The biophysical properties of intracellular diffusion and trafficking of soluble proteins in the axon of a living organism remains largely unknown,in spite of numerous findings from the cultured cells.In the present study,we have used in vivo time-lapse imaging and fluorescent recovery after photobleaching (FRAP) to study the mobility of diffusion of green fluorescence protein (GFP) and active transportation of vesicle associate membrane protein 2 (VAMP2-GFP) in the axon of Mauthner cells and motoneurons in zebratish larvae.We found that a diffusion barrier emerged in the cytoplasm of axonal initial segment (IS),by comparing the recovery rate of GFP and VAMP2-GFP at the IS and main shaft of the axon after the photobleaching.This barrier was susceptible to latrunculin A,which is known to disrupt the actin cytoskeleton in the cytoplasm.Furthermore,the VAMP2-GFP trafficking rate was significantly faster in the anterograde direction than that for retrograde direction.Thus,our results suggest that the actin-based cytoplasmic barrier may exist in the axonal IS and attenuate the mobility of anterograde diffusion or active transportation of axonal proteins.