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. 2011 Apr 27;31(17):6277-88.
doi: 10.1523/JNEUROSCI.0450-11.2011.

β-Adrenergic receptor antagonism prevents anxiety-like behavior and microglial reactivity induced by repeated social defeat

Eric S Wohleb  1 , Mark L Hanke, Angela W Corona, Nicole D Powell, La'Tonia M Stiner, Michael T Bailey, Randy J Nelson, Jonathan P Godbout, John F Sheridan
Affiliations

β-Adrenergic receptor antagonism prevents anxiety-like behavior and microglial reactivity induced by repeated social defeat

Eric S Wohleb et al. J Neurosci. .

Abstract

Psychosocial stress is associated with altered immune function and development of psychological disorders including anxiety and depression. Here we show that repeated social defeat in mice increased c-Fos staining in brain regions associated with fear and threat appraisal and promoted anxiety-like behavior in a β-adrenergic receptor-dependent manner. Repeated social defeat also significantly increased the number of CD11b(+)/CD45(high)/Ly6C(high) macrophages that trafficked to the brain. In addition, several inflammatory markers were increased on the surface of microglia (CD14, CD86, and TLR4) and macrophages (CD14 and CD86) after social defeat. Repeated social defeat also increased the presence of deramified microglia in the medial amygdala, prefrontal cortex, and hippocampus. Moreover, mRNA analysis of microglia indicated that repeated social defeat increased levels of interleukin (IL)-1β and reduced levels of glucocorticoid responsive genes [glucocorticoid-induced leucine zipper (GILZ) and FK506 binding protein-51 (FKBP51)]. The stress-dependent changes in microglia and macrophages were prevented by propranolol, a β-adrenergic receptor antagonist. Microglia isolated from socially defeated mice and cultured ex vivo produced markedly higher levels of IL-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1 after stimulation with lipopolysaccharide compared with microglia from control mice. Last, repeated social defeat increased c-Fos activation in IL-1 receptor type-1-deficient mice, but did not promote anxiety-like behavior or microglia activation in the absence of functional IL-1 receptor type-1. These findings indicate that repeated social defeat-induced anxiety-like behavior and enhanced reactivity of microglia was dependent on activation of β-adrenergic and IL-1 receptors.

