Drosophila electrophysiology from the neuromuscular junction (NMJ)

Sharp-electrode recordings were made from muscle 6 in abdominal segments 2 and 3 from third-instar larvae using an Axoclamp 900A amplifier (Molecular Devices), as described previously (Frank et al. 2006, Muller et al. 2012). Recordings were made in HL3 saline containing the following components: NaCl (70 mM), KCl (5 mM), MgCl₂ (10 mM), NaHCO₃(10 mM), sucrose (115 mM), trehalose (5 mM), HEPES (5 mM), and CaCl₂ (as indicated in figures). For acute pharmacological homeostatic challenge, unstretched larva were incubated in Philanthotoxin-433 (PhTX; 15 μM; Sigma-Aldrich) for 10 min. (Frank et al. 2006). Recordings were excluded if the resting membrane potential (RMP) was more depolarized than −55mV. A threshold 40% decrease in mEPSP amplitude, below average baseline, was used to confirm the activity of PhTX. Miniature spontaneous events were analyzed using MiniAnalysis 6.0.0.7 (Synaptosoft), averaging at least 100 individual mEPSP (or mEPSC) events. EPSP (or EPSC) amplitudes were analyzed in IGOR Pro (Wave-Metrics) with previously routines (Müller et al., 2015). Quantal content was calculated by dividing mean EPSP (or EPSC) by mean mEPSP (or mEPSP). Estimates of RRP were achieved by quantifying cumulative EPSC amplitudes during prolonged high-frequency stimulation and normalization to the amplitude of the underlying spontaneous mEPSC amplitude, as previously performed (Müller et al., 2012, 2015; Weyhersmüller et al., 2011; Wang et al., 2016). Best-fit curves for mEPSP amplitude versus quantal content were fit in Prism 9 (GraphPad) using a power function for all wild-type data points +/−PhTX. 95% data intervals were fit in IGOR Pro (Wave-Metrics) using a power function. Each recording paradigm (i.e. genetic mutant an or treatment) was biologically replicated at least 3 times. Muscle membrane potentials were held at −65 mV during two-electrode voltage clamp experiments.