Transient receptor potential vanilloid 4 channels contribute to the initiation of water-induced swallowing reflexes

Transient receptor potential vanilloid 4 channels contribute to the initiation of water-induced swallowing reflexes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Transient receptor potential vanilloid 4 channels contribute to the initiation of water-induced swallowing reflexes Mohammad Zakir Hossain, Hiroshi Ando, Rita Rani Roy, Junichi Kitagawa This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8706920/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Apr, 2026 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract The swallowing reflex is essential for the safe transfer of food and liquids from the oral cavity to the esophagus. Water is a strong stimulus for triggering this reflex, yet the molecular mechanisms underlying water-induced activation remain unclear. The aim of this study was to evaluate whether transient receptor potential vanilloid 4 (TRPV4) channels contribute to the water-evoked swallowing reflex. We applied distilled water (DW) or saline to superior laryngeal nerve (SLN)-innervated swallowing-related regions in anesthetized rats, measured swallowing reflexes and SLN activity, and conducted immunohistochemical analyses of TRPV4 expression. DW elicited more frequent swallowing and greater SLN activation than saline. TRPV4 immunoreactivity was observed in epithelial cells, taste bud–like structures, and nerve fibers within the laryngopharyngeal and laryngeal mucosa. DW stimulation also induced c-Fos expression in the nodose–petrosal–jugular ganglionic complex, with ~ 60% of activated neurons coexpressing TRPV4. Pretreatment with the TRPV4 antagonist RN9893 significantly attenuated DW-evoked swallowing and SLN activity. These findings provide integrated physiological, pharmacological, and immunohistochemical evidence that TRPV4 channels contribute as molecular sensors initiating the water-induced swallowing reflex. Our study provides enhanced understanding of TRPV4-mediated sensory regulation of swallowing and suggests that TRPV4 is a potential therapeutic target for oropharyngeal dysphagia. Biological sciences/Neuroscience Biological sciences/Physiology TRPV4 channels Water-induced swallowing reflex Superior laryngeal nerve-afferents Nodose–petrosal–jugular ganglionic complex Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The swallowing reflex is a critical component of the digestive process, facilitating the passage of food and liquids from the oral cavity to the esophagus 1 . Previous studies have demonstrated that applying water to the pharyngeal and laryngeal regions can elicit swallowing in both animals 2 – 4 and humans 5 – 7 . However, the molecular mechanisms underlying this water-induced swallowing reflex remain unclear. Patients with dysphagia often experience difficulty swallowing liquids 8 – 10 , highlighting the need to better understand the molecular basis of this reflex in order to develop effective therapeutic interventions 11 , 12 . The laryngopharynx and associated laryngeal regions, which are innervated by the superior laryngeal nerve (SLN)—a branch of the vagus nerve—have been shown to play a key role in initiating the swallowing reflex in response to water stimulation 2 – 4 , 11 , 13 – 17 . Applying water to these regions increases SLN activity and triggers the swallowing reflex 2 – 4 , 13 – 15 , 17 . TRPV4 channels are non-selective cation channels that function as polymodal receptors that respond to various stimuli, including hypoosmotic conditions 18 – 22 . In a previous study, we observed that activation of TRPV4 channels in SLN-innervated regions using the chemical agonist GSK1016790A effectively induced the swallowing reflex 23 . Given that water is inherently hypoosmotic, it is plausible that it can activate TRPV4 channels when applied to swallowing-related regions. Supporting this hypothesis, earlier studies suggest that the responsiveness of laryngeal receptors to water is partially mediated by its hypoosmotic properties 24 – 26 . In addition, water entry into the cell can activate TRPV4 channels, which respond to changes in cell volume 27 , 28 . The objective of this study was to determine whether TRPV4 channels contribute to initiation of the water-induced swallowing reflex. Methods Animals Experiments were conducted using 64 male Sprague-Dawley rats, weighing approximately 300–450 g. Of these, nine rats were used for immunohistochemistry and fifty-five for assessment of the swallowing reflex. All experimental procedures were approved by the Intramural Animal Care and Veterinary Science Committee of Matsumoto Dental University (Ref. No. 394). Every effort was made to minimize animal suffering and reduce the number of animals used. Animals were housed in the Matsumoto Dental University animal facility under controlled conditions (temperature: 22 ± 2 °C; relative humidity: 40 ± 5%; light/dark cycle: 12/12 hours). Food and water were available ad libitum . All procedures adhered to the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines established by the National Centre for the Replacement, Refinement, and Reduction of Animals in Research. Surgical procedure for swallowing reflex recording Rats were anesthetized with urethane (1.0–1.5 g/kg, intraperitoneally) and positioned in a supine posture. A midline incision was made in the ventral neck region to expose the trachea. Respiration was maintained via a custom-made cannula inserted into the trachea and directed toward the lungs. A small ventral portion of the trachea was surgically removed just behind the cricoid cartilage to create a window for solution delivery. To isolate the SLNs as the primary neural input for the swallowing reflex, the pharyngeal (IX-ph) and lingual (IX-li) branches of the glossopharyngeal nerve, as well as the pharyngeal (X-ph) and recurrent laryngeal nerve (RLN) branches of the vagus nerve, were bilaterally transected. The bilateral SLNs were left intact during the main experiments to assess their role in triggering the swallowing reflex. In a separate experimental series, the bilateral SLNs were also transected, along with the aforementioned nerves, to confirm the specific involvement of SLNs in the initiation of the swallowing reflex under the experimental conditions. Stimulating solutions The solutions used for stimulation were 0.9% saline (NaCl; Otsuka Pharmaceutical Co. Ltd., Tokyo, Japan) and DW. Each solution was delivered to the swallowing-related regions innervated by the SLN in a 50-μl volume using a syringe equipped with a blunted 21-gauge needle. All solutions were at room temperature (22–24 °C) when applied. Following each application, reflex responses were recorded for 20 seconds. The interval between successive solution deliveries was 2–3 minutes. During this period, the previously administered solution was aspirated, and the target region was thoroughly rinsed with saline to prevent cross-contamination. Electromyographic identification of the swallowing reflex Electromyographic (EMG) recordings from the mylohyoid muscle, combined with visual confirmation of laryngeal movements, were used to identify and quantify swallowing reflex events. The mylohyoid muscle, along with other supra- and infrahyoid muscles, is activated during initiation of the swallowing reflex 1 . Recording the activity of one or more of these muscles is a well-established method that has been widely adopted as a standardized method for detecting the swallowing reflex. Previous studies, including those from our group, have reliably identified swallowing reflexes based on high-amplitude EMG activity in the mylohyoid muscle in conjunction with visible laryngeal elevation 17,23,29–32 . EMG signals were recorded using bipolar fine-wire electrodes (stainless steel, urethane-coated; Unique Medical Co., Ltd., Tokyo, Japan). A loudspeaker connected to the EMG system facilitated auditory monitoring of mylohyoid muscle activity during data collection. To ensure that the observed high-amplitude EMG bursts in the mylohyoid muscle represented swallowing reflexes—rather than coughing or other airway defense responses—respiratory chest movements and EMG activity from both the mylohyoid and rectus abdominis muscles were simultaneously recorded in some animals (Supplemental Figure 1A, B). The rectus abdominis muscle was included because it is characteristically activated during the cough reflex 33 . Respiratory chest movements were recorded using a piezoelectric transducer (ADInstruments, Inc., Colorado Springs, CO). EMG activity from the rectus abdominis muscle was monitored using bipolar fine-wire electrodes of the same type (stainless steel, urethane-coated; Unique Medical Co., Ltd., Tokyo, Japan). All signals were amplified and filtered using a bioelectric amplifier (MEG-5200; Nihon Kohden, Tokyo, Japan), digitized via a Power 1401 data acquisition system (Cambridge Electronic Design Ltd., Cambridge, UK), and stored on a hard drive for subsequent analysis. Upon DW application to SLN-innervated swallowing-related regions, repetitive high-amplitude EMG bursts were observed in the mylohyoid muscle, accompanied by a temporary cessation of respiration (apnea) (Supplemental Figure 1B). Notably, no EMG activity was detected in the rectus abdominis muscle. The absence of both respiratory chest movements and rectus abdominis activity confirmed that the EMG bursts in the mylohyoid muscle were due to swallowing reflexes. This is because the cough reflex is typically characterized by inspiratory chest movements followed by forceful expiratory movements accompanying the contractions of abdominal muscles, including the rectus abdominis 33,34 . As respiration gradually resumed, additional swallowing reflexes occurred at increasing intervals, again without rectus abdominis involvement. Saline application induced only a single swallowing reflex, associated with a brief pause in respiration (Supplemental Figure 1A). These observations confirm that the repetitive EMG bursts observed following DW application specifically represent swallowing reflex activity. Immunohistochemistry of superior laryngeal nerve-innervated swallowing-related regions Deeply anaesthetized rats were subjected to transcardial perfusion with saline followed by 4% paraformaldehyde. The swallowing-related regions were dissected then immersed in 4% paraformaldehyde for 3 hours. The specimens were subsequently incubated in 30% sucrose until fully submerged to ensure adequate cryoprotection. The samples were then embedded in Tissue-Tek Optimal Cutting Temperature Compound (Sakura Finetek, Tokyo, Japan) and sectioned in the sagittal plane (10 or 50 μm) before being mounted on glass slides. The sections were incubated with 5% normal goat serum in 0.01 M phosphate-buffered saline containing 0.05% Triton X-100 for 1 hour to minimize nonspecific binding. Subsequently, the sections were incubated with rabbit monoclonal anti-TRPV4 (1:100; Cat# ab259361; Abcam, Cambridge, UK) at 4°C overnight, followed by incubation with a fluorescent dye-conjugated secondary antibody (Goat anti-rabbit Alexa Fluor 594; Cat# A-11037; Thermo Fisher Scientific, Waltham, MA) for 1 hour at room temperature. The sections were then treated with 4ʹ,6-diamidino-2-phenylindole (DAPI) for 10 minutes to visualize the cell nuclei. Finally, coverslips were applied using mounting medium (PermaFluor; Thermo Fisher Scientific), and the specimens were examined by fluorescence microscopy (BZ-X700; Keyence Corp., Osaka, Japan). Immunohistochemistry analysis of TRPV4 expression in the nodose–petrosal–jugular ganglionic complex in relation to distilled water-induced c-Fos expression The rats were anesthetized with urethane (1.0–1.5 g/kg, intraperitoneally), the trachea was carefully exposed, and a small window was created just behind the cricoid cartilage for DW delivery. The SLN, IX-ph, IX-li, X-ph, and RLN were left intact to avoid nerve transection–induced c-Fos expression. Following surgery, the rats were maintained in a supine position for 4 hours to minimize surgery-related c-Fos expression, as reported previously 35,36 . Four hours after surgery, DW was applied to the SLN-innervated swallowing-related regions 15 times (50 μL per application, every 2 minutes). Each application was maintained for 30 seconds and then aspirated, for a total duration of 30 minutes. Rats were subsequently kept in the supine position for 1 hour because c-Fos expression is known to peak approximately 1 hour following