The three otolithic endorgans of the inner ear are known to be involved in sound detection in different teleost fishes, yet their relative roles for auditory–vestibular functions within the same species remain uncertain. In zebrafish (Danio rerio), the saccule and utricle are thought to play key functions in encoding auditory and vestibular information, respectively, but the biological function of the lagena is not clear. We hypothesized that the zebrafish saccule serves as a primary auditory endorgan, making it more vulnerable to noise exposure, and that the lagena might have an auditory function given its connectivity to the saccule and the dominant vestibular function of the utricle. We compared the impact of acoustic trauma (continuous white noise at 168 dB for 24 h) between the sensory epithelia of the three otolithic endorgans. Noise treatment caused hair cell loss in both the saccule and lagena but not in the utricle. This effect was identified immediately after acoustic treatment and did not increase 24 h post-trauma. Furthermore, hair cell loss was accompanied by a reduction in presynaptic activity measured based on ribeye b presence, but mainly in the saccule, supporting its main contribution for noise-induced hearing loss. Our findings support the hypothesis that the saccule plays a major role in sound detection and that the lagena is also acoustically affected, extending the species hearing dynamic range.

The inner ear of teleost fishes consists of three orthogonal semi-circular canals and three otolith endorgans. The otolithic endorgans – the utricle, saccule and lagena – consist of a sensory epithelium populated with hair cells (HCs) that are overlaid by a gelatinous membrane and are mechanically coupled to a dense calcareous otolith (Lu and Popper, 1998). Movement of the sensory HCs causes the opening of transduction channels to create a receptor potential, thus exciting the afferent fibers along the axis of stimulation. As a result of the difference in inertia between the sensory macula and the associated otolith, these endorgans function as biological accelerometers, thus encoding linear acceleration, which can include orientation with respect to gravity, but also particle motion within the auditory range (Popper and Fay, 1999; Popper and Hawkins, 2018, 2021).

The sensory role of the inner ear endorgans can be vestibular and/or auditory, depending on the response frequencies and how the brain uses the information encoded in the VIIIth nerve afferents that project into the first-order octaval nuclei in the brainstem (McCormick, 1999). The saccule has been described as the main auditory endorgan in teleost fishes (Brown et al., 2019; Coffin et al., 2012; Ladich and Schulz-Mirbach, 2016; Lu and DeSmidt, 2013; Smith et al., 2011), although the auditory sensitivity of the utricle (Denton and Gray, 1979; Lu et al., 2004; Rogers and Sisneros, 2020) and the lagena (Lu et al., 2003; Meyer et al., 2012; Sand, 1974; Vetter et al., 2019) has also been reported in a few species.

In later diverging vertebrates, such as birds and mammals, the inner ear endorgans typically show a well-defined segregation of their biological function (see Manley, 2000, for review). In fish, the extent to which the different endorgans serve an auditory versus vestibular role remains to be clarified. According to the ‘mixed function’ hypothesis, each otolithic endorgan serves both senses to varying extents (Platt and Popper, 1981; Popper and Fay, 1993; reviewed in Schulz–Mirbach et al., 2019), but this is only based on a limited number of studies and might be species specific. To date, the auditory contribution of single otolith endorgans has only been tested in a few fish species and the studies available mostly support the ‘mixed-function’ hypothesis [e.g. goby Dormitator latifrons (Eleotridae) (Lu et al., 2010); midshipman/toadfish (Batrachoididae) (Fay and Edds-Walton, 1997; Sisneros, 2007; Vasconcelos et al., 2015); goldfish Carassius auratus (Cyprinidae) (Fay, 1984); and catfish Ictalurus punctatus (Ictaluridae) (Moeng and Popper, 1984)]. A well-investigated species is the midshipman fish Porichthys notatus, in which all three endorgans are sensitive to the frequency range of conspecific calls but with distinct auditory thresholds, thus probably complementing their function for acoustic communication and directional hearing (Sisneros, 2007, 2009). Considering the vast morphological diversity observed in fish auditory systems, potentially indicating adaptations for hearing and vestibular functions, it is essential to evaluate the functional contribution of each otolithic endorgan at the species level.

