Mars T, Yu K J, Tang X M, Miranda A F, Grubic Z, Cambi F, King M P
Institute of Pathophysiology, School of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia.
J Comp Neurol. 2001 Sep 17;438(2):239-51. doi: 10.1002/cne.1312.
Motor axons extending from embryonic rat spinal cord explants form fully mature neuromuscular junctions with cocultured human muscle. This degree of maturation is not observed in muscle innervated by dissociated motor neurons. Glial cells present in the spinal cord explants seem to be, besides remaining interneurons, the major difference between the two culture systems. In light of this observation and the well documented role of glia in neuronal development, it can be hypothesized that differentiated and long-lived neuromuscular junctions form in vitro only if their formation is accompanied by codifferentiation of neuronal and glial cells and if this codifferentiation follows the spatial and temporal pattern observed in vivo. Investigation of this hypothesis necessitates the characterization of neuronal and glial cell development in spinal cord explant-muscle cocultures. No such study has been reported, although these cocultures have been used in numerous studies of neuromuscular junction formation. The aim of this work was therefore to investigate the temporal relationship between neuromuscular junction formation and the differentiation of neuronal and glial cells during the first 3 weeks of coculture, when formation and development of the neuromuscular junction occurs in vitro. The expression of stage-specific markers of neuronal and glial differentiation in these cocultures was characterized by immunocytochemical and biochemical analyses. Differentiation of astrocytes, Schwann cells, and oligodendrocytes proceeded in concert with the differentiation of motor neurons and neuromuscular junction formation. The temporal coincidence between maturation of the neuromuscular junction and lineage progression of neurons and glial cells was similar to that observed in vivo. These findings support the hypothesis that glial cells are a major contributor to maturity of the neuromuscular junction formed in vitro in spinal cord explant-muscle cocultures.
从胚胎大鼠脊髓外植体延伸出的运动轴突与共培养的人类肌肉形成完全成熟的神经肌肉接头。在由解离的运动神经元支配的肌肉中未观察到这种成熟程度。脊髓外植体中存在的神经胶质细胞似乎是这两种培养系统之间的主要差异，除此之外还有残留的中间神经元。鉴于这一观察结果以及神经胶质细胞在神经元发育中已得到充分记录的作用，可以推测，只有当神经肌肉接头的形成伴随着神经元和神经胶质细胞的共同分化，并且这种共同分化遵循体内观察到的空间和时间模式时，才会在体外形成分化良好且持久的神经肌肉接头。对这一假设的研究需要对脊髓外植体 - 肌肉共培养物中神经元和神经胶质细胞的发育进行表征。尽管这些共培养物已被用于众多神经肌肉接头形成的研究中，但尚未有此类研究报道。因此，这项工作的目的是研究在共培养的前三周内神经肌肉接头形成与神经元和神经胶质细胞分化之间的时间关系，此时神经肌肉接头的形成和发育在体外发生。通过免疫细胞化学和生化分析对这些共培养物中神经元和神经胶质细胞分化的阶段特异性标志物的表达进行了表征。星形胶质细胞、雪旺细胞和少突胶质细胞的分化与运动神经元的分化和神经肌肉接头的形成同步进行。神经肌肉接头成熟与神经元和神经胶质细胞谱系进展之间的时间巧合与体内观察到的相似。这些发现支持了以下假设：神经胶质细胞是脊髓外植体 - 肌肉共培养物中体外形成的神经肌肉接头成熟的主要贡献者。
