Effects of innervation on myoblastic progenitor cells using embryonic chick cocoltures

In this work we report a description of a muscular and neuronal coculture preparation performed on chick embryos with the aim of studying the effects of axonal connections on muscle determination and differentiation. We obtained neuronal cells by spinal cord (sc) explants of chicks at Embryonic Day 5 (ED 5) by means of enzymatic digestion. Small sc fragments were added in cultured myoblasts isolated from the chick adductor muscle at ED12. The validation of the experimental model was confirmed by a remarkable spreading pattern of neuronal cells, labeled with a NF200 antibody, and the presence of high concentration of myotubes, marked by an -actinin antibody. The novel neuronal junctions were highlighted by -bungarotoxin and synaptophysin1 antibodies. The effects of innervation on myogenic mechanisms were assessed by Real-Time PCR, immunohistochemistry, SDS-gel electrophoresis on MyHC isoforms and calcium imaging analysis with fluo-4 dye. The presence of spinal cord on myotubes undoubtedly accelerates myogenesis; the expression of all myogenic factors analyzed increased in coculture, including MEF-2A and myostatin. Moreover, in our model the excitation-contraction related [Ca2+]i signals presented an altered kinetics. Overall, the experimental model presented in this work might be a useful tool in order to study the cascade of myogenic signals activated by paracrine neuronal factors.