additional research is needed with interventions that better mimic these conditions. The present study aimed to investigate the cumulative effects of acoustic exposures that individually do not shift auditory thresholds permanently but may do so if administered twice in a relatively short period of time, and this prior to any impact of aging. We detected characteristic differences among single-exposure, dualexposure, and control animals, including a pattern of changes in ABR waves, a loss of IHC ribbon synapses, changes in the morphology and quantity of both hair cells, and function of outer hair cells (OHCs). Thus, we report that the repetitive TTS-inducing NE have cumulative effects on hearing function and synaptic

plasticity which lead to PTS and irreversible ribbon synaptic loss without causing premature ARHL.

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EXPERIMENTAL PROCEDURES

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Animals and groups

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A total of 45 adult 6-week-old C57BL/6J male mice with normal hearing purchased from Vital River Laboratories (VRL Animal Technology, Beijing, China) were used in the study. No outer or middle ear pathology was observed in any of the animals. The mice were grouphoused under a 12:12 h light–dark cycle with a constant climate and free access to water and food. Groups of mice were exposed to a 2-h 100 dB sound pressure level (SPL) broadband white noise for a single or double administrations, while cagemates served as unexposed age-matched control (Fig. 1). The animals were handled and treated according to the guidelines of the Institutional Animal Care and Use Committee of Capital Medical University of China (AEEI-2017-112).

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NE

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Awake and unrestrained C57BL/6J mice (n = 30) at 6wk of age in separate acoustically transparent steel wire cages (4 * 4 * 7 cm3) were exposed to a white noise at 100 dB SPL binaurally for 2 h, which has been shown to produce reversible reduction of synaptic ribbons and TTS (Shi et al., 2015b; Wang et al., 2015). In addition, half of the mice (n = 15) went through a second NE with identical parameter (100 dB white noise for 2 h) at the age of 10 weeks. Exposures were performed in a reverberant chamber and the level of sound was consistent throughout the field during the exposure. The noise waveform with a bandwidth from 2 Hz to 20 kHz was generated and equalized by an audio editing software (Audition 3; Adobe Systems, San Jose, CA, USA) driven by a power amplifier (model XTi4002; Crown Audio by HARMAN, Elkhart, IN, USA) coupling to a mixing console (model

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Fig. 1. Noise exposure protocol. Mice entered into experiments at 6 weeks of age and exposed to a 100 dB SPL white noise for 2 h once or twice depending on the group assignment. ABR recordings and histological examination were performed within 24 h after NE and at each time points marked in the graph.

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MG10XU; YAMAHA, Buena Park, CA, USA) and delivered by a pair of opposed loudspeakers (model KP6000; JBL by HARMAN, Elkhart, IN, USA) fitted in the chamber. SPLs for NE were calibrated with a sound level meter (Model AWA5661; Hangzhou Aihua Instruments, China) at multiple locations within the sound chamber to ensure uniformity of the sound field (SPL varied by ±1 dB) and are monitored continuously during the exposure to ensure stability. Age matched control mice (n = 15) were kept in silence (with the loudspeaker turning off) within the same chamber for 2 h.

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Assessment of auditory function

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ABR audiograms were performed at the age of 6 w (the same day after exposure), 7 w, 8 w, 10 w (the same day after re-exposure), 11 w, 14 w. Mice were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg, Sigma, USA) plus xylazine (10 mg/kg, Sigma, USA), and then placed in an electrically shielded and soundproofed audiometric chamber (Shanghai Shengnuo Acoustic Equipment, China). Meanwhile body temperature was maintained with a constanttemperature heating pad. The needle electrodes were placed subcutaneously, with a reference electrode () beneath the pinna of the tested ear; a recording electrode (+) at the junction of anterior edges of both auricles and the midline of the cranial apex; a ground electrode in the contralateral ear. Only the right ears were examined. Acoustic stimuli were delivered monaurally by an earphone attached to a customized plastic speculum inserted into the ear canal. Calibrated tone bursts with 5-ms duration and 0.5 ms rise-fall time were synthesized and presented using TDT System 3 hardware and SigGen/Biosig software (Tucker-Davis Technologies, Alachua, FL, USA). ABRs were measured at 4, 8, 16 and 32 kHz. A total of 1024 responses were averaged near the threshold at various intensities with 5-dB intervals. The lowest level at which ABR waves could be clearly detected was defined as the threshold. Wave I and IV (analogous to wave V of human) amplitudes of 4, 8, 16 and 32 kHz at 80 dB SPL stimuli were analyzed. The amplitude of the wave I was identified as the difference between the first peak in the waveform and the baseline, and the same as wave IV. All ABR measurements were conducted by the same experimenter. Distortion Product Otoacoustic Emission (DPOAE) audiograms were measured using a low-noise microphone ER-10B+ (Etymotic Research, Elk Grove Village, IL, USA) coupled with two separate speakers (EC1 close-field speakers, Tucker-Davis Technologies, Alachua, FL, USA). Two level (L1 = 65 dB SPL, L2 = 55 dB SPL) primary signals f1 and f2 (f2/f1 = 1.2), were generated and attenuated digitally (200 kHz sampling) with test frequencies ranging from 2 to 32 kHz. Stimuli of two primary tones were introduced into the sealed ear canal of the anesthetized mouse through an insert earphone speculum. The ear canal sound pressure was preamplified and digitized. After spectral and waveform averaging, the noise floor was determined by averaging seven spectral points on either

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side of 2f1–f2. Amplitudes of the responses at 2f1–f2 were measured based on the noise floor. A peak at 2f1– f2 in the spectrum was accepted as a DPOAE if the pressure level was 6 dB above the baseline.

