Long-term in vivo three-photon imaging reveals region-specific differences in healthy and regenerative oligodendrogenesis

Abstract

The generation of new myelin-forming oligodendrocytes in the adult CNS is critical for cognitive function and regeneration following injury. Oligodendrogenesis varies between gray and white matter regions suggesting that local cues drive regional differences in myelination and the capacity for regeneration. Yet, the determination of regional variability in oligodendrocyte cell behavior is limited by the inability to monitor the dynamics of oligodendrocytes and their transcriptional subpopulations in white matter of the living brain. Here, we harnessed the superior imaging depth of three-photon microscopy to permit long-term, longitudinal in vivo three-photon imaging of an entire cortical column and underlying subcortical white matter without cellular damage or reactivity. Using this approach, we found that the white matter generated substantially more new oligodendrocytes per volume compared to the gray matter, yet the rate of population growth was proportionally higher in the gray matter. Following demyelination, the white matter had an enhanced population growth that resulted in higher oligodendrocyte replacement compared to the gray matter. Finally, deep cortical layers had pronounced deficits in regenerative oligodendrogenesis and restoration of the MOL5/6-positive oligodendrocyte subpopulation following demyelinating injury. Together, our findings demonstrate that regional microenvironments regulate oligodendrocyte population dynamics and heterogeneity in the healthy and diseased brain.

Extended Data Fig. 1 ∣. Custom three-photon light path and modifications for longitudinal in vivo imaging.

3P excitation source and light path, including motorized half-wave plate for power modulation (λ/2), Glan-Thompson polarizer (GT), dual prism compressor, beam expanding telescope, ALPAO deformable mirror, beam reduction lens relay, galvo-galvo scan mirrors, scan lens, tube lens, dichroic mirror (FF-488-di02, cut at 488 nm), and moveable objective microscope system with collection filters for third harmonic generation signal (430/25 nm, PMT1) and EGFP emission (520/70 nm, PMT2).

Extended Data Fig. 2 ∣. MOBP-EGFP and THG labeling of mature oligodendrocytes and myelin in the cortex and subcortical white matter.

a) Confocal image of a tissue section from the deep posterior parietal cortex stained for transgenic MOBP-EGFP and ASPA. Arrowheads show large, putative newly-generated oligodendrocytes that are MOBP-EGFP-positive and ASPA-negative. b) Confocal image of ASPA/MOBP-EGFP immunofluorescence in the subcortical white matter beneath the posterior parietal cortex (dotted white lines). Arrowheads show MOBP-EGFP-positive and ASPA-negative white matter oligodendrocytes of varying sizes and brightness. c) 99.5% (cortex) and 99.6% (white matter) of ASPA-positive cells express MOBP-EGFP. d) 95.6% (cortex) vs. 86.5% (white matter) of MOBP-EGFP-positive oligodendrocytes were also positive for ASPA (Unpaired, two-tailed Student’s t-test for equal variance, p = 0.016). n = 4 mice, 2 sections per mouse. e) 9 μm z-projection of layer 1 in the posterior parietal cortex showing MOBP-EGFP labeling of myelin sheaths (green) and THG-labeling of myelin sheaths and blood vessels (magenta). f) Zoom image of an isolated EGFP/THG-labeled myelin sheath from the white box in (e) excited with 1300 nm three-photon excitation. g) Isolated MOBP-EGFP sheath from layer 1 of a separate mouse visualized with 920 nm two-photon excitation combined with Spectral Confocal Reflectance (SCoRe) microscopy. Note the decreased THG/SCoRe labeling at putative nodes of Ranvier in (f) and (g) (arrowheads). h) 9 μm z-projection of a single mature oligodendrocyte in layer 1 from a third mouse showing EGFP-labeled processes connected to multiple THG-labeled sheaths. i) 5-pixel line intensity plot from the white line drawn in (d) showing relative fluorescence intensity of EGFP- and THG-labeled myelin sheaths and a THG-labeled blood vessel (arrowhead). All images pixel size = 0.36 μm. *p <0.05, box plots represent the median, interquartile ranges and minimum/maximum values

Extended Data Fig. 3 ∣. Adaptive optics improves SBR and axial resolution in the subcortical white matter.

a) System adaptive optics (AO) correction with a two-dimensional 1.0 μm red polychromatic microsphere sample. Lateral (top) and axial (bottom) projections show a slight enhancement in peak signal after AO correction for system aberrations (8% increase, lateral; 9% increase, axial). The bead sample was diluted 1:10000, coverslipped with Prolong Gold, and the average power at the sample was <0.3 mW. System resolution = 0.88 μm (lateral) and 2.55 μm (axial) as calculated by the full width at half maximum of the gaussian fit after system AO correction. b) Deformable mirror (DM) amplitude plot showing the optimized stroke amplitudes (units = μm RMS) for each Zernike Mode in the system AO correction. c) In vivo, indirect, modal adaptive optics (AO) corrections were made at 775 - 825 μm depth (just above the white matter) by optimizing on the mean intensity of the third harmonic generation channel. The in vivo AO correction revealed cross sections through myelinated axons just dorsal to the corpus callosum (white dotted selection, top, 800 μm) and increased the mean intensity of EGFP-positive oligodendrocytes in the white matter ventral to the plane of the AO correction (bottom, 835 μm). Example images are single-slice images at the depth of the AO correction, 0.14 μm / pixel. d) Deformable mirror (DM) amplitude plot showing the optimized stroke amplitudes (units = μm RMS) for each Zernike Mode in the in vivo AO correction shown in (c). e) XY (top) and XZ (bottom) projections for a single EGFP-positive OL cell body from the example image shown in (c). The AO correction enhanced the normalized 3P signal by 68% (lateral) and 40% (axial). n = 2 mice, 11 OLs at > 800 μm depth. f) The peak intensity of the EGFP-oligodendrocyte signal was significantly enhanced by the AO correction (Paired, two-tailed Student’s t-test, p = 0.0002, n = 2 mice, 11 cells). g) The axial resolution of EGFP- oligodendrocyte cell bodies was significantly enhanced by the AO correction (Paired, two-tailed Student’s t-test, p = 0.014, n = 2 mice, 11 cells). h) AO correction enhances the peak intensity of OL processes by 202% (n = 2 mice, 4 processes). i) Average power vs. depth plot shows the average power at the sample through the depth of the white matter after AO correction (black, left), and the theoretical power curve (gray, right) necessary to maintain the same signal to background ratio without AO. Data are represented as individual points and mean ± SEM. Curves in a, e, and h represent the mean of the gaussian fits for each analyzed region (cell bodies in a, e, cell processes in h) with 95% confidence intervals. The intensity was integrated over a line plotted through the center of the cell structure (10 μm wide, cell bodies, 1 μm wide, processes). Zernike modes in (b), (d) = Spherical (12), Horizontal Trefoil (9), Horizontal Coma (8), Vertical Coma (7), Oblique Trefoil (6), Astigmatism (5), Oblique Astigmatism (3). For detailed statistics, see Supplementary Table 3.