The zebrafish Danio rerio is an otophysian teleost with Weberian ossicles connecting the inner ear to the swim bladder, expanding the auditory sensitivity range. This species has become a well established model organism in hearing research to investigate inner ear development and HC regeneration, and to test ototoxic agents and drug treatments for auditory impairments (Bever and Fekete, 2002; Chiu et al., 2008; Ou et al., 2010; Varshney et al., 2016; Wang et al., 2015; Whitfield et al., 2002). As in other teleost fish, the saccule and utricle of zebrafish are thought to play key functions in encoding auditory and vestibular information, respectively (Schulz–Mirbach et al., 2019; Whitfield et al., 2002). Removal of the utricle, but not the saccule, leads to impaired postural equilibrium (Bianco et al., 2012; Riley and Moorman, 2000). Moreover, larval zebrafish (first week post-fertilization) without the utricle showed only slightly reduced auditory sensitivity at low frequencies <100 Hz, while manipulation of the saccule caused >30 dB hearing loss within 100–400 Hz (Yao et al., 2016). In zebrafish, the lagena is the last endorgan to develop, i.e. around 15 days post-fertilization, and it is attached to the saccule on the posterior part at the adult stage (Bever and Fekete, 2002). In otophysian fishes, it is common for the lagena to be similar or even larger in size compared with the saccule, but its biological function remains unclear (Popper et al., 2003; Schulz-Mirbach and Ladich, 2016).

We hypothesize that the saccule is the main auditory endorgan and that the lagena serves a complementary auditory function given its connectivity to the saccule in zebrafish. The utricle, instead, might serve a predominantly vestibular function in this species. The major goal of this study was to compare the impact of noise exposure on the HCs and presynaptic function between the three otolithic endorgans in the adult zebrafish. By inducing structural–functional damage through noise trauma, we gained insights into the potential sensitivity of these endorgans to acoustic stimuli, similar to the study conducted by Smith et al. (2011). Furthermore, this allowed us to assess their relative contribution to noise-induced hearing loss.

Animals

Wild-type zebrafish (AB line) were originally obtained from China Zebrafish Resource Center (CZRC, Wuhan, China) and reared at the zebrafish facility of the University of Saint Joseph, Macao S.A.R. Fish were initially maintained in 10 l tanks in a standalone housing system (model AAB-074-AA-A, Yakos65, New Taipei City, Taiwan) with filtered, aerated water (pH balanced 7–8; 400–550 μS conductivity) at 28±1°C and under a 12 h:12 h light:dark cycle. Specimens were fed twice a day with both dry food powder (Zeigler, PA, USA) and live artemia. A total of 44 adult zebrafish, 7–8 months old, 2.7–3.2 cm total length and 180–240 mg body mass, were used in this study.

All experimental procedures complied with the ethical guidelines regarding animal research and welfare enforced at the Institute of Science and Environment, University of Saint Joseph, and approved by the Division of Animal Control and Inspection of the Civic and Municipal Affairs Bureau of Macao (IACM), license AL017/DICV/SIS/2016.

Acoustic treatments

Prior to noise exposure, all fish were transferred to 4 l tanks for 7 days in a walk-in soundproof chamber (120a-3, IAC Acoustics, North Aurora, IL, USA) for isolation to reduce potential effects from captive noise (Lara and Vasconcelos, 2019; Vasconcelos et al., 2023). In these tanks, sound pressure level (SPL) was around 103–108 dB re. 1 μPa (LZS, RMS sound level with Z-linear frequency weighting between 6.3 and 20 kHz and measured at S-slow time rate <2 s) and conditions (light, temperature water quality and feeding schedule) were maintained similar to stock.

After the isolation period, specimens were allocated randomly into three experimental groups defined as ‘control’, ‘noise’ or ‘noise+24 h’ (N=16, 16 and 12 fish total for control, noise and noise+24 h treatments, respectively). The acoustic treatments were performed using glass tanks (50 cm length×30 cm width×25 cm height; 38 l) that were placed on top of a sponge base and two Styrofoam layers. The sound treatment was generated via an underwater speaker (UW30, Electro-Voice, Burnsville, MN, USA) positioned in the center of the tank bottom on top of a sponge base that prevented direct contact with the glass. The speaker was connected to an amplifier (ST-50, Ai Shang Ke, Hangzhou, China), which was connected to a laptop running Adobe Audition 3.0 for windows (Adobe Systems Inc., San Jose, CA, USA). In each trial, two specimens were treated at the same time by placing them inside a net box (dimensions: 15×15×15 cm, mesh size 1 mm) that was suspended underwater at 1 cm distance above the speaker (Fig. 1).

Fig. 1.

Acoustic treatment tank used to expose zebrafish inside a net box to sound playback. Sound playback consisted of white noise at 168 dB re. 1 μPa for 24 h.

Fig. 1.

Acoustic treatment tank used to expose zebrafish inside a net box to sound playback. Sound playback consisted of white noise at 168 dB re. 1 μPa for 24 h.