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Cochlear tissue processing and immunohistology

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Animals were sacrificed by decapitation under deep anesthesia. The cochlea was quickly removed from the skull and dissected in ice bath. With stapes removed, the round and oval windows and the apex of the cochlea were opened, through which the cochlea was perfused with 4% paraformaldehyde and fixed overnight at 4 °C. After fixation, the cochlea shell was decalcified with 10% EDTA for 4–6 h. Then the apical, middle and basal turn of the basilar membrane was separated in phosphate-buffered saline (PBS) solutions with vestibular membrane and the tectorial membrane removed. After rinsing in PBS three times for 5 min each, the separated basilar membrane was permeabilized with 0.3% Triton X-100 (Sigma, USA) in PBS for 30 min, and blocked in 10% normal goat serum (ZSGB-BIO, China) in PBS for 1 h at room temperature. Antibodies included mouse anti-CtBP2 (C-terminal binding protein 2; AB204663, BD Biosciences, USA) at 1:200 and rabbit anti-GluR2/3 (glutamate receptor 2/3; AB1506, Millipore, USA) at 1:300 to allow quantification of both presynaptic ribbons in IHCs and postsynaptic elements in the inner hair cell area. Primary incubations were at 4 °C overnight and followed by incubation at room temperature for 60 min in species-appropriate secondary antibodies: goat anti-mouse Alexa Fluor 568 and goat anti-rabbit Alexa Fluor 488 (diluted 1: 200; A11031, A11078, Invitrogen/Molecular Probes, Carlsbad, CA, USA). Three times of rinsing in PBS were performed before and after secondary incubation. A drop (40 ll) of fluorescent mounting media with DAPI (4, 6-diamidino-2-phenylindole; ZLI9557, ZSGB-BIO, China) was placed on the slide and the basilar membrane was tiled under a dissecting microscope and sealed with a coverslip.

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Imaging and quantification

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Images were obtained using a Leica scanning laser confocal microscope (model TCS SP8 II, Leica, Wetzlar, Germany). A 63 oil-immersion objective lens with a 2 digital zoom was used to capture whole mounts images. The excitation wavelength was set at 358 nm, 488 nm and 568 nm. Sequence scanning was performed on cochlear IHCs with a scanning interval of 0.2 lm to ensure that each synapse would be marked, since the size of mature IHC ribbon synapses usually ranges from 150 to 200 nm. For cochlear whole mounts, lengths of the dissected cochlear pieces were measured in each case and located structures to relevant frequency regions (4, 8, 16, 32 kHz) according to a cochlear frequency-place map (Ou et al., 2000). Pictures were cropped, labeled and spaced using the Leica LAS X software (Leica, Wetzlar, Germany). Z stacks of the IHC base were captured, taking care to include all ribbon synapses, and then analyzed off-line with Amira software (Version

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5.4.3, Visage Imaging, Richmond, Australia). Individual ribbons were isolated within Amira with the connected components function and isosurface tools, counted, and expressed as number of synaptic ribbons per IHC. A ribbon synapse is characterized by a presynaptic ribbon with its associated halo of neurotransmitters vesicles and a postsynaptic active zone containing glutamate receptors, to which released neurotransmitter binds. For counting the number of IHCs and OHCs, cochlear tissue was processed in accordance with methods described above. Incubation of primary antibody: Myosin VIIa to visualize cytoplasm of inner and OHCs (rabbit anti-Myosin VIIa at 1:300; 25-6790, Proteus Biosciences, USA). Secondary antibody was applied as: goat anti-rabbit Alexa Fluor 568 (diluted 1:200; A11079, Invitrogen/Molecular Probes, USA); followed by phalloidin 488 (1: 300; A12379, Invitrogen/Molecular Probes, USA) to label stereocilia of IHCs and OHCs. Cell counts from confocal images were performed using Image J software (Version 1.37, NIH, Wayne Rasband, USA). Total numbers of IHCs and OHCs were counted in a 160 * 65 mm area imaged with a 63*/1.4 NA objective on the confocal microscope. A cell was considered a hair cell if there were positive Dapi, Myosin VIIa and phalloidin labeling. IHCs and OHCs were distinguished by their morphology and relative position to their corresponding support cell. In our study, three parts of each cochlea (apex, middle and base) were counted.

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Statistical analyses

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All data are presented as mean ± standard error of the mean (SEM). After satisfying the assumptions of normality (and homogeneity of variance), the parameter test was used. Differences between groups were compared statistically by two-way analysis of variance (ANOVA) followed by Bonferroni multiple comparisons test. A p value of <0.05, <0.01 or <0.001 was considered statistically significant. The statistical significance in the graphs is represented by *p < 0.05; **p < 0.01; ***p < 0.001. Statistical analyses were performed using SPSS software (Version 18.0, IBM, Chicago, IL, USA). GraphPad prism (Version 5.0, GraphPad Software, La Jolla, CA, USA) software was used for preparation of figures.

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RESULTS

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ABR thresholds

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Six-week-old mice exposed to white noise at 100 dB SPL for 2 h showed significant threshold shifts at 4, 8, 16, and 32 kHz within 24 h of exposure, ranging from 5 to 9 dB, at 8 kHz, the threshold shift was least significant (p < 0.05). The shifts were more significant in all other groups (p < 0.01) (Fig. 2B, E). This was followed by complete threshold recovery within 1–2 weeks (p > 0.05), in agreement with previous reports of TTS induced by similar NE (Shi et al., 2015b; Wang et al., 2015). At 10 weeks of age, re-exposure to the same noise conditions again resulted in significant auditory threshold shifts,

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but these shifts remained stable or worsened over 7– 28 days post-exposure at all four frequencies (Fig. 2A– D). The first NE induced a TTS of 5–9 dB (Fig. 2E), while the second induced a PTS (i.e., a persistent hearing impairment) with a maximum of 18 dB (Fig. 2F, G), comparing with age-matched controls. Comparing frequencies after two acoustic exposures, the ABR threshold elevation was minimal (10 dB) at 8 kHz and increased to a maximum of 18 dB at 32 kHz at 14 weeks of age (Fig. 2E, F). We found no significant changes in the control group throughout the entire observation period. The effects of NE on auditory thresholds were highly significant by ANOVA (n = 10 in all groups) at all 4 frequencies, as follows: 4 kHz, F (2, 135) = 25.5, p < 0.0001; 8 kHz, F (2, 135) = 16, p < 0.0001; 16 kHz, F (2, 135) = 59, p < 0.0001; 32 kHz, F (2, 135) = 47.3, p < 0.0001. ANOVA also showed a significant aging effect on threshold shifts at higher frequencies, as follows: 4 kHz, F (5, 135) = 1.26, p = 0.2837; 8 kHz, F (5, 135) = 2.66, p = 0.025; 16 kHz, F (5, 135) = 6.78, p < 0.0001; 32 kHz, F (5, 135) = 5.23, p = 0.0002.

