Activation of Muscarinic Receptors Mediated Varying Cholinergic Effects on Medullar 5-HT Neurons and Locomotor Activities in ePet-EYFP Mice

Activation of Muscarinic Receptors Mediated Varying Cholinergic Effects on Medullar 5-HT Neurons and Locomotor Activities in ePet-EYFP Mice | 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 Activation of Muscarinic Receptors Mediated Varying Cholinergic Effects on Medullar 5-HT Neurons and Locomotor Activities in ePet-EYFP Mice Yue Dai, Yi Cheng, Renkai Ge, Qiang Zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6559559/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Nov, 2025 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Abstract Serotonergic neurons (5-HT) in medulla play an essential role in initiating locomotion. These neurons receive cholinergic input and display varying response to acetylcholine (ACh). However, the mechanism behind remains unknown. Using ePet-EYFP transgenic mice (P3–P6) and multiple approaches, we investigated mechanisms underlying cholinergic modulation of medullar 5-HT neurons and generating locomotion. Our results included: (1) Significant morphological difference in 5-HT neurons was shown between parapyramidal region (PPR) and midline raphe nuclei (MRN). (2) ACh induced triphasic effects (excitatory, inhibitory, and neutral) on 5-HT neurons, which could be duplicated by muscarine, with PPR neurons more excitable and MRN neurons less sensitive to ACh. (3) ACh enhanced excitability via mAChR-M3 receptors, while M2&M4 receptors mediated inhibitory effects. (4) Blocking M3 pathway reduced gait frequency and disrupted locomotion, whereas antagonizing M2&M4 pathways increased gait frequency. This study unveiled interactions between medullary cholinergic and serotonergic systems in modulating neuronal excitability and generating locomotion. Biological sciences/Neuroscience/Neuronal physiology/Intrinsic excitability Biological sciences/Neuroscience/Motor control/Spinal cord Biological sciences/Neuroscience/Motor control/Central pattern generators Biological sciences/Neuroscience/Neuronal physiology/Inhibition–excitation balance Motor Control Locomotion Medulla Neuromodulation Serotonergic Neurons Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryTablesnewversion.docx Supplementary Tables SupplementaryFigureswithLegends.pdf Supplementary Figures Cite Share Download PDF Status: Published Journal Publication published 25 Nov, 2025 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6559559","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":451169620,"identity":"18e8343c-ad46-4922-9a1c-c268a44b23d7","order_by":0,"name":"Yue Dai","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIie3NsQqCQBjA8S8Obrp0VQTrEQwhgupdPhFycailsQTBqQfQtxB6AeUGF2l2dWlqEIRoLG1quWoLuj8c93F3Pw5AJvvJELLm5SD7gOQxfksI+4pYhVvz5Y07aUbydhOBqVQ4aNciUp4t7iO39YC6RhKBrVdIjFhEKuyJqQKbkmEETlohJUxIvIbPkDMK6rUj+w+Ib3F4/kI7gtY7opeXdX5YebYeUttgJ22SlHVoiIhSeMfmtpg7aRHWLdsuRkrh5q2IjLN+CwFIP2iPNQgEAGD0vN4JH8lkMtmfdwe6m0uxmMrzNAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-1773-5228","institution":"East China Normal University","correspondingAuthor":true,"prefix":"","firstName":"Yue","middleName":"","lastName":"Dai","suffix":""},{"id":451169621,"identity":"161555e1-38d2-439c-9c46-68a7be7cd844","order_by":1,"name":"Yi Cheng","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Cheng","suffix":""},{"id":451169622,"identity":"1dc9d61e-21f8-4bf7-839c-c551a78531f1","order_by":2,"name":"Renkai Ge","email":"","orcid":"","institution":"East China Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Renkai","middleName":"","lastName":"Ge","suffix":""},{"id":451169623,"identity":"4166a424-4bdc-4c8e-a82b-8bef1ae69739","order_by":3,"name":"Qiang Zhang","email":"","orcid":"","institution":"Jiangsu University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Qiang","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-04-29 21:40:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6559559/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6559559/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s42003-025-09217-y","type":"published","date":"2025-11-25T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82054291,"identity":"93fdb617-c329-4a6d-932c-7a88b76641b4","added_by":"auto","created_at":"2025-05-06 10:21:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1924091,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of 5-HT Neurons \u003cstrong\u003eA. \u003c/strong\u003eDistribution of serotonergic neurons.\u003cstrong\u003e A1. \u003c/strong\u003eDistribution of 5-HT neurons in a coronal slice of the medulla. The dotted circles represent the parapyramidal region (PPR) and midline raphe nuclei (MRN). \u003cstrong\u003eA2.\u003c/strong\u003e Distribution of 5-HT neurons in a sagittal slice of the brainstem. The dashed line divides the brainstem into the midbrain, pons, and medulla. \u003cstrong\u003eB1.\u003c/strong\u003e Schematic view of two 5-HT neurons (cells 1 and 2) from the PPR (B2, dotted rectangle) and one 5-HT neuron (cell 3) from the MRN (B3, dotted rectangle). \u003cstrong\u003eC.\u003c/strong\u003e Morphology of cells 1, 2, and 3 from panel B. The diameter of the 5-HT neurons in the PPR (n=17) was larger than that in the MRN (n=14) (P \u0026lt; 0.05). \u003cstrong\u003eD.\u003c/strong\u003e Summary diagrams showing the diameter (D1), soma area (D2), soma volume (D3), total branch length (D4), and the number of primary segments, branch points, and branches (D5) of 5-HT neurons from the MRN (n=14) and PPR (n=17). D6. Statistical results show significant differences in intersections between PPR and MRN 5-HT neurons, especially in the range of 50–100 µm (P \u0026lt; 0.05). Error bars represented SD; paired t-test performed; ∗: P \u0026lt; 0.05, ∗∗: P \u0026lt; 0.01, ∗∗∗: P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6559559/v1/fc0edeacc067a67bcc6e3298.png"},{"id":82054290,"identity":"262b418b-8b90-40cf-842b-fc4331cf23f1","added_by":"auto","created_at":"2025-05-06 10:21:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":754079,"visible":true,"origin":"","legend":"\u003cp\u003eMorphology and membrane properties of three types of 5-HT neurons \u003cstrong\u003eA.\u003c/strong\u003e Based on morphological characteristics, 5-HT neurons were divided into pyramidal neuron (cell 1 and cell 2), stellate neuron (cell 3 and cell 4) and bipolar neuron (cell 5 and cell 6). \u003cstrong\u003eB.\u003c/strong\u003e Morphological differences among three types of 5-HT neurons.\u003cstrong\u003e \u003c/strong\u003eB1\u003cstrong\u003e.\u003c/strong\u003e Statistic results showed that intersections were obviously different in pyramidal, stellate and bipolar 5-HT neurons especially in the range of 50–100 µm (p \u0026lt; 0.05). B2. Graphs show the numbers of primary segments, branch points and branches of pyramidal,stellate and bipolar neuron. B3-B6. Summary diagrams show the diameter length (B3), soma area (B4), soma volume (B5), total branches length (B6) of pyramidal (n=7), stellate (n=16) and bipolar (n=8) 5-HT neuron. \u003cstrong\u003eC.