The noise treatment consisted of white noise at 168 dB re. 1 μPa (bandwidth: 100–1500 Hz) adjusted to the tank acoustic properties using Adobe Audition software tools to deliver a relatively flat spectrum (Breitzler et al., 2020). Noise level was calibrated before each treatment so that the intended sound level (LZS) was reached at the center of the net box containing the fish. A SPL variation of ±8 dB was registered inside the net box. In the control treatment, the amplifier connected to the speaker was switched on but without playback and specimens were left in the tank for 24 h. In the noise treatment, specimens were exposed to noise playback for 24 h. In the noise+24 h treatment, fish were firstly exposed to noise playback and then remained in the tank for additional 24 h under lab silent conditions. Sound recordings and SPL measurements were made with a hydrophone (Bruel & Kjær 8104, Naerum, Denmark; frequency range: 0.1 Hz to 120 kHz, sensitivity of – 205 dB re. 1 V μPa−1) connected to a hand-held sound level meter (Bruel & Kjær 2270).

As the fish inner ears detect sound through particle motion and the ability to detect sound pressure is only developed in specific teleost groups, such as otophysans (Popper and Hawkins, 2018), the acoustic treatment was further calibrated with a tri-axial accelerometer to quantify particle acceleration (M20-040, GeoSpectrum Technologies, Dartmouth, NS, Canada; frequency range 13 kHz). The sensor's acoustic center was placed at the same position as the hydrophone, corresponding to the midpoint of the netbox. The sound playback generated was about 140 dB re. 1 m s2, and most energy was in the vertical axis perpendicular to the speaker. Calculations were based on previously described methods using the MATLAB script paPAM (Nedelec et al., 2015).

At the end of the treatments, specimens were immediately euthanized with a MS-222 overdose (300 mg l−1) buffered with sodium bicarbonate. Fish heads were subsequently removed and fixed overnight in 10% neutral buffered formalin solution (Sigma-Aldrich, St Louis, MO, USA), thoroughly rinsed with PBS for 5 min, at least 3 times, and finally stored in PBS at 4°C.

Inner ear morphological analysis

We followed previously described procedures to extract zebrafish inner ear sensory epithelia from the three endorgans (Liang and Burgess, 2009). Sensory epithelia were dissected from both ears in each specimen under a stereomicroscope (Stemi 2000CS, Zeiss, Jena, Germany) and then immediately stained to quantify HC bundles and ribeye b puncta based on previously described protocols (Chaves et al., 2017; Wang et al., 2015; Wong et al., 2022). Ribeye b protein is present in presynaptic ribbons that enhance neurotransmitter release and it is a measure of synaptic function (Nicolson, 2015).

Epithelia were firstly permeabilized with 1% Triton X-100 (Sigma- Aldrich) at room temperature for 2 h, then blocked with 10% goat serum in PBS and incubated with primary antibody against ribeye b protein (1:5000 mouse anti-zebra sh Ribeye b monoclonal antibody provided by T. Nicolson, Stanford University, CA, USA) at 4°C overnight. The following day, samples were rinsed with 1% PBST for 2 h, incubated with Alexa Fluor 647 IgG2a secondary antibody (1:500, Invitrogen, Thermo Fisher Scientific Co., Waltham, MA, USA) and Alexa Fluor 488 phalloidin (1:500, Invitrogen) for 2 h, rinsed for 30 min in PBS and subsequently stained for 1 min in DAPI solution. Finally, the samples were whole mounted with Fluoromount-G (Southern Biotech, Birmingham, AL, USA) on glass slide, imaged with a confocal system (Stellaris 5 LIA, Leica Microsystems, Buffalo Grove, IL, USA), and analyzed with Leica Application Suite software X (LAS X, Leica Microsystems) and ImageJ (version 1.53e, National Institutes of Health, Bethesda, MD, USA).

HC bundles were quantified in different squared regions of 900 mm2 across the sensory epithelia of the three endorgans (Fig. 2). Ribeye b puncta were also quantified in the same regions with 1–2 additional regions on the edge of the saccule and lagena, where a higher amount of puncta was typically found. Additionally, HC nuclei were quantified from 57 saccular epithelial regions from 14 fish exposed to different treatments (N=6, 4 and 4 fish for control, noise and noise+24 h treatment, respectively) to verify its correlation with the number of HC bundles. The epithelial regions were selected based on similar histological studies on fish inner ears from Wang et al. (2015) and Coffin et al. (2012). For the saccule, they were located at 5%, 25%, 37.5%, 50% and 75% across the length of the rostral–caudal axis. In the lagena, the regions were roughly at 25%, 50%, 75% and 95% across the rostral–caudal axis. For the utricle, four regions were selected based on a rectangular projection (see Fig. 2). All HC bundles, nuclei and ribeye b puncta within or overlapping the square outlines were included in the counts. When possible, we examined both inner ears of every fish and used the averaged data from each endorgan, resulting in one value per specimen.

Fig. 2.

