Cortical samples were identified by morphology and then dissociated into single cell suspensions. Tissue was minced (approx. 0.25 – 0.5 mL total volume) with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (Thermo Fisher) and digested with 2 mL trypsin solution for 20 min at 37 °C (Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 2 mM MgCl2, 0.25 mg/mL bovine pancreatic trypsin (EMD Millipore), 10 μg/mL DNase I (Roche), 100 nM TTX (Tocris), 20 μM DNQX (Tocris), and 50 μM DL-AP5 (Tocris), pH 7.6). Digestion was quenched with 6 mL of ice-cold Quenching Buffer (440 mL Leibovitz L-15 medium, 50 mL water, 5 mL 1M HEPES pH 7.3–7.4, 5 mL 100× Pen-Strep, 20 mg/mL bovine serum albumin (Sigma), 100 μg/mL trypsin inhibitor (Sigma), 10 μg/mL DNase I, 100 nM TTX, 20 μM DNQX, and 50 μM DL-AP5. Samples were resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles, and then diluted in 30 mL in Staining Medium (440 mL Leibovitz L-15
