Fig. 4. Phenotypic analysis of BrdU-labeled cells. (A) Four weeks after the last injection of BrdU, cells double-labeled for BrdU and NeuN could be detected. This image confirms previous findings that numerous BrdU/NeuN-positive cells, which are morphologically indistinguishable from the surrounding granule cells can be found 4 weeks after BrdU injection. The inset highlights the double-labeling by showing the immunoreaction for NeuN (arrow) in the BrdU-labeled cell. BrdU/NeuN-positive cells can be found in the entire GCL, not only in the SGZ, indicating early expression of a mature neuronal marker and migration soon after division. NeuN, green; BrdU, red; astrocytic marker S100ß, blue. Scale bar: 80 µm. (B) Eleven months after BrdU injection, BrdU/NeuN-labeled cells could be found. They were morphologically the same as they were 4 weeks after BrdU injection (compare with B). Generally, the staining intensities are lower in the older animals. The whitish granules in and between the cells are age pigment lipofuscin. NeuN, green; BrdU, red; astrocytic marker S100ß, blue. Scale bar: 20 µm. (C) BrdU-labeled cells can be detected as early as 1 day after the last (of 12) injection of BrdU (see inset for display of NeuN-immunoreaction, arrow). NeuN, green; BrdU, red; doublecortin (DCX, see below), blue. Scale bar: 25 µm. (D) Twenty-four hours after a single injection of BrdU, no BrdU/NeuN-labeled cells can be detected (arrow in upper inset; compare with top left inset). Furthermore, NeuN cannot be detected in cells that express intermediate filament nestin as a marker of stem or progenitor cells. Nestin was detected in these mice by expressing green fluorescent protein (GFP) under the nestin promoter (Yamaguchi et al., 2000). The nestin-GFP-expressing putative stem or progenitor cells form clusters along the SGZ (see lower inset). GFP, green; BrdU, red; NeuN, blue. Scale bar: 15 µm. (E) DCX is a protein expressed in young, maturing neurons. Soon after division, many BrdU-labeled nestin-GFP expressing cells become DCX positive. The lower panel shows the fluorescence intensities along the horizontal line drawn through one of the nuclei. BrdU, red; nestin-GFP, green; DCX, blue. The marked cell is triple-labeled, but the fluorescence intensity is lower than in the right neighboring cell that expresses nestin-GFP only. Scale bar: 25 µm. (F) Colocalization of BrdU and DCX can be further visualized in z-series through the section (z-distance is 12 µm). The reconstructed views along the yz-axis (right panel) and xz-axis (bottom panel) demonstrate that red BrdU immunoreaction is surrounded by blue DCX immunoreaction. NeuN, green. Scale bar: 20 µm. (G) The overview shows that along the SGZ BrdU-positive cells (red) can be found that express DCX (blue) and appear pink (arrow, see also F). Similar to C, some BrdU/NeuN-positive cells (orange, arrowhead) can be seen. DCX-labeled cells are restricted to the SGZ. NeuN, green; BrdU, red; DCX, blue. Scale bar: 80 µm. (H) Many DCX-expressing cells are NeuN-positive, indicating a temporal overlap between the two markers. DCX-expressing cells often have long processes, extending into the molecular layer. The inset indicates how cytoplasmic DCX expression (blue) engulfs nuclear and perinuclear NeuN-expression (green). BrdU, red. Scale bar: 10 µm. (I) ß-III-tubulin can serve as another marker for immature neurons. At early time-points after BrdU, clusters of BrdU/ß-III-tubulin marked cells can be found. However, ß-III-tubulin yields a weaker staining, does not extend into the neurites and cannot be combined with NeuN. ß-III-tubulin, green; BrdU, red; S100ß, blue. Scale bar: 20 µm. (K) New astrocytes can be identified by colocalizing immunoreactivity for BrdU (red) and S100ß (blue). NeuN, green. Scale bar: 15 µm. Insets in I and K show controls.

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