Environmental enrichment and gut inflammation modify stress-induced c-Fos expression in the mouse corticolimbic system

Abstract

Environmental enrichment (EE) has a beneficial effect on rodent behaviour, neuronal plasticity and brain function. Although it may also improve stress coping, it is not known whether EE influences the brain response to an external (psychological) stressor such as water avoidance stress (WAS) or an internal (systemic) stressor such as gastrointestinal inflammation. This study hence explored whether EE modifies WAS-induced activation of the mouse corticolimbic system and whether this stress response is altered by gastritis or colitis. Male C67BL/6N mice were housed under standard or enriched environment for 9 weeks, after which they were subjected to a 1-week treatment with oral iodoacetamide to induce gastritis or oral dextran sulfate sodium to induce colitis. Following exposure to WAS the expression of c-Fos, a marker of neuronal activation, was measured by immunocytochemistry. EE aggravated experimentally induced colitis, but not gastritis, as shown by an increase in the disease activity score and the colonic myeloperoxidase content. In the brain, EE enhanced the WAS-induced activation of the dentate gyrus and unmasked an inhibitory effect of gastritis and colitis on WAS-evoked c-Fos expression within this part of the hippocampus. Conversely, EE inhibited the WAS-evoked activation of the central amygdala and prevented the inhibitory effect of gastritis and colitis on WAS-evoked c-Fos expression in this region. EE, in addition, blunted the WAS-induced activation of the infralimbic cortex and attenuated the inhibitory effect of gastritis and colitis on WAS-evoked c-Fos expression in this area. These data reveal that EE has a region-specific effect on stress-induced c-Fos expression in the corticolimbic system, which is likely to improve stress resilience. The response of the prefrontal cortex - amygdala - hippocampus circuitry to psychological stress is also modified by the systemic stress of gut inflammation, and this interaction between external and internal stressors is modulated by the housing environment.

Figure 1. Environmental enrichment enhances spatial learning and memory in the Morris water maze.

The graph displays the effects of environmental enrichment, relative to standard housing, for 9 weeks on the behaviour of mice in this task. Mice were tested for 3 consecutive days (6 trials per day) at the end of the respective housing period. The average latency to find the hidden platform on each trial day is shown. The values represent means ± SEM, n = 10.

Figure 2. Pre-absorption of the c-Fos antibody with c-Fos blocking peptide prevents c-Fos staining in all brain regions under study.

The left column (panels A, C, E, G, I, K, M) shows representative micrographs of the brain regions stained with thionine acetate to reveal their morphology. Parallel sections processed for c-Fos immunocytochemistry in which the c-Fos antibody was pre-absorbed with excess c-Fos blocking peptide (BP) are shown in the right column (panels B, D, F, H, J, L, N). No specific c-Fos staining was detected in any of the brain regions when the pre-absorbed antibody was used. Abbreviations: BLA  =  Basolateral amygdala; CA1  =  CA1 field of the hippocampus; CA3  =  CA3 field of the hippocampus; CC  =  Cingulate cortex; CeA  =  Central amygdala; DGgl  =  Dentate gyrus, granular cell layer; DGml  =  Dentate gyrus, molecular cell layer; DGpl  =  Dentate gyrus, polymorph cell layer; EE  =  Enriched environment; ILC  =  Infralimbic cortex; MeA  =  Medial Amygdala; PVH  =  Paraventricular nucleus of the hypothalamus; SE  =  Standard environment; TT  =  Tenia tecta.

Figure 3. Environmental enrichment increases dextran sulfate sodium (DSS)-induced weight loss and disease severity.

Mice were treated for 7 days with iodoacetamide (IAA, 0.1%, added to the drinking water) or DSS (2%, added to the drinking water) or kept under control conditions (CO, plain drinking water) after standard or enriched housing for 9 weeks. Body weight was measured before and after the treatment period, and the weight loss (panel A) during the treatment period was expressed as a percentage of the body weight measured pre-treatment. The disease activity score (DAS, panel B), a measure of the animal's health status, was assessed at the end of the treatment period. The values represent means − SEM (A) and means + SEM (B), n = 10.

Figure 4. Environmental enrichment increases the dextran sulfate sodium (DSS)-induced rise of colonic myeloperoxidase (MPO) content, but does not influence the iodoacetamide (IAA)-induced rise of gastric MPO content.

Mice were treated for 7 days with IAA (0.1%, added to the drinking water) or DSS (2%, added to the drinking water) or kept under control conditions (CO, plain drinking water) after standard or enriched housing for 9 weeks. The gastric (panel A) and colonic (panel B) MPO content was determined at the end of the treatment period. Note that the scale of the ordinate is different between panel A and B. The values represent means + SEM, n = 10.

Figure 5. Environmental enrichment increases the dextran sulfate sodium (DSS)-induced rise of post-stress corticosterone (CORT) levels.

