Calcium ion distribution in nascent pioneer axons and coupled preaxonogenesis neurons in situ.

The "calcium hypothesis" of regulation of growth cone motility and neurite elongation has derived from analysis of a variety of neurons growing in vitro. It proposes that calcium ion concentration within growth cones is an important regulator of motility and growth. We now extend this analysis by investigating calcium concentrations within growth cones and nascent neurites of identified embryonic neurons growing on their normal substrate in situ. The pair of Ti1 pioneer neurons are the first to extend axons in limb buds of grasshopper embryos. Their growth cones migrate along a stereotyped pathway, where they encounter a series of guidance cues, including preaxonogenesis afferent neurons (guidepost cells). By injecting a pioneer neuron with fura-2 dye, we measured calcium concentration in the injected neuron, and in cells to which it became dye-coupled, at successive stages of pioneer outgrowth through the limb. Pioneer neurons undergoing axonogenesis had calcium concentrations in the above-100-nM range characteristic of a variety of neuron types studied in vitro. In pioneer neurons not yet in contact with guidepost cells, a shallow calcium concentration gradient, highest at the growth cone, was often observed. Guidepost cells that had not begun axonogenesis had lower calcium concentrations, in the 65 nM range. Therefore, an increase in cytosolic calcium concentration may be associated with the onset of axonogenesis. Nascent pioneer neurons were fura-2 dye-coupled to each other and established coupling with contacted guidepost cells. Calcium concentration measurements along pioneer neurites suggest that calcium ions also are transferred from pioneer neurons to these coupled guidepost cells.(ABSTRACT TRUNCATED AT 250 WORDS)