Fig 1.
Morphometric analysis of corneal nerve surface-to-volume ratio using a cycloid grid.
A single image from an SBF-SEM series showing a nerve that has fused with a basal epithelial cell (A). A micrograph from this series was selected at random and a cycloid grid was randomly cast onto the image while maintaining the orientation of the grid (defined by the vertical white arrow) parallel to the epithelial basal lamina (B). The intersection of the grid lines with the surface of the nerve bundle are marked with blue dots (surface area) while grid points falling within the nerve bundle are marked with green dots (volume); the inset, enlarged in panel (C), offers a magnified view of the grid. Scale bars = 2 μm.
Fig 2.
Application of DiI to the trigeminal ganglia of the mouse.
A dissected view of the mouse cranial cavity showing trigeminal ganglia (*) straddling the optic chiasm. The trigeminal branches are labeled as 1, 2 and 3 and correspond to the ophthalmic, maxillary, and mandibular branches. DiI crystal was applied to the severed ophthalmic branch.
Fig 3.
Stromal nerve fusion with the basal epithelium.
A stromal nerve fused with two basal epithelial cells (black arrowheads) through two distinct pores in the basal lamina (Electron density; white arrowheads) (A). Enlargement of panel (A) inset showing magnified view of one basal lamina pore (B). Note the continuity between neuronal and epithelial plasma membranes at the site of fusion (arrows). Electron dense Schwann cell nuclei (N) were visible near the fusion site. The axoplasm (*) was electron translucent, lacked mitochondria and mixing between axoplasm and epithelial cell cytoplasm (E) was not evident. Scale bars = 2 μm.
Fig 4.
Neuronal-epithelial cell fusion involved mixed bundles of fusing and penetrating axons.
Four examples of neuronal-epithelial cell fusion (A-D). The electron translucent portion of each nerve bundle (*) was fused with a basal epithelial cell. Penetrating nerves that continued into the epithelium (visible in Panel A) contributed to the sub-basal plexus and were recognized by their greater electron density (arrow). Scale bar = 2 μm.
Fig 5.
3D reconstruction of nerve penetration through the epithelial basal lamina.
A series of SBF-SEM images showing a penetrating electron dense corneal nerve (*; A-F) that entered the epithelium through a discontinuity in the basal lamina (Panels B-E; arrows). A continuous basal lamina was present on either side of the penetration point (A & F). 3D reconstruction of the penetrating nerve (white) as it passed through the basal lamina (green; G & H). The nerve bifurcated prior to penetration (H; arrowheads). After penetrating into the corneal epithelium, both nerve branches underwent ramification. Scale bar = 2 μm.
Fig 6.
3D reconstruction of neuronal-epithelial cell fusion at the epithelial basal lamina.
A series of SBF-SEM images showing a mixed nerve bundle in which fusing (*) and penetrating axons (arrows) were evident (A-F). The electron dense penetrating axons passed through the basal lamina and contributed to the sub-basal plexus (F). 3D reconstruction of a mixed nerve bundle showing fusing (purple) and penetrating (white) axons (G). A large fusion area (blue) denotes neuronal fusion involving three separate epithelial cells and both penetrating (H) and fusing (I) axons. Scale bar = 2 μm.
Fig 7.
Fusing nerves had smaller surface-to-volume ratios than penetrating nerves.
A penetrating nerve bundle which has passed through the epithelial basal lamina giving rise to the sub-basal plexus (A). The axoplasm was electron dense and contained numerous mitochondria. A nerve bundle containing fusion that has merged with a basal epithelial cell (B). The axoplasm was electron translucent and devoid of mitochondria. The surface-to-volume ratio of nerve bundles containing fusion were significantly smaller than that of penetrating nerve bundles consistent with their “swollen” appearance (C). The diameter of the basal lamina pores through which these nerves interact with the corneal epithelium was similar (D). Scale bars = 2 μm.
Fig 8.
3D reconstruction confirmed fusing axons lack mitochondria at the site of fusion.
Segmentation and 3D reconstruction of penetrating and fusing nerves (A-D). Mitochondria (yellow), penetrating axons (white), fusing axons (purple), and basal lamina (green) are shown. Conventional nerve penetration of the basal lamina involving multiple axons (A) or a single axon (B). In both cases, mitochondria were present throughout the nerve bundle on either side of the basal lamina. Mixed nerve bundle at the basal lamina showing penetrating and fusing axons (C). Mitochondria are clearly absent from the fusing axons. Isolation of the penetrating axons shows mitochondria to be distributed throughout the axoplasm (D).
Fig 9.
3D reconstruction showed mitochondria are present within the distal portion of fusing axons.
Serial images show three levels (A-C) within the 3D reconstruction (D) of the distal portion of the mixed nerve bundle shown in Fig 4. The most distal portion of the nerve within the image series (A) was located ~60 μm distal to the site of fusion and it contained numerous mitochondria and an electron dense axoplasm. As the nerve bundle approached the fusion site, it increased in diameter (B & C). At ~35 um distance from the fusion site, mitochondria (blue) were no longer present in the fusing axons whereas mitochondria (yellow) were retained within the penetrating axons (D). White arrowheads denote the locations of panels A-C within the reconstructed nerve.
Fig 10.
High resolution TEM showed an absence of microtubules in fusing neurons.
A conventional stromal nerve bundle (A) in which the inset is enlarged (B) to show cross-sectional views of microtubules identified by their size and distinctive hollow-ring appearance (arrows). Mitochondria are also present and identified by their double-membranes and internal cristae (*). A fusing nerve bundle (C) with an electron translucent axoplasm in which the inset is enlarged (D) to show the distinct lack of microtubules and mitochondria. Scale bar for panels A & C = 2 μm. Scale bar for panels B & D = 0.2 μm.
Fig 11.
DiI applied to the trigeminal ganglion labeled corneal axons and a sub-population of basal epithelial cells.
While conventional nerve penetration (A) showed DiI labeling extending from the stromal nerve to the sub-basal plexus, the overlying basal epithelium remained unlabeled. In addition, DiI-labeled stromal nerves approached the epithelium and labeled a sub-population of basal epithelial cells (B-D). DAPI staining confirmed the basal location of these epithelial cells and the arrows (column 1) denote nuclei belonging to DiI labeled basal epithelial cells (column 2). Scale bar = 2 μm.
Fig 12.
Orthogonal projection confirmed DiI transfer from corneal neuron to a single basal epithelial cell.
Two fluorescence images from a Z-stack showing a DiI (red) labeled basal epithelial cell (A) located above a DiI labeled stromal nerve (B). An orthogonal slice through the stack taken between the two dashed lines is shown in panel (C) where the DiI labeling extended uninterrupted from the neuronal plasma membrane into the epithelial cell membrane. DAPI (blue) staining denotes cell nuclei. Scale bars = 10 μm.