Abstract
Psychological and physical stressors have been implicated in gastric disorders in humans. The mechanism coupling the brain to the stomach underlying stress-induced gastric dysfunction has remained elusive. Here, we show that the stomach directly receives acetylcholinergic inputs from the dorsal motor nucleus of the vagus (AChDMV), which are innervated by serotonergic neurons in the dorsal raphe nucleus (5-HTDRN). Microendoscopic calcium imaging and multi-tetrode electrophysiological recordings reveal that the 5-HTDRN → AChDMV → stomach circuit is inhibited with chronic stress accompanied by hypoactivate gastric function. Artificial activation of this circuit reverses the gastric dysfunction induced by chronic stress in both male and female mice. Our study demonstrates that this 5-HTDRN → AChDMV → stomach axis drives gastric dysfunction associated with stress, thus providing insights into the circuit basis for brain regulation of the stomach.
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Data availability
All data are available in the main text or the supplementary materials. The second edition of The Mouse Brain in Stereotaxic Coordinates was used in this study44. Source data are provided with this paper.
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Acknowledgements
This work was supported by the National Key Research and Development Program of China (STI2030-Major Projects 2021ZD0203100), National Natural Science Foundation of China (grant nos. 32025017, 32241013, 32100808 and 32121002), Chinese Academy of Science Project for Young Scientists in Basic Research (YSBR-013), China National Postdoctoral Program for Innovative Talents (BX2021286), China Postdoctoral Science Foundation (2020M682018), Fundamental Research Funds for the Central Universities (WK9100000005), the Institute of Health and Medicine (QYZD20220007) and the Excellent Youth Project of Anhui Universities (2022AH030065). It was also supported by the Innovative Research Team of High-level Local Universities in Shanghai. We thank the Confocal Imaging Unit at the Core Facility Centre for Life Science of the University of Science and Technology of China and Z. Liu for valuable technical expertise and assistance.
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W.-Y.D., X.Z., H.-D.T., J.-Y.H., W.-J.T. and Z.Z. initiated and designed the research. X.Z., H.-D.T., J.-Y.H., P.-K.C., M.-Y.Z. and W.-Y.D. performed most of the experiments and analysed and interpreted the results. M.-Y.Z. and P.-K.C. conducted the behavioural experiments and data analysis. Hao Wang and X.-Y.W. conducted the gastric motility recordings. Haitao Wang conducted the whole-cell recordings. W.-Y.D., X.Z., H.-D.T., J.-Y.H., W.-J.T. and Z.Z. wrote the manuscript. Y.M., W.Z. and Y.Z. were involved in the overall design of the study and the revision of the final manuscript.
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Nature Metabolism thanks Shi-Bin Li and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Ashley Castellanos-Jankiewicz, in collaboration with the Nature Metabolism team.
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Extended data
Extended Data Fig. 1 Gastric emptying, body weight and depression-like behavioral tests in chronic stress mice.
a, Total distances of control and CS mice in the open field test (control, n = 6 mice; CS, n = 9 mice). b, Gastric emptying rates in control and CS mice in awake mice (n = 5 mice per group). c, Time course of the body weight in control and CS mice (control, n = 6 mice; CS, n = 9 mice). d, Performance of control and CS mice in the sucrose preference test (SPT), tail suspension test (TST), forced swim test (FST) (n = 10 mice per group). Significance was assessed by two-way repeated-measures ANOVA with post hoc comparisons between groups in (b and c), and two-tailed unpaired Student’s t-tests in (a and d). All data are presented as the mean ± s.e.m. * P < 0.05, ** P < 0.01. CS, chronic stress. SPT, sucrose preference test. TST, tail-suspension test. FST, forced-swim test.
Extended Data Fig. 2 DMV neurons project to the stomach.
a, Schematic of stomach injection with Fluoro-gold (FG). b, Representative images showing the co-localization of FG signals and TUBB3 antibody in the stomach. Scale bar, 100 µm. c,d, FG signals expressed in DMV neurons at different bregma sites. Scale bars, 100 µm.
Extended Data Fig. 3 Chronic stress inhibits AChDMV neuronal activity.