Extended Data Fig. 4 ∣. Mechanical, scanning, and optical modifications to increase 3P signal and decrease average power.

a) The third harmonic generation (THG) signal at the surface of the cranial window was used to align the preparation orthogonally to the excitation light. b) Scanning modifications to increase SBR and decrease average power to mitigate risk of tissue damage. Frame averaging was advantageous compared to increasing the pixel dwell time to reduce risk of nonlinear damage. Z-stack acquisition was paused periodically to allow for heat dissipation (1 min. pause per 3 min. scanning). Laser pulses were blanked on the galvanometer overscan to reduce the average power applied to the preparation at each z-plane. c) Imaged-based AO correction increased SBR at depth (left) and modulating the spherical aberration correction linearly with depth improved SBR throughout the imaging volume (right). For longitudinal imaging, the AO correction was made before acquiring each time point at the same z-plane just above the scattering white matter (750-850 μm depth).

Extended Data Fig. 5 ∣. Calculation of mouse-specific effective attenuation lengths in the posterior parietal cortex.

a) The mean intensity of the top 1% of the brightest pixels (experimental 3P signal) plotted with depth beneath the brain surface for an example mouse. b) Data from the mouse shown in (a) normalized to the cube of the pulse energy shows the decay of the 3P signal with depth in the mouse brain. c) Logarithm of the data in (b) shows a linear decrease with depth. The single mouse-specific effective attenuation length (EAL) can be calculated from the slope of the linear fit (gray = gray matter, blue = white matter). A steeper slope represents a shorter EAL due to increased scattering. d) Semilogarithmic plot showing gray matter and white matter EALs for n = 11 mice at the first time point of longitudinal imaging. Mean EAL (GM) = 249 +/− 12.7 μm, Mean EAL (WM) = 169 +/− 8.9 μm. e) Distributions of experimental mouse-specific EALs in the gray matter (gray) and white matter (blue). Linear fits (black line plots in c,d) were calculated separately by region. Box plots represent the median, interquartile ranges and minimum/maximum values.

Extended Data Fig. 6 ∣. Optimized longitudinal three-photon imaging does not increase glial, neuronal, or vascular reactivity.

a) Related to Fig. 3. Coronal brain section from the imaged (right) and contralateral (left) posterior parietal cortex (PPC) of a mouse that was perfused 24 hrs. following 10 weeks of chronic 3P imaging. b) Coronal brain section from the imaged (right) and contralateral (left) posterior parietal cortex (PPC) of a mouse that received supra-threshold excitation across the cortex and white matter to generate laser-induced positive control tissue (laser injury, right). c) High-resolution confocal images of the contralateral (left), longitudinal imaging field (middle) and laser-induced injury (right) regions in the deep cortical layers of the PPC, stained for transgenic MOBP-EGFP expression (mature oligodendrocytes), Iba-1 (microglia), GFAP (reactive astrocytes), 8-hydroxyguanosine (8-OHG, neuronal RNA oxidation), and CD13/Lectin-49 (pericytes, vasculature, respectively). All images were taken with identical power settings, processed with a 100 pixel rolling ball background subtraction and 0.7 pixel gaussian blur, and brightness/contrast correction was applied identically across channels. The bottom row shows an example of the trainable WEKA segmentation to measure pericytes (green) and vascular coverage (magenta, see Methods). Note the Lectin-positive microglia-like cells present in the laser injury vasculature image (white arrowheads). d) No difference in the density of MOBP-EGFP OLs between the contralateral and imaged regions in healthy mice. e) No difference in the density of MOBP-EGFP OLs between the contralateral and imaged regions in laser-induced injury tissue. f) The ratio of the EGFP mean intensity for the imaged:contralateral (contra.) hemispheres was ~1 for healthy mice and did not change significantly in the positive control tissue. g) No difference in the density of Iba-1 microglia (MG) between the contralateral and imaged regions in healthy mice. h) Density of Iba-1 MG is significantly increased on the ipsilateral side of laser-induced injury tissue (Paired, two-tailed Student’s t-test, t(4) = 3.93, p = 0.016). i) The ratio of the imaged : contralateral Iba-1 mean intensity was ~1 for healthy mice and significantly increased in positive controls (Unpaired, two-tailed Student’s t-test for equal variance, t(8) = 2.73, p = 0.026). j) No difference in the density of GFAP-positive reactive astrocytes (rAstros) between the contralateral and imaged regions in healthy mice. k) Density of GFAP+ rAstros is significantly increased on the damaged side in laser-induced injury tissue (Paired, two-tailed Student’s t-test, t(4) = 3.94, p = 0.017). l) The ratio of the imaged : contralateral (contra.) GFAP mean intensity was ~1 for healthy mice and was significantly increased in positive controls (Wilcoxon rank sum test, Z = 2.09, p = 0.037). m) No difference in the density of 8-OHG-positive neurons after auto-thresholding (see Methods) between the contralateral and imaged regions in healthy mice. n) No difference in the density of 8-OHG-positive neurons between contralateral and imaged regions in laser-induced injury tissue. o) No difference in the imaged:contralateral ratio of 8-OHG mean intensities between healthy vs. laser-damaged mice. p) No difference in vessel coverage (% positive pixels) between the contralateral and imaged hemispheres of healthy multi-month longitudinal 3P imaging mice. q) Increase in vessel coverage (%) on the ipsilateral hemisphere of laser-induced injury positive control mice (Paired, two-tailed Student’s t-test, p = 0.022). r) No difference in the density of pericytes between the contra. and ipsi. hemispheres of healthy long-term 3P mice. s) No difference in the density of pericytes between the contra. and ipsi. hemispheres of laser-induced injury mice. *p < 0.05, **p < 0.01, n.s., not significant; n = 5 mice (healthy longitudinal), 5 mice (laser-induced injury), 2 sections, 4 hemispheres per condition. Box plots represent the median, interquartile ranges and minimum/maximum values. For detailed statistics, see Supplementary Table 3.