Diagram of representative inner ear endorgans (saccule, lagena and utricle) from a zebrafish adult showing the squared regions defined for hair cell (HC) bundle and ribeye b puncta quantification. Each indicated region was 900 µm2. D, dorsal; A, anterior.

Fig. 2.

Diagram of representative inner ear endorgans (saccule, lagena and utricle) from a zebrafish adult showing the squared regions defined for hair cell (HC) bundle and ribeye b puncta quantification. Each indicated region was 900 µm2. D, dorsal; A, anterior.

Statistical analysis

The differences in HC bundles and ribeye b puncta between experimental groups were tested with two-way ANOVA with acoustic treatment and epithelial regions or endorgans as independent factors, followed by Fisher's LSD post hoc multiple comparisons test to detect specific differences between treatments. The relationship between the HC bundles and nuclei was determined with Pearson correlation and fitted with a regression line.

All assumptions for parametric analyses were confirmed through the inspection of normal probability plots and by performing Levene's test for homogeneity of variances. All statistical tests and graphics were performed using Matlab (MathWorks, Inc., Natick, MA, USA) and Prism 9 (GraphPad, San Diego, CA, USA).

Noise exposure for 24 h caused significant HC loss in the saccule (F2,161=68.790, P<0.0001, N=14, 15 and 9 for control, noise and noise+24 h treatment, respectively; Fig. 3) for all epithelial regions analyzed (S1: t161=6.905, P<0.0001; S2: t161=4.274, P< 0.0001; S3: t161=4.733, P<0.0001; S4: t161=2.138, P=0.034; S5: t161=5.412, P<0.0001; Fig. 4), with an overall reduction of 31–57% (mean range) in HC bundles (HC loss in rostral region: 47–57%, central–caudal: 31–47%). Such noise-induced HC loss did not vary with the epithelial location (F8,161=1.749, P=0.091), meaning that the acoustic treatment similarly affected the whole saccule.

Fig. 3.

Representative images of the entire sensory epithelia of the three inner ear endorgans of adult zebrafish. Images were reconstructed with the 3D function of LAS X software (Leica Microsystems) and show phalloidin-stained HCs from the control and noise-treated group. Noise-induced HC bundle loss is clearly visible in the saccule and to some extent in the lagena (arrowheads). Utricular epithelia did not reveal noise-induced morphological changes. Scale bars: 100 μm. A, anterior, D, dorsal.

Fig. 3.

Representative images of the entire sensory epithelia of the three inner ear endorgans of adult zebrafish. Images were reconstructed with the 3D function of LAS X software (Leica Microsystems) and show phalloidin-stained HCs from the control and noise-treated group. Noise-induced HC bundle loss is clearly visible in the saccule and to some extent in the lagena (arrowheads). Utricular epithelia did not reveal noise-induced morphological changes. Scale bars: 100 μm. A, anterior, D, dorsal.

Fig. 4.

Noise-induced changes in HC bundles and ribeye b in the zebrafish saccule. (A) Variation in the number of HC bundles and ribeye b puncta in the zebrafish saccular epithelia under different experimental conditions: control, noise treatment (24 h) and noise treatment followed by a 24 h silent period. A diagram of the saccule is shown at the top to indicate the epithelial regions (S1–6) analyzed. Values are means±s.e.m.; N=14, 15 and 9 for control, noise and noise+24 h, respectively; each data point represents a mean value based on two saccules per fish. Two-way ANOVA, HC: F2,161=68.790, P<0.0001; ribeye b: F2,195=43.870, P<0.0001. Post hoc tests *P<0.05, **P<0.01, ***P<0.001. (B) Correlation between the number of saccular HC bundles and the number of nuclei (r57=0.923, P<0.0001) fitted with a regression line (y=0.828x+3.364, R2=0.852). (C) Representative images of saccular epithelia (from the S2 region) showing phalloidin-stained HCs (green), presynaptic ribeye b puncta (red) and DAPI-stained HC nuclei (blue) for the three experimental groups.

Fig. 4.

Noise-induced changes in HC bundles and ribeye b in the zebrafish saccule. (A) Variation in the number of HC bundles and ribeye b puncta in the zebrafish saccular epithelia under different experimental conditions: control, noise treatment (24 h) and noise treatment followed by a 24 h silent period. A diagram of the saccule is shown at the top to indicate the epithelial regions (S1–6) analyzed. Values are means±s.e.m.; N=14, 15 and 9 for control, noise and noise+24 h, respectively; each data point represents a mean value based on two saccules per fish. Two-way ANOVA, HC: F2,161=68.790, P<0.0001; ribeye b: F2,195=43.870, P<0.0001. Post hoc tests *P<0.05, **P<0.01, ***P<0.001. (B) Correlation between the number of saccular HC bundles and the number of nuclei (r57=0.923, P<0.0001) fitted with a regression line (y=0.828x+3.364, R2=0.852). (C) Representative images of saccular epithelia (from the S2 region) showing phalloidin-stained HCs (green), presynaptic ribeye b puncta (red) and DAPI-stained HC nuclei (blue) for the three experimental groups.

