Cultures of midbrain dopaminergic neurons on top of cortical astrocytes were made from P1-P2 Wistar rats or DAT-IRES-Cre mice (Jackson Laboratory)36 (link). Briefly, tissue was dissected from the ventral midbrain and digested in a papain solution oxygenated with a carbogen (95% O2 + 5% CO2) at 37 °C for 30 min. The digested tissue was brought to a single cell suspension by trituration through pipette tips of increasingly smaller sizes and centrifuged at 500 g for 10 min. The neurons were resuspended in a prewarmed neuron medium (Neurobasal A (10888022, Gibco) with 1% GlutaMAX (35050061, Gibco), 2% B-27 plus (A3582801, Gibco), 200 µM ascorbic acid, 500 µM kynurenic acid, and 0.1% Pen-Strep solution (P0781, Sigma)).
The cells were plated in neuron medium in six-well plates on a monolayer of glia cells grown on poly-d -lysine coated coverslips (⌀ = 25 mm). Two hours after plating neurons, rat glial cell-derived neurotrophic factor (SRP3239, Sigma) was added for a final concentration of 10 ng/mL. The cultures were used for experiments 14–21 days after the neurons were plated out. The neurons obtained from DAT-IRES-Cre mice were transduced 5 days post dissection with pAAV-FLEX-tdTomato (Addgene).
The cells were plated in neuron medium in six-well plates on a monolayer of glia cells grown on poly-