Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Gut enterochromaffin cells drive visceral pain and anxiety

Nature volume 616, pages 137–142 (2023)Cite this article

Subjects

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 epithelium3,4,5. EC cells detect and transduce noxious stimuli to nearby mucosal nerve endings3,6 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 inflammation7,8. 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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: EC cells mediate sex-dependent response to irritants via serotonergic signalling.
Fig. 2: Silencing EC cells attenuates colonic sensitivity to irritants and mechanical distension.
Fig. 3: Activating EC cells increases afferent output in males and induces long-term hypersensitivity.
Fig. 4: EC cells do not modulate distension-sensitive afferents.
Fig. 5: Manipulating EC cell–mucosal afferent activity induces anxiety-like behaviour.

Similar content being viewed by others

Data availability

All data generated or analysed during this study are included in this published article (and its Supplementary Information).

References

  1. Enck, P. et al. Irritable bowel syndrome. Nat. Rev. Dis. Primers 2, 16014 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  2. Grundy, L., Erickson, A. & Brierley, S. M. Visceral pain. Annu. Rev. Physiol. 81, 261–284 (2019).

    Article  PubMed  Google Scholar 

  3. Bellono, N. W. et al. Enterochromaffin cells are gut chemosensors that couple to sensory neural pathways. Cell 170, 185–198 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Racke, K. & Schworer, H. Characterization of the role of calcium and sodium channels in the stimulus secretion coupling of 5-hydroxytryptamine release from porcine enterochromaffin cells. Naunyn Schmiedebergs Arch. Pharmacol. 347, 1–8 (1993).

    Article  CAS  PubMed  Google Scholar 

  5. Strege, P. R. et al. Sodium channel NaV1.3 is important for enterochromaffin cell excitability and serotonin release. Sci. Rep. 7, 15650 (2017).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  6. Gershon, M. D. Serotonin is a sword and a shield of the bowel: serotonin plays offense and defense. Trans. Am. Clin. Climatol. Assoc. 123, 268–280 (2012).

    PubMed  PubMed Central  Google Scholar 

  7. Koh, A., De Vadder, F., Kovatcheva-Datchary, P. & Bäckhed, F. From dietary fiber to host physiology: short-chain fatty acids as key bacterial metabolites. Cell 165, 1332–1345 (2016).

    Article  CAS  PubMed  Google Scholar 

  8. Farup, P. G., Rudi, K. & Hestad, K. Faecal short-chain fatty acids—a diagnostic biomarker for irritable bowel syndrome? BMC Gastroenterol. 16, 51 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Gribble, F. M. & Reimann, F. Enteroendocrine cells: chemosensors in the intestinal epithelium. Annu. Rev. Physiol. 78, 277–299 (2016).

    Article  CAS  PubMed  Google Scholar 

  10. Gribble, F. M. & Reimann, F. Function and mechanisms of enteroendocrine cells and gut hormones in metabolism. Nat. Rev. Endocrinol. 15, 226–237 (2019).

    Article  CAS  PubMed  Google Scholar 

  11. Liddle, R. A. Neuropods. Cell Mol. Gastroenterol. Hepatol. 7, 739–747 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Kaelberer, M. M. et al. A gut–brain neural circuit for nutrient sensory transduction. Science 361, eaat5236 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Treichel, A. J. et al. Specialized mechanosensory epithelial cells in mouse gut intrinsic tactile sensitivity. Gastroenterology 162, 535–547 (2022).

  14. Nozawa, K. et al. TRPA1 regulates gastrointestinal motility through serotonin release from enterochromaffin cells. Proc. Natl Acad. Sci. USA 106, 3408–3413 (2009).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  15. Mawe, G. M. & Hoffman, J. M. Serotonin signalling in the gut—functions, dysfunctions and therapeutic targets. Nat. Rev. Gastroenterol. Hepatol. 10, 473–486 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Osteen, J. D. et al. Selective spider toxins reveal a role for the NaV1.1 channel in mechanical pain. Nature 534, 494–499 (2016).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  17. Sadeghi, M. et al. Contribution of membrane receptor signalling to chronic visceral pain. Int. J. Biochem. Cell Biol. 98, 10–23 (2018).