Noise-induced morphological changes were accompanied by reduced pre-synaptic activity quantified based on ribeye b puncta (F2,195=43.870, P<0.0001, N=14, 15 and 9 for control, noise and noise+24 h treatment, respectively) for most epithelial regions analyzed (S1: t195=2.369, P=0.019; S2: t195=5.572, P<0.0001; S3: t195=3.830, P=0.0002; S5: t195=4.725, P<0.0001; S6: t195=2.598, P=0.010; Fig. 4) with an overall 22–69% decrease in the protein marker. This change varied with the saccular epithelial region (F10,195=1.927, P=0.044). In this case, a more significant decrease (55–69%) was registered in the anterior saccular epithelium (S2 and S3), and no significant difference was found in the S4 region.

In general, an additional 24 h period post-trauma caused no changes in either saccular HC (t161=0.115, P=0.910) or ribeye b (t195=0.012, P=0.990). However, the S5 region located in the middle of the posterior saccular epithelia revealed a slight increase in ribeye b (t195=2.604, P=0.010).

Moreover, the number of HC bundles was significantly correlated with HC nuclei in the saccule (r57=0.923, P<0.0001; Fig. 4B,C). Noise-induced HC bundle loss was associated with a lower amount of HC nuclei, suggesting that acoustic trauma caused cell death rather than only stereocilia damage and/or bundle loss.

Noise treatment also induced significant HC loss in the lagena (F2,114=9.564, P=0.0001, N=15, 15 and 10 for control, noise and noise+24 h treatment, respectively; Figs 3, 5), specifically in regions L1, L2 and L3, which were located in the anterior, middle and posterior epithelia, respectively (L1: t114=2.275, P=0.025; L2: t114=2.947, P=0.004; L3: t106=2.372, P=0.019; Fig. 5). Noise exposure caused about 21–23% of HC bundle loss in these regions. Such cellular damage was accompanied by significant changes in ribeye b (F2,169=3.235, P=0.042, N=15, 15 and 8 for control, noise and noise+24 h treatment, respectively), but significant differences were only found in L3 (t146=2.542, P=0.012, as shown in Fig. 5).

Fig. 5.

Noise-induced changes in HC bundles and ribeye b in the zebrafish lagena. (A) Variation in the number of HC bundles and ribeye b puncta in the zebrafish lagena under different experimental conditions: control, noise treatment (24 h) and noise treatment followed by an additional 24 h silent period. A diagram of the lagena is shown at the top to indicate the epithelial regions analyzed (L1–5). Values are means±s.e.m.; N=14, 15 and 10 for control, noise and noise+24 h, respectively; each data point represents a mean value based on two lagenas per fish. Two-way ANOVA, HC: F2,114=9.564, P=0.0001; ribeye b: F2,169=3.235, P=0.042. Post hoc tests *P<0.05, **P<0.01. (B) Representative images of lagena epithelia (from the L3 region) showing phalloidin-stained HCs (green), presynaptic ribeye b puncta (red) and DAPI-stained HC nuclei (blue) for the three experimental groups.

Fig. 5.

Noise-induced changes in HC bundles and ribeye b in the zebrafish lagena. (A) Variation in the number of HC bundles and ribeye b puncta in the zebrafish lagena under different experimental conditions: control, noise treatment (24 h) and noise treatment followed by an additional 24 h silent period. A diagram of the lagena is shown at the top to indicate the epithelial regions analyzed (L1–5). Values are means±s.e.m.; N=14, 15 and 10 for control, noise and noise+24 h, respectively; each data point represents a mean value based on two lagenas per fish. Two-way ANOVA, HC: F2,114=9.564, P=0.0001; ribeye b: F2,169=3.235, P=0.042. Post hoc tests *P<0.05, **P<0.01. (B) Representative images of lagena epithelia (from the L3 region) showing phalloidin-stained HCs (green), presynaptic ribeye b puncta (red) and DAPI-stained HC nuclei (blue) for the three experimental groups.

Finally, the utricle was not significantly affected by the acoustic treatment, in terms of either HC loss (F2,79=2.270, P=0.110) or presynaptic marker (F2,79=2.966, P=0.057) (N=10, 8 and 10 for control, noise and noise+24 h treatment, respectively; Figs 3, 6).

Fig. 6.

