Motoneurons purified by cell sorting respond to two distinct activities in myotube-conditioned medium

Abstract

Spinal motoneurons from chick embryos were purified by retrograde transport and fluorescence-activated cell sorting. Growth conditions for motoneurons were studied, with experiments focused on the effects of conditioned media from chick myotubes, fibroblasts, and spinal cord dividing cells. Motoneurons rapidly extended neurites when plated onto polylysine-coated dishes that had been exposed to these conditioned media. Enzymatic analysis of the substratum-binding, neurite outgrowth-promoting activity from myotube-conditioned medium indicated that it contained heparan sulfate and protein. The neurite outgrowth-promoting activity sedimented as a peak centered at a density of 1.34 in associative cesium chloride gradients, and eluted near the void volume of a Sepharose CL-6B column. Inclusion of myotube conditioned medium in the culture medium of motoneurons also enhanced their survival over periods greater than 2 days in culture. This enhancement of survival could not be explained by myotubeconditioned medium providing motoneurons with a continuous supply of the neurite outgrowth-promoting activity. Media conditioned by spinal cord dividing cells and fibroblasts supported motoneuron survival to some extent, but this effect was not as great as that of myotube-conditioned medium.

Trophic Support of Motoneurons: Physiological, Pathophysiological, and Therapeutic Implications

“…Experimental achievements were confined to the demonstration that skeletal muscle extract or conditioned medium of primary cultures of skeletal muscle my ob lasts trophically supported motoneurons in vivo (75) and in vitro (11,21,24,25,57,73,89,99).…”

Response of purified chick motoneurons to myotube conditioned medium: Laminin is essential for the substratum-binding, neurite outgrowth-promoting activity

“…Knowledge of this work has led many investigators to examine the effects of muscle extracts or medium conditioned over cultures of myotubes on the growth of cultured whole spinal cord cells or explants, in an attempt to discover molecules important for motoneuron development [ The development of techniques for purifying embryonic chick spinal motoneurons has recently made cultures of purified motoneurons available for study [3,16]. We have reported that purified motoneurons respond to two distinct activities in myo- tube-conditioned medium (MCM): a neurite outgrowth-promoting activity (NOPA), which binds to the culture substratum and acts to promote rapid, profuse outgrowth of motoneuron processes; and a survival-enhancing activity, which acts over the course of a week in culture to promote motoneuron survival [3]. Further characterization of the substratum-binding NOPA by biochemical and immunochemical techniques, reported below, has demonstrated that laminin (LA) is essential for the ability of MCM to promote motoneuron neurite outgrowth.…”

“…Motoneurons were purified from chick embryos by retrograde axonal transport of a fluorescent dye and fluorescence-activated cell sorting [3]. Tissue culture plates (96-well; Costar) were exposed to poly-D-lysine and treated with samples to be tested for NOPA (50 #l/well) overnight at 4°C.…”

“…Tissue culture plates (96-well; Costar) were exposed to poly-D-lysine and treated with samples to be tested for NOPA (50 #l/well) overnight at 4°C. Wells were washed and motoneurons plated into them at a density of 3000-5000 cells/well and maintained for 24 h in serum-free growth medium [3].…”

“…MCM was obtained from chick myotube cultures established according to the method of Konigsberg [7] and maintained as described [3]. After fusion of myotubes, cultures were periodically treated with 10 #M cytosine-~-D-arabinofuranoside to suppress fibroblast growth.…”