Ruthel G, Hollenbeck P J
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907, USA.
J Neurosci. 2000 Mar 15;20(6):2266-74. doi: 10.1523/JNEUROSCI.20-06-02266.2000.
Hippocampal neurons developing in culture exhibit two types of differential, seemingly competitive, process outgrowth in the absence of external cues. During the initial acquisition of polarity, one of several equivalent undifferentiated minor neurites preferentially grows to become the axon. Once the axon has formed, it typically branches, and the branches grow differentially rather than concurrently. In axons with only two branches, growth alternates between branches. In both axon establishment and branch growth alternation, growth among sibling processes or branches must be differentially regulated. We found that elaborate and dynamic growth cones were associated with growth, whereas diminished growth cones were associated with nongrowing processes or branches. To test whether growth cones were necessary for differential growth, growth cone motility was eliminated by application of cytochalasin E. Although cytochalasin treatment before axon formation yielded longer processes overall, a similar percentage of both treated and untreated neurons had one process that grew more rapidly and became much longer than its sibling processes. Immunostaining to visualize dephospho-tau, an axonal marker, demonstrated that these single dominant processes were axons. Axons that formed in cytochalasin were thicker and showed more intense anti-tubulin staining than their sibling processes. Branched axons deprived of growth cones retained a pattern of differential growth and often included alternation. These results indicate that neither formation of a single axon nor differential growth of branches are dependent on growth cone motility and suggest that the neuron can regulate neurite elongation at sites other than at the growth cone.
在体外培养条件下发育的海马神经元，在没有外部线索的情况下会表现出两种不同的、看似相互竞争的突起生长方式。在极性初步形成过程中，几个等效的未分化小神经突中的一个会优先生长成为轴突。一旦轴突形成，它通常会分支，并且分支的生长是不同步的，而非同时进行。在只有两个分支的轴突中，生长在两个分支之间交替进行。在轴突形成和分支生长交替过程中，同源突起或分支之间的生长必须受到不同的调节。我们发现，精细且动态的生长锥与生长相关，而缩小的生长锥则与不生长的突起或分支相关。为了测试生长锥对于差异生长是否必要，我们应用细胞松弛素E消除了生长锥的运动性。尽管在轴突形成前进行细胞松弛素处理总体上产生了更长的突起，但处理组和未处理组中都有相似比例的神经元有一个突起比其同源突起生长得更快且长得多。通过免疫染色来观察轴突标记物去磷酸化tau，结果表明这些单一的优势突起就是轴突。在细胞松弛素存在的情况下形成的轴突比其同源突起更粗，并且抗微管蛋白染色更强。剥夺生长锥的分支轴突保留了差异生长模式，并且常常包括交替生长。这些结果表明，单个轴突的形成和分支的差异生长都不依赖于生长锥的运动性，这表明神经元可以在生长锥以外的部位调节神经突的伸长。
