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. 2021 Jul 12;10:e61173. doi: 10.7554/eLife.61173

Figure 2. Microglia rapidly repopulate the visual cortex after partial depletion.

(A) A field of microglia during depletion and repopulation imaged in vivo in the same awake mouse. (B) The number of microglia (normalized to control day for each animal) during depletion (PLX) and with repopulation (day 1 day 30). Each line represents an individual animal (n=10, 70–80 microglia per mouse). (C) 3D nearest-neighbor quantification showed a large increase during depletion and the early stages of repopulation before returning to control numbers (n=4, 5–80 microglia per mouse; a subset of mice from (C) that could be imaged throughout the control, depletion, and repopulation time points were used for this analysis). (D) Depletion and repopulation dynamics were similar in the absence of P2Y12 (n=four animals, 5–80 microglia per mouse). (E) 3D nearest neighbor analysis shows similar changes in microglial distribution during repopulation in the absence of P2Y12 (n=3 animals 5-80 microglia per mouse). (F) The ratio of microglia numbers observed on D7 PLX to control. (G) Repopulation was slightly delayed in P2Y12-KO mice as compared to WT, as the change in microglial numbers from depletion (PLX) to day 2 of repopulation was significantly smaller in the absence of P2Y12. (H) By day 5 of repopulation, the change in microglial numbers had normalized between WT and P2Y12-KO mice. (I) Microglial numbers never fully recovered to control conditions in either WT or P2Y12-KO mice (F-I, T-test ns, n=4, 5–80 microglia per mouse). Scale bar, 50 μm. Figure 2—source data 1: Source data of microglial repopulation after partial depletion.

Figure 2—source data 1. Source data of microglial repopulation after partial depletion.
Figure 2—source data 2. Source data for the GFAP expression following microglial depletion.

Figure 2.

Figure 2—figure supplement 1. GFAP expression is unchanged following microglial depletion.

Figure 2—figure supplement 1.

Representative confocal images of astrocytes immunoreacted for GFAP in fixed brain sections from mice in control conditions (A) after PLX treatment (B) and after 30 days of repopulation (C). Qualitatively astrocyte morphology and expression of GFAP did not change following microglial depletion. (D) The proportion of V1 area covered by GFAP-positive astrocytes was unchanged with PLX treatment. n=3–5 animals, one-way ANOVA, Dunnett post-hoc test, ns. Graphs show means ± s.e.m. Points represent individual animals. Scale bar, 20 µm. Figure 2—source data 2: Source data for the GFAP expression following microglial depletion.
Figure 2—figure supplement 2. Comparison of microglial number over the course of repopulation in the presence and absence of P2Y12.

Figure 2—figure supplement 2.

(A) Number of microglia for CX3CR1-GFP (red) and P2Y12-KO (black) animals for each imaging normalized to first control time point. (B–J) There was no significant difference over the course of repopulation. n=4–13 mice per group. T-test, ns. Graphs show mean ± s.e.m. Points represent individual animals. Please note that these comparisons include all animals imaged even if the imaging series was not complete. For the comparisons in Figure 2F–I only animals which had complete imaging were used so that changes between time points in individual animals could be compared directly.
Figure 2—figure supplement 3. Comparison of nearest neighbor distance over the course of repopulation in the presence and absence of P2Y12.

Figure 2—figure supplement 3.

(A) The nearest neighbor quantification for CX3CR1-GFP (red) and P2Y12-KO (black) animals for each imaging time point normalized to the first control time point. (B–J) Differences between genotypes were small but significant at D4 and D21 of repopulation. n=4–13 mice per group, T-test, *p<0.05, **p<0.01. Graphs show mean ± s.e.m. Points represent individual animals.