stimulation 37,38 . One hour after the final DW application, the deeply anesthetized rats were perfused transcardially with saline, followed by 4% paraformaldehyde. Each ganglion of the nodose–petrosal–jugular ganglionic complex (NPJc) was identified based on established anatomical criteria 39 . The nodose ganglion (NG) was distinguished by its attachment to the main trunk of the vagus nerve, oriented toward the thorax. The jugular ganglion (JG) was located in the direction of the brainstem, opposite the vagal trunk, whereas the petrosal ganglion (PG) was positioned between the NG and JG. To preserve anatomical orientation during embedding, the right or left NPJc was excised, along with a small segment of the vagus nerve trunk. The excised tissue was fixed in 4% paraformaldehyde, immersed in 30% sucrose until it sank, and then embedded in Tissue-Tek O.C.T. Compound (Sakura Finetek, Tokyo, Japan), maintaining its orientation. The tissue was sectioned into 16-μm-thick slices and mounted onto glass slides, allowing reliable identification of each ganglion after sectioning. Every third section was processed for fluorescent immunohistochemistry, using a combination of indirect and direct methods 40 to detect TRPV4 expression in c-Fos–expressing neurons. First, the sections were blocked in 0.01 M phosphate-buffered saline containing 5% normal goat serum and 0.05% Triton X-100 for 1 hour to minimize nonspecific binding. They were then incubated overnight at 4°C with rabbit polyclonal anti-c-Fos antibody (1:1000; Cat# ab190289; Abcam, Cambridge, UK), followed by goat anti-rabbit Alexa Fluor 594 secondary antibody (Cat# A-11037; Thermo Fisher Scientific) for 1 hour at room temperature. To prevent nonspecific binding of the prior secondary antibody, the sections were subsequently blocked with normal rabbit serum for 1 hour at room temperature 40 , followed by incubation with Alexa Fluor 488–conjugated rabbit monoclonal anti-TRPV4 antibody (1:100; Cat# ab315136; Abcam) for 48 hours at 4°C. Cell nuclei were visualized by counterstaining with DAPI for 10 minutes. The slides were coverslipped and imaged using a fluorescence microscope (BZ-X700; Keyence Corp.). Immunoreactive cells were quantified using ImageJ software (NIH, Bethesda, MD). Because c-Fos is a nuclear marker of neuronal activation expressed 41,42 , only neurons showing nuclear c-Fos expression were included in the counts, although nonspecific cytoplasmic c-Fos staining was also noted. Similar nonspecific cytoplasmic signals were observed on both positive and negative control slides (Supplemental Figure 2A, B). Therefore, only neurons with nuclear c-Fos expression were included in the counts, even if cytoplasmic expression was also present. Cells with cytoplasmic, but no nuclear, c-Fos expression were excluded. TRPV4 expression was not observed in nuclei. Neurons that were positive for both c-Fos (nuclear) and TRPV4, as well as those that were positive for c-Fos (nuclear) but negative for TRPV4, were quantified in the NG, PG, JG, and across the entire NPJc. The trigeminal ganglion (TG) was used as a positive control for the anti-c-Fos and anti-TRPV4 antibodies (Supplemental Figure 2A). To induce c-Fos expression in the TG, capsaicin (0.05 mM, 50 μL) was injected into the right vibrissal pad and lower lip in anesthetized rats. One hour later, rats were deeply anesthetized and perfused transcardially with saline, followed by 4% paraformaldehyde. The right TG was excised, fixed, cryoprotected, embedded in Tissue-Tek O.C.T Compound, sectioned at 16 μm intervals, and processed identically to the NPJc sections. As a negative control, sections of the NG, laryngopharynx, and associated laryngeal regions were incubated with a universal negative control reagent (Cat# ADI-950-231-0025; Enzo Life Sciences, Inc., Farmingdale, NY) instead of primary antibodies (Supplemental Figure 2B, C). TRPV4 antagonist A TRPV4 antagonist RN-9893 [(2E)-N-(2,3-dihydro-1,4-benzodioxin-6-yl)-3-[4-(1,1-dimethylethyl)phenyl]-2-propenamide] was obtained from Tocris Bioscience, Bristol, UK. The efficacy of RN-9893 as a selective TRPV4 antagonist has been validated previously 43,44 . RN-9893 was dissolved in a solution containing 3% dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO) and 1% Tween 80 (Sigma-Aldrich), then diluted with saline to the final concentration. The vehicle control consisted of the same DMSO-Tween 80-saline mixture without the antagonist. The prepared solution was instilled into the SLN-innervated swallowing-related region and left in place for 15 minutes. The solution was aspirated before applying DW. Recording superior laryngeal nerve activity The sternothyroid muscle was bluntly dissected, and the SLN was carefully freed from the surrounding tissues. Both SLNs were transected distally, near their junction with the vagus nerve. A pair of bipolar silver wire electrodes (0.1 mm diameter) was placed unilaterally beneath the SLN. Liquid silicone was applied to secure the electrodes to the nerve, insulate them from surrounding tissues, and prevent desiccation, thereby enabling stable long-term recordings. SLN activity was amplified and integrated with a time constant of 0.3 s. The signals were digitized using a Power 1401 data acquisition system (Cambridge Electronic Design Ltd., Cambridge, UK) and stored for subsequent analysis. Lidocaine To determine whether sensory fibers in the SLN-innervated swallowing region contribute to the DW–induced swallowing reflex, 2% lidocaine (2-(diethylamino)-N-(2,6-dimethylphenyl)acetamide; Xylocaine®, AstraZeneca Ltd., Osaka, Japan) was applied topically. Data and statistical analysis Swallowing reflexes elicited by stimulating solutions were counted over a 20-second period. SLN responses were analyzed as the area of the integrated response above baseline using Spike2 software (Cambridge Electronic Design Ltd.). The integrated SLN response was calculated in 2-second bins from the onset of solution infusion for 20 seconds following stimulation. The stable baseline activity recorded for 2 seconds prior to stimulation was subtracted from each corresponding 2-second response. For statistical evaluation, data were first tested for normality and homogeneity of variance to determine whether parametric or non-parametric tests were appropriate. A paired t-test was used to compare the number of swallowing reflexes triggered by saline versus DW. One-way repeated-measures analysis of variance (ANOVA), followed by Tukey’s post hoc test, was used to compare the number of swallowing reflexes induced by DW with or without prior application of the TRPV4 antagonist or its vehicle. Nerve responses to different stimulating solutions were compared using one-way repeated-measures ANOVA with Tukey’s post hoc test. In addition, paired t-tests were used to compare the number of DW-induced swallowing reflexes before and after lidocaine application or SLN transection. A significance level of P < 0.05 was considered statistically significant. Data are presented as mean ± standard deviation (SD). Statistical analyses were performed using SigmaPlot software (version 14.0; Systat Software Inc., San Jose, CA). Graphs were generated using GraphPad Prism software (version 10.4; GraphPad Software, San Diego, CA). Results Topical application of distilled water facilitated the swallowing reflex We first examined whether DW application facilitated the swallowing reflex. Application of DW to SLN-innervated swallowing-related regions elicited significantly more swallowing reflexes (15 ± 1.58) than application of saline (1 ± 0.00) (Figure 1A, B). TRPV4 expression in peripheral swallowing-related regions We next investigated TRPV4 distribution in SLN-innervated swallowing-related regions (Figure 2) TRPV4 immunoreactivity was observed in both intraepithelial and subepithelial nerve fibers (Figure 2B–D). In addition, TRPV4 expression was detected in epithelial cells located near the base of the epiglottis, aryepiglottic folds, arytenoids, and vestibular folds (Figure 2E–G). Furthermore, TRPV4 immunoreactivity was present in the cells of taste bud–like structures, as well as in associated nerve fibers and neurogenic plaques (Figure 2H–J). TRPV4 expression in distilled water-induced c-Fos–expressing neurons in the nodose–petrosal–jugular ganglionic complex We then assessed whether TRPV4 was expressed in DW-induced c-Fos–positive neurons within the NPJc. Following DW application, c-Fos–positive neurons were predominantly observed in the JG (36.67 ± 20.85) and PG (19.83 ± 16.03), with fewer detected in the NG (6.33 ± 7.09) (Figure 3A, B). Notably, approximately 60% of c-Fos–positive neurons in the NPJc also expressed TRPV4 (Figure 3C). Treatment with a TRPV4 antagonist significantly reduced distilled water-induced swallowing reflexes On the basis of the identification of TRPV4 in peripheral swallowing-related regions and in DW-induced c-Fos–expressing neurons within the NPJc, we next examined whether a treatment with a TRPV4 antagonist could attenuate DW-induced swallowing reflexes. Various concentrations of the TRPV4 antagonist RN9893 were applied to the swallowing-related regions 15 minutes prior to DW application. The effects of treatment with a vehicle control (corresponding to the highest concentration of RN9893 used) were also evaluated. Administration of RN9893 at concentrations of 10 mM, 20 mM, 50 mM, and 100 mM significantly reduced the number of DW-induced swallowing reflexes (Figure 4A, B). In contrast, the vehicle had no significant effect. Lower concentrations of RN9893 (1 mM and 5 mM) likewise produced no significant changes (Figure 4A, B). Notably, even at the highest concentration, RN9893 did not completely abolish DW-induced reflexes. Treatment with a TRPV4 antagonist significantly reduced distilled water-induced superior laryngeal nerve activity We next asked whether treatment with the TRPV4 antagonist would reduce DW-induced SLN activity. Application of DW markedly increased SLN activity compared with saline application (Figure 5A, B). Pretreatment with RN9893 (10 mM) 15 minutes prior to DW application significantly reduced this activity (Figure 5A, B). In contrast, the vehicle had no effect (Figure 5A, B). Local anesthetic or bilateral superior laryngeal nerve transection abolished distilled water-induced swallowing reflexes To confirm that DW-induced swallowing reflexes were mediated by excitation of afferent nerves within swallowing-related regions, a local anesthetic (2% lidocaine) was applied 2 minutes before DW stimulation. This treatment completely abolished DW-induced swallowing reflexes (Figure 6A, B). In addition, no apnea was observed following local anesthetic application (Supplemental Figure 3A). Bilateral transection of the SLNs prior to DW application also eliminated the swallowing reflex, demonstrating that SLN afferents mediated the DW-induced response under our experimental conditions (Figure 6A, C). Similar to the effect of local anesthetic application, no apnea was observed following bilateral SLN transection (Supplemental Figure 3B). Discussion To the best of our knowledge, this study provides the first evidence that TRPV4 channels are involved in triggering the water-induced swallowing reflex. The enhanced reflex activity elicited by water, compared with saline, supports the hypothesis that water activates specific sensory mechanisms responsible for initiating the swallowing reflex. Our findings collectively demonstrate that TRPV4 channels play a pivotal role in this process. Immunohistochemical analysis showed that TRPV4 was expressed by epithelial cells, taste bud cells, and nerve fibers within the laryngopharyngeal and laryngeal mucosa—regions that have previously been identified as playing key roles in triggering swallowing. These findings indicate that TRPV4 is strategically positioned to act as a peripheral sensor that responds to mucosal surface changes following water application. Moreover, c-Fos expression induced by water stimulation was observed in the NPJc, with a substantial proportion of activated neurons coexpressing TRPV4. This suggests that TRPV4-expressing vagal afferent neurons within the NPJc are recruited by water stimulation in swallowing-related areas. TRPV4 localization in peripheral swallowing-related regions is consistent with previous reports describing its expression in nerve fibers of the laryngopharynx and associated laryngeal regions 23 , in epithelial cells of the larynx 45 , 46 , trachea 47 , and esophagus 48 , and in taste bud cells