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Dynamics of Nerve Growth Cone Guidance and Adhesion

神经生长锥引导和粘附的动力学

基本信息

批准号：
10044248
负责人：
KATAYAMA Yoshifumi
金额：
$ 5.06万
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 Scientific Research (A).
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

Using recent advance in optical technology we investigated functions of growth cones of cultured neurons isolated from either the central or peripheral nervous system.Inward currents were recorded from DRG cell bodies in response to mechanical stimuli to growth cones with the whole-cell patch clamp method at the holding potential of -60mV. Their reversal potential changed according to the Nernst equation for ClィイD1-ィエD1. They were completely abolished with extracellular application of NPPB. It is concluded that the inward currents are due to activation of mechanosensitive ClィイD1-ィエD1 channels preferentially locating on the growth cones.CaィイD12+ィエD1 channels on the growth cones were visualized by using imuno-gold particles. Cultured DRG neurons pretreated with CaィイD12+ィエD1 blockers, ω-agatoxin, ω-conotoxin and nicardipine, were incubated with the respective antibody and finally with gold-conjugated antibody. Observations with the scanning and transmission electron microscopy showed that … More ω-agatoxin- and ω-contoxin- sensitive CaィイD12+ィエD1 channels were immunologically localized on the growth cones.The [CaィイD12+ィエD1]ィイD2iィエD2-increase evoked by cell body stimulation was inhibited by ω-agatoxin and ω-conotoxin, but not by nicardipine. The [CaィイD12+ィエD1]ィイD2iィエD2-increase induced by 47mM-KィイD1+ィエD1 solution even in the presence of both peptide toxins was blocked by nicardipine, suggesting that nicardipine-sensitive CaィイD12+ィエD1 channels may localize on the growth cones.Neurites in the frog spinal cord grew toward the negative electrode on untreated Falcon tissue culture plastic or on laminine substrate (negatively charged), but neurites growing on poly-lysine (positively charged) turned toward the positive electrode. The charge of the growth surface influenced the frequency of anodal gavanotropism. It is concluded that the direction of neurite growth in an electric field is influenced by both substratum charge and growth cone-to substratum adhesivity.Dynamic changes of growth cones stained with DiI were observed with a total internal reflection fluorescence microscope (TIRFM). High KィイD1+ィエD1 stimulation to growth cone regions increased DiI-TIRFM intensity in those regions, demonstrating that the basal membrane of the growth cone approached the glass substrate. The changes in the fluorescence intensity of fluo 3-loaded SCG neurons were observed with the TIRFM ; TIRFM images showed a pattern of spotted area of which brightness may indicate the [CaィイD12+ィエD1]ィイD2iィエD2 in submembrane space. Less

利用光学技术的最新进展，我们研究了从中枢或周围神经系统分离的培养神经元的生长锥的功能。使用全细胞膜片钳方法在-60mV的保持电位下记录了DRG细胞体对生长锥的机械刺激的内向电流。它们的逆转势根据 ClィイD1-ィエD1 的能斯特方程而变化。通过细胞外应用 NPPB，它们被完全消除。结论是，内向电流是由于优先位于生长锥上的机械敏感ClィイD1-ィエD1通道的激活所致。使用免疫金粒子对生长锥上的CaィイD12+ィエD1通道进行可视化。将培养的 DRG 神经元用 CaィイD12+ィD1 阻滞剂、ω-琼脂毒素、ω-芋螺毒素和尼卡地平预处理，与相应的抗体一起孵育，最后与金缀合抗体一起孵育。扫描和透射电子显微镜观察表明，对 ω-agatoxin 和 ω-contoxin 敏感的 CaィイD12+ィエD1 通道在免疫学上定位于生长锥上。细胞体刺激引起的 [CaィイD12+ィエD1]ィイD2iィエD2 增加被 ω-agatoxin 抑制，并且ω-芋螺毒素，但不是尼卡地平。即使在两种肽毒素存在的情况下，47mM-KィイD1+ィエD1 溶液诱导的 [CaィイD12+ィエD1]ィイD2iエD2 增加也会被尼卡地平阻断，表明尼卡地平敏感的 CaィイD12+ィエD1 通道可能定位于生长青蛙脊髓中的神经突向负极生长未经处理的 Falcon 组织培养塑料或在层粘连蛋白基质上（带负电），但在聚赖氨酸（带正电）上生长的神经突转向正极。生长表面的电荷影响阳极正反性的频率。得出电场中神经突生长方向受基质电荷和生长锥与基质粘附力共同影响。采用全内反射荧光显微镜(TIRFM)观察DiI染色生长锥的动态变化。对生长锥区域的高 KィイD1+ィD1 刺激增加了这些区域的 DiI-TIRFM 强度，表明生长锥的基底膜接近玻璃基板。用TIRFM观察加载fluo 3的SCG神经元荧光强度的变化； TIRFM 图像显示出斑点区域的图案，其亮度可能表明膜下空间中的[CaィイD12+ィエD1]ィイD2iエD2。较少的