Neuron Culture and Drug Treatment.

Cortical neurons obtained from Sprague–Dawley rats or C57BL/6 mice at embryonic day 18 were plated onto poly-l-lysine–coated tissue culture dishes or glass coverslips and grown in glia-conditioned neurobasal media (Invitrogen) supplemented with 2% (vol/vol) B-27, 2 mM Glutamax, 50 U/mL PenStrep, and 1% horse serum (Invitrogen). Cultured cortical neurons were fed twice per week. For all experiments cortical neurons (grown for 13–14 d in vitro, DIV) were plated at a density of 600,000 cells per well into standard 6-well tissue culture plates or 250,000 cells per well into 12-well tissue culture plates. In some experiments cortical neurons were treated with FR (5 μM/100 nM), PMA (1 μM), or Iso (5 μM) for 10 min, as indicated (drugs added to the culture media). For cLTD, neurons were incubated in artificial cerebrospinal fluid (ACSF) (143 mM NaCl, 5 mM KCl, 10 mM Hepes pH 7.4, 10 mM glucose, 2 mM CaCl₂, 1 mM MgCl₂) for 30 min followed by ACSF containing NMDA 40 μM for 3 min. For cLTP, neurons were pretreated for 30 min in ACSF, treated for 5 min with Mg²⁺-free ACSF containing 200 μM glycine, and then returned to Mg²⁺-containing ACSF without glycine for 20 min before lysis. All incubation steps for cLTP contained 0.5 μM tetrodotoxin, 20 μM bicuculline, and 1 μM strychnine. Hippocampal neurons were obtained from Sprague–Dawley rates at embryonic day 18, plated onto poly-l-lysine–coated glass coverslips, and grown in neurobasal media (Invitrogen) supplemented with 2% (vol/vol) B27 (Invitrogen), 0.5 mM glutamine, and 12.5 μM glutamate, 50 U/mL PenStrep (Gibco).