Extended Data Fig. 7 ∣. Immunofluorescent and in situ hybridization techniques allow for probing OPC proliferation and oligodendrocyte subpopulations in the cortex and subcortical white matter.

a) Experimental timeline of tissue collection for EdU and RNAScope analyses in healthy and cuprizone mice. Healthy MOBP-EGFP mice were injected with 5 mg/kg of the thymidine analog EdU twice a day (10-12 hr. interval) for seven days starting at P70 and perfused the following day. Healthy MOBP-EGFP mice were perfused at P60 and P140 to assess aging-dependent changes in adult oligodendrocyte subpopulations. MOBP-EGFP mice that were fed 0.2% cuprizone for three weeks were perfused at 4 days post-cuprizone removal (peak demyelination, matched to in vivo imaging data, Fig. 4), and 7 weeks post-cuprizone removal (regeneration, matched to the final time point of in vivo imaging, see timeline, Fig. 4). b) EdU-positive proliferated OPCs in the posterior parietal cortex (top, GM) and subcortical white matter (bottom, WM, dashed border). c) The density of PDGFR-α-positive OPCs was significantly increased in the WM compared to the GM (248.6 ± 23.8 vs. 181.2 ± 7.7, Unpaired, two-tailed Student’s t-test for equal variance, t(6) = 2.69, p = 0.036). d) The percentage PDGFR-α+/EdU+ OPCs is increased in the WM compared to the GM (Unpaired, two-tailed Student’s t-test for equal variance, 51.6 ± 6.6 vs. 14.6 ± 2.5, t(6) = 5.29, p = 0.002). e) Coronal sections from the mid-thoracic spinal cord of healthy MOBP-EGFP mice were taken at P140 and run in parallel with brain sections to confirm the labeling specificity of our oligodendrocyte subpopulation probe-set (Egr2, MOL1, cyan; Klk6, MOL2/3, magenta; Ptgds, MOL5/6, orange). f) Egr2 and Ptgds preferentially label oligodendrocytes in the spinal gray matter, while Klk6 predominantly labels oligodendrocytes in the spinal white matter (Unpaired, two-tailed students t-test for unequal variance). g) Coronal sections from the posterior parietal cortex of MOBP-EGFP mice (−1.7 to −2.3 mm posterior and 1 to 3 mm lateral to bregma) showing the pattern of OL subpopulation labeling at the experimental time points described in (a). *p < 0.05, **p<0.01, n = 4 mice, 4 sections per mouse. Boxplots represent the median, interquartile range and the minimum and maximum values. For detailed statistics, see Supplementary Table 3.

Extended Data Fig. 8 ∣. Modeling oligodendrocyte growth, loss, and regeneration in adult mouse cortex and white matter.

a-b) Cumulative oligodendrocyte population growth (% gain over time) in the healthy brain was modeled using asymptote-restricted exponential mechanistic growth curve-fitting. a) Cumulative OL gain (%) and mechanistic growth fit in the gray matter (left) and the white matter (right) for an example mouse. b) Cumulative OL gain (%) and mechanistic growth fits in the gray matter (left) and the white matter (right) for the healthy group (n = 6 mice). c-d) Cumulative oligodendrocyte population loss (% loss over time) due to cuprizone administration was modeled using three-parameter Gompertz Sigmoid curve-fitting. c) Cumulative OL loss (%) and three-parameter Gompertz curves in the gray matter (left) and the white matter (right) for an example mouse. d) Cumulative OL loss (%) and three-parameter Gompertz curves in the gray matter (left) and the white matter (right) for the cuprizone de/remyelination group (n = 6 mice). e-f) Cumulative oligodendrocyte population regeneration (% cell replacement over time) following cuprizone cessation was modeled using three-parameter Gompertz Sigmoid curve-fitting. e) Cumulative OL replacement (%) and three-parameter Gompertz curves in the gray matter (left) and the white matter (right) for an example mouse. f) Cumulative OL replacement (%) and three-paramter Gompertz curves in the gray matter (left) and the white matter (right) for the cuprizone de/remyelination group (n = 6 mice). Modeled rate and timing metrics in Main Figs. 4-7 were calculated by fitting curves to data from individual mice and then extracting summary data (e.g. timing of inflection point).

Extended Data Figure 9 ∣. Layer and region-dependent proportions of MOL1, MOL2/3, and MOL5/6 across healthy aging and cuprizone treatment.

