Functional disruption of cortical cingulate activity attenuates visceral hypersensitivity and anxiety induced by acute experimental colitis

Abstract

Visceral pain is a highly complex experience and is the most common pathological feature in patients suffering from inflammatory gastrointestinal disorders. Whilst it is increasingly recognized that aberrant neural processing within the gut-brain axis plays a key role in development of neurological symptoms, the underlying mechanisms remain largely unknown. Here, we investigated the cortical activation patterns and effects of non-invasive chemogenetic suppression of cortical activity on visceral hypersensitivity and anxiety-related phenotypes in a well-characterized mouse model of acute colitis induced by dextran sulfate sodium (DSS). We found that within the widespread cortical network, the mid-cingulate cortex (MCC) was consistently highly activated in response to innocuous and noxious mechanical stimulation of the colon. Furthermore, during acute experimental colitis, impairing the activity of the MCC successfully alleviated visceral hypersensitivity, anxiety-like behaviors and visceromotor responses to colorectal distensions (CRDs) via downregulating the excitability of the posterior insula (PI), somatosensory and the rostral anterior cingulate cortices (rACC), but not the prefrontal or anterior insula cortices. These results provide a mechanistic insight into the central cortical circuits underlying painful visceral manifestations and implicate MCC plasticity as a putative target in cingulate-mediated therapies for bowel disorders.

Figure 1

Visceral hypersensitivity in the DSS-induced colitis mice. (A) Gradual loss of weight and (B) display of disease symptoms in control (given water) and colitis (given DSS) mice (n = 8/group). DSS was given over 5 days (indicated by gray bars). (C) Morphological features of healthy (left) versus DSS-inflamed colon tissue (right). Extensive edema, infiltrations (indicated by *), loss of goblet cells (triangles) and crypt (open arrow) malformation are observed in the DSS animals but not in healthy animals. Scalebar, 100 µm (D) Quantification of morphological damage in the distal colon post-DSS compared to pre-DSS (n = 5–8/group). (E,F) Abdominal withdrawal to mechanical stimulation (0.008–1 g) is significantly increased and 40% mechanical thresholds significantly decreased in DSS-exposed mice (n = 8/group). (G) Total number of visceromotor responses to colorectal distension increases after DSS exposure and (H) activity bouts decreases after DSS exposure (n = 8/group). (A,B) *p < 0.05, **p < 0.01, ***p < 0.001 compared to respective Day 0 value and ^###p < 0.001 compared between groups; (E) *p < 0.05, ***p < 0.001 compared to pre-DSS value; (G, H) *p < 0.05, **p < 0.01, ***p < 0.001 compared to respective 20 mmHg value and ^#p < 0.05 compared between groups (indicated by corresponding line in graph), all with two-way ANOVA repeated measures with Bonferroni’s multiple comparison; in panels (D,F), **p < 0.01, ***p < 0.001, unpaired Student’s t-test. p values in panels (A,B) indicate significance between entire curves. CM circular muscle, SM submucosa.

Figure 2

Cortical activation patterns to non-noxious and noxious colorectal distension applied in vivo. (A) Quantification of the Fos positive counts in several cortical regions after non-noxious colorectal distension (black) and after noxious colorectal distension (blue). Fos expression was also upregulated after noxious colorectal distension within these regions (B,C) Example images of Fos (green) and Hoescht (blue) expression in the (B) MCC and (C) PI. Scale bar, 100 µm. ***p < 0.001 compared to MCC (non-noxious); ^##p < 0.01, ^###p < 0.001 compared to MCC (noxious); ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001 compared with corresponding non-noxious group, all with one-way ANOVA with Dunn ‘s multiple comparison; MCC midcingulate cortex, PI posterior insula, AI anterior insula, S1Tr primary somatosensory trunk region, S2 secondary somatosensory, rACC rostral anterior cingulate cortex, PrL prelimbic cortex, IL infralimbic cortex.

Figure 3

Chemogenetic targeting of MCC activity during experimental colitis. (A) Experimental scheme (left) and representative images (right) of DREADD targeting in the bilateral MCC. (B) Quantification of Fos positive counts in the MCC in response to noxious CRD (80 mmHg) in the absence or presence of DSS and CNO (n = 20–28 sections/group from 3 mice). (C) Representative example of non-overlapping hM4Di-expressing cells (red) and Fos expression (green, indicated by arrows). (D) Examples of Fos expression (green) in hM4Di-expressing animals under different DSS conditions. Scale bars, 200 µm (A), 100 µm (C,D). ***p < 0.001 compared to DSS + noxious, one-way ANOVA with Dunn’s multiple comparison test; n.s. not significant.

Figure 4

Behavioral and visceromotor responses to functional suppression of MCC activity. (A) Total number of visceromotor responses during colorectal distension (CRD) is attenuated during MCC suppression in hM4Di-expressing DSS animals and (B) total number of visceromotor responses during inter-CRD periods is attenuated during MCC suppression in hM4Di-expressing DSS animals (same n = 5–6/group in (A,B)). (C,D) Withdrawal counts to low intensity (in C) and high intensity (in D) mechanical forces applied to the abdominal skin (same n = 8/group in C,D). (E) Scheme depicting changes in cortical excitability from MCC activity suppression. Blue and gray regions indicate significantly reduced or unaffected activity, respectively. (F) Quantification of Fos levels (from panel E) in hM4Di-expressing DSS mice after noxious CRD applications (with and without CNO; n = 15–25 sections/group from 3 mice). (G) Tracking plot (upper row) and time map (lower row) examples (obtained from ANY-maze software version 6.1, http://www.anymaze.co.uk) of mCherry- and hM4Di-expressing animals performing the open field exploratory task before and after DSS treatment (post-CNO). Quantification of the time spent in the center open area (H), total distance ran (I) and mean speed (J) of mCherry and hM4Di groups before and after DSS treatment (post-CNO) (n = 8/group in H–J). In panels (A–C), *p < 0.05, **p < 0.01, ***p < 0.001, two-way repeated ANOVA with Tukey’s compared to respective pre-DSS values and ^#p < 0.05, ^##p < 0.01, two-way repeated ANOVA with Tukey’s compared between groups (indicated by corresponding line in graph); in panels (F,H–J), *p < 0.05, **p < 0.01, one-way ANOVA with Dunn’s multiple comparison test. MCC midcingulate cortex, PI posterior insula, AI anterior insula, S1Tr primary somatosensory (trunk) cortex, rACC rostral anterior cingulate cortex, PrL prelimbic cortex, IL infralimbic cortex, mPFC medial prefrontal cortex (PrL and IL).