The diffusion of membrane molecules is restricted by DIV5 in hippocampal neurons. (A) Experimental design for the developmental time course in cultured primary hippocampal neurons. (1) Neurons were maintained in gridded glass-bottom Petri dishes and (2) transfected with the membrane-probe GPI-GFP. (3) Individual neurons were relocated on DIV3, 5, 7, and 10, respectively, based on their position on the grid. (4) Single-particle tracking (SPT) experiments were conducted by sparsely labeling transfected neurons with anti-GFP nanobodies conjugated to fluorescent organic dyes. (5) After SPT, neurons were fixed and immunostained for cytoskeletal and or AIS marker. The soma (i) and the proximal axon with the AIS (ii), identified by AnkG staining (inset micrograph), are outlined. (B) Results from the developmental SPT time course on a typical neuron. The AIS was identified by live immunolabeling of neurofascin. (left box) The proximal axon was neurofascin negative on DIV3 and neurofascin positive from DIV5 onwards. (middle box) Plot of trajectories of mobile particles tracked in SPT experiments color-coded for instantaneous diffusion coefficients D. (right box) Histogram of D on the proximal axon where the AIS assembles (white dashed line). Number of trajectories: DIV3, n = 787; DIV5, n = 1,815; DIV7, n = 2005; and DIV10, n = 1,067. (C) Plots of the cumulative D in the AIS (left, shades of green) and a portion of the distal axon (right, shades of blue) of the neuron shown in B. (D) Graph of the median D for all neurons (n = 4) between DIV3 and DIV10. Statistical analysis of median D by paired t test; *, P < 0.01; ns, not significant. Bars, 5 µm.