of the circumvallate papillae 49 . The presence of TRPV4 in these structures suggests its potential role in mediating swallowing-related sensory processes. In our previous study, topical application of the TRPV4 agonist GSK1016790A facilitated the swallowing reflex in a dose-dependent manner 23 . The findings from the present study extend these observations by demonstrating that TRPV4 activation is physiologically relevant for triggering the water-induced swallowing reflex. Pharmacological blockade of TRPV4 with a selective antagonist significantly attenuated both the number of water-induced swallowing reflexes and the associated SLN activity. This attenuation indicates that TRPV4 activation contributes directly to the sensory input driving the water-induced swallowing reflex. Several mechanisms may underlie TRPV4 activation following water application (Fig. 7 ). As a hypo-osmotic sensor 18 – 20 , TRPV4 can be activated by osmotic tension across the cell membrane resulting from the hypoosmotic nature of water. In addition, TRPV4 may be activated by cellular and axonal swelling caused by water entry through aquaporins or by other mechanisms that induce mechanical stretching of the plasma membrane 27 , 28 . Previous studies have shown that airway epithelial cells undergo volume changes in response to alterations in luminal osmolarity 50 , 51 . TRPV4 has been demonstrated to respond to both osmotic and mechanical stimuli 20 – 22 , 52 , 53 . In our SLN recordings, water application produced a gradual increase in neural activity (Fig. 5 ), suggesting that membrane stretching due to cell or axonal volume expansion may account, at least in part, for the delayed phase of activation. This interpretation is further supported by the greater inhibitory effect of the TRPV4 antagonist on the late component of SLN activity, which is consistent with stretch-induced TRPV4 activation. In agreement with this notion, previous studies have reported that long-latency and long-duration water-responsive SLN afferents did not respond to isotonic solutions, indicating that their activation depends on the hypoosmolarity of water 24 – 26 . Previous studies have shown that laryngeal taste buds contribute to water-induced swallowing, likely through ATP release and the consequent activation of purinergic receptors on afferent nerve fibers 54 . However, the specific receptor mediating water-induced activation remained unidentified 55 . Our finding that TRPV4 is expressed in taste bud cells suggests that water may activate TRPV4, leading to intracellular calcium influx and subsequent ATP release. The released ATP could then activate purinergic receptors on afferent fibers, thereby initiating neural signaling. Similarly, a previous study reported that laryngeal neuroendocrine cells—a specialized subset of epithelial cells—are activated by water and release ATP to excite afferent nerves 56 . That study also demonstrated that the neuroendocrine cells are sensitive to hypoosmotic stimulation below 75 mOSm/L 56 . Although neuroendocrine cells were not specifically labeled in the present study, TRPV4 expression in epithelial cells suggests that these populations may also participate in DW-induced signaling. This model is further supported by our previous finding that P2X3 receptors are predominantly expressed on nerve fibers that innervate the epithelium and taste buds in swallowing-related regions 39 . Other studies have also reported P2X2, P2X3, and P2Y1 receptors expression in these areas 54 , 55 , 57 – 61 . Moreover, water failed to evoke swallowing reflexes in P2X2/P2X3 double knockout mice 54 , reinforcing the importance of purinergic signaling in this process. In addition, application of hypoosmotic solutions has been shown to augment ATP release in animal and human airways 62 – 65 , while TRPV4 antagonists or gene knockdown impaired hypoosmotic-evoked ATP release from airway epithelial cells 63 . Together with our previous observation that topical application of exogenous ATP elicits swallowing reflexes 39 , these findings support a mechanism in which TRPV4 activation in epithelial and taste bud cells induces ATP release, thereby activating purinergic receptors on afferent fibers (Fig. 7 ). The ensuing excitation of these afferents enhances sensory input to the swallowing central pattern generator (sCPG) in the brainstem, initiating the swallowing reflex (Fig. 7 ). Furthermore, TRPV4 expression by intraepithelial and subepithelial nerve fibers suggests that water may also directly activate these fibers (Fig. 7 ), given that the airway epithelium permits water movement through both transcellular and paracellular pathways 66 , 67 . Supporting this notion, previous studies have reported that hypoosmotic stimulation depolarized vagal sensory nerves in guinea pigs, mice, and humans—effects that were inhibited by TRPV4 antagonists and attenuated in vagal preparations from Trpv4−/− mice 68 . Although treatment with the TRPV4 antagonist significantly reduced the water-induced swallowing reflex in a dose-dependent manner, it did not completely abolish the reflex, even at concentrations of up to 100 mM. This partial inhibition indicates that, in addition to TRPV4, other receptors or ion channels may contribute to the water-induced swallowing reflex. In this context, earlier electrophysiological and behavioral studies have demonstrated that laryngeal and pharyngeal water-sensitive receptors are influenced by the ionic composition of the stimulating solution, particularly by anions such as chloride 5 , 6 , 15 . Furthermore, anion channels, including volume-regulated anion channels and calcium-activated chloride channels, have been implicated in epithelial osmosensation and fluid homeostasis, which raises the possibility that such channels may also modulate sensory responses to water 69 – 71 . Further studies are warranted to elucidate the contributions of these additional mechanisms. Our findings regarding TRPV4 involvement in the water-induced swallowing reflex have important clinical implications. Patients with oropharyngeal dysphagia often experience difficulty swallowing liquids. Current management strategies primarily rely on compensatory techniques (e.g., modifying bolus viscosity or employing postural adjustments such as the chin-tuck maneuver) and rehabilitative exercises, which have demonstrated limited efficacy 10 , 72 , 73 . Consequently, therapeutic strategies that target the sensory modulation of swallowing are urgently required. Recently, chemical neurostimulation, which targets chemosensory ion channels in the peripheral swallowing-related regions including TRP channels, has shown promising results in improving swallowing safety and efficiency in both preclinical and clinical studies 11 , 12 , 17 , 74 , 75 . Understanding the molecular mechanisms underlying water-induced swallowing reflexes may therefore provide valuable insights that could help develop pharmacological interventions for oropharyngeal dysphagia. Our previous observation that TRPV4 agonists facilitate swallowing 23 further suggests that these compounds may represent promising therapeutic candidates for patients who experience difficulty in swallowing liquids. Such approaches merit further investigation in clinical contexts. Finally, topical application of a local anesthetic to the peripheral swallowing-related regions completely abolished the water-induced swallowing reflex, underscoring the essential role of sensory afferents in mediating this response. To delineate the specific contribution of the SLN, we recorded swallowing reflexes in rats with intact SLNs while transecting other nerves, including bilateral IX-ph, X-ph, IX-li, and RLN. In a separate experiment, bilateral transection of the SLNs completely abolished the swallowing reflex elicited by water application to SLN-innervated regions. These findings confirm that SLN afferents play a critical role in transmitting the sensory input needed to initiate the water-induced swallowing reflex under our experimental conditions. In conclusion, our findings identify TRPV4 as a molecular sensor responsible for water-induced activation of the swallowing reflex. By showing that TRPV4 is involved in this response, the present study enhances our understanding of the sensory regulation of swallowing and highlights TRPV4 as a potential therapeutic target for improving swallowing function in patients with oropharyngeal dysphagia. Declarations Author contributions MZH and JK: Conceptualize and design the work. MZH, HA, RRR, and JK: acquisition, analysis, or interpretation of data. MZH, HA, RRR, and JK: Draft or revise the manuscript critically. All authors have approved the final version of the manuscript. All persons designated as authors qualify for authorship. Funding This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI, grant numbers # 24K08797 to Junichi Kitagawa, # 25K09007 to Hiroshi Ando, # 24K19847 to Rita Rani Roy, and # 24K12880 to Mohammad Zakir Hossain. The grant-providing society had no role in the study design, data collection, analysis, or interpretation. Competing interests The authors declare no competing interests. Data availability statement The data underlying this article will be made available to other researchers from the authors upon reasonable request. References Jean, A. Brain stem control of swallowing: Neuronal network and cellular mechanisms. Physiological Reviews vol. 81 929–969 (2001). Shingai, T. & Shimada, K. Reflex swallowing elicited by water and chemical substances applied in the oral cavity, pharynx, and larynx of the rabbit. Jpn. J. Physiol. 26 , 455–469 (1976). Miller, F. R. & Sherrington, C. S. 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Oropharyngeal dysphagia in older persons – from pathophysiology to adequate intervention: A review and summary of an international expert meeting. Clin. Interv. Aging 11 , 189–208 (2016). Langmore, S. E. & Pisegna, J. M. Efficacy of exercises to rehabilitate dysphagia: A critique of the literature. Int. J. Speech. Lang. Pathol. 17 , 222–229 (2015). Cheng, I., Sasegbon, A. & Hamdy, S. Effects of pharmacological agents for neurogenic oropharyngeal dysphagia: A systematic review and meta-analysis. Neurogastroenterol. Motil. 34 , e14220 (2022). Clavé, P., Ortega, O., Rofes, L., Alvarez-Berdugo, D. & Tomsen, N. Brain and Pharyngeal Responses Associated with Pharmacological Treatments for Oropharyngeal Dysphagia in Older Patients. Dysphagia vol. 38 1449–1466 (2023). Additional Declarations No competing interests reported. Supplementary Files Supplementaryfigureswithfigurelegends.docx Supplementary Figure 1. Respiratory chest movements and electromyographic (EMG) activity of the mylohyoid and rectus abdominis muscles following saline and distilled water (DW) administration. (A) Representative recordings of respiratory chest movements and EMG activity of the mylohyoid and rectus abdominis muscles after saline administration. The black arrowhead indicates when the saline was delivered. Saline administration evoked a single high-amplitude EMG burst in the mylohyoid muscle, corresponding to the swallowing reflex. This reflex was accompanied by a brief respiratory pause (apnea) without any detectable EMG activity in the rectus abdominis muscle. The inset shows an expanded view of the section outlined by the dotted line. (B) Representative recordings following DW administration. The black arrowhead indicates when the DW was delivered. DW application elicited multiple high-amplitude EMG bursts in the mylohyoid muscle within a short period, accompanied by complete cessation of respiratory movements. No EMG activity was observed in the rectus abdominis muscle. The absence of inspiratory and expiratory chest movements, together with the lack of rectus abdominis activity, confirmed that the high-amplitude EMG bursts represented swallowing reflexes rather than cough reflexes. As respiration gradually resumed after repeated reflex triggering, subsequent swallowing reflexes occurred at longer intervals, again without rectus abdominis activation. The inset shows an expanded view of the section outlined by the dotted line. Laryngeal movements associated with the swallowing reflex were also visually observed. Chest movements were recorded using a piezoelectric transducer, which also detected small, heartbeat-associated movements that became evident during respiratory cessation induced by repetitive swallowing reflexes following DW administration. I, inspiratory direction of chest