Investigation of the molecular mechanism of the neurite-outgrowth using a contrast enhanced camera and intracellular perfusion methods.

使用对比增强相机和细胞内灌注方法研究神经突生长的分子机制。

• 批准号: 02807014
• 负责人: YAMAGUCHI Kazuhiko
• 金额: $ 0.96万
• 依托单位: Okazaki National Research Institutes National institute for Physiological Sciences Grant-in-Aid for Scientific Research (C)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02807014/
• 关键词: neurite-outgrowth; axoplasmic flow; contrast enhancing camera; whole cell clamp; filopodia; intracellular perfusion; nifedipine; L-type Ca-channel; Caチャネル; パッチクランプ法; 後根神経節; 高コントラストカメラ; 無血清培養

The present project has been undertaken to clarify the molecular mechanism of the neurite-outgrowth in the higher vertebrate by using a contrast enhancing CCD camera and intracellular perfusion techniques. A high-magnification video microscopy using a contrast enhanced CCD camera could detect the axoplasmic flow in the fine filopodia around the growth cone region of the cultured adult rat dorsal root ganglion neuron. The direction of the axoplasmic flow was retrograde against neurite-elongation direction in the most cases. The velocity of the retrograde axoplasmic flow in filopodia distributed between 1 to 5 mum at 28ﾟC.Biophysical properties of the ionic channels of the growth cone membrane was investigated by means of whole cell patch clamp methods. Especially, properties of Ca-channel was extensively analysed in Na^+-free, TEA-containing external solution. Cs^+ was perfused intracellularly to block K-channel. Two types of high voltage activated Ca-channels, nifedipine-sensitive L-ty … More pe Ca-channel and omega-conotoxin sensitive N-type Ca-channels, were demonstrated at the growth cone region. So far, low voltage activated T-type Ca-channel has not been demonstrated in this region. Then, effects of Ca-channel blockers on the neurite-outgrowth were examined. La^<3+> (5 muM) and nifedipine (5 muM), added in a serum-free cultured medium, suppressed sprouting and outgrowth of the neurite, but omega-conotoxin (5 muM) did not. Close relationship between nifedipine-sensitive L-type Ca-channel activity and neurite-elongation was suggested.Intracellular free Ca^<2+> concentration was measured by fluorometry using fura-2 under the presence of Ca-channel blockers. Though measured [Ca^<2+>] in of the cell body was not different between nifedipine-treated and omega-conotoxin-treated groups, "hotspots" of higher [Ca^<2+>] in were observed near the sprouting filopodia under the presence of omega-conotoxin. These results suggested close relationship between the nifedipine-sensitive Ca-channel and the neurite-sprouting. Less

本研究采用对比度增强的CCD摄像机和细胞内灌注技术来阐明高等脊椎动物神经突生长的分子机制。用对比度增强的CCD摄像机在高放大倍数的视频显微镜下可以检测到培养的成年大鼠背根神经节神经元的生长锥区域周围的细丝状伪足中的轴浆流。大多数情况下，轴浆的流动方向与轴突延伸方向相反。逆行轴浆流在丝状伪足的速度分布在1至5 μ m之间，在28 ℃。生物物理特性的生长锥膜的离子通道的全细胞膜片钳方法进行了研究。特别地，在无Na^+、含TEA的外部溶液中，对Ca通道的性质进行了广泛的分析。细胞内灌注Cs^+以阻断K通道。两种高电压激活的钙通道，硝苯地平敏感的L型 ...更多信息 pe钙通道和ω-芋螺毒素敏感的N-型钙通道。到目前为止，低电压激活的T型钙通道尚未在该区域得到证实。然后，钙通道阻滞剂对神经突起生长的影响进行了检查。在无血清培养基中加入La^<3+>（5 μ M）和硝苯地平（5 μ M），可以抑制神经突的发芽和生长，但ω-芋螺毒素（5 μ M）则没有。在钙通道阻断剂存在下，用Fura-2荧光法测定细胞内游离Ca^2+浓度，结果表明，硝苯地平敏感的L型钙通道活性与神经突起的伸长密切相关。虽然硝苯地平处理组和ω-芋螺毒素处理组之间细胞体的[Ca^<2+>] in测量值没有差异，但在ω-芋螺毒素存在下，在萌发的丝状伪足附近观察到较高[Ca^<2+] in的“热点”。提示硝苯地平敏感性钙通道与神经突出芽密切相关。少

项目成果

Yamaguchi,Kazuhiko: "Voltage gated ionic channels in the growth cone membrane of the cultured rat sensory ganglion neuron." Journal of Newoscience.

Yamaguchi，Kazuhiko：“培养的大鼠感觉神经节神经元生长锥膜中的电压门控离子通道。”

Yamaguchi,Kazuhiko: "Voltage gated and chemically gated ion channel in the cultured cochlear ganglion neurone of the chick." Journal of Physiology. 420. 185-206 (1990)

Yamaguchi，Kazuhiko：“培养的小鸡耳蜗神经节神经元中的电压门控和化学门控离子通道。”

Yamaguchi, K.: "Acetylcholine reduces K-current in the cultured chick cochlea ganglion neuron." Neurosci Research. Supp114. S132 (1991)

Yamaguchi, K.：“乙酰胆碱可降低培养鸡耳蜗神经节神经元中的 K 电流。”

Yamaguchi, K.: "Intracellular mechanisms of K-current suppression by ACh in the cultured chick cochlear ganglion neuron." Japanese Journal of Physiology. 41 Suppl. S214 (1991)

Yamaguchi, K.：“培养的小鸡耳蜗神经节神经元中 ACh 抑制 K 电流的细胞内机制。”

Yamaguchi,Kazuhiko: "Modulation of inwardly rectifying channels by substance P in cholinergic brain neurones culture." Journal of Physiology. 426. 499-520 (1990)

Yamaguchi，Kazuhiko：“胆碱能脑神经元培养物中 P 物质对内向整流通道的调节。”