An enriched environment reduces chronic stress-induced visceral pain through modulating microglial activity in the central nucleus of the amygdala

Abstract

Cognitive behavioral therapy (CBT) improves the quality of life for patients with brain-gut disorders; however, the underlying mechanisms of CBT remain to be explored. Previously, we showed that environmental enrichment (EE), an experimental paradigm that mirrors positive behavioral intervention, ameliorates chronic stress-induced visceral hypersensitivity in a rodent model via mechanisms involving altered activity in the central nucleus of amygdala (CeA). In the present study, we investigated whether microglia-mediated synaptic plasticity in the CeA is a potential mechanism underlying the protective effects of EE against stress-induced visceral hypersensitivity. We stereotaxically implanted corticosterone (CORT) micropellets onto the dorsal margin of the CeA shown previously to induce colonic hypersensitivity. Animals were housed in EE cages or standard cages for 14 days after CORT implantation. Visceral sensitivity was assessed via visceromotor behavioral response to colorectal distension. Microglial morphology, microglia-mediated synaptic engulfment, and the expression of synaptic pruning-related signals complement component 1q (C1q), complement component 3 (C3), and C3 receptor (C3R) were measured using immunofluorescence and RNAscope assay. We found that housing CORT implanted rats in EE cages for 14 days attenuated visceral hypersensitivity in both male and female rats as compared with control rats maintained in standard housing. EE reduced CORT-induced microglial remodeling and microglia-mediated synaptic pruning with reduced C1q and CR3, but not C3, expression. Our data suggest that exposure to EE is sufficient to ameliorate stress-induced visceral pain via reducing amygdala microglia-modulated neuronal plasticity.NEW & NOTEWORTHY Clinical studies show that cognitive behavioral therapy (CBT) is effective in ameliorating visceral pain in patient with irritable bowel syndrome (IBS), yet the underlying mechanisms remain unexplored. By using environmental enrichment (EE), an experimental paradigm that mirrors positive behavioral intervention, we demonstrated that microglia-mediated synaptic plasticity in the CeA explains, plays a role, at least in part, in the positive effects of EE to reduce visceral hypersensitivity.

Keywords: abdominal pain; cognitive behavioral therapy (CBT); complement component 3; complement component C1q; irritable bowel syndrome (IBS).

Graphical abstract

Figure 1.

Housing in enriched conditions reduced visceral sensitivity after amygdalar CORT implants. A: experimental design. Experimental day is listed in each circle. B: schematic diagram of SH and EE cages. C: visceral sensitivity in animals implanted with CORT/CHOL (CHOL SH, n = 7; CORT SH, n = 7; two-way ANOVA with Tukey’s post hoc test). D: visceral sensitivity in rats housed in EE and SH cages after CORT implants (CORT SH, n = 7; CORT EE, n = 8; two-way ANOVA with Tukey’s post hoc test). Data are presented as means ± SD. CHOL, cholesterol; CORT, corticosterone; EE, environmental enrichment; SH, standard-housed; VMR, visceromotor behavioral response.

Figure 2.

EE reduced microglial activity after CORT implants. A–C: representative images of microglial morphology in the CeA. D and E: enlarged images of an original and skeletonized microglia in the CeA. F: an overlay of generated microglial skeleton to the original fluorescent image. G: the skeletonized images were processed by using the analyze skeleton plugin. Tagged skeletonized processes were colored brown, endpoints were blue, and junctions were purple. H and I: quantification of microglial endpoints (H) and branch length (I) in the CeA after CORT implants (one-way ANOVA with Tukey’s post hoc test). Data are presented as means ± SD. Scale bars: 20 μm. CeA, central nucleus of the amygdala; CHOL, cholesterol; CORT, corticosterone; EE, environmental enrichment; SH, standard-housed.

Figure 3.

EE reduced microglia-mediated synaptic engulfment in the CeA. A–C: selected slices from z-stack images indicate PSD95-positive puncta overlapping (arrows) with Iba-1 in the CeA of animals in CHOL SH (A, A’), CORT SH (B, B’), and CORT EE (C, C’) group. Orthogonal views shown (XY, YZ, and XZ). D: quantification of PSD95-positive puncta overlapping with Iba1 immunostaining in the CeA (one-way ANOVA with Tukey’s post hoc test). Data are presented as mean ± SD. Scale bars: 20 μm. CeA, central nucleus of the amygdala; CHOL, cholesterol; CORT, corticosterone; EE, environmental enrichment; PSD95, postsynaptic density protein 95; SH, standard-housed.

Figure 4.

EE reduced microglial C1qA and ITGAM mRNA expression in the CeA. A–I: Colabeling of C1qA (A, D, and G) and ITGAM mRNA (B, E, and H) with Iba1 antibody (merged in C, F, and I) in the CeA of animals in CHOL SH (A–C), CORT SH (B–E), and CORT EE (G–I) group. J: RNAscope quantification of C1qA mRNA expression in the CeA (one-way ANOVA with Tukey’s post hoc test). K: RNAscope quantification of ITGAM mRNA in the CeA (one-way ANOVA with Tukey’s post hoc test). Data are presented as means ± SD. Scale bars: 20 μm. CeA, central nucleus of the amygdala; CHOL, cholesterol; CORT, corticosterone; EE, environmental enrichment; ITGAM, integrin subunit α-M; SH, standard-housed.

Figure 5.

EE did not change complement component 3 expression in the CeA. A–D: colabeling of C3 (A) with antibody Iba1 (B) and neuronal marker NeuN (C) (merged in D) in the CeA. E–H: colabeling of C3 (E) with antibody Iba1 (F) and astrocyte marker GFAP (C) (merged in H) in the CeA. I–K: microglial C3 expression (left panels, and merged with Iba1 in right panels) in the CeA of animals in CHOL SH (I), CORT SH (J), and CORT EE (K) group. L: RNAscope quantification of C3 mRNA expression in the CeA (one-way ANOVA with Tukey’s post hoc test). Data are presented as means ± SD. Scale bars: 20 μm. CeA, central nucleus of the amygdala; CHOL, cholesterol; CORT, corticosterone; EE, environmental enrichment; SH, standard-housed.