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. 2019 Dec 25;123(2):658–669. doi: 10.1152/jn.00642.2019

Intense noise exposure alters peripheral vestibular structures and physiology

C E Stewart 1,✉, D S Bauer 1, A C Kanicki 1, R A Altschuler 1, W M King 1
PMCID: PMC7052639  PMID: 31875485

Abstract

The otolith organs play a critical role in detecting linear acceleration and gravity to control posture and balance. Some afferents that innervate these structures can be activated by sound and are at risk for noise overstimulation. A previous report demonstrated that noise exposure can abolish vestibular short-latency evoked potential (VsEP) responses and damage calyceal terminals. However, the stimuli that were used to elicit responses were weaker than those established in previous studies and may have been insufficient to elicit VsEP responses in noise-exposed animals. The goal of this study was to determine the effect of an established noise exposure paradigm on VsEP responses using large head-jerk stimuli to determine if noise induces a stimulus threshold shift and/or if large head-jerks are capable of evoking VsEP responses in noise-exposed rats. An additional goal is to relate these measurements to the number of calyceal terminals and hair cells present in noise-exposed vs. non-noise-exposed tissue. Exposure to intense continuous noise significantly reduced VsEP responses to large stimuli and abolished VsEP responses to small stimuli. This finding confirms that while measurable VsEP responses can be elicited from noise-lesioned rat sacculi, larger head-jerk stimuli are required, suggesting a shift in the minimum stimulus necessary to evoke the VsEP. Additionally, a reduction in labeled calyx-only afferent terminals was observed without a concomitant reduction in the overall number of calyces or hair cells. This finding supports a critical role of calretinin-expressing calyceal-only afferents in the generation of a VsEP response.

NEW & NOTEWORTHY This study identifies a change in the minimum stimulus necessary to evoke vestibular short-latency evoked potential (VsEP) responses after noise-induced damage to the vestibular periphery and reduced numbers of calretinin-labeled calyx-only afferent terminals in the striolar region of the sacculus. These data suggest that a single intense noise exposure may impact synaptic function in calyx-only terminals in the striolar region of the sacculus. Reduced calretinin immunolabeling may provide insight into the mechanism underlying noise-induced changes in VsEP responses.

Keywords: calretinin, calyx, continuous noise, sacculus, vestibular

INTRODUCTION

The effects of noise on hearing are well established. Noise overstimulation causes temporary or permanent damage to sensory cells (hair cells) and their synapses, resulting in temporary or permanent hearing threshold shifts and/or hidden hearing loss (Hawkins and Schacht 2005; Kujawa and Liberman 2009; Liberman and Kujawa 2017). It is known that vestibular responses can be elicited with sound stimulation and that the otolith organs are most sound sensitive (e.g., Curthoys et al. 2016; Murofushi et al. 1995; Zhu et al. 2011, 2014). Despite this background, the susceptibility of the otolith organs to noise overstimulation and concomitant functional consequences are not well understood. Animal studies demonstrate that peripheral vestibular damage can occur after noise exposure and lead to anatomical and/or physiological changes (Fetoni et al. 2009; Hsu et al. 2008; Perez et al. 2002; Stewart et al. 2016, 2018; Tamura et al. 2012). However, the reports are inconsistent, and there is little evidence of a direct link between noise-induced otolith organ injury and impaired vestibular nerve function (Akdogan et al. 2009; Fetoni et al. 2009; Hsu et al. 2008; Tamura et al. 2012).

Balance deficits are a significant public health problem in the United States. Agrawal and colleagues (Agrawal et al. 2009, 2013) reported that 35% of adults older than 40 yr had evidence of postural instability and that balance dysfunction increased with age so that by 80 yr, 85% of adults reported balance problems. Although the causes of balance dysfunction are complex and multifactorial, multiple studies suggest there may be a linkage between hearing loss and vestibular dysfunction in humans (Akin et al. 2012; Guest et al. 2011; Heitz et al. 2019; Lin and Ferrucci 2012; Manabe et al. 1995; Oosterveld et al. 1982; Shupak et al. 1994; Yilmaz et al. 2018; Ylikoski and Ylikoski 1994; Ylikoski et al. 1988; Zuniga et al. 2012). Noise exposure as a risk factor for vestibular loss has obvious public health implications, but there is a lack of evidence for a causal connection between noise exposure and vestibular loss.

The sacculus is vulnerable to noise by virtue of the anatomical proximity of this structure to the oval window (Backous et al. 1999). The irregular afferents that innervate the sacculus may be particularly vulnerable to intense noise exposure because of the known sensitivity of this population of afferents to acoustic stimulation (Curthoys 2017; Curthoys et al. 2016; Curthoys and Grant 2015; Curthoys and Vulovic 2011; Murofushi et al. 1995; Zhu et al. 2011, 2014). These afferents can be activated by loud sounds and overstimulated by intense or persistent noise (McCue and Guinan 1997; Shupak et al. 1994; Stewart et al. 2016, 2018; Tamura et al. 2012). Action potentials conducted by irregular afferents are believed to be a major source of the vestibular short-latency evoked potential (VsEP). We previously showed that intense noise exposure can reduce VsEP responses to undetectable levels when evoked by relatively weak linear acceleration head-jerks (shortened to “head-jerks” for the remainder of this paper; Stewart et al. 2018). These changes were associated with decreased numbers of calyceal endings in the sacculus; however, the effect of noise on vestibular hair cells was not quantitatively examined (Stewart et al. 2018). We therefore could not rule out that damage or loss of vestibular hair cells contributed to the functional changes observed in the previous study (Stewart et al. 2018).

The current study extends previous neurophysiological findings by using strong VsEP head-jerk stimuli to determine if large stimuli can elicit measurable responses after noise exposure. We also quantitatively determined if hair cell numbers and calyceal terminals in the striolar region of the sacculus were reduced or altered by noise. We hypothesized that noise exposure would reduce the number of calyx-only afferent terminals that could be identified with calretinin immunolabeling and that this loss would be associated with an increased stimulus threshold and/or attenuation of VsEP responses.

