Axonal varicosities or swellings are enlarged structures along axon shafts and profoundly affect action potential propagation and synaptic transmission. These structures, which are defined by morphology, are highly heterogeneous and often investigated concerning their roles in neuropathology, but why they are present in the normal brain remains unknown. Combining confocal microscopy and cryo-electron tomography (Cryo-ET) with in vivo and in vitro systems, we report that non-uniform mechanical interactions with the microenvironment can lead to 10-fold diameter differences within an axon of the central nervous system (CNS). In the brains of adult Thy1-YFP transgenic mice, individual axons in the cortex displayed significantly higher diameter variation than those in the corpus callosum. When being cultured on lacey carbon film-coated electron microscopy (EM) grids, CNS axons formed varicosities exclusively in holes and without microtubule (MT) breakage, and they contained mitochondria, multivesicular bodies (MVBs), and/or vesicles, similar to the axonal varicosities induced by mild fluid puffing. Moreover, enlarged axon branch points often contain MT free ends leading to the minor branch. When the axons were fasciculated by mimicking in vivo axonal bundles, their varicosity levels reduced. Taken together, our results have revealed the extrinsic regulation of the three-dimensional ultrastructures of central axons by the mechanical microenvironment under physiological conditions.
Keywords: axon branch point; axon fasciculation; axonal varicosity; cryo-electron tomography (Cryo-ET); microtubule (MT); mitochondria; multivesicular body (MVB); primary neuron culture.