P0 p1 c57bl 6j pups

Hippocampal cultures were made using previously established methods (Chander et al., 2019) . Briefly, brains from P0-P1 C57Bl/6J pups (The Jackson Laboratory, Bar Harbor, ME) were isolated, and the hippocampus was dissected in ice-cold HBSS solution (Sigma, St. Louis, MO) supplemented with 20% FBS and NaHCO3 (4.2mM), HEPES (1mM; Sigma), pH 7.4. Dissected hippocampi were digested for 10 min with 0.25% Trypsin (Thermo Fisher Scientific), then washed and dissociated using fire polished Pasteur pipettes of decreasing diameter in ice cold HBSS containing DNase (1500 U; Sigma). The cells were pelleted, resuspended in plating media and plated at a density of 4-5×105 cells/12-mm coverslip (Electron Microscopy Sciences, Hatfield, PA) coated with poly-Ornithine (0.1mg/ml; Sigma; Cat. #4638) and laminin (5μg/ml; Thermo Fisher Scientific). Cells were allowed to adhere for 15 min before addition of 0.5ml of plating media containing Neurobasal supplemented with 1X B27, 2mM Glutamax, 0.5mg/ml Pen/Strep and 5% FBS (all from Thermo Fisher Scientific) for the first 24h. Half of the media was removed and replaced with serum-free media after 24h. Half of the media was removed and replaced after 48h supplemented and with 4µM cytosine 1-β-d-arabinofuranoside (Ara-C; Sigma). Neurons were fed by replacing half the volume of spent media with fresh media without serum or Ara-C every week thereafter.