Related to Figs. 7,8. Raw counts of the total # of segmented MOBP-EGFP OLs (top), MOL1+ OLs (row 2), MOL2/3+ OLs (row 3) and MOL5/6+ OLs (bottom) across cortical and subcortical layers (x-axes) and experimental time points (shaded background colors). Data presented in the main figures were expressed either as the percentage of total OLs for each probe (Figs. 7-8), or the change in proportion of each marker from the healthy P140 time point (Figs. 4-6).

Extended Data Figure 10 ∣. Layer-dependent differences in the temporal dynamics of cuprizone induced loss and regeneration.

a-e) Related to Fig. 7. a) OL population replacement (%) plotted over weekly time bins following cuprizone cessation for each anatomically defined region. Replacement rate is increased from healthy baseline from 2-4 weeks post-cuprizone in L1-3 and CC, and 3-4 weeks post-cuprizone in L4 and L5/6 (Kruskal-Wallis test followed by Steel method for comparison with control, p = 0.034 for L1-3, Weeks 2-4 vs. Healthy; p = 0.034 for L4 Weeks 3-4 vs. Healthy; p = 0.034 for L5-6, Weeks 3-4 vs. Healthy; p = 0.034 for CC Weeks 2-4 vs. Healthy). b) First-derivative of the modeled growth curves for healthy baseline and OL replacement (Mechanistic Growth, Gompertz 3P, respectively) showing the response duration of regeneration across regions. c) The regeneration response duration, calculated as the full width at half maximum of the rate curves in (b), is significantly longer in the superficial layers 1-3 compared to the CC (One-way ANOVA followed by Tukey’s HSD, p = 0.048). d) The magnitude of the regeneration response, as calculated by the area under the curve above the healthy baseline rate, is significantly suppressed in L5-6 compared to the CC (One-way ANOVA followed by Tukey’s HSD, p = 0.037). e-f) No significant differences in the duration or magnitude of the cuprizone cell loss response across regions. Data in (a) were calculated based on the raw % change in cell population from baseline. Data in b-f were derived from the modeled and scaled growth curves. *p < 0.05, **p < 0.01, n.s. = not significant; cumulative growth curves represent cubic splines with 95% confidence intervals, box plots represent the median, interquartile ranges and minimum/maximum values. For detailed statistics, see Supplementary Table 3.

Figure 1 ∣. Three-photon microscopy enhances the detection of oligodendrocytes and myelin deep in the adult mouse brain.

a) xz projection of a 2-photon z stack of oligodendrocytes and myelin in a high-quality chronic cranial window acquired with the Olympus 25X objective. Power was modulated from 8-160 mW. b) xz projection of a 3-photon z stack of MOBP-EGFP and third harmonic generation (THG) signal shows high SBR through the PPC, subcortical corpus callosum (CC), and the alveus of the hippocampus. The average power at the sample was modulated from 0 – 45 mW. c) Two-photon (2P) SBR of oligodendrocyte cell bodies is significantly increased in the superficial 100 μm of cortex compared to three-photon (3P, unpaired, two-tailed Student’s t-test, t(6) = −2.45, p = 0.043), while 3P imaging at depths greater than 400 μm has enhanced SBR (unpaired, two-tailed Student’s t-test, t(6) = 2.92, p = 0.027 (401 - 500 μm), t(6) = 4.72, p = 0.003 (501 - 600 μm), t(3.07) = 4.41, p = 0.021 (601 - 700 μm). d) 2P and 3P allow for the detection of similar numbers of oligodendrocytes in layers 1-4, yet significantly fewer cells are detected with 2P in layer 5 (unpaired, two-tailed t-test, t(10) = 2.29, p = 0.045. e) Stacked bar chart showing the cortical (gray) and total (blue) imaging depth across the PPC for n = 8 mice at 10 weeks of age. f) The corpus callosum (CC) can be differentiated from the alveus by the orientation of THG-positive fibers (t(5.57) = −3.24, p = 0.026) for n = 6 mice. *p < 0.05, **p < 0.01, n.s., not significant; cumulative growth curves represent cubic splines with 95% confidence intervals, bar plots represent mean +/− SEM, box plots represent the median, interquartile ranges and minimum/maximum values. For detailed statistics, see Supplementary Table 3.

Figure 2 ∣. Optimization of excitation parameters permits long-term three-photon microscopy without tissue damage.

a) Example imaging time points from a mouse exposed to 2.7 nJ pulse energy at the focus. Dotted circle represents the area of the lesion as defined by an increase in THG intensity; white arrowhead shows a newly-generated OL in response to cell ablation. Depth of lesion = 594 – 786 μm. b) Cumulative OL population cell loss (%) and cell gain (%) in lesioned regions shows rapid OL cell death that proceeded for up to 40 days following the time point of max THG intensity increase. OL cell regeneration was rapid and biphasic for lesion 1 (black) but delayed by ~20 days and linear for lesions 2 and 3. Mean OL population loss was 32.9 +/− 5.2 %; Mean replacement of the lost OLs was 50.0 +/− 16.9 % (mean +/− SEM). c) Mean intensity of the THG signal over time in the lesion area (dotted circle), aligned to the time point of max intensity change for n=3 mice lesioned mice. d) Similar exponential power vs. depth curves were applied at all time points in lesioned vs. healthy mice that underwent long-term 3P imaging. The excitation laser was blanked on the galvanometer overscan. Data are from n=3 mice with laser-induced injury and n= 11 mice with successful longitudinal imaging without THG damage or cell death. e) The pulse energy at the focus vs. z-depth was determined for each mouse by calculating mouse-specific effective attenuation lengths (EALs) at the first time point. Mouse-specific differences in cortical EAL, and not average power (d), drove the increase in pulse energy that caused cellular damage. The minimum damage threshold was 2.7 nJ at the focus (red dotted line). Healthy imaged mice had a mean pulse energy at the focus of less than 2 nJ across the cortical depth (cyan). f) Example imaging time points from a healthy mouse with optimized settings and ~1.4 nJ excitation at the focus across the cortical depth. Note the detection of newly generated oligodendrocytes (white dotted circles) without cell death or changes in THG intensity. THG, power, and pulse energy curves are cubic splines with 95% confidence intervals. Mean z-width of the lesions was 149 +/− 26.1 μm.

