Experiments were conducted using the Tübingen strain of zebrafish of either sex provided by the McDermott zebrafish core facility. Transgenic zebrafish stably expressing GFP in the hair cell body (pvalb3b::GFP) were previously generated (McDermott et al., 2010), and cdh23^tj264a mutant (Söllner et al., 2004) was a kind gift from Dr. Teresa Nicolson (Oregon Health and Science University). Fish were maintained and bred at 28°C according to standard procedures (Nüsslein-Volhard and Dahm, 2002). This study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and animal welfare guidelines of the Committee of Case Western Reserve University (CWRU), USA. The protocol was approved by the Institutional Animal Care and Use Committee at CWRU (Protocol Number: 2012-0187).

Zebrafish larvae at days post fertilization (dpf) 5–6, were placed in varying concentrations of cisplatin (50–200 μM, ThermoFisher Scientific, Waltham, MA, USA) and/or mdivi-1 (1–10 μM, Enzo Life Sciences, Farmingdale, NY, USA) overnight for 16 h. The next day, the larvae were transferred to another dish, anesthetized with MS-222 (Sigma-Aldrich, St. Louis, MO, USA), and secured in a recording chamber using strands of dental floss tie downs (Ricci and Fettiplace, 1997) and placed under the microscope, an upright Olympus BX51WI microscope equipped with 100× 1NA objective for observation. To assess viability, blood flow and heart rate were visually monitored. Images were observed with a Grasshopper3 CMOS camera (Point Grey, Richmond, BC, Canada) and captured with manufacturer provided software. Starting with the eye neuromasts and moving caudal, the number of hair bundles were counted in approximately 10 neuromasts per fish.

After overnight treatment with cisplatin and/or Mdivi-1, fish were placed into wells containing FM1-43 (ThermoFisher Scientific, Waltham, MA, USA) in fish water. After 30 s, fish were transferred to fish solution containing MS-222 and BSA. The larvae were then secured in a recording chamber and placed under the microscope for imaging as described above. Approximately 3–4 neuromasts were imaged, and maximal projection images were generated using ImageJ (NIH, Bethesda, MD, USA). For lateral line neuromasts, raw images were gathered using an Olympus BX51WI microscope and a Grasshopper3 CMOS camera as described above. Fluorescence measurements were obtained using ImageJ. A region of interest was used to obtain measurements from the cells in each neuromast (I_cell) and an area without cells (I_background) in the same image. Fluorescence intensity of FM1-43FX (I_load) for each neuromast was normalized (I_load = I_cell − I_background).

We anesthetized zebrafish larvae (5–7 dpf) using MS-222 dissolved in a standard bath solution containing (in mM): NaCl (120), KCl (2), HEPES (10), CaCl₂ (2), NaH₂PO₄ (0.7), adjusted to pH ~7.2. The larvae were secured in a recording chamber and placed under the microscope for observation as described above. Viability, blood flow and heart rate of larvae were visually monitored. Images were observed with a Grasshopper3 CMOS camera and captured with manufacturer provided software. We recorded from posterior neuromasts; kinocilia tufts were deflected with a fluid jet (Nicolson et al., 1998; Trapani and Nicolson, 2010) delivered via a glass pipette with a diameter of approximately 5–7 μm and controlled by HSPC-1 (ALA Scientific Instruments, Farmingdale, NY, USA). Fluid jet pipette was placed approximately 75 μm near the neuromast and used to deliver sinusoidal stimuli of 50 Hz frequency. The microphonic potentials were recorded at room temperature (22°C). We used borosilicate glass electrodes with a resistance of 3–6 MΩ, which were filled with standard bath solution and placed near the apical edges of the lateral line neuromasts. We recorded microphonic potentials using a PC-505B amplifier (Warner Instruments, Hamden, CT, USA) and a PCI-6221 digitizer (National Instruments, Austin, TX, USA). Microphonic potentials were amplified by 20 (SIM983, Stanford Research, Sunnyvale, CA, USA), measured by a jClamp (Scisoft, Yale University, New Haven, CT, USA) in a current-clamp mode, and low-pass filtered at 100 Hz. All records represent an average of at least 500 trials.