METHODS

Animals

Long-Evans rats weighing 350–400 g (Charles River Laboratories) at the start of the experiment were pair housed on a 12:12-h light-dark cycle (lights on at 0800 and off at 2000) with ad libitum access to food and water. Data from a total of 60 rats are included in this article. Exact numbers of rats are provided in the specific methods for each procedure. All procedures were carried out in accordance with National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use Committee at the University of Michigan.

Surgical Preparation

Head posts were attached to the skull of all rats included in this study. Rats’ heads were positioned on a stereotaxic frame under isoflurane anesthesia, and a dorsal cranial midline incision was made to expose bregma and lambda. The skull was leveled with respect to the stereotaxic frame by adjusting the pitch of the head until the Z-axis coordinate of bregma and lambda was equal. After the skull was leveled, two anchor screws were placed into the skull and a custom head bolt was bonded to the skull with C&B Metabond cement (Parkell, Inc., Edgewood, NY). After the cement was set, the head bolt was fused to the anchor screws in the skull with dental acrylic and rats were allowed to recover for 10 days.

Noise Exposure Parameters

The noise exposure booth was fitted with a 52-Hz-resonance loudspeaker (Kappalite 3012H0; Eminence Speaker LLC, Eminence, KY), mounted in a custom-built 1.7-ft3 sealed enclosure. The loudspeaker was driven by a simultaneous high-voltage, high-current magnetic field power amplifier (TFM-35; Carver Corporation, Lynnwood, WA), which received an audio signal from an audio CD player (PDM320; Marantz America, LLC, Mahwah, NJ). The audio CD was created using Adobe Audition version 1.5 software. Sound level and spectrum were measured with a fast Fourier transform (FFT) spectrum analyzer (SRS760; Stanford Research Systems, Sunnyvale, CA), Bruel & Kjaer type 4136 microphone, type 2619 preamplifier, and type 2804 power supply (Bruel & Kjaer Sound and Vibration Measurement, Naerum, Denmark).

In this study rats were either not exposed to noise or exposed to noise on a single day and allowed to recover for 28 days. These groups were used for auditory brain stem response, VsEP, and tissue measurements. A continuous free-field noise stimulus (1.5-kHz, 3-octave band noise) designed to impact the most apical 20% of the rat cochlea or the lower end of the rat hearing frequency range (Greenwood 1996; Müller 1991; Viberg and Canlon 2004). The noise was delivered at a maximum intensity of 120 dB SPL (Fig. 1), which is comparable in intensity to a loud rock concert or sporting event. Unanesthetized Long-Evans rats were placed into individual wire mesh cages and held in a ventilated sound exposure booth for 6 hours. After noise exposure, the rats were returned to their home cages to recover.

Fig. 1.

Fig. 1.

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Noise exposure frequency spectrum. The peak intensity of the continuous noise spectrum falls at 120 dB SPL, at a frequency of ~2 kHz. The continuous noise spectrum produced for this study falls at intensities greater than 105 dB SPL across a frequency range of ~0.5–4.0 kHz.

Auditory Brain Stem Response

Auditory function was evaluated in control (n = 15; 8 male, 7 female) and noise-exposed rats (n = 15; 8 male, 7 female) using the auditory brain stem response (ABR; a far-field potential reflecting electrically or acoustically induced auditory nerve activity) with tones delivered at 8, 4, and 1.5 kHz. ABRs were recorded in an electrically and acoustically shielded chamber (Acoustic Systems, Austin, TX). Needle electrodes were placed at the vertex (active), below the test ear (reference), and in the hip (ground). Tucker Davis Technologies (TDT) System III hardware and SigGen/BioSig software (TDT, Alachua, FL) were used to present the stimulus and record responses. Tones were delivered through an EC1 driver (aluminum-shielded enclosure made in house), with the speculum placed just inside the tragus. The stimulus was presented in 15-ms-duration tone bursts with 1-ms rise and fall times, at a rate of 10/s. The sound stimulus was calibrated with an FFT spectrum analyzer (SRS760 Stanford Research Systems) connected to a microphone (4136; Bruel & Kjaer), preamplifier (2619; Bruel & Kjaer), and power supply (2804; Bruel & Kjaer). The microphone was coupled to the sound-source speculum, and the unattenuated level of each tone was measured. This measurement was used to create a file to equalize the levels of all tones. The levels were then verified using the output generated by the BioSig ABR program. Responses (1,024) were averaged for each stimulus intensity, and this procedure was repeated at each frequency. Response waveforms were collected for stimulus levels in 10-dB steps at higher stimulus levels with additional 5-dB steps near threshold. Thresholds were interpolated between the lowest stimulus level where a response was observed and 5 dB lower where no response was observed. Measurements were taken in the non-noise-exposed rats and the noise-exposed rats 28 days after noise exposure. Group data at each frequency were compared with two-tailed unpaired t tests assuming equal variances in MATLAB.

