Primary cultures of mouse cerebellar granule cells

CD-1 mice were obtained from Envigo RMS S.r.l. (S. Pietro al Natisone, Udine, Italy). Primary cultures of cerebellar granule cells were prepared from 7-day old mice of mixed sexes, using trypsin digestion (0.25% w/v), as previously described [34]. Dissociated cells were mixed with horse serum (final concentration 20%) (Euroclone, Milan, Italy) and collected by centrifugation. Cells were resuspended in Neurobasal-A medium containing N-2 supplement (Gibco-Thermo Fisher Scientific, Waltham, MA, USA), 0.5 mM L-glutamine, 50 µM β-mercaptoethanol and penicillin/streptomycin. Cells were plated on either 6-well plates or glass coverslips (Electron Microscopy Sciences, Fort Washington, PA, USA) pre-coated with 0.01% poly-L-lysine (Sigma-Aldrich) at the density of ~ 1 × 10⁶ and 1.0 × 10⁴ cells/well, respectively. After 5–6 h, the medium was replaced with fresh medium supplemented with 25 mM KCl. Cultures were used 5–7 days after plating and contained ~ 90% neurons as assessed by immunofluorescence staining with anti-neurofilament 160/200 (NF160/200) and anti-glial fibrillary acidic protein antibodies (Sigma-Aldrich). Experiments were performed according to the recommendations of the European Commission (EU Directive 2010/63/EU for animal experimentation) and were approved by the Institutional Ethical Committee.