Williams D K, Cohan C S
Department of Anatomy and Cell Biology, School of Medicine and Biomedical Sciences, State University of New York at Buffalo 14214, USA.
J Neurobiol. 1995 May;27(1):60-75. doi: 10.1002/neu.480270107.
Accumulating evidence indicates that cytosolic calcium levels regulate growth cone motility and neurite extension. The purpose of this study was to determine if intracellular calcium levels also influence the initiation of neurite extension induced by growth-promoting factors. An in vitro preparation of axotomized neurons that can be maintained in the absence of growth-promoting factors was utilized. The distal axons of cultured Helisoma neurons plated into defined medium do not extend neurites until they are exposed to Helisoma brain-conditioned medium. This provided the opportunity to study the intracellular changes associated with neurite extension. Cytosolic calcium levels were monitored with the calcium-sensitive dye fura 2 at the distal axon. In control medium calcium levels in the distal axon were constant. However, transient elevations in cytosolic calcium in the axonal growth cone occurred after addition of conditioned medium and coincident with the initiation of neurite extension. Application of calcium channel blockers showed that the transients resulted from calcium influx across the neuronal membrane. The transients, however, were not required for neurite extension, although they did influence the rate and extent of neurite outgrowth. Simultaneous extracellular patch recordings demonstrated that the calcium transients were correlated temporally with an increase in rhythmic spontaneous electrical activity of cells, suggesting that conditioned medium influences ionic membrane properties of these neurons.
越来越多的证据表明，胞质钙水平调节生长锥的运动性和神经突的延伸。本研究的目的是确定细胞内钙水平是否也影响由生长促进因子诱导的神经突延伸的起始。我们利用了一种体外制备的轴突切断神经元，这种神经元可以在没有生长促进因子的情况下维持。培养在特定培养基中的海兔神经元的远端轴突在暴露于海兔脑条件培养基之前不会延伸神经突。这为研究与神经突延伸相关的细胞内变化提供了机会。用钙敏染料fura 2监测远端轴突的胞质钙水平。在对照培养基中，远端轴突中的钙水平是恒定的。然而，添加条件培养基后，轴突生长锥中的胞质钙出现短暂升高，且与神经突延伸的起始同时发生。应用钙通道阻滞剂表明，这些瞬变是由钙通过神经元膜内流引起的。然而，这些瞬变对于神经突延伸不是必需的，尽管它们确实影响神经突生长的速率和程度。同时进行的细胞外膜片钳记录表明，钙瞬变在时间上与细胞节律性自发电活动的增加相关，这表明条件培养基影响这些神经元的离子膜特性。