VsEP Protocol

The VsEP is a far-field electrical potential produced by action potentials in the vestibular nerve and evoked by linear acceleration of the head (Jones et al. 2002; Jones and Jones 1999; Nazareth and Jones 1998; Weisleder et al. 1990). The VsEP selectively samples large irregular afferent fibers that fire synchronously in response to rapid head-jerks (Jamali et al. 2019; Jones et al. 1998, 2011). The VsEP was measured in control (n = 33; 26 male, 7 female) and noise-exposed rats (n = 20; 9 male, 11 female) according to the method established by Jones et al. (2011) and described previously by Stewart et al. (2018). Briefly, anesthetized Long-Evans rats were attached to a shaker using the implanted head bolt so that motion of the shaker was dependably transmitted to the rat’s skull and the head was linearly accelerated in the naso-occipital direction (Stewart et al. 2018). Five head-jerk stimulus waveforms were developed based on criteria established by Jones et al. (2011). Each stimulus waveform consisted of brief head-jerks (0.65–1.35 ms) ranging from ~0.32 to 5.5 g/ms, equal to a range of −4.9 to 7.4 dB (referenced to 1 g/ms; Fig. 2). We chose to vary the stimulus jerk levels with a constant peak acceleration (isoacceleration) based on evidence that VsEP response amplitudes are dependent on jerk amplitude but independent of peak acceleration (Jones et al. 2011). Electrodes (stainless steel needles) placed subcutaneously at the vertex (noninverting), mastoid (reference), and hip (ground) recorded the VsEP. Stimuli were delivered as successive positive and negative head-jerks (200 of each) and averaged together to minimize electromagnetic artifacts from the shaker. With the use of custom software (MATLAB), the average was synchronized, using the stimulus onset as a trigger. Figure 2 shows the stimuli used to elicit VsEP responses and Fig. 3 illustrates typical averaged VsEP recordings obtained in our laboratory with these stimuli.

Fig. 2.

Fig. 2.

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Vestibular short-latency evoked potential (VsEP) head-jerk stimuli. Five head-jerks were used to elicit VsEP responses. Each stimulus waveform consisted of brief head-jerks (0.65–1.35 ms) ranging from 0.32 (solid black trace) to 5.5 g/ms (gray trace). Decibels corresponding to each stimulus in g/ms can be calculated by dividing the stimulus used in g/ms by the reference stimulus (1 g/ms) and calculating dB as 10 times the log of the quotient (see key, top right).

Fig. 3.

Fig. 3.

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Vestibular short-latency evoked potential (VsEP) traces from 2 representative rats. A rat with a relatively small control VsEP response (A) has almost no response to any of the 5 stimuli employed in the study 28 days after noise exposure (B). A rat with a large control VsEP response (C) has responses to the 4 largest stimuli, although they are severely attenuated after noise exposure. There is no detectable response to the smallest stimulus, suggesting a threshold shift 28 days after noise exposure (D).

Signals from the electrodes were amplified (200,000 times), filtered (300–10,000 Hz), and digitized by a CED data acquisition system (Cambridge Electronic Design, Cambridge, UK) at 20 kHz. An accelerometer, epoxied to the shaker arm, recorded the head acceleration; its output was also digitized at 20 kHz. Data were transferred to a personal computer for storage and offline analysis. VsEP waveform amplitude values were determined as the amplitude of the positive peak (P1 or P2) less the amplitude of the negative (N1 or N2) valley. VsEP waveform latency values were determined as the time at which each peak (P1 and P2) occurred relative to the stimulus peak. Group data at each frequency were compared with two-tailed unpaired t tests assuming equal variances in MATLAB.

Quantitative Analysis of Hair Cells and Calyceal Terminations

Following a lethal dose of pentobarbital sodium, rats were decapitated and inner ears were rapidly removed. Inner ears then received intrascalar fixation with 4% paraformaldehyde and were immersed in 4% paraformaldehyde overnight, washed with PBS, and decalcified with 5% EDTA for dissection.

Control (n = 16) and noise-exposed (n = 22, calretinin and neurofilament; n = 23, myosin 7a) vestibular sensory epithelia were blocked and permeabilized with 5% normal donkey serum in a solution of 0.3% Triton X in 1× PBS (17000121; Jackson Laboratories) and then incubated in primary antibodies: mouse anti-calretinin (CR; 1:1,000; Millipore MAB1568, 29 kDa), chicken anti-neurofilament H (NF; 1:1,000; Millipore AB5539, 200 kDa), and rabbit anti-myosin 7a (Myo7a; 1:200; Proteus Bioscience Rab PAB 25-6790, 200 kDa). CR is used to identify calyx-only afferents (Dechesne et al. 1991; Leonard and Kevetter 2002), while neurofilament labels all fibers projecting to and from the vestibular ganglion (afferent and efferent fibers; Demêmes et al. 1992; Hafidi and Romand 1989). Myo7a is used to label hair cell bodies (Hasson et al. 1997). Tissue was washed three times in PBS and incubated with respective secondary antibodies, washed again with PBS, and mounted with ProLong Diamond mounting medium.

Imaging was carried out on a confocal microscope (Leica TCS SP8). First, a ×20 z series of the upper half of the sacculus containing the bend of the sacculus and upper portions of the long and short limbs was collected to determine the region of interest (ROI) for ×40 imaging (Fig. 4A). Next, a z series was collected at ×40 magnification, in 1-µm steps, for quantification of calyces and hair cells and to produce a two-dimensional z-stack-collapsed image of the bend of the sacculus using methods similar to those described previously (Stewart et al. 2018).

Fig. 4.

Fig. 4.

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Region of interest in the sacculus used for quantitative analysis of noise-induced vestibular injury. A: ×20 confocal image of a non-noise-exposed control sacculus (left). The region containing the bend of the sacculus and upper portions of the long and short limbs was used to determine a 100 × 200-μm2 region of interest for ×40 imaging (right). B. the same 100 × 200-μm2 region of interest was used to analyze the distribution of calyx-only afferent terminals. Distribution was analyzed by dividing the region of interest into 10 equal 100 × 20-μm2 subregions and counting calyx-only afferent terminals, identified with calretinin, within each subregion.

Images collected from noise-exposed and control sacculi were analyzed in Fiji (ImageJ). Images were qualitatively examined to determine the absence or presence of calretinin-positive calyces. After identification of the upper and lower limits in the z plane of the area of interest within the sacculus, steps at which calyces were present were selected for analysis. Steps were selected for the z-stack-collapsed image if they contained calyces, defined here as neurofilament-positive circles, ≥5 µm in diameter, that appeared below the cuticular plate of the sensory epithelium. A separate channel showing calretinin immunoreactivity was merged with the neurofilament channel, and all structures that met the criteria for a calyx and were also colocalized with calretinin were considered calyx-only afferents (Leonard and Kevetter 2002). A difference between the present method and previous methods (Stewart et al. 2018) is that no normalization was necessary, since all areas were 100 × 200 µm2. Additionally, because the data were normally distributed, control and noise-exposed tissue were compared with a two-tailed unpaired t test in MATLAB.

