Calcium as a second messenger in developing nerve cells

Calcium is thought to play a crucial role in the regulation of nerve cell development and growth cone behaviour. In this context I studied three aspects of calcium as a second messenger. - In growth cones of the N1E-115 cell line I studied the properties and distribution of calcium channels and the pattern of calcium flux through these channels. - In the same cells the effect of the cytosolic calcium concentration [Ca2+]i on neurite outgrowth was investigated. - Finally I monitored spontaneous calcium changes in life zebrafish embryos. 1) In imaging experiments, using the calcium indicator dye Fluo-3, I studied the calcium influx into club-shaped growth cones which are characteristically found on advancing neurites. Depolarisation caused the highest calcium influx near the distal, leading tip. This gradient is not altered by blocking calcium induced calcium release from internal stores. Using the voltage clamp technique in the cell attached mode I identified and characterised T type and L type calcium channels on the growth cones of this cell line. L channel density was significantly higher at the distal tip than at the more proximal growth cone. The calcium gradient in the depolarised growth cone can thus be explained by a gradient of calcium channel density. 2) In N1E-115 cells decreasing [Ca2+]i below the resting value promoted neurite outgrowth monotonically unlike the situation in some other celltypes which show rather a bell shaped dependence. Surprisingly, neurite outgrowth was also promoted when [Ca2+]i was raised much above the resting levels. 3) In an attempt to study calcium dynamics in nerve cells in an intact, in vivo, preparation the calcium indicator dye calcium-green dextran was injected into one of the blastomeres of a 2 to 3 hour old zebra fish embryo which was then allowed to develop further. Studying 18 to 24 hour old embryos, using a confocal microscope, labelled nerve cells among the daughter cells of the injected blastomere could be clearly identified according to their shape and position. When such 24 hour old embryos were dissociated and plated on culture dishes labelled nerve cells responded to a depolarisation with a fluorescence increase demonstrating that the dye was retained in the cytoplasm and still capable of reporting calcium changes. Spontaneous calcium changes were observed in developing motoneurons in situ