Figure 3 ∣. Long-term three-photon imaging does not perturb oligodendrocyte damage markers or healthy oligodendrogenesis.

a) Coronal brain section from the imaged (right) and contralateral (left) posterior parietal cortex (PPC) of a mouse that was perfused 24 hrs. following 10 weeks of chronic 3P imaging (top) stained for MOBP-EGFP, heat shock proteins 70/72 and the gamma-H2ax marker of DNA double stranded breaks. Coronal brain section from the imaged (right) and contralateral (left) posterior parietal cortex (PPC) of a mouse that received supra-threshold excitation across the cortex and white matter to generate laser-induced positive control tissue (laser injury, right). b) To generate laser-damage tissue, the power at the sample was increased to saturate the THG PMT signal without causing tissue ablation or bubbling and the mouse was perfused 24 hrs. following laser exposure. c) Example images of oligodendrocyte (OL) cell body segmentation and cell-specific intensity analyses. d) High-resolution confocal images of the contralateral (left), longitudinal imaging field (middle) and laser-induced injury (right) regions in the deep cortical layers of the PPC of MOBP-EGFP mice, stained for heat shock proteins 70/72 (HSP70/72, thermal damage), and the phosphorylated histone protein H2A.X (gamma-H2A.X, DNA double-strand breaks). e) No difference in the density of HSP70/72 – positive segmented OLs (HSP70/72 intensity > 2 * background intensity) between the contralateral and ipsilateral (imaged) hemispheres of healthy mice that underwent multi-month longitudinal 3P imaging with optimized scan and excitation settings. f) Increased density of HSP70/72 – positive OLs on the ipsilateral hemisphere of laser-induced injury positive control mice (Paired, two-tailed Student’s t-test, p = 0.041). g) Increase in the ratio of the full-field ipsilateral : contralateral hemisphere mean intensity of HSP70/72 in laser-induced injury vs. long-term 3P mice (Wilcoxon rank sum test, z = 2.089, p = 0.037). h) No difference in the density of γ–H2a.X – positive OLs (γ–H2a.X intensity > 2 * background intensity) between the contralateral and ipsilateral (imaged) hemispheres of healthy multi-month longitudinal 3P imaging mice. i) No difference in the density of γ–H2a.X – positive OLs between the contralateral and ipsilateral hemispheres of laser-induced injury positive control mice. j) No difference in the ratio of the full-field ipsilateral : contralateral hemisphere mean intensity of γ–H2a.X in laser-induced injury vs. long-term 3P mice. k) Two-photon imaging of stable (white) and newly generated (orange) oligodendrocytes in layer 2/3 of the PPC (top). Three-photon imaging of stable (white) and newly generated (blue) oligodendrocytes in layer 2/3 of the PPC (bottom). l) Comparison of cumulative oligodendrocyte gain (%) in the healthy PPC over ~7 weeks with standard 2P settings (orange) vs. 3P (blue). No significant differences in the mean rate of healthy oligodendrocyte gain per week (Unpaired, two-tailed Student’s t-test for equal variance, t(6) = 0.82, p = 0.440), or the total cumulative OL gain (%) (Unpaired, two-tailed Student’s t-test for equal variance, t(6) = 0.491, p = 0.641) between the two imaging modalities. m) Mature oligodendrocytes are stable over time in the adult mouse brain, and 3P imaging does not increase mature cell death. Layers 1-3 (L1-3) represents ~0-340 μm depth; Layers 4–corpus callosum (L4-CC) represents ~341-1000 μm depth, as defined by the Allen Brain Atlas. Images in (c) are confocal maximum z-projections of 16 μm (2 μm step), pixel size = 0.57 μm. Images in (m) are maximum z-projections of 30 μm (3 μm z step), pixel size (2P) = 0.39 μm; pixel size (3P) = 0.75 μm. *p < 0.05, n.s., not significant; cumulative growth curves represent cubic splines with 95% confidence intervals, box plots represent the median, interquartile ranges and minimum/maximum values. For detailed statistics, see Supplementary Table 3.

Figure 4 ∣. Gray versus white matter differences in population growth correlate with transcriptional heterogeneity.

