Gut enterochromaffin cells drive visceral pain and anxiety

Abstract

Gastrointestinal (GI) discomfort is a hallmark of most gut disorders and represents an important component of chronic visceral pain¹. For the growing population afflicted by irritable bowel syndrome, GI hypersensitivity and pain persist long after tissue injury has resolved². Irritable bowel syndrome also exhibits a strong sex bias, afflicting women three times more than men¹. Here, we focus on enterochromaffin (EC) cells, which are rare excitable, serotonergic neuroendocrine cells in the gut epithelium^3-5. EC cells detect and transduce noxious stimuli to nearby mucosal nerve endings^3,6 but involvement of this signalling pathway in visceral pain and attendant sex differences has not been assessed. By enhancing or suppressing EC cell function in vivo, we show that these cells are sufficient to elicit hypersensitivity to gut distension and necessary for the sensitizing actions of isovalerate, a bacterial short-chain fatty acid associated with GI inflammation^7,8. Remarkably, prolonged EC cell activation produced persistent visceral hypersensitivity, even in the absence of an instigating inflammatory episode. Furthermore, perturbing EC cell activity promoted anxiety-like behaviours which normalized after blockade of serotonergic signalling. Sex differences were noted across a range of paradigms, indicating that the EC cell-mucosal afferent circuit is tonically engaged in females. Our findings validate a critical role for EC cell-mucosal afferent signalling in acute and persistent GI pain, in addition to highlighting genetic models for studying visceral hypersensitivity and the sex bias of gut pain.

Extended Data Figure 1. Sex-specific sensitization of mucosal afferents and visceromotor responses by isovalerate.

a, Graphical scheme of ex vivo mucosal afferent recordings (evMAR) from Na_V1.8-ChR2 mice. b-c, Representative pelvic mucosal afferent fiber responses showing that isovalerate lowers the threshold to optogenetic activation (0.05 – 34 mW/mm²) in males whereas females display heightened baseline sensitivity. d, Fluorescent channelrhodopsin-eYFP signal in L6 DRG sections from Na_V1.8-ChR2 mice, showing no difference between sexes. Scale bars=100μm. e, Heatmaps from two representative males showing in vivo Na_V1.8-GCaMP6s responses of L6 DRG neurons to intracolonic application of an isovalerate bolus (200μM) after pretreatment with alosetron (10μM). f, Responses of dissociated DRG neurons from male (N=3) and female (N=3) Na_V1.8-GCaMP6s mice showing no sex differences in responsiveness to ionomycin 5μM (females: 514 neurons from 15 coverslips, males: 521 neurons from 18 coverslips), potassium chloride (KCl 30mM; females: 478 neurons from 11 coverslips, males: 629 neurons from 18 coverslips) or capsaicin 20nM (females: 246 neurons from 11 coverslips, males: 363 neurons from 18 coverslips). g, Data from individual mice showing total area under the curve for all colonic distension pressures. VMRs are significantly increased in male but not female mice following intracolonic application of isovalerate (100μl bolus of 200μM) versus vehicle; male: N=12 vehicle, N=12 isovalerate, female: N=12 vehicle, N=12 isovalerate). h, total AUC compared across cohorts, similarly demonstrating increased VMRs over baseline (grey) following application of isovalerate (purple) in male but not female mice (male: N=15 vehicle, N=9 isovalerate, female: N=7 vehicle, N=7 isovalerate). i, Colonic compliance is unchanged with isovalerate in both male and female mice. Unpaired t-test, 2-tailed in panel d, Unpaired Mann Whitney U test, 2-tailed in panels f, h, Wilcoxon matched-pairs signed-rank test 2-tailed in panel g and two-way ANOVA in panel i. **p< 0.01, ***p< 0.001, error bars represent mean ± SEM. Graphical element in panel a is licensed from BioRender.