movement; E, expiratory direction of chest movement; S, seconds. Supplementary Figure 2. Positive and negative controls for the TRPV4 and c-Fos antibodies. (A) Representative photomicrograph of a rat trigeminal ganglion (TG) section used as a positive control for the anti-c-Fos (rabbit polyclonal anti-c-Fos antibody; Cat# ab190289; Abcam, Cambridge, UK) and anti-TRPV4 (Alexa Fluor 488–conjugated rabbit monoclonal anti-TRPV4 antibody; Cat# ab315136; Abcam, Cambridge, UK) antibodies. White arrows indicate cells positive for both c-Fos and TRPV4; yellow arrows indicate neurons positive for TRPV4 but negative for c-Fos; and white arrowheads indicate cells negative for TRPV4 but positive for c-Fos. Nonspecific cytoplasmic c-Fos signals are visible in the positive control section. High-magnification images of the cells outlined by the dotted circles are shown in the right panels; the nuclei were counterstained with 4ʹ,6-diamidino-2-phenylindole (DAPI). DAPI stained the nuclei of both the neurons and the surrounding satellite cells. To induce c-Fos expression in the TG, capsaicin (0.05 mM, 50 μL) was injected into the right vibrissal pad and lower lip. (B) Representative photomicrograph of a nodose ganglion section that was used as a negative control for the anti-c-Fos and anti-TRPV4 antibodies. A universal negative control reagent containing a mixture of purified rabbit, mouse, and goat immunoglobulins was substituted for the primary antibodies. High-magnification images of the cells outlined by the dotted circles are shown in the right panels, with DAPI nuclear counterstaining. As in the positive control, DAPI stained the nuclei of both the neurons and the surrounding satellite cells. Nonspecific cytoplasmic c-Fos signals were observed in the negative control section. (C) Representative photomicrographs of a section from the swallowing-related region (epiglottis) innervated by the superior laryngeal nerve that was used as an additional negative control for the anti-TRPV4 antibody. DAPI staining was used to visualize cell nuclei. As with the nodose ganglion, a universal negative control reagent containing a mixture of purified rabbit, mouse, and goat immunoglobulins was substituted for the primary antibody. Supplementary Figure 3. Prior topical lidocaine application or bilateral superior laryngeal nerve (SLN) transection prevented distilled water (DW) from inducing swallowing and apnea. (A) Representative recordings of respiratory chest movements and electromyographic (EMG) activity of the mylohyoid and rectus abdominis muscles following DW administration after topical lidocaine application. The black arrowhead indicates when the DW was delivered. No swallowing reflexes or apnea were observed following DW application. The inset shows an expanded view of the section outlined by the dotted line. (B) Representative recordings of respiratory chest movements and EMG activity of the mylohyoid and rectus abdominis muscles following DW administration after bilateral transection of the SLNs. The black arrowhead indicates when the DW was delivered. Consistent with panel A, no swallowing reflexes or apnea were observed after DW application. I, inspiratory direction of chest movement; E, expiratory direction of chest movement; S, seconds. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8706920","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":585146904,"identity":"809dfb92-2e5f-4d21-a516-f643ef76770b","order_by":0,"name":"Mohammad Zakir Hossain","email":"data:image/png;base64,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","orcid":"","institution":"Matsumoto Dental University","correspondingAuthor":true,"prefix":"","firstName":"Mohammad","middleName":"Zakir","lastName":"Hossain","suffix":""},{"id":585146906,"identity":"6d384306-0d4a-4e90-9a65-e592431b9311","order_by":1,"name":"Hiroshi Ando","email":"","orcid":"","institution":"Matsumoto Dental University","correspondingAuthor":false,"prefix":"","firstName":"Hiroshi","middleName":"","lastName":"Ando","suffix":""},{"id":585146911,"identity":"b9de3d25-af9d-40d7-8dc3-5d26082ce1eb","order_by":2,"name":"Rita Rani Roy","email":"","orcid":"","institution":"Matsumoto Dental University","correspondingAuthor":false,"prefix":"","firstName":"Rita","middleName":"Rani","lastName":"Roy","suffix":""},{"id":585146912,"identity":"4b4fdcdf-9ddb-4c5d-9058-572d4d68f0ca","order_by":3,"name":"Junichi Kitagawa","email":"","orcid":"","institution":"Matsumoto Dental University","correspondingAuthor":false,"prefix":"","firstName":"Junichi","middleName":"","lastName":"Kitagawa","suffix":""}],"badges":[],"createdAt":"2026-01-27 07:09:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8706920/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8706920/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-026-51222-6","type":"published","date":"2026-04-29T15:58:11+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":101876376,"identity":"e23602f0-fac2-4479-bf3f-d7d4869f7643","added_by":"auto","created_at":"2026-02-04 14:13:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":261572,"visible":true,"origin":"","legend":"\u003cp\u003eTopical application of distilled water (DW) facilitates swallowing. \u003cstrong\u003e(A) \u003c/strong\u003eRepresentative recordings of swallowing reflexes, as detected by electromyographic (EMG) activity of the mylohyoid muscle, following topical application of saline or DW. The black arrowheads indicate when the stimulating solution was delivered. \u003cstrong\u003e(B)\u003c/strong\u003e Comparison of the number of swallowing reflexes evoked by saline and DW. Statistical analysis was performed using a paired t-test (n = 5). Data are presented as the mean ± standard deviation, with individual values shown as points. S, seconds.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8706920/v1/442454d1f6dbc27b28c9aa4a.png"},{"id":101876365,"identity":"0786c82a-a6e5-45d5-a223-a05f89155a33","added_by":"auto","created_at":"2026-02-04 14:13:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":7201158,"visible":true,"origin":"","legend":"\u003cp\u003eTRPV4 expression in peripheral swallowing-related regions. \u003cstrong\u003e(A)\u003c/strong\u003e Schematic illustration of the peripheral swallowing-related regions innervated by the superior laryngeal nerve. The boxes in the enlarged image outline the areas from which photomicrographs were obtained. \u003cstrong\u003e(B–J)\u003c/strong\u003eRepresentative photomicrographs showing TRPV4 (red) immunoreactivity in the \u003cstrong\u003e(B, C)\u003c/strong\u003e epiglottis (EP), \u003cstrong\u003e(D)\u003c/strong\u003e epiglottic vallecula (EV), \u003cstrong\u003e(E)\u003c/strong\u003e base of the epiglottis, \u003cstrong\u003e(F, G)\u003c/strong\u003e vestibular fold (VeF), and \u003cstrong\u003e(H–J)\u003c/strong\u003e taste bud–like structures. The white arrowheads indicate intraepithelial nerve fibers expressing TRPV4. The thick white arrows denote larger nerves expressing TRPV4. The yellow arrowheads indicate nerve fibers expressing TRPV4 located between the epithelium and lamina propria. The thin white arrows indicate epithelial cells expressing TRPV4. The yellow arrows highlight TRPV4-expressing taste bud cells. The curved white arrows indicate nerve fibers or subgemmal neurogenic plaques associated with taste buds expressing TRPV4. TRPV4 expression in peripheral swallowing-related regions was analyzed in three rats, and the photomicrographs shown are representative images. LAR, larynx; T, tongue; ES, Esophagus.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8706920/v1/fb611613a7e8e2cd4f849818.png"},{"id":101876348,"identity":"a2006785-5b00-49de-afee-2781837f4cdf","added_by":"auto","created_at":"2026-02-04 14:13:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4942525,"visible":true,"origin":"","legend":"\u003cp\u003eTRPV4 expression in c-Fos–expressing NPJc neurons induced by distilled water application to peripheral swallowing-related regions. \u003cstrong\u003e(A)\u003c/strong\u003eRepresentative photomicrographs showing TRPV4 expression in c-Fos–expressing neurons within the nodose ganglion (NG), petrosal ganglion (PG), and jugular ganglion (JG). White arrows indicate neurons that are positive for both TRPV4 and c-Fos, yellow arrows indicate neurons that are positive for TRPV4 but negative for c-Fos, and white arrowheads indicate neurons that are negative for TRPV4 but positive for c-Fos. High-magnification images of the neurons within the dotted circles are shown in the right panels; the nuclei were counterstained with 4ʹ,6-diamidino-2-phenylindole (DAPI). Note that DAPI stained the nuclei of both the neurons and the surrounding satellite cells. \u003cstrong\u003e(B)\u003c/strong\u003e Quantification of c-Fos–stained, TRPV4-positive, and TRPV4-negative neurons per animal in the NG, PG, and JG and across the entire NPJc. Data are presented as the mean ± standard deviation (n = 6). Every third section (16 μm) of the right or left NPJc from each animal was processed for immunohistochemistry, and the total number of c-Fos–stained TRPV4-positive and TRPV4-negative neurons was counted for each animal. Each dot represents an individual data point. \u003cstrong\u003e(C)\u003c/strong\u003ePercentage of c-Fos–stained, TRPV4-positive, and TRPV4-negative neurons in the NG, PG, and JG and across the entire NPJc. c-Fos(+)/TRPV4(+), c-Fos–stained neurons immunopositive for TRPV4; c-Fos(+)/TRPV4(−), c-Fos–stained neurons immunonegative for TRPV4.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8706920/v1/4cfb4fab85d0997d2126d163.png"},{"id":101876333,"identity":"9a2fc059-6ed5-41ad-b89d-c743678b01f1","added_by":"auto","created_at":"2026-02-04 14:13:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":844273,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of topical pretreatment with the TRPV4 antagonist RN9893 on the number of distilled water (DW)-induced swallowing reflexes. \u003cstrong\u003e(A) \u003c/strong\u003eRepresentative recordings of swallowing reflexes induced by DW following pretreatment with various concentrations of RN9893 or vehicle. Black arrowheads indicate when the stimulating solution was delivered. \u003cstrong\u003e(B)\u003c/strong\u003eThe number of swallowing reflexes induced by DW with and without pretreatment with different concentrations of RN9893 or vehicle. Statistical analysis was performed using one-way repeated measures analysis of variance followed by Tukey’s post hoc test (n = 5 for each group). Data are presented as the mean ± standard deviation. Individual data points are shown as dots. S, seconds; EMG, electromyography.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8706920/v1/c8de88bcc3f3fee90094bf79.png"},{"id":101876340,"identity":"77198977-bfe4-4d17-b02f-23619821e7b7","added_by":"auto","created_at":"2026-02-04 14:13:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1127388,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of topical pretreatment with the TRPV4 antagonist RN9893 on distilled water (DW)-induced superior laryngeal nerve (SLN) activity. \u003cstrong\u003e(A) \u003c/strong\u003eRepresentative recordings of SLN activity induced by saline, DW alone, DW following pretreatment with vehicle, and DW following pretreatment with the TRPV4 antagonist. Black arrowheads indicate when the stimulating solution was delivered. \u003cstrong\u003e(B)\u003c/strong\u003e SLN activity induced by saline, DW alone, DW following pretreatment with vehicle, and DW following pretreatment with the TRPV4 antagonist. SLN activity was defined as the area of the integrated response above baseline. The integrated SLN response was calculated in 2-second bins beginning with solution infusion and continuing for 20 seconds following stimulation. The stable baseline activity recorded for 2 seconds prior to stimulation was subtracted from each corresponding 2-second response value. Statistical analysis was performed using one-way repeated-measures analysis of variance followed by Tukey’s post hoc test for each 2-second bin among the different groups (n = 5). Data are presented as the mean ± standard deviation. S, seconds.