Mobility of neuronal growth-cones and formation of synapses

将光学技术的最新进展应用于细胞生理学研究是十分必要的。利用一些新技术，我们证明了培养的大鼠神经元生长锥可以释放神经递质，并对机械和电场刺激敏感。通过使用急性游离海马神经元作为生物检测器检测生长锥的谷氨酸释放，并从中进行全细胞膜片钳记录。释放依赖于Ca^<2+>，并被ω -agatoxin消除。培养的神经元用毒素预处理，然后用毒素抗体孵育，再用金偶联抗体染色。通过扫描电镜和透射电镜观察到生长锥膜上有免疫金颗粒簇，提示对ω - agat毒素敏感的Ca^<2+>通道在生长锥上有免疫定位。采用激光共聚焦扫描显微镜观察小生理电场作用下神经元的细胞骨架元件、第二信使信号元件和细胞核，探讨了培养DRG神经细胞的阴极定向生长机制。使用了三种类型的荧光探针，a)罗丹明荧光素染色丝状肌动蛋白，b)荧光素偶联单克隆抗体标记酪氨酸残基磷酸化的蛋白质，c)线粒体标记物通过排除清楚地描绘细胞核的位置，仅存在于细胞质中。生理电场诱导丝状肌动蛋白骨架和生长锥中酪氨酸残基磷酸化蛋白的不对称，并改变细胞核的位置。采用全细胞膜片钳技术，对DRG神经元生长锥施加机械刺激，记录其胞体在-60mV保持电位下向内电流。当除板状基底外的其他部位受到刺激时，几乎没有电反应记录。根据Cl^-通道的电生理特性，认为向外的电流主要是由于Cl^-通道的激活。在高分辨率视频增强微分干涉对比显微镜中引入了倏逝光照明。用荧光染料Dil对玻璃板上培养的神经元的生长锥和细胞体进行染色，用内全反射倏逝光显微镜观察。消失图像显示，生长锥和一些有限区域的体细胞附着在培养玻璃板表面。少

It is necessary to apply recent advance in optical technology to studying cellular physiology. Using some new techniques, we demonstrated that growth cones of rat cultured neurons can release neurotransmitter and be sensitive to both mechanical and electric field stimulation.Glutamate release from growth cones was detected by using an acutely dissociated hippocampal neuron as a biodetector from which whole cell patch clamp recordings were made. The release was dependent on Ca^<2+> and was abolished by omega-agatoxin. Cultured neurons were pretreated with the toxin, then incubated with the antibody to the toxin and further stained with gold-conjugated antibody. Since clusters of immuno-gold particles were observed over the membrane of growth cones by means of scanning and transmission electron microscpes, it is suggested that omega-agatoxin-sensitive Ca^<2+> channels were immunologically localized on growth cones.The mechanism underlying cathodally-directed growth of cultured DRG neurit … More es was investigated by using confocal laser scanning microscopy to observe cytoskeletal elements, second messenger signaling elements and the nucleus in neurons exposed to small physiological electric fields. Three types of fluorescent probes were used, a) Rhodamine halloidin stains filamentous actin, b) a fluorescein-conjugated monoclonal antibody labelled proteins phosphorylated on tyrosine residues and c) a marker for mitochondria clearly delineated the position of the nucleus by exclusion, being present only in the cytoplasma. Physiological electric fields induce asymmetries of the filamentous actin cytoskeleton and proteins phosphorylated on tyrosine residues in growth cones, and alter the positioning of cell nucleus.When mechanical stimulation was applied to growth cones of DRG neurons, inward currents were recorded, from their soma at the holding potential of -60mV,using whole cell patch clamp technique. Almost no electrical response was recorded when other sites except lamellipodium were stimulated. The outward currents were supposed to be due mainly to activation of Cl^- channels according to their electrophysiological properties.Evanescent light illumination was introduced into a high resolution video-enhanced differential interference contrast microscope. Growth cones and soma of neurons which were cultured on glass plates were stained with a fluorescent dye, Dil, and were observed with an internal total reflection evanescent light microscope. The evanescent images showed that growth cones and some limited areas of soma attached to the surface of culture glass plate. Less