a) Oligodendrogenesis (cyan) over 4 weeks of imaging in layer 5 of the posterior parietal cortex (PPC). b) Cumulative OL gain curves for individual mice over 10 weeks in the entire depth of the gray matter (GM). c) Oligodendrogenesis in the dorsal corpus callosum over ~6 weeks of imaging in the white matter (WM). d) Cumulative OL gain curves for individual mice in the WM. e) The WM gained substantially more OLs per imaging volume at a faster rate than the GM (Unpaired two-tailed Student’s t-test for unequal variance, t(4.45) = 4.03, p = 0.013 for total gain at 66 d; Unpaired two-tailed Student’s t-test for unequal variance t(6.86) = 5.14, p = 0.002 for mean rate). f) The cumulative percentage gain relative to the initial population of OLs and the rate of OL population gain (%) were higher in the GM vs. WM. g) Total population gain was increased in the GM vs. the WM (Unpaired two-tailed Student’s t-test for equal variance, t(8) = −2.83, p = 0.022). h) The rate of OL population growth during the fifth week of imaging (~P100) was increased in the GM vs. WM (Unpaired two-tailed Student’s t-test for equal variance, t(10) = −2.756, p = 0.020). i) The time at 50% of the maximum cell gain of the mechanistic growth curves did not differ between GM and WM. j) The rate of white matter population gain (% per week) decreased more rapidly with age than in the gray matter (Dunnett’s comparison with control, p = 0.037 for Weeks 5-6 vs. Weeks 1-2; p = 0.022 for Weeks 9-10 vs. Weeks 1-2). k-m) Spinning disk confocal images of the distribution of MOL1+ (EGFP/Egr1+), MOL2/3+ (EGFP/Klk6+), and MOL5/6+ (EGFP/Ptgds+) oligodendrocyte populations in the PPC and subcortical WM at P60 (left) and P140 (right). n) No differences in the percent change of population proportions for MOL1, MOL2/3, and MOL5/6 between P60 and P140. o) Differences in transcriptional heterogeneity between the GM and WM. The proportion of MOL1+ OLs was higher in the GM vs. WM (Unpaired two-tailed Student’s t-test for unequal variance, t(7.033) = −4.804, p = 0.002). The proportion of MOL2/3+ OLs was lower in the GM vs. WM (Wilcoxon rank sum test, z = 2.363, p = 0.018). The proportion of MOL5/6+ OLS was lower in the GM vs. WM (Unpaired two-tailed Student’s t-test for equal variance, t(14) = 3.283, p = 0.005). Data in h,i were calculated from the slope and inflection point of the mechanistic growth curves, respectively. Data in j were calculated as the percentage of the initial cell population generated per week (not modeled). Data in a-j represent n = 6 mice / group. Data in n-o represent n = 8 mice, two sections per mouse. *p < 0.05, **p < 0.01, n.s., not significant; cumulative growth curves represent cubic splines with 95% confidence intervals, box plots represent the median, interquartile ranges and the minimum/maximum values. Line plots represent the mean at each time point. For detailed statistics, see Supplementary Table 3.

Figure 5 ∣. Cuprizone affects oligodendrocyte population decline and molecular heterogeneity similarly across the gray and white matter.

a) 3P imaging timeline to track oligodendrocyte (OL) loss and gain induced by cuprizone administration. b) Depth of PPC and subcortical WM that was imaged over 66 days for n = 6 mice (healthy) and n= 6 mice (cuprizone). c) Percentage of the corpus callosum (CC) that was imaged and analyzed longitudinally did not differ by treatment group. d) Example time series of cuprizone-mediated OL loss in the deep cortex. e) Cumulative OL loss (%) over time in the GM for individual mice. f) Example time series of cuprizone-mediated OL loss in the corpus callosum (CC) ventral to the PPC. g) Cumulative OL loss (%) over time in the CC for individual mice. h) Cumulative number of OLs lost per 350 x 350 x 60 μm imaging volume was lower in the GM vs. WM (Unpaired, two-tailed Student’s t-test for unequal variance, t(5.25) = 3.15, p = 0.024). i) Similar dynamics of cumulative % loss of the initial population between regions. j) No difference in the total % OL loss for the GM vs. WM (Unpaired, two-tailed Student’s t-test for equal variance t(10) = 0.04, p = 0.970). k) No difference in the weekly rate of % OL loss for the GM vs. WM during the loss phase (−21 to +23 days, Unpaired, two-tailed Student’s t-test for equal variance, t(10) = 0.16, p = 0.876). l) No difference in the inflection point of the Gompertz 3-parameter cumulative % OLloss curve (Wilcoxon rank sum test, Z = 1.04, p = 0.298). m) The rate of GM and WM population loss (% per week), binned by 2-3 week intervals with respect to the 3-week cuprizone administration and late plateau phases. No significant differences were found between the GM and WM at each time point. GM cell loss is increased compared to the first week of cuprizone from −2 to +2 weeks post-cuprizone (Steel method for comparison with control, p = 0.046 for Weeks −2-0 vs. Weeks −3; p = 0.046 for Weeks 1 to 2 vs. Week −3). WM cell loss was increased at 1-2 weeks post-cuprizone compared to the first week of cuprizone (Steel method for comparison with control, p = 0.045 for Weeks 1-2 vs. Weeks 5-7; p = 0.019 for Weeks −2 to 0 vs. Weeks 5-7; p = 0.019 for Weeks 1-2 vs. Week −3). n-p) Spinning disk confocal images of the distribution of MOL1+ (EGFP/Egr1+), MOL2/3+ (EGFP/Klk6+), and MOL5/6+ (EGFP/Ptgds+) OL populations in the PPC and subcortical WM at 4 days post-cuprizone removal. q) The percent change of population proportions for MOL1, MOL2/3, and MOL5/6 (Healthy P140 vs. 4 days post-cuprizone) were reduced for all markers in the GM and WM (Wilcoxon rank sum test, Z = −2.71, p = 0.007, MOL1 GM; Wilcoxon rank sum test, Z = −2.07, p = 0.038, MOL1 WM; Wilcoxon rank sum test, Z = −2.90, p = 0.004, MOL2/3 GM; Wilcoxon rank sum test, Z = −2.86, p = 0.004, MOL2/3 WM; Unpaired, two-tailed Student’s t-test for unequal variance, t(8.39) = −6.75, p =0.0001, MOL5/6 GM; Unpaired, two-tailed Student’s t-test for unequal variance, t(8.50) = −8.40, p <0.0001, MOL5/6 WM).. r) Differences in transcriptional heterogeneity between the GM and WM 4 days following cessation of cuprizone. The proportion of MOL1+ OLs was higher in the GM vs. WM (Wilcoxon rank sum test, z = −2.538, p = 0.011). The proportion of MOL2/3+ OLs was lower in the GM vs. WM (Wilcoxon rank sum test, z = 2.586, p = 0.0097). No difference in the proportion of MOL5/6+ OLS in the GM vs. WM. Data in k,l were calculated from the slope and inflection point of the mechanistic growth curves, respectively. Data in m were calculated as the percentage of the initial cell population generated per week (not modeled). Data in a-m represent n = 6 mice / group. Data in q-r represent n = 5 mice, two sections per mouse. *p < 0.05, **p < 0.01, ***p<0.001, n.s., not significant; cumulative growth curves represent cubic splines with 95% confidence intervals, box plots represent the median, interquartile ranges and the minimum/maximum values. Line plots represent the mean at each time point. For detailed statistics, see Supplementary Table 3.

