Primary hippocampal neurons were cultured from P0 to P1 pups from APP+/− bred to either APP+/− or APP−/− to obtain the desired genotypes. Individual hippocampi from each pup were dissected and processed separately. Genotyping by PCR was performed from a sample of the tail from each pup. Following genotyping, hippocampal cultures remained separate regardless of the genotype (APP+/+, APP+/−, or APP−/−) and were analyzed separately. The hippocampal cultures were prepared at a density of 300 cells/mm2 on poly-L-lysine-coated coverslips and maintained in neurobasal medium with B-27 supplement (Calabrese et al., 2007). The neurons were transfected with pEGFP-N3 plasmid to visualize dendritic spines (Clontech, Mountain View, CA, USA) using calcium phosphate precipitation at 16-21 days in vitro (DIV). The neurons were fixed and analyzed within 24 hours after the transfection.
