INTRACELLULAR CALCIUM LEVELS INFLUENCE APOPTOSIS IN MATURE SENSORY NEURONS AFTER TROPHIC FACTOR DEPRIVATION

Embryonic dorsal root ganglion (DRG) neurons require nerve growth fact or (NGF) for survival in vitro. Withdrawal of NGF results in an apopto tic death in these immature DRG neurons. After time in culture, DRG ne urons become progressively less dependent upon NGF for survival. Immat ure embryonic DRG: neurons remain highly dependent upon NGF during the ir first 14 days in cell culture but by Day 21 the majority lose their NGF dependence for survival, During this period of maturation, the in tracellular calcium concentration ([Ca2+](i)) progressively increases from the lower levels found in immature DRG neurons to the higher leve ls 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 foun d in immature neurons. These mature neurons (e,g., E-15 DRG neurons gr own for 21 days in culture), normally NGF independent, became highly d ependent upon NGF for survival. The onset of DNA fragmentation is a ma rker of apoptotic cell death, We measured the onset of DNA fragmentati on in apoptotic neurons with use of the fluorescent dye, Hoechst, in n eurons maintained in either standard calcium medium (1800 mM) or in th e low calcium medium (0.35 mM). A higher percentage of neurons with lo wered [Ca2+](i) showed initial signs of apoptosis, i.e., DNA condensat ion, at earlier times after NGF deprivation. This work provides furthe r evidence to support a ''set-point'' hypothesis regarding the relatio nship between intracellular calcium concentration and NGF dependence f or survival in DRG neurons. (C) 1996 Academic Press, Inc.