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. 2026 May 26;109:102384. doi: 10.1016/j.molmet.2026.102384

Vagal control of the brain-esophagus axis ameliorates stress-induced esophageal motility dysfunction in male mice

Kun Zhang 1,2,4,7,8,#, Dake Song 2,7,8,#, Yingying Zhang 2,3,7,8, Shanbo Ma 4,5, Hengxin Gong 2,7,8, Rui Zhang 6, Min Wang 2,7,8, Xubo Li 2,7,8, Xinshang Wang 1,2,7,8, Yumei Wu 2,7,8, Minggao Zhao 2,3,7,8,⁎, Shui-bing Liu 1,2,7,8,∗∗
PMCID: PMC13265709  PMID: 42203107

Abstract

Mental stress serves as a major contributor to a spectrum of esophageal motility disorders, including distal esophageal spasm (DES), a condition marked by premature contractile activity of the lower esophageal sphincter (LES). These spasms are often exacerbated by psychiatric conditions such as distress, anxiety, and depression. This study investigates the impact of mental stress on LES contractile function in mice. Chronic unpredictable mild stress (CUMS) was observed to reduce food intake, prolong esophageal emptying time, and increase LESP, along with elevated levels of inflammatory factors, IL-1β and TNFα in mice. Retrograde viral tracing identified a neuronal projection from the hypothalamus paraventricular nucleus (PVN) to the LES via the dorsal motor nucleus of the vagus (DMV) and the vagus nerve. Optogenetic activation of the PVN-DMV-vagus pathway increased the frequency and amplitude of LES electromyogram signals in mice, an effect negated by vagotomy or acetylcholine receptor antagonists. Conversely, chemogenetic inhibition of the PVN-DMV-vagus pathway alleviated CUMS-induced LES contraction abnormalities. High-throughput RNA sequencing revealed an upregulation of myosin binding protein C2 (MYBPC2) in the gastroesophageal junction (GEJ) following sustained activation of the PVN-DMV-vagus pathway. Similarly, cultured GEJ cells exposed to acetylcholine exhibited increased MYBPC2 levels. Notably, downregulation of MYBPC2 expression in the GEJ restored normal function in mice with PVN-DMV-vagus-acetylcholine pathway activation. In summary, this study demonstrates that mental stress induces LES contraction disorders through a PVN-DMV-vagus dependent pathway, which drives an increase in MYBPC2 expression in the GEJ.

Keywords: Diffuse esophageal spasm, Chronic unpredictable mild stress, Paraventricular nucleus of hypothalamus, Vagus, Myosin binding protein C2

Highlights

  • •

    We verified PVN-originated neuronal projections to the LES routed via the DMV–vagus axis.

  • •

    Inhibition of the PVN-DMV-vagus pathway alleviated mental stress induced LES contraction abnormalities.

  • •

    Downregulation of MYBPC2 in LES rescues LES function upon PVN-DMV-vagus cholinergic pathway activation.

1. Introduction

Normal esophageal motility, particularly physiological function of the gastroesophageal junction (GEJ)—which consists of three anatomical components: the distal esophagus, the lower esophageal sphincter (LES, a specialized circular smooth muscle), and the gastric cardia—is essential for the transportation of food from the mouth to the stomach. Psychological and psychiatric disturbances frequently contribute to functional esophageal motility disorders, such as distal esophageal spasm (DES), which is clinically manifested as dysphagia and chest pain [1,2]. Therefore, elucidating the association between psychological stress and GEJ function, as well as developing more efficacious, safer and economical therapeutic strategies, has become a core research focus in contemporary biomedical studies.

Psychiatric conditions, including anxiety, depression, and chronic distress, are associated with distinct esophageal contraction abnormalities [3,4]. Long-term psychological stress exacerbates symptoms in DES patients, highlighting the importance of stress-responsive physiological systems [5], such as the autonomic nervous system (ANS) and the hypothalamic-pituitary-adrenal (HPA) axis, in the disorder's development. The ANS, which includes the parasympathetic (e.g. vagus), sympathetic, and enteric divisions, plays a crucial role in regulating esophageal peristalsis and sphincter function. Specifically, acetylcholine (ACh)-mediated vagal pathways are critical for LES contraction [6]. An imbalance between excitation and inhibition of cholinergic neurons may contribute to esophageal motility disorders. However, the precise underlying neuronal circuits and molecular mechanisms remain to be further elucidated.

In this study, we found chronic unpredictable mild stress (CUMS) induced esophageal motility disorder. Retrograde viral tracing revealed a neuronal projection from the hypothalamus paraventricular nucleus (PVN) to the LES via the dorsal motor nucleus of the vagus (DMV) and the vagus nerve. Optogenetic activation of the PVN-DMV-vagus pathway enhanced the frequency and amplitude of LES electromyograms, whereas chemogenetic inhibition of this pathway alleviated CUMS-induced LES dysfunction without improving depression-like behaviors. Sustained activation of the PVN-DMV-vagus pathway elevated MYBPC2 levels in the GEJ through a ACh-dependent mechanism. Notably, downregulation of MYBPC2 expression in the GEJ restored normal function in mice with PVN-DMV-vagus activation. These results provide novel insights into the neural pathways that regulate esophageal dysfunction under stress, offering potential targets for therapeutic intervention.

2. Results

2.1. CUMS decreases food intake in mice

As reported previously, CUMS could add the risk factor for esophagus motility disorders through peripheral and central aspects [7], thus we used this model to explore the relationship between stress and esophageal motility disorder. Stressors included cold water swimming, light/dark inversion, tail pinching, wet bedding, and cage tilting. Behavioral assays were performed to evaluate stress-related emotional behaviors, including the open field test (OFT), elevated plus maze (EPM), forced swimming test (FST), tail suspension test (TST), and novelty-suppressed feeding test (NSFT) (Figure 1A). After 4-week stress exposure, CUMS mice exhibited a significant reduction in center time during the OFT, while total distance traveled remained unchanged (Figure 1B). In the EPM, stressed mice showed fewer open arm entries, although total entries were comparable to control (Con) group (Figure 1C). Furthermore, CUMS increased immobility time in both the FST and TST (Figure 1D,E). The NSFT revealed prolonged latency to food in CUMS mice (Figure 1F). Additionally, CUMS led to reductions in body weight, daily food consumption, and time spent in the food zone during free-feeding tests compared to Con mice (Figure 1G–J). No significant difference in food intake was found between the Con and CUMS groups after fasting (Figure 1K). Furthermore, CUMS markedly elevated heart rate (Figure 1l), arterial blood pressure (Figure 1M), serum corticosterone (CORT) levels (Figure 1N), and corticotropin-releasing hormone (CRH) expression in the paraventricular nucleus (PVN) (Figure 1O). These findings demonstrate that CUMS-induced emotional disturbance coupled with feeding dysfunction reduces food intake under both baseline daily feeding and ad libitum feeding conditions, with no significant effect on refeeding behavior after fasting.

Figure 1.

Figure 1

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Chronic unpredictable mild stress (CUMS) induces food intake dysfunction. a, Schematic of procedure for establishing a CUMS model in mice. b, Sample traces and summarized data in the OFT. n = 5. c, Sample traces and summarized data in EPM. d, Immobility time in the FST. e, Immobility time in the tail suspension test. f, Latency to food in the NSFT. g, Body weight gain in 4-week CUMS. h, Food intake during 28 days of CUMS. i, Meal number and meal duration within a 1-hour observational period. j, Traces and statistical analysis of the time spent in the food zone. k, The food consumption in 1 h after fasting overnight. l-o, Changes in heart rate (l), SAP, DBP and MAP (m), serum CORT levels (n), and PVN CRH (o) after 28-day CUMS. Heart rate, SAP, DBP and MAP were measured by the tail-cuff method, with all measurements conducted from 9:00 A.M. to 10:00 A.M. Significance was assessed using a two-way repeated-measures ANOVA with post hoc comparisons between groups (h), a two-tailed unpaired Student's t-test (b-g and i-o). All data are presented as the mean ± s.e.m. n = 5. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. OFT, open field test; EPM, elevated plus maze; FST, forced swimming test; TST, tail suspension test; NSFT, novelty suppressed feeding test; SAP, systolic blood pressure; DAP, diastolic blood pressure; MAP, mean arterial blood pressure; CORT, corticosterone; CRH, corticotropin-releasing hormone.

2.2. CUMS induces esophageal motility disorder and epithelial hyperkeratotic remodeling

The esophagus is a tubular organ connecting the mouth to the stomach, and its physiological function is highly susceptible to mental stress [[8], [9], [10]]. However, the precise effects of CUMS on esophageal motility disorder remain unclear. To assess the impact of CUMS on esophageal motility, esophageal emptying time and LESP were measured. Esophageal emptying time reflects swallowing function, while LESP evaluates the sphincter's ability to resist food during swallowing [11]. Elevated LESP is a hallmark of esophageal motility disorders, such as distal esophageal spasm [12]. Our results revealed that after 4-week CUMS, esophageal transit time was significantly prolonged, although esophageal width remained unaffected (Figure 2A). CUMS also increased LESP (Figure 2B), indicating impaired esophageal motility. Occasionally, exploratory analyses revealed that the levels of the inflammatory markers interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were significantly elevated in the gastroesophageal junction (GEJ) of CUMS mice compared with control mice (Supplementary Fig. 1a and b), indicating the initiation of local inflammation induced by chronic stress. Histological analysis of esophageal tissue using hematoxylin-eosin (HE) staining showed increased thickness of the esophageal epithelial and keratinous layers in CUMS mice (Figure 2C). These findings collectively indicate that CUMS induces esophageal motility dysfunction and epithelial hyperkeratotic remodeling.

