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Functional alteration of ribbon synapses in inner hair cells by noise exposure causing hidden hearing loss
Neuroscience Letters 707 (2019) 134268
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Research article
Functional alteration of ribbon synapses in inner hair cells by noise exposure causing hidden hearing loss
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Huihui Liua,b,c,1, Jiawen Lua,b,c,1, Zhongying Wanga,b,c, Lei Songa,b,c, Xueling Wanga,b,c,⁎, Geng-Lin Lia,b,c,⁎, Hao Wua,b,c,⁎ a
Department of Otolaryngology-Head and Neck Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Ear Institute, Shanghai Jiao Tong University School of Medicine, Shanghai, China c Shanghai Key Laboratory of Translational Medicine on Ear and Nose Diseases, Shanghai, China b
ARTICLE INFO
ABSTRACT
Keywords: Hidden hearing loss Inner hair cell Ribbon synapse Electrophysiology Exocytosis
For decades, studies on noise-induced hearing loss have been focusing on the loss of sensory hair cells and/or auditory afferent fibers following severe noise exposure. Recently, a condition of hidden hearing loss was characterized, in which moderate noise exposure that causes only temporary threshold elevation could induce persistent reduction in auditory brainstem response (ABR) amplitudes and loss of ribbon synapses in inner hair cells (IHCs). However, it is not clear whether and how moderate noise exposure alters the functionality of surviving and/or recovering ribbon synapses in IHCs. To address this issue, we applied moderate noise exposure to mice and combined auditory systems physiology, whole-mount immunofluorescence staining and patchclamp electrophysiology to characterize changes of ribbon synapse functions in IHCs. After the noise exposure, the ABR threshold was elevated and then recovered, while the ABR Wave I amplitude was reduced but did not recover. Coincidently, whole-mount cochlea staining revealed the loss and recovery of ribbon synapses in IHCs. We then performed whole-cell patch-clamp recording in IHCs and we found that the Ca2+ current, the sustained exocytosis of synaptic vesicles, and the replenishment of synaptic vesicles were all significantly reduced one day after the noise exposure. Fourteen days after the noise exposure, however, only the sustained exocytosis failed to recover, and further examination revealed that this persistent reduction is due to a decrease in the Ca2+ efficiency of triggering exocytosis. In conclusion, our results suggest temporary and persistent alterations of ribbon synapse functions in IHCs contribute to the hidden hearing loss.
1. Introduction Sensorineural hearing loss inflicts millions of people worldwide and it deeply impacts people’s daily communication and quality of life. While some forms of hearing loss are genetically determined at birth, others can be acquired later in life. Many risk factors have been identified, including acoustic overexposure [1], aging [2], infection [3], ototoxic drugs [4], etc. In particular, acoustic noise in the environment could cause damages in our auditory system, leading to noise-induced hearing loss (NIHL). Furthermore, it has been shown that people at different ages can all suffer from NIHL [5]. However, the underlying mechanisms for NIHL are not clearly understood and there are no effective treatments to cure or mitigate NIHL. Along the mammalian auditory pathways, many neurons and
synapses can be involved in NIHL, but the inner hair cells (IHCs) and their ribbon synapses with auditory afferent fibers in the cochlea are particularly vulnerable. Severe noise exposure induces intense metabolic stress within the cochlea and makes IHCs undergo intracellular Ca2+ overload [6], ATP depletion [7] and overproduction of reactive oxygen species [8], leading to the loss of IHCs and permanent threshold shift. In contrast, moderate noise exposure has been shown to cause only temporary threshold shift. Further examination of cochleae from animals treated with moderate noise exposure showed that IHCs are all intact but the numbers of ribbon synapses and afferent fiber terminals are significantly reduced [9,10]. Consistent with these morphological changes, the ABR Wave I amplitude is also reduced [11]. Given that this form of hearing loss is often overlooked by audiological examination, it is widely recognized as the hidden hearing loss.
Corresponding authors at: Department of Otolaryngology-Head and Neck Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. E-mail addresses: [email protected] (X. Wang), [email protected] (G.-L. Li), [email protected] (H. Wu). 1 These two authors contributed equally to this study. ⁎
https://doi.org/10.1016/j.neulet.2019.05.022 Received 27 February 2019; Received in revised form 23 April 2019; Accepted 13 May 2019 Available online 16 May 2019 0304-3940/ © 2019 Published by Elsevier B.V.
Neuroscience Letters 707 (2019) 134268
H. Liu, et al.
It is unclear, however, if and how the functionality of ribbon synapses in IHCs is altered in the hidden hearing loss. These ribbon synapses are specialized for accurate transmission [12,13], and any deviation from optimal operation would have significant impact on the overall hearing performance [14]. In this study, we applied moderate noise exposure to mice and established an animal model of temporary threshold shift without causing significant loss of IHCs. We examined ribbon synapse functions in IHCs following the noise exposure and uncovered a combination of temporary and persistent changes, which could be involved in the hidden hearing loss.
