Differential outgrowth of axons and their branches is regulated by localized calcium transients

During development axon outgrowth and branching are independently regulated such that axons can stall or retract while their interstitial branches extend toward targets. Previous studies have shown that guidance cues and intracellular signaling components can promote branching of cortical axons without affecting axon outgrowth. However, the mechanisms that regulate differential outgrowth of axons and their branches are not well understood. Based on our previous work showing the importance of localized repetitive calcium transients in netrin-1-induced cortical axon branching, we sought to investigate the role of calcium signaling in regulating differential outgrowth of axons and their branches. Using fluorescence calcium imaging of dissociated developing cortical neurons, we show that localized spontaneous calcium transients of different frequencies occur in restricted regions of axons and their branches. Higher frequencies occur in more rapidly extending processes whereas lower frequencies occur in processes that stall or retract. Direct induction of localized calcium transients with photolysis of caged calcium induced rapid outgrowth of axonal processes. Surprisingly outgrowth of one axonal process was almost invariably accompanied by simultaneous retraction of another process belonging to the same axon, suggesting a competitive mechanism for differential process outgrowth. Conversely, reducing frequencies of calcium transients with nifedipine and TTX reduced the incidence of differential process outgrowth. Together these results suggest a novel activity-dependent mechanism whereby intrinsic localized calcium transients regulate the competitive growth of axons and their branches. These mechanisms may also be important for the development of cortical connectivity in vivo.