Primary cortical neuron culture and transduction

Pregnant rats (E17) of the Sprague-Dawley strain were purchased from Charles River. Embryos were surgically removed from the mothers and cortices dissected in Hanks Balanced salt solution (Gibco). The meninges were removed and cells dissociated using a papain dissociation system (Worthington) before being resuspended in Neurobasal medium A supplemented with antibiotic-antimycotic solution (Gibco), L-glutamine substitute (GlutaMAX™; Gibco) and factor B27 (Gibco). Cells were plated on poly-D-lysine coated glass coverslips at a density of 5 × 10⁵ cells/well and incubated at 37 °C in 5% CO₂ with half media changes every 3 days. Cells were transduced with AAV-A53T, AAV-Empty and AAV-Ub^G76V-GFP 2 days post-isolation at a multiplicity of infection (MOI) of 3000. Media containing AAV vectors were removed after 72 h and cells were fixed with 4% PFA for immunofluorescence staining at 8 days post-isolation.