Recording of mEPC in the NMJ was performed as previously described with some modifications (47 (link)). Skinned larvae were pinned down to the recording chamber coated with Sylgard and immobilized by bathing in 10 μM nifedipine (Merck, Kenilworth, NJ). Patch-clamp recordings were made by the whole-cell ruptured technique of muscle cells. The pipette solution used for the voltage-clamp recording was 120 mM KCl, 5 mM BAPTA, and 5 mM Hepes (pH 7.2). The extracellular solution contained 112 mM NaCl, 2 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 3 mM glucose, and 5 mM Hepes (pH 7.4). Membrane currents were recorded with an EPC10 amplifier and PatchMaster. For mEPC recordings, muscle cells were voltage-clamped at −90 mV, and 1 μM Tetrodotoxin (TTX) was added to the recording solution. The currents were sampled at 50 kHz and filtered at 3 to 5 kHz before analysis. Capacitive transients were compensated manually.
For puff application, a glass electrode [opening, ∼30 μm; filled with bath solution containing 30 μM ACh and dextran–Alexa Fluor 488 (Thermo Fisher Scientific, Waltham, MA)] was placed near the voltage-clamped muscle cell and positive pressure was applied using Picospritzer II (Parker Hannifin; 30 ms, 1 psi). ACh-induced currents were recorded by the whole-cell ruptured technique.
For puff application, a glass electrode [opening, ∼30 μm; filled with bath solution containing 30 μM ACh and dextran–Alexa Fluor 488 (Thermo Fisher Scientific, Waltham, MA)] was placed near the voltage-clamped muscle cell and positive pressure was applied using Picospritzer II (Parker Hannifin; 30 ms, 1 psi). ACh-induced currents were recorded by the whole-cell ruptured technique.
