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

本研究利用对比度增强型CCD相机和细胞内灌流技术，对高等脊椎动物轴突生长的分子机制进行了研究。用高倍率视频显微镜，结合对比增强的CCD摄像机，可以检测到培养的成年大鼠背根神经节神经元生长锥区周围的细丝状足中的轴浆流。多数情况下轴浆流动方向与轴突延长方向相反。在28ﾟC下，丝状伪足中轴质逆行流动的速度在1~5微米之间。用全细胞膜片钳方法研究了生长锥膜离子通道的生物物理性质。特别是在无Na~+、含TEA的外液中，对钙通道的性质进行了广泛的分析。CS^+细胞内灌流阻断K-通道。两种高压激活的钙通道--硝苯地平敏感的L…在生长锥区有较多的PE钙通道和欧米茄毒素敏感的N型钙通道。到目前为止，该区域还没有发现低电压激活的T型钙通道。观察钙通道阻滞剂对神经突起生长的影响。在无血清培养液中加入La^~(3+)和硝苯地平(5um)可抑制神经突起的萌发和生长，但欧米茄毒素(5um)对此无明显影响。提示硝苯地平敏感的L型钙通道活动与轴突延长密切相关。在钙通道阻滞剂存在下，用Fura-2荧光法测定细胞内游离钙离子浓度。硝苯地平处理组和芋螺毒素处理组细胞胞体[Ca^&lt；2+&gt；]的测定值无明显差异，但在芋毒素存在下，在萌发的丝状孢子附近观察到[Ca^&lt；2+&gt；]较高的“热点”。这些结果提示，硝苯地平敏感的钙通道与轴突萌发密切相关。较少

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