Noise-induced changes in HC bundles and ribeye b in the zebrafish utricle. (A) Variation in the number of HC bundles and ribeye b puncta in the zebrafish utricle under different experimental conditions: control, noise treatment (24 h) and noise treatment followed by an additional 24 h silent period. A diagram of the utricle is shown at the top to indicate the specific epithelial regions (U1–4) analyzed. Values are means±s.e.m.; N=10, 8 and 10 for control, noise and noise+24 h, respectively; each data point represents a mean value based on two utricles per fish. Two-way ANOVA, HC: F2,79=2.270, P>0.05; ribeye b: F2,79=2.970, P>0.05. (B) Representative images from utricular epithelia (U2 region) showing phalloidin-stained HCs (green), presynaptic ribeye b puncta (red) and DAPI-stained HC nuclei (blue) for the three experimental groups.

Fig. 6.

Noise-induced changes in HC bundles and ribeye b in the zebrafish utricle. (A) Variation in the number of HC bundles and ribeye b puncta in the zebrafish utricle under different experimental conditions: control, noise treatment (24 h) and noise treatment followed by an additional 24 h silent period. A diagram of the utricle is shown at the top to indicate the specific epithelial regions (U1–4) analyzed. Values are means±s.e.m.; N=10, 8 and 10 for control, noise and noise+24 h, respectively; each data point represents a mean value based on two utricles per fish. Two-way ANOVA, HC: F2,79=2.270, P>0.05; ribeye b: F2,79=2.970, P>0.05. (B) Representative images from utricular epithelia (U2 region) showing phalloidin-stained HCs (green), presynaptic ribeye b puncta (red) and DAPI-stained HC nuclei (blue) for the three experimental groups.

Overall comparison of noise-induced HC loss and ribeye b confirmed the differences between the endorgans (HC: F1,46=28.10, P<0.0001; ribeye b: F1,39=5.409, P=0.025; N=14 and 15 for control and noise, respectively; Fig. 7).

Fig. 7.

Impact of noise exposure on the otolithic endorgans of the zebrafish inner ear. Comparison of the mean HC bundles (A) and ribeye b puncta (B) based on the selected regions (900 µm2 each) for the saccule (5–6 epithelial regions), lagena (5 epithelial regions) and utricle (4 epithelial regions) of zebrafish exposed to control and noise treatments. Values are means±s.e.m. HC: F1,46=28.10, P<0.0001; ribeye b: F1,39=5.409, P=0.025. Post hoc tests *P<0.05, ***P<0.001.

Fig. 7.

Impact of noise exposure on the otolithic endorgans of the zebrafish inner ear. Comparison of the mean HC bundles (A) and ribeye b puncta (B) based on the selected regions (900 µm2 each) for the saccule (5–6 epithelial regions), lagena (5 epithelial regions) and utricle (4 epithelial regions) of zebrafish exposed to control and noise treatments. Values are means±s.e.m. HC: F1,46=28.10, P<0.0001; ribeye b: F1,39=5.409, P=0.025. Post hoc tests *P<0.05, ***P<0.001.

The present work compared the effects of acoustic trauma between the sensory epithelia of the three otolithic endorgans of the zebrafish inner ear. Our results showed the highest effects at the morphological and synaptic level in the saccule. The lagena was also acoustically affected but with less damage, contrasting with the utricle, which did not reveal noise-induced epithelial changes at the noise level investigated (168 dB re. 1 μPa for 24 h). Our study supports the hypothesis that zebrafish inner ear endorgans have distinct auditory sensitivities, probably to extend species' hearing dynamic range and to complement their differential roles in auditory–vestibular function.

Noise-induced HC loss

The impact of noise exposure on the fish inner ear has been addressed in a few studies. Most researchers have focused on the saccule, which is considered to play a major role in hearing in this taxon (for reviews, see Ladich and Schulz-Mirbach, 2016; Schulz-Mirbach et al., 2019), and reported evidence of a tonotopic organization of the saccular epithelia, with the rostral region being more sensitive to high-frequency noise and the caudal region affected by low frequencies (Breitzler et al., 2020; Enger, 1981; Han et al., 2022; Schuck and Smith, 2009; Smith et al., 2006, 2011).

