Calcium signals and FGF-2 induced neurite growth in cultured parasympathetic neurons: spatial localization and mechanisms of activation.

The growth of neuritic processes in developing neurons is tightly controlled by a wide set of extracellular cues that act by initiating downstream signaling cascades, where calcium signals play a major role. Here we analyze the calcium dependence of the neurite growth promoted by basic fibroblast growth factor (bFGF or FGF-2) in chick embryonic ciliary ganglion neurons, taking advantage of dissociated, organotypic, and compartmentalized cultures. We report that signals at both the growth cone and the soma are involved in the promotion of neurite growth by the factor. Blocking calcium influx through L- and N-type voltage-dependent calcium channels and transient receptor potential canonical (TRPC) channels reduces, while release from intracellular stores does not significantly affect, the growth of neuritic processes. Simultaneous recordings of calcium signals elicited by FGF-2 at the soma and at the growth cone show that the factor activates different patterns of responses in the two compartments: steady and sustained responses at the former, oscillations at the latter. At the soma, both voltage-dependent channel and TRPC blockers strongly affect steady-state levels. At the growth cone, the changes in the oscillatory pattern are more complex; therefore, we used a tool based on wavelet analysis to obtain a quantitative evaluation of the effects of the two classes of blockers. We report that the oscillatory behavior at the growth cone is dramatically affected by all the blockers, pointing to a role for calcium influx through the two classes of channels in the generation of signals at the leading edge of the elongating neurites.

Differential repression by the transcription factor REST/NRSF of the various Ca2+ signalling mechanisms in pheochromocytoma PC12 cells.

Expression of the nerve cell phenotype is orchestrated by the REST/NRSF transcription repressor, working on hundreds of genes recognized at a specific regulatory binding sequence. Most PC12 clones, the most frequently employed neuronal model, maintain low levels of REST; however a few, defective of neurosecretion, express high levels. To investigate the role of REST in Ca2+ signalling we studied the [Ca2+](i) changes in single cells of four clones, two wild-type and two defective, pre-treated for 5 days with NGF. We focused on Ca2+ influxes induced by depolarization and ATP. Only a subpopulation ( approximately 15%) of the defective, high REST cells responded to depolarization (Ca(V) expression approximately 10%). The ATP-induced intracellular Ca2+ release was little changed, whereas influx via ionotropic P2X receptors was decreased, in agreement with the decreased expression of P2X2 receptors. The percentage of defective cells expressing store-operated calcium entry (SOCE) following ATP stimulation was also lower. The responses of the defective clones were little affected by their differentiated state. In conclusion, our results revealed important new aspects of REST control of Ca2+ homeostasis, of potential physiological importance. The mechanisms of this control remain to be investigated.

A quantitative approach to the dynamics of neurite sprouting induced by a neurotrophic factor.

The sprouting, stabilization and growth of neurites is a dynamic process by which developing neurons establish connections with the other elements of the nervous system; this process is under the control of extracellular cues, among which neurotrophic factors play a crucial role. Due to the complexity of the spatiotemporal evolution of the neurite network, particularly in the early stages of growth, it is not easy to obtain information about the relevant parameters from qualitative observations. We have developed a quantitative description of the dynamics of production and stabilization of neuritic processes in a well-characterized experimental model of peripheral neurons in culture, and we have combined it with a simulation approach to extract the differences between the behaviour in the absence and in the presence of the neurotrophic factor basic Fibroblast Growth Factor (bFGF). We show that the factor rapidly stabilizes the neuronal morphology to a bipolar phenotype, by shifting the sprouting process from a disordered phase to a more ordered and organized one.

A simple method to study cellular migration.

We describe here a simple and fast method for the characterisation of cell motion. By projecting on a single plane different positions of the cell a ribbon is generated, whose characteristics can be related to the type of motion. The proposed method allows both to determine, very quickly, the motility of a population of cells and to investigate and characterise properties of a single cell's motion. The methodology presented here can be applied to a large range of cell movement and also adapted and extended to other problems involving biological motion.