SKELETAL MUSCLE-DERIVED TROPHIC FACTORS PREVENT MOTONEURONS FROM ENTERING AN ACTIVE CELL-DEATH PROGRAM IN-VITRO

The purpose of the experiments reported here is to provide evidence th at motoneurons (MTNs) isolated from chick embryo spinal cords go throu gh an active process of cell death when deprived of trophic support in vitro. In order to analyze and characterize this process, MTNs were i solated with a metrizamide gradient technique and cultured in the pres ence of saturating concentrations of soluble muscle extract. When musc le extract was washed off from the cultures, MTNs entered a process of cell death that could be blocked with inhibitors of mRNA and protein synthesis. Two other additional criteria were used to define this proc ess as an active one. First, ultrastructural analysis of MTNs dying as a consequence of muscle extract deprivation showed that some, but not all, of the MTNs displayed clear signs of apoptotic cell death. Those included cytoplasm condensation, fragmentation of chromatin, and pres ervation of cytoplasmic organelles. Second, internucleosomal degradati on of DNA was detected in MTNs deprived of muscle extract. When DNA wa s analyzed by Southern hybridization techniques using digoxigenin-labe led genomic probes, a clear ladder pattern could be identified on musc le extract-deprived MTNs. The degradation of DNA upon trophic deprivat ion could be prevented by cycloheximide (CHX). In an attempt to charac terize further the process of active cell death in MTNs, we found a ti me point of commitment to cell death of similar to 10 hr by using thre e different approaches: muscle extract deprivation plus readdition of muscle extract, muscle extract deprivation plus addition of CHX, and m uscle extract deprivation plus addition of actinomycin D. Moreover, we show that MTNs deprived of trophic support from muscle extract but ma intained alive with CHX could not be rescued from cell death by readdi ng muscle extract if CHX was washed off the cultures within the first 15 hr of muscle extract deprivation. However, muscle extract alone was able to rescue MTNs that had been kept alive with CHX for periods of time longer than 24 hr after muscle extract deprivation. From these re sults we postulate that the activation of the cell death program after trophic deprivation is transient.