DEATH OF DEVELOPING SEPTAL CHOLINERGIC NEURONS FOLLOWING NGF WITHDRAWAL IN-VITRO - PROTECTION BY PROTEIN-SYNTHESIS INHIBITION

Fetal septal neurons were grown in vitro under glass coverslips. This sandwich culture method significantly increased general neuronal survi val, reduced glial proliferation, and permitted the removal of serum f rom the growth medium after 5 d in vitro. Thereafter, a simple, and co mpletely defined, medium was used, and the effects of NGF, NGF withdra wal, and protein synthesis inhibition were examined on septal choliner gic neurons. NGF added to septal cultures at the time of plating resul ted in a threefold increase in the number of cholinergic neurons seen at 14 d in vitro but had no effect on the survival of non-cholinergic cells. Cholinergic neurons identified by staining for AChE, ChAT, and p75(NGFR) could be maintained in serum-free, NGF-supplemented medium f or over 40 d. When NGF was removed and NGF antibodies added to 14-d-ol d cultures, less than 30% of cholinergic neurons survived a further 4 d, but when NGF was similarly withdrawn from 35-d-old cultures, over 7 5% of cholinergic neurons survived. Reapplication of NGF after 3 but n ot after 12 or more hours of NGF withdrawal from 14-d-old cultures pre vented the death of most cholinergic neurons. When NGF was withdrawn f rom 14-d-old cultures in the presence of the protein synthesis inhibit or cycloheximide, over 75% of the cholinergic neurons survived. These findings suggest that septal cholinergic neurons are dependent on NGF for survival only during a critical period of development and that gro wth factor-regulated developmental cell death may occur in CNS neurons by activation of programmed cell death requiring protein synthesis.