Intracellular calcium levels influence apoptosis in mature sensory neurons after trophic factor deprivation

Embryonic dorsal root ganglion (DRG) neurons require nerve growth factor (NGF) for survival in vitro. Withdrawal of NGF results in an apoptotic death in these immature DRG neurons. After time in culture, DRG neurons become progressively less dependent upon NGF for survival. Immature embryonic DRG neurons remain highly dependent upon NGF during their first 14 days in cell culture but by Day 21 the majority lose their NGF dependence for survival. During this period of maturation, the intracellular calcium concentration ([Ca²⁺](i)) progressively increases from the lower levels found in immature DRG neurons to the higher levels that are characteristic of older or mature DRG neurons. By changing the cell culture medium to one with very low calcium, we were able to lower [Ca²⁺](i) in the mature neurons to levels similar to those found in immature neurons. These mature neurons (e.g., E-15 DRG neurons grown for 21 days in culture), normally NGF independent, became highly dependent upon NGF for survival. The onset of DNA fragmentation is a marker of apoptotic cell death. We measured the onset of DNA fragmentation in apoptotic neurons with use of the fluorescent dye, Hoechst, in neurons maintained in either standard calcium medium (1800 mM) or in the low calcium medium (0.35 mM). A higher percentage of neurons with lowered [Ca²⁺](i) showed initial signs of apoptosis, i.e., DNA condensation, at earlier times after NGF deprivation. This work provides further evidence to support a 'set-point' hypothesis regarding the relationship between intracellular calcium concentration and NGF dependence for survival in DRG neurons.