Preparation of Rat Neuronal Cultures

Rat neurons were prepared as described previously (31 (link)) with some modifications. In brief, cerebella of 7-day-postnatal Sprague-Drawly rats were dissociated in Versene solution (1:5000, Invitrogen) and total cells (1 × 10⁶ cells per 35 mm²) were plated in poly-D-Lysine (Sigma, St. Louis, MO) coated plates or dishes for five minutes after which, the non-adherent cell suspension was removed and used for preparing glial cells. Adherent cells were maintained in MEM (Mediatech, Herndon, VA) supplemented with heat-inactivated 10% fetal bovine serum (FBS, Atlas Biologicals, Fort Collins, CO), 25 mM KCl, 3gm/500 glucose (Sigma), and 1% antibiotic-antimycotic solution (Sigma). Cytosine-D-arabinoside (10 μM Ara-C, Sigma) was added to cultures 24 hrs after plating to block the proliferation of non-neuronal cells. These neurons were routinely used during 9–12 days in vitro (DIV). Ara-C was avoided to obtain neuron-glial mix culture (where glial growth was allowed in the neuronal culture). Rat hippocampal and cortical neurons were obtained from E18 embryos. The hippocampi or the cortices were dissected and dissociated in HBSS solution. Then they were plated in Poly-D-Lysine (Sigma) coated plates or dishes in Neurobasal media supplemented with B27 (Invitrogen). These neurons were routinely used during 14–21 days in vitro (DIV).