Extended Data Figure 2. Intersectional genetic strategy targets gene expression to gut EC cells.

a, Histologic sections of EC^PFTox (Pet1Flp;Tac1^Cre;RC::PFTox) colon demonstrating colocalization of PFTox allele GFP reporter (green) and 5-HT (magenta); representative of 93 fields from at least 2 animals Scale bars = 50μm (upper) and 10μm (lower). Colon EC cell density is unchanged by expression of PFTox allele (n = 6, 6 independent fields). b, Histologic sections of EC^hM3Dq (Pet1Flp;Tac1Cre;RC::FL-hM3Dq) colon and duodenum demonstrating colocalization (white arrowheads) of the hM3Dq allele reporter (red) and 5-HT (magenta); representative of 129 fields from at least 2 animals. Scale bars=50μm (upper) and 10μm (lower). Colon EC cell density is unchanged following three weeks of DREADD agonist treatment (n = 6, 6 independent fields). c, Whole-mount small intestine (jejunum) of EC^hM3Dq demonstrating expression of single-(Pet1Flp-GFP, green) and double-recombination (Pet1Flp::Tac1Cre, mCherry, red) hM3Dq allele reporters and 5HT (magenta); representative of 10 fields from at least 2 animals. Scale bar 50μm. d, Whole-mount DRG from spinal segments L6/S1 of EC^hM3Dq demonstrating the absence of mCherry (red) expression; representative of 8 fields from 1 animal. Scale bars=50μm. e, Images of EC^hM3Dq dorsal raphe (DR) and median raphe (MnR) nuclei showing 5HT-expressing neurons (yellow) and lack of mCherry (red) expression; representative of 6–8 fields from each of 3 animals. Scale bar=100μm. f, Images of EC^hM3Dq lumbosacral spinal cord (L6-S1) showing the absence of mCherry (red) and 5-HT (yellow) expression; representative of 10 fields from each of 3 animals. Scale bar=100μm. g, Quantitation of specificity and penetrance of intersectional genetic approach demonstrating ≥ 95% (Tac1^Cre, light grey) and 80% (Vil1^Cre, dark grey) EC cell specificity and ~60% penetrance in the colon from male mice (data collected from 20–30 random fields). Student’s t-test (unpaired, 2-tailed) in panels a, b; ns = not significant, error bars represent mean ± SEM.

Extended Data Figure 3. Silencing EC cells attenuates responses to irritants and noxious colonic distension.

a, Representative examples of pelvic mucosal afferents firing more action potentials in response to stroking with 10 mg or 500 mg von frey hairs (vfh) in the presence of isovalerate for control (Tac^Cre, left panel) but not EC^PFTox (right panel) mice. b,c, Group data showing before and after isovalerate (200 μM) application response to increasing mechanical stimulation with vfh for males (b) and females (c) for control (Tac^Cre, upper panels) and EC^PFTox (lower panels) mice. d, Group data showing total area under the curve for all colonic distension pressures showing VMRs significantly reduced after comparing Tac^Cre and EC^PFTox males (N = 6, 12) and females (N = 9, 8). e, Colonic compliance is unchanged in EC^PFTox animals. Wilcoxon matched-pairs signed-rank 2-tailed test in panels b, c; unpaired 2-tailed Mann Whitney U test for panel d; two-way ANOVA in panel e. *p< 0.05, **p< 0.01, ns = not significant, error bars represent mean ± SEM.

Extended Data Figure 4. Activating EC cells increases afferent output and VMR to colorectal distension.

a, DREADD agonist DCZ (1.7μM) elicits Ca²⁺ responses in EC^hM3Dq intestinal organoids as detected by a change in GCaMP fluorescence ratio. b, Representative examples of pelvic mucosal afferents firing action potentials in response to stroking with 10mg or 500mg von frey hairs (vfh) in the presence of vehicle (black/grey) or CNO (100μM; green) for control (upper panels) and EC^hM3Dq (lower panels) mice. c,d, Group data showing before and after CNO (100μM) application response to increasing mechanical stimulation with vfh for males (c) and females (d) for control (Tac^Cre upper panels) and EC^hM3Dq (lower panels) mice. e, Group data showing total area under the curve for all colonic distension pressures showing VMRs significantly increased in Tac^Cre and EC^hM3Dq male mice (N = 6, 7) following DCZ (75μg/kg i.p.). f, Colonic compliance is unchanged in EC^hM3Dq animals. Wilcoxon matched-pairs signed-rank 2-tailed test in panels c, d; Student’s t-test (unpaired, 2-tailed) in panel e; two-way ANOVA in panel f. *p< 0.05, **p< 0.01, ns = not significant, error bars represent mean ± SEM.

Extended Data Figure 5. Gastrointestinal transit is unchanged in EC manipulation models.

a, Total gastrointestinal and colonic transit times are similar between Tac^Cre control (black) and EC^PFTox (red) male (N = 5, 3) and female (N = 5, 4) mice. b, Total gastrointestinal transit times trend faster in DCZ-treated (75μg/kg) Tac^Cre and EC^hM3Dq male (N = 14, 8) and female (N = 4, 4) mice, as did colonic transit times for male (N = 3, 4) and female (N = 7, 7) mice. Transit measurements started 15 min after DCZ i.p. injection. Unpaired 2-tailed Mann Whitney U test in panels a, b. ns = not significant.

