Fig. 8. Reduced cell proliferation and impaired neurogenesis in adult mutant neurogenic regions. (A) Decreased BrdU labelling in mutant (right) versus wild-type (left) hippocampus dentate gyrus (DG, top) and lateral ventricle (LV, bottom). BrdU immunoreactivity was revealed by diaminobenzydine (DAB) staining (brown) in the hippocampus, and by rhodamine fluorescence (red) in the lateral ventricle. In the lateral ventricle, the upper panel shows the top lateral corner, and the lower panel shows the ventrolateral wall of the ventricle. (B) Quantitation of BrdU labelling. The average number of BrdU-positive cells in wild-type mice was set equal to 100. Each bar corresponds to the total number of BrdU-positive nuclei in the lateral ventricle walls (LV, left) or in the hippocampus dentate gyrus germinative layer (DG, right) of an individual mouse (white bars, wild type; black bars, mutants). (C) Double fluorescence immunohistochemistry for BrdU (red) and calbindin (CB, green, marking mature granule neurones) on hippocampus dentate gyrus (confocal microscopy). Mice were treated with BrdU for 12 days. BrdU/CB double-positive cells (yellow) are newborn neurones still retaining the label (BrdU) of a recently divided precursor. White arrows indicate BrdU-positive, CB-negative precursors at the base of the granule cell layer; the white arrowhead indicates a BrdU/CB double-positive cell within the granule cell layer (yellow). Quantitative assessment is shown in E. (D) Double fluorescence immunocytochemistry for BrdU (red) and ß-tubulin (green, marking neuroblasts) on dissociated cells from the periventricular region of the lateral ventricle of mice treated with BrdU for 7 days (blue, DAPI nuclear staining). Note that ß-tubulin staining is cytoplasmic. An example of five different cells, stained with all three labels and viewed as a merged image, is shown on the left (ß-tub, BrdU, DAPI). The corresponding single fluorescence images are shown in the panels to the right. Double-positivity for BrdU and ß-tubulin results in a pink nuclear image (due to overlap of the red BrdU staining with the blue DAPI) partially surrounded by a blue-green halo (top left cell). Single positivity for BrdU results in a pink image (top right cell), whereas single positivity for ß-tubulin results in a blue-green colour (bottom left cell). At the bottom right, note the doublet consisting of a doubly positive cell and a single ß-tubulin-positive cell. (E) Quantitation of BrdU/CB double-positive cells (left histogram), and of the proportion of CB-positive cells among BrdU-positive cells, in the dentate gyrus of wild-type (white bars) and mutant (black bars) mice (see C). The average number of BrdU/CB double-positive cells counted in wild-type mice was 71 (5 sections per mouse counted), and was set equal to 100 (left histogram). The average proportion of CB-positive cells among BrdU-positive cells was 31% in wild-type mice. Each bar corresponds to one mouse, identified by numbers on the x axis (see also panel B). (F) Quantitation of BrdU/ß-tubulin double-positive cells in dissociated periventricular cells of the lateral ventricle from wild-type and mutant mice (see panel D). The average value for wild-type mice (corresponding to 60 double-positive cells/four chambered wells counted) was set equal to 100.