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8706920/v1/ca16e1fae5e1cf7d6d78ffbc.png"},{"id":101876379,"identity":"36191749-6ebc-4cb6-a773-672305cdf8d4","added_by":"auto","created_at":"2026-02-04 14:13:13","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":624136,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of topical lidocaine administration or bilateral superior laryngeal nerve (SLN) transection on subsequent distilled water (DW)-induced swallowing reflexes. \u003cstrong\u003e(A) \u003c/strong\u003eRepresentative recordings of swallowing reflexes triggered by DW with or without prior topical application of 2% lidocaine or bilateral SLN transection. Black arrowheads indicate when DW was delivered. \u003cstrong\u003e(B)\u003c/strong\u003e The number of swallowing reflexes triggered by DW with and without prior topical lidocaine application. \u003cstrong\u003e(C) \u003c/strong\u003eThe number of swallowing reflexes triggered by DW with and without prior bilateral SLN transection. Statistical analysis was performed using paired t-tests (n = 5). Data are presented as the mean ± standard deviation, and individual data points are represented by dots. EMG, electromyography.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8706920/v1/03f1473f78c75e1076450192.png"},{"id":101876378,"identity":"daf52b99-5d49-46b2-a82a-1940a4f152e0","added_by":"auto","created_at":"2026-02-04 14:13:12","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2189942,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the proposed mechanisms by which TRPV4 contributes to water-induced swallowing reflexes. Water may generate osmotic tension across the membranes of epithelial cells, taste bud cells, and afferent nerve axons, thereby activating the TRPV4 channels expressed on these structures. In addition, water entry into these cells and axons can induce swelling, leading to mechanical membrane stretching and further TRPV4 activation. TRPV4 activation in epithelial and taste bud cells promotes cation (e.g., Ca²⁺) influx, which triggers ATP release from these cells. The released ATP subsequently activates purinergic receptors on sensory afferent fibers, initiating action potential generation. Furthermore, TRPV4 activation on sensory nerve fibers directly causes cation influx and neuronal excitation. The increased activity of these afferents enhances sensory input to the sCPG and to cortical and subcortical swallowing-related regions, ultimately triggering the swallowing reflex.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8706920/v1/6cf079ccbecd62c66723d1c1.png"},{"id":108440293,"identity":"ecfb0c9a-a375-4aa5-bb4b-c7fe51239592","added_by":"auto","created_at":"2026-05-04 16:35:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":15984689,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8706920/v1/80a2faf1-ebab-43ce-8840-bd5d6588498f.pdf"},{"id":101881598,"identity":"293a63d2-16d1-4e10-9d7f-49f6290c5cb1","added_by":"auto","created_at":"2026-02-04 15:13:43","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":859922,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1.\u003c/strong\u003e Respiratory chest movements and electromyographic (EMG) activity of the mylohyoid and rectus abdominis muscles following saline and distilled water (DW) administration.\u003cstrong\u003e (A)\u003c/strong\u003e Representative recordings of respiratory chest movements and EMG activity of the mylohyoid and rectus abdominis muscles after saline administration. The black arrowhead indicates when the saline was delivered. Saline administration evoked a single high-amplitude EMG burst in the mylohyoid muscle, corresponding to the swallowing reflex. This reflex was accompanied by a brief respiratory pause (apnea) without any detectable EMG activity in the rectus abdominis muscle. The inset shows an expanded view of the section outlined by the dotted line. \u003cstrong\u003e(B)\u003c/strong\u003e Representative recordings following DW administration. The black arrowhead indicates when the DW was delivered. DW application elicited multiple high-amplitude EMG bursts in the mylohyoid muscle within a short period, accompanied by complete cessation of respiratory movements. No EMG activity was observed in the rectus abdominis muscle. The absence of inspiratory and expiratory chest movements, together with the lack of rectus abdominis activity, confirmed that the high-amplitude EMG bursts represented swallowing reflexes rather than cough reflexes. As respiration gradually resumed after repeated reflex triggering, subsequent swallowing reflexes occurred at longer intervals, again without rectus abdominis activation. The inset shows an expanded view of the section outlined by the dotted line. Laryngeal movements associated with the swallowing reflex were also visually observed. Chest movements were recorded using a piezoelectric transducer, which also detected small, heartbeat-associated movements that became evident during respiratory cessation induced by repetitive swallowing reflexes following DW administration. I, inspiratory direction of chest movement; E, expiratory direction of chest movement; S, seconds.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2.\u003c/strong\u003e Positive and negative controls for the TRPV4 and c-Fos antibodies. \u003cstrong\u003e(A)\u003c/strong\u003e Representative photomicrograph of a rat trigeminal ganglion (TG) section used as a positive control for the anti-c-Fos (rabbit polyclonal anti-c-Fos antibody; Cat# ab190289; Abcam, Cambridge, UK) and anti-TRPV4 (Alexa Fluor 488–conjugated rabbit monoclonal anti-TRPV4 antibody; Cat# ab315136; Abcam, Cambridge, UK) antibodies. White arrows indicate cells positive for both c-Fos and TRPV4; yellow arrows indicate neurons positive for TRPV4 but negative for c-Fos; and white arrowheads indicate cells negative for TRPV4 but positive for c-Fos. Nonspecific cytoplasmic c-Fos signals are visible in the positive control section. High-magnification images of the cells outlined by the dotted circles are shown in the right panels; the nuclei were counterstained with 4ʹ,6-diamidino-2-phenylindole (DAPI). DAPI stained the nuclei of both the neurons and the surrounding satellite cells. To induce c-Fos expression in the TG, capsaicin (0.05 mM, 50 μL) was injected into the right vibrissal pad and lower lip. \u003cstrong\u003e(B)\u003c/strong\u003e Representative photomicrograph of a nodose ganglion section that was used as a negative control for the anti-c-Fos and anti-TRPV4 antibodies. A universal negative control reagent containing a mixture of purified rabbit, mouse, and goat immunoglobulins was substituted for the primary antibodies. High-magnification images of the cells outlined by the dotted circles are shown in the right panels, with DAPI nuclear counterstaining. As in the positive control, DAPI stained the nuclei of both the neurons and the surrounding satellite cells. Nonspecific cytoplasmic c-Fos signals were observed in the negative control section. \u003cstrong\u003e(C)\u003c/strong\u003e Representative photomicrographs of a section from the swallowing-related region (epiglottis) innervated by the superior laryngeal nerve that was used as an additional negative control for the anti-TRPV4 antibody. DAPI staining was used to visualize cell nuclei. As with the nodose ganglion, a universal negative control reagent containing a mixture of purified rabbit, mouse, and goat immunoglobulins was substituted for the primary antibody.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 3.\u003c/strong\u003e Prior topical lidocaine application or bilateral superior laryngeal nerve (SLN) transection prevented distilled water (DW) from inducing swallowing and apnea. \u003cstrong\u003e(A)\u003c/strong\u003e Representative recordings of respiratory chest movements and electromyographic (EMG) activity of the mylohyoid and rectus abdominis muscles following DW administration after topical lidocaine application. The black arrowhead indicates when the DW was delivered. No swallowing reflexes or apnea were observed following DW application. The inset shows an expanded view of the section outlined by the dotted line. \u003cstrong\u003e(B)\u003c/strong\u003e Representative recordings of respiratory chest movements and EMG activity of the mylohyoid and rectus abdominis muscles following DW administration after bilateral transection of the SLNs. The black arrowhead indicates when the DW was delivered. Consistent with panel A, no swallowing reflexes or apnea were observed after DW application. I, inspiratory direction of chest movement; E, expiratory direction of chest movement; S, seconds.\u003c/p\u003e","description":"","filename":"Supplementaryfigureswithfigurelegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-8706920/v1/0d19d725e114552c06d21471.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Transient receptor potential vanilloid 4 channels contribute to the initiation of water-induced swallowing reflexes","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe swallowing reflex is a critical component of the digestive process, facilitating the passage of food and liquids from the oral cavity to the esophagus \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Previous studies have demonstrated that applying water to the pharyngeal and laryngeal regions can elicit swallowing in both animals \u003csup\u003e\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e and humans \u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. However, the molecular mechanisms underlying this water-induced swallowing reflex remain unclear. Patients with dysphagia often experience difficulty swallowing liquids \u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, highlighting the need to better understand the molecular basis of this reflex in order to develop effective therapeutic interventions \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe laryngopharynx and associated laryngeal regions, which are innervated by the superior laryngeal nerve (SLN)\u0026mdash;a branch of the vagus nerve\u0026mdash;have been shown to play a key role in initiating the swallowing reflex in response to water stimulation \u003csup\u003e\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan additionalcitationids=\"CR14 CR15 CR16\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Applying water to these regions increases SLN activity and triggers the swallowing reflex \u003csup\u003e\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTRPV4 channels are non-selective cation channels that function as polymodal receptors that respond to various stimuli, including hypoosmotic conditions \u003csup\u003e\u003cspan additionalcitationids=\"CR19 CR20 CR21\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In a previous study, we observed that activation of TRPV4 channels in SLN-innervated regions using the chemical agonist GSK1016790A effectively induced the swallowing reflex \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Given that water is inherently hypoosmotic, it is plausible that it can activate TRPV4 channels when applied to swallowing-related regions. Supporting this hypothesis, earlier studies suggest that the responsiveness of laryngeal receptors to water is partially mediated by its hypoosmotic properties \u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. In addition, water entry into the cell can activate TRPV4 channels, which respond to changes in cell volume \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe objective of this study was to determine whether TRPV4 channels contribute to initiation of the water-induced swallowing reflex.\u003c/p\u003e"},{"header":"Methods","content":"\u003ch2\u003eAnimals\u003c/h2\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp;Experiments were conducted using 64 male Sprague-Dawley rats, weighing approximately 300–450 g. Of these, nine rats were used for immunohistochemistry and fifty-five for assessment of the swallowing reflex. All experimental procedures were approved by the Intramural Animal Care and Veterinary Science Committee of Matsumoto Dental University (Ref. No. 394). Every effort was made to minimize animal suffering and reduce the number of animals used. Animals were housed in the Matsumoto Dental University animal facility under controlled conditions (temperature: 22 ± 2 °C; relative humidity: 40 ± 5%; light/dark cycle: 12/12 hours). Food and water were available \u003cem\u003ead libitum\u003c/em\u003e. All procedures adhered to the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines established by the National Centre for the Replacement, Refinement, and Reduction of Animals in Research.