Neuron Subpopulations with Different Elongation Rates and DCC Dynamics Exhibit Distinct Responses to Isolated Netrin-1 Treatment

Abstract

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 sub-populations based on different elongation behavior and different DCC membrane dynamics. Netrin-1 reduced the elongation rates in both sub-populations, where the effect was more pronounced in the slow growing sub-population. Both the source of Ca 2+ influx and the basal cytosolic Ca 2+ levels regulated the effect of Netrin-1, e.g., 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.

Charge and topography patterned lithium niobate provides physical cues to fluidically isolated cortical axons

“…26 The culture medium consisted of DMEM-Glutamax supplemented with 10% fetal bovine serum, 1% Penicillin/Streptomycin solution (P/S), and B 27 neuron supplement (all from Gibco). At 3 days in vitro (DIV), the culture medium was replaced with Neurobasal (Gibco) supplemented with 1% P/S and B 27 .…”

“…Cells were aldehyde-fixed on 3-6 DIV, stained to reveal b3-tubulin, F-actin, and nuclei, and mounted on microscope slides as previously described. 26 Neurite length was quantified from tubulin staining using the NeuronJ plug-in 28 in ImageJ (NIH, Bethesda, MD). Cell clustering was quantified by dividing the number of clusters to the number of nuclei.…”

Neuronal Cell Bodies Remotely Regulate Axonal Growth Response to Localized Netrin-1 Treatment via Second Messenger and DCC Dynamics

“…These devices consist of axonal and somatic compartments that are fluidically isolated for at least 4 h, as demonstrated using a fluorescent tracer (Blasiak et al, 2015). Axons reached the axonal compartment after 3 days and extended toward its distal wall after 5–6 days (Figure 1B).…”

“…(E) Comparison of axon growth in the first (early) and last (late) 0.5 h of imaging. Data for axonal Netrin-1 treatment are re-presented with permission from Blasiak et al (2015). Copyright 2015 American Chemical Society.…”

“…In contrast to extensive studies on axon turning, fewer efforts have focused on Netrin-1's effects on axon elongation rate. We recently showed that when applied locally to the axons, uniform concentrations of Netrin-1 increase membranous DCC level and slow down cortical axons (Blasiak et al, 2015). The roles of second messengers specific to these processes are poorly understood.…”