The Mechanical Microenvironment Regulates Axon Diameters Visualized by Cryo-Electron Tomography

Abstract

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.

Handling difficult cryo-ET samples: A study with primary neurons fromDrosophila melanogaster

“…When neurons were cultured on grids with either 2 µm holes spaced 1 µm apart (R 2/1) or 1.2 µm holes spaced 20 µm (R 1.2/20) apart, swellings were spotted along the neurite (Figure 2). These swellings resembled the varicosities that have been reported in past studies (Ma et al, 2022, Fischer et al, 2018, Schrod et al, 2018. We noted that at both the neurite and cell body, membrane blebs, defined as spherical, blister-like membrane protrusions commonly seen in processes of apoptosis and cytotoxicity, were frequently seen surrounding the periphery, (Ponuwei, 2017, Lesort et al, 1997.…”

“…With the observation that chemically fixed Drosophila neurons had considerably better cellular integrity and reduced frequency of varicosities, we decided to quantitatively examine the nature of these varicosities. We defined varicosities as oblong-shaped swellings that have diameters at least three times larger than the flanking neurites, based on past work that examined these objects present in tomograms of mouse neurons (Ma et al, 2022). Such swellings can become as large as 6 µm, which can be easily resolved by light microscopy yet were rarely seen by FLM before plunge-freezing.…”

“…In addition, the thinness and length-spans of neurites when compared with other cellular structures, may lend them to being more fragile during the processes of grid blotting and plunge-freezing. Axonal varicosities, defined as enlarged, oval-shaped swellings along narrow neurite shafts, have been noticeably present in most cryo-ET studies done on primary neurons (Foster et al, 2022, Schrod et al, 2018, Ma et al, 2022, Wu et al, 2023, Liu et al, 2020, Tao et al, 2018, Fischer et al, 2018. When such swellings appear in vivo or in vitro, they have been associated with neuronal damage, such as neurodegeneration or mild traumatic brain injury (Sun et al, 2022a).…”

“…When such swellings appear in vivo or in vitro, they have been associated with neuronal damage, such as neurodegeneration or mild traumatic brain injury (Sun et al, 2022a). It was also demonstrated that puffs of buffer at low pressure were enough to induce varicosity formation in cultured neurons, suggesting that it may be possible that processes of blotting and plunge-freezing could induce varicosities due to contact pressure (Gu et al, 2017, Ma et al, 2022. Whether neurite varicosities in cryo-EM and cryo-ET studies are physiologically relevant or are artifacts of sample preparation is unknown.…”

High-resolution spatiotemporal analysis of single serotonergic axons in an in vitro system

“…Early studies in other systems have suggested that varicosities can form directly from “stopped” growth cones ( Hatada et al, 1999 ), and that their distribution can be described by random point-processes such as the Poison process ( Hellwig et al, 1994 ). More recent studies have shown that varicosities strongly respond to their mechanical and biological environment ( Shepherd et al, 2002 ; Ma et al, 2022 ) and are generally plastic, including pathological states ( Gu, 2021 ). In the mouse brain, serotonergic axons appear to undergo a developmental transition from “dot-like” enlargements, with virtually undetectable connections, to a smooth morphology by the end of the first developmental month ( Maddaloni et al, 2017 ).…”