Quantitative analysis of long-term survival and neuritogenesis in vitro: Cochleovestibular ganglion of the chick embryo in BDNF, NT-3, NT-4/5, and insulin

The dynamics of survival and growth were examined for cochleovestibular ganglion (CVG) cells maintained in long-term cultures. CVG cells were explanted from chick embryos after 90 h of incubation into a defined-medium containing BDNF, NT-3, or NT-4/5 and an insulin, transferrin, selenium, and progesterone supplement. Explant survival and neuritogenesis was measured for 23 to 24 days in vitro. All three neurotrophins prolonged CVG survival in a dose-dependent manner although insulin acted as a cofactor. In 0.872 mu M insulin-containing medium the ED50 for BDNF and NT-3 was 100 pg/ml, whereas the ED50 for NT-4/5 was 600-1200 pg/ml. However, at later ages in vitro, survival decreased with concentrations of BDNF greater than 2 ng/ml. In insulin-free medium, concentrations of 5-200 ng/ml of BDNF or 30-200 ng/ml of NT-4/5 maintained the survival of explants at a rate that was equivalent to or less than the survival rate of cultures treated with insulin but not with neurotrophin. In contrast, NT-3-treated explants in insulin-free medium did not survive the duration of the experiment. Dose-dependent effects of BDNF and NT-3 on explant neuritogenesis were reflected as an initial delay in outgrowth, whereas NT-4/5 had no effect. Insulin regulation of neuritogenesis was suggested when outgrowth decreased in the presence of an antibody to the insulin receptor. These data suggest that while all three of these neurotrophins protect the CVG from death the long-term consequences of cofactors and certain dose levels should be considered when treating CVG cells in vivo. (C) 1997 Academic Press.