The generation of cultures were approved by the Animal Experimentation Inspectorate, Denmark (Permission 2017–15–0201–01177). All efforts were made to minimize animal suffering and to reduce the number of animals used. Wistar rat pups (Charles River) at postnatal day 1 to 2 were used as described (36 (link)) for generation of cultures of a monolayer of cortical astrocytes with midbrain dopaminergic neurons atop. The cultures were plated on 25 mm poly-D-lysine coated coverslips in 6-well plates using a serum-free medium (Neurobasal A (Gibco) with 1% GlutaMAX (Gibco), 2% B-27 plus (Gibco), 200 μM ascorbic acid, 500 μM kynurenic acid and 0.1% Pen-Strep solution (Sigma-Aldrich) and half the medium was changed twice a week. The astrocytes were grown until 70 to 80% confluent following the addition of 6.7 μg/ml 5-fluoro-2′-deoxyuridine to inhibit further cell division. Rat glial cell-derived neurotrophic factor was included two hours after plating midbrain neurons for a final concentration of 10 ng/ml. The cultures were used for imaging after 14 to 21 days in vitro.
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