Primary Rat Hippocampal Neuron Culture

Cell suspensions were prepared from rat hippocampus as described previously [21 (link)]. Coverslips (for immunofluorescence staining and calcium imaging) and culture dishes (for any other application) were coated with poly-D-lysine hydrobromide (Sigma-Aldrich, Taufkirchen, Germany). Cell suspension was preplated onto an uncoated flask and incubated at 37 °C in 5% CO₂ for 1 h. During this time glial cells settled down and adhered to the bottom of the flask, while neurons remained in the supernatant. Supernatant was collected thereafter and centrifuged at 800 rpm for 8 min at room temperature. Cell pellets were resuspended and cultured in serum-free Neurobasal-A medium supplemented with 2% B27, 0.5 mM GlutaMAX and 1% penicillin-streptomycin (all Life Technologies, Darmstadt, Germany). Cells were plated on poly-D-lysine coated dishes or coverslips at a density of 2.5 × 10⁵ onto 3.5 cm². Cells were maintained at 37 °C in a fully humidified incubator containing 5% CO₂. After 24 h Cytosine β-D-arabinofuranoside hydrochloride (AraC; Sigma-Aldrich) was added to inhibit proliferation of remaining glial cells. Neurons were maintained in dispersed culture with the original media up to 40 days in vitro (DIV).