Dissociation Buffer avoided activity-induced toxicity by 1) excluding extracellular Ca2+ and 2) utilizing ionic concentrations that maintain voltage-gated Na channels in an inactivated state [Vm = −30.5 mV, estimated by the Goldman-Hodgkin-Katz equation using the following parameters: Inside(mM): K+ = 140, Na+ = 4, Cl− = 24; Outside(mM): K+ = 30, Na+ = 82, Cl− = 5; P: K+ = 1, Na+ = 0.05, Cl− = 0.45; T = 34°C]. Regions of interest were gently dissected under visual guidance of a stereoscope (Leica MZ10). Dissection also served as a wash step between buffers. Chunks of tissue containing the regions of interest were then transferred into 5 ml of “Dissociation + Enzyme Buffer” in a 15 ml falcon tube. “Dissociation + Enzyme Buffer” consists of “Dissociation Buffer” with 3 mg/ml of Protease XXIII (Sigma-Aldrich, P5380) and 10 units/ml of Papain, 0.5 mM L-Cysteine and 0.25 mM EDTA (Worthington, LK003153). Digestion was performed at 34 C using durations that were optimized for each region in a separate set of experiments (Table S1; see below). Tubes containing digested tissue were transferred onto ice and the “Dissociation Buffer + Enzyme” replaced with 10 ml of “Dissociation Buffer + Stop Solution” containing “Dissociation Buffer” and
