Evaluating the Death and Recovery of Lateral Line Hair Cells Following Repeated Neomycin Treatments

Acute chemical ablation of lateral line hair cells is an important tool to understand lateral line-mediated behaviors in free-swimming fish larvae and adults. However, lateral line-mediated behaviors have not been described in fish larvae prior to swim bladder inflation, possibly because single doses of ototoxin do not effectively silence lateral line function at early developmental stages. To determine whether ototoxins can disrupt lateral line hair cells during early development, we repeatedly exposed zebrafish larvae to the ototoxin neomycin during a 36 h period from 3 to 4 days post-fertilization (dpf). We use simultaneous transgenic and vital dye labeling of hair cells to compare 6-h and 12-h repeated treatment timelines and neomycin concentrations between 0 and 400 µM in terms of larval survival, hair cell death, regeneration, and functional recovery. Following exposure to neomycin, we find that the emergence of newly functional hair cells outpaces cellular regeneration, likely due to the maturation of ototoxin-resistant hair cells that survive treatment. Furthermore, hair cells of 4 dpf larvae exhibit faster recovery compared to 3 dpf larvae. Our data suggest that the rapid functional maturation of ototoxin-resistant hair cells limits the effectiveness of chemical-based methods to disrupt lateral line function. Furthermore, we show that repeated neomycin treatments can continually ablate functional lateral line hair cells between 3 and 4 dpf in larval zebrafish.