    Article  CAS  PubMed  Google Scholar 

  18. Lu, V. B., Gribble, F. M. & Reimann, F. Free fatty acid receptors in enteroendocrine cells. Endocrinology 159, 2826–2835 (2018).

    Article  CAS  PubMed  Google Scholar 

  19. Mars, R. A. T. et al. Longitudinal multi-omics reveals subset-specific mechanisms underlying irritable bowel syndrome. Cell 182, 1460–1473 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Alcaino, C. et al. A population of gut epithelial enterochromaffin cells is mechanosensitive and requires Piezo2 to convert force into serotonin release. Proc. Natl Acad. Sci. USA 115, e7632–e7641 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Wang, F. et al. Mechanosensitive ion channel Piezo2 is important for enterochromaffin cell response to mechanical forces. J. Physiol. 595, 79–91 (2017).

    Article  CAS  PubMed  Google Scholar 

  22. Brierley, S. M., Jones, R. C. 3rd, Gebhart, G. F. & Blackshaw, L. A. Splanchnic and pelvic mechanosensory afferents signal different qualities of colonic stimuli in mice. Gastroenterology 127, 166–178 (2004).

    Article  PubMed  Google Scholar 

  23. Daou, I. et al. Remote optogenetic activation and sensitization of pain pathways in freely moving mice. J. Neurosci. 33, 18631–18640 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Kim, J. C. et al. Linking genetically defined neurons to behavior through a broadly applicable silencing allele. Neuron 63, 305–315 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Jensen, P. et al. Redefining the serotonergic system by genetic lineage. Nat. Neurosci. 11, 417–419 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Erspamer, V. & Asero, B. Identification of enteramine, the specific hormone of the enterochromaffin cell system, as 5-hydroxytryptamine. Nature 169, 800–801 (1952).

    Article  ADS  CAS  PubMed  Google Scholar 

  27. Spohn, S. N. & Mawe, G. M. Non-conventional features of peripheral serotonin signalling—the gut and beyond. Nat. Rev. Gastroenterol. Hepatol. 14, 412–420 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Brierley, S. M., Hibberd, T. J. & Spencer, N. J. Spinal afferent innervation of the colon and rectum. Front. Cell Neurosci. 12, 467 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Uhlig, F. et al. Identification of a quorum sensing-dependent communication pathway mediating bacteria–gut–brain cross talk. iScience 23, 101695 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  30. Makadia, P. A. et al. Optogenetic activation of colon epithelium of the mouse produces high-frequency bursting in extrinsic colon afferents and engages visceromotor responses. J. Neurosci. 38, 5788–5798 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Grundy, L. et al. Chronic linaclotide treatment reduces colitis-induced neuroplasticity and reverses persistent bladder dysfunction. JCI Insight 3, e121841 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Najjar, S. A. et al. Optogenetic inhibition of the colon epithelium reduces hypersensitivity in a mouse model of inflammatory bowel disease. Pain 162, 1126–1134 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Jones, R. C. 3rd, Xu, L. & Gebhart, G. F. The mechanosensitivity of mouse colon afferent fibers and their sensitization by inflammatory mediators require transient receptor potential vanilloid 1 and acid-sensing ion channel 3. J. Neurosci. 25, 10981–10989 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Castro, J. et al. Activation of pruritogenic TGR5, MrgprA3, and MrgprC11 on colon-innervating afferents induces visceral hypersensitivity. JCI Insight 4, e131712 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  35. Fothergill, L. J. & Furness, J. B. Diversity of enteroendocrine cells investigated at cellular and subcellular levels: the need for a new classification scheme. Histochem. Cell Biol. 150, 693–702 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Koo, A., Fothergill, L. J., Kuramoto, H. & Furness, J. B. 5-HT containing enteroendocrine cells characterised by morphologies, patterns of hormone co-expression, and relationships with nerve fibres in the mouse gastrointestinal tract. Histochem. Cell Biol. 155, 623–636 (2021).

