Imaging calcium dynamics in developing neurons.
Here we describe the techniques developed to image Ca2+ signals in motile nerve growth cones both in culture and in the developing Xenopus spinal cord. We have used these methods to identify two spatially and temporally distinct classes of Ca2+ transients in growth cones. Imaging Ca2+ in morphologically complex migratory cells allows for analysis and correlation of discrete signals with a wide variety of cellular behaviors. For example, we find that localized Ca2+ changes at the tips of individual filopodia correlate with reduced filopodial motility. Further, rapid fixation after Ca2+ imaging made it possible to determine that transients occur at integrin receptor clusters that may generate and in turn be regulated by these local signals. We describe the use of caged-Ca2+ to locally impose Ca2+ transients in individual filopodia and find this treatment sufficient to repel neurite outgrowth. Calcium signals across broad spatial and temporal dimensions are universal regulators of numerous complex and varied cellular functions. The imaging methods we describe here begin to view growth cones over a range of spatial resolutions and temporal frequencies necessary to detect different types of Ca2+ transients, however it is clear that not all dimensions have been examined. In particular, imaging cells more rapidly and at higher magnification may one day allow us to detect more elemental events such as single-channel openings, as has been achieved in nonneuronal cells. We also describe techniques used to examine Ca2+ signals in growth cones migrating within the spinal cord. These types of studies are ultimately necessary to confirm the relevance of in vitro findings. Although designed for the Xenopus spinal cord, the methods we outline should be applicable to other tissues and organisms. Finally, we use caged Ca2+ as a tool to reproduce very precise changes in cytosolic Ca2+ levels. This is a powerful means to test the function of different types of Ca2+ transients and assess the downstream regulators of those signals. These types of manipulations can also be used with other types of caged compounds, many of which are commercially available (Molecular Probes) or readily synthesized.