Culturing Primary Insect Neuronal Cells

Primary neuron cultures were established as previously described^{19 (link),32 (link),33 (link),60 (link),73 (link)}. In brief, 20 tribolium or 2 locust brains per culture were dissected and collected in Leibowitz 15 medium (Gibco; Life Technologies, Darmstadt, Germany) supplemented with 1% penicillin/streptomycin and 1% amphotericin B (both Sigma-Aldrich, Munich, Germany) (from now referred to as L15 medium). Subsequently, brains were enzymatically digested in collagenase/dispase (2 mg/ml, Sigma-Aldrich, Munich, Germany) for 45 min (T. castaneum) or 30 min (L. migratoria) at 27 °C. Enzymatic reaction was stopped by repeated washing in Hanks’ balanced salt solution and brains were mechanically dissociated by repeated pipetting in L15. The suspension of dissociated brain cells was seeded on Concanavalin A (Sigma-Aldrich, Munich, Germany) coated coverslips and let to rest for 2 h. Afterwards, culture dishes were filled with L15 supplemented with 5% fetal bovine serum gold (FBSG, PAA Laboratories GmbH, Pasching, Austria). Medium was replaced by L15 plus FBSG on day two and by L15 without serum on day four in vitro. Primary cell cultures were maintained at 27 °C without CO₂ buffering.