Calcium Imaging of Mechanosensitive Hair Cells

For calcium imaging, measurements were made as previously described (Kindt et al., 2012 (link); Zhang et al., 2016 (link)). Briefly, larvae were anesthetized with 0.03% 3-amino benzoic acid ethylester (MESAB, Western Chemical, Ferndale, WA) in E3, and mounted with tungsten pins onto a Sylgard recording chamber. Larvae were then microinjected in the heart with 125 μM α-bungarotoxin (Tocris, Bristol, UK) to suppress muscle activity. After paralysis, calcium imaging was performed in extracellular solution in mM: 130 NaCl, 2 KCl, 2 CaCl₂, 1 MgCl₂ and 10 HEPES, pH 7.3, 290 mOsm. A pressure clamp (HSPC-1, ALA Scientific, New York, NY) attached to a glass pipette (tip diameter ~30–50 μm) was filled with extracellular solution and used to mechanically stimulate HCs along the anterior-posterior axis of the fish. Calcium measurements were made on a Nikon Eclipse NiE microscope using a 60 × 1.0 NA CFI Fluor water-immersion objective and the following filter set: excitation: 480/30 and emission: 535/40. The microscope was equipped with an Orca D2 camera (Hamamatsu, Hamamatsu City, Japan), and images were acquired using Elements software (Nikon Instruments Inc., Melville, NY).