Determination of key aspects of precursor cell proliferation, cell cycle length and kinetics in the adult mouse subgranular zone

Abstract

Neurogenesis studies on the adult mouse hippocampal subgranular zone (SGZ) typically report increases or decreases in proliferation. However, key information is lacking about these proliferating SGZ precursors, from the fundamental--what dose of bromodeoxyuridine (BrdU) is appropriate for labeling all S phase cells?--to the detailed--what are the kinetics of BrdU-labeled cells and their progeny? To address these questions, adult C57BL/6J mice were injected with BrdU and BrdU-immunoreactive (IR) cells were quantified. Initial experiments with a range of BrdU doses (25-500 mg/kg) suggested that 150 mg/kg labels all actively dividing precursors in the mouse SGZ. Experiments using a saturating dose of BrdU suggested BrdU bioavailability is less than 15 min, notably shorter than in the developing mouse brain. We next explored precursor division and maturation by tracking the number of BrdU-IR cells and colabeling of BrdU with other cell cycle proteins from 15 min to 30 days after BrdU. We found that BrdU and the Gap2 and mitosis (G2/M) phase protein pHisH3 maximally colocalized 8 h after BrdU, indicating that the mouse SGZ precursor cell cycle length is 14 h. In addition, triple labeling with BrdU and proliferating cell nuclear antigen (PCNA) and Ki-67 showed that BrdU-IR precursors and/or their progeny express these endogenous cell cycle proteins up to 4 days after BrdU injection. However, the proportion of BrdU/Ki-67-IR cells declined at a greater rate than the proportion of BrdU/PCNA-IR cells. This suggests that PCNA protein is detectable long after cell cycle exit, and that reliance on PCNA may overestimate the length of time a cell remains in the cell cycle. These findings will be critical for future studies examining the regulation of SGZ precursor kinetics in adult mice, and hopefully will encourage the field to move beyond counting BrdU-IR cells to a more mechanistic analysis of adult neurogenesis.

Figure 1. Dose response of BrdU in C57BL/6J mice

(a) Tabular representation of BrdU dose response from Experiment I (25, 50, 150, 300 and 500 mg/kg) and Experiment II (50, 100, 150 mg/kg). (b-c) Graphical representation of the total number of BrdU-IR cells or clusters in the SGZ after a single injection of BrdU 2 hr earlier from experiment I (b) and experiment II (c). n=3-4 for each BrdU dose, **p<0.01 and *p<0.05 when compared to 150 mg/kg dose, by one-way ANOVA Tukey’s post-hoc analysis.

Figure 2. Morphology and quantitative analysis of BrdU-IR cells and clusters at different time points after a single injection of BrdU

a-f: Dark brown staining of BrdU-IR cells at 2 hr (b), 8 hr (c), 15 hr (d); light brown with punctate staining of BrdU-IR cells at 96 hr (d), 240 hr (f) and 720 hr (g). Clusters of multiple BrdU-IR cells are seen in proliferation time points (b-d); clusters of single BrdU-IR cells are seen in survival time points (e-g). Arrow points to BrdU-IR cells. g: Schematic of one coronal section of mouse dentate gyrus (bregma -2.18) indicates the different regions examined: Subgranular zone (SGZ, hatched), molecular layer (Mol, dark gray), hilus (Hil, light gray) and outer granule cell layer (oGCL, red not hatched). h-l: Quantitative data of BrdU-IR cells (h-i), clusters (j-k) and cells per cluster (l) in the SGZ. All time points are significantly different from 15 hr time point in (h). All other time points except 96 hr and 240 hr are significantly different from 15 hr time point (j). m-o Quantitative data of BrdU-IR cells (m), clusters (n) and cells per cluster (o) in the Mol, Hilus and oGCL. Data is expressed as mean ± SEM. n = 4-8 animals at each time point. *p<0.05 when compared to 0.25 hr time point by one-way ANOVA, Dunnett’s post-hoc analysis. Scale bar = 10 μm in f, applies a-f.

