AGs differ in relative hair cell toxicity dependent on time of exposure and length of incubation. (A–C) Fish were treated with 200 μM neomycin (Neo) or gentamicin (Gent; red bar) for time indicated, or rinsed into fresh medium (blue bar). (A) Hair cells were exposed for 1 h acute treatment with AGs. Hair cells are effectively killed by Neo, but are largely spared by Gent. n = 9–11 fish, 4 NMs/fish. One-way ANOVA with Dunnet's post-hoc comparison to control, ****P-value < 0.0001. (B) Chronic 24 h treatment with Neo or Gent results in hair cell loss. n = 9–11 fish, 4 NMs/fish. One-way ANOVA with Dunnet's post-hoc comparison to control, ****P-value < 0.0001. (C) Substantial death occurs after 1 h treatment with Neo or Gent, rinsing, and then incubation for 23 h in fresh medium (1+23 h). n = 9–11 fish, 4 NMs/fish. One-way ANOVA with Dunnet's post-hoc comparison to control, ****P-value < 0.0001. (D) Dose-dependent loss of hair cells after treatment with neomycin for 1, 24, or 1+23 h. Differences between treatments were highly significant (2-way ANOVA, Tukey's multiple comparison, p < 0.0005). n = 9–11 fish, 4 NMs/fish for each condition. (E) Dose-dependent loss of hair cells after treatment with gentamicin for 1, 24, or 1+23 h. Differences between treatments were highly significant (2-way ANOVA, Tukey's multiple comparison, p < 0.0001). Post-hoc comparison of individual points are presented in Supplementary Tables 1, 2. n = 9–11 fish, 4 NMs/fish for each condition. Error bars represent Standard Deviation.

The rate of delayed hair cell loss is dependent on initial gentamicin concentration. Fish were treated with doses of gentamicin (25, 50, 100, and 200 μM) for 1 h (red bar), then rinsed and incubated in fresh medium (blue bar). Loss of hair cells was assessed at 5, 11, 17, and 23 h after the 1 h incubation period. Increasing initial dose results in more rapid delayed hair cell loss. Differences between 25, 50, and 200 μM treatments were significant (Tukey's multiple comparison, p < 0.0001). There was no significant difference between 100 and 200 μm treatments. n = 9–11 fish, 4 NMs/fish for each treatment. Error bars represent Standard Deviation.

Different mitochondrial Ca²⁺ responses during acute or delayed hair cell death. (A–C) Fish were incubated in AG (red bar) for time indicated, or rinsed into fresh medium (blue bar). Fluorescence changes above baseline (F/F₀) from mitoGCaMP in response to AG addition were monitored at 30 s intervals by spinning disk microscopy over the interval indicated (black bar). Individual traces represent responses of individual cells. Traces are aligned to time of cell fragmentation. (A) Changes in mitoGCaMP signal in cells undergoing acute death in response to 100 μM neomycin. Hair cells were imaged during the 1st h of neomycin exposure. Increases in mitochondrial Ca²⁺ were observed in 16/16 dying cells. (B) Changes in mitoGCaMP signal in cells undergoing delayed death after exposure to 100 μM G418. Cells were exposed to G418 for 1 h followed by rinse and incubation in fresh embryo medium (EM) for 1.5 h, and then imaged over an additional 2 h period. Increases in mitochondrial Ca²⁺ were observed in 2/11 dying cells. (C) Changes in mitoGCaMP signal in cells undergoing acute death in response to 400 μM G418. Hair cells were imaged during the 1st h of G418 exposure. Increases in mitochondrial Ca²⁺ were observed in 10/14 dying cells. (D) Maximum mitoGCaMP signal compared to baseline for dying cells after neomycin or G418 exposure. P-values from Kruskal-Wallis with Dunn's multiple comparison test. Error bars represent Standard Deviation.

The mitochondrially-targeted antioxidant mitoTEMPO protects against acute neomycin damage from neomycin but not delayed damage from G418. 50 μM mitoTEMPO was added for 30 min before AG (purple bar), co-treated with neomycin for 1 h or co-treated with G418 for 1 h (red bar), rinsed, and incubated with mitoTEMPO alone for 23 h (purple bar). (A) mitoTEMPO partially protected against damage from both 100 and 200 μM neomycin treatment. ****Two-way ANOVA, Sidak's multiple comparison, p < 0.0001. (B) mitoTEMPO offered no protection against damage from either 100 or 200 μM G418 treatment. ns Two-way ANOVA, Sidak's multiple comparison, p = 0.98. n = 9–11 fish, 4 NMs/fish for each treatment group. Error bars represent Standard Deviation.

Neomycin and G418 differentially accumulate in Rab7+ vesicles. (A) G418-TR (magenta) accumulation in Rab7+ vesicles (green) in neuromast from Tg(myosin6b:EGFP-Rab7a) transgenic line. (A') G418-TR signal is mainly in vesicles. (B) Neo-TR (magenta) accumulation in Rab7+ vesicles (green) in neuromast from Tg(myosin6b:EGFP-Rab7a) transgenic line. (B') Neo-TR signal is found in vesicles and cytoplasm. (C) Ratio of G418-TR and Neo-TR found in Rab7+ vesicles compared to cytoplasm. Shading indicates measurements from three separate experiments. **Unpaired T-test, p < 0.01. Error bars represent Standard Deviation.

GPN treatment reduces the number of vesicles accumulating G418. (A) Image of neuromast treated with G418-TR. (A') Mask of segmented hair cells. (A”) Mask of segmented vesicles. (B) Image of neuromast treated with G418-TR and GPN. (B') Mask of segmented hair cells. (B”) Mask of segmented vesicles. (C) Number of vesicles per hair cell, mean for each neuromast analyzed. ****Mann-Whitney test, p < 0.0001. (D) Vesicle volume per hair cell, mean for each neuromast. ns, Mann-Whitney test. (E) Vesicle fluorescence per hair cell, mean for each neuromast analyzed. ns, Mann-Whitney test. Error bars represent Standard Deviation.

GPN treatment protects against delayed gentamicin damage but not acute neomycin damage. 250 μM GPN was added for 30 min before AG (purple bar), co-treated with neomycin for 1 h or co-treated with G418 for 1 h (red bar), rinsed, and incubated with GPN alone for 23 h (purple bar). (A) GPN treatment offers robust protection against delayed death from all concentrations of gentamicin (Two-way ANOVA, Sidak's multiple comparison, p < 0.0001). (B) GPN does not protect against acute death from neomycin at any concentration. n = 9–11 fish, 4 NMs/fish for each treatment group. Error bars represent Standard Deviation.