Buchanan J, Sun Y A, Poo M M
Section of Molecular Neurobiology, Yale University School of Medicine, New Haven, Connecticut 06510.
J Neurosci. 1989 May;9(5):1540-54. doi: 10.1523/JNEUROSCI.09-05-01540.1989.
We have studied the fine structure of nerve-muscle contacts during the first few hours of synaptogenesis in embryonic Xenopus cell cultures. The precise timing of contact was achieved by manipulating isolated spherical myocytes (myoballs) into contact with growth cones or neurites of co-cultured spinal neurons. The contacts were shown to be functional by whole-cell voltage-clamp recording of nerve-evoked synaptic currents in the muscle cell. The ultrastructure of these functional contacts was examined by thin-section electron microscopy. In total, 20 nerve-muscle pairs were studied with contact periods ranging from 20 min to 12 hr, during which time a substantial increase in the amplitude of synaptic currents occurred. The structure of noncontacting cells and of nerve-muscle contacts formed between the cells by natural encounters in 1-d-old cultures were also examined in order to identify the features and the time course of morphological differentiation of early functional contacts. Prominent features of the contact area during the first few hours included: close apposition of the nerve and muscle membranes, greater frequency of coated pits and vesicles, and thickening of postsynaptic muscle membrane. Occasionally, clusters of clear vesicles occurred near presynaptic membrane, but no further sign of active zone differentiation was observed. In comparison, definitive active zone structure, well-formed extracellular basal lamina, and widened cleft were seen in natural contacts less than 24 hr old. This study of the identified functional contacts may help us to understand the structural basis for early nerve-muscle interaction and the functional significance of various synaptic specializations.
我们研究了非洲爪蟾胚胎细胞培养中突触发生最初几个小时内神经 - 肌肉接触的精细结构。通过操控分离的球形肌细胞（肌球）与共培养的脊髓神经元的生长锥或神经突接触，实现了接触时间的精确控制。通过对肌肉细胞中神经诱发的突触电流进行全细胞电压钳记录，证明这些接触具有功能。通过超薄切片电子显微镜检查了这些功能性接触的超微结构。总共研究了20对神经 - 肌肉，接触时间从20分钟到12小时不等，在此期间突触电流幅度大幅增加。还检查了非接触细胞的结构以及1日龄培养物中细胞间自然接触形成的神经 - 肌肉接触的结构，以确定早期功能性接触的形态分化特征和时间进程。最初几个小时内接触区域的突出特征包括：神经和肌肉膜紧密并列、被膜小窝和小泡的频率增加以及突触后肌肉膜增厚。偶尔，在突触前膜附近会出现清亮小泡簇，但未观察到活性区分化的进一步迹象。相比之下，在小于24小时的自然接触中可见明确的活性区结构、良好形成的细胞外基膜和增宽的间隙。对已确定的功能性接触的这项研究可能有助于我们理解早期神经 - 肌肉相互作用的结构基础以及各种突触特化的功能意义。
