Localization of CdSe/ZnS quantum dots in the lysosomal acidic compartment of cultured neurons and its impact on viability: potential role of ion release.

CdSe Quantum Dots (QDs) are increasingly being employed in both industrial applications and biological imaging, thanks to their numerous advantages over conventional organic and proteic fluorescent markers. On the other hand a growing concern has emerged that toxic elements from the QDs core would render the nanoparticles harmful to cell cultures, animals and humans. The interaction between QDs and neuronal cells in particular needs to be carefully evaluated, since nanoparticles could access the nervous system by several pathways, including the olfactory epithelium, even if no data are presently available about QDs. The pH of the environment to which the nanoparticles are exposed may play a crucial role in the stability of QDs coating. For this reason we investigated the release of metal ions from CdSe/ZnS QDs in artificial media reproducing the cytosolic and lysosomal cellular compartments characterized respectively by a neutral and an acidic pH. In the latter significant amounts of both Cd(2+) and Zn(2+) were released. We provide evidence that these QDs are internalized in the GT1-7 neuronal cell line and located in the lysosomal compartment. These findings can be related to a slight but significant reduction in cell survival and proliferation.

TRPC channels are involved in calcium-dependent migration and proliferation in immortalized GnRH neurons.

Gonadotropin-releasing hormone (GnRH)-secreting neurons are key regulators of the reproductive behaviour in vertebrates. These neurons show a peculiar migratory pattern during embryonic development, and its perturbations have profound impact on fertility and other related functional aspects. Changes in the intracellular calcium concentration, [Ca(2+)](i), induced by different extracellular signals, play a central role in the control of neuronal migration, but the available knowledge regarding GnRH neurons is still limited. Our goal was to investigate mechanisms that may be involved in the Ca(2+) dependence of the migratory behaviour in these neurons. We focused on the "classical" Transient Receptor Potential (TRPC) subfamily of Ca(2+)-permeable cation channels, recently shown to be involved in other aspects of neuronal development. Using GN11 cells, immortalized early stage GnRH neurons, we set to investigate Ca(2+) signals under basal conditions and in the presence of a well-established motogen, fetal calf serum (FCS), and the effect of pharmacological TRPC agonists and antagonists on Ca(2+) oscillations, cell motility and proliferation. We have found that a subpopulation of GN11 cells shows spontaneous Ca(2+) transients and that this activity is increased in the presence of serum. Quantitative real-time PCR showed that transcripts of some TRPC members are expressed in GN11 cells. Interestingly, pharmacological experiments with inhibitors, SKF-96365, lanthanum, anti-TRPC1 antibody, and activators, 1-oleil 2-acetyl-sn-glycerol, of TRPCs suggested that the activation of these channels can account for both the basal Ca(2+) oscillations and the increased activity in the presence of FCS. Moreover, functional studies using the same pharmacological tools supported their involvement in the control of motility and proliferation. Thus, our data provide evidence for the involvement of Ca(2+)-permeable channels of the TRPC subfamily in the control of functional properties of neurosecretory cells and neuronal motility.

Expression and localisation of TRPC channels in immortalised GnRH neurons.

Calcium-permeable cation channels of the transient receptor potential (TRP) superfamily are involved in agonist-induced calcium influx in several cell types. In this work we evaluated expression and localisation of classical TRP (TRPC) channels in two immortalised cell lines derived from the gonadotrophin releasing hormone (GnRH) neuroendocrine system, at different developmental stages: GT1-7 cells display many characteristics of mature hypothalamic GnRH neurons and are a suitable model to study neuritogenesis and neurosecretion, whereas GN11 cells retain a more immature phenotype with migratory activity. Immunoblotting analysis demonstrates that GN11 and GT1-7 cells differentially express several members of the TRPC family: TRPC1 and TRPC5 are expressed at high levels in GN11 cells, and TRPC4 is expressed at higher levels in GT1-7 cells. Immunocytochemical experiments show a widespread localisation for TRPC proteins in GN11 cells and a characteristic staining along neurites in GT1-7 cells. These data suggest that different TRPC proteins could play specific functional roles at different developmental stages of the GnRH system.

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.