\u003c/strong\u003e Summary diagrams show the resting membrane potential (RMP, C1), rheobase (C2), action potential (AP) height (C3), afterhyperpolarization (AHP) depth (C4), and input resistance (Rin, C5) of pyramidal (n=11), stellate (n=13) and bipolar (n=11) 5-HT neuron. Error bars represented SD; paired t-test performed; ∗: P \u0026lt; 0.05, ∗∗: P \u0026lt; 0.01, ∗∗∗: P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6559559/v1/e070de0df0f91dacb30cb0a6.png"},{"id":82055874,"identity":"89726305-fb54-450d-954f-7f72226841c1","added_by":"auto","created_at":"2025-05-06 10:29:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":749654,"visible":true,"origin":"","legend":"\u003cp\u003eThe modulation of acetylcholine (ACh) on 5-HT neuron excitability \u003cstrong\u003eA. \u003c/strong\u003e\u0026nbsp;ACh increased the excitability of 5-HT neurons. \u003cstrong\u003eA1.\u003c/strong\u003e ACh induced membrane potential depolarization.\u003cstrong\u003e A2.\u003c/strong\u003e ACh decreased AP height and amplitude of AHP. \u003cstrong\u003eA3. \u003c/strong\u003eACh decreased Rin. \u003cstrong\u003eA4–A8\u003c/strong\u003e. Summary diagrams showed ACh-induced significant changes in the membrane properties including RMP, rheobase, AP height, AHP depth and Rin recorded in control (black) and presence of 15µM ACh (red) (n=38). \u003cstrong\u003eB. \u003c/strong\u003eACh decreased the excitability of 5-HT neurons. \u003cstrong\u003eB1.\u003c/strong\u003e ACh induced membrane potential hyperpolarization.\u003cstrong\u003e B2.\u003c/strong\u003eACh decreased AP height and amplitude of AHP. \u003cstrong\u003eB3. \u003c/strong\u003eACh decreased Rin. \u003cstrong\u003eB4–B8\u003c/strong\u003e. Summary diagrams showed ACh -induced significant changes in the membrane properties including RMP, rheobase, AP height, AHP depth and Rin recorded in control (black) and presence of 15µM ACh (blue) (n=8). \u003cstrong\u003eC. \u003c/strong\u003eACh did not substantially changed the excitability of 5-HT neurons. ACh did not induced significant change in membrane potential (\u003cstrong\u003eC1\u003c/strong\u003e), AP height and amplitude of AHP\u003cstrong\u003e (C2\u003c/strong\u003e) and Rin (\u003cstrong\u003eC3\u003c/strong\u003e). \u003cstrong\u003eC4–C8\u003c/strong\u003e. Summary diagrams showed ACh did not induce substantial change in the membrane properties of RMP, rheobase, AP height, AHP depth and Rin which were recorded in control (black) and presence of 15µM ACh (grey) (n=22). \u003cstrong\u003eD.\u003c/strong\u003e Muscarinic receptor was involved the modulation of ACh on 5-HT neurons. \u003cstrong\u003eD1.\u003c/strong\u003e ACh-mediated RMP depolarization were blocked by atropine. \u003cstrong\u003eD2. \u003c/strong\u003eStatistical results listed for the RMP, Rheobase, AP height AHP depth and Rin of 5-HT neurons in control (black), 15mM ACh (red) and 3mM atropine (green), respectively. \u003cstrong\u003eD3.\u003c/strong\u003e ACh-mediated RMP hyperpolarization were blocked by atropine. \u003cstrong\u003eD2. \u003c/strong\u003eStatistical results summarized for the RMP, Rheobase, AP height AHP depth and Rin of 5-HT neurons recorded in control (black), 15 mM ACh (blue) and 3 mM atropine (green). \u003cstrong\u003eE1.\u003c/strong\u003e Three type effects induced by ACh on the RMP of 5-HT neurons and their medullary distribution. \u003cstrong\u003eE2.