Fig. 2

Vesicular release under the myelin. a Myelin loaded with DiOC₆ (green) and the Ca²⁺-indicator X-rhod-1 (red) in adult mouse RON. Note the loading into myelin profiles (e.g., arrow). b Top: X-rhod-1 intensity (relative to initial mean) is elevated following depolarization with 50 mMK⁺ (black line) consistent with activation of NMDA receptors in the myelin, an effect blocked by pre-treatment with the vesicular loading blocker bafilomycin (red line). b Bottom: data summary. Asterisks on the error bars indicate P = 0.0000 significance vs., the initial fluorescence mean; on the bar they indicate P = 0.0000 significance vs., the two conditions. n = 5 mice in each protocol, 1–3 slices/mouse, ANOVA with Holm−Šídák post test. Bar = 8 μm. c 20–50 nm vesicles are present in clusters in the sub-myelinic axoplasm (e.g., arrows) (Ax = axons; Ast = astrocyte process containing glial filaments; bar = 1 μm). d Sub-myelinic vesicles are less common in RONs fixed after 30 min of OGD and can be seen juxtaposed on the axolemma (black arrow). White boxed area shown at higher magnification above. Note the absence of vesicles in the astrocyte process identified by the presence of glial filaments (Gf), and the retention of microtubules in myelinated axons (white arrows). Bar = 1 μm. e A single vesicle (black arrow) docked with the axolemma (arrow heads) beneath the myelin following OGD. Note the presence of focal myelin injuries in both (d) and (e) (white asterisks). Bar = 500 nm. f, g The distribution of vesicle clusters in axon profiles by number of vesicles in control and 30 min OGD RONs (per field of view). Note the large number of axons with zero vesicles following OGD (scales differ). h, i Focal myelin injury scores are shifted to higher values in myelin regions adjacent to axoplasmic vesicles post OGD compared to control RONs