Mice were treated for 7 days with IAA (0.1%, added to the drinking water) or DSS (2%, added to the drinking water) or kept under control conditions (CO, plain drinking water) after standard or enriched housing for 9 weeks. Plasma CORT was measured 2 h after the beginning of a 30-min exposure to water avoidance stress at the end of the treatment period. The values represent means + SEM, n = 10.

Figure 6. Environmental enrichment alters stress-induced c-Fos expression in the forebrain.

The effect of water avoidance stress (30 min) on the cerebral c-Fos expression was examined in mice kept for 9 weeks under standard (SE) or enriched environment (EE). The left column panels (A, C, E, G, I, K, M) show representative micrographs of 9 forebrain regions taken from SE mice while the right column panels (B, D, F, H, J, L, N) depict micrographs of the same brain regions taken from EE mice. Relative to SE animals, EE increased stress-induced c-Fos expression in the granular cell layer of the dentate gyrus (A, B), but decreased it in the CA1 region of the hippocampus (C, D), the central amygdala (E, F) and the infralimbic cortex (M, N). In contrast, EE failed to modify stress-induced c-Fos expression in the CA3 region of the hippocampus (C, D), the basolateral and medial amygdala (E, F, G, H), the paraventricular nucleus of the hypothalamus (I, J) and the cingulate cortex (K, L). Abbreviations: BLA  =  Basolateral amygdala; CA1  =  CA1 field of the hippocampus; CA3  =  CA3 field of the hippocampus; CC  =  Cingulate cortex; CeA  =  Central amygdala; DGgl  =  Dentate gyrus, granular cell layer; DGml  =  Dentate gyrus, molecular cell layer; DGpl  =  Dentate gyrus, polymorph cell layer; ILC  =  Infralimbic cortex; MeA  =  Medial Amygdala; PVH  =  Paraventricular nucleus of the hypothalamus; TT  =  Tenia tecta.

Figure 7. Environmental enrichment and visceral inflammation modify stress-induced c-Fos expression in the hippocampus.

Environmental enrichment increases stress-induced expression of c-Fos in (A) the granular cell layer of the dentate gyrus, decreases c-Fos expression in (B) the hippocampal CA1 region, but does not alter c-Fos levels in (C) the hippocampal CA3 region. Gastritis evoked by iodoacetamide (IAA) and colitis evoked by dextran sulphate sodium (DSS) dampen stress-induced c-Fos expression in (A) the dentate gyrus and (C) the hippocampal CA1 region of mice kept both under standard (SE) or enriched environment (EE). Mice were maintained for 9 weeks under SE or EE and then treated for 7 days with IAA (0.1% added to the drinking water) or DSS (2% added to the drinking water), while control (CO) mice drank plain water. Expression of c-Fos was visualized 2 h after the beginning of a 30-min exposure to water avoidance stress at the end of the treatment period. The values represent means + SEM, n = 6–7.

Figure 8. Environmental enrichment and visceral inflammation modify stress-induced c-Fos expression in the amygdala.

Environmental enrichment decreases stress-induced c-Fos expression in (B) the central amygdala but does not alter stress-induced c-Fos levels in (A) the basolateral and (C) medial amygdala. Gastritis evoked by iodoacetamide (IAA) and colitis evoked by dextran sulphate sodium (DSS) dampen stress-induced c-Fos expression in all 3 amygdalar nuclei of mice kept under standard environment (SE), an effect that is largely absent in mice kept under enriched environment (EE). Mice were maintained for 9 weeks under SE or EE and then treated for 7 days with IAA (0.1% added to the drinking water) or DSS (2% added to the drinking water), while control (CO) mice drank plain water. Expression of c-Fos was visualized 2 h after the beginning of a 30-min exposure to water avoidance stress at the end of the treatment period. The values represent means + SEM, n = 7–8.

Figure 9. Environmental enrichment and visceral inflammation modify stress-induced c-Fos expression in the medial prefrontal cortex and the hypothalamus.

Environmental enrichment decreases stress-induced c-Fos expression in (C) the infralimbic cortex but does not alter stress-induced c-Fos levels in (B) the cingulate cortex and (A) paraventricular nucleus of the hypothalamus. Gastritis evoked by iodoacetamide (IAA) and colitis evoked by dextran sulphate sodium (DSS) dampen stress-induced c-Fos expression in mice kept under standard environment (SE) in a region-dependent manner, an effect that is largely absent in mice kept under enriched environment (EE). Mice were maintained for 9 weeks under SE or EE and then treated for 7 days with IAA (0.1% added to the drinking water) or DSS (2% added to the drinking water), while control (CO) mice drank plain water. Expression of c-Fos was visualized 2 h after the beginning of a 30-min exposure to water avoidance stress at the end of the treatment period. The values represent means + SEM, n = 7–8.