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 ϳ10 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.

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.

Abbreviations: TESL, total eye scanning length; TNGP, total numbers of gaze points. Edited by RYOJI YAMAKAWA A Life Span Study of Exploratory Eye Movements in Healthy Subjects Summary:To evaluate age and gender differences as well as effects of affection, we examined exploratory eye movements. Exploratory eye movements were recorded in healthy subjects (57 women and 57 men) ranging from 9 to 74 years. All subjects were divided into three groups as pre puberty, young, and older adults to study the influences of age and gonadal hormones. Exploratory eye movements were analyzed for total eye scanning length (TESL), and total numbers of gaze points (TNGP) as subjects viewed neutral or affectively charged pictures. TESL and TNGP in older adults were significantly larger than that in both pre puberty and young adults for crying babies. TESL and TNGP in pre puberty were significantly smaller than that in both young and older adults for circles. TESL and TNGP in pre puberty were significantly smaller than that in older adults for smiling babies. Pre puberty and young adult of both genders for crying babies showed significantly shorter TESL than for when smiling babies. When viewing circles, young adult women had shorter TESL than men. TNGP in young adult women was smaller than in men for circles or crying babies. TNGP of young adult women in the visual right field was significantly smaller than in men. TNGP for crying babies was significantly smaller than that for smiling babies in young adults of both genders for the left field. Exploratory eye movements thus are a useful marker of visual cognitive function. Gender differences were limited to younger adults, suggesting influences of gonadal hormones.

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