A 100 × 200-µm2 region of interest containing the striolar and juxtastriolar regions of the bend of the sacculus was selected to isolate an ROI for each sample (Fig. 4A). After selection of the proper ROI in each sample, the region outside of the ROI for each z stack was cleared and quantification of calyx-only endings, total calyces, and total hair cell bodies was carried out using the Cell Counter plugin (Fiji, ImageJ). Calyx-only afferent endings were CR positive, did not colocalize with Myo7a, and were restricted to the striolar region of the sacculus. Calyceal endings (dimorphic and calyx only) were neurofilament positive and were analyzed morphologically to confirm calyceal shape (e.g., not efferent or bouton endings). Calyces were morphologically defined to surround a hair cell with a flask-like shape. Therefore, within steps containing calyceal endings, a neurofilament-positive circle that was empty in the middle was identified as a calyx. Hair cells were Myo7a positive, and while some colocalized with calretinin (type II hair cell bodies), no attempt was made to evaluate the ratio of type I to type II hair cells. Counts were compared with a two-tailed unpaired t test assuming equal variances in MATLAB.

Distribution of Calyx-Only Afferent Terminals

We next determined if the number of calretinin-stained calyces was uniformly reduced across the width of the ROI after noise exposure vs. control. Desai et al. (2005) described the striolar region of the otolith organs as the region that contained 99% of the calyx-only afferent terminals, identified with calretinin. We determined the distribution of calyx-only afferent terminal counts within the ROI by dividing it into 10 identical subregions measuring 100 × 20 µm2. Calretinin-positive calyces were counted within each subregion of the ROI. Calyces that were split between two ROI subregions were only counted in the first subregion in which they were identified (Fig. 4B). Because each ROI was not perfectly centered over the line of polarity reversal, subregions were shifted so that the subregion with the highest number of calyx-only afferent terminals was considered the center for later quantification of calyx distribution across the region of interest. By centering the subregion with the highest number of calyx-only afferent terminals (setting this to subregion 7), empty cells were included at the edges of each data set, leading to a uniform number of 14 subregions in the shifted data.

Each noise-exposed subregion was compared with control using a two-tailed unpaired t test in MATLAB (see Table 4). Means and SE for each of the 14 subregions (control, n = 16; noise, n = 22) are plotted in Fig. 9.

Table 4.

Calretinin-immunolabeled calyx data divided by subregion

Control
 Noise
 Summary
Subregion n Mean SE n Mean SE %Control Variance P value Effect size
1 2 0 0 2 0.5 0.5 0.42
2 4 0.5 0.5 4 2.5 1.4 500 1.0 0.24 −4.00
3 10 1.4 0.7 9 1.6 0.7 116 5.2 0.96 −0.28
4 13 3.9 1.0 14 1.9 0.7 49 12.7 0.08 2.02
5 16 6.1 0.9 18 3.9 0.8 63 12.8 0.06 2.46
6 16 8.5 0.5 22 6.4 0.5 75 3.3 0.006 4.60
7 16 11.8 0.7 22 9.1 0.5 77 8.5 0.005 3.71
8 16 7.6 0.8 22 6.2 0.5 81 11.1 0.20 1.68
9 16 5.5 0.9 22 5.2 0.4 95 12.7 0.88 0.34
10 16 4.3 0.9 22 3.5 0.5 81 12.1 0.38 0.92
11 13 2.8 0.8 19 1.8 0.3 61 7.8 0.13 1.29
12 10 1.9 0.8 17 1.1 0.2 56 6.3 0.16 1.01
13 4 3.5 1.8 12 0.7 0.4 20 12.3 0.03 1.59
14 2 2.5 1.5 7 0.3 0.3 13 4.5 0.04 1.47
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Values are group data (control, 16 rats; noise exposure, 22 rats), compared by subregion with a two-tailed unpaired t test, where n is the number of sacculi analyzed and means and SE refer to the number of calyces counted in each subregion for each group. Mean %control, variance, and effect size could not be calculated for subregion 1 because the mean and SD of the control subregion was 0. Boldface indicates a statistically significant reduction in calretinin-labeled calyces. While the most significant reduction in calretinin-labeled calyces was observed centrally, there was also a significant reduction at the edge of the region of interest in subregions 13 and 14. While these subregions have a smaller n value in both groups than was observed in more central subregions, the effect size is adequate.

Fig. 9.

Fig. 9.

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Distribution of calyx-only afferent terminals in the striolar region of the sacculus. The 100 × 200-μm2 region of interest (ROI) was divided into 10 subregions to determine the distribution of calretinin-immunolabeled calyceal terminals in control and noise-exposed sacculi. The peak of each set of subregions (subregion with greatest number of calyces) was shifted to subregion 7, and group data were averaged. Means are plotted with error bars indicating SE. Twenty-eight days after noise exposure, there was a significant reduction in the average number of calretinin-positive terminals in central regions (subregions 6 and 7) and at the edge of the ROI (subregions 13 and 14) compared with controls. *P < 0.05; **P < 0.01.

RESULTS

Larger Stimuli are Required to Evoke Auditory and Vestibular Responses After Noise Exposure

Auditory brain stem response.

In controls, responses to pure tones could be elicited at intensities below 50 dB SPL (see Table 1). Following a 28-day recovery from noise exposure, ABR thresholds were significantly elevated at all three measured intensities, indicating substantial noise-induced hearing loss (see Table 1). Data are sorted by frequency and are means ± SD.

Table 1.

ABR threshold shifts in noise-exposed rats

Control Noise
8 kHz
    Mean 45.000 78.000*
    SD 2.673 12.071
4 kHz
    Mean 46.667 80.333*
    SD 3.086 10.259
1.5 kHz
    Mean 48.667 71.667*
    SD 5.164 8.997
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Values are auditory brain stem response (ABR) measurements expressed in dB SPL for tones delivered at 8, 4, and 1.5 kHz.

