Journal of Neuroscience, Vol 9, 3645-3656, Copyright © 1989 by Society for Neuroscience
Characterization of two factors enhancing choline acetyltransferase activity in cultures of purified rat motoneurons
JC Martinou, A Le Van Thai, G Cassar, F Roubinet and MJ Weber
Laboratoire de Pharmacologie et de Toxicologie Fondamentales, Centre National de la Recherche Scientifique 205, Toulouse, France.
Motoneurons from E14 rat embryos have been retrogradely labeled with the
carbocyanine derivative dil and purified 12-fold by centrifugation on a
density gradient made of Nycodenz, as assessed by the increase in CAT
activity per cell and in the percentage of dil-labeled cells. A 20- to
36-fold purification was achieved by the microdissection of the labeled
lumbar motor columns followed by density-gradient centrifugation.
Motoneurons were then purified to near homogeneity by
fluorescence-activated cell sorting. In certain experiments, motoneurons
were purified by a novel cell-sorting procedure that only uses
light-scatter parameters and does not necessitate retrograde labeling. In
cultures of motoneurons enriched by centrifugation, muscle- conditioned
media (CM) had only a marginal effect on the overall neuronal survival but
increased up to 10-fold the number of labeled motoneurons surviving after 7
d in culture. CAT activity per culture was quantitatively stimulated to the
same extent, suggesting that the increase in CAT activity caused by CM
mostly reflected better survival of motoneurons. In cultures of motoneurons
purified by cell sorting, CM increased both survival and CAT expression per
motoneuron. We have partially purified 2 factors from CM that stimulate CAT
activity per motoneuron 2- to 3-fold without affecting their survival. One
of these factors is probably identical to the factor involved in the
cholinergic/noradrenergic choice of sympathetic neurons (Fukada, 1985).
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