mouse IgG1 and Alexa Fluor 647-conjugated IgG2a from Invitrogen, 1:1000) at room temperature for 2 h. Confocal images were acquired on a laser scanning confocal microscope (Leica Microsystems, Germany) with a 63X oil immersion objective and 3X digital zoom. Z-axis stacks of 2D images were taken with a step size of 0.5 μm. CtBP2 and GluR2 immunofluorescence puncta were counted in the z-stacks and divided by the number of IHCs. 2.4. Patch-clamp recording Patch-clamp recording was performed in IHCs from the apical turn of the organ of Corti, estimated to be corresponding to 5–8 kHz, according to a protocol described previously [18]. Whole-cell recordings were made at room temperature with an EPC10/2 amplifier (HEKA Electronics, Germany). Recording pipettes were pulled from borosilicate glass (World Precision Instruments) to a resistance of 5–6 MΩ and coated with dental wax. The pipette solution contained (in mM): 135 Cs-methanesulfonate, 10 CsCl, 10 TEA-Cl, 10 HEPES, 2 EGTA, 3 Mg-ATP and 0.5 Na-GTP, pH adjusted to 7.20 and osmolarity adjusted to 290 mOsm. The extracellular solution contained (in mM): 130 NaCl, 2.8 KCl, 10 CaCl2, 1 MgCl2, 10 HEPES, and 10 D-glucose, pH adjusted to 7.40 and osmolarity adjusted to 300 mOsm. Liquid junction potential (−10 mV) was corrected offline. The current-voltage relationship (I–V curve) for the Ca2+ current in IHCs was obtained from current responses to ramp depolarization from −80 mV to 70 mV, and fitted to the following equation to obtain the half activation potential (Vhalf) and the slope of activation (k):
2. Materials and methods 2.1. Animals and moderate noise exposure Male CBA/CaJ mice of four weeks old were purchased from the SIPPR-BK Laboratory Animal Ltd (Shanghai, China) and housed under the standard laboratory conditions. We chose CBA/CaJ mice because they maintain excellent hearing sensitivity even at old ages [15]. We used animals of 4 weeks old because the morphology of IHCs reaches the mature stage and the hearing threshold is stabilized [16,17]. A total of about 80 mice were used, and handling of animals was carried out in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, USA), approved by the University Committee of Laboratory Animals of Shanghai Jiao Tong University. Animals were divided into one control group (Pre) and three experimental groups (1d, 7d and 14d). For each group, animals were sacrificed at the end of ABR tests and cochleae were dissected for either staining or patch-clamp experiments. For moderate noise treatment, the animals were exposed to a broadband noise (2–20 kHz) at 98 dB sound pressure level (SPL) for 2 h in a soundproof chamber. During the exposure, the animals were awake and unrestrained in a wire-mesh cage with a loudspeaker placed 10 cm above to deliver noise through free field to both ears. The loudspeaker was driven with signals from a TDT RZ6 system (Tucker-Davis Technologies, USA) and calibrated with a quarter inch microphone to the targeted sound pressure level before each exposure session.
I(V) = ( V
Vrev) ×
Gmax 1 + exp( ( V Vhalf )/ k )
where Vrev is the reversal potential and Gmax is the maximum conductance. Whole-cell membrane capacitance measurements were made with the “Sine + DC” method in Patchmaster (HEKA) [18,22], and the increase of capacitance before and after stimulation (ΔCm) was used to assess exocytosis of synaptic vesicles from IHCs. 2.5. Data analysis and statistical tests Data were analyzed in the Igor Pro (WaveMetrics, USA) and statistical tests were performed in the Prism (GraphPad, USA). Statistical significance was assessed by one-way or two-way ANOVA followed by Bonferroni post-hoc test, or unpaired Student’s t-test. Data are presented in Mean ± SEM and the level of statistical significance was set to be p < 0.05.
2.2. Auditory brainstem response recording Auditory brainstem responses (ABRs) were recorded from anesthetized mice according to a protocol described previously [18]. Briefly, animals were anesthetized by intraperitoneal injection of 480 mg/kg chloral hydrate, and pure tone pips of 3 ms with 1 ms of rise and 1 ms of fall at 4, 5.65, 8, 11.31, 16, 22.62 and 32 kHz were presented at a rate of 20 pips per second. For each frequency and each SPL, a total of 400 responses were recorded and averaged in the TDT RZ6 system. The ABR threshold was defined as the lowest SPL that was able to evoke an appropriate ABR response, in 5 dB steps descending from 90 dB [19,20], and was assessed by an independent researcher who was blind to the conditions and time point. The ABR Wave I amplitude was measured from the positive peak to the subsequent negative trough in the waveform and used as an index of the ABR amplitude [21].
3. Results 3.1. Noise-induced temporary threshold shift To induce temporary threshold shift in mice, we exposed them to a single session of moderate noise treatment. The hearing threshold was measured before (Pre) and 1, 7 and 14 days after the noise exposure (1d, 7d and 14d), based on the averages of ABR responses (Fig. 1A), and the results are consistent with previously published studies [10]. As shown in Fig. 1B, the ABR thresholds were elevated significantly across all the frequencies tested for 1d (n = 10, two-way ANOVA, p < 0.001). Across different frequencies, the threshold shift ranged from 17 to 25 dB. For 7d and 14d, however, a gradual recovery of the ABR thresholds was observed. For 7d, the ABR thresholds remained elevated at 4 kHz (n = 7, p < 0.05) and 32 kHz (n = 7, p < 0.001) but recovered at the other frequencies tested. For 14d, the ABR thresholds became indistinguishable from Pre for all frequencies tested (n = 10, p > 0.05). We therefore demonstrated in our hands that moderate noise exposure causes temporary threshold shift in mice. We then measured the ABR Wave I amplitude at 5.65 and 8 kHz in the apical turn [23]. We chose to focus on the apical turn because in the
2.3. Immunohistochemistry staining The mice were deeply anesthetized and sacrificed, and the cochleae were quickly removed from the skull and perfused with 4% paraformaldehyde at 4 °C overnight. The next day, the organ of Corti was dissected out and permeabilized with 5% Triton X-100 for 30 min and then incubated in 5% BSA for 60 min. The tissue was then doublestained with the mixture of two primary antibodies directed against a synaptic ribbon protein (mouse anti-CtBP2 IgG1 from BD Biosciences, 1:200) and an AMPA-type glutamate receptor (mouse anti-GluR2 IgG2a from Millipore, 1:100) overnight at 4 °C. Next, the tissue was incubated with the secondary antibodies (Alexa Fluor 568-conjugated goat anti 2
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Fig. 1. Changes of hearing performance following moderate noise exposure. A), representative traces of auditory brainstem response (ABR) before the noise exposure, evoked by brief pure tones at 8.0 kHz, and the hearing threshold was determined as 35 dB. B), after the noise exposure, the hearing thresholds were elevated across all frequencies tested, began to recover after 7 days and recovered fully after 14 days. C and D), ABR Wave I amplitudes, evoked by suprathreshold tones at 5.656 kHz (C) and 8.0 kHz (D), were significantly reduced and did not recover fully even after 14 days. Data are presented as Mean ± SEM, statistical significance was assessed with two-way ANOVA followed by Bonferroni post-hoc test, * means p < 0.05, ** means p < 0.01 and *** means p < 0.001.
adult cochlea only this region permits patch-clamp analysis on IHCs, an approach we would like to take for later experiments. For both frequencies, the mean amplitude of the ABR Wave I was significantly reduced after the noise exposure (Fig. 1C and D). Unlike the ABR thresholds, however, the ABR Wave I amplitude did not recover. Even 14 days after the noise exposure, it was still significantly smaller when compared to that of Pre. Again, these results are consistent with previously published studies on hidden hearing loss with temporary threshold shift [1].
