Axons are the principle site of white matter vesicular fusion. a Thy-1-YFP expression in adult coronal section (box: corpus callosum). b, c YFP(+) corpus callosum axons (b) and FM4-64 vesicles (c overlaid red). d Single axons (overlaid at higher gain in the box). e, f Corpus callosum astrocyte (e) in adult GFAP-GFP mouse and FM4-64 staining (f overlaid red). g K⁺-evoked FM4-64 de-staining in whole corpus callosum, axons, and astrocytes. h Corpus callosum axon FM4-64 de-staining in aCSF and during ischemia. i K⁺-evoked [glutamate]_e release is not inhibited by TBOA. j Both the first (top) and second (bottom) 50 mMK⁺-evoked glutamate rise is reduced by bafilomycin (50 nM, red trace). k Data summary shown on the bottom. Scale = 1 mm (a), 10 μm (b, d, f). Stars indicate the first mean to reach significance. Grouped analysis ANOVA; correct for multiple comparisons using the Holm−Šídák method. P values: g top *0.016, **0.001, ***0.0000; g middle *0.038, **0.002, ***0.0004; h *0.021, **0.009, ***0.0013. Unpaired t-test P values: k Peak 1 ns 0.1119; Peak 2 *0.048

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

White matter ischemic glutamate release is primarily vesicular. a Ischemia-evoked [glutamate]_e elevation in adult rat RON is Na⁺-independent/TBOA-insensitive. b The ischemic-[glutamate]_e rise is Ca²⁺-dependent (ANOVA with Holm−Šídák post test: P = 0.012) and diltiazem-sensitive (P = 0.010). c, d In juvenile rat RON, the ischemic [glutamate]_e elevation (P = 0.001 vs., control aCSF) is Ca²⁺-dependent (P = 0.009 vs., ischemia)/Na⁺-independent and resistant to inhibitors of non-vesicular glutamate release pathways. e, f Ischemic-[glutamate]_e elevation in juvenile RON is significantly inhibited by blockers of vesicular glutamate loading bafilomycin (P = 0.004); rose bengal (P = 0.013) and combined bafilomycin + TBOA (P = 0.004)

Myelin injury is NMDA-receptor-mediated. a CAP recordings from adult rat RON showing elevated functional recovery from ischemia following 120 min MK-801 pre-treatment. b Ischemia-evoked loss and recovery of function following different periods of MK-801 pre-treatment (insert: data summary). ANOVA with Holm−Šídák post test. P values: 90 min **0.002; 120 min ***0.0001; 165 min ***0.0000. c 120 min pre-treatment with selective NMDA receptor GluN2C/D blockers QNZ-46 (P = 0.0013) or PPDA (P = 0.0009), or the TRPA1 blocker A967076. d Oligodendrocyte cytoplasmic domain (PLP-GFP mouse, arrows) is distinct from the myelin domain (FM: fluoromyelin red) in live adult mouse RON. Highlighted square shown in insert as rendered stack. e Myelin structure is protected from ischemic damage by 120 min pre-treatment with QNZ-46, imaged via FM vital staining. f–h Ischemia evoked myelin swelling (P = 0.000 in aCSF) is prevented by 120 min QNZ-46 pre-treatment (P = 0.047 vs. aCSF). Scale = 10 μm

Myelin protection in adult mouse RON. a Oligodendrocyte (Nuc = nucleus) and myelinated axons (e.g., arrows) in control RON have a normal healthy structure. b, c Following OGD there is glial disruption including nuclear condensation and process degeneration (e.g., short arrows) with myelin disruption in the axon population. Note that the inner myelin layers are often separated and may form bubbles. For example in (c) (higher power image of the boxed area in (b)) where a region of myelin on the left side of the axon has five layers (small arrows) and no bubbles but the inner layer has four layers with a series of bubble profiles on the right side (e.g., *). Note retention of microtubules within the axon profile (Ax). d–f Uniform protection of myelin structure (e.g., arrows) throughout RONs fixed after OGD + QNZ-46. Note that glial cell soma (d, e) and processes (f) also retain normal structure in this protocol. g–j Myelin thickness assessed as G-ratio under the three conditions, showing myelin expansion following OGD (ANOVA with Holm−Šídák post test; P = 0.0001) and prevention of this effect by perfusion with QNZ-46 (P = 0.0007). j, k Mean data showing the changes in G-ratio under these conditions (j); which are not accounted for by significant axonal shrinking (k). l Myelin stained with fluoroMyelin (FM) under these conditions (left) and (right) the mean FM intensity decline evoked by OGD (P = 0.00027), which is prevented by MK801 (P = 0.00062). Bar a, b, e, f = 1 μm; d = 5 μm; c, l = 100 nm

