Primary Hippocampal Culture Preparation

Primary hippocampal cultures were prepared by dissecting both hippocampi of P0-P1 C57BL/6JOlaHsd (α-Syn^−/−) mouse brains, as described previously (80 (link), 81 (link)). After trituration in 20 units/ml of papain solution (Worthington Lakewood, NJ, USA), 1 × 10⁵ cells were plated on coverslips that were pre-coated with 5 μg/ml of poly-D-lysine (Sigma-Aldrich, Rehovot, Israel) in a 24-well–plate containing 1.5 ml of Neurobasal-A medium (Gibco, ThermoFisher Scientific, Petah Tikva, Israel) supplemented with 5% fetal bovine serum, 2% B-27 (Gibco, ThermoFisher Scientific), 1% Glutamax I, and 1 μg/ml of gentamicin. After 1 day, the solution was replaced to 1 ml of Neurobasal-A supplemented with 2% B-27 and 1% Glutamax I. To slow the proliferation of glial cells, 1 μm cytosine β-d-arabinofuranoside (Ara-C; Sigma-Aldrich) was added to the culture at 2 DIV. Cultures were maintained at 37 °C in a 5% CO₂ humidified incubator until used at 8-13 DIV as indicated. For measurements of Tf-568 endocytosis, neurons were prepared and grown as previously describe (5 (link)).