Hippocampal Cell Culture and conditions
Rat primary hippocampal neuronal cells were purchased from QBM Cell Science (Montreal, Canada) and cultured in Neuro basal medium supplemented with 10% FBS, 2 mM GlutaMAX-1, 1 mM sodium pyruvate, 100 U/ml penicillin G, and 100 μg/ml streptomycin and 2% B-27 supplement at 37°C in a humidified 10% CO2 environment according to manufacturer’s instructions. Half part of the medium was changed every 3 days. The cells were grown under the above conditions (control condition) for 7 d. Cultured hippocampal neurons were grown and treated for 24 hours under specific pharmacologic conditions. Treated hippocampal cells were divided into experimental groups: (1) Control group; (2) M1145-treated group (100nM); (3) Y1R agonist-treated group receiving an NPYY1R agonist [Leu31,Pro34]NPY (100nM); (4) GAL+Y1R: Group administered with both substances; (5) GAL+Y1+M871: Group injected with GAL, [Leu31,Pro34]NPY and the GALR2 antagonist (M871; 1μM). Cells were grown on poly-D-lysine-coated glass coverslips and fixed with 4% formaldehyde solution for 20 min followed by two washes with PBS containing 20 mM glycine to quench the aldehyde groups.