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The Journal of Neuroscience, September 10, 2008, 28(37):9117-9121; doi:10.1523/JNEUROSCI.1731-08.2008

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Brief Communications
Role of RhoA in Activity-Dependent Cortical Axon Branching

Soichiro Ohnami,¹ Mitsuharu Endo,² Satoshi Hirai,¹ Naofumi Uesaka,¹ Yumiko Hatanaka,¹ Toshihide Yamashita,² and Nobuhiko Yamamoto¹

¹Laboratory of Cellular and Molecular Neurobiology, Graduate School of Frontier Biosciences, and ²Department of Molecular Neuroscience, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan

Correspondence should be addressed to Nobuhiko Yamamoto, Laboratory of Cellular and Molecular Neurobiology, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan. Email: nobuhiko{at}fbs.osaka-u.ac.jp

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.

Key words: branch formation; cytoskeleton; RhoA; axon guidance; cerebral cortex; organotypic culture

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Received April 21, 2008; revised July 5, 2008; accepted July 29, 2008.

Correspondence should be addressed to Nobuhiko Yamamoto, Laboratory of Cellular and Molecular Neurobiology, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan. Email: nobuhiko{at}fbs.osaka-u.ac.jp

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