Microglia enhance neurogenesis and oligodendrogenesis in the early postnatal subventricular zone

Abstract

Although microglia have long been considered as brain resident immune cells, increasing evidence suggests that they also have physiological roles in the development of the normal CNS. In this study, we found large numbers of activated microglia in the forebrain subventricular zone (SVZ) of the rat from P1 to P10. Pharmacological suppression of the activation, which produces a decrease in levels of a number of proinflammatory cytokines (i.e., IL-1β, IL-6, TNF-α, and IFN-γ) significantly inhibited neurogenesis and oligodendrogenesis in the SVZ. In vitro neurosphere assays reproduced the enhancement of neurogenesis and oligodendrogenesis by activated microglia and showed that the cytokines revealed the effects complementarily. These results suggest that activated microglia accumulate in the early postnatal SVZ and that they enhance neurogenesis and oligodendrogenesis via released cytokines.

Keywords: cytokine; microglia; neurogenesis; neurosphere; oligodendrogenesis; subventricular zone.

Figure 1.

There is a population of activated microglia accumulated in the early postnatal SVZ. Aa1, Distribution of microglia in the postnatal VZ/SVZ (P1, P4, P10, P14, P30). Sagittal sections of forebrains were immunostained with anti-Iba1 (green: microglia) and anti-GFAP antibodies (red: neural stem cells and astrocytes). Aa2, Magnified images of the hatched squares in Aa1. The accumulation in the SVZ in P4 and P10 was distinctive. Aa3, Magnified images of the hatched squares in Aa2. Morphological changes of microglia with age from amoeboid shape to more ramified shape is remarkable (P4, P10, P30). Bb1, Activation of microglia in P10 SVZ. Sagittal sections immunostained with anti-CD11b (red: activated microglia) and anti-Iba1 antibodies (green: microglia). Right panel, Magnified image of the hatched square in the left panel. The microglia in the SVZ have an amoeboid shape and positive for CD11b (white arrowheads), whereas those outside SVZ have more ramified shape and are negative for CD11b (black arrowheads). Bb2, Sagittal sections immunostained with anti-CD68 (red: activated microglia) and anti-Iba1 antibodies (green: microglia). Right panel, Magnified image of the hatched square in the left panel. The microglia in the SVZ have an amoeboid shape and positive for CD68 (white arrowheads), whereas those outside SVZ have more ramified shape and are negative for CD68 (black arrowheads). Similar results were obtained in three independent experiments.

Figure 2.

The temporal and spatial dynamics of activated microglia in the postnatal SVZ. A, A schematic of the rostrocaudal levels in this experiment. B, The distribution of microglia in the rostral, medial, and caudal SVZ at P1, P4, P10, and P30. Coronal sections of forebrains at rostral (2 mm anterior to the bregma), medial (0.7 mm anterior to the bregma), and caudal (0.5 mm posterior to the bregma) levels were immunostained with anti-Iba1 (green: microglia) followed by DAPI staining (blue: cell nuclei). A population of activated microglia accumulated within the SVZ at P1-P10. Cc1, Typical morphology of microglia in the middle SVZ at P1, P4, P10, and P30. Morphological change of microglia with age from amoeboid shape to more ramified shape is remarkable. Cc2, The middle SVZ sections immunostained with anti-CD68 (red: activated microglia) and anti-Iba1 antibodies (green: microglia). The microglia at P1, P4, and P10 in the SVZ have an amoeboid shape and are positive for CD68 (representative cells: white arrowheads), whereas those at p30 have a more ramified shape and are negative for CD68. D, The quantification of the number of Iba1⁺ cells in the SVZ. d1, Time course of the Iba1⁺ microglia in the middle SVZ. The number peaked at P10. d2, d3, The comparison of the numbers of microglia among the rostral, middle, and caudal SVZ at P4 (d2) and P10 (d3). *p < 0.05 versus p1 or rost group (Tukey's test by ANOVA). Data are mean ± SEM. E, The cell density of Iba1⁺ microglia at different rostrocaudal levels at P1, P4, P10, and P30. The cell density of microglia in the SVZ paralleled with that of the number of microglia throughout a period of the observation. F, The ratio of activated microglia in the SVZ (CD68⁺/Iba1⁺). During the experimental period, the highest ratio was obtained at P1. We confirmed the similar results in three independent experiments.

