Optical plastic dishes

Superior cervical ganglia (SCG) and dorsal root ganglia (DRG) were isolated from E16–17 Sprague-Dawley rat embryos (Hilltop Labs, Inc., Scottsdale, PA) and neurons were cultured in tri-chambers as described (Ch’ng and Enquist, 2005 ). All animal work was performed in accordance with the Princeton Institutional Animal Care and Use Committee (protocols 1947–13 and 1851–14). Multiwell dishes (Falcon), 35-mm plastic tissue culture dishes (Falcon) or optical plastic dishes (Ibidi) were coated with 500 μg/ml of poly-DL-ornithine (Sigma Aldrich) and 10 μg/ml of natural mouse laminin (Life Technologies). To prepare compartmented neuronal cultures, two sets of evenly spaced parallel grooves were etched on the dishes before a silicone grease-coated tri-chamber (Tyler Research) was placed. SCGs were trypsinized and triturated before plating in the Soma (S) compartment. Neurons were maintained in neurobasal medium (Gibco) supplemented with 100 ng/ml nerve growth factor 2.5S (Invitrogen), 2% B27 (Gibco) and 1% penicillin and streptomycin with 2 mM glutamine (Life Technologies). Two days after plating, 1 mM cytosine-D-arabinofuranoside (Sigma-Aldrich) was added to eliminate non-neuronal dividing cells. Neurons were cultured for 14–21 days.