Figure 2.

Figure 2

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CUMS induces esophageal dysmotility and squamous epithelial hyperplasia with hyperkeratosis. a, CUMS prolonged esophageal transit time in the esophageal emptying test, with no significant change in esophageal width. b, Lower esophageal sphincter pressure (LESP) was increased after 4-week CUMS. c, Hematoxylin and Eosin staining (HE) staining showing the thicknesses of the epithelial cell layer and keratin layer in the distal esophagus following CUMS exposure. The blue line demarcates the epithelial cell layer, and the yellow line marks the keratin layer. All data are presented as the mean ± s.e.m. n = 5. Significance was assessed using a Student's t-test. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

2.3. PVN-DMV-vagus projection dominates LES contraction through a cholinergic pathway

In the end of esophagus, the LES plays a vital role in coordinating the passage of food into the stomach, releasing gas after meals, and preventing the reflux of harmful stomach contents into the esophagus [13]. To explore potential connections between the LES and the brain, retrograde trans-synaptic virus tracing was performed by injecting PRV-EGFP (1 μL, 8 × 109 PFU/mL) into the LES. EGFP signals were subsequently detected in several brain regions, including the hypothalamus PVN, central amygdala (CeA), nucleus tractus solitarii (NTS), lateral parabrachial nucleus (LPB), medial parabrachial nucleus (MPB), parasubthalamic nucleus (PSTN), and DMV (Figure 3A). Previous studies have identified the PVN as a critical hub in the interaction between mental stress and the brain-gut axis [14,15]. Furthermore, the PVN has been shown to project to DMV acetylcholinergic neurons, regulating gastric motility [16]. To confirm the role of PVN-DMV pathway in controlling LES function, a series of specific viral injections were performed. Retrograde virus expressing Cre recombinase (Retro-Cre) was injected into the LES, while antrograde virus expressing Flp recombinase (Antro-Flp) was injected into the PVN. Additionally, Cre/Flp dual-dependent virus encoding channelrhodopsin-2 (CreOn/FlpOn-ChR2) was delivered into the DMV to specifically activate the PVN-DMV-vagus pathway (Figure 3B, D). Electrophysiological recordings revealed that activation of the PVN-DMV-vagus pathway significantly increased electromyogram (EMG) activity in the LES (Figure 3C). Importantly, denervation of the esophagus completely abolished the PVN-DMV-vagus activation-induced increase in EMG activity, confirming direct neural regulation rather than involvement of the HPA axis (F3,16 = 30.68, P < 0.0001 for frequency; F3,16 = 13.77, P = 0.0001 for amplitude; Figure 3D). The LES receives innervation from both sympathetic and vagus nerves, which release norepinephrine and ACh, respectively [17,18]. To determine the neurotransmitter responsible for the observed effects, various antagonists were tested. Only anisodamine, a non-selective antagonist of ACh receptors, successfully blocked the PVN-DMV-vagus-induced increase in EMG activity, indicating a ACh-dependent mechanism (F5, 24 = 33.8, P < 0.0001 for frequency; F5,24 = 25.05, P < 0.0001 for amplitude; Figure 3E). These results collectively demonstrate that the PVN-DMV-vagus pathway regulates LES contraction via a cholinergic mechanism.

Figure 3.

Figure 3

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The paraventricular nucleus (PVN)-dorsal motor nucleus of the vagus (DMV)-vagal nerve projection governs LES contraction via a cholinergic pathway. a, Schematic of pseudorabies virus-enhanced green fluorescent protein (PRV-EGFP) injection and representative images showing viral EGFP expression in selected brain regions. b, Schematic of EMG recording in LES during optogenetic activation of the PVN-DMV-vagus pathway. c, Optogenetic activation of the PVN-DMV-vagus pathway increased the frequency and amplitude of LES EMG. d, Esophageal vagotomy abolished PVN-DMV-vagus pathway activation induced LES contraction. e, EMG frequency and amplitude of yohimbine, propronolol, prazosin, or anisodamine intraperitoneally injected mice during optogenetic activation of the PVN-DMV-vagus pathway. Significance was assessed using a two-tailed unpaired Student's t-test (c), a two-way ANOVA with post hoc comparisons between groups (d), and a one-way ANOVA with post hoc comparisons (e). n = 5. All data are presented as the mean ± s.e.m. n = 5. ∗∗P < 0.01. LES, lower esophageal sphincter; PVN, paraventricular nucleus of hypothalamus; CeA, central amygdala; BLA, basal lateral amygdala; LA, lateral amygdala; NTS, nucleus of solitary tract; LPBS, lateral parabrachial nucleus; MPB, medial parabrachial nucleus; PSTN, parasubthalamic nucleus; DMV, dorsal nucleus of vagus nerve; VX, vogatomy; EMG, electromyography; Yohimbine, α-adrenergic receptor blocker; Propronolol, β adrenergic receptor blocker; Prazosin, α-adrenergic receptor blocker; Anisodamine, a non-selective antagonist of ACh receptors.

2.4. CUMS induces esophageal motility disorder through a vagus-dependent pathway

To determine whether CUMS induces esophageal motility dysfunction via a vagus-dependent pathway, the effects of vagotomy were evaluated. The increased esophageal transit time (F3,16 = 32.42, P < 0.0001) and LESP (F3,16 = 41.06, P < 0.0001) observed in CUMS mice were significantly reversed by vagotomy (Figure 4A, B). Additionally, CUMS-induced thickening of the distal esophageal epithelial layer (F3,16 = 39.74, P < 0.0001) and keratinous layers (F3,16 = 12.33, P < 0.0001) was abolished following vagotomy (Figure 4C). The vagus nerve releases ACh to regulate the contraction of esophageal smooth muscle [19]. In this study, anisodamine treatment did not further decrease esophageal transit time (F4,20 = 14.42, P < 0.0001) or LESP (F4,20 = 29.9, P < 0.0001) in vagotomized CUMS mice (Figure 4D, E), suggesting that vagotomy and anisodamine share a similar anticholinergic mechanism. These findings indicate that CUMS induces esophageal motility dysfunction through a vagus-dependent, ACh-mediated pathway.

Figure 4.

Figure 4

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Acetylcholinergic vagus inhibition ameliorates CUMS induced esophageal motility disorder. a, Schematic of esophageal function tests in mice subjected to CUMS following esophageal vagotomy. b, Esophageal transmit time and LESP in vagotomized CUMS mice. c, HE staining of the distal esophageal sections. d, Schematic of anisodamine intraperitoneally injection daily during CUMS. e, Effect of anisodamine on esophageal transmit time and LESP in vagotomized CUMS mice. EFT, esophageal function test (esophageal transit time test and LESP). Significance was assessed using a two-way ANOVA with post hoc comparisons between groups (b and c), and a one-way ANOVA with post hoc comparisons (e). All data are presented as the mean ± s.e.m. n = 5. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Inhibition of the PVN-DMV-vagus pathway ameliorates CUMS-induced esophageal motility dysfunction.

The PVN directly projects to autonomic regions and plays a central role in integrating autonomic and digestive system responses to stress [20,21]. To investigate whether the PVN mediates both behavioral and esophageal dysfunction in CUMS mice, we pharmacogenetically inhibited PVN neuronal activity via AAV-mediated chemogenetic manipulation (Figure 5A). Following CUMS, clozapine-N-oxide (CNO, 0.3 mg/kg) was administered intraperitoneally daily to suppress hM4Di-EGFP-labeled PVN neurons (Fig. 5B). Increased c-Fos+ cells in the PVN observed after CUMS were significantly reduced by chemogenetic inhibition of the PVN (F3,16 = 93.27, P < 0.0001, Figure 5C). PVN inhibition also reversed CUMS-induced behavioral changes, including decreased center distance in the OFT (F3,16 = 6.467, P = 0.0045, Figure 5D), reduced open arm entries in the EPM (F3,16 = 12.83, P = 0.0002, Figure 5E), and increased immobility time in the TST (F3,16 = 7.727, P = 0.0021) and FST (F3,16 = 29.51, P < 0.0001) (Figure 5F, G). These findings indicate that anxiety- and depression-like behaviors were alleviated by PVN inhibition. Furthermore, LESP (F3,16 = 31.25, P < 0.0001) and esophageal transit time (F3,16 = 11.3, P < 0.0003) were also significantly reduced following PVN inhibition in CUMS mice (Figure 5H). Additionally, food intake (F3,16 = 32.88, P < 0.0001) and meal duration (F3,16 = 23.5, P < 0.0001) were notably restored in CUMS mice upon PVN inhibition (Figure 5I, J). Collectively, these findings demonstrate that neuronal inhibition of the PVN alleviates CUMS-triggered emotional disturbances and esophageal motility dysfunction.