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Reduction and recovery of wave I peak amplitudes

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The amplitudes of wave I peaks evoked by 80 dB SPL suprathreshold tone bursts at 4, 8, 16, and 32 kHz were measured to provide a measure of the synchronous, sound-evoked activity of cochlear nerve fibers. The amplitudes of ABR wave I were significantly reduced (p < 0.001) after the first acoustic insult at 6 weeks of age, which indicates an elevation in hearing threshold. The effect of aging on wave I amplitude was not significant by ANOVA, F (5, 468) = 1.50, p = 0.1911 (Fig. 3A). After the first NE at 6 weeks, amplitudes were significantly different from those of controls at all observed frequencies (Fig. 3B). However, the changes in wave I peak amplitude did not completely parallel the alterations in ABR thresholds; although the thresholds fully recovered within 1 week, we did not observe complete recovery of the ABR wave I peak amplitudes by post-exposure day 14 (p < 0.05), suggesting that auditory nervous function had not completely recovered (Fig. 3A, C). After the second NE, wave I peak amplitudes were dramatically reduced (p < 0.001) and did not exhibit signs of recovery, consistent with the observed elevations in cochlear threshold (Fig. 3A). The maximum and minimum reductions were observed at 16 kHz (p < 0.001) and 4 kHz (p < 0.05), respectively (Fig. 3C). At 14 weeks of age, the reduction in amplitude in the dual-NE group was most significant at 32 kHz (p < 0.001). No significant differences were observed between the single-NE group and controls (Fig. 3D). Wave I amplitudes did not vary significantly with age in the control group (n = 10 in all groups).

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ABR wave IV amplitudes and wave IV/I ratios

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We also measured the amplitudes of ABR wave IV. Compared with controls, wave IV was slightly increased in both NE groups. Differences among time points were not statistically significant (p > 0.05) except for dual-NE mice at 14 weeks (p < 0.05). ANOVA revealed a small

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Fig. 2. ABR thresholds and threshold shifts. Shown are the auditory thresholds at 4, 8, 16, and 32 kHz against time (n = 10 in all groups). (A–D) Mice in the single-noise exposure (single-NE) group show a significant increase in hearing threshold only one day after exposure, whereas mice in the two-noise exposures (dual-NE) group show highly significant auditory threshold shifts after the second NE, at 10, 11, and 14 weeks of age. (E) At 6 weeks, the first NE induces significant threshold shifts at all 4 frequencies. (F) At 10 weeks, mice in the single-NE group have fully recovered from the temporary threshold shift (TTS) with no significant difference in acuity from age-matched controls, while in the dual-NE group, threshold shifts are dramatically elevated after the second NE relative to the first at all frequencies. (G) At 14 weeks of age, the single-NE group shows no significant acuity differences from control mice, whereas the dual-NE group shows exacerbated threshold shifts at all frequencies. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

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but significant effect of NE number on wave IV amplitudes, F (2, 585) = 3.22, p = 0.0435 but no significant effect of aging, F (5, 585) = 0.68, p = 0.6358 (Fig. 4A). We calculated the amplitude ratios of ABR wave IV to wave I, and found a significant increase at 2 weeks after the first exposure in the single-NE group (p < 0.05). Values then returned to normal over subsequent weeks. At 10 weeks of age, mice were administered a second NE and showed a persistently higher IV/I ratio than did age-matched controls (p < 0.05). This difference remained significant until 14 weeks of age. At this point, the IV/I ratio of the dual-NE group was dramatically increased compared with the NE and control groups (p < 0.01 and p < 0.001, respectively). No significant changes were observed in the two comparison groups. ANOVA revealed an extremely significant effect of NE number on IV/I ratio, F (2, 585) = 10.87, p < 0.0001, and a small but significant effect of aging, F (5, 585) = 2.31, p = 0.0447 (Fig. 4B). Comparing frequencies at

14 weeks, the IV/I ratio of the dual-NE group was significantly increased compared with controls, particularly at 16 and 32 kHz, whereas the ratio was not significantly increased in the single-NE group (p > 0.05) (Fig. 4C) (n = 10 in all groups).

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Changes in ribbon synapses

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To identify and quantify ribbon synapses, organs of Corti were immunostained with antibodies to CtBP2 (a prominent component of the presynaptic ribbon) (Khimich et al., 2005) and subunits of GluR2/3 to identify postsynaptic receptor patches (Matsubara et al., 1996) (Fig. 5A). Presynaptic ribbons and postsynaptic patches were counted using Amira software to generate threedimensional reconstructed image from confocal image sequence (Fig. 5B). Unexposed (Fig. 5C–C0 ), singleexposed (Fig. 5D–D0 ) and double-exposed (Fig. 5E–E0 ) IHCs revealed a loss and subsequent recovery of ribbon synapses.

