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. 2022 Sep 1;11:e77775. doi: 10.7554/eLife.77775

Figure 3. Cav1.3a and otofb mutant hair cells have normal neomycin uptake and retention.

(A-A’) Example of hair cells in a wildtype neuromast after incubation in 25 µM neomycin-Texas Red solution (Neo-TR) for 10 min. GCaMP6s outlines hair cells in A. (B) Average dot plots of Neo-TR fluorescence intensity in neuromasts from cav1.3a-/- (blue) and otofb-/- (orange) mutants and siblings (black and grey, respectively) after a 10-min incubation in 25 µM Neo-TR. No differences in overall neomycin uptake are seen between mutants and siblings. (C–E) Average Neo-TR fluorescence intensity over the course of 10 min 25 µM exposure in cav1.3a-/- mutant neuromasts (blue) and siblings (black) (C) and otofb-/- mutant neuromasts (orange) and siblings (grey) (E). No differences are seen between mutants and siblings in the time course of neomycin uptake. (D–F) Average Neo-TR fluorescence intensity over the course of 30 min following Neo-TR washout in cav1.3a-/- mutant neuromasts (blue) and siblings (black) (D) and otofb-/- mutant neuromasts (orange) and siblings (grey) (F). No significant differences were detected in neomycin clearance or retention between mutants and siblings. Each point in B represents one neuromast. A minimum of 14 neuromasts are averaged for the plots in C-F. A minimum of five animals at 5 dpf were examined per treatment group. Error bars: SEM. For comparisons, unpaired t-tests were used in B, and a two-way ANOVA with a Sidak’s correction for multiple comparisons was used in C-F. Scale bar = 5 µm.

Figure 3—source data 1. Mean numbers and statistics for ototoxicity analyses.

Figure 3.

Figure 3—figure supplement 1. Time course of neomycin uptake and packaging in cav1.3a and otofb mutants and siblings.

Figure 3—figure supplement 1.

(A–C) Representative examples of hair cells in wildtype (A), cav1.3a mutant (B), and otofb mutant (C) during Neo-TR uptake and clearance. Shown at t=0 are neuromasts immediately following application of a 25 µM Neo-TR solution. These images are followed by images taken at 5 and 10 min after application. After 10 min, the Neo-TR solution was washed out, images were acquired from each neuromast (in embryo media) at 17.5, 25, and 40 min from the initial addition of Neo-TR solution. Scale bar = 5 µm.