Isradipine

Cells were treated for 48 h beginning at day 2 of differentiation with DMSO (vehicle control, ATCC), aceclidine (Sigma-Aldrich), acyclovir (Sigma-Aldrich), alloxazine (Sigma-Aldrich), carbadox (Sigma-Aldrich), felodipine (Sigma-Aldrich), GW5074 (Sigma-Aldrich), isoproterenol (Sigma-Aldrich), isradipine (Sigma-Aldrich), lacidipine (Sigma-Aldrich), nafadotride (Tocris Bioscience), nandrolone (Sigma-Aldrich), nifedipine (Sigma-Aldrich), nilvadipine (Sigma-Aldrich), alloxazine (Sigma-Aldrich) diluted in cell-type specific differentiation media at doses listed in figures.

Zebrafish (Danio rerio) work was performed under animal study protocol #1362–13 approved by the Animal Care and Use Committee at the NIH. Strains were maintained in TAB5 or Tubingen wild-type background. Zebrafish larvae were raised in E3 embryo media (5 mM NaCl, 0.17 KCl mM, 0.33 mM CaCl₂ and 0.33 mM MgSO₄) at 30 °C. Larvae were examined at 4–6 days post fertilization (dpf). To image ribbon synapses, the previously described transgenic line Tg(-6.5myosin6b:Ribeye b-GFP)vo67 was used^{25 (link)}. Despite having enlarged ribbons compared to wildtype siblings, transgenic fish have no observable auditory or vestibular defects, and therefore represent a viable model to study ribbon dynamics. For our analyses only basally localized ribbons were examined, rather than small ectopic cytosolic Ribeye b-EGFP aggregates. For microscopy, larvae were anaesthetized with 0.01% ethyl 3-aminobenzoate methanesulfonate (MS-222, Sigma-Aldrich). Larvae were then oriented in glass-bottomed dishes for imaging in 1% low melting point agarose and immersed in E3 embryo media containing 0.01% MS-222. To block synaptic function, 10 μM isradipine (Sigma-Aldrich) and 0.1% DMSO was added to the low melting point agarose and the E3 solution.