Figure 5.

Figure 5

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PVN-DMV-vagus pathway inhibition relieves esophageal motility disorder without affecting depressive behaviors by CUMS. a, Schematic for chemogenetic inhibition of the PVN by injecting virus expressing hM4Di. b, representative image of hM4Di-EYFP signals in PVN. c. Representative images and quantification analysis showing the c-Fos signals in the PVN after CUMS and PVN inhibition. d-e, Summarized data of CUMS mice after PVN inhibition in the open field test (d), the elevated plus maze test (e). f-g, Summarized data of CUMS mice after PVN inhibition the tail suspension test (f) and the forced swimming test (g). h, The LESP and esophageal transit time of CUMS mice after PVN inhibition. i, Food intake in the last day of CUMS. j, Number and duration of meals in free feeding test after PVN inhibition in CUMS mice. k, Schematic of procedure for chemogenetic inhibition of PVN-DMV-vagus pathway during CUMS. l, Representative images showing the virus expression in the PVN and DMV but not in ambiguus nucleus (AN). m-q, Summarized data of PVN-DMV-vagus pathway inhibiting mice during CUMS in the open field test (m), the elevated plus maze test (n), the tail suspension test (o) and the forced swimming test (p), the LESP and esophageal transit time (q). r, Food intake in the last day of CUMS in PVN-DMV-vagus pathway inhibition mice. s, Number and duration of meals in free feeding test after PVN-DMV-vagus pathway inhibition in CUMS mice. Significance was assessed using a two-way ANOVA with post hoc comparisons between groups. All data are presented as the mean ± s.e.m. n = 5. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Notably, when the PVN-DMV-vagus pathway was specifically suppressed in CUMS mice (Figure 5K, L), only esophageal motility dysfunction (F3,16 = 31.25, P < 0.0001 for LESP; F3,16 = 6.553, P = 0.0042 for esophageal transit time) was rescued, while anxiety- and depression-like behaviors remained unaffected (F3,16 = 12.19, P = 0.0002 for open field test; F3,16 = 20.01, P < 0.0001 for elevated plus maze; F3,16 = 20.58, P < 0.0001 for tail suspension test; F3,16 = 14.52, P < 0.0001 for forced swimming test; Figure 5M-Q). Meanwhile, food intake (F3,16 = 16, P < 0.0001), meal number (F3,16 = 7.605, P = 0.0022), and meal duration (F3,16 = 26.49, P < 0.0001) in CUMS mice was also rescued by inhibiting PVN-DMV-vagus pathway (Figure 5R, S). These findings highlight the PVN as a critical intersection between mental stress and esophageal motility dysfunction. Inhibiting PVN-DMV-vagus pathway specifically alleviated esophageal motility dysfunction without influencing depression-like behaviors in CUMS mice.

2.5. Sustained PVN-DMV-vagus pathway activation increased MYBPC2 level in GEJ

To explore the mechanisms underlying esophageal motility dysfunction induced by PVN-DMV-vagus pathway activation, we chemogenetically activated this pathway via the hM3Dq tool for one week. Then, the GEJ was dissected and analyzed via high-throughput bulk mRNA sequencing (Figure 6A). Several genes, including neutrophilic granule protein (Ngp), E3 ubiquitin-protein ligase TRIM50 (TRIM50), actin-binding Rho-activating protein (Abra), cell division cycle protein 20 homolog (CDC20), G2/mitotic-specific cyclin-B2 (CCNB2), and MYBPC2, were differentially expressed between control (Con) and CUMS mice. To identify key genes, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis on the differentially expressed genes was performed. Functional groups related to synaptic activity, such as actin binding, ubiquitin ligase activator activity, telethonin binding, and MRF binding, were significantly enriched (Figure6 6B). Among these, MYBPC2, a protein known to bind myosin heavy chains, F-actin, and native thin filaments—modulates actin-activated myosin ATPase activity [22], potentially influencing muscle contraction or serving a structural role. To validate MYBPC2 expression in PVN-DMV-vagus pathway-activated mice, Western blot analysis was used to examine the expression of MYBPC2, CCNB2, and CDC20 in mCherry, hM3Dq + saline, and hM3Dq + CNO groups. Consistent with the RNA sequencing results, MYBPC2 expression was increased (F2,12 = 38.83, P < 0.0001), while CDC20 (F2,12 = 26.15, P < 0.0001) and CCNB2 (F2,12 = 14.95, P = 0.0006) expression were decreased in CUMS mice (Figure 6C). Immunofluorescence staining further confirmed higher MYBPC2 levels in the GEJ after PVN-DMV-vagus pathway activation (Figure 6D). These findings reveal that MYBPC2, a myosin-binding protein, is upregulated in PVN-DMV-vagus pathway-activated mice and may play a role in GEJ dysfunction.

Figure 6.

Figure 6

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Sustained activation of PVN-DMV-vagus pathway increases MYBPC2 level in the gastroesophageal junction (GEJ). a, Schematic of procedure for high-throughput RNA sequencing of esophagus tissue and GEJ function after sustained PVN-DMV-vagus pathway activation. b, Heatmap, volcano map, and KEGG analysis of differently expressed gene in distal esophagus between CUMS mice and Con mice. c, Representative images and quantification analysis showing the CDC20, CCNB2, and MYBPC2 levels in GEJ after sustained PVN-DMV-vagus pathway. d, Representative images and quantification analysis of MYBPC2 levels in GEJ after PVN-DMV-vagus pathway activation by immunohistochemistry staining. TRIM50, E3 ubiquitin-protein ligase TRIM50; Abra, actin-binding Rho-activating protein; CDC20, cell division cycle protein 20 homolog; CCNB2, G2/mitotic-specific cyclin-B2; MYBPC2, myosin binding protein C2; Nnat, neuronatin. Significance was assessed using a two-tailed unpaired Student's t-test (a and d), and a one-way ANOVA with post hoc comparisons between groups (c). All data are presented as the mean ± s.e.m. n = 5. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Down regulating MYBPC2 expression relieved PVN-DMV-vagus pathway activation induced esophageal motility dysfunction.

Our data strongly suggest that MYBPC2 plays a critical role in PVN-DMV-vagus pathway activation-induced esophageal motility dysfunction. Since the vagus nerve releases ACh as a neurotransmitter to regulate esophageal motility [23,24], the relationship between ACh and increased MYBPC2 levels was investigated. Cultured GEJ cells were incubated with exogenous ACh for 24 h, resulting in a significant increase in MYBPC2 expression (Figure 7A). This increase was reversed by anisodamine, as confirmed by Western blot analysis (F2,12 = 38.83, P < 0.0001; Figure 7B). Furthermore, downregulating MYBPC2 expression by shRNA in GEJ (F3,16 = 73.3, P < 0.0001) successfully alleviated the increased LESP (F3,16 = 33.22, P < 0.0001) and prolonged esophageal transit time (F3,16 = 8.987, P = 0.001) observed in PVN-DMV-vagus pathway-activated mice (Figure 7C–E). These findings identify MYBPC2 as a critical molecular mediator in PVN-DMV-vagus pathway activation-induced esophageal motility disorder.

Figure 7.

Figure 7

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Downregulating MYBPC2 expression eases PVN-DMV-vagus activation induced esophageal motility dysfunction. a, Representative images and quantification analysis of MYBPC2 level in primary cultures of GEJ cells after ACh treatment by immunohistochemistry staining. b, ACh increased the MYBPC2 level in primary cultures of GEJ cells by Western blot. c, Schematic of local injecting virus expressing Mybpc2 shRNA in distal esophagus and PVN-DMV-vagus pathway activation by chemogenetic tools. d, Mybpc2 shRNA injection decreased PVN-DMV-vagus pathway activation induced MYBPC2 elevation in GEJ. e, Mybpc2 shRNA injection in GEJ rescued the increased LESP and esophageal transit time by sustained PVN-DMV-vagus pathway activation. Significance was assessed using a two-tailed unpaired Student's t-test (a) and a one-way ANOVA with post hoc comparisons between groups (b) a two-way ANOVA with post hoc comparisons between groups (c). All data are presented as the mean ± s.e.m. n = 5. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

3. Discussion

Mental stress and functional esophageal disorders are prevalent medical conditions with significant morbidity and economic burdens, often co-occurring [25,26]. Chronic stress has been shown to exacerbate functional esophageal disorders, disrupting peristalsis and worsening esophageal dysfunction [27,28]. While antidepressant medications may alleviate both depression and esophageal dysfunction [29,30], their connections remain poorly understood, complicating effective treatment strategies. Stress induces a complex physiological response involving the HPA axis, the nervous system, and various neurotransmitters that modulate vagal nerve projections to the digestive system [[31], [32], [33]]. Accumulating studies have focused on the regulatory role of the PVN-DMV pathway in gastric and intestinal functions [34,35]. Herein, using multiple neuronal tracers and chemogenetic approaches, we directly identified a PVN-DMV-esophagus axis that mediates the interplay between emotional activity and esophageal function, which broadens our understanding of brain–esophagus communication.

