Article Figures & Data
Figures
- Figure 1.
A, Timeline depicting the experimental paradigm used. To induce fluorophore expression, mice were injected with tamoxifen three times during postnatal week 7 and subsequently underwent treatment with either pilocarpine or saline solution on postnatal week 8. Mice were killed on postnatal week 16. B, Example of a three-dimensional reconstruction of the mouse hippocampus. The Scale-cleared 300 µm sections were imaged, aligned, and reconstructed into a three-dimensional reconstruction of the hippocampus with single-cell resolution. C.1, Brainbow fluorophore expression was absent from animals not treated with tamoxifen. C.2, A small cohort of animals was killed 2 d after the last tamoxifen injection (in week 7), and analysis of their dentate gyri revealed that the tamoxifen treatment induced, on average, two type 1 cells (indicated by white arrows) per 300 µm hippocampal section. C.3, C.4, Clonal clusters were observed in both control (C.3) and pilocarpine-treated (C.4) animals. D, The number of cells per cluster increased in pilocarpine-treated SE animals. Scale bars: B (three-dimensional reconstruction), 600 µm; C.1, C.2, 250 µm; C.3, C.4, 200 µm.
- Figure 2.
A, Cells present in clonal clusters were classified based on morphology (see Materials and Methods) into type 1 progenitor cells (A.1); type 2/3 progenitor cells (A.2); immature granule cells (A.3); mature granule cells (A.4), with spiny apical dendrites (A.5); or astrocytes (A.6). B, Immunocharacterization of the different cell types. Type 1 cells were shown to express nestin and GFAP; type 2/3 cells expressed doublecortin (DCX); immature DGCs expressed calretinin; mature DGCs expressed calbindin; and astrocytes were shown to express GFAP. C, The number of clonal clusters per mouse hippocampus was significantly increased in female mice that underwent SE relative to female controls. Female SE mice also had more clusters than male SE mice. D, Graph shows the composition of cell types in clonal clusters from control and SE animals. There was a significant decrease in the number of type 1 cells and a trend (p = 0.06) toward an increase in the number of mature cells in mice exposed to status. E, The percentage of clusters containing either type 1 or type 2/3 progenitors was decreased in SE mice relative to controls, while the percentage of fully differentiated clusters increased. *p < 0.05; **p < 0.01. Scale bars: A.1–A.3, A.6, 25 µm; A.4, A.5, 50 µm; B, 20 µm.
- Figure 3.
Ectopic dentate granule cells are derived from a small number of clonal clusters. Shown is an image of clonal cluster composed entirely of hilar ectopic dentate granule cells (higher-magnification image is outlined in purple in the inset). The graph shows quantification of all the clusters from SE animals that contained ectopic cells. Additionally, for comparison, nine randomly selected clusters containing no ectopic cells are shown. Orange bars show the total number of cells in the cluster, whereas the blue bars show the number of ectopic cells. Ectopic DGCs tended to occur in clusters in which majority of the cells are ectopic. GCL, granule cell layer; H, hilus. Scale bars: A, 150 µm; A inset, 40 µm.
- Figure 4.
Dentate granule cells with basal dendrites arise from diverse clonal clusters. Shown is a neuronal reconstruction of a granule cell (red) with basal dendrites (white arrows) within a clonal cluster. The axon is denoted by the arrowhead. Adjacent cells in the cluster are shown in blue. The graph shows quantification of all the clusters from SE animals that contained cells with basal dendrites (blue bars) relative to total cluster size (orange bars). For comparison, a subset of randomly selected clusters that contained only normal DGCs is shown. Scale bar, 50 µm.
- Figure 5.
Graphs show the distribution of mature granule cells (top) and type 1 cells (bottom) along the dorsal–ventral axis of the hippocampus. Black dots depict the total number of cells at each bregma level (top graph only), whereas blue dots depict the number of mature or type 1 cells, respectively. Red triangles give the percentage of mature or type 1 cells at each level. No relationship between mature cells and bregma level was evident, while higher numbers and proportions of type 1 cells were present at more dorsal levels.
- Figure 6.
Summary of the key findings of the study. The first panel shows five type 1 progenitor cells labeled with either RFP (red) or YFP (yellow), numbered from 1 to 5. Under control conditions, most of the type 1 cells remain quiescent (progenitor cells 1, 2, 4, and 5 remain quiescent); however, a proportion of type 1 cells will enter the mitotic cycle to give rise to differentiated cells (only progenitor cell 3 undergoes terminal differentiation). After epileptogenesis, the following three key changes occur: (1) the number of clusters containing type 1 cells decreases in epileptic animals relative to controls, and the number of clusters composed of differentiated DGCs and astrocytes increases (progenitors 1, 2, 4, and 5 terminally differentiate); (2) progenitor cells either produce all correctly located offspring, or ectopic offspring (progenitor cell 5 gives rise to a cluster composed entirely of ectopic DGCs); and (3) progenitor cells that produce correctly located offspring occasionally produce cells with a basal dendrite, but mostly produce cells with normal dendrites (progenitor cells 1 and 2 give rise to clusters that contain DGCs with basal dendrites and normal DGCs).
Tables
Data Data structure Type of test Power Number of clonal clusters per hippocampus Normality test (Shapiro-Wilk): passed (p = 0.561)Equal variance test (Brown-Forsythe): passed (p = 0.614) Two-way ANOVA with treatment and sex as factors Treatment: 0.372Treatment × sex: 0.579 Mean clone size Normality test (Shapiro-Wilk): passed(p = 0.658)Equal variance test (Brown-Forsythe): passed (p = 0.399) Two-way ANOVA with treatment and sex as factors Treatment: 0.516Treatment × sex: 0.259 Control vs SE, type 1 Normality test (Shapiro-Wilk): failed (p < 0.050) Mann–Whitney RST 25–75% CIs, control: 7.76–37; SE: 0-3.5 Control vs SE, type 2 Normality test (Shapiro-Wilk): failed (p < 0.050) Mann–Whitney RST 25–75%, control: 0-21.6; SE: 0–6.1 Control vs SE, immature cells Normality test (Shapiro-Wilk): failed (p < 0.050) Mann–Whitney RST 25–75%, control: 0–23.4; SE: 0–4.5 Control vs SE, mature cells Normality test (Shapiro-Wilk): failed (p < 0.050) Mann–Whitney RST 25–75%, control: 26.9–83.6; SE: 75.0–93.8 Control vs SE, astrocytes Normality test (Shapiro-Wilk): failed (p < 0.050) Mann–Whitney RST 25–75%, control: 0–10.4; SE: 3.2–7.0 Clusters with progenitors Normality test (Shapiro-Wilk): passed (p = 0.526) t test Power = 0.793 Self-renewing (2 type 1) clusters Normality test (Shapiro-Wilk): failed (p < 0.050) Mann–Whitney RST 25–75%, control: 0–0.15; SE: 0–0 Fully differentiated clusters Normality test (Shapiro-Wilk): passed (p = 0.536) t test Power = 0.790 Ectopic cells Normality test (Shapiro-Wilk): failed (p < 0.050) Mann–Whitney RST 25–75%, control: 0–0; SE: 0–0 Cells with basal dendrites Normality test (Shapiro-Wilk): failed (p < 0.050) Mann–Whitney RST 25–75%, control: 0–0; SE: 0–0
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