Extended Data Figure 6. EC cells do not modulate distension-sensitive afferents.

Group data showing afferent firing to increasing distension pressures in colonic preparations from Vil^Cre control mice at baseline and following isovalerate. Two-way ANOVA (Šidák’s multiple-comparisons test); ns = not significant, error bars represent mean ± SEM.

Extended Data Figure 7. Activation and silencing of EC cells in male and female mice increase anxiety-like behaviors.

a, Time spent in open or closed arms of EPM following DCZ treatment (75 μg/kg i.p.) 10 min prior to testing. The total time mobile and total distance traveled remain unchanged between Tac^Crecontrol and EC^hM3Dq mice (N = 12, 9). b, Time spent in open or closed arms of EPM for EC^PFTox and Tac^Cre control animals (N = 18, 12). The total time mobile and total distance traveled remain unchanged between EC^PFToxand Tac^Cre animals (N = 18, 12). c, EC^PFTox mice show significantly reduced marble-burying behavior compared to Tac^Crecontrols (N = 17, 12). d, EC^PFTox mice do not show differences in nestlet shredding behavior (N = 14, 12). e, Contextual and cued fear conditioning in Tac^Cre and EC^PFTox mice (N = 9, 8). Two-way ANOVA (Šidák’s multiple-comparisons test) for panel a. Unpaired 2-tailed Mann Whitney test for panels b-e. **p< 0.01, ns = not significant, error bars represent mean ± SEM.

Fig. 1. EC cells mediate sex-dependent response to irritants via serotonergic signaling

a, Schematic of ex vivo ‘flat sheet’ colon pelvic nerve mucosal afferent recordings (evMAR) from Na_V1.8-ChR2 mice stimulated with 470nm light. b-c, Percentage of male or female afferents responding at indicated light intensities, and activation thresholds of mucosal afferents before and after isovalerate (ISV, 200μM) treatment (male: n=15; female: n=14). d, Illustration of intravital calcium imaging from DRGs (outlined by dashed lines) at baseline and after intracolonic delivery of isovalerate; white arrowheads indicate responsive neurons. Images are representative of data from 53 animals. Scale bars=100μm. e-f, Heatmaps from two representative males (e) or females (f) showing responses of L6 DRG neurons to intracolonic application of saline followed by an isovalerate bolus (200μM) with examples of fluorescence changes shown in respective righthand panels (purple traces depict isovalerate-sensitive neurons). g, Group data comparing magnitude of basal activity in neurons from males (n=67) and females (n=124). h-i, Percentage of DRG neurons showing (h) basal activity, or (i) responses to isovalerate or isovalerate plus alosetron (ALS; 10μM) in males and females; n = total neurons analyzed per category. j, Experimental setup for measuring electromyography visceromotor response (VMR) of abdominal muscles to colorectal distension following intra-colonic administration of vehicle followed by isovalerate (200 μM). k-l, VMR responses for post-pubertal wild-type healthy males (k) or females (l). Biological replicates (N’s) indicated in graphs and representative traces shown in respective righthand panels. Non-linear regression, Chi-squared, and Wilcoxon 2-tailed test in panels b, c; unpaired 2-tailed Mann Whitney test in panels g, h; one-way ANOVA in panel i, and two-way ANOVA in panels k and l (Repeated measures, Šidák’s multiple-comparisons test). *p< 0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001, ns = not significant, error bars represent mean ± SEM. Graphical elements in panel d are licensed from BioRender or Adobe Stock.

Fig. 2. Silencing EC cells attenuates colonic sensitivity to irritants and mechanical distension