Our study demonstrates that CUMS activates the PVN, which projects to the DMV and enhances vagal nerve activity. The augmented vagal input promotes LES contraction, thereby contributing to esophageal motility dysfunction under stress-induced depressive states. Global inhibition of PVN activity ameliorates both behavioral abnormalities and esophageal irregularities in CUMS mice. These results identify the PVN as a critical crosstalk hub linking psychological stress and esophageal dysfunction. Notably, selective suppression of the PVN-DMV-vagus pathway effectively relieves esophageal motility dysfunction but exerts no obvious influence on anxiety- and depression-like behaviors. Collectively, these findings may provide a theoretical basis for developing targeted therapies to simultaneously alleviate stress-related emotional deficits and esophageal dysfunction.

In this study, the FST and TST were primarily employed to assess depression-like phenotypes in mice. While parallel Con mice were included in these tests, it is important to acknowledge that both the FST and TST inherently introduce emotional stress. This aspect represents a notable limitation of our research, as the imposed stress may confound the interpretation of results related to depression-like behaviors. Notably, our investigation focused on neuronal ACh signaling in esophageal dysfunction, as well as the therapeutic potential of vagus nerve inhibition for esophageal motility disorder in a CUMS model with comorbid anxiety- and depression-like behaviors. This model closely recapitulates the clinical co-occurrence of emotional disturbances and functional esophageal diseases in humans, thereby strengthening the translational relevance of our findings. While the anticholinergic drug anisodamine is widely used clinically to treat esophageal motility disorders, our study offers novel insights into its underlying mechanisms of action.

In humans, the distal 50–60% of the esophagus consists of smooth muscle, which is innervated by two major populations of effector neurons: excitatory cholinergic neurons and nonadrenergic noncholinergic inhibitory neurons [36]. ACh signaling governs swallow-induced peristalsis in the smooth muscle esophagus, with central and peripheral control mechanisms coordinating the activity of excitatory acetylcholinergic neurons [37]. Typically, the central mechanism predominates, initiating and regulating peristalsis in the smooth muscle esophagus [38]. In contrast, contraction of striated muscle is driven by sequential excitation via vagal acetylcholinergic fibers under central control [16,39]. Anatomical studies have established direct projections from the PVN to the DMV, while physiological studies have shown that electrical stimulation of the PVN activates a specific subset of DMV neurons [40,41]. The PVN can influence up to 80% of DMV neurons that innervate the gastrointestinal tract, with excitatory effects predominating over inhibitory ones [42,43]. PVN neurons that project to the DMV synthesize and release a variety of neurotransmitters, including glutamate, bombesin, oxytocin (OXT), apelin, vasopressin, CRH, somatostatin, Leu-enkephalin, and Met-enkephalin [44]. Different neuronal subtypes within the PVN exert distinct regulatory control over the DMV-gut axis. Under chronic stress conditions, apelin and CRH mediates stress-induced impairments in gastric and colonic motility [16,45], whereas OXT exerts the protective effects [16,46,47]. Rodent studies have revealed that stress adaptability is linked to increased levels of OXT and catecholamines in specific brain regions, which play crucial roles in restoring gastrointestinal function [48,49]. As CRH acts as a classic stress mediator in the PVN, we speculated that it may also participate in CUMS-induced esophageal motility dysfunction. We detected significant differences in fluorescence intensity between the rostral and caudal subregions of the DMV following PRV injection into the LES (Fig. 3A), implying that the heterogeneous distribution of vagal neurons within the DMV contributes to the regulation of LES contractility and warrants further in-depth investigation [50]. Moreover, as shown in Figure 5K–S, we specifically inhibited DMV neurons that receive input from the PVN and project to the LES; nevertheless, these neurons may still exert regulatory effects on other brain regions, which represents a limitation of the present study. Further investigations are therefore required to characterize the specific neuronal subtypes and neurotransmitters within the PVN-DMV circuit that mediate this regulatory process.

We used optogenetic approaches to activate the PVN-DMV-vagus pathway and observed rapid changes in EMG activity, indicating direct neuronal regulation of GEJ function. To mimic sustained activation of this pathway under CUMS conditions, we employed chemogenetic manipulation, which effectively recapitulated CUMS-induced functional changes in the GEJ over a prolonged time course. Furthermore, high-throughput RNA sequencing identified multiple differentially expressed genes in the GEJ, including Mybpc2, Cdc20, Ccnb2, Trim50, and Abra. Among them, MYBPC2 is enriched in esophageal striated and fast-twitch skeletal muscle, maintaining sarcomeric structure, regulating contractility and Ca2+ sensitivity, suppressing hypercontraction, and modulating GEJ esophageal peristalsis [51]. Consistently, ACh treatment increased MYBPC2 levels in primary cultured GEJ cells. Furthermore, shRNA-mediated knockdown of MYBPC2 effectively rescued esophageal motility dysfunction in CUMS mice. In mice, the striated muscle of the esophagus intermingles with the smooth muscle of the LES within the GEJ [52], rendering it technically infeasible to isolate the LES purely as an independent tissue. Accordingly, we collected the entire GEJ for bulk mRNA sequencing. Given that MYBPC2 is recognized as a striated muscle-specific marker and is not expressed in smooth muscle (e.g., Human Protein Atlas data), the cellular origin of upregulated MYBPC2—whether derived from the distal esophagus or the LES—remains to be further clarified. We therefore speculate that MYBPC2 acts as a key effector mediating CUMS-induced esophageal motility dysfunction. By contrast, the roles of other differentially expressed proteins, including CDC20, CCNB2, TRIM50, and ABRA, warrant further investigation.

In fact, the causal relationship between emotional states and eating behaviors is a well-recognized phenomenon in daily life. Nevertheless, the underlying neuronal circuits involved in this relationship warrant further in-depth exploration. The successful conveyance of food and other ingestibles from the mouth to the stomach hinges upon the orderly and exquisitely coordinated propulsion of food. Our study introduces a novel brain-esophagus pathway implicated in the effects of mental stress on swallowing function, offering a foundation for further investigations into the intricate relationships between the brain and visceral organs.

4. Methods

4.1. Animals

All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University. C57BL/6J mice were purchased from Charles River (Beijing) Biotechnology. Unless otherwise noted, mice were group-housed at 18–23 °C with light on at 7 a.m.-7 p.m. and were provided with ad libitum water and chow unless otherwise specified. Male mice (8-13-week old) were randomly selected for all experiments. All experimental data were collected by individuals blind to experimental conditions.

4.2. CUMS procedure

The CUMS procedure was performed as previously described [53] with a slight modification. Mice in the CUMS were randomly exposed to different stressors: cage tilting for 24 h, cold water swimming for 3 min (at 0 °C), water or food deprivation for 24 h, level shaking for 15 min, tail nip for 1 min (1 cm from the end of the tail), 45 °C heat stress for 5 min and inversion of the light/dark cycle for 24 h. These stressors were applied for 28 days, during which each stressor was applied 4 times. The mice were exposed to different stressors at random every day, making it impossible for the animals to predict the stimulus. The same stressor was not applied on consecutive days. The Con group were undisturbed except for necessary procedures.

4.3. Behavioural tests

All behavioural tests were carried out in a dimly lit testing room (approximately 20 lux); mice were habituated for at least half a day before the tests. All behaviors were videotaped using a video tracking system (DigBehv software, Jiliang, Shanghai, China) and measured offline. OFT, EPM, TST, and FST were carried out as reported previously [54].

4.4. Food intake measurement

Before measurement, mice were adapted to the cage (25 cm long, 15 cm wide, 10 cm high) alone for five days. For long-term measurements of total food intake and body weight during CUMS, food and body weights were measured at 09:00. For detecting food consumption in mice after CUMS, each animal was single-housed and fasted for 16 h (overnight) from 5:00 pm to the next day 9:00 am. Subsequently, each animal was provided with 5 food pellets for refeeding. As previously reported, food intake was quantified 1 h post-refeeding [55]. For calculation of the cumulative food intake, the latter weight of feed was subtracted from the former value. For the feeding patterns (Figure 1I), a new meal was defined when the time without feeding was longer than 5 min from the previous response.

4.5. Free feeding test

All mice used for the free feeding tests were placed in a behaviour box (50 cm long, 25 cm wide, 30 cm high, with a partition in the middle) for two days, 20 min per day. The fake food was made of odorless and tasteless agarose gel, with the same shape and size as regular chow. It was used as a visual control excluding food odor and nutritional reward interference in the free-feeding behavioral test. Fake food and food pellets were placed in the lower left and lower right corner of the box, respectively. All free feeding tests were conducted during the light phase and mice were fed ad libitum before the tests. During the free feeding test, each mouse was placed in the center of the box and freely travelled for 1 h. The food pellet was weighed before and after the test. Time spent in the food zone (%) was calculated as the time spent in the food zone/total time × 100. After each session, the box was thoroughly cleaned with 75% ethanol and water.

