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Journal of Neuroscience, Vol 14, 2674-2686, Copyright © 1994 by Society for Neuroscience
Skeletal muscle-derived trophic factors prevent motoneurons from entering an active cell death program in vitro
JX Comella, C Sanz-Rodriguez, M Aldea and JE Esquerda
Departament de Ciencies Mediques Basiques, Facultat de Medicina, Universitat de Lleida, Spain.
The purpose of the experiments reported here is to provide evidence that
motoneurons (MTNs) isolated from chick embryo spinal cords go through an
active process of cell death when deprived of trophic support in vitro. In
order to analyze and characterize this process, MTNs were isolated with a
metrizamide gradient technique and cultured in the presence of saturating
concentrations of soluble muscle extract. When muscle 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 process 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 preservation of cytoplasmic organelles.
Second, internucleosomal degradation of DNA was detected in MTNs deprived
of muscle extract. When DNA was analyzed by Southern hybridization
techniques using digoxigenin-labeled genomic probes, a clear ladder pattern
could be identified on muscle extract-deprived MTNs. The degradation of DNA
upon trophic deprivation could be prevented by cycloheximide (CHX). In an
attempt to characterize further the process of active cell death in MTNs,
we found a time point of commitment to cell death of approximately 10 hr by
using three different approaches: muscle extract deprivation plus
readdition of muscle extract, muscle extract deprivation plus addition of
CHX, and muscle extract deprivation plus addition of actinomycin D.
Moreover, we show that MTNs deprived of trophic support from muscle extract
but maintained alive with CHX could not be rescued from cell death by
reading 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 results we
postulate that the activation of the cell death program after trophic
deprivation is transient.
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