Fig. 7. Testosterone enhances neurite outgrowth. Morphological changes in neuroblastoma cells were monitored after 3 days of treatment with testosterone. Cells were incubated with Cell Tracker green and visualized by confocal microscopy. (A) Neuroblastoma cells grown under control conditions have short neurites. (B) Cells treated with testosterone exhibit an increase in the neurite outgrowth compared with non-stimulated control cells. Bar, 30 µm. (C) Neurite outgrowth was normalized by calculating the ratio of the neurite length to the soma length (neurite/soma). Testosterone-induced neurite outgrowth was 2.5-fold higher than control cells. Neurite elongation induced by testosterone was inhibited in cells with the cytosolic Ca²⁺ buffered by the expression of PV-NES-DSR. Buffering the nuclear pool of Ca²⁺ with PV-NLS-DSR induced a smaller increase in the neurite outgrowth compared with non-transfected cells. Cells expressing the mutated form of the nuclear localized parvalbumin showed a response to testosterone similar to the response observed in control cells. (D) Neurite elongation was smaller after inhibition of the iAR pathway (siRNA-AR or cyproterone) or when using T-BSA compared with the response with testosterone alone. These results suggest that genomic and non-genomic mechanisms for testosterone-induced neurite outgrowth in neuroblastoma cells are inter-dependent. Values are mean ± s.e.m., ^*P<0.05; ^**P<0.01.