In the current work, continuous exposure to white noise at 168 dB for 24 h caused a significant reduction in HC bundles throughout the whole saccule. Our results did not reveal a significant variation in HC loss with epithelia region, but we found a higher impact in the rostral region of the saccule (47–57%) compared with the central and caudal regions (<47%). This pattern is similar to prior studies that investigated the same species after white noise exposure. In our previous study (Breitzler et al., 2020), we exposed zebrafish to white noise at 150 dB re. 1 μPa for 24 h and identified significant HC loss (up to 27%) mostly in the rostral region of the saccule. More recently, Han et al. (2022) treated zebrafish with white noise generated by a woofer air speaker at 140 dB re. 1 μPa for 6 h and reported up to a 22% decrease in saccular HCs also in the rostral region. Both studies found the highest auditory threshold shifts at 1000 Hz (Han et al., 2022) and 1000–2000 Hz (Breitzler et al., 2020), and such high frequencies are typically detected in the rostral saccular region. These frequencies fall within the best hearing range of the species (Breitzler et al., 2020; Han et al., 2022; Monroe et al., 2016), and it is likely that white noise exposure may induce greater damage in the epithelial regions that detect the most sensitive frequencies. The higher amplitude used in our study (168 dB re. 1 μPa) is probably the cause of the higher and extended HC loss detected compared with Han et al. (2022) and Breitzler et al. (2020).

In the present study, we also evaluated the impact of noise on the lagena and utricle. We found about 21–23% HC loss in the lagena in three different epithelial regions across the anterior-posterior axis, contrary to the utricle that did not reveal any cellular damage. This is one of the first reports comparing the impact of noise among the three inner ear endorgans in a fish species. Han et al. (2022) investigated the effect of 140 dB white noise for 6 h on zebrafish and identified over 40% HC loss in the caudal epithelia of the lagena and up to 25% HC loss in both saccule and utricle. The results obtained by these authors are difficult to compare with the present work as they used a very different treatment setup with a woofer air speaker and they did not provide information on particle motion. Additionally, the authors identified higher noise-induced damage in the lagena compared with the saccule, which is difficult to interpret. Our findings agree with the work by Smith et al. (2006) that exposed goldfish to white noise (170 dB re. 1 μPa rms) for 48 h and identified highest levels of apoptotic cells in the saccule immediately following noise exposure but also in the lagena 24 h post-treatment. According to this study, the utricle did not reveal any noise-induced apoptotic cells. Furthermore, Wang et al. (2019) reported an increase in apoptosis and cell proliferation in the zebrafish inner ear after a blast wave exposure that caused visceral hemorrhage. The saccule and lagena were the most affected endorgans, both having around 400 labeled apoptotic cells, contrasting with the utricle, which only revealed about 60 dead cells.

We analyzed noise-induced changes immediately after acoustic treatment and 24 h post-treatment. The results showed that this additional period caused no changes in either saccular HCs or presynaptic activity. In addition to the primary auditory HC death that occurs during noise exposure, secondary cellular death can be induced post-trauma due to reactive oxygen and nitrogen species (ROS and RNS) (Yamashita et al., 2004). Nevertheless, in this study, no further HC bundle loss was found in the saccule 1 day after noise exposure, similar to the result reported by Schuck and Smith (2009). This might be due to different time delays in ROS and RNS formation, or because the intense damage on the saccule masked the effect of such oxidative processes.

Noise-induced synaptopathy

The present work also identified a significant noise-induced reduction in presynaptic activity based on ribeye b quantification mostly in the saccule. The lagena also showed a slight reduction but only in one epithelial region. Ribeye b is known to be present in presynaptic ribbons that promote fast signal transmission between the auditory receptors and postsynaptic neurons, and thus is critical in sound encoding (Kindt and Sheets, 2018; Nicolson, 2015). A reduction in ribeye b protein has been associated with synaptopathy in HCs after acoustic trauma mainly in mammals (Fernandez et al., 2015; Liberman and Kujawa, 2017; Valero et al., 2017) but also in fish (Uribe et al., 2018; Wang et al., 2015; Wong et al., 2022), leading to auditory threshold shifts and noise-induced hearing loss (Kindt and Sheets, 2018; Wong et al., 2022).

In zebrafish, a previous study reported a decrease in ribeye b along with HC loss in the saccule associated with increasing size and aging (Wang et al., 2015). Other researchers have also identified a reduction in the amount of ribeye b ribbons in the neuromasts of noise-treated larval zebrafish (Holmgren and Sheets, 2021; Uribe et al., 2018). Recently, our lab (Wong et al., 2022) exposed zebrafish adults to white noise (150 dB re. 1 μPa) of varying temporal patterns for 24 h and found significant ribeye b reduction in the saccule mostly with continuous noise (up to 40–50%) compared with intermittent regimes. In the present study, we found up to 69% reduction in protein amount, probably as a result of the higher noise amplitude used.

Our findings support the hypothesis that damage to the saccule makes a major contribution to noise-induced hearing loss and that the sensory function of the lagena is also acoustically affected but to a lesser degree.