    Article  CAS  PubMed  Google Scholar 

  37. Lumsden, A. L. et al. Sugar responses of human enterochromaffin cells depend on gut region, sex, and body mass. Nutrients 11, 234 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Bohórquez, D. V. et al. Neuroepithelial circuit formed by innervation of sensory enteroendocrine cells. J. Clin. Invest. 125, 782–786 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  39. Brenner, D. M. & Sayuk, G. S. Current US Food and Drug Administration-approved pharmacologic therapies for the treatment of irritable bowel syndrome with diarrhea. Adv. Ther. 37, 83–96 (2020).

    Article  PubMed  Google Scholar 

  40. Bradesi, S. et al. Dual role of 5-HT3 receptors in a rat model of delayed stress-induced visceral hyperalgesia. Pain 130, 56–65 (2007).

    Article  CAS  PubMed  Google Scholar 

  41. Miranda, A., Peles, S., McLean, P. G. & Sengupta, J. N. Effects of the 5-HT3 receptor antagonist, alosetron, in a rat model of somatic and visceral hyperalgesia. Pain 126, 54–63 (2006).

    Article  CAS  PubMed  Google Scholar 

  42. El-Ayache, N. & Galligan, J. J. 5-HT3 receptor signaling in serotonin transporter-knockout rats: a female sex-specific animal model of visceral hypersensitivity. Am. J. Physiol. Gastrointest. Liver Physiol. 316, G132–G143 (2019).

    Article  CAS  PubMed  Google Scholar 

  43. Hicks, G. A. et al. Excitation of rat colonic afferent fibres by 5-HT(3) receptors. J. Physiol. 544, 861–869 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Ji, Y., Tang, B. & Traub, R. J. The visceromotor response to colorectal distention fluctuates with the estrous cycle in rats. Neuroscience 154, 1562–1567 (2008).

    Article  CAS  PubMed  Google Scholar 

  45. Gustafsson, J. K. & Greenwood-Van Meerveld, B. Amygdala activation by corticosterone alters visceral and somatic pain in cycling female rats. Am. J. Physiol. Gastrointest. Liver Physiol. 300, G1080–G1085 (2011).

    Article  CAS  PubMed  Google Scholar 

  46. Ji, Y., Murphy, A. Z. & Traub, R. J. Estrogen modulates the visceromotor reflex and responses of spinal dorsal horn neurons to colorectal stimulation in the rat. J. Neurosci. 23, 3908–3915 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Balasuriya, G. K., Hill-Yardin, E. L., Gershon, M. D. & Bornstein, J. C. A sexually dimorphic effect of cholera toxin: rapid changes in colonic motility mediated via a 5-HT3 receptor-dependent pathway in female C57Bl/6 mice. J. Physiol. 594, 4325–4338 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Törnblom, H. & Drossman, D. A. Psychopharmacologic therapies for irritable bowel syndrome. Gastroenterol. Clin. North Am. 50, 655–669 (2021).

    Article  PubMed  Google Scholar 

  49. Galligan, J. J. et al. Visceral hypersensitivity in female but not in male serotonin transporter knockout rats. Neurogastroenterol. Motil. 25, e373–e381 (2013).

    Article  CAS  PubMed  Google Scholar 

  50. Wang, Y. C. et al. The ETS oncogene family transcription factor FEV identifies serotonin-producing cells in normal and neoplastic small intestine. Endocr. Relat. Cancer 17, 283–291 (2010).