4.6. Esophageal manometry

As reported previously [56], LESP was measured 18 h after fasting. In order to avoid asphyxia, we used a single-channel pressure gauge and kept mice at a position of 60° during the experiment. Intraluminal esophageal manometry was performed using a specially designed micro-sized catheter with one micro-transducer (MMS-G-84300, SAR-MED. S.R.L, Iglesias, Italia). Mice were lightly anesthetized with 3% isoflurane inhalation for induction before intubation and then anesthetized at concentrations of 1.5% for maintenance during the examination. The manometric tracings were recorded by a water perfusion pressure measurement system (MMS, Rotterdam, The Netherlands). Manometry was carried out by a stationary pull-through method with the catheter placed trans-orally into the stomach of the spontaneously breathing mouse. The water infusion rate was 0.15 mL/min. The mean LESP for each mouse was calculated by measuring the pressure of the LESP in resting state for 1 min three times (the average of the sum of three LESP); for each time, the tube was relocated with the tube rotated 60° clock wise. The evaluation of the tracings was blinded and assessed by a senior gastroenterologist. The duration of esophageal manometry for each mouse was about 4–5 min.

4.7. Esophageal radiography for esophageal transit time measurement

As reported previously [56], after fasting and water deprivation for 18 h, the mice were kept in supine position and lightly anesthetized with 3% isoflurane inhalation for induction and 1.5% for maintenance (methoxy-fluorane; Schering-Plough Animal Health, Union, NJ, USA). After that, a 12-gauge blunt-ended stainless steel animal feeding needle (Beijing Jingkaida Instrument Co., Ltd., Beijing, China) was inserted trans-orally to the upper part of esophagus at the level of sternum angle and 0.1 mL iohexol (320 mg/mL, Beijing Beilu Pharmaceutical Co., Ltd., Beijing, China) was injected into the stomach through the esophagus. The width of the widest esophagus under X-ray fluoroscopy is defined as the width of the esophagus. Then, another gavage needle (12-gauge, Beijing Jingkaida Instrument Co., Ltd., Beijing, China) with modified tip to maintain an X-ray opaque marker (stainless steel ball with 1 mm in diameter, Yuncheng County Kangda Steel Ball Co., Ltd., Yuncheng, China) was inserted trans-orally to the upper part of esophagus (at the level of sternum angle). Then, 0.1 mL of air was rapidly injected into the stomach via the gavage needle, driving the release of the marker to the esophagus at the sternal angle. The contour of esophageal cavity, the location of the marker and the whole process of its passing through the esophagus were monitored and recorded by X-ray fluoroscopy (Siemens AXIOMIconos R200, Siemens, Co. Ltd., Berlin, Germany). The time required for the marker to reach or pass through the cardia was recorded as esophageal transit time and cardia passing time, respectively. X-ray fluoroscopy was performed by an experienced radiologist. The duration of esophageal emptying test for each mouse was about 15–30 min. The data was assessed by two senior gastroenterologists who were blind to grouping. Any disagreement was settled by consensus (Figure 3).

5. Measurement of heart rate and blood pressure

Systolic blood pressure, diastolic blood pressure, mean arterial pressure, and heart rate were measured in mice using a non-invasive tail-cuff system. Prior to detection, mice were prewarmed at 37 °C for 10–15 min to dilate tail blood vessels and acclimated to the experimental environment for another 5–10 min. For each mouse, 5 to 10 consecutive measurements were recorded; outliers were excluded, and the remaining valid data were averaged for subsequent analysis.

5.1. Histological staining

Mice were killed humanely and perfused with 10 mL of phosphate-buffered saline (PBS) prior to necropsy. The GEJ with distal esophagus were soaked in 4% paraformaldehyde in 0.1 M PBS (pH 7.4) for 24 h, dehydrated and embedded in paraffin, cut into 5 μm slices transversely with a microtome, stained with HE and analyzed by Olympus BX53 microscope. For histological analysis, 3–5 randomly selected non-overlapping visual fields were captured for each tissue section, with 5 mice included per experimental group. Fields exhibiting obvious tissue damage or folding were excluded from subsequent analysis. The thicknesses of the keratinized layer and epithelial cell layer were measured at three sites per section at an interval of 20 μm using ImageJ software, and the mean values were calculated for statistical evaluation. All image quantifications were conducted independently by two blinded researchers to eliminate subjective bias.

5.2. Enzyme-linked immunosorbent assay (ELISA)

The concentrations of Corticotropin-releasing hormone (CRH, catlog E-EL-M0351, Elabscience, Wuhan, China), Corticosterone (CORT, catlog E-OSEL-M0001, Elabscience), interleukin-1β (IL-1β, catlog RK04878, Abclonal, Wuhan, China) and tumor necrosis factor-α (TNFα, catlog E-EL-M3063, Elabscience) in distal esophagus were measured with validated specific ELISA assays according to the manufacturer's instructions.

5.3. Virues

AAV2/9-hSyn-CreOn/FlpOn-ChR2-mCherry, AAV2/Retro-hSyn-Cre, AAV2/Antro-hSyn-Flp, AAV2/9-hSyn-hM4Di-EGFP, AAV2/Antro-hSyn-Cre, AAV2/Retro-hSyn-Flp, AAV2/9-hSyn-CreOn/FlpOn-hM4Di-mCherry, AAV2/9-hSyn-CreOn/FlpOn-hM3Dq-mCherry andAAV-hSyn-EYFP were purchased from Taitool Bioscience (Shanghai, China). For MYBPC2 knockdown, engineered AAVs carrying Mybpc2-shRNA (sequence: 5′-ACGAATTCTTACCATCAACAAGT-3′) or negative control (sequence: 5′-CTCGCTTGGGCGAGAGTAAG-3′) were also produced by Obio. All AAV titers ranged from 0.2 to 3.0 × 1013 V. G./mL.

5.4. Local virus injection

Stereotaxic injection was performed according to our published work [54]. Mice were anesthetized with isoflurane. Standard surgery was performed to expose the brain surface above the PVN. Coordinates used for PVN injection were: bregma - 0.82 mm, lateral ±0.25 mm, and dura −4.75 mm. Coordinates used for DMV injection were: bregma - 7.32 mm, lateral ±0.3 mm, and dura −4.4 mm. The AAVs with a total volume of 200 nL were stereotaxically injected with a glass pipette connected to a Nanoliter Injector 201 (World Precision Instruments, Inc.) at a slow flow rate of 30 nL/min to avoid potential damage to local brain tissue. The pipette was withdrawn at least 20 min after viral injection. Next, the scalp was sutured, and animals were placed on a plate at 37 °C for recovery. Three weeks later, when required, optical fibers (Inper, Hangzhou, China) were implanted into the PVN. Behavioral tests were conducted four weeks after viral injection. After the behavioral tests, the mice were euthanized, and the injection sites in the PVN were examined via frozen-section analysis. Only the data from the mice with accurate injection sites were statistically analyzed. For retrograde tracing from the LES, mice were anaesthetized and the abdomen was clipped and cleaned with 75% alcohol followed by laparotomy to expose the GEJ. Then, PRV-CAG-EGFP (BrainVTA, Wuhan, China, catlog. P03001) was injected into the LES of mice at two different sites, using a Hamilton microsyringe; the pipette was left in place for an additional 5 min after the injection and then slowly withdrawn.

5.5. In vivo optogenetic and chemogenetic manipulations

For the optogenetic manipulation, three weeks after virus injection, optic fibres (200 μm diameter, Newdoon) were implanted into the PVN. Tissue glue and dental cement were used to ensure that the embedded optical fibre could be firmly attached to the skull. The embedded fibres were connected using optic fibre sleeves to a laser generator. Power was set to deliver a 30 min pulse of 473 nm blue light (5 mW, 20 Hz) by a Master-8 pulse stimulator (A.M.P.I.). Mice were allowed to recover for at least one weeks before the experiment and then given 30 min of light along with EMG recording in esophagus. Con mice injected with AAV-hSyn-EYFP (1.6 × 1013 V G./mL, 400 nL) were subjected to the same surgery. For chemogenetic activation, CNO (1 mg/kg) was given daily with an intraperitoneal injection and the last injection was 30-min before behavioral test.

5.6. EMG electrode implantation and EMG recording

For physiological validation of contraction of smooth muscle in esophagus when PVN activation, EMGs of distal esophagus was simultaneously recorded. The EMG recording method followed previously published work [57]. Mice were anesthetized with an intraperitoneal injection of urethane (1.25 g/kg body weight). Satisfactory anesthesia was verified by the lack of hindlimb retraction to a toe pinch. Supplementary doses of 100 mg/kg were administered as needed. Each mouse was placed in a stereotaxic frame in a prone position. Body temperature was maintained at 37 °C using a program-controlled heating pad. To monitor esophageal smooth muscle activity, we implanted acute EMG electrodes in the distal esophagus. The EMG electrode was made of flexible multi-strand stainless steel wires (No. 793200, A-M Systems, Sequim, WA, USA). The insulation of a small segment of the wire (0.5 mm) was removed to expose the electrode to the muscle. During the surgical procedure, the wires were threaded through and anchored with a knot in the muscle. The wires were then attached to ground electrodes on the forelimb. After surgery, mice were connected to flexible EMG connection cables and EMG signals were recorded using a Microelectrode AC Amplifier Model 1800 (A-M System), filtered (10–500 Hz EMG recordings) and digitized at 250 Hz using Spike2 software. The ChR2 expressed PVN activated by a 3-second blue light at an 5-second intervals was simultaneously recorded to synchronize the video and EMG signals. A muscle was considered activated when the amplitude of the EMG trace increased by at least 30% above the basal level.

