Hair Cell Assessment

The majority of our hair cell assessment was done using 2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide (DASPEI; ThermoFisher, Eugene, OR, USA, D426), a mitochondrial dye that robustly labels the hair cells in the lateral line of zebrafish and other fishes (Harris et al., 2003; Coffin et al., 2009; Brown et al., 2010). After aminoglycoside and/or analog treatments, zebrafish were incubated for 15 min at 28°C with 0.005% DASPEI. The fish were subsequently rinsed 2× with EM and anesthetized with 0.001% buffered tricaine methanesulfonate (MS-222, Syndel, Ferndale, WA, USA) before visualization. We assessed the relative fluorescence intensity for 10 head neuromasts on every zebrafish (IO1, IO2, IO3, IO4, M2, O2, MI1, MI2, SO1, SO2; Raible and Kruse, 2000) using 50x magnification on a Leica M165F fluorescent dissecting microscope. Each neuromast was given a score to quantify the fluorescence: 2 (bright labeling), 1 (modest labeling), and 0 (no labeling); the individual neuromast scores were added to provide an overall score between 0–20 per fish (Harris et al., 2003; Coffin et al., 2009; Owens et al., 2009; Kruger et al., 2016). Scores were averaged per treatment group. To normalize the data, all treatment group averages were divided by the average value of the controls for that experiment and multiplied by 100 to arrive at the percentage of surviving hair cells. DASPEI labeling is a viable method of assessing hair cells with comparable results to directly counting immunolabeled hair cells (Harris et al., 2003; Coffin et al., 2013a; Kruger et al., 2016). For a subset of experiments, we used Tg(Brn3c:GFP) larvae, where hair cells express membrane-specific GFP. These experiments provided observation of cellular morphology following aminoglycoside and analog treatment. Animals were treated as described above for analog and aminoglycoside exposure, then euthanized with 0.002% buffered MS-222, fixed in 4% paraformaldehyde, and imaged on a Leica SP8 confocal microscope.