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During neurodevelopment, neurons form growth cones, F-actin rich extensions located at the distal end of the neurites. Growth cones allow dendrites and axons to build synaptic connections through a process of neurite guidance whose mechanisms have not been fully elucidated. Calcium is an important element in this process by inducing F-actin reorganization. We hypothesized that other biologically active elements might be involved in the growth cone-mediated neurite guidance mechanisms. We performed super resolution and confocal microscopy of F-actin, followed by synchrotron X-ray fluorescence microscopy of phosphorous, sulfur, chlorine, potassium, calcium, iron and zinc on growth cones from primary rat hippocampal neurons. We identified two main patterns of element organization. First, active growth cones presenting an asymmetric distribution of Ca co-localized with the cytoskeleton protein F-actin. In active growth cones, we found that the distributions of P, S, Cl, K, and Zn are correlated with Ca. This correlation is lost in the second pattern, quiescent growth cones, exhibiting a spread elemental distribution. These results suggest that Ca is not the only element required in the F-actin rich active regions of growth cones. In addition, highly concentrated Fe spots of submicrometer size were observed in calcium-rich areas of active growth cones. These results reveal the need for biological active elements in growth cones during neural development and may help explain why early life deficiencies of elements, such as Fe or Zn, induce learning and memory deficits in children.

Imaging the structural organization of chemical elements in growth cones of developing hippocampal neurons