\u003c/p\u003e\n\u003ch2\u003eSurgical procedure for swallowing reflex recording\u003c/h2\u003e\n\u003cp\u003eRats were anesthetized with urethane (1.0–1.5 g/kg, intraperitoneally) and positioned in a supine posture. A midline incision was made in the ventral neck region to expose the trachea. Respiration was maintained via a custom-made cannula inserted into the trachea and directed toward the lungs. A small ventral portion of the trachea was surgically removed just behind the cricoid cartilage to create a window for solution delivery. To isolate the SLNs as the primary neural input for the swallowing reflex, the pharyngeal (IX-ph) and lingual (IX-li) branches of the glossopharyngeal nerve, as well as the pharyngeal (X-ph) and recurrent laryngeal nerve (RLN) branches of the vagus nerve, were bilaterally transected. The bilateral SLNs were left intact during the main experiments to assess their role in triggering the swallowing reflex. In a separate experimental series, the bilateral SLNs were also transected, along with the aforementioned nerves, to confirm the specific involvement of SLNs in the initiation of the swallowing reflex under the experimental conditions.\u003c/p\u003e\n\u003ch2\u003eStimulating solutions\u003c/h2\u003e\n\u003cp\u003eThe solutions used for stimulation were 0.9% saline (NaCl; Otsuka Pharmaceutical Co. Ltd., Tokyo, Japan) and DW. Each solution was delivered to the swallowing-related regions innervated by the SLN in a 50-μl volume using a syringe equipped with a blunted 21-gauge needle. All solutions were at room temperature (22–24 °C) when applied.\u003c/p\u003e\n\u003cp\u003eFollowing each application, reflex responses were recorded for 20 seconds. The interval between successive solution deliveries was 2–3 minutes. During this period, the previously administered solution was aspirated, and the target region was thoroughly rinsed with saline to prevent cross-contamination.\u003c/p\u003e\n\u003ch2\u003eElectromyographic identification of the swallowing reflex\u003c/h2\u003e\n\u003cp\u003eElectromyographic (EMG) recordings from the mylohyoid muscle, combined with visual confirmation of laryngeal movements, were used to identify and quantify swallowing reflex events. The mylohyoid muscle, along with other supra- and infrahyoid muscles, is activated during initiation of the swallowing reflex \u003csup\u003e1\u003c/sup\u003e. Recording the activity of one or more of these muscles is a well-established method that has been widely adopted as a standardized method for detecting the swallowing reflex. Previous studies, including those from our group, have reliably identified swallowing reflexes based on high-amplitude EMG activity in the mylohyoid muscle in conjunction with visible laryngeal elevation \u003csup\u003e17,23,29–32\u003c/sup\u003e. EMG signals were recorded using bipolar fine-wire electrodes (stainless steel, urethane-coated; Unique Medical Co., Ltd., Tokyo, Japan). A loudspeaker connected to the EMG system facilitated auditory monitoring of mylohyoid muscle activity during data collection.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;To ensure that the observed high-amplitude EMG bursts in the mylohyoid muscle represented swallowing reflexes—rather than coughing or other airway defense responses—respiratory chest movements and EMG activity from both the mylohyoid and rectus abdominis muscles were simultaneously recorded in some animals (Supplemental Figure 1A, B). The rectus abdominis muscle was included because it is characteristically activated during the cough reflex \u003csup\u003e33\u003c/sup\u003e. Respiratory chest movements were recorded using a piezoelectric transducer (ADInstruments, Inc., Colorado Springs, CO).\u0026nbsp;EMG activity from the rectus abdominis muscle was monitored using bipolar fine-wire electrodes of the same type (stainless steel, urethane-coated; Unique Medical Co., Ltd., Tokyo, Japan). All signals were amplified and filtered using a bioelectric amplifier (MEG-5200; Nihon Kohden, Tokyo, Japan), digitized via a Power 1401 data acquisition system (Cambridge Electronic Design Ltd., Cambridge, UK), and stored on a hard drive for subsequent analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUpon DW application to SLN-innervated swallowing-related regions, repetitive high-amplitude EMG bursts were observed in the mylohyoid muscle, accompanied by a temporary cessation of respiration (apnea) (Supplemental Figure 1B). Notably, no EMG activity was detected in the rectus abdominis muscle. The absence of both respiratory chest movements and rectus abdominis activity confirmed that the EMG bursts in the mylohyoid muscle were due to swallowing reflexes. This is because the cough reflex is typically characterized by inspiratory chest movements followed by forceful expiratory movements accompanying the contractions of abdominal muscles, including the rectus abdominis \u003csup\u003e33,34\u003c/sup\u003e.\u0026nbsp;As respiration gradually resumed, additional swallowing reflexes occurred at increasing intervals, again without rectus abdominis involvement. Saline application induced only a single swallowing reflex, associated with a brief pause in respiration (Supplemental Figure 1A). These observations confirm that the repetitive EMG bursts observed following DW application specifically represent swallowing reflex activity.\u003c/p\u003e\n\u003ch2\u003eImmunohistochemistry of superior laryngeal nerve-innervated swallowing-related regions\u003c/h2\u003e\n\u003cp\u003eDeeply anaesthetized rats were subjected to transcardial perfusion with saline followed by 4% paraformaldehyde. The swallowing-related regions were dissected then immersed in 4% paraformaldehyde for 3 hours. The specimens were subsequently incubated in 30% sucrose until fully submerged to ensure adequate cryoprotection. The samples were then embedded in Tissue-Tek Optimal Cutting Temperature Compound (Sakura Finetek, Tokyo, Japan) and sectioned in the sagittal plane (10 or 50 μm) before being mounted on glass slides.\u003c/p\u003e\n\u003cp\u003eThe sections were incubated with 5% normal goat serum in 0.01 M phosphate-buffered saline containing 0.05% Triton X-100 for 1 hour to minimize nonspecific binding. Subsequently, the sections were incubated with rabbit monoclonal anti-TRPV4 (1:100; Cat# ab259361; Abcam, Cambridge, UK) at 4°C overnight, followed by incubation with a fluorescent dye-conjugated secondary antibody (Goat anti-rabbit Alexa Fluor 594; Cat# A-11037; Thermo Fisher Scientific, Waltham, MA) for 1 hour at room temperature. The sections were then treated with 4ʹ,6-diamidino-2-phenylindole (DAPI) for 10 minutes to visualize the cell nuclei. Finally, coverslips were applied using mounting medium (PermaFluor; Thermo Fisher Scientific), and the specimens were examined by fluorescence microscopy (BZ-X700; Keyence Corp., Osaka, Japan).\u003c/p\u003e\n\u003ch2\u003eImmunohistochemistry analysis of TRPV4 expression in the nodose–petrosal–jugular ganglionic complex\u0026nbsp;in relation to distilled water-induced c-Fos expression\u003c/h2\u003e\n\u003cp\u003eThe rats were anesthetized with urethane (1.0–1.5 g/kg, intraperitoneally), the trachea was carefully exposed, and a small window was created just behind the cricoid cartilage for DW delivery. The SLN, IX-ph, IX-li, X-ph, and RLN were left intact to avoid nerve transection–induced c-Fos expression. Following surgery, the rats were maintained in a supine position for 4 hours to minimize surgery-related c-Fos expression, as reported previously \u003csup\u003e35,36\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFour hours after surgery, DW was applied to the SLN-innervated swallowing-related regions 15 times (50 μL per application, every 2 minutes). Each application was maintained for 30 seconds and then aspirated, for a total duration of 30 minutes. Rats were subsequently kept in the supine position for 1 hour because c-Fos expression is known to peak approximately 1 hour following stimulation \u003csup\u003e37,38\u003c/sup\u003e. One hour after the final DW application, the deeply anesthetized rats were perfused transcardially with saline, followed by 4% paraformaldehyde.\u003c/p\u003e\n\u003cp\u003eEach ganglion of the nodose–petrosal–jugular ganglionic complex (NPJc) was identified based on established anatomical criteria \u003csup\u003e39\u003c/sup\u003e. The nodose ganglion (NG) was distinguished by its attachment to the main trunk of the vagus nerve, oriented toward the thorax. The jugular ganglion (JG) was located in the direction of the brainstem, opposite the vagal trunk, whereas the petrosal ganglion (PG) was positioned between the NG and JG. To preserve anatomical orientation during embedding, the right or left NPJc was excised, along with a small segment of the vagus nerve trunk. The excised tissue was fixed in 4% paraformaldehyde, immersed in 30% sucrose until it sank, and then embedded in Tissue-Tek O.C.T. Compound (Sakura Finetek, Tokyo, Japan), maintaining its orientation. The tissue was sectioned into 16-μm-thick slices and mounted onto glass slides, allowing reliable identification of each ganglion after sectioning.\u003c/p\u003e\n\u003cp\u003eEvery third section was processed for fluorescent immunohistochemistry, using a combination of indirect and direct methods \u003csup\u003e40\u003c/sup\u003e to detect TRPV4 expression in c-Fos–expressing neurons. First, the sections were blocked in 0.01 M phosphate-buffered saline containing 5% normal goat serum and 0.05% Triton X-100 for 1 hour to minimize nonspecific binding. They were then incubated overnight at 4°C with rabbit polyclonal anti-c-Fos antibody (1:1000; Cat# ab190289; Abcam, Cambridge, UK), followed by goat anti-rabbit Alexa Fluor 594 secondary antibody (Cat# A-11037; Thermo Fisher Scientific) for 1 hour at room temperature. To prevent nonspecific binding of the prior secondary antibody, the sections were subsequently blocked with normal rabbit serum for 1 hour at room temperature \u003csup\u003e40\u003c/sup\u003e, followed by incubation with Alexa Fluor 488–conjugated rabbit monoclonal anti-TRPV4 antibody (1:100; Cat# ab315136; Abcam) for 48 hours at 4°C. Cell nuclei were visualized by counterstaining with DAPI for 10 minutes.\u003c/p\u003e\n\u003cp\u003eThe slides were coverslipped and imaged using a fluorescence microscope (BZ-X700; Keyence Corp.). Immunoreactive cells were quantified using ImageJ software (NIH, Bethesda, MD). Because c-Fos is a nuclear marker of neuronal activation expressed \u003csup\u003e41,42\u003c/sup\u003e, only neurons showing nuclear c-Fos expression were included in the counts, although nonspecific cytoplasmic c-Fos staining was also noted. Similar nonspecific cytoplasmic signals were observed on both positive and negative control slides (Supplemental Figure 2A, B). Therefore, only neurons with nuclear c-Fos expression were included in the counts, even if cytoplasmic expression was also present. Cells with cytoplasmic, but no nuclear, c-Fos expression were excluded. TRPV4 expression was not observed in nuclei. Neurons that were positive for both c-Fos (nuclear) and TRPV4, as well as those that were positive for c-Fos (nuclear) but negative for TRPV4, were quantified in the NG, PG, JG, and across the entire NPJc.\u003c/p\u003e\n\u003cp\u003eThe trigeminal ganglion (TG) was used as a positive control for the anti-c-Fos and anti-TRPV4 antibodies (Supplemental Figure 2A). To induce c-Fos expression in the TG, capsaicin (0.05 mM, 50 μL) was injected into the right vibrissal pad and lower lip in anesthetized rats. One hour later, rats were deeply anesthetized and perfused transcardially with saline, followed by 4% paraformaldehyde. The right TG was excised, fixed, cryoprotected, embedded in Tissue-Tek O.C.T Compound, sectioned at 16 μm intervals, and processed identically to the NPJc sections. As a negative control, sections of the NG, laryngopharynx, and associated laryngeal regions were incubated with a universal negative control reagent (Cat# ADI-950-231-0025; Enzo Life Sciences, Inc., Farmingdale, NY) instead of primary antibodies (Supplemental Figure 2B, C).\u003c/p\u003e\n\u003ch2\u003eTRPV4 antagonist\u003c/h2\u003e\n\u003cp\u003eA TRPV4 antagonist RN-9893 [(2E)-N-(2,3-dihydro-1,4-benzodioxin-6-yl)-3-[4-(1,1-dimethylethyl)phenyl]-2-propenamide] was obtained from Tocris Bioscience, Bristol, UK. The efficacy of RN-9893 as a selective TRPV4 antagonist has been validated previously \u003csup\u003e43,44\u003c/sup\u003e. RN-9893 was dissolved in a solution containing 3% dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO) and 1% Tween 80 (Sigma-Aldrich), then diluted with saline to the final concentration. The vehicle control consisted of the same DMSO-Tween 80-saline mixture without the antagonist. The prepared solution was instilled into the SLN-innervated swallowing-related region and left in place for 15 minutes. The solution was aspirated before applying DW.