Figure 1.
Analyses of cell-type specific markers in BrdU-positive cells in the hippocampal neurogenic area and non-neurogenic area.
(A, B) Immunostaining with anti-BrdU, anti-nestin (A), and anti-DCX (B) antibodies in the neurogenic area. White arrows indicate BrdU-positive cells labeled with neural stem/progenitor cell markers. (C to E) Double immunostaining with anti-BrdU and anti-Olig2 (C), anti-PDGFRα (D), or anti-NG2 (E) antibodies in the non-neurogenic area. White arrows indicate BrdU-positive cells labeled with each OPC marker (C to E). Note that the dividing OPCs extend processes. (F to I) Double immunostaining with anti-BrdU and anti-GFAP (F), anti-Iba1 (G), anti-nestin (H) and anti-DCX (I) antibodies in the non-neurogenic area. White arrowheads indicate BrdU-positive cells that are negative for GFAP (F), Iba1 (G), nestin (H), and DCX (I). Yellow arrows indicate Iba1-positive cells (G). (J) The percentage of cells positive for each marker among all BrdU-positive cells. Scale bars: 20 µm (A to I).
Figure 2.
Daily variation in the number of BrdU-positive cells and PH3-positive cells in the neurogenic area and the non-neurogenic area of the hippocampus.
(A) Representative actogram of the locomotor activity of a mouse reared under a constant L-D cycle. The white and black horizontal bars represent the light and dark periods, respectively, across two days. Black bars on each line represent the amount of locomotor activity. (B) Total number of BrdU-positive cells in the neurogenic area at various ZT (mean ± s.e.m., n = 8 to 10 for each time point). White and black bars represent light and dark periods of the day, respectively. There was no significant variation in the number of BrdU-positive cells between each time point ZT (p = 0.322 by one-way ANOVA). (C) Total number of PH3-positive cells in the neurogenic area at various ZT. The number of PH3-positive cells was the highest at ZT18 and the lowest at ZT6 (mean ± s.e.m., n = 8 for each time point, p<0.001 by one-way ANOVA, ***p<0.001 and by a post hoc Tukey test). (D) BrdU-positive cells labeled with NG2 in the non-neurogenic area. (E) Total number of BrdU-positive cells in the non-neurogenic area at various ZT (mean ± s.e.m., n = 8 to 10 for each time point). The number of BrdU-positive cells was the highest at ZT6 and the lowest at ZT21 (p = 0.021 by one-way ANOVA, *p<0.05 by a post hoc Tukey test). (F) PH3-positive cell labeled with NG2 in the non-neurogenic area. (G) Total number of PH3-positive cells in the non-neurogenic areas at various ZT. The number of PH3-positive cells was the highest at ZT18 and the lowest at ZT6 (mean ± s.e.m., n = 8 for each time point, p = 0.004 by one-way ANOVA, **p<0.01 by a post hoc Tukey test).
Figure 3.
Cyclin D1 is expressed in OPCs in the hippocampus.
(A) Immunostaining with anti-cyclin D2 antibody in the hippocamous. Immunoreactive signals were seen along the SGZ of the DG. (B) Immunostaining with anti-cyclin D1 antibody in the hippocampus. White dots show the margin of the DG. The bright band of immunoreactivity seen above the DG is the cluster of pyramidal neurons in CA1 as previously reported [47]. (C and D) Double immunostaining with anti-PDGFRα and anti-cyclin D1 antibodies in the hippocampus. White arrows indicate PDGFRα-positive cells that are positive for cyclin D1. Higher magnitude image of double positive cell (D). Scale bar: 20 µm. (E) The percentage of cells positive for each marker among all PDGFRα-positive cells.