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Fig. 3. Reduction and recovery of ABR wave I amplitudes. (A) The single-NE group shows significant reductions in the amplitude of wave I of the auditory brainstem response (ABR) at 6, 7, and 8 weeks of age superimposed on a gradual recovery (n = 10 in all groups). In the dual-NE group, the second NE produces significant and stable decreases in wave I amplitudes. (B) After an NE at 6 weeks of age, significant differences are observed between the single-NE group and controls at all tested frequencies, with the most significant results observed at 16 kHz. (C) At 10 weeks, amplitudes in the single-NE group have fully recovered and are not significantly different from those of age-matched controls, while the dual-NE group shows a greater reduction in amplitudes after the second NE. (D) At 14 weeks of age, the wave I amplitude in the single-NE group is not significantly different from that of age-matched controls, whereas the dual-NE group shows significant differences from controls at all four frequencies. Data are presented as mean ± SEM, *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 4. ABR wave IV amplitude and IV/I amplitude ratio. Shown are the effects of single- and dual-NE on ABR wave IV amplitudes by frequency and time (n = 10 in all groups). (A) Significant differences (enhancements) in wave IV are only observed in the dual-NE group at 14 weeks old. (B) In the dual-NE group, the ABR wave IV/I amplitude ratio is significantly increased after a second NE compared with controls, while in the single-NE group, a significant difference is only observed at 8 weeks of age. (C) At 14 weeks, the dual- and single-NE groups show significant increases compared with age-matched controls at 16 and 32 kHz, and at 32 kHz, respectively. Data are presented as mean ± SEM, *p < 0.05; **p < 0.01; ***p < 0.001. 411 412 413 414 415 416 417

The single-NE mice were sacrificed on the day following the first NE, and the cochlear basilar membrane was harvested for immunofluorescence staining. We observed that the number of ribbon synapses in IHCs was significantly lower than that in control mice (p < 0.001). Subsequently, at 7 weeks of age (1 week after NE) and later, we observed no

significant difference between the single-NE and control groups (p > 0.05). To investigate the limit of ribbon synapse plasticity, half of the mice were administered a second NE at 10 weeks of age (4 weeks after the first NE). As expected, the count of synapses again decreased significantly on the day of the second NE (p < 0.001). Unlike the previous NE, a significant

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the cochlea in control and both NE groups at 14 weeks of age. No significant differences among groups were observed for either IHC or OHC counts across all frequency areas on the basilar membrane regardless of the number of NEs compared with controls at 6 weeks of age (p > 0.05). No significant changes in cell or ciliary morphology were observed (Fig. 7A–C) (n = 8 in all groups). To explore whether repetitive NE disrupts the function of OHCs, we measured the DPOAEs of both NE groups at 14 weeks of age for possible changes. We found no significant differences among groups across all frequencies (Fig. 7D). Our results suggested that neither single- nor dual-NE causes permanent alterations in OHC function (n = 5 in all groups).

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DISCUSSION

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In animal models, periods of up to 3 weeks are required for recovery from TTS (Ryan et al., 2016). It may thus be premature to identify Fig. 5. Expression pattern of ribbon synapses in IHCs. Shown is a three-dimensional reconstruction a threshold shift as temporary until image, CtBP2 (presynaptic structures) is in red, GluR2/3 (postsynaptic) is in green, Dapi is in blue, at least 3 weeks have elapsed and the dotted lines are the approximate outlines of IHCs. (A) High-power thumbnails show ribbon post-exposure. Only then can the synapses with all pre- and post-synaptic elements as well as orphan ribbons with only pre-synaptic terminals (red arrow) or post-synaptic glutamate receptor patches (green arrow). (B) Complete ribbon presence of a PTS be decided. In synapses are observed as paired red-green puncta in the basal zones of IHCs, just above the our study, we observed no tenterminals of the spiral bundle. (C–E0 ) Representative images of positively stained ribbon synapses dency to hearing recovery in the 0 from control, single- and dual- NE mice. Scale bars: A = 1 lm; B–E = 20 lm. (For interpretation of dual-NE mice by the fourth week the references to color in this figure legend, the reader is referred to the web version of this article.) after the second NE, implying that a PTS occurred. A previous study statistical difference remained between the dual-NE and on enduring changes in neural control groups at the age of 11 and 14 weeks responsiveness suggested that each TTS episode pro(p < 0.001). We did not observe any obvious recovery duces an increment of damage to the ear that eventually trend subsequently (Fig. 6A). For the single-NE group, contributes to a measurable PTS. That experiment significant losses of ribbon synapses were found at all involved repeatedly exposing monkeys to short-lasting four frequencies evaluated, and by 14 weeks old, no TTS-inducing noise over many months (Lonsbury-Martin evident differences remained between the single-NE et al., 1987). Wang and Ren administered a repeated mice and control groups (Fig. 6B). However, after the 100 dB narrow-band noise centered at 12 kHz to CBA second NE, the dual-NE mice showed no sign of mice elicited a PTS on the third exposure (Wang and synaptic recovery at any frequency (Fig. 6C). ANOVA Ren, 2012). In the present study, the first NE induced a revealed extremely significant effect of both NE number, TTS, and the hearing of the mice recovered to normal F (2, 132) = 134.5, p < 0.0001 and aging, F (5, 132) levels 1 week later. However, after the second NE, the = 8.06, p < 0.0001 on the quantitative ribbon synapse mice exhibited an increase in threshold that was signifidata (n = 6 in all groups). cantly higher than that following the first NE. Moreover, their hearing acuity had decreased further by 11 and Morphology and quantification of hair cells and 14 weeks of age. Our results support the evidence that OHCs function inducing repeated TTSs may result in a PTS (Wang and Damage or loss of hair cells is characteristic of different Ren, 2012). types of auditory impairment. We compared the number It is well established that the pattern of threshold-shifts of IHCs and OHCs in apical, middle and basal turns of in the aging human ear is a progressive high-tone hearing Please cite this article in press as: Luo Y et al. Repeated Moderate Sound Exposure Causes Accumulated Trauma to Cochlear Ribbon Synapses in Mice. Neuroscience (2020), https://doi.org/10.1016/j. neuroscience.2019.12.049