[24], the CtBP2 antibody for presynaptic ribbons also recognized the nuclei, and further examination of the number of nuclei indicated no apparent loss of IHCs after the noise exposure (Fig. 2A). As shown in Fig. 2B, the number of ribbon synapses per IHC was significantly reduced from 15.1 ± 0.30 (Mean ± SEM, n = 18) for Pre to 12.7 ± 0.35 for 1d (n = 19, one-way ANOVA, p < 0.001), coinciding with the greatest ABR threshold shift. For 7d, the number of ribbon synapses recovered partially, and a full recovery was achieved after 14 days when the number of ribbon synapses per IHC became indistinguishable from that of Pre.
3.2. Loss of ribbon synapses and recovery
3.3. Alteration of Ca2+ current
Considering that the ABR Wave I amplitude represents the soundevoked discharges of all responding afferent fibers, we wondered if its reduction is due to the loss of ribbon synapses between inner hair cells (IHCs) and auditory afferent fibers. In accordance with the ABR Wave I amplitude analysis earlier and patch-clamp analysis on IHCs later, we chose to focus on the apical turn and carried out whole-mount immunostaining of the sensory epithelium in this region. Tissues were double labeled with an antibody against presynaptic ribbons [24] and an antibody against postsynaptic glutamate receptors. Merged confocal images were obtained and each closely juxtaposed fluorescent punctum was defined as a single ribbon synapse [25]. As previously described
To examine possible functional changes of ribbon synapses, we performed whole-cell patch-clamp recording in IHCs of all the four animal groups. We focused on the apical turn because only this region of the hearing epithelium can be readily excised from the adult cochlea for patch-clamp recording. We applied a voltage ramp from −80 mV to +70 mV and recorded the Ca2+ current responses (Fig. 3A). We found that the Ca2+ current amplitude (ICa) was significantly reduced from −150 ± 4.80 pA for Pre (n = 14) to -114 ± 3.24 pA for 1d (n = 13, one-way ANOVA, p < 0.001, Fig. 3B). It recovered after 7 days, as the values for both 7d (-135 ± 6.07 pA, n = 15) and 14d (-151 ± 9.39 pA, Fig. 2. Loss and recovery of ribbon synapses in inner hair cells (IHCs) following moderate noise exposure. A), whole-mounted cochleae double stained for CtBP2 (red) and GluR2 (green), showing the two overlapped (yellow). The IHC nuclei were also labeled due to the nuclear expression of CtBP2. Puncta of co-labeling are presumably ribbon synapses between IHCs and auditory afferent fibers. B), number of ribbon synapses in IHCs from different animal groups. Note that the number of ribbon synapses was significantly reduced following noise exposure but recovered fully after 14 days (one-way ANOVA, p < 0.001).
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Fig. 3. Changes of Ca2+ influx in IHCs following moderate noise exposure. A), representative I–V curves of the Ca2+current in IHCs before (Pre, black) and 1 day after the noise exposure (1d, red). The current response was induced by a voltage ramp from -80 mV to 70 mV and then leak subtracted. The dashed gray line depicts the curve fitting to obtain the half activation potential (Vhalf) and the slope of activation (k). B), after the noise exposure, the Ca2+ current amplitude (ICa) was significantly reduced and then gradually recovered. C and D), voltage dependence of the Ca2+ current, assessed based on Vhalf and k, was not significantly altered by the noise exposure.
n = 10) are indistinguishable from that of Pre (one-way ANOVA, p > 0.05). Meanwhile, we found no significant changes in the resting capacitance of IHCs before and after the noise exposure (Pre: 8.12 ± 0.35 pF; 1d: 7.92 ± 0.20 pF; 7d: 8.04 ± 0.21; 14d: 8.78 ± 0.70 pF; one-way ANOVA, p > 0.05). We then analyzed the voltage dependence of the Ca2+ current, and we found that neither the half activation potential (Vhalf) nor the slope of activation (k) was significantly altered by the noise exposure (Fig. 3C and D). Although we cannot rule out the changes of single channel conductance and/or the open probability, we favor the simple interpretation of the reduction and recovery of the Ca2+ current in IHCs are due to the changes in the number of Ca2+ channels accompanied with the loss and recovery of ribbon synapses.
3.4. Alteration of exocytosis We next examined exocytosis from IHCs before and after noise exposure. We performed whole-cell membrane capacitance measurements and used the capacitance increase before and after stimulation (ΔCm, see Fig. 4A) to quantify exocytosis. To examine both the rapid and sustained release of synaptic vesicles, we applied strong depolarizing pulses (i.e. 0 mV, the potential gives the maximum of Ca2+ current) and calculated ΔCm for different stimulation durations from 10 ms to 1 s [26,27]. For stimulation of 10, 20 and 50 ms, ΔCm was similar for 1d when compared to Pre (n = 11–20, two-way ANOVA, p > 0.05, Fig. 4B), suggesting that the noise exposure did not affect the rapid exocytosis significantly. For intermediate stimulation durations, including 100, 200 and 400 ms, exocytosis was not significantly altered, either. For stimulation of 600 ms, ΔCm was significantly reduced for 1d (n = 7–9, p < 0.01) and 7d (n = 9–13, p < 0.05), and it recovered Fig. 4. Changes of exocytosis in IHCs following moderate noise exposure. A), representative Ca2+ currents (ICa) and the resulting capacitance jumps (ΔCm) recorded from IHCs before (Pre, black) and 14 days after the noise exposure (14d, green). B), ΔCm and the Ca2+ charge (QCa) evoked by stimulations of different durations from 10 ms to 1 s. ΔCm for prolonged stimulations was significantly reduced by the noise exposure and failed to recover. Statistical significance was assessed with two-way ANOVA followed by Bonferroni posthoc test. C), the Ca2+ efficiency of triggering exocytosis, assessed based on the ratio of ΔCm/ QCa, was reduced significantly for stimulation of 1 s and did not recover even after 14 days. Statistical significance was assessed with unpaired Student’s t-test.