*

P < 0.001.

Vestibular short-latency evoked potential.

Figure 3 shows representative VsEP responses from two rats obtained before and 28 days after noise exposure (Fig. 1). Figure 3, A and C, shows that each rat produced robust responses to head-jerks of 1.1 to 5.5 g/ms before noise exposure. Although smaller and delayed in time, there were also clear responses to the weakest head-jerk stimulus (0.32 g/ms, top traces in Fig. 3, A and C). As described by previous studies (e.g., Jones et al. 2011; Stewart et al. 2018), each VsEP response is characterized by two positive and negative peaks, P1N1 and P2N2 (Fig. 5A, bottom trace). The initial peak typically occurs with a latency of 0.97–1.3 ms depending on head-jerk amplitude. The P2 peak occurs ~0.8 ms later and likely is produced by postsynaptic currents and action potentials induced in secondary vestibular neurons (Shimazu and Precht 1965). Figure 3, B and D, shows that there is a marked attenuation of the VsEP responses from these rats 28 days after noise exposure. In particular, a response to the weakest head-jerk amplitude is not discernable in Fig. 3B (rat 88) and is arguably missing in rat 80 (Fig. 3D), suggesting larger head-jerk amplitudes are needed after noise exposure to produce a detectable response (“threshold shift”).

Fig. 5.

Fig. 5.

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Individual traces for all rats included in this report. The vestibular short-latency evoked potential response measured in this report has 2 distinct peaks (P1, P2) and troughs (N1, N2; see A, bottom traces). P1N1 is thought to represent eighth nerve activity, while P2N2 is thought to represent activity from secondary vestibular neurons. A: when control responses (n = 30) are grouped by stimulus intensity and overlaid, the waveforms are similar across all rats. B: after noise exposure (n = 20), there is virtually no response from any animal measured in response to the smallest stimulus (top traces). In response to the larger 4 head-jerk stimuli, there are severely attenuated peripheral and central responses from all but 2 rats (arrows). These 2 rats appear to have no deficit after noise exposure.

Figure 5 shows that noise exposure caused significant changes in VsEP responses for nearly every rat in our study. In Fig. 5A, responses are shown for 30 rats that were not noise exposed. Although there is variability in response amplitude and latency, VsEP waveforms are remarkably similar across multiple rats. Figure 5B shows responses for 20 rats obtained 28 days after noise exposure. With the exception of two rats (indicated by arrows in Fig. 5B), noise exposure significantly attenuated or abolished VsEP responses. The two rats whose VsEP responses were relatively normal after noise exposure (arrows, Fig. 5B) were excluded from the statistical analysis (Tables 2 and 3) as outliers. However, these two rats apparently experienced an intense noise exposure, since they had tissue damage that was comparable to that of other noise-exposed rats. Specifically, the calretinin-positive calyx counts for these rats fell within 1 SD of the mean of the noise-exposed data and below the mean of the control data.

Table 2.

Comparison of VsEP response amplitudes in controls and 28 days after noise exposure

P1N1 Amplitude
 P2N2 Amplitude
Control
 Noise
 Control
Noise
Stimulus Intensity Mean CI Mean CI t Statistic Mean CI Mean CI t Statistic
5.5 g/ms 0.458 0.107 0.141 0.031 7.83 0.522 0.095 0.160 0.025 10.2
3.2 g/ms 0.408 0.097 0.114 0.054 8.08 0.487 0.089 0.138 0.052 10.3
2.2 g/ms 0.342 0.087 0.109 0.047 7.16 0.454 0.093 0.120 0.045 9.58
1.1 g/ms 0.221 0.066 0.068 0.043 6.02 0.397 0.096 0.087 0.034 8.73
0.32 g/ms 0.072 0.025 0.025 0.015 4.86 0.250 0.060 0.046 0.017 9.36
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Values are vestibular short-latency evoked potential (VsEP) amplitudes (in µV), averaged by group and compared. Responses were collected from control (n = 33) and noise-exposed rats (n = 18). Means are shown with 99.9% confidence intervals (CI). P1N1 and P2N2 amplitudes are significantly reduced in response to all stimulus intensities (P < 0.001).

Table 3.

Comparison of VsEP response latencies in controls and 28 days after noise exposure

P1 Latency
 P2 Latency
Control
 Noise
 Control
 Noise
Stimulus Intensity Mean CI Mean CI t Statistic Mean CI Mean CI t Statistic
5.5 g/ms 0.970 0.033 1.066 0.023 −6.52 1.762 0.063 1.926 0.129 −4.77
3.2 g/ms 1.012 0.040 1.127 0.049 −6.61 1.811 0.062 1.973 0.197 −3.57
2.2 g/ms 1.052 0.042 1.183 0.043 −7.54 1.849 0.062 1.974 0.239 −2.36
1.1 g/ms 1.115 0.053 1.288 0.063 −7.54 1.931 0.071 2.179 0.332 −3.49
0.32 g/ms 1.296 0.081 1.646 0.179 −7.60 2.183 0.088 2.462 0.424 −3.09
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Values are vestibular short-latency evoked potential (VsEP) response latencies (in ms), averaged by group and compared. Responses were collected from control (n = 33) and noise-exposed rats (n = 18). Means are shown with 99.9% confidence intervals (CI). P1 and P2 latencies are significantly increased in response to all stimulus intensities. Bold text indicates a P value below 0.05 (2.2 g/ms) or 0.01 (1.1 and 0.32 g/ms); all other P values are below 0.001.

Figure 6 graphically illustrates a significant noise-induced attenuation of the VsEP waveform amplitudes (Fig. 6, A and B) and a significant increase in response latencies (Fig. 6, C and D). Values for control and noise-exposed rats were compared with a two-tailed unpaired t test assuming equal variances in MATLAB.