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Fig. 6. Recovery and loss of ribbon synapses. (A) The single-NE group exhibits a significant loss of ribbon synapses at 6 weeks old that subsequently recovers. In the dual-NE group, the second NE causes significant and persistent synaptic loss (n = 6 in all groups). (B) In the singleNE group, significant losses of ribbon synapses are observed at all frequencies evaluated, but by 14 weeks of age, no significant differences remain between single-NE mice and controls. (C) In the dual-NE group, significant synaptic loss persists at 14 weeks of age. Data are presented as mean ± SEM, *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 7. Morphology and numbers of hair cells. (A) The cytoplasm of the cochlear hair cells is labeled with anti-myosin VIIa (red), the stereocilia with phalloidin (green), and the nuclei with DAPI (blue), Scale bar = 30 lm. (B, C) At 14 weeks of age, no statistical differences among groups are observed in counts of either OHCs or IHCs across all frequency areas on the cochlear basilar membrane (n = 8 in all groups). (D) At the same timepoint, DPOAEs shows no significant differences among control, single-NE or dual-NE groups across all frequencies (n = 5 in all groups). Data are presented as mean ± SEM. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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loss with smaller shifts at low frequencies (Pearson et al., 1995; Kujawa and Liberman, 2019). Existing researches on repetitive TTS-inducing NE have suggested that the pattern of PTS induction is reminiscent of the ARHL (Wang and Ren, 2012; Mannstrom et al., 2015; Alvarado et al., 2019). Wang and Ren reported that a third exposure of CBA mice to 100 dB octave-band noise (8– 16 kHz, 2 h, 2 weeks apart) resulted in high-frequency hearing impairments toward the base of the cochlea (Wang and Ren, 2012). However, the ABR results of the present study did not show the frequency characteristics of ARHL; significant threshold-shifts and reductions in wave I amplitude were observed across all frequencies tested (4–32 kHz). This discrepancy may be due to methodological differences. In our study, white noise of bandwidth 2 Hz to 20 kHz at 100 dB SPL was selected, because this is a superior representation of noise from occupational and recreational sources (Turcot et al.,

2015), and, being random, has a uniform power spectral density over the entire frequency domain. Therefore, any damage it causes to the basement membrane of the cochlea should theoretically be the same in all parts of the membrane. Consistent with these speculative features of white noise-induced hearing damage (Uran et al., 2014), the same hearing loss (manifesting as a TTS after the first NE and a PTS after the second) was observed at all four frequency ranges selected for our experimental design. These results are not in agreement with the known frequency dependence of ARHL. Unlike the frequency pattern of ARHL in aging humans, those observed in animal models are more complex due to species differences (Kujawa and Liberman, 2019). For instance, rats display age-related elevations of ABR thresholds that are more evenly distributed across frequencies (Balogova et al., 2017; Cai et al., 2018). Mannstrom et al. used Sprague-Dawley rats

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at least 10 weeks old, and exposed them to different levels of white noise ranging from 101 to 110 dB for 90 min every 6 weeks (Mannstrom et al., 2015). They found that 104 dB SPL was the highest intensity that could be repeated up to six times without causing a PTS. Alvarado et al. reported that repeated exposures to relatively loud noise over extended time periods could accelerate the progression of ARHL in Wistar rats (Alvarado et al., 2019). In that study, the stimulation protocol consisted of 1 h of continuous white noise at 110 dB, 5 days a week, from 3 months of age to 18 months. In aging C57BL/6J mice, the threshold shifts begin in the high frequencies and progress toward lower frequencies (Willott et al., 1995; Parham, 1997). ARHL is known to start early in this strain, which is commonly considered a mouse model of presbycusis, but the reported time of onset of presbycusis varies among studies. Spongr et al. reported that the C57BL/6 strain develops a sensorineural hearing loss beginning around 3–6 months of age (Spongr et al., 1997). However, according to Parham and Zheng et al., the onset of ARHL in C57BL/6J mice begins at 32 or 33 weeks (7–8 months) at the earliest (Parham, 1997; Zheng et al., 1999). Moreover, in our previous studies of these mice, hearing disorders were initially identified at 6–10 months of age (Chen et al., 2012). In the present study, the entire experimental period was therefore confined to a period of 8 weeks (6– 14 weeks of age), to minimize as much as possible the influence of aging on the results (The control group did not show any sign of hearing disorder during the entire experimental period). The first NE was administered at 6 weeks of age. At that time, the early vulnerable period had passed, and the cochlea was fully mature (Song et al., 2008). The observed PTS was thus due to NE rather than to young or old age. Another reason our study used C57BL/6J mice was that this strain is notably sensitive to noise, and exhibit a degree of reversibility of loss of ribbon synapses that is much greater than that previously reported in other strains (Wang et al., 2015). The suprathreshold amplitudes of wave I have been shown to be more sensitive metrics of primary neural degeneration than ABR thresholds (Melcher et al., 1996). This is largely because the high-threshold cochlear nerve fibers, which provide minimal contributions to the threshold determination but contribute more to the growth of response amplitudes with increasing SPLs, may be more susceptible to noise insults (Furman et al., 2013). ABR wave I represents the discharge potential at the junction between IHCs and type-I spiral ganglion cells in the cochlea (Zuccotti et al., 2012). In this study, on the day after the first NE, the ABR wave I amplitudes evoked by 80 dB SPL pure tone stimulation were found to be significantly decreased, indicating attenuated auditory nerve excitability induced by stimulation at suprathreshold levels. Moreover, subsequent dynamic observations revealed that the trend of wave I amplitude was not completely synchronized with changes in hearing threshold. Although the hearing threshold of mice returned to normal within 1 week after the first NE, the amplitudes of the ABR wave I had not recovered. This indicates that auditory function had not completely recovered at this time. Similar

