The diffusion barrier is formed in DIV 10 but not in DIV 4 neurons. Comparison of the diffusion behavior of a mutant deficient for ankyrin binding (Kv-Nav EA4SA) at two development stages, DIV 4 (A–C) and DIV 10 (D–F), and in two distinct compartments of the plasma membrane, the AIS and proximal dendrites. For DIV 4, n = 18 and 20 trajectories from three and two independent experiments at the AIS and at proximal dendrites, respectively. For DIV 10, n = 36 and 41 trajectories from three and two independent experiments at the AIS and at proximal dendrites, respectively. (A and D) Cumulative frequencies of the instantaneous diffusion coefficients at the AIS and in proximal dendrites in DIV 4 (A) and DIV 10 (D) neurons. KS: ***, P < 0.001. (B and E) Histogram of the mean values ± SEM of the immobile population percentage in DIV 4 (B) and DIV 10 (E) neurons at the AIS and in proximal dendrites. MW: *, P < 0.05. (C and F) MDC (25–75% IQR) of the mobile population at the AIS and in proximal dendrites. MW: **, P < 0.01. (G) Mean plot of the MSD of the mobile population versus time (solid line) ± SEM (dotted line) for the indicated period for the mobile population at the AIS of DIV 4 and DIV 10 neurons. (H) Magnification of the boxed area in G.