    Article  CAS  PubMed  Google Scholar 

  51. Hennessy, M. L. et al. Activity of Tachykinin1-expressing Pet1 raphe neurons modulates the respiratory chemoreflex. J. Neurosci. 37, 1807–1819 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Madison, B. B. et al. Cis elements of the villin gene control expression in restricted domains of the vertical (crypt) and horizontal (duodenum, cecum) axes of the intestine. J. Biol. Chem. 277, 33275–33283 (2002).

    Article  CAS  PubMed  Google Scholar 

  53. Salvatierra, J. et al. NaV1.1 inhibition can reduce visceral hypersensitivity. JCI Insight 3, e121000 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  54. Hockley, J. R. F. et al. Single-cell RNAseq reveals seven classes of colonic sensory neuron. Gut 68, 633–644 (2019).

    Article  CAS  PubMed  Google Scholar 

  55. Cantu, D. A. et al. EZcalcium: open-source toolbox for analysis of calcium imaging data. Front. Neural Circuits 14, 25 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    Article  CAS  PubMed  Google Scholar 

  57. Sato, T. et al. Single Lgr5 stem cells build crypt–villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).

    Article  ADS  CAS  PubMed  Google Scholar 

  58. Becker, L. et al. Age-dependent shift in macrophage polarisation causes inflammation-mediated degeneration of enteric nervous system. Gut 67, 827–836 (2018).

    Article  CAS  PubMed  Google Scholar 

  59. Li, Z. S., Schmauss, C., Cuenca, A., Ratcliffe, E. & Gershon, M. D. Physiological modulation of intestinal motility by enteric dopaminergic neurons and the D2 receptor: analysis of dopamine receptor expression, location, development, and function in wild-type and knock-out mice. J. Neurosci. 26, 2798–2807 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank J. Poblete for expert assistance with animal husbandry and genotyping; the Garvan Institute, Australia, for genotyping services; the Preclinical, Imaging and Research Laboratories (SAHMRI) for the use of their small animal facility; and the University of South Australia and the UCSF Nikon Imaging Core for use of their confocal imaging facilities. We thank K. Yackle and all members of our groups for many helpful suggestions and critical comments and N. Bellono for strategic contributions in the early stages of this project. We thank B. Yu for assistance in transmitter surgeries and animal care. This work was supported by NIH training grant T32 DK007762 and postdoctoral fellowship from the A.P. Giannini Foundation (R.D.M.) and the Damon Runyon Cancer Research Foundation (K.K.T.), a Simons Foundation Autism Research Initiative Pilot Award (514791 to D.J.), a Rainin Foundation Innovator Award (20191150 to D.J.), grants from the US National Institutes of Health (HEAL-SPARC Initiative U01NS113869 to H.A.I., D.J. and S.M.B., R35 NS105038 to D.J., R01 DK121657 and GCRLE0320 to H.A.I., and R03 DK121061 and R01 DK128346 to J.R.B.), National Health and Medical Research Council of Australia (NHMRC) Investigator Leadership Grant (APP2008727 to S.M.B.), an NHMRC Development Grant (APP2014250 to S.M.B), an NHMRC Ideas Grant (APP1181448 to J.C.) and the Hospital Research Foundation PhD scholarship (SAPhD000242018 to J.M.).

Author information

Author notes

  1. Ryan D. Morrie

    Present address: Maze Therapeutics, San Francisco, CA, USA

  2. Kristina N. Braverman

    Present address: Jansen, Johnson & Johnson, San Diego, CA, USA

  3. These authors contributed equally: James R. Bayrer, Joel Castro

Authors and Affiliations

  1. Department of Pediatrics, University of California, San Francisco, San Francisco, CA, USA

    James R. Bayrer, Kristina N. Braverman & Carla Bueno-Silva

  2. College of Medicine and Public Health, Flinders Health and Medical Research Institute, Adelaide, South Australia, Australia