Figure 6 ∣. Replacement of lost oligodendrocytes following cuprizone is enhanced in the corpus callosum and regional heterogeneity is partially restored through regeneration.

a) Timelapse of 24-day period after cuprizone cessation showing oligodendrocyte (OL) cell body compaction and loss (red), followed by the generation of mature myelinating OLs (cyan) in the deep posterior parietal cortex. b) Cumulative OL replacement (% gain normalized to % loss) across the cortex for individual mice. c) Same time points and mouse as in (a) in the corpus callosum. d) Cumulative OL replacement (% gain normalized to % loss) for individual mice in the corpus callosum. e) Density of newly-generated OLs was significantly increased in the WM compared to the GM (Wilcoxon rank sum test, Z = 2.80, p = 0.005). Note the high variability in the dynamics of OL cell gain calculated by volumetric density. f) Cumulative OL replacement over time in WM vs. GM. Significance asterisks represent data in (g) and (h). g) OL replacement (%) is significantly increased at 45 days post-cuprizone cessation in the WM (Unpaired, two-tailed Student’s t-test for unequal variance t(5.97) = 2.57, p = 0.043). h) Rate of replacement is enhanced in the WM compared to the GM (Unpaired, two-tailed Student’s t-test for unequal variance t(5.72) = 2.59 p = 0.043). i) No difference in the timing of OL replacement between GM and WM. j) The rate of GM and WM population replacement (% per week), binned by 1-3 week intervals with respect to the 3-week cuprizone administration and late plateau phases. OL replacement rate (% per week) is significantly increased between the WM vs. GM at the 3-4 week post-cuprizone phase (Two-way ANOVA followed by piecewise Student’s t comparison with Bonferroni correction for multiple comparisons, p = 0.0009). Both the WM and the GM are significantly increased at the 3–4-week phase compared to the 7-week plateau phase (Dunnett’s method for comparison with control, p = 0.002 for Weeks 3-4 vs. Week 7 in the GM; p = 0.0052 for Weeks 3-4 vs. Week 7 in the WM). k-m) Spinning disk confocal images of the distribution of MOL1+ (EGFP/Egr1+), MOL2/3+ (EGFP/Klk6+), and MOL5/6+ (EGFP/Ptgds+) OL populations in the PPC and subcortical WM at 7 weeks post-cuprizone removal. n) The percent change of population proportions did not differ for MOL1 and MOL2/3 (Healthy P140 vs. 7 weeks post-cuprizone), yet MOL5/6 remained significantly decreased from healthy levels (Unpaired, two-tailed Student’s t-test for equal variance t(13) = −3.712, p = 0.003, GM; Wilcoxon rank sum test, z = −2.720, p = 0.007, WM). o) Differences in transcriptional heterogeneity between the GM and WM 7 weeks following cuprizone cessation. The proportion of MOL1+ OLs was higher in the GM vs. WM (Unpaired, two-tailed Student’s t-test for unequal variance t(6.118) = −4.234, p = 0.005). The proportion of MOL2/3+ OLs was unchanged in the GM vs. WM (Unpaired, two-tailed Student’s t-test for unequal variance t(7.560) = 2.187, p = 0.063). The proportion of MOL5/6+ OLs was lower in the GM vs. WM (Unpaired, two-tailed Student’s t-test for equal variance t(12) = 2.432, p = 0.032). Data in h,i were calculated from the slope and inflection point of the Gompertz 3-parameter growth curves respectively. Data in j were calculated as the percentage of lost cell population that was replaced per week (not modeled). *p < 0.05, **p < 0.01, n.s., not significant; cumulative growth curves represent cubic splines with 95% confidence intervals, box plots represent the median, interquartile ranges and minimum/maximum values. Line plots represent the mean at each time point. For detailed statistics, see Supplementary Table 3.

Figure 7 ∣. Layer-dependent differences in healthy and regenerative oligodendrogenesis correlate with changes in molecular heterogeneity.