All statistical analyses were performed using GraphPad Prism 7. Data are reported as mean ± SEM. Comparisons between groups were analyzed by ANOVA with Tukey post hoc testing.

Our data show that functional MET potentiate cisplatin-induced hair cell toxicity in lateral line neuromasts in a zebrafish (Figure 1), in accordance with published reports (Thomas et al., 2013; Stawicki et al., 2014). cdh23^{tj264a/tj264a} mutant zebrafish do not have functional MET in hair cells, because Cdh23 is an integral part of mechanosensitive stereocilia bundles in hair cells (Siemens et al., 2004; Söllner et al., 2004; Kazmierczak et al., 2007; Indzhykulian et al., 2013). Notably, cdh23 mutants have smaller numbers of hair cells per neuromast in comparison to wild-type or heterozygous fish (Figure 1). Despite the fact that treatment with 50 μM of cisplatin did not significantly change the number of hair cells in neuromasts of cdh23^{tj264a/tj264a} zebrafish, whereas in wild-type fish this dose of cisplatin considerably reduced the number of hair cells (Figure 1A). This result indicates that MET channels may be involved in cisplatin entry into the hair cell. Alternatively, cisplatin entry into the hair cell is largely independent of the MET channel, but the ion flow carried out by functional MET potentiates cisplatin-induced damage.

If cisplatin enters hair cells via MET channels, it could interact with the channel directly and attenuate ion flow through the channel. To test this hypothesis, the microphonic potentials of neuromast hair cells (Figures 2A,B) were measured with and without application of 50 μM or 100 μM of cisplatin. The microphonic potential is an evoked electrical potential elicited by hair bundle deflections. The microphonic potential results from modulation of the cationic current flowing into stimulated hair cells via functional MET channels. Our results show that microphonic potentials were not affected by cisplatin application (Figures 2A,B). An alternate approach using FM1-43FX was also employed to test the hypothesis. FM1–43FX is a derivative of FM1-43, an amphipathic styryl dye that is known to rapidly accumulate in sensory hair cells via the MET channels that are partially open at rest in non-stimulated hair bundles (Gale et al., 2001; Meyers et al., 2003). Loading of FM1-43FX in live hair cells of lateral line neuromasts of controls and after 100-μM-cisplatin was not significantly different (Figures 2C,D). Our results did not reveal any evidence that cisplatin enters hair cells via MET channels. It is known that aminoglycosides enter hair cells via MET channels and are permeant blockers of these channels. Our results, however, do not rule out the possibility cisplatin may enter hair cells via the MET channel but this amount may not be sufficient to affect measured microphonic potentials.

When MET is functional, substantial amounts of calcium can enter hair cells through MET channels. Intracellular calcium balance is critical for hair cell function; it was found that calcium homeostasis is rapidly disrupted following ototoxic aminoglycoside exposure (Esterberg et al., 2014). It is possible that hair cell mitochondria continuously buffer calcium entering cell via functional MET channels, causing hair cells to become more vulnerable to toxic insult. Drugs that could reduce mitochondrial stress and/or protect mitochondria in other ways, may potentially increase hair cell viability when faced with ototoxic drugs.

Here we tested whether mdivi-1 can protect hair cells against cisplatin induced toxicity in neuromast hair cells. Mdivi-1 is an inhibitor of mitochondrial division that selectively attenuates dynamin-related protein 1 activity, a fission protein that involved in the constriction and cleavage of mitochondria (Cassidy-Stone et al., 2008). First, we tested different doses of mdivi-1 for neuromast hair cell toxicity. High doses of mdivi-1, more than 10 μM, were toxic to the 5–6 dpf larvae (Figure 3A); therefore, we used lower doses of mdivi-1, 3 and 7 μM. Our data show that these doses of mdivi-1 protected hair cells of lateral line neuromast against toxicity of 50 μM of cisplatin (Figures 3B,C). These data demonstrated that modulating mitochondria dynamics may increase viability of hair cells against cisplatin toxicity in a zebrafish model. This finding is interesting also because it is known that mdivi-1 assists the abilities of cisplatin to trigger apoptosis in certain platinum-resistant tumor cells (Qian et al., 2014). Future studies, incorporating mammalian models, will be of further value in corroborating our results and revealing the mechanism of mdivi-1-mediated protection.