Dissociated Hippocampal Neuron Treatment Protocol

All procedures were approved by the Yale University Institutional Animal Care and Use Committee. Primary dissociated hippocampal cultures were prepared from Sprague–Dawley rat at embryonic day 18 and plated on poly-D-lysine-coated plates (1 × 10⁶ cells/well) in Neurobasal supplemented with 2% B27 (Invitrogen, San Diego, CA) and grown for 18–21 days as described33 (link). At 18–21 days in vitro, neurons were treated with muscarine (10 μM) plus NMDA (50 μM) dissolved in ECS for 10 min. In some groups, inhibitors and antagonists (pirenzepine: 10 μM; EGTA/AM: 0.1 or 1 mM; TC-2153: 1 μM; xestospongin C: 10 μM; PP2: 10 μM or PP3: 10 μM) or vehicle (0.1% DMSO) were added 30 min before and throughout the treatment with muscarine and NMDA. After treatments, neurons were lysed in 1 × RIPA buffer (Pierce Biotechnology, Rockford, IL) with complete phosphatase and protease inhibitors (Roche, Indianapolis, IN).