a) Confocal image of the cortex and subcortical white matter showing myelo-, neuronal, axonal, and thalamic input architecture of the posterior parietal cortex (PPC). There is a Vglut2-positive layer 4 in the PPC (+2 mm lateral, −2 mm posterior, from Bregma). b) Spatial map of MOL1+ (cyan), MOL2/3+ (magenta), and MOL5/6+ (orange) OLs across the PPC and subcortical white matter at the Healthy P140, Cuprizone + 4 days, and Cuprizone + 7 weeks time points. Approximately ~50% of the mature OLs were not labeled for any of the three markers (None, gray) and single OLs could have multiple identities in these analyses. c) Cumulative healthy OL growth curves plotted by sub-region across the cortical and subcortical depth. d) Healthy oligodendrogenesis is increased in layer 4 vs. the corpus callosum (CC, One-way ANOVA followed by Tukey’s HSD, p = 0.012). e) The percentage of MOBP-EGFP OLs that were positive for Egr1 (%MOL1) decreased with depth in the healthy brain and was significantly increased in L1-3 and L4 compared to the WM (One-way ANOVA followed by Tukey’s HSD, p = 0.0004, L1-3 vs. CC; p = 0.006, L4 vs. CC). f) The percentage of Klk6-positive OLs (%MOL2/3) increased with depth in the healthy brain and was significantly increased in L5-6 and CC vs. L1-3 and L4 (Kruskal-Wallis test followed by Dunn’s test for multiple comparisons, p = 0.0034 (L4 vs. L5-6); p = 0.0034 (L1-3 vs. L5-6); p = 0.0009 (L4 vs. CC); p = 0.0009 (L5-6 vs. CC)). g) The percentage of Ptgds-positive OLs (%MOL5/6) was greater than 22% in all analyzed regions, but also increased with depth in the healthy brain (One-way ANOVA followed by Tukey’s HSD, p = 0.042 (L5-6 vs. CC); p = 0.005 (L4 vs. CC); p = 0.0005 (L1-3 vs. CC)). h) Cumulative OL loss curves plotted by subregion across the cortical and subcortical depth. i) No layer-dependent differences in cuprizone OL loss rate per week between L1-3, L4, L5-6, and the CC. Note the increased variability of demyelination in the superficial layers L1-4. j) Layer-specific differences in %MOL1-positive OLs across cortical and subcortical layers at 4 days post-cuprizone removal (Kruskal-Wallis test followed by Dunn’s test for multiple comparisons, p = 0.016, L1-3 vs. CC). k) Layer-specific differences in %MOL2/3-positive OLs across cortical and subcortical layers at 4 days post-cuprizone (Kruskal-Wallis test followed by Dunn’s test for multiple comparisons, p = 0.022 L5-6 vs. CC; p = 0.004 L4 vs. CC; p = 0.004 L1-3 vs. CC). l) No differences across layers for the %MOL5/6-positive OLs at 4 days post-cuprizone (One-way ANOVA). m) Cumulative OL replacement curves plotted by sub-region across the cortical and subcortical depth. n) OL replacement (% of lost cell population) is decreased specifically in L5-6 vs. the CC (One-way ANOVA followed by Tukey’s HSD, p = 0.035). o) The percentage of MOL1-positive OLs was specifically increased in L4 compared to the WM at 7 weeks post-cuprizone cessation (One-way ANOVA followed by Tukey’s HSD, p = 0.012). p) The percentage of MOL2/3-positive OLs was increased in L5-6 and the CC compared to the superficial layers 1-4 (Kruskal-Wallis test followed by Dunn’s test for multiple comparisons, p = 0.025 (L1-3 vs. L5-6); p = 0.025 (L4 vs. L5-6); p = 0.003 (L1-3 vs. CC); p = 0.003 (L4 vs. CC)). q The percentage of MOL5/6-positive OLs was only significantly decreased in L5-6 vs. CC (One-way ANOVA followed by Tukey’s HSD, p = 0.015)). Data in c-d, h-I, m-n were calculated with the raw % growth or % replacement, n=6 mice per treatment group. E-g, j-l, o-q, n= 5-8 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant. Cumulative growth curves represent cubic splines with 95% confidence intervals, box plots represent the median, interquartile ranges and minimum/maximum values. For detailed statistics, see Supplementary Table 3.

Figure 8 ∣. Decreased oligodendrogenesis and insufficient recovery of molecular identity in deep layers 5-6 drive diminished cortical regeneration.

a) Growth curves plotted by cortical sub-region and scaled to the maximum % gain or replacement to enable between groups comparisons of healthy vs. regenerative oligodendrogenesis. Significance asterisks are related to data plotted in (b-c). b) Significant decrease in scaled total OL replacement (%, modeled maximum at 66d) in layers 5-6 after demyelination relative to healthy OL gain (%, modeled maximum at 66d,Two-way ANOVA followed by pairwise comparisons with Bonferroni correction within cortical layers, p = 0.006). c) Significant decrease in scaled rate of OL replacement in L5-6 compared to healthy OL gain. (Two-way ANOVA followed by pairwise comparisons with Bonferroni correction, p = 0.0025) d) No differences in the scaled inflection points across regions. e) (Top) Differences in the number of molecular subtypes (MOL1, MOL2/3, MOL5/6) represented at the Healthy P140, Cup. + 4d. and Cup. + 7-weeks time points (classes were counted if the %-positive cells were greater than the minimum detected proportion in healthy mice; Kruskal-Wallis test followed by Dunn’s test for joint ranks). (Bottom) Stacked bar chart showing changes in the relative proportion of MOL1-, MOL2/3- and MOL5/6-positive OLs at 7 weeks post-cuprizone treatment. f) No differences in the percentage of MOL1-positive OLs within individual cortical layers, compared between the three represented time points (Two-way ANOVA). g) No differences in the percentage of MOL2/3-positive OLs within individual cortical layers, compared between the three represented time points (Two-way ANOVA). h) The percentage of MOL5/6-positive OLs decreased significantly at four days post-cuprizone cessation in L4, L5-6, and the CC (Healthy P140, purple vs. Cuprizone + 4 days, red; Two-Way ANOVA followed by Tukey’s HSD; p = 0.001, L4; p = 0.0001, L5-6; p = 0.0001, CC). The percentage of MOL5/6-positive OLs returned significantly at 7 weeks post-cuprizone only in the CC (Cup. 4d (red) vs. Cup. 7w (green), Two-way ANOVA with Tukey’s HSD, p = 0.012). The percentage of MOL5/6-positive OLs returned to Healthy P140 levels in the superficial cortical layers 1-4, but remained decreased in L:5-6 and the CC (Healthy P140 (purple) vs. Cup. + 7wks. (green) Two-way ANOVA followed by Tukey’s HSD, p = 0.0007, L5-6; p = 0.001, CC). Data in a-d were derived from the modeled and scaled growth curves. *p < 0.05, **p < 0.01,***p < 0.001, n.s., not significant; Cumulative growth curves represent cubic splines with 95% confidence intervals, box plots represent the median, interquartile ranges and minimum/maximum values. For detailed statistics, see Supplementary Table 3.