Primary culture of mouse cortical neurons

Dissociated cultures of mixed neuronal and glial populations were prepared from unsexed P0-P1 neonatal mice. These mice are wild-type mice from a colony of Rab3A+/− mice housed at Wright State University. The Rab3A+/− mice are derived from C57BL/6J, 129 Sv-Rab3a^(tm1Sud) from The Jackson Laboratory, originally crossed three times to C57BL/6J (IMSR catalog #JAX:002443, RRID:IMSR_JAX:002443); we subsequently backcrossed the line 11 times to wild-type mice from a Rab3A^+/Ebd colony. Rab3A^Ebd/Ebd are mice with a point mutation in Rab3A (Kapfhamer et al., 2002), identified in an ENU-mutagenesis screen of C57BL/6J, crossed once to C3H/HeJ for mapping, and backcrossed three times to C57BL/6J (MGI catalog #2388809, RRID:MGI:2388809). Pups were euthanized by rapid decapitation with sharp scissors, as approved by the Wright State University Institutional Animal Care and Use Committee. Brains were removed, and cortices were collected after removal and discarding of hippocampi. Tissue digestion was conducted by the addition of papain (Worthington Biochemical) at 20 units/ml in Neurobasal-A media (Invitrogen), osmolarity adjusted to 270 mOsm, and incubated in a 37°C H₂O bath for 20 min with gentle stirring. After digestion, cortices were gently triturated with a fire-polished Pasteur pipette, filtered through a 100-μm cell strainer (Corning Falcon), and transferred to a sterile 15-ml conical tube. The cell suspension was centrifuged at 1100 rpm for 2 min, supernatant was removed, and the pellet re-suspended in room temperature Neurobasal-A media (osmolarity adjusted to 270 mOsm), supplemented with 5% fetal bovine serum (FBS; promotes glial growth), 2% B-27 supplement to promote neuronal growth, L-glutamine (2 mm), and gentamicin (0.01 mg/ml; all from Invitrogen). Neurons were plated onto 12-mm coverslips pre-coated with poly-L-lysine (Corning) at a density of 0.15 × 10⁶ cells/coverslip. The culture media for the first day (0 DIV) was Neurobasal-A media supplemented with FBS, B-27, L-glutamine, and gentamicin, and was switched at 24 h (1 DIV) to media consisting of Neurobasal-A (270 mOsm), 2% B-27, and L-glutamine, without FBS. FBS was excluded to avoid its toxic effects on neuronal viability and health (Stellwagen and Malenka, 2006). Cells were maintained in culture for 13–14 DIV in a humidified 5% CO₂ incubator at 37°C. Culture media was changed twice weekly by replacing half with freshly prepared media. Two days before experiments, culture dishes containing four 12-mm coverslips were randomly chosen to receive TTX (500 nm; Tocris) to chronically silence all network activity and induce homeostatic synaptic plasticity mechanisms, while sister cultures not receiving TTX treatment served as untreated controls.