Interaction of spherical silica nanoparticles with neuronal cells: size-dependent toxicity and perturbation of calcium homeostasis.

The effects of Stöber silica nanoparticles on neuronal survival, proliferation, and on the underlying perturbations in calcium homeostasis are investigated on the well-differentiated neuronal cell line GT1-7. The responses to nanoparticles 50 and 200 nm in diameter are compared. The 50-nm silica affects neuronal survival/proliferation in a dose-dependent way, by stimulating apoptotic processes. In contrast, the 200-nm silica does not show any toxic effect even at relatively high concentrations (292 µg mL−1). To identify the mechanisms underlying these effects, the changes in intracellular calcium concentration elicited by acute and chronic administration of the two silica nanoparticles are analyzed. The 50-nm silica at toxic concentrations generates huge and long-lasting increases in intracellular calcium, whereas the 200-nm silica only induces transient signals of much lower amplitude. These findings provide the first evidence that silica nanoparticles can induce toxic effects on neuronal cells in a size-dependent way, and that these effects are related to the degree of perturbation of calcium homeostasis.

Neuregulin1/ErbB4-induced migration in ST14A striatal progenitors: calcium-dependent mechanisms and modulation by NMDA receptor activation.

BACKGROUND: A number of studies have separately shown that the neuregulin1 (NRG1)/ErbB4 system and NMDA-type glutamate receptors (NMDARs) are involved in several aspects of neuronal migration. In addition, intracellular calcium fluctuations play central roles in neuronal motility. Stable expression of the tyrosine kinase receptor ErbB4 promotes migratory activity in the neural progenitor cell line ST14A upon NRG1 stimulation. In this work we analyzed the potential interactions between the NRG1/ErbB4 system and NMDARs in the ST14A migratory process as well as its calcium dependence. RESULTS: RT-PCR studies have shown that both native ST14A cells (non-expressing ErbB4), as well as ErbB4-transfected cells express low levels of a restricted number of NMDAR subunits: NR1, NR2C, NR2D and NR3B. The resulting NMDAR would form Ca(2+) channels characterized by low Mg(2+)-sensitivity and low Ca(2+)-permeability, generating small, long-lasting currents. Ca(2+)-imaging experiments showed slow [Ca(2+)](i) increases in 45% of the cells following 8 µM NMDA stimulation. Basal migration of ErbB4-transfected ST14A cells was unaffected by 18 hrs NMDA incubation. However, over the same incubation time, NMDA was able to significantly enhance NRG1-induced migration. Pre-incubation with the intracellular calcium chelator BAPTA-AM reduced both NRG1- and NRG1/NMDA-stimulated migration, suggesting the involvement of Ca(2+) in these processes. NRG1 stimulation of ErbB4-transfected ST14A cells induced a sustained, long-lasting increase in [Ca(2+)](i), in 99% of the cells. These intracellular Ca(2+) signals could be ascribed to both release from intracellular stores and influx from the extracellular medium trough a mechanism of store-operated calcium entry (SOCE). Short-time co-incubation of NMDA and NRG1 did not substantially modify the NRG1-induced intracellular calcium signals. CONCLUSIONS: In summary, NRG1 stimulation of the ErbB4 receptor exerts a sustained [Ca(2+)](i) increase in ST14A neural progenitors; NRG1-induced migration is Ca(2+)-dependent and can be positively modulated by activation of the NMDA receptor.

Specificity of the second messenger pathways involved in basic fibroblast growth factor-induced survival and neurite growth in chick ciliary ganglion neurons.

Basic fibroblast growth factor (bFGF) exerts multiple neurotrophic actions on cultured neurons from the ciliary ganglion of chick embryo, among them promotion of neuronal survival and of neurite outgrowth. To understand the specificity of the signal transduction cascades involved in the control of these processes, we used pharmacological inhibitors of the three main effectors known to act downstream of the bFGF receptor (FGFR): phospholipase Cgamma (PLCgamma), mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3-K). Neuronal survival was assessed at 24 and 48 hr; neurite growth was analyzed both on dissociated neurons and on explants of whole ganglia. Our data show that only the PI3-K pathway is involved in the survival-promoting effect of bFGF; on the other hand, all three effectors converge on the enhancement of neurite outgrowth, both on isolated neurons and in whole ganglia.

