Spira M E, Dormann A, Ashery U, Gabso M, Gitler D, Benbassat D, Oren R, Ziv N E
Dept. of Neurobiology, Life Sciences Institute, The Hebrew University of Jerusalem, Israel.
J Neurosci Methods. 1996 Oct 21;69(1):91-102. doi: 10.1016/S0165-0270(96)00024-6.
The present report describes the experimental advantages offered by the combined use of Aplysia neurons and contemporary techniques to analyze the cellular events associated with nerve injury in the form of axotomy. The experiments were performed by transecting, under visual control, the main axon of identified Aplysia neurons in primary culture while monitoring several related parameters. We found that in cultured Aplysia neurons axotomy leads to the elevation of the [Ca2+]i in both the proximal and distal axonal segments from a resting level of 100 nM up to the millimolar range for a duration of 3-5 min. This increase in [Ca2+]i led to identical alterations in the cytoarchitecture of the proximal and distal segments. The formation of a membrane seal over the transected ends by their constriction and the subsequent fusion of the membrane is a [Ca2+]i-dependent process and is triggered by the elevation of [Ca2+]i to the microM level. Seal formation was followed by down-regulation of the [Ca2+]i to control levels. Following the formation of the membrane seal an increase in membrane retrieval was observed. We hypothesize that the retrieved membrane serves as an immediately available membrane reservoir for growth cone extension.
本报告描述了结合使用海兔神经元和当代技术来分析以轴突切断形式出现的与神经损伤相关的细胞事件所具有的实验优势。实验是在视觉控制下，切断原代培养中已鉴定的海兔神经元的主要轴突，同时监测几个相关参数来进行的。我们发现，在培养的海兔神经元中，轴突切断会导致近端和远端轴突段的细胞内钙离子浓度（[Ca2+]i）从100 nM的静息水平升高到毫摩尔范围，持续3 - 5分钟。[Ca2+]i的这种增加导致近端和远端段的细胞结构发生相同的变化。通过切断端的收缩在其切断端形成膜密封以及随后膜的融合是一个依赖[Ca2+]i的过程，由[Ca2+]i升高到微摩尔水平触发。密封形成后，[Ca2+]i下调至对照水平。在膜密封形成后，观察到膜回收增加。我们推测回收的膜作为生长锥延伸的即时可用膜库。
