Imaging and manipulating calcium transients in developing Xenopus spinal neurons

Many forms of electrical excitability expressed in the embryonic nervous system depend on Ca(2+) influx. This discovery has stimulated investigation of the functions of spontaneous elevations of intracellular Ca(2+) and their roles in neuronal development. We present a protocol for imaging different classes of intracellular Ca(2+) transients in embryonic Xenopus (amphibian) spinal neurons grown in dissociated cell culture and in the intact neural tube (the developing spinal cord), focusing on early stages of neuronal differentiation around the time of neural tube closure. The protocol describes methods for gain-of-function and loss-of-function experiments to reveal the functions of these Ca(2+) transients. The methods can also be applied to explant and organotypic cultures. The procedures are sufficiently simple that they can be further adapted for dissociated neuronal cell cultures from other developing embryos, embryonic spinal cords of vertebrates such as zebrafish, and ganglia in the developing nervous systems of invertebrates.