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Fig. 5. Synaptic vesicle replenishment was largely unaltered by moderate noise exposure. A), representative Ca2+ responses to paired pulse stimulation and capacitance measurements from IHCs before (Pre, black) and 1 day after the noise exposure (1d, red). B), following the noise exposure, synaptic vesicle replenishment, assessed based on the ratio of ΔCm2/ ΔCm1, remained indistinguishable from that of Pre, except the transient reduction for 1d with the shortest inter-pulse interval only.
relies on tight control of Ca2+ influx through voltage-gated Ca2+ channels [32]. We therefore characterized the biophysical properties of Ca2+ channels in IHCs [33]. After moderate noise exposure, the Ca2+ current amplitude was reduced and then gradually recovered. Furthermore, we assessed the voltage dependence of the Ca2+ current with the half activation potential (Vhalf) and the slope of activation (k), and we found no significant change in either parameter (Fig. 3). Following a previous study [34], we evaluated the number and morphological appearance of ribbon synapses in IHCs to address potential mechanisms. We found that the temporary reduction of the Ca2+ current in IHCs is in close agreement quantitatively with the decrease in the number of ribbon synapses in IHCs. Under physiological conditions, IHCs are partially depolarized even in the absence of acoustic stimulation, and they must faithfully encode a wide dynamic range of graded membrane potentials [35]. Accordingly, IHCs have been shown to be capable of both rapid and prolonged exocytosis of synaptic vesicles [36] and fast replenishment of synaptic vesicles [33]. The rapid exocytosis lasts less than 50 ms [37], representing release of the readily releasable pool of synaptic vesicles. The sustained exocytosis could last for seconds, reflecting the fast and efficient recycling of synaptic vesicles [38]. In our mouse model for the hidden hearing loss, the sustained exocytosis was reduced one day after the noise exposure. The reduction recovered slowly for intermediate stimulation, and failed to recover for long stimulation of 800 ms and 1 s. This stimulation duration-dependent reduction of exocytosis cannot be attributed exclusively to the loss and recovery of ribbon synapses, as similar changes of Ca2+ influx was not observed (Fig. 4B). At least for exocytosis of 1 s stimulation, the reduction is due in part to a decrease of the Ca2+ efficiency in triggering synaptic vesicle release, as the ratio of ΔCm/QCa was significantly decreased after the noise exposure and did not recover (Fig. 4C). The temporary and persistent alterations of ribbon synapse functions in IHCs could bear significant consequences in auditory signal coding. The rapid exocytosis is critically important for reliable and precise coding for brief sounds with short gaps [25,39]. The sustained release of hundreds of synaptic vesicles is a peculiar feature of ribbon synapses that is required to drive auditory afferent fibers to fire spikes at hundreds of Hz [40]. Fast and efficient vesicle replenishment is required to maintain this high rate of exocytosis for prolonged period of time [41]. Changes in these key ribbon synapse functions could be a new direction for research in the hidden hearing loss.
partially for 14d (n = 8–9, p > 0.05, Fig. 4B). For stimulation of 800 ms and 1 s, ΔCm was reduced and failed to recover (n = 8–15, p < 0.05, Fig. 4B). Meanwhile, the Ca2+ influx, assessed with the Ca2+ charge (QCa) was not altered except for stimulation of 800 ms and 1 s, both of which recovered quickly. We next examined the Ca2+ efficiency of triggering exocytosis for 14d by calculating the ratio of ΔCm/QCa, and we found that ΔCm/QCa was significantly smaller only for stimulation of 1 s (Pre: 1.82 ± 0.85 fF/pC, n = 10; 14d: 1.21 ± 0.36 fF/pC, n = 15; unpaired Student’s t-test, p < 0.05), suggesting the persistent reduction of ΔCm for prolonged stimulation is due to a decrease of the Ca2+ efficiency in triggering exocytosis. Lastly, we investigated synaptic vesicle replenishment in IHCs by applying a paired pulse protocol for stimulation. We depolarized IHCs for 500 ms to maximally deplete synaptic vesicles [28], followed by another depolarization for 500 ms with a varying interval in between (Fig. 5A). We used the ratio of the first and second capacitance increase (ΔCm2/ΔCm1) to assess the synaptic vesicle replenishment. As shown in Fig. 5B, the synaptic vesicle replenishment was slowed only with an interval as short as 100 ms for the 1d group only (Pre: 0.51 ± 0.049, n = 9; 1d: 0.29 ± 0.026, n = 10; two-way ANOVA, p < 0.05), and the slowed replenishment recovered quickly as no significant difference was observed for the replenishment between the 7d group and the Pregroup (7d, 0.44 ± 0.034, n = 15, p > 0.05). 4. Discussion Studies on noise-induced hearing loss (NIHL) have been focusing primarily on the death and survival of IHCs following intensive noise exposure. However, it is not clear if and how ribbon synapse functions in IHCs are altered by moderate noise exposure. In the present study, we found that following moderate noise exposure that causes temporary threshold shift, the Ca2+ current, the sustained exocytosis of synaptic vesicles and the replenishment of synaptic vesicles in IHCs are all significantly reduced but only the sustained exocytosis fails to recover after 14 days. We suggest the combination of temporary and persistent alterations of ribbon synapse functions in IHCs contributes to the hidden hearing loss following moderate noise exposure. It has been shown that moderate noise exposure could cause significant loss of ribbon synapses with no apparent loss of IHCs [10]. In this study, we revisited how moderate noise exposure damages ribbon synapses in the cochlea. We first focused on morphological changes. Our immunostaining results suggest that within days after the moderate noise exposure, the number of ribbon synapses per IHC in the apical region was significantly reduced and then fully recovered (Fig. 2). In contrast, no significant change was observed in the same region in previous studies by the Liberman group [1,10]. This discrepancy could be due to a combination of factors, including the animal age (4 weeks for our study vs 8–16 weeks for their studies) and noise frequency band (2–20 kHz vs 8–16 kHz). Indeed, significant loss of IHC ribbon synapses was observed in CBA/CaJ mice of 16 weeks old without noise exposure [29] and significant recovery of IHC ribbon synapses was also reported by other groups using a variety of experimental conditions [30,31]. Precise transmission of auditory signals in IHC ribbon synapses
Author contributions HW, G-LL designed and supervised the whole study. HL, JL and ZW performed the experiments. HL, JL and LS analyzed the data and wrote the manuscript. XW edited the revised manuscript. Acknowledgement The work was supported by two research grants from the National Natural Science Foundation of China to Dr. Hao Wu (81330023 and 81730028). Preliminary results were presented in the ARO 41st 5
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MidWinter meeting in 2018, and we thank Xu Ding for helping us prepare the poster. The authors declare no competing financial interests.
