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Mobility of neuronal growth-cones and formation of synapses

神经元生长锥的移动性和突触的形成

基本信息

批准号：
08044257
负责人：
KATAYAMA Yoshifumi
金额：
$ 5.5万
依托单位：
Tokyo Medical and Dental University
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

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

将光学技术的最新进展应用于细胞生理学的研究是必要的。本研究采用全细胞膜片钳技术，以急性分离的海马神经元为生物检测器，检测了生长锥中谷氨酸的释放。这种释放依赖于Ca^<2+>，可被ω-蛇毒素所阻断。用毒素预处理培养的神经元，然后用毒素的抗体孵育，并进一步用金标记抗体染色。通过扫描和透射电镜观察到生长锥细胞膜上有免疫金颗粒簇，提示生长锥上存在对蛇毒毒素敏感的ω-Ca^<2+>通道。 ...更多信息应用激光共聚焦扫描显微镜观察了生理小电场作用下神经元细胞骨架元件、第二信使信号元件和细胞核的变化。使用了三种类型的荧光探针，a）罗丹明卤代菌素染色丝状肌动蛋白，B）荧光素缀合的单克隆抗体标记的酪氨酸残基上磷酸化的蛋白质和c）线粒体的标记物通过排除清楚地描绘了核的位置，仅存在于细胞质中。生理电场可引起生长锥内丝状肌动蛋白骨架和酪氨酸磷酸化蛋白质的不对称性，改变细胞核的位置，当机械刺激DRG神经元生长锥时，用全细胞膜片钳技术在-60 mV的保持电位下记录到从其索马胞体发出的内向电流。当刺激除板状伪足以外的其他部位时，几乎没有记录到电反应。根据Cl^-通道的电生理学特性，推测外向电流主要是由于Cl^-通道的激活引起的。在高分辨率视频增强微分干涉显微镜中引入倏逝光照明。用荧光染料Dil对培养在玻璃板上的神经元的生长锥和索马体进行染色，并用内全反射消逝光显微镜观察。隐失像显示生长锥和部分有限区域的索马体附着于培养玻璃板表面。少