Fig. 6.

Fig. 6.

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Vestibular short-latency evoked potential (VsEP) amplitude (A and B) and latency (C and D) in non-noise-exposed controls (A and C) and 28 days after noise exposure (B and D). After noise exposure, averaged measured P1N1 (A) and P2N2 (B) waveform amplitudes are significantly reduced (n = 18; red squares) vs. control responses (n = 31; black squares), and average measured P1 (C) and P2 (D) waveform latencies are significantly increased (n = 18; red squares) vs. control responses (n = 31; black squares). *P < 0.05; ***P < 0.001. Means are plotted with 95% confidence intervals. Ampl, amplitude; Lat, latency; Stim Accel, stimulus acceleration.

Figure 6 and Table 2 show that exposure to noise reduced P1N1 amplitude by ~60% across all stimulus intensities. The attenuation was highly significant regardless of head-jerk intensity (P < 0.001). A similar, statistically significant (P < 0.001) reduction in P2N2 amplitude (~61%) occurred for head-jerk amplitudes of 2.2–5.5 g/ms (Fig. 6B and Table 2). However, for the weaker head-jerk stimuli (0.32 and 1.1 g/ms), P2N2 was even more attenuated (~71%; Fig. 6B and Table 2). Figure 6 and Table 3 show there was a significant increase in the latencies of the P1 (Fig. 6C) and P2 (Fig. 6D) responses after noise exposure. P1 latency increased ~10% for the strongest three head-jerk stimuli (Table 3). However, for the weaker head-jerks, the latency increase was much larger, 14% for the 1.1 g/ms head-jerk and 24% for the 0.32 g/ms head-jerk. Changes in P2 latency followed a similar pattern, ~7–9% increase for the strongest three head-jerks and ~12% increase for the weakest two head-jerks (Table 3). The change in P2 latencies in response to the largest two head-jerks were significant to P < 0.001, and the P2 latencies in response to the smallest three head-jerks were significant to P < 0.05. When the two outliers were included, reductions in P1N1 amplitudes for head-jerk amplitudes of 0.32 and 1.1 g/ms stimuli were not significant; however, all other measurements remained significant (P < 0.01) with or without the outliers.

The ~60% attenuation of the VsEP response measured 28 days postexposure could represent a partial recovery of a more severely attenuated VsEP immediately after noise exposure. To examine this possibility, we measured VsEP responses longitudinally in seven rats from 1 to 28 days after noise exposure (Fig. 7). The results, presented in Fig. 7, show that there was no statistically significant recovery of VsEP amplitude, and, with the exception of a modest recovery of P1 latency in response to 1.1 g/ms (P = 0.038), no recovery in latency when measured 1 day and 28 days after noise exposure.

Fig. 7.

Fig. 7.

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Vestibular short-latency evoked potential (VsEP) amplitude and latency 1 day after noise exposure does not recover 28 days after noise exposure. One day (gray squares) and 28 days after noise exposure (red squares), average measured P1N1 (A) and P2N2 (B) waveform amplitudes are significantly reduced, and P1 (C) and P2 (D) latencies are significantly increased from baseline responses (black squares) in a sample of 7 rats with longitudinal measurements (P < 0.001). There is no significant difference between measurements taken 1 or 28 days after noise exposure except for P1 latency in response to the 1.1 g/ms stimulus, which appeared to partially recover by 28 days after noise exposure (C; *P = 0.038). Means are plotted with 95% confidence intervals. Lat, latency; Stim Accel, stimulus acceleration.

Noise Exposed Rats Have Fewer Calretinin Immunoreactive Calyces

Calretinin was used to label and count calyx-only afferent terminals (Dechesne et al. 1991; Leonard and Kevetter 2002; Stewart et al. 2018). Within a 100 × 200-μm2 region of interest (Fig. 8A), calretinin-immunoreactive calyces were counted and compared in control and noise-exposed sacculi with a two-tailed unpaired t test in MATLAB. This analysis revealed a reduction in calretinin-immunoreactive calyx-only afferent terminals in noise-exposed rats (Fig. 8B; n = 22, mean = 44, SD = 11) vs. control rats (Fig. 8A; n = 16, mean = 62, SD = 16) that was statistically significant (Fig. 8C; P < 0.001, t = 4.093, df = 36).

Fig. 8.

Fig. 8.

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Two-dimensional z-stack-collapsed images showing immunoreactivity in controls (A, D, and G) and 28 days after noise exposure (B, E, and H). Calretinin-immunolabeled calyces (A and B) are significantly reduced 28 days after noise exposure compared with controls (C; ***P < 0.001). Hair cells, labeled with myosin 7a (D and E), are unaltered by noise exposure (F). Total calyces, labeled with neurofilament (G and H), are also unaltered by noise exposure (I). Box plots represent the entire range of the data, with a center (red) line representing median values for each group with upper and lower quartiles (blue upper and lower boxes). Scale bars, 50 µm.

Hair Cell Numbers and Total Calyx Counts are Unchanged

Myosin 7a was used to label and count hair cells within the 100 × 200-μm2 region of interest (Fig. 8D) (Hasson et al. 1997). Counts acquired from control and noise-exposed sacculi were compared with a two-tailed unpaired t test in MATLAB. This analysis revealed that hair cell counts in the region of interest in control sacculi (Fig. 8D; n = 16, mean = 207, SD = 38) and noise-exposed sacculi (Fig. 8E; n = 23, mean = 203, SD = 44) were not statistically different (Fig. 8F; P = 0.767, t = 0.299, df = 37).

Neurofilament H labels all fibers projecting to and from the vestibular ganglion (Demêmes et al. 1992; Hafidi and Romand 1989). Neurofilament H was used to label and count all afferents with calyceal terminations, identified morphologically within the 100 × 200-μm2 region of interest (Fig. 8, G and H) and as described previously (Stewart et al. 2018). Counts acquired from control and noise-exposed sacculi were compared with a two-tailed unpaired t test in MATLAB. This analysis revealed that the number of calyceal terminals in the region of interest in control sacculi (Fig. 8G; n = 16, mean = 144, SD = 34) and noise-exposed sacculi (Fig. 8H; n = 22, mean = 141, SD = 29) was not statistically different (Fig. 8I; P = 0.767, t = 0.299, df = 36).