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Functional alteration of ribbon synapses in inner hair cells by noise exposure causing hidden hearing loss
T
Huihui Liua,b,c,1, Jiawen Lua,b,c,1, Zhongying Wanga,b,c, Lei Songa,b,c, Xueling Wanga,b,c,⁎, Geng-Lin Lia,b,c,⁎, Hao Wua,b,c,⁎ a
Department of Otolaryngology-Head and Neck Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Ear Institute, Shanghai Jiao Tong University School of Medicine, Shanghai, China c Shanghai Key Laboratory of Translational Medicine on Ear and Nose Diseases, Shanghai, China b
ARTICLE INFO
ABSTRACT
Keywords: Hidden hearing loss Inner hair cell Ribbon synapse Electrophysiology Exocytosis
For decades, studies on noise-induced hearing loss have been focusing on the loss of sensory hair cells and/or auditory afferent fibers following severe noise exposure. Recently, a condition of hidden hearing loss was characterized, in which moderate noise exposure that causes only temporary threshold elevation could induce persistent reduction in auditory brainstem response (ABR) amplitudes and loss of ribbon synapses in inner hair cells (IHCs). However, it is not clear whether and how moderate noise exposure alters the functionality of surviving and/or recovering ribbon synapses in IHCs. To address this issue, we applied moderate noise exposure to mice and combined auditory systems physiology, whole-mount immunofluorescence staining and patchclamp electrophysiology to characterize changes of ribbon synapse functions in IHCs. After the noise exposure, the ABR threshold was elevated and then recovered, while the ABR Wave I amplitude was reduced but did not recover. Coincidently, whole-mount cochlea staining revealed the loss and recovery of ribbon synapses in IHCs. We then performed whole-cell patch-clamp recording in IHCs and we found that the Ca2+ current, the sustained exocytosis of synaptic vesicles, and the replenishment of synaptic vesicles were all significantly reduced one day after the noise exposure. Fourteen days after the noise exposure, however, only the sustained exocytosis failed to recover, and further examination revealed that this persistent reduction is due to a decrease in the Ca2+ efficiency of triggering exocytosis. In conclusion, our results suggest temporary and persistent alterations of ribbon synapse functions in IHCs contribute to the hidden hearing loss.
1. Introduction Sensorineural hearing loss inflicts millions of people worldwide and it deeply impacts people’s daily communication and quality of life. While some forms of hearing loss are genetically determined at birth, others can be acquired later in life. Many risk factors have been identified, including acoustic overexposure [1], aging [2], infection [3], ototoxic drugs [4], etc. In particular, acoustic noise in the environment could cause damages in our auditory system, leading to noise-induced hearing loss (NIHL). Furthermore, it has been shown that people at different ages can all suffer from NIHL [5]. However, the underlying mechanisms for NIHL are not clearly understood and there are no effective treatments to cure or mitigate NIHL. Along the mammalian auditory pathways, many neurons and
synapses can be involved in NIHL, but the inner hair cells (IHCs) and their ribbon synapses with auditory afferent fibers in the cochlea are particularly vulnerable. Severe noise exposure induces intense metabolic stress within the cochlea and makes IHCs undergo intracellular Ca2+ overload [6], ATP depletion [7] and overproduction of reactive oxygen species [8], leading to the loss of IHCs and permanent threshold shift. In contrast, moderate noise exposure has been shown to cause only temporary threshold shift. Further examination of cochleae from animals treated with moderate noise exposure showed that IHCs are all intact but the numbers of ribbon synapses and afferent fiber terminals are significantly reduced [9,10]. Consistent with these morphological changes, the ABR Wave I amplitude is also reduced [11]. Given that this form of hearing loss is often overlooked by audiological examination, it is widely recognized as the hidden hearing loss.
Corresponding authors at: Department of Otolaryngology-Head and Neck Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. E-mail addresses: [email protected] (X. Wang), [email protected] (G.-L. Li), [email protected] (H. Wu). 1 These two authors contributed equally to this study. ⁎
https://doi.org/10.1016/j.neulet.2019.05.022 Received 27 February 2019; Received in revised form 23 April 2019; Accepted 13 May 2019 Available online 16 May 2019 0304-3940/ © 2019 Published by Elsevier B.V.
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It is unclear, however, if and how the functionality of ribbon synapses in IHCs is altered in the hidden hearing loss. These ribbon synapses are specialized for accurate transmission [12,13], and any deviation from optimal operation would have significant impact on the overall hearing performance [14]. In this study, we applied moderate noise exposure to mice and established an animal model of temporary threshold shift without causing significant loss of IHCs. We examined ribbon synapse functions in IHCs following the noise exposure and uncovered a combination of temporary and persistent changes, which could be involved in the hidden hearing loss.
mouse IgG1 and Alexa Fluor 647-conjugated IgG2a from Invitrogen, 1:1000) at room temperature for 2 h. Confocal images were acquired on a laser scanning confocal microscope (Leica Microsystems, Germany) with a 63X oil immersion objective and 3X digital zoom. Z-axis stacks of 2D images were taken with a step size of 0.5 μm. CtBP2 and GluR2 immunofluorescence puncta were counted in the z-stacks and divided by the number of IHCs. 2.4. Patch-clamp recording Patch-clamp recording was performed in IHCs from the apical turn of the organ of Corti, estimated to be corresponding to 5–8 kHz, according to a protocol described previously [18]. Whole-cell recordings were made at room temperature with an EPC10/2 amplifier (HEKA Electronics, Germany). Recording pipettes were pulled from borosilicate glass (World Precision Instruments) to a resistance of 5–6 MΩ and coated with dental wax. The pipette solution contained (in mM): 135 Cs-methanesulfonate, 10 CsCl, 10 TEA-Cl, 10 HEPES, 2 EGTA, 3 Mg-ATP and 0.5 Na-GTP, pH adjusted to 7.20 and osmolarity adjusted to 290 mOsm. The extracellular solution contained (in mM): 130 NaCl, 2.8 KCl, 10 CaCl2, 1 MgCl2, 10 HEPES, and 10 D-glucose, pH adjusted to 7.40 and osmolarity adjusted to 300 mOsm. Liquid junction potential (−10 mV) was corrected offline. The current-voltage relationship (I–V curve) for the Ca2+ current in IHCs was obtained from current responses to ramp depolarization from −80 mV to 70 mV, and fitted to the following equation to obtain the half activation potential (Vhalf) and the slope of activation (k):
2. Materials and methods 2.1. Animals and moderate noise exposure Male CBA/CaJ mice of four weeks old were purchased from the SIPPR-BK Laboratory Animal Ltd (Shanghai, China) and housed under the standard laboratory conditions. We chose CBA/CaJ mice because they maintain excellent hearing sensitivity even at old ages [15]. We used animals of 4 weeks old because the morphology of IHCs reaches the mature stage and the hearing threshold is stabilized [16,17]. A total of about 80 mice were used, and handling of animals was carried out in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, USA), approved by the University Committee of Laboratory Animals of Shanghai Jiao Tong University. Animals were divided into one control group (Pre) and three experimental groups (1d, 7d and 14d). For each group, animals were sacrificed at the end of ABR tests and cochleae were dissected for either staining or patch-clamp experiments. For moderate noise treatment, the animals were exposed to a broadband noise (2–20 kHz) at 98 dB sound pressure level (SPL) for 2 h in a soundproof chamber. During the exposure, the animals were awake and unrestrained in a wire-mesh cage with a loudspeaker placed 10 cm above to deliver noise through free field to both ears. The loudspeaker was driven with signals from a TDT RZ6 system (Tucker-Davis Technologies, USA) and calibrated with a quarter inch microphone to the targeted sound pressure level before each exposure session.
