The role of calcium activity in cortical axon growth and guidance

In the developing nervous system axons tipped with motile growth cones navigate to targets by responding to environmental guidance cues. Calcium signaling plays an important role in these events. However, target innervation in the mammalian central nervous system often occurs by interstitial branching but the role of calcium signaling in this process was unknown. By measuring calcium activity in dissociated cortical neurons I found that higher frequency localized calcium transients occurred in advancing axon processes while lower frequencies were associated with retraction. Using photolysis of caged calcium, I found that calcium activity evoked in one process promoted outgrowth while unstimulated processes of the same axon stalled or retracted. This suggested a competitive activity dependent mechanism for differentially regulating the outgrowth of axons and their branches.;Does calcium signaling play a role in the growth and guidance of cortical axons in vivo? To investigate this question, I devised a cortical slice model in which I transfected cortical neurons with a calcium biosensor and imaged calcium activity in growing corpus callosal axons. Measurements of calcium activity showed that higher frequencies of calcium transients were associated with faster rates of outgrowth, as I had found in vitro. We previously found that Wnt5a, a morphogen that regulates axon guidance, evokes calcium signaling pathways to increase axon outgrowth of dissociated neurons and induce repulsive growth cone turning. Wnt5a gradients surround the corpus callosum in vivo. Therefore, I asked whether Wnt/calcium signaling mechanisms regulate the growth and guidance of callosal axons. In dissociated cultures we found that calcium release from stores through IP3 receptors and calcium entry through TRP channels differentially regulates Wnt induced axon outgrowth and repulsive turning. Similar pharmacological approaches in cortical slices showed that blocking these channels reduced axon outgrowth rates and caused guidance errors. We found that CaMKII is an important signaling component in the Wnt/calcium pathway. Inhibiting CaMKII reduced axon outgrowth and resulted in guidance errors in the callosum. Remarkably, knockdown of the Wnt receptor Ryk reduced rates of callosal axon outgrowth and caused guidance errors by attenuating calcium signaling, revealing the importance of Wnt/calcium signaling in vivo.