\u003c/p\u003e\n\u003ch2\u003eRecording superior laryngeal nerve activity\u003c/h2\u003e\n\u003cp\u003eThe sternothyroid muscle was bluntly dissected, and the SLN was carefully freed from the surrounding tissues. Both SLNs were transected distally, near their junction with the vagus nerve. A pair of bipolar silver wire electrodes (0.1 mm diameter) was placed unilaterally beneath the SLN. Liquid silicone was applied to secure the electrodes to the nerve, insulate them from surrounding tissues, and prevent desiccation, thereby enabling stable long-term recordings. SLN activity was amplified and integrated with a time constant of 0.3 s. The signals were digitized using a Power 1401 data acquisition system (Cambridge Electronic Design Ltd., Cambridge, UK) and stored for subsequent analysis.\u003c/p\u003e\n\u003ch2\u003eLidocaine\u003c/h2\u003e\n\u003cp\u003eTo determine whether sensory fibers in the SLN-innervated swallowing region contribute to the DW–induced swallowing reflex, 2% lidocaine (2-(diethylamino)-N-(2,6-dimethylphenyl)acetamide; Xylocaine®, AstraZeneca Ltd., Osaka, Japan) was applied topically. \u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eData and statistical analysis\u003c/h2\u003e\n\u003cp\u003eSwallowing reflexes elicited by stimulating solutions were counted over a 20-second period. SLN responses were analyzed as the area of the integrated response above baseline using Spike2 software (Cambridge Electronic Design Ltd.). The integrated SLN response was calculated in 2-second bins from the onset of solution infusion for 20 seconds following stimulation. The stable baseline activity recorded for 2 seconds prior to stimulation was subtracted from each corresponding 2-second response.\u003c/p\u003e\n\u003cp\u003eFor statistical evaluation, data were first tested for normality and homogeneity of variance to determine whether parametric or non-parametric tests were appropriate. A paired t-test was used to compare the number of swallowing reflexes triggered by saline versus DW. One-way repeated-measures analysis of variance (ANOVA), followed by Tukey’s post hoc test, was used to compare the number of swallowing reflexes induced by DW with or without prior application of the TRPV4 antagonist or its vehicle. Nerve responses to different stimulating solutions were compared using one-way repeated-measures ANOVA with Tukey’s post hoc test. In addition, paired t-tests were used to compare the number of DW-induced swallowing reflexes before and after lidocaine application or SLN transection.\u003c/p\u003e\n\u003cp\u003eA significance level of P \u0026lt; 0.05 was considered statistically significant. Data are presented as mean ± standard deviation (SD). Statistical analyses were performed using SigmaPlot software (version 14.0; Systat Software Inc., San Jose, CA). Graphs were generated using GraphPad Prism software (version 10.4; GraphPad Software, San Diego, CA).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eTopical application of distilled water facilitated the swallowing reflex\u003c/p\u003e\n\u003cp\u003eWe first examined whether DW application facilitated the swallowing reflex. Application of DW to SLN-innervated swallowing-related regions elicited significantly more swallowing reflexes (15 \u0026plusmn; 1.58) than application of saline (1 \u0026plusmn; 0.00) (Figure 1A, B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTRPV4 expression in peripheral swallowing-related regions\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe next investigated TRPV4 distribution in SLN-innervated swallowing-related regions (Figure 2) TRPV4 immunoreactivity was observed in both intraepithelial and subepithelial nerve fibers (Figure 2B\u0026ndash;D). In addition, TRPV4 expression was detected in epithelial cells located near the base of the epiglottis, aryepiglottic folds, arytenoids, and vestibular folds (Figure 2E\u0026ndash;G). Furthermore, TRPV4 immunoreactivity was present in the cells of taste bud\u0026ndash;like structures, as well as in associated nerve fibers and neurogenic plaques (Figure 2H\u0026ndash;J). \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTRPV4 expression in distilled water-induced c-Fos\u0026ndash;expressing neurons in the nodose\u0026ndash;petrosal\u0026ndash;jugular ganglionic complex\u003c/p\u003e\n\u003cp\u003eWe then assessed whether TRPV4 was expressed in DW-induced c-Fos\u0026ndash;positive neurons within the NPJc. Following DW application, c-Fos\u0026ndash;positive neurons were predominantly observed in the JG (36.67 \u0026plusmn; 20.85) and PG (19.83 \u0026plusmn; 16.03), with fewer detected in the NG (6.33 \u0026plusmn; 7.09) (Figure 3A, B). Notably, approximately 60% of c-Fos\u0026ndash;positive neurons in the NPJc also expressed TRPV4 (Figure 3C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTreatment with a TRPV4 antagonist significantly reduced distilled water-induced swallowing reflexes\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOn the basis of the identification of TRPV4 in peripheral swallowing-related regions and in DW-induced c-Fos\u0026ndash;expressing neurons within the NPJc, we next examined whether a treatment with a TRPV4 antagonist could attenuate DW-induced swallowing reflexes. Various concentrations of the TRPV4 antagonist RN9893 were applied to the swallowing-related regions 15 minutes prior to DW application. The effects of treatment with a vehicle control (corresponding to the highest concentration of RN9893 used) were also evaluated.\u003c/p\u003e\n\u003cp\u003eAdministration of RN9893 at concentrations of 10 mM, 20 mM, 50 mM, and 100 mM significantly reduced the number of DW-induced swallowing reflexes (Figure 4A, B). In contrast, the vehicle had no significant effect. Lower concentrations of RN9893 (1 mM and 5 mM) likewise produced no significant changes (Figure 4A, B). Notably, even at the highest concentration, RN9893 did not completely abolish DW-induced reflexes.\u003c/p\u003e\n\u003cp\u003eTreatment with a TRPV4 antagonist significantly reduced distilled water-induced superior laryngeal nerve activity\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; We next asked whether treatment with the TRPV4 antagonist would reduce DW-induced SLN activity. Application of DW markedly increased SLN activity compared with saline application (Figure 5A, B). Pretreatment with RN9893 (10 mM) 15 minutes prior to DW application significantly reduced this activity (Figure 5A, B). In contrast, the vehicle had no effect (Figure 5A, B).\u003c/p\u003e\n\u003cp\u003eLocal anesthetic or bilateral superior laryngeal nerve transection abolished distilled water-induced swallowing reflexes\u003c/p\u003e\n\u003cp\u003eTo confirm that DW-induced swallowing reflexes were mediated by excitation of afferent nerves within swallowing-related regions, a local anesthetic (2% lidocaine) was applied 2 minutes before DW stimulation. This treatment completely abolished DW-induced swallowing reflexes (Figure 6A, B). In addition, no apnea was observed following local anesthetic application (Supplemental Figure 3A).\u003c/p\u003e\n\u003cp\u003eBilateral transection of the SLNs prior to DW application also eliminated the swallowing reflex, demonstrating that SLN afferents mediated the DW-induced response under our experimental conditions (Figure 6A, C). Similar to the effect of local anesthetic application, no apnea was observed following bilateral SLN transection (Supplemental Figure 3B). \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTo the best of our knowledge, this study provides the first evidence that TRPV4 channels are involved in triggering the water-induced swallowing reflex. The enhanced reflex activity elicited by water, compared with saline, supports the hypothesis that water activates specific sensory mechanisms responsible for initiating the swallowing reflex. Our findings collectively demonstrate that TRPV4 channels play a pivotal role in this process.\u003c/p\u003e \u003cp\u003eImmunohistochemical analysis showed that TRPV4 was expressed by epithelial cells, taste bud cells, and nerve fibers within the laryngopharyngeal and laryngeal mucosa\u0026mdash;regions that have previously been identified as playing key roles in triggering swallowing. These findings indicate that TRPV4 is strategically positioned to act as a peripheral sensor that responds to mucosal surface changes following water application. Moreover, c-Fos expression induced by water stimulation was observed in the NPJc, with a substantial proportion of activated neurons coexpressing TRPV4. This suggests that TRPV4-expressing vagal afferent neurons within the NPJc are recruited by water stimulation in swallowing-related areas.\u003c/p\u003e \u003cp\u003eTRPV4 localization in peripheral swallowing-related regions is consistent with previous reports describing its expression in nerve fibers of the laryngopharynx and associated laryngeal regions \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, in epithelial cells of the larynx \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, trachea \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, and esophagus \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, and in taste bud cells of the circumvallate papillae \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. The presence of TRPV4 in these structures suggests its potential role in mediating swallowing-related sensory processes. In our previous study, topical application of the TRPV4 agonist GSK1016790A facilitated the swallowing reflex in a dose-dependent manner \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The findings from the present study extend these observations by demonstrating that TRPV4 activation is physiologically relevant for triggering the water-induced swallowing reflex. Pharmacological blockade of TRPV4 with a selective antagonist significantly attenuated both the number of water-induced swallowing reflexes and the associated SLN activity. This attenuation indicates that TRPV4 activation contributes directly to the sensory input driving the water-induced swallowing reflex.\u003c/p\u003e \u003cp\u003eSeveral mechanisms may underlie TRPV4 activation following water application (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). As a hypo-osmotic sensor \u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, TRPV4 can be activated by osmotic tension across the cell membrane resulting from the hypoosmotic nature of water. In addition, TRPV4 may be activated by cellular and axonal swelling caused by water entry through aquaporins or by other mechanisms that induce mechanical stretching of the plasma membrane \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Previous studies have shown that airway epithelial cells undergo volume changes in response to alterations in luminal osmolarity \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. TRPV4 has been demonstrated to respond to both osmotic and mechanical stimuli \u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. In our SLN recordings, water application produced a gradual increase in neural activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), suggesting that membrane stretching due to cell or axonal volume expansion may account, at least in part, for the delayed phase of activation. This interpretation is further supported by the greater inhibitory effect of the TRPV4 antagonist on the late component of SLN activity, which is consistent with stretch-induced TRPV4 activation. In agreement with this notion, previous studies have reported that long-latency and long-duration water-responsive SLN afferents did not respond to isotonic solutions, indicating that their activation depends on the hypoosmolarity of water \u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePrevious studies have shown that laryngeal taste buds contribute to water-induced swallowing, likely through ATP release and the consequent activation of purinergic receptors on afferent nerve fibers \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. However, the specific receptor mediating water-induced activation remained unidentified \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Our finding that TRPV4 is expressed in taste bud cells suggests that water may activate TRPV4, leading to intracellular calcium influx and subsequent ATP release. The released ATP could then activate purinergic receptors on afferent fibers, thereby initiating neural signaling. Similarly, a previous study reported that laryngeal neuroendocrine cells\u0026mdash;a specialized subset of epithelial cells\u0026mdash;are activated by water and release ATP to excite afferent nerves \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. That study also demonstrated that the neuroendocrine cells are sensitive to hypoosmotic stimulation below 75 mOSm/L \u003csup\u003e56\u003c/sup\u003e. Although neuroendocrine cells were not specifically labeled in the present study, TRPV4 expression in epithelial cells suggests that these populations may also participate in DW-induced signaling.