Characterization of two factors enhancing choline acetyltransferase activity in cultures of purified rat motoneurons.
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).
Martinou
J C
JC
Laboratoire de Pharmacologie et de Toxicologie Fondamentales, Centre National de la Recherche Scientifique 205, Toulouse, France.
Le Van Thai
A
A
Cassar
G
G
Roubinet
F
F
Weber
M J
MJ
D000818:Animals; D002232:Carbocyanines; D002454:Cell Differentiation / Q000187:drug effects; D002469:Cell Separation; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D002795:Choline O-Acetyltransferase / Q000378:metabolism*; D003470:Culture Media / Q000032:analysis / Q000494:pharmacology; D004622:Embryo, Mammalian; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D009046:Motor Neurons / Q000187:drug effects / Q000201:enzymology* / Q000502:physiology; D009132:Muscles / Q000166:cytology; D051381:Rats; D013116:Spinal Cord / Q000166:cytology; D013347:Subcellular Fractions / Q000201:enzymology
C035005:3,3'-dihexadecylindocarbocyanine; D002232:Carbocyanines; D003470:Culture Media; D005456:Fluorescent Dyes; D002795:Choline O-Acetyltransferase