a, Intersectional genetic strategy to selectively express tetanus toxin (TeNT) in EC cells. b, Images of EC cells showing overlapping expression of TeNT (GFP) and 5-HT (magenta); representative of >800 EC cells examined from at least 2 animals. Scale bars = 10μm. c, 5-HT release as detected by ELISA is blunted in EC^PFTox intestinal organoids compared to Tac^Cre controls upon stimulation with the TRPA1 agonist allyl isothiocyanate (AITC, 20μM; n = 6 or 8 organoid cultures per group) (left panel). Serum 5-HT levels from Tac^Cre controls and EC^PFTox female (N = 8, 3) and male (N = 7, 5) mice (right panel). d, Group data from mucosal afferent recordings (evMAR) at baseline and following isovalerate (200μM) application for Tac^Cre control and EC^PFTox male and female cohorts. e, VMR to colorectal distension is shown for male and female EC^PFTox mice compared to littermate controls. f, Representative traces for control Tac^Cre (left) or EC^PFTox (right) male (upper) or female (lower) mice. g, Percent change in total VMR for Tac^Cre control and EC^PFTox male mice (N = 12, 12) and female mice (N = 10, 11), showing loss of isovalerate sensitization in EC^PFTox males. One-way ANOVA in panel c; two-way ANOVA (Šidák’s multiple-comparisons test) in panels c-e; Wilcoxon 2-tailed test in panel g. *p< 0.05, ****p< 0.0001, ns = not significant, error bars represent mean ± SEM.

Fig. 3. Activating EC cells increases afferent output in males and induces long-term hypersensitivity

a, Intersectional genetic strategy for expressing DREADD-hM3Dq in EC cells. b, EC cell showing DREADD (mCherry) and 5-HT (magenta) co-expression; representative of >300 EC cells examined from at least 2 animals. Scale bar = 15μm. c, 5-HT release from Tac^Cre control or EC^hM3Dq intestinal organoid cultures (n = 5 per group) following treatment with DCZ (1.7μM). Serum 5-HT levels in Tac^Cre or EC^hM3Dq males at 15 min (N = 5, 6) and 30 min (N = 5, 3) and females at 15 min (N = 5, 5) and 30 min (N = 6, 6) after DCZ (75μg/kg) administration. d, Group data from mucosal afferent recordings (evMAR) for EC^hM3Dq mice at baseline and following clozapine N-oxide (CNO, 100μM) application show sensitization in males but not females. e, VMR data following acute stimulation with DCZ (75μg/kg, 15 min) in male (upper) and female (lower) Tac^Cre and EC^hM3Dq cohorts. f, Administration of alosetron (ALS; 100μg/kg) 10 min prior to DCZ (75μg/kg) prevents sensitization to colorectal distension. Individual total AUCs are shown for DCZ, and DCZ/alosetron (ALS) experiments completed one week apart in the same mice. g, Timeline for chronic administration of DCZ (75μg/kg) for 21 days followed by a 3-day washout. h, VMR data showing persistent visceral hypersensitivity in male EC^hM3Dq mice compared to Tac^Cre controls following DCZ washout. i, Representative traces at each distension pressure for a male Tac^Cre and EC^hM3Dq mouse. One-way ANOVA in panel c; two-way ANOVA (Šidák’s multiple-comparisons test) in panels d, e, f (top) and h; Wilcoxon 2-tailed in f (lower). *p< 0.05, **p< 0.01, ns = not significant, error bars represent mean ± SEM.

Fig. 4. EC cells do not modulate distension-sensitive afferents.

a, Graphical illustration of different afferent subtypes innervating the colon and representative ex vivo ‘intact’ colonic afferent recordings showing low threshold (LT), wide dynamic range (WDR), and high threshold (HT) distension sensitive afferents from the pelvic nerve. b-c, Group data showing afferent firing to increasing distension pressures in preparations from EC^PFTox mice (N = 3 females and 2 males combined) at baseline or following intraluminal application of isovalerate (200μM), or from EC^hM3Dq mice (N = 7 females and 2 males) at baseline or following intraluminal application of CNO (100μM), as indicated. Recordings were performed with mice in which EC cell-selective expression of tetanus toxin or DREADD receptor was directed by Pet1Flp;Vil1Cre recombination. Two-way ANOVA (Šidák’s multiple-comparisons test) for panels b, c. ns = not significant, error bars represent mean ± SEM.

Fig. 5. Manipulating EC cell-mucosal afferent activity induces anxiety-like behavior.

Distance traveled and time mobile in open arms of an elevated plus-maze using naive male and female cohorts (combined) comparing a, Tac^Cre control and EC^hM3Dq mice, 10 min post-DCZ (75μg/kg, N = 12, 9) or with or without alosetron (ALS; 100μg/kg, N = 12, 10) treatment 15 min prior to DCZ administration, or b, comparing Tac^Cre to EC^PFTox mice (N = 18, 12). Unpaired 2-tailed Mann Whitney test for panels a, b. *p< 0.05, **p< 0.01, ***p< 0.001, ns = not significant, error bars represent mean ± SEM.