Correct wiring of the nervous system requires guidance cues, diffusible or substrate-bound
proteins that steer elongating axons to their target tissues.
Netrin-1, the best characterized member of the
Netrins family of guidance molecules, is known to induce axon turning and modulate axon elongation rate; however, the factors regulating the axonal response to
Netrin-1 are not fully understood. Using microfluidics, we treated fluidically isolated axons of mouse primary cortical neurons with
Netrin-1 and characterized axon elongation rates, as well as the membrane localization of deleted in
colorectal cancer (DCC), a well-established receptor of
Netrin-1. The capacity to stimulate and observe a large number of individual axons allowed us to conduct distribution analyses, through which we identified two distinct neuron subpopulations based on different elongation behavior and different DCC membrane dynamics.
Netrin-1 reduced the elongation rates in both subpopulations, where the effect was more pronounced in the slow growing subpopulation. Both the source of Ca(2+) influx and the basal cytosolic Ca(2+) levels regulated the effect of
Netrin-1, for example, Ca(2+) efflux from the endoplasmic reticulum due to the activation of
Ryanodine channels blocked Netrin-1-induced axon slowdown.
Netrin-1 treatment resulted in a rapid membrane insertion of DCC, followed by a gradual internalization. DCC membrane dynamics were different in the central regions of the growth cones compared to filopodia and axon shafts, highlighting the temporal and spatial heterogeneity in the signaling events downstream of
Netrin-1. Cumulatively, these results demonstrate the power of microfluidic compartmentalization and distribution analysis in describing the complex axonal
Netrin-1 response.