GDNF and bFGF are differentially involved in glial cell differentiation and neurite bundle formation in cultures from chick embryonic ciliary ganglia.

We have shown that the neurotrophic factors glial cell line-derived neurotrophic factor (GDNF) and basic fibroblast growth factor (bFGF) exert different effects on glial cells in cultures from chick embryo ciliary ganglia. bFGF acts as a mitogen on glial cells, and induces their aggregation to neuronal bodies; after 48 h of culture no glial cells could be observed along neurites. GDNF has no proliferative role; in contrast, it promotes the expression of the differentiative marker O4 and the association of glial cell bodies to neurites to form robust bundles.

A K(+) channel activated by cholinergic muscarinic receptors in chick ciliary ganglion neurons at early developmental stage.

Embryonic chick ciliary ganglion (CG) neurons obtained from E7-E8 ganglia maintained in serum-free medium were stimulated with 50 microM muscarine. A fast hyperpolarization of the membrane potential was observed in 25% of the cells tested, that in some cases was associated with a slower depolarization. Accordingly, in voltage clamp experiments, either an outward current or a biphasic current response could be observed. Single-channel experiments provide evidence that these signals can be associated to the activation of a K(+) channel whose conductance is 20 pS.

Calcium signals induced by FGF-2 in parasympathetic neurons: role of second messenger pathways.

Basic Fibroblast Growth Factor, or FGF-2, has been shown to promote neuronal survival and neurite outgrowth in dissociated neurons from the embryonic chick ciliary ganglion; in these effects the three main signal transduction pathways downstream the activated FGFR receptor, i.e. the MAPK, the PI3-K and the PLCγ ones, are differentially involved. While it has been shown that FGF-2 can elicit long lasting elevations in intracellular calcium concentration, [Ca(2+)](i), the role of the three pathways in this process has not been elucidated. Here we show, by means of pharmacological inhibitors, that all three are involved, at a different extent, in the generation of the [Ca(2+)](i) increase induced by FGF-2; in particular, inhibition of the PLCγ pathway, in addition to reducing the number of responsive cells, induces, in a significant population of cells, basal calcium oscillations in the absence of the growth factor and interferes with calcium signals elicited by depolarization. We propose that this complex behaviour can be due to a perturbation in PIP(2) levels at the plasmamembrane.

Temporal dynamics of neurite outgrowth promoted by basic fibroblast growth factor in chick ciliary ganglia.

Basic fibroblast growth factor (bFGF) is a potent and multifunctional neurotrophic factor that can influence neuronal survival and differentiation. It has been shown to modulate growth and orientation of neuritic processes both in intact organs and in neuronal cultures, with a wide spectrum of effects on different preparations. Here we report that it promotes neurite growth in developing parasympathetic neurons from the chick ciliary ganglion. We have used both organotypic cultures and dissociated neurons, and we have combined assessment of global neurite growth by immunocytochemical techniques with evaluation of dynamic parameters of single neurites via time-lapse microscopy. We show that laminin, a molecule of the extracellular matrix that has been associated with stimulation of neurite extension, has only a limited and short-lived effect on neurite outgrowth. In contrast, bFGF can promote global growth of the neuritic network both in whole ganglia and in dissociated cultures for times up to 48 hr, and this effect is related to an increase in the growth rate of single neurites. Moreover, the effect can be observed even in enriched neuronal cultures, pointing to a direct action of bFGF on neurons.

In vitro analysis of neuron-glial cell interactions during cellular migration.

We used time-lapse microscopy to study the in vitro migration of neuronal cells from developing chick ciliary ganglion. These cells, when dissociated and cultured in a chemically defined medium, are able to migrate and to associate into clusters. We focused our attention on the study of the distribution of neuronal velocity components. Quantitative analysis of cell trajectories allowed us to demonstrate that, in many cells, velocities are well described by the Langevin equation, when deterministic components of the forces acting on the cells are taken into account. We also have shown that the majority of neurons whose movement is not purely random migrate in association with glial cells. We conclude that glial cells, by guiding neurons during migration, play an important role in the cell organization in vitro.