QNZ-46 is absorbed by myelin. a QNZ-46 emission shows accumulation and retention in adult rat RON. b Confocal imaging of vital QNZ-46 fluorescence (120 min treatment) in adult corpus callosum (left) and the white matter–gray matter border (right). c Vital QNZ-46/FM co-staining of myelinated axon profiles. d, e Vital QNZ-46/FM co-staining in brain slice (d) and RON (e) following systemic QNZ-46 injection 4 h pre-sacrifice. f 60 min QNZ-46 pre-treatment + 60 min wash-out is functionally protective of adult rat RON (ANOVA with Holm−Šídák post test; P = 0.037). g Systemic QNZ-46 pre-treatment is functionally protective of mouse RON perfused with aCSF (blue, P = 0.047), and is not significantly different when QNZ-46 is included in the bath (green). Note, proportional recovery was similar to that found in Fig.  (insert). Merged images recolored for clarity. Scale bars = 5 μm

A single QNZ-46 pre-treatment produces high levels of white matter and gray matter neuro-protection. a–c Brain lesion volume 24 h post-tMCAO. d–f Functional recovery prior to the killing. Drug treatment significantly improved the outcome in all measures. ANOVA with Holm−Šídák post test. P values: b ***0.0000; c **0.000; e **0.004; f *0.011. Mann−Whitney test; d *<0.05. g–j YFP expression (green) and luxol fast-blue/cresyl violet (blue/purple: myelin) in Thy-1-YFP mice. g YFP(+) axons project within the external capsule which is extensively myelinated in contralateral (Contra) and ipsilateral (Ipsi) hemispheres in sham-operated mice. Higher power micrographs are shown at the bottom. h Mice treated with vehicle show disruption and loss of axonal YFP that extends to both hemispheres and loss of myelin within the ipsilateral white matter. i Drug-treated mice retain YFP(+) axons and myelin staining in both hemispheres. j Myelin stain density is significantly reduced in the vehicle-treated group (***0.0002), and preserved by QNZ-46 pre-treatment (**0.005). Scale bars = 100 μm

Structural protection following systemic QNZ-46 pre-treatment. a, b Brain lesions in vehicle- and QNZ-46-treated mice subject to the standard MCAO protocol. Boxed areas (external capsule-motor cortex border) were examined via TEM. c Cortical neuronal somata (nuclei indicated by short arrows), neuropil (arrow heads), and myelinated axons (long arrows) in contralateral QNZ-46-treated mice appeared normal with no unusual features. d, e Ipsilateral vehicle-treated external capsule-motor cortex border showed wide-scale cellular destruction and loss of structure including disrupted myelinated axon tracts (arrows d, e top left) and necrotic neuronal soma (arrow heads d, e bottom left). White matter glial cell soma and process were also necrotic (e, top right, short arrow) and occasional distorted myelin profiles were apparent (e, top right, arrow). Cell processes in the neuropil were generally disrupted with free-floating mitochondria present (e, bottom right). f–h Ipsilateral QNZ-46-treated external capsule-motor cortex border showed almost normal structural features in both white matter and gray matter areas. Myelinated axon tracts showed no myelin damage (f, g top left, h arrows) and both neuronal and glial soma (f, g top right, h short arrows) appeared largely intact. Glial processes were often swollen (g, bottom left and right white arrows) and in many cases could be identified as astrocytic containing glial filaments (g, bottom right, white arrow). Neuronal, neuropil, and myelinated axon profiles are shown at higher gain in the expanded boxed area in h (lower panel). i Cell viability was severely compromised (ANOVA with Holm−Šídák post test; ***P = 0.0000) in ipsilateral vehicle-treated mice (where white matter and gray matter, neuron and glia could not always be distinguished and are grouped together). QNZ-46 pre-treatment significantly improved cell viability in both white matter and gray matter (***P = 0.0000 vs. untreated ipsilateral injury), with no significant difference between ipsi- and contralateral cells in white matter regions. j–k G-ratio was not significantly different in ipsi- and contralateral white matter axons in QNZ-46 pre-treated mice. Note axon disruption precluded the measurement of G-ratio in ipsilateral vehicle-treated white matter