Brain-derived neurotrophic factor, acidic and basic fibroblast growth factors, insulin-like growth factor-I, and various antioxidants do not prevent the apoptotic death of developing septal cholinergic neurons following nerve growth factor withdrawal in vitro

Abstract

The degree to which growth factors act alone or in combination to influence neuronal survival during the developmental of the central nervous system is not well understood. In this study, we investigated whether multiple growth factors might interact to regulate the survival of developing basal forebrain cholinergic neurons in vitro, in the rat. We have previously shown that most embryonic septal cholinergic neurons grown in sandwich cultures in serum-free, completely defined medium are dependent on nerve growth factor during a critical period of their development, such that nerve growth factor withdrawal during this period results in the protein synthesis-dependent, apoptotic death of most, but not all, of these neurons. Here we report that brain-derived neurotrophic factor, acidic and basic fibroblast growth factors, and insulin-like growth factor-I applied individually in serum-free, completely defined medium, were not able either to support the development of septal cholinergic neurons from plating at embryonic day 16, or to prevent the cell death of these neurons induced by nerve growth factor withdrawal during days 14–18 after plating. We also found that the apoptotic death of developing septal cholinergic neurons induced by nerve growth factor withdrawal was not prevented by a number of antioxidants, with the exception of a high concentration (50mM) of ascorbic acid. However, this effect of ascorbic acid was prevented when pH was buffered, and is likely to have been mediated via a proton-induced sustained neuronal depolarization.These findings suggest that in the absence of serum and other additives, brain-derived neurotrophic factor, acidic and basic fibroblast growth factors, and insulin-like growth factor-I do not interact with nerve growth factor to regulate the survival of septal cholinergic neurons during the developmental period spanned by this in vitro model. In addition, the findings suggest that the apoptotic death of septal cholinergic neurons induced by nerve growth factor withdrawal is not mediated by oxidative stress or free radical generation.