Muscle-derived factors that support survival and promote fiber outgrowth from embryonic chick spinal motor neurons in culture

Abstract

The purpose of the experiments reported is to provide an unambiguous demonstration that embryonie skeletal muscle contains factors that act directly on embryonie spinal motor neurons both to support their survival and to stimulate the outgrowth of neurites. Cells of lumbar and brachial ventral spinal cords from 6-day-old chick embryos were separated by centrifugation in a two-step metrizamide gradient, and a motor neuron enriched fraction was obtained. Motor neurons were identified by retrogradely labeling with rhodamine isothiocyanate, and were enriched fourfold in the motor neuron fraction relative to unfractionated cells. In culture, the isolated motor neurons died within 3-4 days unless they were supplemented with embryonie chick skeletal muscle extract. Two functionally distinct entities separable by ammonium sulfate precipitation were responsible for the effects of muscle extracts on motor neurons. The 0-25% ammonium sulfate precipitate contained molecules that alone bad no effect on neuronal survival but when bound to polyornithine-coated culture substrata, stimulated neurite outgrowth and potentiated the survival activity present in muscle. Mostofthis activity was due to a laminin-like molecule being immunoprecipitated with antisera against laminin, and immunoblotting demonstrated the presence of both the A and B chains of laminin. A long-term survival activity resided in the 25-70% ammonium sulfate fraction, and its apparent total and specific activities were strongly dependent on the culture substrate. In contrast to the motor neurons, the cells from the other metrizamide fraction (including neuronal cells) could be kept in culture for a prolonged time without addition of exogenous factor(s).

A Globular, Not Asymmetric, Form of Acetylcholinesterase Is Expressed in Chick Motor Neurons: Down‐Regulation Toward Maturity and After Denervation

“…We first separated motor neurons from other cells of E6 chick spinal cords. The separation procedure resulted in two fractions, one of which was enriched with motor neurons, the other enriched with nonmotor neurons (Dohrmann et al, 1986). Figure 4 shows that the AChE catalytic subunit was recognized by ACB-1 antibody at -~105kDa as previously described for chick AChE (Randall et a!., 1987;Tsim et al, 1988a-c).…”

“…Motor neurons were isolated from chick spinal cords as described by Dohrmann et al (1986). In brief, cervical and lumbar regions of spinal cords from embryonic day 6 (E6) chick spinal cords were removed and incubated in 0.5 mg ml -' trypsin for 30 mm.…”

Extracellular Heat Shock Protein 70: A Critical Component for Motoneuron Survival

“…Neurotrophic factors are defined by their ability to support the survival of developing and/or mature neurons, and the dependence of motoneurons on target-derived trophic support is well documented (Dohrmann et al, 1986;Oppenheim et al, 1988;Crews and Wigston, 1990;Eagleson et al, 1990). A deficit of trophic factors has been proposed as an underlying cause of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) (Appel, 1981).…”

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).…”