Culturing and Maintaining Sympathetic Neurons

Sympathetic neurons were harvested from P0 to P1 Sprague Dawley rats or P0 to P4 TrkA^R685A or TrkA^WT mice, enzymatically dissociated, and grown in mass cultures or compartmentalized cultures as described previously (17 (link), 36 (link)). Cells were maintained in culture with high-glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin (1 U/mL), and NGF purified from mouse submaxillary glands (100 ng/mL) (37 (link)). For NGF deprivation, neurons were placed in high-glucose DMEM containing 1.0% FBS with anti-NGF (1:1,000) and BAF (50 μM) for 36 h. For adenoviral infections, neuronal cultures were infected with high-titer CsCl-purified or VivaPure AdenoPack20-purified pAdenoX-Tet3G adenoviruses for 36 h as previously described (9 (link)). Adenovirus-mediated protein expression was induced by adding doxycycline (Sigma, 100 ng/mL) to culture media.