a, Schematic of recording gastric vagus nerve activities during optogenetic activation of AChDMV neurons. b,c, Representative images of ChR2-EYFP signals colocalized with ChAT antibody in the DMV of ChAT-Cre mice. Scale bars, 100 µm (b) and 20 µm (c). d, Representative images showing co-localization of the EYFP signals and TUBB3 antibody. Scale bar, 100 µm. e, Schematic of microendoscopic imaging in freely moving mice; the inset shows magnified example field of view. f, Sample traces (left) and data (right) showing neural activity of GCaMP6m-expressing DMV neurons (n = 25 cells per group, P < 0.0001). g, Expression of GCaMP6m and GRIN lens tract implanted in the DMV. Scale bar, 100 µm. h, Schematic of microendoscopic imaging in freely moving mice; the inset shows magnified example field of view. i, Expression of GCaMP6m and GRIN lens tract implanted in the DMV. Scale bar, 100 µm. j, Schematic of recording stomach-projecting DMV neurons by whole-cell recordings. k, Sample traces (left) and firing rates (right) recorded from GFP+ DRN neurons (control, n = 23 cells; CS, n = 29 cells). l, Rheobase values recorded from GFP+ DRN neurons detected by depolarized continuous current stimuli (control, n = 23 cells; CS, n = 20 cells). Significance was assessed by two-way repeated-measures ANOVA with post hoc comparisons between groups in (k), two-tailed paired Student’s t-test in (f), and two-tailed Mann-Whitney U tests in (l). All data are presented as the mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001.
Extended Data Fig. 4 Activation of AChDMV neurons reverses chronic stress-induced gastric dysfunction.
a, Schematic of procedure for chemogenetic activation of gastric-projecting DMV neurons. b, A typical image showing the injection site in the DMV. Scale bar, 100 µm. c, Representative images (left) and quantification analysis (right) showing gastric-projecting neurons (red) labeled by ChAT antibody (green) in the DMV (n = 6 brain slices). Scale bar, 20 µm. d, Whole-cell recordings showing the effect of CNO on AAV-DIO-mCherry or AAV-DIO-hM3Dq-mCherry-expressing DMV neurons (n = 3 mice per group). e, Acid secretion and pepsin enzyme activity of CS mice after chemogenetic activation of gastric-projecting DMV neurons (n = 5 mice per group, left, P = 0.0002; right, P = 0.0013). f, Heatmaps showing the locations of mCherry and hM3Dq-mCherry mice in the free feeding tests. g, Statistical analysis of time spent in the food zone and food intake in mCherry and hM3Dq-mCherry mice (n = 7 mice per group). Significance was assessed by two-way repeated-measures ANOVA with post hoc comparisons between groups in (d), and two-tailed unpaired Student’s t-test in (e and g). All data are presented as the mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001.
Extended Data Fig. 5 5-HT2AR expressed in AChDMV neurons.
a-d, Representative images showing Htr2a mRNA expression in AChDMV neurons at the indicated bregma sites. Scale bars, 100 μm.
Extended Data Fig. 6 The 5-HTDRN → AChDMV→stomach circuit controls gastric dysfunction induced by chronic stress.
a, Schematic of deep-brain calcium imaging in freely moving mice; the inset shows magnified example field of view. b, A representative image showing the expression of GCaMP6m and the GRIN lens tract in the DRN. Scale bar, 100 µm. c, Example traces (left) and data (right) showing neural activity of GCaMP6m-expressing DRN neurons (n = 25 cells per group, P < 0.0001). d, Schematic of procedure for optogenetic activation. e, Typical images (left, scale bar, 100 µm) showing the viral expression in the DRN of Pet1-Cre mice, representative images (middle, scale bar, 20 µm) and quantification analysis (right) showing the EYFP (green) colocalized with 5-HT antibody (red) in the DRN (n = 5 brain slices). f,g, Heatmaps (f) and IVIS quantitation (g) of gastric emptying rates in CS mice with optogenetic activation of 5-HTDRN terminals in the DMV (n = 5 mice per group). h, Heatmaps showing the locations of CS mice using the strategy indicated in (d) in the free feeding tests. i, Statistical analysis of time spent in the food zone and food intake in in mice as indicated in (h) (n = 5 mice per group, left, P = 0.0004; right, P = 0.0104). Significance was assessed by two-way repeated-measures ANOVA with post hoc comparisons between groups in (g), two-tailed paired Student’s t-test in (c), and two-tailed unpaired Student’s t-test in (i). All data are presented as the mean ± s.e.m. * P < 0.05, *** P < 0.001.
Extended Data Fig. 7 Acute stress increases 5-HTDRN neuronal activity.
a, Schematic of deep-brain calcium imaging in freely moving mice. b, Example traces (left) and data (right) showing neural activity of GCaMP6m-expressing DRN neurons (n = 32 cells per group, P < 0.0001). c,d, Representative images (c) and quantification analysis (d) showing the c-Fos signals in the DRN (n = 5 brain slices per group, P < 0.0001). Scale bar, 100 µm. e, Representative images (left) and quantification analysis (right) showing the c-Fos-labeled neurons in the DRN colocalized with 5-HT antibody (n = 7 brain slices). Scale bar, 20 µm. Significance was assessed by two-tailed paired Student’s t-tests in (b), and two-tailed unpaired Student’s t-tests in (d). All data are presented as the mean ± s.e.m. *** P < 0.001.