Differential role of the inner ear otolithic endorgans

According to our results, the saccule was the most sensitive endorgan to noise exposure based on the higher HC loss and synaptopathy observed. These findings agree with most literature on fish auditory systems, which report the saccule as the major auditory endorgan (Brown et al., 2019; Coffin et al., 2012; Ladich and Schulz-Mirbach, 2016), including in zebrafish (Brown et al., 2019; Coffin et al., 2012; Ladich and Schulz-Mirbach, 2016; Lu and DeSmidt, 2013; Smith et al., 2011). Fish species that have accessory hearing structures and improved auditory abilities, such as otophysans that include zebrafish, possess Weberian ossicles connecting the saccule to the swim bladder, which is typically correlated with their higher auditory sensitivities and/or expanded hearing range (reviewed in Braun and Grande, 2008). Otolith-removal experiments (Lu and Xu, 2002) and single-unit or field potential recordings from saccular HCs (Lu et al., 1998; Lu and Xu, 2002; Vasconcelos et al., 2015; Yao et al., 2016) also provided evidence that the saccule plays a major role in sound detection. As field potential recordings from the zebrafish endorgans would be very difficult to perform because of the difficulties in accessing the inner ear compared with other fish models (e.g. midshipman fish, toadfish and gobies; Brown et al., 2019; Lu et al., 2010; Vasconcelos et al., 2015), the present work provides key evidence supporting the main hearing function of the saccule for acoustic stimuli in the adult zebrafish.

We further demonstrated that the lagena was also acoustically affected but comparatively less so, in terms of HC loss and reduction in the presynaptic marker. Lower noise-induced effects in the lagena suggest higher auditory thresholds, which implies that this endorgan probably aids fish in sound detection, especially at high sound levels close to the sound source when the saccule becomes saturated. This extended hearing dynamic range would occur mostly in the vertical plane, as the HCs of both the lagena and saccule are typically oriented along this plane (Schulz-Mirbach et al., 2019). Currently, the functional role of the lagena is not clear and it probably varies among different fish species, given the high diversity in morphology and connection/proximity to the swimbladder (Schulz-Mirbach and Ladich, 2016). Evidence of an auditory function of the lagena was reported in only a few studies. For example, Vetter et al. (2019) showed that the lagena of the midshipman P. notatus was sensitive to low frequencies but with higher thresholds compared with the saccule. Other studies focusing on lagenar function (either microphonics or afferent recordings) also demonstrated its acoustic sensitivity and suggested an important role for this endorgan in directional hearing in the vertical plane (Fay, 1984; Lu et al., 2003; Sand, 1974).

Finally, the present work showed the absence of HC loss and synaptopathy in the utricle. This endorgan is known to be mainly involved in detecting gravitational forces for vestibular sensing (Riley and Moorman, 2000; Roberts et al., 2017). Nevertheless, a few studies support the idea that the utricle may serve an auditory role to some extent (Lu et al., 2004; Rogers and Sisneros, 2020), including in larval zebrafish (Yao et al., 2016).

To date, the auditory contribution of single otolith endorgans has been tested only in a few fish species (Fay, 1984; Fay and Edds-Walton, 1997; Lu et al., 2010; Moeng and Popper, 1984; Sisneros, 2007; Vasconcelos et al., 2015) and such studies mostly support the ‘mixed-function’ hypothesis. Our results partially agree with this hypothesis, providing evidence that the saccule is the major auditory endorgan and the lagena is also acoustically sensitive. As the utricle was not affected by the acoustic stimuli tested, our findings suggest a low sensitivity and/or a dominant vestibular function for this endorgan (Schulz-Mirbach et al., 2019; Whitfield et al., 2002).

Given the vast diversity in structure and function of fish auditory systems, it is important that future research considers distinct taxonomic groups, as well as the contribution of each otolithic endorgan for hearing within each species and their potential differences in sensitivity to acoustic trauma.

We thank Dr Teresa Nicolson (Stanford University, CA, USA) and Dr Jiping Wang (Affiliated Sixth People's Hospital of Shanghai Jiao Tong University, Otolaryngology Institute of Shanghai Jiao Tong University, Shanghai, China) for providing the ribeye b antibody. We are also grateful to the Institute of Science and Environment, University of Saint Joseph, Macao, for support with animal maintenance and imaging facilities.

Author contributions

Conceptualization: R.O.V.; Methodology: R.O.V.; Formal analysis: I.H.L.; Investigation: I.H.L., R.O.V.; Writing - original draft: I.H.L., R.O.V.; Writing - review & editing: R.O.V.; Project administration: R.O.V.; Funding acquisition: R.O.V.

Funding

This study was supported by the Science and Technology Development Fund (FDCT), Macao (ref. 046/2018/A2 and 0068/2020/A2).

Data availability

Data supporting the findings of this study are available from Dryad (Vasconcelos and Lau, 2023): https://doi.org/10.5061/dryad.gf1vhhmvp.

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Competing interests

The authors declare no competing or financial interests.