Effect of cycloheximide and mRNA synthesis inhibition on death of trophically deprived ciliary ganglion neurons in culture

J. L. Bruses and G. R. Pilar Department of Physiology and Neurobiology, University of Connecticut, Storrs 06269, USA. 1. The relationship between cycloheximide (CHX) and RNA synthesis inhibitors on trophic-deprived neuronal survival was studied with the use of primary cultures of stage (St) 34 chick ciliary ganglion (CG) neurons, to analyze the biological process of neuronal death caused by trophic factor withdrawal. Tissue culture conditions were refined by characterizing the additional medium components required to obtain 100% survival, for at least 1 wk, in the presence of an eye extract [choroid, ciliary body, iris, and pigment epithelium (CIPE)] as a trophic support for the neurons. Highly enriched neuronal cultures almost devoid of nonneuronal cells were used. 2. The time at which trophically deprived neurons cannot be rescued by the addition of trophic support, "commitment point," was established to be between 11 and 17 h after trophic deprivation. 3. CHX, an inhibitor of protein translation, reduced 3H-leucine incorporation by 90-95%, at a concentration of 10-100 micrograms/ml. The effect of the RNA transcription blockers actinomycin D (Act-D), alpha-amanitin, and 5.6 dichlorobenzimidazole riboside (DRB) on 3H-uridine incorporation into macromolecules was evaluated. Total RNA synthesis was inhibited by 10-25% by alpha-amanitin, whereas Act-D and DRB inhibited 80-97.5% of the 3H-uridine incorporation. 4. The effect of short- and long-term incubation with CHX on neuronal survival was analyzed. Continuous application of CHX promoted survival for 2-3 days, but thereafter neurons died regardless of whether CIPE was present or absent. Application of CHX for 6 h from the onset of the culture was enough to delay the commitment point up to 24 h after plating, and the addition of CIPE at this time maintained survival and promoted differentiation of CHX treated neurons. 5. The RNA transcription blockers Act-D, alpha-amanitin, and DRB were applied to both trophically deprived and trophically supported neurons, and the survival of each was evaluated. Neither drug was effective in supporting the survival of trophically deprived neurons in culture, and in most cases neurons even when cultured with CIPE died within 1-2 days in the presence of either drug. 6. Experiments using both CHX and mRNA synthesis blockers were performed to determine the effect of blocking mRNA transcription in trophically deprived neurons rescued by CHX. The addition of mRNA synthesis inhib