\u003c/p\u003e \u003cp\u003eThis model is further supported by our previous finding that P2X3 receptors are predominantly expressed on nerve fibers that innervate the epithelium and taste buds in swallowing-related regions \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Other studies have also reported P2X2, P2X3, and P2Y1 receptors expression in these areas \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e,\u003cspan additionalcitationids=\"CR58 CR59 CR60\" citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Moreover, water failed to evoke swallowing reflexes in P2X2/P2X3 double knockout mice \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e, reinforcing the importance of purinergic signaling in this process. In addition, application of hypoosmotic solutions has been shown to augment ATP release in animal and human airways \u003csup\u003e\u003cspan additionalcitationids=\"CR63 CR64\" citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e, while TRPV4 antagonists or gene knockdown impaired hypoosmotic-evoked ATP release from airway epithelial cells \u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. Together with our previous observation that topical application of exogenous ATP elicits swallowing reflexes \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, these findings support a mechanism in which TRPV4 activation in epithelial and taste bud cells induces ATP release, thereby activating purinergic receptors on afferent fibers (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The ensuing excitation of these afferents enhances sensory input to the swallowing central pattern generator (sCPG) in the brainstem, initiating the swallowing reflex (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Furthermore, TRPV4 expression by intraepithelial and subepithelial nerve fibers suggests that water may also directly activate these fibers (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), given that the airway epithelium permits water movement through both transcellular and paracellular pathways \u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e,\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. Supporting this notion, previous studies have reported that hypoosmotic stimulation depolarized vagal sensory nerves in guinea pigs, mice, and humans\u0026mdash;effects that were inhibited by TRPV4 antagonists and attenuated in vagal preparations from \u003cem\u003eTrpv4\u0026minus;/\u0026minus;\u003c/em\u003e mice \u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAlthough treatment with the TRPV4 antagonist significantly reduced the water-induced swallowing reflex in a dose-dependent manner, it did not completely abolish the reflex, even at concentrations of up to 100 mM. This partial inhibition indicates that, in addition to TRPV4, other receptors or ion channels may contribute to the water-induced swallowing reflex. In this context, earlier electrophysiological and behavioral studies have demonstrated that laryngeal and pharyngeal water-sensitive receptors are influenced by the ionic composition of the stimulating solution, particularly by anions such as chloride \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Furthermore, anion channels, including volume-regulated anion channels and calcium-activated chloride channels, have been implicated in epithelial osmosensation and fluid homeostasis, which raises the possibility that such channels may also modulate sensory responses to water \u003csup\u003e\u003cspan additionalcitationids=\"CR70\" citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. Further studies are warranted to elucidate the contributions of these additional mechanisms.\u003c/p\u003e \u003cp\u003eOur findings regarding TRPV4 involvement in the water-induced swallowing reflex have important clinical implications. Patients with oropharyngeal dysphagia often experience difficulty swallowing liquids. Current management strategies primarily rely on compensatory techniques (e.g., modifying bolus viscosity or employing postural adjustments such as the chin-tuck maneuver) and rehabilitative exercises, which have demonstrated limited efficacy \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e,\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e. Consequently, therapeutic strategies that target the sensory modulation of swallowing are urgently required. Recently, chemical neurostimulation, which targets chemosensory ion channels in the peripheral swallowing-related regions including TRP channels, has shown promising results in improving swallowing safety and efficiency in both preclinical and clinical studies\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e,\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e. Understanding the molecular mechanisms underlying water-induced swallowing reflexes may therefore provide valuable insights that could help develop pharmacological interventions for oropharyngeal dysphagia. Our previous observation that TRPV4 agonists facilitate swallowing \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e further suggests that these compounds may represent promising therapeutic candidates for patients who experience difficulty in swallowing liquids. Such approaches merit further investigation in clinical contexts.\u003c/p\u003e \u003cp\u003e Finally, topical application of a local anesthetic to the peripheral swallowing-related regions completely abolished the water-induced swallowing reflex, underscoring the essential role of sensory afferents in mediating this response. To delineate the specific contribution of the SLN, we recorded swallowing reflexes in rats with intact SLNs while transecting other nerves, including bilateral IX-ph, X-ph, IX-li, and RLN. In a separate experiment, bilateral transection of the SLNs completely abolished the swallowing reflex elicited by water application to SLN-innervated regions. These findings confirm that SLN afferents play a critical role in transmitting the sensory input needed to initiate the water-induced swallowing reflex under our experimental conditions.\u003c/p\u003e \u003cp\u003eIn conclusion, our findings identify TRPV4 as a molecular sensor responsible for water-induced activation of the swallowing reflex. By showing that TRPV4 is involved in this response, the present study enhances our understanding of the sensory regulation of swallowing and highlights TRPV4 as a potential therapeutic target for improving swallowing function in patients with oropharyngeal dysphagia.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;MZH and JK: Conceptualize and design the work. MZH, HA, RRR, and JK: acquisition, analysis, or interpretation of data. MZH, HA, RRR, and JK: Draft or revise the manuscript critically.\u0026nbsp;All authors have approved the final version of the manuscript. All persons designated as authors qualify for authorship.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI, grant numbers #\u0026nbsp;24K08797 to Junichi Kitagawa, # 25K09007 to Hiroshi Ando, #\u0026nbsp;24K19847 to Rita Rani Roy, and #\u0026nbsp;24K12880 to Mohammad Zakir Hossain. The grant-providing society had no role in the study design, data collection, analysis, or interpretation.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003eData availability statement\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; The data underlying this article will be made available to other researchers from the authors upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eJean, A. Brain stem control of swallowing: Neuronal network and cellular mechanisms. \u003cem\u003ePhysiological Reviews\u003c/em\u003e vol. 81 929\u0026ndash;969 (2001).\u003c/li\u003e\n \u003cli\u003eShingai, T. \u0026amp; Shimada, K. 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Brain and Pharyngeal Responses Associated with Pharmacological Treatments for Oropharyngeal Dysphagia in Older Patients. \u003cem\u003eDysphagia\u003c/em\u003e vol. 38 1449\u0026ndash;1466 (2023).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"TRPV4 channels, Water-induced swallowing reflex, Superior laryngeal nerve-afferents, Nodose–petrosal–jugular ganglionic complex","lastPublishedDoi":"10.21203/rs.3.rs-8706920/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8706920/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe swallowing reflex is essential for the safe transfer of food and liquids from the oral cavity to the esophagus. Water is a strong stimulus for triggering this reflex, yet the molecular mechanisms underlying water-induced activation remain unclear. The aim of this study was to evaluate whether transient receptor potential vanilloid 4 (TRPV4) channels contribute to the water-evoked swallowing reflex. We applied distilled water (DW) or saline to superior laryngeal nerve (SLN)-innervated swallowing-related regions in anesthetized rats, measured swallowing reflexes and SLN activity, and conducted immunohistochemical analyses of TRPV4 expression. DW elicited more frequent swallowing and greater SLN activation than saline. TRPV4 immunoreactivity was observed in epithelial cells, taste bud\u0026ndash;like structures, and nerve fibers within the laryngopharyngeal and laryngeal mucosa. DW stimulation also induced c-Fos expression in the nodose\u0026ndash;petrosal\u0026ndash;jugular ganglionic complex, with ~\u0026thinsp;60% of activated neurons coexpressing TRPV4. Pretreatment with the TRPV4 antagonist RN9893 significantly attenuated DW-evoked swallowing and SLN activity. These findings provide integrated physiological, pharmacological, and immunohistochemical evidence that TRPV4 channels contribute as molecular sensors initiating the water-induced swallowing reflex. Our study provides enhanced understanding of TRPV4-mediated sensory regulation of swallowing and suggests that TRPV4 is a potential therapeutic target for oropharyngeal dysphagia.\u003c/p\u003e","manuscriptTitle":"Transient receptor potential vanilloid 4 channels contribute to the initiation of water-induced swallowing reflexes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-04 14:12:08","doi":"10.21203/rs.3.rs-8706920/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-23T10:30:06+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-20T19:37:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-20T13:00:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"7976842213969796453539830841421804560","date":"2026-02-02T15:45:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"48602134885039835463992380080186987307","date":"2026-02-02T11:57:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-02T10:06:32+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-30T09:26:45+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-28T10:37:46+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-28T10:35:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-01-27T06:56:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5cbc6c86-8ca0-435f-a790-8993c151073d","owner":[],"postedDate":"February 4th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":62250825,"name":"Biological sciences/Neuroscience"},{"id":62250826,"name":"Biological sciences/Physiology"}],"tags":[],"updatedAt":"2026-05-04T16:35:38+00:00","versionOfRecord":{"articleIdentity":"rs-8706920","link":"https://doi.org/10.1038/s41598-026-51222-6","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-04-29 15:58:11","publishedOnDateReadable":"April 29th, 2026"},"versionCreatedAt":"2026-02-04 14:12:08","video":"","vorDoi":"10.1038/s41598-026-51222-6","vorDoiUrl":"https://doi.org/10.1038/s41598-026-51222-6","workflowStages":[]},"version":"v1","identity":"rs-8706920","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8706920","identity":"rs-8706920","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}