5.7. Subdiaphragmatic vagotomy

Subdiaphragmatic vagotomy was performed according to research published previously [58]. In brief, mice were anaesthetized with 2.5% isoflurane and underwent surgical procedures in ventral recumbency on a heating pad. The abdominal cavity was exposed by surgery, followed by the stomach and esophagus without damaging blood vessels or the liver. The esophagus was exposed by pulling the costal arc using a curved glass rod. The ventral and dorsal branches of the vagus nerve were visualized, isolated from the esophagus, and excised with a microsurgical scissor (S11036, RWD Life Science Co., Ltd., Shenzhen, China) under a dissecting microscope (M60, Leica Biosystems, Wetzlar, Germany) to achieve total subdiaphragmatic vagotomy. For sham surgery, the trunks of the vagus nerves were similarly exposed but not cut. To confirm the effectiveness of subdiaphragmatic vagotomy, PRV-CAG-EGFP (1 μL) was injected into the vagotomized esophagus. The abdominal muscle layer and skin were then sutured. For all mice received vagotomy, extreme care was taken to avoid any potential injuries to the subdiaphragmatic esophagus. After vagotomy, the mice were allowed 2 weeks for recovery before subsequent experiments, and an increased stomach size after feeding also indicated a successful vagotomy. The presence of EGFP in the PVN was histologically examined in the mice.

5.8. PVN and GEJ immunohistochemistry

c-Fos-like immunoreactivity was visualized as described previously [59]. In brief, 24 h after the last CUMS, mice were anesthetized using isoflurane. Following euthanasia, the mice were transcardially perfused with 0.1 M PBS of pH 7.4 (25 mL), followed by 4% buffered paraformaldehyde (25 mL). The brain or GEJ were postfixed overnight in 4% buffered paraformaldehyde at 4 °C. Then the tissues were dehydrated for 48 h in 20% sucrose at 4 °C. Serial coronal PVN sections and coronal/sagittal distal esophagus sections of 20 μm thickness were cut with a cryostat. The coordinates of the PVN region under study were bregma −0.58 mm to −1.22 mm according to Allen-Brain-Atlas. Only every second section was used. Immunohistochemistry was performed with free-floating sections which first were washed once in 0.1 M PBS, then washed twice in washing buffer (WB; 0.1 M PBS with 0.01 % Triton X 100), and incubated in 0.3 % H2O2 for 30 min. After three further washes (each for 10 min in WB), the tissues were incubated for 1.5 h with a blocking serum (2.5 % goat serum) at room temperature and then with the primary antibody (rabbit polyclonal anti-c-Fos, 1:200, catlog 2250, Cell Signaling Technology, USA; rabbit polyclonal anti-MYBPC2, 1:200, catlog A13331, ABclonal; mouse monoclonal anti-Myosin, 1:200, monoclonal [A4.1025], catlog ab37484, Abcam, Cambridge, UK; anti-F-actin, Phalloidin-iFluor 488, catlog ab176753, Abcam) for overnight at 4 °C. Both the blocking serum and primary antibody were dissolved in 0.1 M PBS containing 0.3 % Triton-X100 and 1 % bovine serum albumin. Afterwards the sections were washed three times in WB and incubated for 1.5 h in a solution containing the CY3 secondary antibody (goat anti-rabbit IgG, 1:200, catlog A10520, Thermo, USA), FITC secondary antibody (goat anti-mouse IgG, 1:200, catlog F-2761, Thermo, USA). After three washes, the slices were mounted on slides with DAPI/Antifade solution (catlog S7113, Sigma–Aldrich, St. Louis, MO, USA). The immunohistochemistry processed sections were examined with a fluorescence microscope (BX53, Olympus, Japan) coupled to a computerized image analysis system.

5.9. Extraction of tissue and RNA sequencing

RNA sequencing was performed on three samples from each of the groups: Control and CUMS as we reported previously [54]. A P-value adjustment was calculated by categorizing the items in KEGG and calculating the FDR. The log2 ratio was used to convert the original expression value for serial cluster analysis. We identified several unique profiles by employing the clustering strategy on short-term series gene expression data. In this case, the probability of observing a distinct significant pattern was higher than expected based on Fisher's exact test and multiple comparison tests.

5.10. Western blot assay

Protein lysates were added into the gastric tissues of mice after rinsing with pre-cooled PBS for 3 times, lysed at 4 °C and centrifuged (10,000 r/min) for 15 min. Supernatant proteins were then extracted and mixed with SDS-PAGE (sodium dodecyl-sulfate-polyacrylamide gel electrophoresis) loading buffer. Primary antibodies of CDC20 (catlog A15656, 1:1000, Abclonal), CCNB2 (catlog A7956, 1:1000, Abclonal), MYBPC2 (catlog A13331, 1:1000, Abclonal), and anti-β-actin (catlog A2228, 1:10,000, Sigma) were put into them after SDS-PAGE gel electrophoresis and transfer to a membrane, and the proteins were placed overnight at 4 °C. Then horseradish peroxidase-conjugated secondary antibodies were incubated with the proteins at room temperature. At last, immunoreactive proteins were tested with a chemiluminescent enhanced chemiluminescence assay kit (Mishushengwu, Xi'an, China) and observed with a Tannon5200 luminescent image analyzer (Tannon, Shanghai, China) with β-actin as internal reference.

5.11. Drug infusion

For intraperitoneal (i.p.) drug infusion, yohimbine (0.2 mg/kg, catlog HY-N0127, MCE, Shanghai, China), propronolol (10 mg/kg, catlog HY-B0573, MCE), prazosin (1 mg/kg, catlog HY-B0193, MCE), and anisodamine (20 mg/kg, catlog HY-N0584, MCE) were dissolved in DMSO, and further diluted using 0.9% sterile saline, and EMG recording were performed 30 min after i. p. injection. For Figure 4E, anisodamine was injected i. p. daily during CUMS. For in vitro use in cultured esophagus smooth cell, anisodamine was diluted by Dulbecco's Modified Eagle Medium (DMED) at 10 μM final concentration 24 h before harvesting. For Figure 7A and B, ACh (catlog HY-B0282, MCE) was diluted by DMED at 100 μM final concentration 24 h before harvesting.

5.12. Culture of the EGJ cells

Primary culture of the GEJ cells were carried out as described previously [60] with modifications. The GEJ cells from two-week-old C57BL/6J mice, which is identifiable as a distinct thickening of circular muscle at the gastroesophageal junction, was dissected free from adjacent tissue under an anatomical microscope. After the mucosal layer was removed, the gastroesophageal junction dissected were minutely chopped and incubated with 0.25% trypsin-Ethylenediaminetetraacetic acid (EDTA) solution (catlog 25200056, Gibco, Thermo Fisher Scientific, Grand Island, NY, USA) at 37 °C for 15 min. They were then centrifuged at 800 rpm for 5 min, and the precipitated cells were resuspended and cultured in DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin/streptomycin, and 2 mM l-glutamine at 37 °C in a humidified atmosphere of 5% CO2. Adherent and proliferating esophagus smooth cell on the dish were passaged at least three times and used for analysis.

5.13. Quantification and statistical analysis

All analyses were performed using Prism (GraphPad), and data sets were assessed for normality and group variance prior to statistical analysis. Student's t-test was used to perform simple statistical comparisons. For the experimental groups with multiple comparisons, data were analysed using one-way or two-way ANOVA followed by Tukey's post hoc test. Significance levels were ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001. The results are expressed as the mean ± s. e.m. Analyses and graphing were conducted in Prism 8 (GraphPad software). At least three experimental repeats were used for each micrograph.

CRediT authorship contribution statement

Kun Zhang, Dake Song, and Yingying Zhang initiated and designed the research. Kun Zhang and Dake Song, performed most of the experiments and analysed and interpreted the results. Shanbo Ma, Hengxin Gong, and Rui Zhang conducted the behavioral experiments and data analysis. Min Wang, Xubo Li, and Xinshang Wang conducted the EMG recordings. Yumei Wu and Kun Zhang wrote the manuscript. Minggao Zhao, Shui-bing Liu, and Kun Zhang were involved in the overall design of the study and the revision of the final manuscript.

Funding

This research was supported by Shaanxi science and technology innovation team plan (No. 2023-CX-TD-63), Technology Innovation Talent Engineering Project (2023RCZZ003), National Natural Science Foundation of China (No. 82471537, 82571721), Science and Technology Research Projects (2024GJJH03-02), Xijing Hospital (No. LHJJ2023-YX08).

