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.