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Figures

Figure 1.
Figure 1.
Repeated social defeat-induced c-Fos activation in the CNS was β-adrenergic-dependent. Male C57BL/6 mice were subjected to one or three cycles of SDR, brains were collected immediately following the final cycle, and c-Fos staining was determined in the PFC, LS, BNST, PVN, MeA, and HPC. A, Representative pictures of c-Fos staining from the prefrontal cortex (20×) are shown. B, Average number of c-Fos-positive cells. C, D, In a related study, male C57BL/6 mice were injected subcutaneously with vehicle or propranolol (10 mg/kg) before each of the six cycles of social defeat. Brains were collected immediately after the sixth cycle of social disruption and c-Fos staining was determined as above. C, Representative pictures of c-Fos staining from the prefrontal cortex (20×) are shown. D, Average number of c-Fos-positive cells. Error bars represent the mean ± SEM (n = 6–8). Means with different letters (a, b, or c) are significantly different (p < 0.05) from each other.
Figure 2.
Figure 2.
Repeated propranolol pretreatment blocked repeated social defeat-induced anxiety-like behavior. Male C57BL/6 mice were injected subcutaneously with vehicle or propranolol (10 mg/kg) before each of the six cycles of SDR. Light/dark preference was determined 14 h after the sixth cycle of social defeat. A, Representative motion paths of the mice used in the light/dark testing paradigm. B, Average time to enter the dark zone. C, Average time spent in the dark zone. Bars represent the mean ± SEM (n = 10–16). Mice were treated as above. D, E, Spleen weight (D) and plasma IL-6 levels (E) were determined. Bars represent the mean ± SEM (n = 9). Means with different letters (a or b) are significantly different (p < 0.05) from each other.
Figure 3.
Figure 3.
Repeated social defeat increased the percentage of CD11b+/ CD45high/Ly6Chigh macrophages in the CNS. Male C57BL/6 mice were injected subcutaneously with vehicle or propranolol (10 mg/kg) before each of the six cycles of SDR. Brains were collected 14 h after the sixth cycle of social defeat, CD11b+ cells were enriched by Percoll gradient separation, and CD11b, CD45, and Ly6C expression levels were determined through flow cytometry. A, Representative bivariate dot plots of CD11b/CD45 staining. B, Average number of CD11b+/CD45high macrophages (n = 10). C, Representative bivariate dot plots of Ly6C/CD45 staining. D, Average number of CD11b+/CD45high/Ly6Chigh macrophages detected (n = 5). Bars represent the mean ± SEM. Means with different letters (a or b) are significantly different (p < 0.05) from each other. E, In a related study, mice were subjected to repeated social defeat and half the mice were perfused before collecting brains for flow cytometric analysis (n = 6). Bars represent the mean ± SEM. Means with different letters (a or b) are significantly different (p < 0.05) from each other. APC, Allophycocyanin; PerCP, peridinin chlorophyll protein.
Figure 4.
Figure 4.
Increased surface expression of inflammatory markers on microglia and CNS macrophages after repeated social defeat. Male C57BL/6 mice were subjected to six cycles of SDR. Brains were collected 14 h after the sixth cycle of social defeat, CD11b+ cells were enriched by Percoll gradient separation, and CD11b, CD45, CD14, TLR4, and CD86 expression levels were determined. Cells were gated on microglia (CD45low) or macrophages (CD45high). A, Representative bivariate dot plots are shown for CD11b/CD14. B–E, Average percentages of positive cells for CD14 (B), TLR4 (C), CD86 (D), and MHC-II (E) are shown. Bars represent the mean ± SEM (n = 9). Asterisks indicate HCC and SDR within cell types are significantly different (p < 0.05) from each other. In a separate but related study, male C57BL/6 mice were injected subcutaneously with vehicle or propranolol (10 mg/kg) before each of the six cycles of social defeat. Brains were collected 14 h after the sixth cycle of social defeat and CD11b, CD45, and CD14 levels were determine as above. F, G, Representative bivariate dot plots and the average percentage of positive cells for CD14. Bars represent the mean ± SEM (n = 10–12). Means with different letters (a, b, or c) are significantly different (p < 0.05) from each other. APC, Allophycocyanin; FITC, fluorescein isothiocyanate.
Figure 5.
Figure 5.
Increased activated morphology of microglia after repeated social defeat. Male C57BL/6 mice were injected subcutaneously with vehicle or propranolol (Prop; 10 mg/kg) before each of the six cycles of SDR. Mice were perfused and brains were collected 14 h after the sixth cycle of social defeat for Iba-1 staining. A, Representative pictures of Iba-1 staining [fluorescent (i–iv) and DAB (v–viii) from the medial amygdala (40×) are shown). Inset includes enlarged image of Iba-1+ cell indicated by arrow. B–E, Proportional area for Iba-1 staining in the medial amygdala (B, n = 3), prefrontal cortex (C, n = 3), hippocampus (D, n = 3), and paraventricular nucleus (E, n = 3). Bars represent the mean ± SEM. Means with different letters (a or b) are significantly different (p < 0.05) from each other.
Figure 6.
Figure 6.
Repeated social defeat increased the reactivity of microglia. Male C57BL/6 mice were subjected to six cycles of SDR. Brains were collected 14 h after the sixth cycle of social defeat. Enriched microglia were collected by Percoll gradient separation and cultured ex vivo. A–C, Cells were incubated with LPS and corticosterone (Cort) (0, 0.1, or 5 μm) and IL-6 (A), TNF-α (B), MCP-1 (C) protein levels were determined in supernatants collected 18 h later. Bars represent the percentage increase over controls ± SEM. Means with different letters (a, b, or c) are significantly different (p < 0.05) from each other.
Figure 7.
Figure 7.
Repeated social defeat enhanced c-Fos activation, but did not affect behavior or microglial activation in IL-1r1−/− mice. Male IL-1r1−/− C57BL/6 mice were subjected to six cycles of SDR. A, Representative pictures of c-Fos staining from MeA (20×). B, Average number of c-Fos-positive cells in the MeA (n = 5). C–F, In a separate set of studies, WT or IL-1r1−/− C57BL/6 mice were subjected to repeated social defeat and light/dark preference was determined 14 h after the sixth cycle of social defeat. C, Average time to enter the dark zone. D, Average time spent in the dark zone (n = 6–8). Following behavioral testing, mice were perfused and brains were collected for Iba-1 immunohistology. E, Representative images of Iba-1 staining from MeA (40×). Insets include enlarged image of Iba-1+ cell indicated by arrow. F, Proportional area for Iba-1 staining in the MeA (n = 3). Bars represent the mean ± SEM. Asterisks indicate significant differences within the genotype (p < 0.05). n.s., Not significant.
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