Figure 6

Increasing neuronal activity induces the reorganization of F-actin rings into longitudinal fibers in dendrites but not in axons. (a) Representative images of the periodical F-actin rings in axons of neurons exposed to 4 different treatments modulating neuronal activity. The area of F-actin rings segmented by the FCN \(\mathrm {h}_{\rm {a}}\) is shown in green. (b) The detected area for axonal rings in 13 DIV neurons remains unchanged upon stimulation (one-sided ANOVA, \(p = 0.2525\), numbers above each boxes indicate the number of neurons from 4 independent cultures). (c) Representative images of the periodical F-actin lattice and the longitudinal fibers in dendrites of 13 DIV neurons for four different treatments modulating neuronal activity: Top STED Images of F-actin stained with Phalloidin-STAR635 overlayed with the confocal images of the dendritic (MAP2, yellow) and axonal (phosphorylated neurofilaments, cyan) makers. Bottom: Predictions of the FCN for F-actin rings (green) and fibers (magenta) inside the dendritic mask (white line). (d) Bivariate kernel density estimate of the raw data (grey cross) for 8 DIV and 13 DIV neurons treated with (i) high \(\text {Mg}^{2+}\)/low \(\text {Ca}^{2+}\) for 10 min (blue), (ii) 0\(\text {Mg}^{2+}\)/Gly/Bic for 10 min (green), (iii) high \(\text {K}^{+}\) for 2 min (orange) and iv) Glu/Gly for 2 min (violet). (e) Mean distributions using bootstrapping. The formation of F-actin fibers is enhanced for 13 DIV neurons compared to 8 DIV neurons for the synaptic stimulation 0\(\text {Mg}^{2+}\)/Gly/Bic or Glu/Gly, but not for high \(\text {K}^{+}\) stimulations. Shown are the regions comprising 95%, 99% and 99.9% of the data point distribution. Scale bars 1 \(\upmu\)m. d,e) Number of independent cultures (N): high \(\text {Mg}^{2+}\)/low \(\text {Ca}^{2+}\) \(N_{\mathrm{8 DIV}} = 6, N_{\mathrm{13 DIV}} = 9\); 0\(\text {Mg}^{2+}\)/Gly/Bic \(N_{\mathrm{8 DIV}} = 4, N_{\mathrm{13 DIV}} = 8\); high \(\text {K}^{+}\) \(N_{\mathrm{8 DIV}} = 6, N_{\mathrm{13 DIV}} = 9\); Glu/Gly \(N_{\mathrm{8 DIV}} = 5, N_{\mathrm{13 DIV}} = 6\). Note that the detected areas for dendrite and axons cannot be compared since the detection of F-actin rings was performed with two different FCNs and using different foreground masks. Only a comparison between the stimulation conditions for each experiment (dendrites or axons) is possible. Scale bars 1 \(\upmu\)m. For the raw image without overlay see Supplementary Fig. 10.