The Subventricular Zone Continues to Generate Corpus Callosum and Rostral Migratory Stream Astroglia in Normal Adult Mice

Lack of immature properties of nestin fate-mapped astrocytes in the adult corpus callosum. A, B, Images of the posterior corpus callosum at 270 d post-TM immunostained for EYFP and GFAP. C, The percentages of EYFP⁺ cells that were GFAP⁺ in the anterior, middle, and posterior corpus callosum, showing that the vast majority of EYFP⁺ cells were astrocytes. D, E, Images of the middle corpus callosum in close proximity to the SVZ at 120 d post-TM. Arrows indicate EYFP⁺/vimentin⁺/GFAP⁺ cells in the corpus callosum. Note that all EYFP⁺/vimentin⁺ cells were also GFAP⁺ and no EYFP⁺/vimentin⁺/GFAP⁻ cells (i.e., presumably immature astrocytes) were detected in the corpus callosum. F, G, EYFP⁺/GFAP⁺ cells in the corpus callosum do not express nestin. H, Colocalization of nestin and PECAM (i.e., endothelial cells) expressions in the corpus callosum of adult wild-type mice. I, J, Orthogonal images of EYFP⁺/GFAP⁺/Ki67⁺ cell in the SVZ at 120 d post-TM, showing the presence of a fate-mapped mitotic astroglial cell (i.e., presumably type B cell). K, EYFP⁺/GFAP⁺ astrocytes in the corpus callosum do not proliferate. BV, Blood vessel; CC, corpus callosum; LV, lateral ventricle. Dotted lines in (A, B, D, E) demarcate the corpus callosum. Scale bars, 25 μm. Results are mean ± SEM (n = 3–4 brains).

Formation of perivascular endfeet and glutamate transporters by nestin fate-mapped astrocytes in the adult corpus callosum. A, Low-magnification image of the posterior corpus callosum at 270 d post-TM immunostained for EYFP, GFAP, and AQP4. A red rectangle in the schematic drawing shows the location of the low-magnification image. Arrows indicate the contacts between EYFP⁺/AQP4⁺ astrocytic endfeet and PECAM⁺ endothelial cells. Dotted lines demarcate the corpus callosum. B–D, Higher magnifications of the area marked by the white rectangle in A, showing EYFP⁺/AQP4⁺ astrocytic endfeet completely enwrapping a PECAM⁺ blood vessel. E, The density of EYFP⁺/AQP4⁺ perivascular astrocytes in the adult corpus callosum increased with increasing time after TM and toward more posterior regions of the corpus callosum (anterior CC: *p < 0.01 vs 40 d, **p < 0.005 vs 40 d, #p < 0.05 vs 120 d; middle CC: *p < 0.01 vs 40 d, **p < 0.005 vs 40 d, #p < 0.05 vs 120 d; posterior CC: **p < 0.005 vs 40 d, ##p < 0.01 vs 120 d). F, The density of PECAM⁺ blood vessels in different anterior–posterior regions of the adult corpus callosum. G, The percentages of PECAM⁺ blood vessels that were in contact with EYFP⁺/AQP4⁺ endfeet in anterior, middle, and posterior corpus callosum with increasing time after TM. Note that at 270 d after TM, 22.5% of total blood vessels in the posterior corpus callosum were associated with EYFP⁺/AQP4⁺ astrocytic endfeet. H, I, Both EYFP⁻/GFAP⁺ (arrowheads) and EYFP⁺/GFAP⁺ (arrows) astrocytes in the corpus callosum expressed excitatory amino acid transporters 1 (EAAT1; H) and EAAT2 (I), astrocyte-specific glutamate transporters. CC, Corpus callosum. Scale bars: A, 100 μm; H, I, 15 μm. Results are mean ± SEM (n = 3–4 brains).

Astrocyte turnover in the adult corpus callosum. A, Image of adult GFAP:GFP mouse corpus callosum immunostained for GFP and GFAP showing colocalization of GFAP:GFP and GFAP. B, Quantification of total numbers of GFAP:GFP⁺ astrocytes in the adult corpus callosum. C, Orthogonal view of a TUNEL⁺/GFAP⁺/DAPI⁺ cell in the adult corpus callosum. Insets below are the magnified images of the area outlined by the rectangle. Note convoluted nucleus with cavitation, a feature of early apoptosis (Johnson et al., 2000). D, Calculated net daily addition of EYFP⁺/GFAP⁺ astrocytes in the adult corpus callosum. CC: corpus callosum. Scale bars, 20 μm. Results are mean ± SEM (n = 3 brains in B, n = 3–4 brains in D).

Nestin fate-mapping of astrocytes in the adult RMS. A, Schematic representation of a coronal forebrain section 1.0 mm anterior to bregma used for analysis (B–E) of the posterior RMS (pRMS). With increasing time post-TM, the density of nestin fate-mapped astrocytes (EYFP⁺/GFAP⁺) increased in the pRMS, as demonstrated by quantifications (B, C) and immunostaining (D, 7 d; E, 120 d post-TM). F–I, Anterior RMS (aRMS) visualized in sagittal (F, G) and coronal (H, I) sections. Note the robust stream of SVZ-derived EYFP⁺ cells in RMS at 120 d post-TM (G–I). Some of the EYFP⁺ cells were colabeled with DCX (H) or GFAP (G, I). Also, note the paucity of EYFP⁺ cells in the aRMS at 3 d post-TM (F), indicating that there were very few nestin⁺ NPCs in RMS, and validating the mapping strategy. J, K, RMS within the olfactory [RMS(ob)] at 120 d post-TM, showing some fate-mapped cells are astrocytes (EYFP⁺/GFAP⁺; K: magnified image of the boxed area in J). L, EYFP⁺ astrocytes are absent in the GCL at 120 d post-TM. Insets in D, E, and G are magnified images of the boxed areas. Arrowheads in G, I, and K indicate fate-mapped astrocytes (EYFP⁺/GFAP⁺). The RMS is highlighted by dotted lines. CC, Corpus callosum; LV, lateral ventricle. Scale bars: D–G, J, 100 μm; H, I, L, 50 μm. *p < 0.01 versus 7 d, #p < 0.05 versus 40 d in B; *p < 0.01 versus 7 d, **p < 0.001 versus 7 d, #p < 0.01 versus 40 d in C. Results are mean ± SEM (n = 3–4 brains).