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

本项目采用CCD成像增强技术和细胞内灌注技术，旨在阐明高等脊椎动物神经突生长的分子机制。采用高倍率视频显微技术，利用CCD相机对培养的成年大鼠背根神经节神经元生长锥区周围细丝状足的轴浆流动进行检测。大多数情况下，轴浆血流方向与神经突伸长方向相反。在28℃时，丝状足逆行轴质血流速度分布在1 ~ 5 μ m之间。采用全细胞膜片钳法研究了生长锥膜离子通道的生物物理性质。特别是在不含Na^+、含有tea的外溶液中，广泛地分析了ca通道的性质。细胞内灌注Cs^+阻断k通道。在生长锥区发现了两种高压激活的钙通道，硝苯地平敏感的L-ty型和ω - concontoxin敏感的n型钙通道。到目前为止，低压激活的t型钙通道尚未在该区域得到证实。然后，研究了钙通道阻滞剂对神经突生长的影响。在无血清培养基中添加La^<3+> (5 muM)和硝苯地平（5 muM）可抑制神经突的发芽和生长，但omega- concontoxin （5 muM）无此作用。硝苯地平敏感的l型钙通道活性与神经突伸长密切相关。在Ca通道阻滞剂存在下，用fura-2荧光法测定细胞内游离Ca^<2+>浓度。虽然在硝苯地平处理组和omega- concontoxin处理组之间测得的细胞体[Ca^<2+>] in没有差异，但在omega- concontoxin存在下，在发芽丝状足附近观察到较高的[Ca^<2+>] in的“热点”。这些结果提示硝苯地平敏感钙通道与神经突发芽密切相关。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