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results were obtained in the analysis of ABR wave I in patients with normal hearing after NE (Stamper and Johnson, 2015). Further, persistent wave I amplitude reduction could be elicited by a TTS-inducing noise (Kujawa and Liberman, 2009; Fernandez et al., 2015). In our study, the reduced amplitudes no longer recovered after the second NE, suggesting a cumulative detrimental effect from individual TTS-inducing NEs. Corresponding to human wave V, wave IV of the rodent ABR reflects sound-evoked activity in higher auditory nerve centers (Melcher and Kiang, 1996), and is the most robust and stable waveform at low intensities for both tone burst and click stimuli (Scimemi et al., 2014). Amplitudes of wave IV are enhanced following NE, suggesting possible enhancement of evoked activity in the central parts of the auditory system, i.e., ‘‘central gain” (Gu et al., 2012; Auerbach et al., 2014). However, in our study, this effect reached significance only at the last observation time point. This may due to the high variation of wave IV amplitude found in mice. In addition, central gain levels are dynamic; the magnitude of the gain enhancement and even the cell-types displaying it vary over time postexposure (Auerbach et al., 2014). For instance, simultaneous measurement of amplitude-level functions in central auditory pathways demonstrated a complex temporal profile of evoked-response enhancement before and up to 2 weeks post-exposure (white noise 120 dB SPL, 1 h) (Syka et al., 1994). Moreover, after removal of 95% of cochlear afferent synapses, which eliminated sound-evoked brainstem responses and acoustic startle reflexes, increasing central gain progressively restored auditory processing (Chambers et al., 2016). Furthermore, a continuous and progressive enhancement of the IV/I ratio, due to both enhancement of wave IV and reduction of wave I, was observed in the present study, but only in dual-NE mice. This may be due to the damage caused by NE being cumulative but not evident after the first exposure. About 5–20 auditory nerve fibers make synaptic junctions with an IHC, called ribbon synapses. Increasingly, ribbon synapses are being viewed as sensitive to noise, and damage to this structure is likely to be the basis for NIHL (Shi et al., 2016). Cochlear synaptic loss, rather than hair cell death, is an earlier sign of damage in both noise- and age-related hearing impairment (Kujawa and Liberman, 2009; Sergeyenko et al., 2013). A reduced amplitude of ABR wave I reflects decreased output from the auditory nerve, and may be due to loss of ribbon synapses (Kujawa and Liberman, 2009). Ribbon synapses are the primary targets of many risk factors for hearing impairment, such as ototoxic drugs (Liu et al., 2013), NE (Shi et al., 2015b), and aging (Sergeyenko et al., 2013). Injury to ribbon synapses, which mainly manifests as a decrease in their numbers, can occur independently of damage or loss of hair cells or auditory nerves (Kujawa and Liberman, 2015). This process may become permanent if it leads to degeneration and necrosis of spiral ganglion cells (Kujawa and Liberman, 2009). Similar results were reported in another paper with the same noise condition administered to CBA mice (Wang and Ren, 2012). In some cases, noise dam-

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age may be reversible (Shi et al., 2013; Shi et al., 2015a; Shi et al., 2015b). Plastic changes in cochlear ribbon synapses may be the molecular basis of the temporary hearing threshold shifts observed in NIHL (Wang et al., 2015; Liberman and Kujawa, 2017; Wang et al., 2019). In the present study, ribbon synapses counts had decreased significantly on the day after the first NE but returned to normal within 1 week. These events were synchronized with changes in hearing threshold. In contrast, the recovery of ABR wave I amplitude was delayed to 2– 4 weeks after exposure, suggesting that ribbon synapses recover earlier than hearing function. A lack of consensus persists as to whether noise-induced damage to ribbon synapses is the cause or result of threshold shifts. Although synaptic loss is a primary and early consequence of NE, not all TTS-inducing exposures are synaptopathic (Fernandez et al., 2015). Moreover, after NE, threshold shifts can fully recover despite a permanent loss of 50% of ribbon synapses (Kujawa and Liberman, 2009). These discrepancies in ribbon synapse recovery after NE require further investigation. In NIHL, the process that manifests as TTS can be reversible under certain conditions. Otherwise PTS results. In contrast, ARHL is usually a slow, irreversible process that progresses to damage to stereocilia and loss of IHCs or OHCs (Ohlemiller, 2008; Sergeyenko et al., 2013). Moreover, hair cells cannot spontaneously regenerate in humans and most mammals (Zheng and Zuo, 2017). For typical noise-induced PTS, which can be defined as a threshold shift that persists after a period of recovery subsequent to exposure (Ryan et al., 2016), the characteristic pathological features are prominent loss of OHCs and limited loss of IHCs. These changes can be followed by apoptosis and degeneration of the cellular parts of the entire organ of Corti if the intensity and duration of the noise are sufficient (Wang et al., 2002). A significant loss of hair cells and increase in apoptosis has been induced by administration of repeated, highintensity noise (3 h, 5–20 kHz, 115 dB SPL) (Frohlich et al., 2017). However, our immunofluorescence results showed persistent synapse loss but no obvious reductions in hair cell numbers or morphological changes. The cumulative damages sustained by ribbon synapses could be the major determinant of the induction of PTS by repetitive NEs. Moreover, this was supported by our observation of unaffected DPOAEs. These findings are consistent with those of a previous study on induction of PTS by repeated exposure to moderate noise, which found no significant loss of either hair cells or DPOAEs (Wang and Ren, 2012). Taken together, these results indicate that hearing function can be impaired permanently preceding the loss of either hair cells or OHC function. A limitation of the current study was that the role of aging cannot be completely ruled out when investigating the hearing impacts of repetitive, moderate NE, even though we elicited PTS in C57BL/6J mice in young adulthood after only two NEs. Indeed, the effects of age on threshold shifts and synaptic loss here showed significance. Determining the long-term effects of repeated, moderate noise will require follow-up

experiments. We hypothesize that the first exposure to TTS-inducing noise causes injuries that were here undetected owing to the limited precision of our testing equipment or methods. These injuries are probably persistent and accumulate over repeated NEs. In summary, we demonstrated that fully recovered, noise-exposed C57BL/6J mice exposed to an identical noise a second time showed exacerbated and persistent threshold shifts and wave I amplitude reductions. In addition, the wave IV/I amplitude ratio was significantly enhanced after the second NE compared with the single NE and control groups. We found that after the second NE, paralleling the changes in threshold, the counts of ribbon synapses failed to recover, proving that the plasticity of this synapse is limited. Our work supports the hypothesis that a PTS can be caused by repeated, moderate NE, and may not manifest after a single exposure. In addition, this process can take place in a relatively short period of time well in advance of ARHL onset, and results from an accumulated trauma to ribbon synapses without obvious quantitative changes in IHC or OHC numbers or in function.

