Calcium as a second messenger in neuronal growth cones

Calcium is thought to be involved in the regulation of motility and growth in the neuronal growth cone. The aim of this work was to investigate the role of cytosolic free calcium concentration ([Ca2+]i) and voltage-gated calcium influx in controlling growth cone behaviour and neurite elongation. [Ca2+]i and voltage-gated calcium influx was measured using digital imaging fluorescence microscopy (DIFM) and the whole-cell patch-clamp technique. The behaviour of growth cones of N IE-115 mouse neuroblastoma was observed and categorized into five classes. The spatial distribution of [Ca2+]i associated with these behavioural states was measured. True growth occurred only at the lowest levels. Any spontaneous elevation of [Ca2+]i above this level was associated with an inhibition of neurite elongation. Large elevations of [Ca2+]i were associated with specific morphological changes. L- and T-type voltage gated calcium currents were measured during differentiation. All cells possessing neurites had T-type currents. T-type current density was higher in cells with neurites than in cells without. There was no other detectable change in the characteristics of either current during differentiation. The spatial distribution of voltage-dependent calcium influx through L- and T-type channels was measured at the growth cone. L-type channels were found to be clustered in the growth cone membrane, whereas T-type channels were uniformly distributed. Activation of L-channel clusters caused [Ca2+]i 'hotspots' in which [Ca2+]i rose to micromolar levels during 1s depolarizations. Localized morphological changes at the site of the hotspot were observed during action potentials