Distribution of Vestibular Calyx-Only Terminal Labeling Within the Striola

In addition to a reduction in the number of calyx-only afferent terminals, there appeared to be a narrowing of the striolar region in noise-exposed sacculi, identified by the presence of calyx-only afferent terminals. However, when the 100 × 200-μm2 region of interest was divided into 10 subregions to determine the distribution of labeled calyx-only terminals, there was a reduction in staining, both in the center and at the edge of the region of interest. Comparison of control (n = 16) and noise-exposed (n = 22) subregions with an unpaired two-tailed t test revealed that while only 4 of the 14 subregions had significantly fewer calretinin-immunolabeled calyces than controls (Table 4 and Fig. 9), there was a trend toward fewer visibly calretinin-immunolabeled calyx-only afferent across the entire ROI after noise exposure. Therefore, although the striolar region of the sacculus appeared to be narrower in noise-exposed tissue, it was not when analyzed quantitatively. The largest reduction in calretinin labeling of calyceal terminals was observed centrally (P < 0.01); however, a significant reduction (P < 0.05) in calretinin-immunolabeled calyces was also observed at one edge of the ROI, supporting fewer stained calyces at that edge of the striolar region of the sacculus.

DISCUSSION

In this study, we built upon previous work, using the VsEP as a metric to evaluate the damaging effects of intense noise on the rat sacculus. The present study compared control VsEP responses with responses from rats 28 days after a single 6-h intense noise exposure (Fig. 1; 120 dB SPL, 0.5–4 kHz). Additionally, we investigated the relationship between deficient VsEP responses and structural changes in a uniformly defined region of the sacculus. We hypothesized that intense noise exposure would inflict damage on the vestibular periphery that would cause persistent attenuation of VsEP responses.

The present study builds on the previous literature by establishing a continuous noise exposure paradigm that alters calyx terminals in the vestibular periphery and reduces or abolishes VsEP responses to head-jerks. We previously reported a complete loss of VsEP responses that persisted up to 3 wk after noise exposure (Stewart et al. 2018). Using the same noise exposure paradigm, we have extended the end point of the study to 4 wk after noise exposure and used larger head-jerk stimuli to determine if there were any residual function that could be detected with the larger stimuli. While intense noise exposure abolished responses to small head-jerks as previously reported, larger stimuli did evoke detectable responses that were severely attenuated compared with control data (Figs. 5 and 6). Reductions in VsEP response amplitudes measured 1 day after noise exposure persisted without recovery up to 28 days after noise exposure (Fig. 6, A and B; Fig. 7, A and B). Additionally, there was a significant increase in the latency of the P1 waveform in response to all stimulus intensities 1 day and 4 wk after noise exposure (Fig. 6, C and D; Fig. 7, C and D; P < 0.001). However, an increase in latency of the P2 waveform was only significant at P < 0.001 in response to the largest two stimulus intensities. At weaker intensities, the significance level was less (P < 0.05; Fig. 6D and Table 3).

The use of vestibular short-latency evoked potentials (VsEPs) to assess vestibular function is well established in a variety of mammalian species including rats (Jones et al. 2011; Jones and Jones 1999; Plotnik et al. 1997, 1999; for review see Jones and Jones 2007). Recent studies have associated abnormal VsEP responses with otopathological evidence of cellular injury in models using genetic mutation (Ono et al. 2019; for review see Jones and Jones 2014), ototoxic drug treatment (Bremer et al. 2014), and impulse noise (Perez et al. 2002). The observed changes in the post-noise VsEP waveforms in the present study could be related to the characteristics of the nerve fibers contributing to the post-noise VsEP. For example, if firing of the most rapidly conducting axons (largest fibers) were disrupted or if synchrony of nerve discharges were reduced by noise, then one would expect reduced VsEP amplitudes and longer latencies. This idea is consistent with the observed reduction of calretinin-stained calyx-only afferents, since these afferents have the largest fiber diameters, fastest conduction times, and greatest sensitivity to head-jerks. The reduction in calretinin staining and reduced VsEP responses together indicate specific noise-induced damage to these afferents that reduces the number of functionally active terminals and/or limits their ability to respond synchronously to head-jerks.

We do not exclude the possibility that at least some dimorphic afferents that produce synchronous responses to head-jerks may also contribute to the VsEP. Our structural analyses were limited to the striolar zone of the sacculus; however, dimorphic afferents synapse throughout the epithelium. The VsEP measured at 1 and 28 days after noise exposure could, therefore, include responses of surviving calyx-only and dimorphic afferents within and outside the striolar zone. Additional studies will be needed to determine the sensitivity of extrastriolar afferents to noise exposure.

After exposure to 120-dB noise, there was no statistically significant recovery of VsEP P1N1 or P2N2 amplitudes for up to 4 wk after exposure (Fig. 7), suggesting there was no central compensatory response of monosynaptically contacted secondary neurons. Lack of recovery of stimulus-dependent activity of monosynaptically contacted vestibular neurons after bilateral labyrinthectomy has been observed previously (Ris and Godaux 1998). This lack of compensation is also supported by the clinical literature on head impulse testing in patients with bilateral vestibular loss. In these subjects, smooth pursuit is still possible, but responses to rapid and unexpected head rotations, thought to be purely vestibular, are permanently altered (for review see Halmagyi et al. 2017). Other work has shown partial recovery of VsEP responses after exposure to intense impulse noise (ten 160-dB gunshots) at 1 and 6 wk after exposure. This study showed a progressive P1 amplitude and latency recovery across the immediate, 1-wk, and 6-wk recovery times. While it was not explicitly reported, there apparently was recovery of the P2 waveform at 6 wk after noise exposure, which appears relatively complete (Perez et al. 2002). Although these findings appear in conflict with the present data, they suggest a difference between the effects of impulse vs. continuous noise exposure on the vestibular sensory end organs.

