Trophic influences on embryonic chicken spinal motor neurons

Abstract

Embryonic spinal motor neurons are generated in excess. Superfluous neurons are subsequently eliminated by a process of naturally occurring cell death which coincides with the innervation of the target tissue, skeletal muscle. From extensive in vivo and in vitro studies, motor neurons have been shown to depend upon a target-derived trophic factor in order to survive the period of cell death. However, such a trophic factor for motor neurons has not been identified and characterized as yet. To investigate the trophic influences of skeletal muscle and of other cells interacting with motor neurons, purified motor neuron cultures were established from 6-day-old chicken embryos using metrizamide step-gradient centrifugation. One of the cell fractions was enriched in spinal motor neurons since it contained cells with a high specific activity of choline acetyltransferase (CAT), a high percentage of retrogradely labeled cells, and which had a large average cell diameter. Our laboratory has isolated a neurite-promoting protein from striated muscles. This protein has been further characterized by various biochemical, biological, and immunological assays. when the protein was tested in neuronal cultures including spinal motor, sensory, and autonomic neurons, it supported survival and neuritic growth of all neurons tested but it was not able to promote CAT activity or choline uptake by motor neurons. Although the biological effect of the protein is not specific for motor neurons, it does promote survival and growth of the neurons in culture. Additional trophic influences on spinal motor neurons were studied using media conditioned by skeletal muscle, heart muscle, sensory neurons, astrocytes, and Schwann cells. These conditioned media promoted the survival, neuritic growth, and CAT activity of the cultured motor neurons. Motor neurons especially exhibited distinct neuritic arborization in different conditioned media. These data suggest that the cells interacting with motor neurons in situ may affect the survival and development of the motor neuron. The present study provides some basic properties of the trophic interactions between motor neurons and their environment. Further characterization of the neurite-promoting protein should help in better understanding the nature of trophic interactions at the molecular level.