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CONFLICT OF INTEREST

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The authors declare that they have no conflict of interest.

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ACKNOWLEDGMENTS

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This work was supported by the National Natural Science Foundation of China (grant number 81770997; 81830030); and the joint funding project of Beijing Natural Science Foundation and Beijing Education Committee (grant number KZ201810025040).

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REFERENCES

752

Albera R, Lacilla M, Piumetto E, Canale A (2010) Noise-induced hearing loss evolution: influence of age and exposure to noise. Eur Arch Otorhinolaryngol 267:665–671. Alvarado JC, Fuentes-Santamaria V, Gabaldon-Ull MC, Juiz JM (2019) Age-related hearing loss is accelerated by repeated shortduration loud sound stimulation. Front Neurosci 13:77. Auerbach BD, Rodrigues PV, Salvi RJ (2014) Central gain control in tinnitus and hyperacusis. Front Neurol 5:206. Balogova Z, Popelar J, Chiumenti F, Chumak T, Burianova JS, Rybalko N, Syka J (2017) Age-related differences in hearing function and cochlear morphology between male and female Fischer 344 rats. Front Aging Neurosci 9:428. Cai R, Montgomery SC, Graves KA, Caspary DM, Cox BC (2018) The FBN rat model of aging: investigation of ABR waveforms and ribbon synapse changes. Neurobiol Aging 62:53–63. Chambers AR, Resnik J, Yuan Y, Whitton JP, Edge AS, Liberman MC, Polley DB (2016) Central gain restores auditory processing following near-complete cochlear denervation. Neuron 89:867–879. Chen L, Xiong S, Liu Y, Shang X (2012) Effect of different gentamicin dose on the plasticity of the ribbon synapses in cochlear inner hair cells of C57BL/6J mice. Mol Neurobiol 46:487–494. Fernandez KA, Jeffers PW, Lall K, Liberman MC, Kujawa SG (2015) Aging after noise exposure: acceleration of cochlear synaptopathy in ‘‘recovered” ears. J Neurosci 35:7509–7520. Frohlich F, Ernst A, Strubing I, Basta D, Groschel M (2017) Apoptotic mechanisms after repeated noise trauma in the mouse medial geniculate body and primary auditory cortex. Exp Brain Res 235:3673–3682.

753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781

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Y. Luo et al. / Neuroscience xxx (2020) xxx–xxx 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848

Furman AC, Kujawa SG, Liberman MC (2013) Noise-induced cochlear neuropathy is selective for fibers with low spontaneous rates. J Neurophysiol 110:577–586. Gu JW, Herrmann BS, Levine RA, Melcher JR (2012) Brainstem auditory evoked potentials suggest a role for the ventral cochlear nucleus in tinnitus. J Assoc Res Otolaryngol 13:819–833. Huang Q, Tang J (2010) Age-related hearing loss or presbycusis. Eur Arch Otorhinolaryngol 267:1179–1191. Khimich D, Nouvian R, Pujol R, Tom DS, Egner A, Gundelfinger ED, Moser T (2005) Hair cell synaptic ribbons are essential for synchronous auditory signalling. Nature 434:889–894. Kujawa SG, Liberman MC (2009) Adding insult to injury: cochlear nerve degeneration after ‘‘temporary” noise-induced hearing loss. J Neurosci 29:14077–14085. Kujawa SG, Liberman MC (2015) Synaptopathy in the noise-exposed and aging cochlea: primary neural degeneration in acquired sensorineural hearing loss. Hear Res 330:191–199. Kujawa SG, Liberman MC (2019) Translating animal models to human therapeutics in noise-induced and age-related hearing loss. Hear Res 377:44–52. Le TN, Straatman LV, Lea J, Westerberg B (2017) Current insights in noise-induced hearing loss: a literature review of the underlying mechanism, pathophysiology, asymmetry, and management options. J Otolaryngol Head Neck Surg 46:41. Liberman MC (2017) Noise-induced and age-related hearing loss: new perspectives and potential therapies. F1000Res 6:927. Liberman MC, Kujawa SG (2017) Cochlear synaptopathy in acquired sensorineural hearing loss: manifestations and mechanisms. Hear Res 349:138–147. Liu K, Jiang X, Shi C, Shi L, Yang B, Shi L, Xu Y, Yang W, et al. (2013) Cochlear inner hair cell ribbon synapse is the primary target of ototoxic aminoglycoside stimuli. Mol Neurobiol 48:647–654. Liu L, Wang H, Shi L, Almuklass A, He T, Aiken S, Bance M, Yin S, et al. (2012) Silent damage of noise on cochlear afferent innervation in guinea pigs and the impact on temporal processing. PLoS ONE 7 e49550. Lonsbury-Martin BL, Martin GK, Bohne BA (1987) Repeated TTS exposures in monkeys: alterations in hearing, cochlear structure, and single-unit thresholds. J Acoust Soc Am 81:1507–1518. Mannstrom P, Kirkegaard M, Ulfendahl M (2015) Repeated moderate noise exposure in the rat–an early adulthood noise exposure model. J Assoc Res Otolaryngol 16:763–772. Matsubara A, Laake JH, Davanger S, Usami S, Ottersen OP (1996) Organization of AMPA receptor subunits at a glutamate synapse: a quantitative immunogold analysis of hair cell synapses in the rat organ of Corti. J Neurosci 16:4457–4467. Melcher JR, Guinan JJ, Knudson IM, Kiang NY (1996) Generators of the brainstem auditory evoked potential in cat. II. Correlating lesion sites with waveform changes. Hear Res 93:28–51. Melcher JR, Kiang NY (1996) Generators of the brainstem auditory evoked potential in cat. III: Identified cell populations. Hear Res 93:52–71. Ohlemiller KK (2008) Recent findings and emerging questions in cochlear noise injury. Hear Res 245:5–17. Ou HC, Harding GW, Bohne BA (2000) An anatomically based frequency-place map for the mouse cochlea. Hear Res 145:123–129. Parham K (1997) Distortion product otoacoustic emissions in the C57BL/6J mouse model of age-related hearing loss. Hear Res 112:216–234. Pearson JD, Morrell CH, Gordon-Salant S, Brant LJ, Metter EJ, Klein LL, Fozard JL (1995) Gender differences in a longitudinal study of age-associated hearing loss. J Acoust Soc Am 97:1196–1205. Ryan AF, Kujawa SG, Hammill T, Le Prell C, Kil J (2016) Temporary and permanent noise-induced threshold shifts: a review of basic and clinical observations. Otol Neurotol 37:e271–e275.

