Brain dissection, slice preparation, stimulation, and lysis

Adult brain slices were prepared from mature adult mice using the protective recovery method (Zhao et al. 2011). Mice were deeply anesthetized with Isofluorane and transcardially perfused with 25–30mL of N-methyl-D-glucamine (NMDG)-protective recovery solution (93 mM NMDG, 2.5 mM KCl, 1.2 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 2 mM thiourea, 5 mM Na-ascorbate, 3 mM Na-pyruvate, 0.5 mM CaCl2.4H2O, and 10 mM MgSO4.7H2O; titrated to pH 7.4 with concentrated hydrochloric acid). Brains were removed and coronal cortical slices were sectioned at 400 μm thickness using a vibratome. Slices were immediately hemisected with a sharp razor blade and each half placed in an alternate treatment group with treatment groups (i.e. KCL vs. aCSF) being arbitrarily assigned. Slices were initially recovered in NMDG-protective recovery solution for 10–15 minutes at 32–34°C, then transferred to a modified HEPES holding solution [92 mM NaCl, 2.5 mM KCl, 1.2 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 2 mM thiourea, 5 mM Na-ascorbate, 3 mM Na-pyruvate, 2 mM CaCl2.4H2O, and 2 mM MgSO4.7H2O; pH 7.4] for an additional 60–90 minute recovery at room temperature. For KCl stimulation, slices were incubated at 37°C in High K⁺ or control HEPES-aCSF for 5 minutes. Following stimulation, tissue was homogenized in 0.32M sucrose in HEPES buffer with protease/phosphatase inhibitor cocktails using 12 strokes of a glass-teflon homogenizer and PSD preparations were made as previously described (Li et al. 2016).