Ohnami Soichiro, Endo Mitsuharu, Hirai Satoshi, Uesaka Naofumi, Hatanaka Yumiko, Yamashita Toshihide, Yamamoto Nobuhiko
Laboratory of Cellular and Molecular Neurobiology, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan.
J Neurosci. 2008 Sep 10;28(37):9117-21. doi: 10.1523/JNEUROSCI.1731-08.2008.
During development, axon branching is influenced by sensory-evoked and spontaneous neural activity. We studied the molecular mechanism that underlies activity-dependent branch formation at horizontally elongating axons (horizontal axons) in the upper cortical layers, focusing on Rho family small GTPases. Axonal labeling with enhanced yellow fluorescent protein showed that horizontal axons formed several branches in organotypic slice cultures. This branch formation was considerably increased by introducing constitutively active RhoA and was slightly inhibited by dominant-negative RhoA. Activators and inhibitors of endogenous RhoA signaling also promoted and inhibited branching, respectively. Daily imaging of horizontal axon growth further demonstrated that constitutively active RhoA increased the dynamic addition and loss of branches. Moreover, the amount of active RhoA relative to the total amount of RhoA was examined by a pull-down assay in cortical slices treated with sodium channel or glutamate receptor blockers to reduce neural activity. Activity blockade significantly decreased active RhoA compared with normal culture conditions, in which spontaneous firing is prominent. These findings suggest that RhoA signaling acts as a positive regulator for activity-dependent axon branching in cortical neurons.
在发育过程中，轴突分支受到感觉诱发和自发神经活动的影响。我们研究了上层皮质水平伸长轴突（水平轴突）上依赖活动的分支形成的分子机制，重点关注Rho家族小GTP酶。用增强型黄色荧光蛋白进行轴突标记显示，水平轴突在器官型脑片培养物中形成了多个分支。通过引入组成型活性RhoA，这种分支形成显著增加，而显性负性RhoA则略有抑制作用。内源性RhoA信号的激活剂和抑制剂也分别促进和抑制分支形成。对水平轴突生长的每日成像进一步表明，组成型活性RhoA增加了分支的动态添加和丢失。此外，在用钠通道或谷氨酸受体阻滞剂处理以降低神经活动的皮质切片中，通过下拉试验检测了活性RhoA相对于RhoA总量的量。与自发放电突出的正常培养条件相比，活动阻断显著降低了活性RhoA。这些发现表明，RhoA信号作为皮质神经元中依赖活动的轴突分支的正调节因子发挥作用。
