Activity dependence of cortical axon branch formation: A morphological and electrophysiological study using organotypic slice cultures
Activity dependence of cortical axon branch formation: A morphological and electrophysiological study using organotypic slice cultures
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DOI：
10.1523/jneurosci.3855-04.2005
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发表时间：
2005-01-05
影响因子：
5.3
通讯作者：
Yamamoto, N
中科院分区：
文献类型：
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作者：
Uesaka, N;Hirai, S;Yamamoto, N
通过对大鼠视皮层器官型切片培养的2/3层神经元轴突成像，研究了神经元活动对皮层轴突分支的影响。共聚焦显微镜下观察经编码黄色荧光蛋白的质粒电穿孔标记的上层神经元。单标记轴突的延时观察显示，在体外培养8 ~ 10 d后轴突开始形成分支。在随后的7 - 10天，分支的复杂性逐渐增加的增长和收缩的分支，导致轴突乔木，形态学上类似于在2至3周龄的动物中观察到的。在上层的神经元活动的电生理记录，使用多电极盘，表明自发放电的频率显着增加类似于10天在体外，并保持在后期阶段升高。为了检查自发放电和突触活动在分支形成中的参与，将各种阻断剂应用于培养基。TTX或APV和DNQX的组合使培养物沉默，但在单独的NMDA型或非NMDA型谷氨酸受体阻断剂存在下，在数天内表现出自发活动的稳态恢复。TTX和AMPA受体阻断剂抑制轴突分支，但不被NMDA受体阻断剂抑制。我们的结论是，皮层轴突分支是高度动态的，神经活动调节上层皮层神经元的早期发育分支通过激活AMPA型谷氨酸受体。
The influence of neuronal activity on cortical axon branching was studied by imaging axons of layer 2/3 neurons in organotypic slice cultures of rat visual cortex. Upper layer neurons labeled by electroporation of plasmid encoding yellow fluorescent protein were observed by confocal microscopy. Time-lapse observation of single-labeled axons showed that axons started to branch after 8 - 10 d in vitro. Over the succeeding 7 - 10 d, branch complexity gradually increased by both growth and retraction of branches, resulting in axon arbors that morphologically resembled those observed in 2- to 3-week-old animals. Electrophysiological recordings of neuronal activity in the upper layers, made using multielectrode dishes, showed that the frequency of spontaneous firing increased dramatically similar to 10 d in vitro and remained elevated at later stages. To examine the involvement of spontaneous firing and synaptic activity in branch formation, various blockers were applied to the culture medium. Cultures were silenced by TTX or by a combination of APV and DNQX but exhibited a homeostatic recovery of spontaneous activity over several days in the presence of blockers of either NMDA-type or non-NMDA-type glutamate receptors alone. Axonal branching was suppressed by TTX and AMPA receptor blockade but not by NMDA receptor blockade. We conclude that cortical axon branching is highly dynamic and that neural activity regulates the early developmental branching of upper layer cortical neurons through the activation of AMPA-type glutamate receptors.