Dissociated Hippocampal Neuron Culture Protocol

The preparation of cultured dissociated hippocampal neurons was described previously27 (link)30 . Before dissection of hippocampi, neonatal rats were sacrificed by decapitation, in accordance to the procedures approved by the animal care committee of Université Laval. The dissociated cells were plated on poly-d-lysine-coated glass coverslips (12 or 18 mm) at a density of 4225 or 860 cells/mm² respectively. Growth media consisted of Neurobasal and B27 (50:1), supplemented with penicillin/streptomycin (50 U/mL; 50 μg/mL) and 0.5 mM L-GlutaMAX (Invitrogen). Fetal bovine serum (2%; Hyclone) was added at time of plating. After 5 days, half of the media was changed without serum and with Ara-C (5 μM; Sigma-Aldrich) to limit proliferation of non-neuronal cells. Twice a week thereon, half of the growth medium was replaced with serum- and Ara-C–free medium. The neurons were transfected at 11–14 days in vitro (DIV) with Lipofectamine 2000 (Invitrogen) as described previously27 (link). For Ca²⁺ imaging, plasmids encoding GCaMP6s21 (link) and RCaMP1.07 (gift of J. Nakai) were transfected 1 day prior to measurements. CaMKII was tagged with a monomeric GFP as described previously27 (link).