I(V) = ( V
Vrev) ×
Gmax 1 + exp( ( V Vhalf )/ k )
where Vrev is the reversal potential and Gmax is the maximum conductance. Whole-cell membrane capacitance measurements were made with the “Sine + DC” method in Patchmaster (HEKA) [18,22], and the increase of capacitance before and after stimulation (ΔCm) was used to assess exocytosis of synaptic vesicles from IHCs. 2.5. Data analysis and statistical tests Data were analyzed in the Igor Pro (WaveMetrics, USA) and statistical tests were performed in the Prism (GraphPad, USA). Statistical significance was assessed by one-way or two-way ANOVA followed by Bonferroni post-hoc test, or unpaired Student’s t-test. Data are presented in Mean ± SEM and the level of statistical significance was set to be p < 0.05.
2.2. Auditory brainstem response recording Auditory brainstem responses (ABRs) were recorded from anesthetized mice according to a protocol described previously [18]. Briefly, animals were anesthetized by intraperitoneal injection of 480 mg/kg chloral hydrate, and pure tone pips of 3 ms with 1 ms of rise and 1 ms of fall at 4, 5.65, 8, 11.31, 16, 22.62 and 32 kHz were presented at a rate of 20 pips per second. For each frequency and each SPL, a total of 400 responses were recorded and averaged in the TDT RZ6 system. The ABR threshold was defined as the lowest SPL that was able to evoke an appropriate ABR response, in 5 dB steps descending from 90 dB [19,20], and was assessed by an independent researcher who was blind to the conditions and time point. The ABR Wave I amplitude was measured from the positive peak to the subsequent negative trough in the waveform and used as an index of the ABR amplitude [21].
3. Results 3.1. Noise-induced temporary threshold shift To induce temporary threshold shift in mice, we exposed them to a single session of moderate noise treatment. The hearing threshold was measured before (Pre) and 1, 7 and 14 days after the noise exposure (1d, 7d and 14d), based on the averages of ABR responses (Fig. 1A), and the results are consistent with previously published studies [10]. As shown in Fig. 1B, the ABR thresholds were elevated significantly across all the frequencies tested for 1d (n = 10, two-way ANOVA, p < 0.001). Across different frequencies, the threshold shift ranged from 17 to 25 dB. For 7d and 14d, however, a gradual recovery of the ABR thresholds was observed. For 7d, the ABR thresholds remained elevated at 4 kHz (n = 7, p < 0.05) and 32 kHz (n = 7, p < 0.001) but recovered at the other frequencies tested. For 14d, the ABR thresholds became indistinguishable from Pre for all frequencies tested (n = 10, p > 0.05). We therefore demonstrated in our hands that moderate noise exposure causes temporary threshold shift in mice. We then measured the ABR Wave I amplitude at 5.65 and 8 kHz in the apical turn [23]. We chose to focus on the apical turn because in the
2.3. Immunohistochemistry staining The mice were deeply anesthetized and sacrificed, and the cochleae were quickly removed from the skull and perfused with 4% paraformaldehyde at 4 °C overnight. The next day, the organ of Corti was dissected out and permeabilized with 5% Triton X-100 for 30 min and then incubated in 5% BSA for 60 min. The tissue was then doublestained with the mixture of two primary antibodies directed against a synaptic ribbon protein (mouse anti-CtBP2 IgG1 from BD Biosciences, 1:200) and an AMPA-type glutamate receptor (mouse anti-GluR2 IgG2a from Millipore, 1:100) overnight at 4 °C. Next, the tissue was incubated with the secondary antibodies (Alexa Fluor 568-conjugated goat anti 2
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Fig. 1. Changes of hearing performance following moderate noise exposure. A), representative traces of auditory brainstem response (ABR) before the noise exposure, evoked by brief pure tones at 8.0 kHz, and the hearing threshold was determined as 35 dB. B), after the noise exposure, the hearing thresholds were elevated across all frequencies tested, began to recover after 7 days and recovered fully after 14 days. C and D), ABR Wave I amplitudes, evoked by suprathreshold tones at 5.656 kHz (C) and 8.0 kHz (D), were significantly reduced and did not recover fully even after 14 days. Data are presented as Mean ± SEM, statistical significance was assessed with two-way ANOVA followed by Bonferroni post-hoc test, * means p < 0.05, ** means p < 0.01 and *** means p < 0.001.
adult cochlea only this region permits patch-clamp analysis on IHCs, an approach we would like to take for later experiments. For both frequencies, the mean amplitude of the ABR Wave I was significantly reduced after the noise exposure (Fig. 1C and D). Unlike the ABR thresholds, however, the ABR Wave I amplitude did not recover. Even 14 days after the noise exposure, it was still significantly smaller when compared to that of Pre. Again, these results are consistent with previously published studies on hidden hearing loss with temporary threshold shift [1].
