Oligodendrocyte dynamics in the healthy adult CNS: evidence for myelin remodeling

Abstract

Oligodendrocyte precursors (OPs) continue to proliferate and generate myelinating oligodendrocytes (OLs) well into adulthood. It is not known whether adult-born OLs ensheath previously unmyelinated axons or remodel existing myelin. We quantified OP division and OL production in different regions of the adult mouse CNS including the 4-month-old optic nerve, in which practically all axons are already myelinated. Even there, all OPs were dividing and generating new OLs and myelin at a rate higher than can be explained by first-time myelination of naked axons. We conclude that adult-born OLs in the optic nerve are engaged in myelin remodeling, either replacing OLs that die in service or intercalating among existing myelin sheaths. The latter would predict that average internode length should decrease with age. Consistent with that, we found that adult-born OLs elaborated much shorter but many more internodes than OLs generated during early postnatal life.

Figure 1

All OPs Proliferate in Forebrain Gray and White Matter at P21 and P60 (A–F) EdU was administered to P21 and P60 mice in the drinking water at 0.2 mg/ml, which we previously determined to be nontoxic (Figures S1 and S2). At various labeling times, coronal brain sections were processed to detect EdU (red) and immunolabeled for Pdgfra (green). Images are shown of the corpus callosum (cc) (P21, A; P60, B and C) and medial (primarily motor) cortex (ctx) (P21, D; P60, E and F). (G) The labeling index was plotted against time of EdU administration (means ±SD, n = 3 mice). The maximum labeling index is the fraction of the OP population that is mitotically active (the growth fraction). In both cc and ctx, all OPs eventually incorporated EdU (GF = 100%). Arrows and arrowheads indicate Pdgfra⁺, EdU⁺ and Pdgfra⁺, EdU-negative cells, respectively. Cell nuclei were stained with Hoechst 33258 (blue). Scale bars represent 25 μm.

Figure 2

All OPs in the Spinal Cord and Optic Nerve Are Proliferating at P21 and P60 (A–F) Dividing OPs were labeled by continuous EdU administration to P21 and P60 mice via their drinking water at 0.2 mg/ml. Images are shown of spinal cord white matter (spinal wm) labeled with Pdgfra and EdU after EdU administration from P21 (A) or P60 (B and C) and of spinal cord gray matter (spinal gm) that was EdU labeled from P21 (D) or P60 (E and F). (G–J) The percentages of Pdgfra⁺ OPs that were EdU labeled (labeling index) were plotted against EdU labeling time (G). Also shown are images of optic nerve labeled to detect Pdgfra and EdU following labeling from P21 (H) or P60 (I and J). (K) The EdU labeling index in optic nerve was plotted against EdU labeling time (means ±SD, n = 3 mice). The maximum labeling index (GF) was 100%. Arrows and arrowheads indicate Pdgfra⁺, EdU⁺ and Pdgfra⁺, and EdU-negative cells, respectively. Cell nuclei were stained with Hoechst 33258 (blue). Scale bars represent 25 μm.

Figure 3

OPs Generate Differentiated OLs throughout the Healthy Adult CNS (A–E) Tamoxifen was administered to Pdgfra-CreER^T2: R26R-YFP transgenic mice at P60. Photomicrographs of the corpus callosum (cc) (A), motor cortex (ctx) (B), spinal cord white matter (spinal wm) (C), spinal cord gray matter (spinal gm) (D), and optic nerve (E) were collected following immunolabeling with CC1 (red) and anti-GFP (green). (F) The percentage of YFP⁺ cells that were differentiated OLs (YFP⁺, CC1⁺/total YFP⁺) was plotted against time after tamoxifen (mean ±SD, n = 3 mice). White arrows indicate YFP⁺, CC1-negative cells. Arrowheads indicate double-labeled cells. Cell nuclei were stained with Hoechst 33258 (Hst, blue). Scale bars represent 15 μm.

Figure 4

Adult-Born Myelinating OLs in Spinal Cord and Optic Nerve (A–F) Sections of cerebral cortex and corpus callosum (A and B), spinal cord gray and white matter (D and E), and optic nerve (C and F) from P60+37 Pdgfra-CreER^T2: Tau-mGFP mice were immunolabeled with anti-GFP (green) and monoclonal CC1 (red). mGFP⁺, CC1⁺ myelinating OLs are shown (arrowheads). Shown are compressed z stacks (A–C) or single confocal scans (D–F). Cortex, ctx; corpus callosum, cc; gray matter, gm; white matter, wm; optic nerve. The corpus callosum and cerebral cortex of Pdgfra-CreER^T2: Tau-mGFP mice were also examined at P45+150 (Figure S3) Cell nuclei were counterstained with Hoechst 33258 (Hst, blue). Scale bars represent 25 μm (A–C) or 8 μm (D–F).