Declaration of competing interest

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.molmet.2026.102384.

Contributor Information

Minggao Zhao, Email: minggao@fmmu.edu.cn.

Shui-bing Liu, Email: liushb1974@aliyun.com.

Appendix A. Supplementary data

The following is the Supplementary data to this article.

Multimedia component 1
mmc1.docx (82KB, docx)

Data availability

Data will be made available on request.

References

  • 1.Guadagnoli L., Mashimo H., Lo W.K. Assessment of post-traumatic stress disorder among objective esophageal motility and reflux phenotypes in symptomatic veterans. J Clin Psychol Med Settings. 2023;30(3):606–617. doi: 10.1007/s10880-022-09920-6. [DOI] [PubMed] [Google Scholar]
  • 2.Tack J., Pandolfino J.E. Pathophysiology of gastroesophageal reflux disease. Gastroenterology. 2018;154(2):277–288. doi: 10.1053/j.gastro.2017.09.047. [DOI] [PubMed] [Google Scholar]
  • 3.Luo Y., Keefer L. The clinical value of brain-gut behavioral therapies for functional esophageal disorders and symptoms. Neuro Gastroenterol Motil. 2022;34(6) doi: 10.1111/nmo.14373. [DOI] [PubMed] [Google Scholar]
  • 4.Camilleri M. Abnormal gastrointestinal motility is a major factor in explaining symptoms and a potential therapeutic target in patients with disorders of gut-brain interaction. Gut. 2023;72(12):2372–2380. doi: 10.1136/gutjnl-2023-330542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Clouse R.E. Psychiatric disorders in patients with esophageal disease. Med Clin. 1991;75(5):1081–1096. doi: 10.1016/s0025-7125(16)30400-x. [DOI] [PubMed] [Google Scholar]
  • 6.Chen J.H. Ineffective esophageal motility and the vagus: current challenges and future prospects. Clin Exp Gastroenterol. 2016;9:291–299. doi: 10.2147/CEG.S111820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Xu T.T., Li L., Geng L., Zhou B.D., Zhu S.L. Peripheral and central pathophysiological changes in a new rat model of acid reflux combined with mental stress. Neuro Gastroenterol Motil. 2022;34(6) doi: 10.1111/nmo.14360. [DOI] [PubMed] [Google Scholar]
  • 8.Yang T., Qiao Y., Xiang S., Li W., Gan Y., Chen Y. Work stress and the risk of cancer: a meta-analysis of observational studies. Int J Cancer. 2019;144(10):2390–2400. doi: 10.1002/ijc.31955. [DOI] [PubMed] [Google Scholar]
  • 9.Ding H., Duan Z., Yang D., Zhang Z., Wang L., Sun X., et al. High-resolution manometry in patients with and without globus pharyngeus and/or symptoms of laryngopharyngeal reflux. BMC Gastroenterol. 2017;17(1) doi: 10.1186/s12876-017-0666-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Cha S., Kim S.S. Comorbidity patterns of mood disorders in adult inpatients: applying Association rule mining. Healthcare (Basel) 2021;9(9) doi: 10.3390/healthcare9091155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Ilhan M., Arabaci E., Turgut S., Karaman O., Danalioglu A., Tasan E. Esophagus motility in overt hypothyroidism. J Endocrinol Investig. 2014;37(7):639–644. doi: 10.1007/s40618-014-0085-6. [DOI] [PubMed] [Google Scholar]
  • 12.Storr M., Allescher H.D. Esophageal pharmacology and treatment of primary motility disorders. Dis Esophagus. 1999;12(4):241–257. doi: 10.1046/j.1442-2050.1999.00059.x. [DOI] [PubMed] [Google Scholar]
  • 13.Hershcovici T., Mashimo H., Fass R. The lower esophageal sphincter. Neuro Gastroenterol Motil. 2011;23(9):819–830. doi: 10.1111/j.1365-2982.2011.01738.x. [DOI] [PubMed] [Google Scholar]
  • 14.Swaab D.F., Bao A.M., Lucassen P.J. The stress system in the human brain in depression and neurodegeneration. Ageing Res Rev. 2005;4(2):141–194. doi: 10.1016/j.arr.2005.03.003. [DOI] [PubMed] [Google Scholar]
  • 15.Stanton L.M., Price A.J., Manning E.E. Hypothalamic corticotrophin releasing hormone neurons in stress-induced psychopathology: revaluation of synaptic contributions. J Neuroendocrinol. 2023;35(4) doi: 10.1111/jne.13268. [DOI] [PubMed] [Google Scholar]
  • 16.Wang X.Y., Chen X.Q., Wang G.Q., Cai R.L., Wang H., Wang H.T., et al. A neural circuit for gastric motility disorders driven by gastric dilation in mice. Front Neurosci. 2023;17 doi: 10.3389/fnins.2023.1069198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Hornby P.J., Abrahams T.P. Central control of lower esophageal sphincter relaxation. Am J Med. 2000;108(Suppl 4a):90S–98S. doi: 10.1016/s0002-9343(99)00345-9. [DOI] [PubMed] [Google Scholar]
  • 18.Sang Q., Goyal R.K. Lower esophageal sphincter relaxation and activation of medullary neurons by subdiaphragmatic vagal stimulation in the mouse. Gastroenterology. 2000;119(6):1600–1609. doi: 10.1053/gast.2000.20234. [DOI] [PubMed] [Google Scholar]
  • 19.Hornby P.J., Abrahams T.P., Partosoedarso E.R. Central mechanisms of lower esophageal sphincter control. Gastroenterol Clin N Am. 2002;31(4 Suppl):S11–S20. doi: 10.1016/s0889-8553(02)00038-9. [v-vi] [DOI] [PubMed] [Google Scholar]
  • 20.Li L., Zhang Y.M., Qiao W.L., Zhang J.F., Wang L. Role of mitogen-activated protein kinases in the regulation of paraventricular nucleus to gastric ischemia-reperfusion injuries. Chin Med J (Engl) 2007;120(12):1082–1087. [PubMed] [Google Scholar]
  • 21.Mitchell C.S., Campbell E.J., Fisher S.D., Stanton L.M., Burton N.J., Pearl A.J., et al. Optogenetic recruitment of hypothalamic corticotrophin-releasing-hormone (CRH) neurons reduces motivational drive. Transl Psychiatry. 2024;14(1) doi: 10.1038/s41398-023-02710-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Chen Z., Li X.Y., Guo P., Wang D.L. MYBPC2 and MYL1 as significant gene markers for Rhabdomyosarcoma. Technol Cancer Res Treat. 2021;20 doi: 10.1177/1533033820979669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Cunningham E.T., Jr., Sawchenko P.E. Central neural control of esophageal motility: a review. Dysphagia. 1990;5(1):35–51. doi: 10.1007/BF02407391. [DOI] [PubMed] [Google Scholar]
  • 24.Sohn U.D., Harnett K.M., De Petris G., Behar J., Biancani P. Distinct muscarinic receptors, G proteins and phospholipases in esophageal and lower esophageal sphincter circular muscle. J Pharmacol Exp Therapeut. 1993;267(3):1205–1214. [PubMed] [Google Scholar]
  • 25.Whitehead W.E. Psychosocial aspects of functional gastrointestinal disorders. Gastroenterol Clin N Am. 1996;25(1):21–34. doi: 10.1016/s0889-8553(05)70363-0. [DOI] [PubMed] [Google Scholar]
  • 26.Zhong C., Liu K., Wang K., Liu H., Su H., Wu J., et al. Developing a diagnostic understanding of GERD phenotypes through the analysis of levels of mucosal injury, immune activation, and psychological comorbidity. Dis Esophagus. 2018;31(10) doi: 10.1093/dote/doy039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Zheng J., Dobner A., Babygirija R., Ludwig K., Takahashi T. Effects of repeated restraint stress on gastric motility in rats. Am J Physiol Regul Integr Comp Physiol. 2009;296(5):R1358–R1365. doi: 10.1152/ajpregu.90928.2008. [DOI] [PubMed] [Google Scholar]
  • 28.Khoo J., Rayner C.K., Feinle-Bisset C., Jones K.L., Horowitz M. Gastrointestinal hormonal dysfunction in gastroparesis and functional dyspepsia. Neuro Gastroenterol Motil. 2010;22(12):1270–1278. doi: 10.1111/j.1365-2982.2010.01609.x. [DOI] [PubMed] [Google Scholar]
  • 29.Gyawali C.P., Fass R. Management of gastroesophageal reflux disease. Gastroenterology. 2018;154(2):302–318. doi: 10.1053/j.gastro.2017.07.049. [DOI] [PubMed] [Google Scholar]
  • 30.Bixquert-Jimenez M., Bixquert-Pla L. [Antidepressant therapy in functional gastrointestinal disorders] Gastroenterol Hepatol. 2005;28(8):485–492. doi: 10.1157/13079000. [DOI] [PubMed] [Google Scholar]
  • 31.Czimmer J., Tache Y. Peripheral corticotropin releasing factor signaling inhibits gastric emptying: mechanisms of action and role in stress-related gastric alterations of motor function. Curr Pharm Des. 2017;23(27):4042–4047. doi: 10.2174/1381612823666170228142428. [DOI] [PubMed] [Google Scholar]