[24], the CtBP2 antibody for presynaptic ribbons also recognized the nuclei, and further examination of the number of nuclei indicated no apparent loss of IHCs after the noise exposure (Fig. 2A). As shown in Fig. 2B, the number of ribbon synapses per IHC was significantly reduced from 15.1 ± 0.30 (Mean ± SEM, n = 18) for Pre to 12.7 ± 0.35 for 1d (n = 19, one-way ANOVA, p < 0.001), coinciding with the greatest ABR threshold shift. For 7d, the number of ribbon synapses recovered partially, and a full recovery was achieved after 14 days when the number of ribbon synapses per IHC became indistinguishable from that of Pre.
3.2. Loss of ribbon synapses and recovery
3.3. Alteration of Ca2+ current
Considering that the ABR Wave I amplitude represents the soundevoked discharges of all responding afferent fibers, we wondered if its reduction is due to the loss of ribbon synapses between inner hair cells (IHCs) and auditory afferent fibers. In accordance with the ABR Wave I amplitude analysis earlier and patch-clamp analysis on IHCs later, we chose to focus on the apical turn and carried out whole-mount immunostaining of the sensory epithelium in this region. Tissues were double labeled with an antibody against presynaptic ribbons [24] and an antibody against postsynaptic glutamate receptors. Merged confocal images were obtained and each closely juxtaposed fluorescent punctum was defined as a single ribbon synapse [25]. As previously described
To examine possible functional changes of ribbon synapses, we performed whole-cell patch-clamp recording in IHCs of all the four animal groups. We focused on the apical turn because only this region of the hearing epithelium can be readily excised from the adult cochlea for patch-clamp recording. We applied a voltage ramp from −80 mV to +70 mV and recorded the Ca2+ current responses (Fig. 3A). We found that the Ca2+ current amplitude (ICa) was significantly reduced from −150 ± 4.80 pA for Pre (n = 14) to -114 ± 3.24 pA for 1d (n = 13, one-way ANOVA, p < 0.001, Fig. 3B). It recovered after 7 days, as the values for both 7d (-135 ± 6.07 pA, n = 15) and 14d (-151 ± 9.39 pA, Fig. 2. Loss and recovery of ribbon synapses in inner hair cells (IHCs) following moderate noise exposure. A), whole-mounted cochleae double stained for CtBP2 (red) and GluR2 (green), showing the two overlapped (yellow). The IHC nuclei were also labeled due to the nuclear expression of CtBP2. Puncta of co-labeling are presumably ribbon synapses between IHCs and auditory afferent fibers. B), number of ribbon synapses in IHCs from different animal groups. Note that the number of ribbon synapses was significantly reduced following noise exposure but recovered fully after 14 days (one-way ANOVA, p < 0.001).
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Fig. 3. Changes of Ca2+ influx in IHCs following moderate noise exposure. A), representative I–V curves of the Ca2+current in IHCs before (Pre, black) and 1 day after the noise exposure (1d, red). The current response was induced by a voltage ramp from -80 mV to 70 mV and then leak subtracted. The dashed gray line depicts the curve fitting to obtain the half activation potential (Vhalf) and the slope of activation (k). B), after the noise exposure, the Ca2+ current amplitude (ICa) was significantly reduced and then gradually recovered. C and D), voltage dependence of the Ca2+ current, assessed based on Vhalf and k, was not significantly altered by the noise exposure.
n = 10) are indistinguishable from that of Pre (one-way ANOVA, p > 0.05). Meanwhile, we found no significant changes in the resting capacitance of IHCs before and after the noise exposure (Pre: 8.12 ± 0.35 pF; 1d: 7.92 ± 0.20 pF; 7d: 8.04 ± 0.21; 14d: 8.78 ± 0.70 pF; one-way ANOVA, p > 0.05). We then analyzed the voltage dependence of the Ca2+ current, and we found that neither the half activation potential (Vhalf) nor the slope of activation (k) was significantly altered by the noise exposure (Fig. 3C and D). Although we cannot rule out the changes of single channel conductance and/or the open probability, we favor the simple interpretation of the reduction and recovery of the Ca2+ current in IHCs are due to the changes in the number of Ca2+ channels accompanied with the loss and recovery of ribbon synapses.
3.4. Alteration of exocytosis We next examined exocytosis from IHCs before and after noise exposure. We performed whole-cell membrane capacitance measurements and used the capacitance increase before and after stimulation (ΔCm, see Fig. 4A) to quantify exocytosis. To examine both the rapid and sustained release of synaptic vesicles, we applied strong depolarizing pulses (i.e. 0 mV, the potential gives the maximum of Ca2+ current) and calculated ΔCm for different stimulation durations from 10 ms to 1 s [26,27]. For stimulation of 10, 20 and 50 ms, ΔCm was similar for 1d when compared to Pre (n = 11–20, two-way ANOVA, p > 0.05, Fig. 4B), suggesting that the noise exposure did not affect the rapid exocytosis significantly. For intermediate stimulation durations, including 100, 200 and 400 ms, exocytosis was not significantly altered, either. For stimulation of 600 ms, ΔCm was significantly reduced for 1d (n = 7–9, p < 0.01) and 7d (n = 9–13, p < 0.05), and it recovered Fig. 4. Changes of exocytosis in IHCs following moderate noise exposure. A), representative Ca2+ currents (ICa) and the resulting capacitance jumps (ΔCm) recorded from IHCs before (Pre, black) and 14 days after the noise exposure (14d, green). B), ΔCm and the Ca2+ charge (QCa) evoked by stimulations of different durations from 10 ms to 1 s. ΔCm for prolonged stimulations was significantly reduced by the noise exposure and failed to recover. Statistical significance was assessed with two-way ANOVA followed by Bonferroni posthoc test. C), the Ca2+ efficiency of triggering exocytosis, assessed based on the ratio of ΔCm/ QCa, was reduced significantly for stimulation of 1 s and did not recover even after 14 days. Statistical significance was assessed with unpaired Student’s t-test.
