Cell suspensions were generated by adapting protocols from single-cell patch-clamp recording (Carter and Bean, 2009) and digest times were optimized for each region (Table S1). Mice were anesthetized by isoflurane inhalation and perfused through the heart with ice-cold Sucrose-HEPES “Cutting Buffer” containing (in mM) 110 NaCl, 2.5 KCl, 10 HEPES, 7.5 MgCl2, and 25 glucose, 75 sucrose (~350 mOsm kg−1). The brain was removed and placed in ice-cold Cutting Buffer. Blocking cuts depended on the region of interest and desired slice orientation (Table S1). Blocked brains were then transferred to a slicing chamber containing ice-cold Cutting Buffer. 400 μm thick brain slabs were cut with a Leica VT1000s vibratome. Slabs containing the regions of interest were gently transferred to a dissection dish with ice-cold “Dissociation Buffer” containing (in mM): 82 Na2SO4, 30 K2SO4, 10 HEPES, 10 glucose and 5 MgCl2. 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+ =