  • 32.Tache Y., Bonaz B. Corticotropin-releasing factor receptors and stress-related alterations of gut motor function. J Clin Investig. 2007;117(1):33–40. doi: 10.1172/JCI30085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Babygirija R., Bulbul M., Yoshimoto S., Ludwig K., Takahashi T. Central and peripheral release of oxytocin following chronic homotypic stress in rats. Auton Neurosci. 2012;167(1–2):56–60. doi: 10.1016/j.autneu.2011.12.005. [DOI] [PubMed] [Google Scholar]
  • 34.Ma J., Peng X., Zhang M., Gao W., Yang X., Wang Z., et al. A brain-to-small intestine circuit mediates morphine-induced constipation in male mice. Nat Commun. 2025;17(1) doi: 10.1038/s41467-025-67765-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Sun H., Zhao P., Liu W., Li L., Ai H., Ma X. Ventromedial hypothalamic nucleus in regulation of stress-induced gastric mucosal injury in rats. Sci Rep. 2018;8(1) doi: 10.1038/s41598-018-28456-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Goyal M., Nagalli S. Disclosure: Shivaraj Nagalli declares no relevant financial relationships with ineligible companies. 2025. Esophageal Motility Disorders. StatPearls: treasure Island (FL) ineligible companies. [Google Scholar]
  • 37.van der Keylen P., Garreis F., Steigleder R., Sommer D., Neuhuber W.L., Worl J. Involvement of catecholaminergic neurons in motor innervation of striated muscle in the mouse esophagus. Histochem Cell Biol. 2016;145(5):573–585. doi: 10.1007/s00418-015-1403-6. [DOI] [PubMed] [Google Scholar]
  • 38.Richards W.G., Sugarbaker D.J. Neuronal control of esophageal function. Chest Surg Clin. 1995;5(1):157–171. [PubMed] [Google Scholar]
  • 39.Nishimura H., Oomura Y. Effects of hypothalamic stimulation on activity of dorsomedial medulla neurons that respond to subdiaphragmatic vagal stimulation. J Neurophysiol. 1987;58(4):655–675. doi: 10.1152/jn.1987.58.4.655. [DOI] [PubMed] [Google Scholar]
  • 40.Willett C.J., Rutherford J.G., Gwyn D.G., Leslie R.A. Projections between the hypothalamus and the dorsal vagal complex in the cat: an HRP and autoradiographic study. Brain Res Bull. 1987;18(1):63–71. doi: 10.1016/0361-9230(87)90034-7. [DOI] [PubMed] [Google Scholar]
  • 41.Swanson L.W., Kuypers H.G. The paraventricular nucleus of the hypothalamus: cytoarchitectonic subdivisions and organization of projections to the pituitary, dorsal vagal complex, and spinal cord as demonstrated by retrograde fluorescence double-labeling methods. J Comp Neurol. 1980;194(3):555–570. doi: 10.1002/cne.901940306. [DOI] [PubMed] [Google Scholar]
  • 42.Berthoud H.R., Carlson N.R., Powley T.L. Topography of efferent vagal innervation of the rat gastrointestinal tract. Am J Physiol. 1991;260(1 Pt 2):R200–R207. doi: 10.1152/ajpregu.1991.260.1.R200. [DOI] [PubMed] [Google Scholar]
  • 43.Leslie R.A., Gwyn D.G., Hopkins D.A. The central distribution of the cervical vagus nerve and gastric afferent and efferent projections in the rat. Brain Res Bull. 1982;8(1):37–43. doi: 10.1016/0361-9230(82)90025-9. [DOI] [PubMed] [Google Scholar]
  • 44.Smith V.C., Dhatt N., Buchan A.M. The innervation of the human antro-pyloric region: organization and composition. Can J Physiol Pharmacol. 2001;79(11):905–918. [PubMed] [Google Scholar]
  • 45.Bulbul M., Izgut-Uysal V.N., Sinen O., Birsen I., Tanriover G. Central apelin mediates stress-induced gastrointestinal motor dysfunction in rats. Am J Physiol Gastrointest Liver Physiol. 2016;310(4):G249–G261. doi: 10.1152/ajpgi.00145.2015. [DOI] [PubMed] [Google Scholar]
  • 46.Jiang Y., Travagli R.A. Hypothalamic-vagal oxytocinergic neurocircuitry modulates gastric emptying and motility following stress. J Physiol. 2020;598(21):4941–4955. doi: 10.1113/JP280023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Babygirija R., Cerjak D., Yoshimoto S., Gribovskaja-Rupp I., Bulbul M., Ludwig K., et al. Affiliative behavior attenuates stress responses of GI tract via up-regulating hypothalamic oxytocin expression. Auton Neurosci. 2012;169(1):28–33. doi: 10.1016/j.autneu.2012.03.001. [DOI] [PubMed] [Google Scholar]
  • 48.Jiang Y., Coleman F.H., Kopenhaver Doheny K., Travagli R.A. Stress adaptation upregulates Oxytocin within hypothalamo-vagal neurocircuits. Neuroscience. 2018;390:198–205. doi: 10.1016/j.neuroscience.2018.08.021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Jiang Y., Browning K.N., Toti L., Travagli R.A. Vagally mediated gastric effects of brain stem alpha(2)-adrenoceptor activation in stressed rats. Am J Physiol Gastrointest Liver Physiol. 2018;314(4):G504–G516. doi: 10.1152/ajpgi.00382.2017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Travagli R.A., Hermann G.E., Browning K.N., Rogers R.C. Brainstem circuits regulating gastric function. Annu Rev Physiol. 2006;68:279–305. doi: 10.1146/annurev.physiol.68.040504.094635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Hessel A.L., Engels N.M., Kuehn M.N., Nissen D., Sadler R.L., Ma W., et al. Myosin-binding protein C regulates the sarcomere lattice and stabilizes the OFF states of myosin heads. Nat Commun. 2024;15(1) doi: 10.1038/s41467-024-46957-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Jiang Y., Bhargava V., Lal H.A., Mittal R.K. Variability in the muscle composition of rat esophagus and neural pathway of lower esophageal sphincter relaxation. Am J Physiol Gastrointest Liver Physiol. 2011;301(6):G1014–G1019. doi: 10.1152/ajpgi.00273.2011. [DOI] [PubMed] [Google Scholar]
  • 53.Li H., Xiang Y., Zhu Z., Wang W., Jiang Z., Zhao M., et al. Rifaximin-mediated gut microbiota regulation modulates the function of microglia and protects against CUMS-induced depression-like behaviors in adolescent rat. J Neuroinflammation. 2021;18(1) doi: 10.1186/s12974-021-02303-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Ma S., Chong Y., Zhang R., Quan W., Gui J., Li L., et al. Glycyrrhizic acid treatment ameliorates anxiety-like behaviour via GLT1 and Per1/2-dependent pathways. J Ethnopharmacol. 2024;328 doi: 10.1016/j.jep.2024.118013. [DOI] [PubMed] [Google Scholar]
  • 55.Ge T., Zhang Z., Lv J., Song Y., Fan J., Liu W., et al. The role of 5-HT(2c) receptor on corticosterone-mediated food intake. J Biochem Mol Toxicol. 2017;31(6) doi: 10.1002/jbt.21890. [DOI] [PubMed] [Google Scholar]
  • 56.Zhao W., Wang B., Zhang L., Jin H. Eosinophils infiltration in esophageal Muscularis Propria induces Achalasia-like esophageal motility disorder in mice. Biomolecules. 2022;12(12) doi: 10.3390/biom12121865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Xie Z., Gu H., Huang M., Cheng X., Shang C., Tao T., et al. Mechanically evoked defensive attack is controlled by GABAergic neurons in the anterior hypothalamic nucleus. Nat Neurosci. 2022;25(1):72–85. doi: 10.1038/s41593-021-00985-4. [DOI] [PubMed] [Google Scholar]
  • 58.Chen J., Cheng M., Wang L., Zhang L., Xu D., Cao P., et al. A Vagal-NTS neural pathway that stimulates feeding. Curr Biol. 2020;30(20):3986–3998 e3985. doi: 10.1016/j.cub.2020.07.084. [DOI] [PubMed] [Google Scholar]
  • 59.Wultsch T., Painsipp E., Thoeringer C.K., Herzog H., Sperk G., Holzer P. Endogenous neuropeptide Y depresses the afferent signaling of gastric acid challenge to the mouse brainstem via neuropeptide Y type Y2 and Y4 receptors. Neuroscience. 2005;136(4):1097–1107. doi: 10.1016/j.neuroscience.2005.08.038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 60.Hubert M.O., Rodriguez-Vita J., Wiedmann L., Fischer A. Isolation of Murine Primary aortic smooth muscle cells. Bio Protoc. 2021;11(3) doi: 10.21769/BioProtoc.3907. [DOI] [PMC free article] [PubMed] [Google Scholar]

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