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Fig. 5. Synaptic vesicle replenishment was largely unaltered by moderate noise exposure. A), representative Ca2+ responses to paired pulse stimulation and capacitance measurements from IHCs before (Pre, black) and 1 day after the noise exposure (1d, red). B), following the noise exposure, synaptic vesicle replenishment, assessed based on the ratio of ΔCm2/ ΔCm1, remained indistinguishable from that of Pre, except the transient reduction for 1d with the shortest inter-pulse interval only.
relies on tight control of Ca2+ influx through voltage-gated Ca2+ channels [32]. We therefore characterized the biophysical properties of Ca2+ channels in IHCs [33]. After moderate noise exposure, the Ca2+ current amplitude was reduced and then gradually recovered. Furthermore, we assessed the voltage dependence of the Ca2+ current with the half activation potential (Vhalf) and the slope of activation (k), and we found no significant change in either parameter (Fig. 3). Following a previous study [34], we evaluated the number and morphological appearance of ribbon synapses in IHCs to address potential mechanisms. We found that the temporary reduction of the Ca2+ current in IHCs is in close agreement quantitatively with the decrease in the number of ribbon synapses in IHCs. Under physiological conditions, IHCs are partially depolarized even in the absence of acoustic stimulation, and they must faithfully encode a wide dynamic range of graded membrane potentials [35]. Accordingly, IHCs have been shown to be capable of both rapid and prolonged exocytosis of synaptic vesicles [36] and fast replenishment of synaptic vesicles [33]. The rapid exocytosis lasts less than 50 ms [37], representing release of the readily releasable pool of synaptic vesicles. The sustained exocytosis could last for seconds, reflecting the fast and efficient recycling of synaptic vesicles [38]. In our mouse model for the hidden hearing loss, the sustained exocytosis was reduced one day after the noise exposure. The reduction recovered slowly for intermediate stimulation, and failed to recover for long stimulation of 800 ms and 1 s. This stimulation duration-dependent reduction of exocytosis cannot be attributed exclusively to the loss and recovery of ribbon synapses, as similar changes of Ca2+ influx was not observed (Fig. 4B). At least for exocytosis of 1 s stimulation, the reduction is due in part to a decrease of the Ca2+ efficiency in triggering synaptic vesicle release, as the ratio of ΔCm/QCa was significantly decreased after the noise exposure and did not recover (Fig. 4C). The temporary and persistent alterations of ribbon synapse functions in IHCs could bear significant consequences in auditory signal coding. The rapid exocytosis is critically important for reliable and precise coding for brief sounds with short gaps [25,39]. The sustained release of hundreds of synaptic vesicles is a peculiar feature of ribbon synapses that is required to drive auditory afferent fibers to fire spikes at hundreds of Hz [40]. Fast and efficient vesicle replenishment is required to maintain this high rate of exocytosis for prolonged period of time [41]. Changes in these key ribbon synapse functions could be a new direction for research in the hidden hearing loss.
partially for 14d (n = 8–9, p > 0.05, Fig. 4B). For stimulation of 800 ms and 1 s, ΔCm was reduced and failed to recover (n = 8–15, p < 0.05, Fig. 4B). Meanwhile, the Ca2+ influx, assessed with the Ca2+ charge (QCa) was not altered except for stimulation of 800 ms and 1 s, both of which recovered quickly. We next examined the Ca2+ efficiency of triggering exocytosis for 14d by calculating the ratio of ΔCm/QCa, and we found that ΔCm/QCa was significantly smaller only for stimulation of 1 s (Pre: 1.82 ± 0.85 fF/pC, n = 10; 14d: 1.21 ± 0.36 fF/pC, n = 15; unpaired Student’s t-test, p < 0.05), suggesting the persistent reduction of ΔCm for prolonged stimulation is due to a decrease of the Ca2+ efficiency in triggering exocytosis. Lastly, we investigated synaptic vesicle replenishment in IHCs by applying a paired pulse protocol for stimulation. We depolarized IHCs for 500 ms to maximally deplete synaptic vesicles [28], followed by another depolarization for 500 ms with a varying interval in between (Fig. 5A). We used the ratio of the first and second capacitance increase (ΔCm2/ΔCm1) to assess the synaptic vesicle replenishment. As shown in Fig. 5B, the synaptic vesicle replenishment was slowed only with an interval as short as 100 ms for the 1d group only (Pre: 0.51 ± 0.049, n = 9; 1d: 0.29 ± 0.026, n = 10; two-way ANOVA, p < 0.05), and the slowed replenishment recovered quickly as no significant difference was observed for the replenishment between the 7d group and the Pregroup (7d, 0.44 ± 0.034, n = 15, p > 0.05). 4. Discussion Studies on noise-induced hearing loss (NIHL) have been focusing primarily on the death and survival of IHCs following intensive noise exposure. However, it is not clear if and how ribbon synapse functions in IHCs are altered by moderate noise exposure. In the present study, we found that following moderate noise exposure that causes temporary threshold shift, the Ca2+ current, the sustained exocytosis of synaptic vesicles and the replenishment of synaptic vesicles in IHCs are all significantly reduced but only the sustained exocytosis fails to recover after 14 days. We suggest the combination of temporary and persistent alterations of ribbon synapse functions in IHCs contributes to the hidden hearing loss following moderate noise exposure. It has been shown that moderate noise exposure could cause significant loss of ribbon synapses with no apparent loss of IHCs [10]. In this study, we revisited how moderate noise exposure damages ribbon synapses in the cochlea. We first focused on morphological changes. Our immunostaining results suggest that within days after the moderate noise exposure, the number of ribbon synapses per IHC in the apical region was significantly reduced and then fully recovered (Fig. 2). In contrast, no significant change was observed in the same region in previous studies by the Liberman group [1,10]. This discrepancy could be due to a combination of factors, including the animal age (4 weeks for our study vs 8–16 weeks for their studies) and noise frequency band (2–20 kHz vs 8–16 kHz). Indeed, significant loss of IHC ribbon synapses was observed in CBA/CaJ mice of 16 weeks old without noise exposure [29] and significant recovery of IHC ribbon synapses was also reported by other groups using a variety of experimental conditions [30,31]. Precise transmission of auditory signals in IHC ribbon synapses
Author contributions HW, G-LL designed and supervised the whole study. HL, JL and ZW performed the experiments. HL, JL and LS analyzed the data and wrote the manuscript. XW edited the revised manuscript. Acknowledgement The work was supported by two research grants from the National Natural Science Foundation of China to Dr. Hao Wu (81330023 and 81730028). Preliminary results were presented in the ARO 41st 5
Neuroscience Letters 707 (2019) 134268
H. Liu, et al.
MidWinter meeting in 2018, and we thank Xu Ding for helping us prepare the poster. The authors declare no competing financial interests.
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