Electrophysiological profiling of exocytosis during early-stage development of the zebrafish lateral line.

Hair cells of the zebrafish lateral line have proven to be a good model for studying hair cell function in a system that is easily genetically manipulated, rapidly develops and is experimentally accessible. However, characterization of potential developmental changes, and possible differences along lateral line position are lacking. Here, we used in vivo patch clamp to investigate the exocytic properties of neuromast hair cells over early development across body location. Long depolarizations led to steady increases in membrane capacitance, presumably due to exocytosis of vesicles localized to ribbon synapses. The magnitude and kinetics of capacitance changes did not vary significantly across the L1 to L6 position of neuromasts along lateral line, but the magnitudes were found to be significantly smaller in hair cells found in the tail region across all developmental time points. For each region, we found no significant changes in capacitance responses between 3 and 7 days after fertilization. Hair cell capacitance responses were greatly reduced in animals injected with CRISPR/Cas9 with gRNAs targeted to otoferlin b. These results confirm the essential role of otoferlin b in neuromast hair cell function, and they demonstrate the fidelity of CRISPR/Cas9 to rapidly mediate genetic removal of critical genes to study their impact on synaptic release.Significance Statement The zebrafish lateral line is a well-established model system for studying hair cell function. In our manuscript, we systematically investigate these properties and find that properties remain consistent across developmental time, despite ongoing maturation of the synapse and discover key differences in hair cell responses in the tail region relative to those along the body. Moreover, we establish that disruption of Otoferlin B by CRISPR/Cas9 dramatically reduces exocytosis. These studies provide important new information that are important for establishing a baseline for investigating the impact of genetic manipulations on hair cell function.