A neurite‐promting factor from muscle supports the survival of cultured chicken spinal motor neurons

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Abstract
During embryonic development, spinal motor neurons require muscle‐derived trophic factors for their survival and growth. We have recently isolated a protein from muscle that is not laminin but that still stimulates neurite outgrowth from embryonic neurons in culture. In the present study, we investigated whether this protein, which we refer to as muscle‐derived neurite‐promoting factor (NPF), could also promote the survival and growth of motor neurons in culture. Spinal motor neurons were isolated from 6‐day‐old chicken embryos by a metrizamide step‐gradient centrifugation protocol. Most large cells (putative motor neurons) were found in the upper metrizamide fraction (0%–6.8% interface; fraction I). Motor neurons were identified by increased specific activity of choline acetyltransferase (CAT) and by their propensity to transport retrogradely either wheat germ agglutininhorseradish peroxidase or the fluorescent dye, 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine per chlorate (diI), when those substances were injected into the target field. Labeled motor neurons were 2.6‐fold enriched in fraction I and the specific CAT activity was 4.4‐fold increased in fraction I as compared to unfractionated cells. When motor neurons were grown on muscle‐derived NPF, the protein supported the survival of at least 21% of the neurons for as long as 6 days in culture. The protein showed no significant effect on either CAT specific activity or on high‐affinity choline uptake by neurons. There was a substantial increase from 21% to 38% of the survival of motor neurons when a combination of muscle‐derived NPF also promoted the survival of sensory neurons and sympathetic neurons in culture. Our results demonstrate that a neurite‐promoting protein derived from muscle promotes both the survival and the outgrowth of neurites from cultured spinal motor neurons as well as from sensory and sympathetic neurons.

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