The current and previous (Stewart et al. 2018) study found a significant loss of calyx-only afferent terminals after noise exposure. However, this study failed to identify any significant change in the number of total calyces despite a reduction in the number of visibly stained calretinin-positive calyces, a finding in conflict with our earlier and more limited study (Stewart et al. 2018). We believe the current findings suggest that while the expression of calretinin in the striolar ROI is downregulated, the number of afferents terminating as calyces is not reduced. We cannot rule out that noise induces a retraction of calyces, similar to what has been reported after ototoxic insult (Sultemeier and Hoffman 2017). The present study used lower magnification and a larger region of interest than our previous study (Stewart et al. 2018). These changes could account for differences in the previous and current data. The hypothesized downregulation of calretinin following noise, suggested by weaker immunolabeling, could account for detection of fewer calyx-only afferent terminals in the ROI. The weaker calretinin immunolabeling, coupled with a reduction in VsEP responses, suggests there is a loss of normal signaling in noise-exposed calyx-only terminals in the vestibular periphery.

Although hair cell loss was not previously investigated as a potential contributing factor to the reported VsEP deficits (Stewart et al. 2018), the present study has established that there was no difference in the number of hair cells within the ROIs between noise-exposed and non-noise-exposed rats. However, it is possible that some of the counted hair cells in noise-exposed tissue are dysfunctional. For example, we could not exclude the possibility that hair bundles were damaged and normal mechanotransduction was compromised. An in vitro study of hair bundle recovery after gentamicin exposure demonstrated an ~2-wk time course for functional recovery of hair bundles (Taura et al. 2006). The lack of recovery of VsEP responses in this study suggests that hair bundle damage is an unlikely source of the persistent VsEP attenuation that we observed. Nonetheless, hair bundle disruption may have contributed to short-term VsEP attenuation and should be investigated in future work.

Our observation of preserved numbers of total calyces, fewer detectable immunostained calyx-only terminals, and persistent VsEP attenuation implies that reduced calretinin expression in the calyx-only afferent terminals is correlated with synaptic dysfunction of those terminals. It is intriguing that the calyx-only afferent population is the most sound sensitive and the most susceptible to noise-induced injury. Whatever the cellular mechanisms, the concurrent changes in VsEP responses and calyx staining suggest decreased excitability, decreased synaptic efficiency, and/or modified synaptic transmission in the affected terminals and their afferent fibers.

The severe post-noise attenuation of VsEP responses suggests that calyx-only afferents make a major contribution to the generation of the VsEP response even though these afferents represent a small fraction of total afferents in the eighth nerve (~10–20% depending on species; Desai et al. 2005). Ono et al. (2019) identified a critical role for the development of central regions of the vestibular sensory epithelia and complex striolar calyces for the generation of VsEP responses. Desai et al. (2005) identified 11–14% of calretinin-immunoreactive calyceal afferents in the mouse sacculus, 10–13% in the guinea pig sacculus, and ~19.5% in the gerbil sacculus. While Desai et al. (2005) did not quantify calretinin-immunoreactive calyceal afferents in the rat, it is reasonable to assume a similar percentage as in the other rodents.

An important limitation of our study is that the VsEP is not a sensitive metric with which to assess the activity of regular afferents, because these afferents are relatively insensitive to head-jerk stimuli (Jones et al. 2011) and are small in diameter, slowly conducting, and unlikely to respond synchronously to the head-jerks used to elicit the VsEP (Jones et al. 2011). Regular afferents are relatively insensitive to acoustic stimulation (Curthoys 2017; Curthoys et al. 2016; Curthoys and Vulovic 2011), so they also may be less vulnerable to direct injury caused by noise exposure. They may, however, be affected by other damaging mechanisms such as oxidative stress secondary to noise-induced ischemia (Fetoni et al. 2009).

Previous studies reported noise-induced damage in the semicircular canal cristae (Fetoni et al. 2013) despite the relative insensitivity of semicircular canal afferents to acoustic stimuli. Noise exposure also caused reductions in vestibuloocular reflex (VOR) gain in guinea pigs (Fetoni et al. 2009). This finding could implicate damage to regular afferents, as VOR gain is believed to be primarily dependent on signals conveyed by regular afferents (Minor and Goldberg 1991). In the cristae, bouton endings associated with regular and dimorphic afferents that are not directly sensitive to acoustic stimulation could be affected by oxidative stress related to noise-induced ischemia (e.g., Fetoni et al. 2013, 2016; Lamm and Arnold 1996; Yamashita et al. 2004; for review see Kurabi et al. 2017). Noise-induced oxidative stress has also been identified in the mouse vestibule (Tamura et al. 2012). Although our analysis was focused on irregular afferents in the rat sacculus, regular afferent bouton endings in the sacculus and other vestibular end organs should be targets for future studies.

GRANTS

This work was supported by National Institutes of Health (NIH) Grants DC000011 (to C. E. Stewart), DC017063 (to C. E Stewart), and DC015097 (to W. M. King) and U.S. Department of Veterans Affairs Grant 1I01RX001986 (to R. A. Altschuler).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

C.E.S., D.S.B., R.A.A., and W.M.K. conceived and designed research; C.E.S., D.S.B., and A.C..K. performed experiments; C.E.S., D.S.B., A.C..K., and W.M.K. analyzed data; C.E.S., D.S.B., A.C..K., R.A.A., and W.M.K. interpreted results of experiments; C.E.S. and W.M.K. prepared figures; C.E.S. and W.M.K. drafted manuscript; C.E.S., D.S.B., A.C..K., R.A.A., and W.M.K. edited and revised manuscript; C.E.S., D.S.B., A.C..K., R.A.A., and W.M.K. approved final version of manuscript.

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