    Joel Castro, Jessica Maddern, Aenea Hendry, Sonia Garcia-Caraballo, Gudrun Schober, Mariana Brizuela & Stuart M. Brierley

  3. Hopwood Centre for Neurobiology, Lifelong Health, South Australian Health and Medical Research Institute (SAHMRI), North Terrace, Adelaide, South Australia, Australia

    Joel Castro, Jessica Maddern, Aenea Hendry, Sonia Garcia-Caraballo, Gudrun Schober, Mariana Brizuela & Stuart M. Brierley

  4. Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA, USA

    Archana Venkataraman, Fernanda M. Castro Navarro & Holly A. Ingraham

  5. Department of Physiology, University of California, San Francisco, CA, USA

    Kouki K. Touhara, Nathan D. Rossen, Ryan D. Morrie & David Julius

  6. Tetrad Graduate Program, University of California, San Francisco, CA, USA

    Nathan D. Rossen

Authors
  1. James R. Bayrer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Joel Castro
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Archana Venkataraman
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kouki K. Touhara
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Nathan D. Rossen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ryan D. Morrie
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jessica Maddern
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Aenea Hendry
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Kristina N. Braverman
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Sonia Garcia-Caraballo
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Gudrun Schober
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Mariana Brizuela
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Fernanda M. Castro Navarro
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Carla Bueno-Silva
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Holly A. Ingraham
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Stuart M. Brierley
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. David Julius
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

R.D.M. developed and implemented transgenic strategies for targeting DREADD and PfTox expression to EC cells. N.D.R., A.V. and R.D.M. characterized DREADD and PfTox expression in these lines histologically. J.C. and S.M.B. designed, performed and analysed ex vivo studies mechanically and optogenetically stimulating colonic mucosal afferents. M.B. and S.M.B. designed, performed and analysed ex vivo studies on colonic distension-sensitive afferents. M.B. performed and analysed in vitro responses of NaV1.8-GCaMP6 DRG neurons. G.S., J.M., J.C., S.M.B., K.B. and J.R.B. designed, performed and analysed VMR to CRD. J.C., A.H. and S.M.B. designed, performed and analysed intravital GCaMP imaging studies. S.G.-C. quantified eYFP expression in male and female NaV1.8-Ch2 DRG neurons. K.K.T. quantified serotonin release from organoids. A.V., F.C.N. and C.B.S. collected blood samples and measured serotonin levels in serum. A.V. performed and analysed experiments to assess anxiety-related behaviours. H.A.I., J.R.B., S.M.B. and D.J. wrote the manuscript with input and suggestions from all authors and provided advice and guidance throughout.

Corresponding authors

Correspondence to James R. Bayrer, Holly A. Ingraham, Stuart M. Brierley or David Julius.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature thanks the anonymous reviewers for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

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

a, Graphical scheme of ex vivo mucosal afferent recordings (evMAR) from NaV1.8-ChR2 mice. b-c, Representative pelvic mucosal afferent fibre 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 NaV1.8-ChR2 mice, showing no difference between sexes. Scale bars = 100 μm. e, Heatmaps from two representative males showing in vivo NaV1.8-GCaMP6s responses of L6 DRG neurons to intracolonic application of an isovalerate bolus (200 μM) after pretreatment with alosetron (10 μM). f, Responses of dissociated DRG neurons from male (N = 3) and female (N = 3) NaV1.8-GCaMP6s mice showing no sex differences in responsiveness to ionomycin 5 μM (females: 514 neurons from 15 coverslips, males: 521 neurons from 18 coverslips), potassium chloride (KCl 30 mM; females: 478 neurons from 11 coverslips, males: 629 neurons from 18 coverslips) or capsaicin 20 nM (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 (100 μl bolus of 200 μM) 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. N  =  number of animals and n  =  number of neurons/sections. The graphical element in panel a was created using BioRender (https://biorender.com).

