Microglia modulate hippocampal neural precursor activity in response to exercise and aging

Abstract

Exercise has been shown to positively augment adult hippocampal neurogenesis; however, the cellular and molecular pathways mediating this effect remain largely unknown. Previous studies have suggested that microglia may have the ability to differentially instruct neurogenesis in the adult brain. Here, we used transgenic Csf1r-GFP mice to investigate whether hippocampal microglia directly influence the activation of neural precursor cells. Our results revealed that an exercise-induced increase in neural precursor cell activity was mediated via endogenous microglia and abolished when these cells were selectively removed from hippocampal cultures. Conversely, microglia from the hippocampi of animals that had exercised were able to activate latent neural precursor cells when added to neurosphere preparations from sedentary mice. We also investigated the role of CX(3)CL1, a chemokine that is known to provide a more neuroprotective microglial phenotype. Intraparenchymal infusion of a blocking antibody against the CX(3)CL1 receptor, CX(3)CR1, but not control IgG, dramatically reduced the neurosphere formation frequency in mice that had exercised. While an increase in soluble CX(3)CL1 was observed following running, reduced levels of this chemokine were found in the aged brain. Lower levels of CX(3)CL1 with advancing age correlated with the natural decline in neural precursor cell activity, a state that could be partially alleviated through removal of microglia. These findings provide the first direct evidence that endogenous microglia can exert a dual and opposing influence on neural precursor cell activity within the hippocampus, and that signaling through the CX(3)CL1-CX(3)CR1 axis critically contributes toward this process.

Figure 1.

Characterization of hippocampal transgene expression and neurosphere formation frequency in Csf1r-GFP mice. A, Photomicrograph showing the adult hippocampus of a Csf1r-GFP mouse. Note that GFP^pos cells display the typical ramified morphology characteristic of brain microglia. B, Double-immunofluorescence staining for Iba1 (red) and GFP (green) confirmed that the GFP^pos cells were indeed microglia. Cell nuclei are shown in blue. C, GFP^pos cells formed a discrete population of cells within hippocampal cell suspensions from Csf1r-GFP mice, constituting ∼10% of all cells. D, Photomicrographs of single Draq5^pos/GFP^pos cells and Draq5^pos/GFP^neg cells from dissociated Csf1r-GFP hippocampus, taken during flow cytometry. Note the morphological homogeneity of sorted microglia. Scale bar, 160 μm.

Figure 2.

Effects of voluntary exercise on microglial proliferation and neural precursor cell activity. A, The total number of GFP^pos hippocampal microglia was not significantly increased as a result of exercise (n = 10). B, Flow cytometry also revealed a significant decrease in the median intensity of GFP fluorescence following a 2 week running period, suggesting reduced Csf1r-driven transgene expression under this experimental condition (*p < 0.05; n = 3). C, Running (R) significantly increased the number of DCX^pos cells in the dentate gyrus of the adult mouse hippocampus compared with nonrunner (NR) controls (*p < 0.05, n = 5 per experimental condition). D, E, Similarly, significantly greater numbers of BrdU^pos cells (D) and neurospheres (E) were observed following voluntary running (*p < 0.05, n = 3–5 per experimental condition). R, Runner mice; NR, Nonrunner mice.

Figure 3.