Figure 5

Differentiated OLs Are Generated in the Optic Nerve after Four Months of Age (A and B) Sections of optic nerve from P120+65 Pdgfra-CreER^T2: R26R-YFP mice were immunolabeled with anti-GFP (YFP⁺ cells; green), monoclonal CC1 (OLs; red), and anti-Olig2 (all OL-lineage cells; blue). Shown are a confocal stack (A) and a single confocal scan (B). (C) Adjacent sections were immunolabeled with anti-GFP (YFP⁺ cells; green) and anti-NG2 (OPs; red). (D) The percentages of YFP⁺ cells that were OL lineage cells (YFP⁺, Olig2⁺), OPs (YFP⁺, NG2⁺), or differentiated OLs (YFP⁺, CC1⁺) were determined (means ±SD, n = 3 mice). Scale bars represent 15 μm.

Figure 6

Late-Born OLs Make Many More, Shorter Internodes than Early-Born OLs (A) Optic nerve sections from P30+30 Pdgfra-CreER^T2: Tau-mGFP mice were immunolabeled with anti-GFP (white) (confocal stack). (B) Optic nerve sections from P120+65 Pdgfra-CreER^T2: Tau-mGFP optic nerves were immunolabeled with anti-GFP (white) (confocal stack). Arrows indicate a single long internode (293 μm in this example). (C) Sections of P120+65 Pdgfra-CreER^T2: Tau-mGFP optic nerves were immunolabeled with anti-GFP (green), anti-Olig2 (blue) and monoclonal CC1 (OLs; red) (confocal stack). A single confocal scan through the cell body is shown on the right. (D) Quantification of the number of internodes per OL (means ±SD). The numbers are corrected for the sampling error introduced by the fact that only a fraction of the internodes of a given OL could be contained within the section; how the correction factor was derived is explained in Figure S4. (E) Distributions of internode lengths for OLs born between P30 and P60 (black bars) or P120 and P185 (red bars) (n = 288 and n = 702 internodes, respectively) and between P50 and P80 (gray bars) or P50 and P115 (blue bars) (n = 164 and n = 146, respectively). The K-S test determined that internode lengths were not normally distributed but that the internode length distribution was significantly different for mGFP⁺ OLs in the P30+30 and P120+65 nerves (p < 10⁻⁶). The internode length distributions of mGFP⁺OLs in P50+30 and P50+65 nerves were indistinguishable (p = 0.1, K-S test). (F) Average lengths of newly formed (mGFP⁺) internodes as a function of age of animal. There is a progressive reduction in internode lengths with age. The blue data point is taken from Butt et al. (1994), assuming that the internodes they measured had been generated at the peak of OL production around P10–P20 (Skoff et al., 1969). (G) Sections of P120+65 Pdgfra-CreER^T2: Tau-mGFP optic nerves were immunolabeled for mGFP (green) and the paranodal protein Caspr (red) (single confocal scan). mGFP⁺ myelin sheaths frequently terminated (136/144 times) at a Caspr⁺ paranode (white arrows). (H) mGFP⁺ Caspr⁺ paranodes were always opposed by mGFP-negative, Caspr⁺ paranodes. X-Z compressed confocal stack (upper panel) and single X-Y confocal scan (bottom panel). (I) Sections from the same nerves were immuno-labeled for mGFP (green), Caspr (red) and Nav1.6 (blue). White arrows indicate Caspr⁺ paranodes and white arrowhead indicates a Nav1.6⁺ node. Scale bars represent 30 μm (A–C).

Figure 7

Adult-Born OLs Make Compact Myelin (A) An illustrative electron micrograph of a myelinated axon in the P60 optic nerve. The external cytoplasmic tongue process has been pseudo-colored yellow. (B) An electron micrograph of an axon that has been myelinated by an mGFP⁺ OL in a P30+30 Pdgfra-CreER^T2: Tau-mGFP optic nerve. The DAB reaction product identifies the mGFP⁺ cytoplasmic tongue of the corresponding myelin internode (indicated by an arrowhead). (C) An electron micrograph of an axon that has been myelinated by an mGFP⁺ OL in a P120+65 Pdgfra-CreER^T2: Tau-mGFP optic nerve. The DAB⁺ cytoplasmic tongue is indicated (arrowhead). (D) A higher-magnification image showing compact myelin synthesized by an mGFP⁺ OL in a P120+65 Pdgfra-CreER^T2: Tau-mGFP optic nerve. (E) A scatterplot of axon diameter versus g-ratio for mGFP-negative myelinated axons in the P60 and P185 mouse optic nerves. (F) The equivalent scatterplot for axons myelinated by recently formed (mGFP⁺) OLs in P30+30 and P120+65 Pdgfra-CreER^T2: Tau-mGFP nerves. (G) Immunogold labeling of late-born mGFP⁺ OLs (born P120-P185) confirms that mGFP is restricted to the external tongue (outlined in yellow) and does not enter the myelin sheath. (H) Lower magnification of (G). Scale bars represent 200 nm.