Cortical neurons were harvested from embryonic day-18 Wistar rats (either sex, Charles River Laboratories, Wilmington, MA). Cultures were prepared according to a previously described procedure with some modifications (Ueno et al., 2012 (link)). Briefly, embryos were removed, and the cerebral cortex dissected, stripped of meninges, and dissociated by a combination of Ca2+- and Mg2+- free Hanks balance salt solution (HBSS) containing 0.125% trypsin digestion for 15 min, then mechanically triturated for ~20 times. The triturated cells were passed through a 40 μm cell strainer and counted to obtain a concentration of 3×107cells/ml.
To separate axons from neuronal soma, a microfluidic chamber (Standard Neuron Device, Cat# SND450, Xona Microfluidics, Temecula, CA) was employed (Taylor et al., 2005 (link); Taylor et al., 2009 (link)). The small dimension of the microgrooves in the chamber allows axons to sprout from the cells seeded compartment into the other compartment of the chamber, but prevents the passage of cell bodies(Taylor et al., 2005 (link); Taylor et al., 2009 (link)). Briefly, cleaned, sterilized, and dried chambers were affixed to poly-D-lysine (PDL) (Sigma-Aldrich, CA) -coated dishes (35mm, Corning). The cortical neurons were plated at a density of 6×105cells/chamber in DMEM with 5% FBS and, incubated for an initial 24 h. After 24 h, cell culture was initiated with the addition of neurobasal growth medium (Invitrogen), 2% B-27 (GIBCO), 2mM GlutaMax, and 1% antibiotic-antimycotic. On day in vitro (DIV) 3, one-half of the medium was replaced with culture medium containing 20 μM 5-fluorodeoxyuridine. The growth media was changed every other day thereafter.
To separate axons from neuronal soma, a microfluidic chamber (Standard Neuron Device, Cat# SND450, Xona Microfluidics, Temecula, CA) was employed (Taylor et al., 2005 (link); Taylor et al., 2009 (link)). The small dimension of the microgrooves in the chamber allows axons to sprout from the cells seeded compartment into the other compartment of the chamber, but prevents the passage of cell bodies(Taylor et al., 2005 (link); Taylor et al., 2009 (link)). Briefly, cleaned, sterilized, and dried chambers were affixed to poly-D-lysine (PDL) (Sigma-Aldrich, CA) -coated dishes (35mm, Corning). The cortical neurons were plated at a density of 6×105cells/chamber in DMEM with 5% FBS and, incubated for an initial 24 h. After 24 h, cell culture was initiated with the addition of neurobasal growth medium (Invitrogen), 2% B-27 (GIBCO), 2mM GlutaMax, and 1% antibiotic-antimycotic. On day in vitro (DIV) 3, one-half of the medium was replaced with culture medium containing 20 μM 5-fluorodeoxyuridine. The growth media was changed every other day thereafter.