Extended Data Fig. 8 Acute stress increases AChDMV neuronal activity.
a, Schematic of microendoscopic imaging in freely moving mice. b, Example traces (left) and data (right) showing neuronal activity of GCaMP6m-expressing DMV neurons (n = 28 cells per group). c, Schematic of viral injection and the recording configuration in acute slices. d, Sample traces (left) for step current-evoked action potentials (10 pA/step) recorded from stomach-projecting DMV neurons and the summarized data (right) (Control, n = 18 cells; CS, n = 17 cells). e, Rheobase values of ramp current injection-evoked action potentials in stomach-projecting DMV neurons (Control, n = 21 cells; CS, n = 18 cells). f,g, Representative images (f) and quantification analysis (g) showing the c-Fos signals in the DMV (n = 6 brain slices, P = 0.0001). Scale bar, 100 µm. h, Representative images (left) and quantification analysis (right) showing the c-Fos-labeled neurons in the DMV colocalized with ChAT antibody (n = 6 brain slices). Scale bar, 20 µm. Significance was assessed by two-way repeated-measures ANOVA with post hoc comparisons between groups in (d), two-tailed paired Student’s t-tests in (b), two-tailed unpaired Student’s t-tests in (g), and two-tailed Mann-Whitney U tests in (e). All data are presented as the mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001.
Extended Data Fig. 9 The 5-HTDRN → AChDMV→stomach circuit controls gastric dysfunction induced by acute stress.
a, Schematic of procedure for chemogenetic inhibition. b, Representative images showing the injection site and viral expression in the DRN (left), and cannula tract in the DMV (right). Scale bars, 100 µm. c, Whole-cell recordings showing the effect of CNO on AAV-DIO-mCherry or AAV-DIO-hM4Di-mCherry-expressing DRN neurons (n = 3 mice per group). d,e, Sample traces (d) and summarized data (e) of the gastric motility in AS mice with chemogenetic inhibition of 5-HTDRN terminals in the DMV (mCherry, n = 6 mice; hM4Di-mCherry, n = 7 mice, P < 0.0001). f, IVIS quantitation of gastric emptying rates in AS mice with chemogenetic inhibition of 5-HTDRN terminals in the DMV (mCherry, n = 5 mice; hM4Di-mCherry, n = 6 mice). g, Acid secretion and pepsin enzyme activity in AS mice with chemogenetic inhibition of 5-HTDRN terminals in the DMV (n = 6 mice per group, left, P = 0.0001, right, P < 0.0001). Significance was assessed by two-way repeated-measures ANOVA with post hoc comparisons between groups in (c and f), and two-tailed unpaired Student’s t-tests in (e and g). All data are presented as the mean ± s.e.m. ** P < 0.01, *** P < 0.001.
Extended Data Fig. 10 Inhibition of gastric hyperfunction alleviates increased food intake following acute stress.
a, Schematic of chemogenetic inhibition design. b, Food intake over 6 hours in AS mice infused with AAV-DIO-mCherry or AAV-DIO-hM4Di-mCherry (mCherry, n = 5 mice; hM4Di-mCherry, n = 6 mice). c, Heatmaps showing the locations of mice in free feeding tests. d, Quantitative analysis of time spent in the food zone and food intake as indicated in (c) (mCherry, n = 6 mice; hM4Di-mCherry, n = 5 mice). e, Schematic of intraperitoneal injection. f, Food intake over 6 hours in AS mice infused with saline or pinaverium bromide (Saline, n = 6 mice; Pinaverium, n = 10 mice). g, Heatmaps showing the locations of mice in free feeding tests. h, Quantitative analysis of time spent in the food zone and food intake as indicated in (g) (Saline, n = 6 mice; Pinaverium, n = 10 mice, left, P < 0.0001; right, P < 0.0001). Significance was assessed by two-way repeated-measures ANOVA with post hoc comparisons between groups in (b and f), and two-tailed unpaired Student’s t-tests in (d and h). All data are presented as the mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001.
Supplementary information
Supplementary Information
Supplementary Figs. 1–6 and statistical analyses related to the figures 1–6, extended data figures 1–10 and supplementary figures 1–6.
Supplementary Video 1
Gastric motility recording in freely moving mice.
Supplementary Video 2
Microendoscopic calcium imaging of AChDMV neurons in freely moving mice.
Supplementary Video 3
Reconstruction imaging of RV-DsRed expression in the DRN. Scale bar, 100 µm.
Supplementary Video 4
Microendoscopic calcium imaging of 5-HTDRN neurons in freely moving mice.
Supplementary Data 1
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Dong, WY., Zhu, X., Tang, HD. et al. Brain regulation of gastric dysfunction induced by stress. Nat Metab 5, 1494–1505 (2023). https://doi.org/10.1038/s42255-023-00866-z
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DOI: https://doi.org/10.1038/s42255-023-00866-z
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