Breaking the code: regulation of neuronal differentiation by spontaneous calcium transients

Calcium ions play critical roles in neuronal development. Stimulation of transient elevations of intracellular calcium (Ca2+i) activates protein kinases, regulates transcription and influences motility and morphology. Embryonic Xenopus spinal neurons exhibit a Ca(2+)-sensitive period in culture; removing extracellular Ca2+ during this period affects several aspects of neuronal differentiation. However, both the mechanisms that generate natural fluctuations in Ca2+i and the signals they transduce are not well understood. Spontaneous, transient and repeated elevations of Ca2+i in embryonic Xenopus spinal neurons have been observed over periods up to 5 h in vitro and in vivo, confocally imaging fluo 3-loaded cells. Developing neurons generate two distinctive types of spontaneous Ca2+i transients, calcium spikes and calcium waves. We have investigated the mechanisms by which they are generated and their roles in directing neuronal differentiation. Spikes are generated by spontaneous action potentials, and thus are rapidly propagated throughout entire neurons. Ca2+ entry triggers Ca2+ release from intracellular stores, and spikes have a characteristic double exponential decay. In contrast, the generation of waves does not involve conventional voltage-dependent Ca2+ channels, but an unknown Ca2+ entry pathway that can be blocked by Ni2+ at a higher concentration than required to block classical voltage-dependent Ca2+ channels. Waves rise and decay slowly, and unlike spikes, are local events. However both spikes and waves are abolished by removal of extracellular Ca2+. Developmentally, the incidence and frequency of spikes decrease while the incidence and frequency of waves are constant. To study the roles of Ca2+ transients, we have imaged Ca2+ in spinal neurons throughout an extended period of early development, and find that spikes and waves are expressed at distinct frequencies. Neuronal differentiation is altered when they are eliminated by preventing Ca2+ influx. By reimposing different frequency patterns of Ca2+ transients, we demonstrate that natural spike activity is sufficient to promote normal neurotransmitter expression and channel maturation, while wave activity at growth cones is sufficient to regulate neurite extension. On the other hand, suppression of spontaneous Ca2+ elevations with BAPTA, a rapid Ca2+ chelator, indicates that they are also necessary to direct differentiation. Ca2+ transients appear to encode information in their frequency. Thus, they act like action potentials, although they are 10(4) times longer in duration and less frequent and implement an intrinsic development program.