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

a, Histologic sections of ECPFTox (Pet1Flp;Tac1Cre;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 = 50 μm (upper) and 10 μm (lower). Colon EC cell density is unchanged by expression of PFTox allele (n = 6, 6 independent fields). b, Histologic sections of EChM3Dq (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=50 μm (upper) and 10 μm (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 EChM3Dq demonstrating expression of single-(Pet1Flp-GFP, green) and double-recombination (Pet1Flp::Tac1Cre, mCherry, red) hM3Dq allele reporters and 5-HT (magenta); representative of 10 fields from at least 2 animals. Scale bar 50 μm. d, Whole-mount DRG from spinal segments L6/S1 of EChM3Dq demonstrating the absence of mCherry (red) expression; representative of 8 fields from 1 animal. Scale bars = 50 μm. e, Images of EChM3Dq dorsal raphe (DR) and median raphe (MnR) nuclei showing 5-HT-expressing neurons (yellow) and lack of mCherry (red) expression; representative of 6-8 fields from each of 3 animals. Scale bar = 100 μm. f, Images of EChM3Dq 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 = 100 µm. g, Quantitation of specificity and penetrance of intersectional genetic approach demonstrating ≥ 95% (Tac1Cre, light grey) and 80% (Vil1Cre, 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 Fig. 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 (TacCre, left panel) but not ECPFTox (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 (TacCre, upper panels) and ECPFTox (lower panels) mice. d, Group data showing total area under the curve for all colonic distension pressures showing VMRs significantly reduced in ECPFTox versus TacCre males (N = 6, 12) and females (N = 9, 8). e, Colonic compliance is unchanged in ECPFTox 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. N  =  number of animals, n  =  number of afferents.

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

a, DREADD agonist DCZ (1.7 μM) elicits Ca2+ responses in EChM3Dq 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 10 mg or 500 mg von frey hairs (vfh) in the presence of vehicle (black/grey) or CNO (100 μM; green) for control (upper panels) and EChM3Dq (lower panels) mice. c,d, Group data showing before and after CNO (100 μM) application response to increasing mechanical stimulation with vfh for males (c) and females (d) for control (TacCre upper panels) and EChM3Dq (lower panels) mice. e, Group data showing total area under the curve for all colonic distension pressures showing VMRs significantly increased in TacCre and EChM3Dq male mice (N = 6, 7) following DCZ (75 μg/kg i.p.). f, Colonic compliance is unchanged in EChM3Dq 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. N  =  number of animals and n  =  number of afferents.

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

a, Total gastrointestinal and colonic transit times are similar between TacCre control (black) and ECPFTox (red) male (N = 5, 3) and female (N = 5, 4) mice. b, Total gastrointestinal transit times trend faster in DCZ-treated (75 μg/kg) TacCre and EChM3Dq 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. N  =  number of animals.

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

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

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

a, Time spent in open or closed arms of EPM following DCZ treatment (75 μg/kg i.p.) 10 min before testing. The total time mobile and total distance travelled remain unchanged between TacCre control and EChM3Dq mice (N = 12, 9). b, Time spent in open or closed arms of EPM for ECPFTox and TacCre control animals (N = 18, 12). The total time mobile and total distance travelled remain unchanged between ECPFTox and TacCre animals (N = 18, 12). c, ECPFTox mice show significantly reduced marble-burying behaviour compared to TacCre controls (N = 17, 12). d, ECPFTox mice do not show differences in nestlet shredding behaviour (N = 14, 12). e, Contextual and cued fear conditioning in TacCre and ECPFTox 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.

Extended Data Table 1 List of statistical tests used and post hoc comparisons for data shown in each panel of Figs. 1 to 5
Full size table

Supplementary information

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Bayrer, J.R., Castro, J., Venkataraman, A. et al. Gut enterochromaffin cells drive visceral pain and anxiety. Nature 616, 137–142 (2023). https://doi.org/10.1038/s41586-023-05829-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41586-023-05829-8

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing