Hippocampal Primary Neuron Culture Protocol

Mouse hippocampal primary cultures were prepared at embryonic day 18 as previously described (Kellner et al., 2014 (link)). Briefly, embryos were rapidly decapitated, and the brains were immersed in ice cold Gey’s Balanced Salt Solution (GBSS) supplemented with glucose and adjusted to pH 7.2. The dissected hippocampi were incubated in Trypsin/EDTA (Sigma-Aldrich) at 37°C for 30 min after which the digestion was stopped. Subsequently, the neurons were dissociated mechanically using a Pasteur pipette and were re-suspended in Gibco Neurobasal medium supplemented with 2% B27, 10% N2 and 0.5 mM L-Glutamine and plated at different densities depending on the experiment performed: a high density (7 × 10⁴cells/cm²; analysis of the architecture of mature neurons and expression of cFos and pERK), middle density (3.5 × 10⁴ cells/cm², measure of TrkB phosphorylation levels), low density (1 × 10⁴cells/cm², analysis of the architecture in developing neurons) on poly-L-lysine-coated coverslips (12 mm, Sigma-Aldrich). The cultures were incubated at 37°C, 5% CO₂, and 99% humidity. Once a week 20% of the medium was exchanged. Cultures were used at DIV7 or at DIV21/22.