917 918 919

11

Scimemi P, Santarelli R, Selmo A, Mammano F (2014) Auditory brainstem responses to clicks and tone bursts in C57 BL/6J mice. Acta Otorhinolaryngol Ital 34:264–271. Sergeyenko Y, Lall K, Liberman MC, Kujawa SG (2013) Age-related cochlear synaptopathy: an early-onset contributor to auditory functional decline. J Neurosci 33:13686–13694. Shi L, Chang Y, Li X, Aiken S, Liu L, Wang J (2016) Cochlear synaptopathy and noise-induced hidden hearing loss. Neural Plast 2016:6143164. Shi L, Guo X, Shen P, Liu L, Tao S, Li X, Song Q, Yu Z, et al. (2015a) Noise-induced damage to ribbon synapses without permanent threshold shifts in neonatal mice. Neuroscience 304:368–377. Shi L, Liu K, Wang H, Zhang Y, Hong Z, Wang M, Wang X, Jiang X, et al. (2015b) Noise induced reversible changes of cochlear ribbon synapses contribute to temporary hearing loss in mice. Acta Oto-Laryngol 135:1093–1102. Shi L, Liu L, He T, Guo X, Yu Z, Yin S, Wang J (2013) Ribbon synapse plasticity in the cochleae of Guinea pigs after noiseinduced silent damage. PLoS ONE 8 e81566. Song L, McGee J, Walsh EJ (2008) Development of cochlear amplification, frequency tuning, and two-tone suppression in the mouse. J Neurophysiol 99:344–355. Spongr VP, Flood DG, Frisina RD, Salvi RJ (1997) Quantitative measures of hair cell loss in CBA and C57BL/6 mice throughout their life spans. J Acoust Soc Am 101:3546–3553. Stamper GC, Johnson TA (2015) Auditory function in normal-hearing, noise-exposed human ears. Ear Hear 36:172–184. Syka J, Rybalko N, Popelar J (1994) Enhancement of the auditory cortex evoked responses in awake guinea pigs after noise exposure. Hear Res 78:158–168. The WHO (2018) Addressing the rising prevalence of hearing loss. Geneva: World Health Organization. Turcot A, Girard SA, Courteau M, Baril J, Larocque R (2015) Noiseinduced hearing loss and combined noise and vibration exposure. Occup Med (Lond) 65:238–244. Uran SL, Gomez-Casati ME, Guelman LR (2014) Long-term recovery from hippocampal-related behavioral and biochemical abnormalities induced by noise exposure during brain development. Evaluation of auditory pathway integrity. Int J Dev Neurosci 37:41–51. Wang H, Zhao N, Yan K, Liu X, Zhang Y, Hong Z, Wang M, Yin Q, et al. (2015) Inner hair cell ribbon synapse plasticity might be molecular basis of temporary hearing threshold shifts in mice. Int J Clin Exp Pathol 8:8680–8691. Wang J, Yin S, Chen H, Shi L (2019) Noise-induced cochlear synaptopathy and ribbon synapse regeneration: repair process and therapeutic target. Adv Exp Med Biol 1130:37–57. Wang Y, Hirose K, Liberman MC (2002) Dynamics of noise-induced cellular injury and repair in the mouse cochlea. J Assoc Res Otolaryngol 3:248–268. Wang Y, Ren C (2012) Effects of repeated ‘‘benign” noise exposures in young CBA mice: shedding light on age-related hearing loss. J Assoc Res Otolaryngol 13:505–515. Willott JF, Erway LC, Archer JR, Harrison DE (1995) Genetics of agerelated hearing loss in mice. II. Strain differences and effects of caloric restriction on cochlear pathology and evoked response thresholds. Hear Res 88:143–155. Zheng F, Zuo J (2017) Cochlear hair cell regeneration after noiseinduced hearing loss: does regeneration follow development? Hear Res 349:182–196. Zheng QY, Johnson KR, Erway LC (1999) Assessment of hearing in 80 inbred strains of mice by ABR threshold analyses. Hear Res 130:94–107. Zuccotti A, Kuhn S, Johnson SL, Franz C, Singer W, Hecker D, Geisler HS, Kopschall I, et al. (2012) Lack of brain-derived neurotrophic factor hampers inner hair cell synapse physiology, but protects against noise-induced hearing loss. J Neurosci 32:8545–8553.

(Received 16 August 2019, Accepted 26 December 2019) (Available online xxxx)

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