Figure 3. BrdU-IR cells express pHisH3 during G₂/M phase of the cell division cycle

(a-i) Confocal single z plane images of proliferating cells labeled with pHisH3 in green (a, d, g; CY2) and BrdU in blue (b, e, h; CY5). Double labeling of BrdU and pHisH3 (a-c): 2 hr after single BrdU injection, (d-f): 8 hr after single BrdU injection, (g-h): 15 hr after single BrdU injection. j: The proportion of pHisH3-IR cells that are BrdU positive in the SGZ. Every pHisH3-IR cell was analyzed from each animal at every time point (average of 26.8 ± 4.9 pHisH3 cells from each animal; n = 4-6 animals for each time point). Inset in j shows extended x axis between 0 and 30 hr. k: Pie chart indicating the cell cycle of proliferating cells of adult mouse SGZ; S phase (white), G₂/M (gray/hatched) and G₁ (black). The ratio of the different phases in the pie chart is proportional to the time line of each phase: total cell cycle 14 hr; S 7.6 hr; G₂/M 4.5 hr; and G₁ 1.9 hr (Hayes and Nowakowski, 2002). The position of the blue arrow is indicative of the position of BrdU-IR cells (cells in S phase at time of BrdU injection) at the time point indicated below each pie chart. The percent of BrdU-IR cells in G₂/M (blue arrow in pie chart) approximately correlates with the percent of BrdU-IR cells labeled with pHisH3 (see j). The small differences between the actual (x-y graph in j) vs. theoretical (pie chart in k) data may be due to the small number of pHisH3-IR cells available for analysis. The number in parenthesis after the time point below each pie chart suggests the approximate number of cell cycles that have occurred since BrdU incorporation. Data is expressed as mean ± SEM. *p<0.05 when compared to the 0.25 hr time point. Scale bar = 10 μm, applies a-i.

Figure 4. Distribution and quantitative analysis of Ki-67 and PCNA-IR clusters in the dentate gyrus of adult mouse brain

a:Representative coronal section of mouse dentate gyrus stained for PCNA; b: stained for DAPI nuclear staining (200X; bregma - 2.18) indicates the different regions examined: Subgranular zone (SGZ), molecular layer (Mol), hilus (Hil) and outer granule cell layer (oGCL). c: Quantitative data of Ki-67-IR clusters in the dentate gyrus. n = 6-8 animals at each time point. d: Quantitative data of PCNA-IR clusters in the dentate gyrus. n = 4-8 animals at each time point. Arrowhead points to a cell in SGZ, thin arrow points to a cell in oGCL and thick arrow points to a cell in the Mol. Data are expressed as mean ± SEM; *p < 0.05 when compared to Ki-67 Mol clusters by one-way ANOVA Bonferroni’s Multiple Comparison Test.

Figure 5. Comparison of endogenous markers Ki-67 and PCNA with exogenous marker BrdU over a BrdU time course

(a-l): Confocal single z plane images of proliferating cells labeled with Ki-67 in red (a, e, i; CY3), BrdU in green (b, f, j; CY3), and PCNA in blue (c, g, k; CY5). Triple labeling of BrdU with Ki-67 and PCNA (a-d): 2 hr after single BrdU injection, (e-h): 15 hr after single BrdU injection, (i-l): 96 hr after single BrdU injection. m: Stacked bar graph indicating ratios of BrdU labeling; white stacked bar- triple labeled BrdU/PCNA/Ki-67-IR cells, orange stacked bar- double labeled BrdU/Ki-67-IR cells, aqua stacked bar- double labeled BrdU/PCNA-IR cells, and green stacked bar- single labeled BrdU-IR cells. 50 to 60 BrdU-IR cells in the SGZ from each mouse were randomly selected for confocal analysis. The ratios of cells are indicated as a percent of 100 and the total sum up to 100% for each group. n: x-y graph of the ratios of BrdU/PCNA and BrdU/Ki-6-IR. The BrdU/PCNA curve is shifted rightward compared to BrdU/Ki-67, the 50% labeling is 15 hr for BrdU/Ki-67 and 80 hr for BrdU/PCNA. n = 4-6 animals for each time point. Data is expressed as mean ± SEM. Scale bar in i = 10 μm, applies a-l.