Figure 3.

Minocycline suppressed microglial activation in vivo. A, Effects of minocycline on the number of Iba1⁺ cells in the SVZ and their morphologies. Minocycline was administered by intraperitoneal injection for 3 d beginning at P2 (30 mg/kg/d, P2-P4, n = 6/group). Sagittal sections of minocycline-treated forebrains were immunostained for Iba1 (green) followed by DAPI staining (cyan). Although the number of Iba1⁺ microglia in the SVZ did not change (graph), their shape shifted from an amoeboid type to a more ramified type by minocycline (bottom). Bb1, Effects of minocycline on the expression of activation markers and the morphologies of microglia. Sagittal sections of minocycline-treated forebrains were immunostained for Iba1 (green), and CD11b (red), and CD68 (red). Minocycline significantly decreased the number of cells positive for CD11b or CD68. The morphologies of the cells were also changed from amoeboid shape to more ramified shape. Bb2, The significant decrease in the expression of CD11b and CD68 was confirmed by Western blotting of the SVZ as well. *p < 0.05 (Student's t test). Data are mean ± SEM. Similar results were obtained in three independent experiments.

Figure 4.

Minocycline decreased the numbers of proliferating cells, neuronal progenitors, and oligodendrocyte progenitors in the early postnatal SVZ. A, Minocycline was administered by intraperitoneal injection for 3 d beginning at P2 (30 mg/kg/d, P2-P4, n = 6/group). Sagittal sections of forebrains were immunostained with antibodies to Ki67, nestin, Dcx, PDGFRα, O1, MBP, ALDH1L1, and S100β. The numbers of cells positive for Ki67, Dcx, MBP, or O1 were counted, whereas the protein levels of nestin, PDGFRα, ALDH1L1, and S100β were examined by Western blotting. Minocycline significantly decreased the number of Ki67⁺ proliferating cells and decreased the level of nestin. The number of cells positive for Dcx was significantly reduced. Minocycline decreased the numbers of cells positive for O1 and MBP, whereas the expression level of PDGFRα tended to increase. *p < 0.05, **p < 0.01 (Student's t test). n = 6 mice/group. Data are mean ± SEM. B, The ratio of the Ki67⁺ cells also positive for respective differentiation markers did not change in the absence or presence of minocycline (left graph). Typical images of the cells positive for Ki67 and Nestin, and the cells positive for Ki67 and Dcx in the control group are shown (right panels). We confirmed the same results in three independent experiments.

Figure 5.

The activated microglia raised the cytokine levels in the SVZ. A, A subpopulation of the microglia express IGF-1 in the early postnatal SVZ, but IGF-1 is not involved in the action of activated microglia during this period. Sagittal sections were immunostained with anti-CD11b (green: microglia) and anti IGF-1 (red) antibodies. Right panel, Magnified image of the square in the left. A subpopulation of microglia is positive for IGF-1 (arrowheads). The percentage of CD11b⁺IGF-1⁺ was 43.42 ± 6.72% in CD11b⁺ cells. B, Minocycline did not affect the amount of IGF-1 in the early postnatal SVZ. Minocycline was administered by intraperitoneal injection for 3 d beginning at P2 (30 mg/kg/d, P2-P4, n = 6/group), and the amount of IGF-1 in the SVZ was quantified by ELISA. C, Minocycline decreased the amount of inflammatory cytokines in the SVZ. IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-γ, and TNF-α levels in the SVZ tissue lysate were measured by BioPlex cytokine detection assay system. *p < 0.05 (Student's t test). n = 6 rats/group. Data are mean ± SEM. Similar results were obtained in two independent experiments.

Figure 6.

