Ivins J K, Raper J A, Pittman R N
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104.
J Neurosci. 1991 Jun;11(6):1597-608. doi: 10.1523/JNEUROSCI.11-06-01597.1991.
When the growth cone of a chick dorsal root ganglion (DRG) neurite contacts the neurite of a chick retinal ganglion cell in vitro, the growth cone typically responds by withdrawing its lamellipodia and filopodia and collapsing. We have used the fluorescent calcium indicator dye fura-2 and digital imaging microscopy to measure calcium levels within DRG growth cones and to determine whether changes in calcium levels are responsible for the collapse of growth cone morphology when a DRG growth cone contacts a retinal ganglion cell neurite. Calcium levels within DRG growth cones were stable during neurite outgrowth. Calcium was typically distributed homogeneously throughout the growth cone, though occasionally gradients of free calcium were present. When calcium gradients were observed, calcium levels appeared higher in the active veil regions than in the central core region. Calcium levels in DRG growth cones appeared to remain stable during the period of contact-mediated growth cone collapse. Low concentrations of the calcium ionophore ionomycin increased calcium levels two- to threefold without having any observable morphological effects on DRG growth cones. Likewise, depolarization with 15 mM KCl caused a transient two- to threefold increase in calcium levels without having any observable morphological effect. These results suggest that changes in calcium levels are not responsible for contact-mediated collapse of growth cone structure. A growth cone collapsing activity has been solubilized from embryonic chick brain (Raper and Kapfhammer, 1990). Application of this material to cultures of DRG neurons caused growth cones to collapse but had no effect on calcium levels within the growth cones. The crude growth cone collapsing activity was not blocked by the presence of cobalt, nickel, lanthanum, nifedipine, or reduced-calcium medium, suggesting that transmembrane calcium fluxes were not required for growth cone collapse. These results suggest that the morphological changes associated with the collapse of growth cone structure can be independent of changes in growth cone calcium levels, and that second messengers other than calcium are likely to be involved in the regulation of many growth cone behaviors.
当鸡背根神经节（DRG）神经突的生长锥在体外与鸡视网膜神经节细胞的神经突接触时，生长锥通常会通过缩回其片状伪足和丝状伪足并发生塌陷来做出反应。我们使用了荧光钙指示剂染料fura-2和数字成像显微镜来测量DRG生长锥内的钙水平，并确定当DRG生长锥与视网膜神经节细胞神经突接触时，钙水平的变化是否是生长锥形态塌陷的原因。在神经突生长过程中，DRG生长锥内的钙水平是稳定的。钙通常均匀地分布在整个生长锥中，不过偶尔会出现游离钙的梯度。当观察到钙梯度时，活跃的边缘区域的钙水平似乎高于中央核心区域。在接触介导的生长锥塌陷期间，DRG生长锥内的钙水平似乎保持稳定。低浓度的钙离子载体离子霉素使钙水平增加了两到三倍，而对DRG生长锥没有任何可观察到的形态学影响。同样，用15 mM KCl进行去极化导致钙水平短暂增加两到三倍，而没有任何可观察到的形态学影响。这些结果表明，钙水平的变化不是接触介导的生长锥结构塌陷的原因。一种生长锥塌陷活性物质已从胚胎鸡脑中溶解出来（Raper和Kapfhammer，1990）。将这种物质应用于DRG神经元培养物中会导致生长锥塌陷，但对生长锥内的钙水平没有影响。粗制的生长锥塌陷活性不受钴、镍、镧、硝苯地平的存在或低钙培养基的影响，这表明生长锥塌陷不需要跨膜钙通量。这些结果表明，与生长锥结构塌陷相关的形态学变化可能与生长锥钙水平的变化无关，并且除钙之外的第二信使可能参与了许多生长锥行为的调节。