Microglia mediate the beneficial effects of exercise on NPC activity. A, Schematic diagram of the experimental paradigm used to assess the role of hippocampal microglia in NPC activation following running. In brief, hippocampi from experimental Csf1r-GFP mice were dissociated, after which GFP^pos microglia were either sorted as a purified cell population or depleted from neurosphere preparations through FACS. B, Hippocampal cell preparations from nonrunner mice (NR) and runner (R) mice were split in half such that microglia MG could be removed from part of the obtained suspension. NR w/o MG, Nonrunner without microglia; R w/o MG, runner without microglia. Depletion of microglia from nonrunner hippocampal cultures did not affect the neurosphere-forming frequency compared with baseline (p > 0.05; n = 5 per experimental condition). Note, however, that depletion of microglia from hippocampal neurosphere cultures completely annulled the positive effect of voluntary exercise on NPC activity (*p < 0.05; n = 5 per experimental condition). Removal of MHCII^pos microglia from cell preparations of runner hippocampi resulted in a further increase in the frequency of neurosphere formation (*p < 0.05; n = 6 per experimental condition), suggesting that MHCII^pos microglia negatively control NPC activity. C, Hippocampal cell preparations were depleted of microglia and cocultured with microglia derived from nonrunners and runners. NR MG, nonrunner microglia; R MG, runner microglia. Addition of microglia from runner mice resulted in a significantly greater number of neurospheres in hippocampal cell cultures from sedentary mice that were depleted of endogenous microglia relative to controls in which microglia from nonrunner mice were added (*p < 0.05; n = 7 per experimental condition).

Figure 4.

CX₃CR1 deficiency negatively affects adult hippocampal neurogenesis. A–D, Cx₃cr1^gfp/gfp (KO) hippocampi had significantly lower BrdU^pos and DCX^pos cell numbers compared with their wild-type (WT) counterparts in young (2-month-old) (A, B) and aged (20-month-old) (C, D) animals (*p < 0.05; n = 3–4 per experimental condition).

Figure 5.

Effect of age and voluntary exercise on CX₃CL1/fractalkine levels in the adult mouse hippocampus. A, Under sedentary conditions, a significant decrease in the levels of soluble (i.e., cleaved) CX₃CL1 was observed in 12- and 18-month-old mice compared with their younger (2-month-old) counterparts (*p < 0.05; n = 4 per experimental condition). B, Voluntary wheel running resulted in a small but significant increase in hippocampal CX₃CL1 levels in young mice (*p < 0.05; n = 6 per experimental condition). C, Exercise also increased hippocampal CX₃CL1 levels in aged mice. Voluntary wheel running for a period of 28 d significantly increased CX₃CL1 levels in 12-month-old mice (*p < 0.05; n = 5 per experimental condition), restoring these to the levels normally observed in young, nonrunner hippocampus (compare with A). R, Runner; NR, nonrunner.

Figure 6.

Signaling through the CX₃CL1–CX₃CR1 axis mediates neural precursor activation. A, Intraparenchymal infusion of a CX₃CR1 blocking antibody into the hippocampus abrogated the effect of running on the frequency of neurosphere formation to a similar extent as microglia depletion (*p < 0.05; n = 3–4 per experimental condition). B, Addition of CX₃CL1 to crude hippocampal cell preparations resulted in a dose-dependent increase in neurosphere numbers, with the optimal dose being 100 ng/ml (*p < 0.05; n = 3 per experimental condition). C, Microglia are critical for mediating the CX₃CL1 effect as their depletion abolished the response. An approximate twofold reduction in the neurosphere-forming frequency was observed in cultures from which microglia were depleted (*p < 0.05; n = 5 per experimental condition), despite the presence of 100 ng/ml CX₃CL1. D, No benefits of CX₃CL1 addition on neural precursor activity were observed in hippocampal neurosphere preparations from Cx₃cr1^gfp/gfp mice, indicating that the effect was specifically mediated via CX₃CR1.

Figure 7.

Microglia in the aged hippocampus negatively control neural precursor cell activity. A, The frequency of neurosphere formation, which is a direct reflection of NPC activity, significantly declines with age between 2 and 9 months of age (*p < 0.05; n = 3 per experimental condition). B, Depletion of microglia from the hippocampi of young (2-month-old) sedentary mice did not affect the neurosphere frequency. Note, however, that microglia depletion at more advanced ages significantly increased neurosphere numbers (*p < 0.05; n = 3 per experimental condition). C, Addition of microglia from aged hippocampus to microglia-depleted hippocampal cell cultures of young mice did not significantly impact on the neurosphere frequency compared with control cultures in which hippocampal microglia from 2-month-old mice were added (p > 0.05; n = 6 per experimental condition); MG, microglia; w/o MG, without microglia.