The reproduction of the enhancement of neurogenesis and oligodendrogenesis by activated microglia in vitro. Microglia cultured independently of neurosphere on transwells were activated by LPS (10 ng/ml, 30 min) in the presence or absence of minocycline (10 μm), washed carefully, and the transwells were set onto the neurospheres or dissociated cells from neurosphere in prodifferentiation conditions. After differentiation periods suitable for neurons (7 d) or oligodendrocytes (11 d), neurospheres were stained for β3-tubulin (green), PDGFRα (green), O4 (green), GFAP (red), and TOTO3 (cyan). To check the effects of minocycline alone, dissociated cells were incubated in the presence of minocycline (10 μm) for 7 d. A, Quantification of the numbers of neurons, oligodendrocyte progenitors, or astrocytes differentiated from neurospheres cocultured with activated microglia in the presence or absence of minocycline. ***p < 0.001 (Tukey's test by ANOVA). n = 12 neurospheres/group. Data are mean ± SEM. B, Representative immunostained images of neurospheres cocultured with activated microglia in the presence or absence of minocycline. C, The effects of activated microglia on differentiation of single cells dissociated from neurospheres in the presence or absence of minocycline. The effects of minocycline alone were also shown (mino-cont in each graph). *p < 0.05, **p < 0.01, ***p < 0.001. (Tukey's test by ANOVA). n = 12 neurospheres/group. Data are mean ± SEM. D, Images of cells immunostained for differentiation markers. Arrowheads indicate the representative cells positive for the differentiation markers.

Figure 7.

The in vitro enhancement of neurogenesis and oligodendrogenesis by activated microglia was suppressed by the mixture of function-blocking antibodies (anti-IL-1β, anti-IL-6, anti-TNF-α, and anti-IFN-γ). A, The release of IL-1β, IL-6, TNF-α, or IFN-γ from activated microglia was suppressed by minocycline. Cultured microglia were activated by LPS (10 ng/ml, 30 min) in the absence and presence of minocycline (10 μm). The concentration of each cytokine in the supernatant was measured by ELISA 24 h after. *p < 0.05 (Student's t test). Data are mean ± SEM. B, Effects of function-blocking antibodies to IL-1β, IL-6, TNF-α, and IFN-γ on enhanced neurogenesis and oligodendrogenesis by the activated microglia. The neurospheres were differentiated in the absence or presence of functional blocking antibodies (goat anti-rat IL-1β antibody, goat anti-rat IL-6 antibody, TNF-α antibody, or goat anti-mouse/rat IFN-γ antibody) (1 μg/ml for each) and a mixture of all of these antibodies. After a differentiation period suitable for neurons (7 d) or oligodendrocytes (11 d), neurospheres were stained for β3-tubulin (green), O4 (green), and TOTO3 (cyan). The data of single function blocking antibodies were compared with the controls, which include the same concentration of isotype-matched control IgGs (1 μg/ml for each). The data of the mixture of function blocking antibodies were compared with the controls, which include the same concentrations of isotype-matched control IgGs (i.e., 3 μg/ml of normal goat IgG control and 1 μg/ml of rabbit IgG control). *p < 0.05. **p < 0.01, versus isotype-matched control IgG group (Tukey's test by ANOVA). Data are mean ± SEM. C, Representative immunostained images of neurospheres cocultured with activated microglia in the absence or presence of the mixture of the function-blocking antibodies. We confirmed the same results in three independent experiments.

Figure 8.

The effect of each cytokine on neurogenesis and oligodendrogenesis. Neurospheres were incubated for differentiation period suitable for neurons (7 d) or oligodendrocytes (11 d) in the presence of each single cytokine (rIL-1β, rIL-6, rTNF-α, or rIFN-γ) at 1–10 ng/ml. Neurospheres were stained for β3-tubulin (green), O4 (green), followed by TOTO3 (cyan). A, Quantification of the effects of cytokines on neurogenesis and oligodendrogenesis. IL-1β and IFN-γ significantly enhanced neurogenesis at 1 ng/ml. IL-1β and IL-6 enhanced oligodendrogenesis at 10 ng/ml. *p < 0.05 versus control (Tukey's test by ANOVA). **p < 0.01 versus control (Tukey's test by ANOVA). n = 8 neurospheres/group. Data are mean ± SEM. B, Representative images of neurospheres immunostained for β3-tubulin and O4 after differentiation in the presence of the cytokine. C, The effect of each cytokine (10 ng/ml) on cell viability. They did not affect cell viability at 10 ng/ml. The same results were obtained in two independent experiments.