\u003c/strong\u003e Proportions of three type effects by ACh on the RMP in PPR and MRN, respectively. Error bars represented SD; paired t-test performed; ∗: P \u0026lt; 0.05, ∗∗: P \u0026lt; 0.01, ∗∗∗: P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6559559/v1/cdfa9325b30ca84dda603d4b.png"},{"id":82054293,"identity":"23e87052-fb61-4ad7-82a3-49dbd7dfee55","added_by":"auto","created_at":"2025-05-06 10:21:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":656230,"visible":true,"origin":"","legend":"\u003cp\u003eThe modulation of muscarine on 5-HT neurons excitability \u003cstrong\u003eA. \u003c/strong\u003eMuscarine increased the excitability of 5-HT neurons. \u003cstrong\u003eA1.\u003c/strong\u003e Muscarine induced membrane potential depolarization and spontaneous firing. \u003cstrong\u003eA2. \u003c/strong\u003eMuscarine decreased the rheobase. \u003cstrong\u003eA3. \u003c/strong\u003eMuscarine reduced the Rin. \u003cstrong\u003eA4. \u003c/strong\u003eSuperimposed action potential recorded from a 5-HT neuron before (black) and after muscarine (red) showed that muscarine decreased AP height and AHP. \u003cstrong\u003eA5-A9. \u003c/strong\u003eStatistical results for the RMP, Rheobase, AP height AHP depth and Rin of 5-HT neurons recorded in control (black) and in the presence of 20mM muscarine (red). \u003cstrong\u003eB. \u003c/strong\u003eMuscarine decreased the excitability of 5-HT neurons. \u003cstrong\u003eB1.\u003c/strong\u003e Muscarine induced membrane potential hyperpolarization. \u003cstrong\u003eB2. \u003c/strong\u003eMuscarine increased the rheobase.\u003cstrong\u003e B3. \u003c/strong\u003eMuscarine reduced the Rin. \u003cstrong\u003eB4. \u003c/strong\u003eSuperimposed action potential recorded from a 5-HT neuron before (black) and after muscarine (blue), muscarine decreased AP height and AHP. \u003cstrong\u003eB5-B9. \u003c/strong\u003eStatistical results listed for the RMP, Rheobase, AP height AHP depth and Rin of 5-HT neurons recorded in control (black) and in the presence of 20mM muscarine (blue). \u003cstrong\u003eC. \u003c/strong\u003eMuscarine did not significantly change the excitability of 5-HT neurons. \u003cstrong\u003eC1- C2.\u003c/strong\u003eMuscarine did not statistically change RMP (\u003cstrong\u003eC1\u003c/strong\u003e) and rheobase (\u003cstrong\u003eC2\u003c/strong\u003e). \u003cstrong\u003eC3. \u003c/strong\u003eMuscarine reduced the Rin. \u003cstrong\u003eC4. \u003c/strong\u003eSuperimposed action potential recorded from a 5-HT neuron before (black) and after muscarine (grey), muscarine did not substantively change AP height and AHP. \u003cstrong\u003eC5-C9. \u003c/strong\u003eStatistical results summarized for the RMP, Rheobase, AP height AHP depth and Rin of 5-HT neurons recorded in control (black) and in the presence of 20mM muscarine (grey). \u003cstrong\u003eD1.\u003c/strong\u003eThree-type effects induced by muscarine on the RMP of 5-HT neurons and their distribution. \u003cstrong\u003eD2.\u003c/strong\u003e Proportions of three type effects by muscarine on the RMP in PPR and MRN, respectively. Error bars represented SD; paired t-test performed; ∗: P \u0026lt; 0.05, ∗∗: P \u0026lt; 0.01, ∗∗∗: P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6559559/v1/b4998d353db9decb12593f89.png"},{"id":82054296,"identity":"6c6ba02f-792a-4bdd-be47-230874274a53","added_by":"auto","created_at":"2025-05-06 10:21:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":776607,"visible":true,"origin":"","legend":"\u003cp\u003eThe\u003cstrong\u003e \u003c/strong\u003emAChR-M3 receptors mediated the muscarine-induced excitatory effect on medullar 5-HT neurons. \u003cstrong\u003eA.\u003c/strong\u003e M1 receptor was not involved the modulation of muscarine on 5-HT neurons. \u003cstrong\u003eA1.\u003c/strong\u003e Muscarine-mediated depolarization and spontaneous firing persisted following application of M1 receptor antagonist telenzepine(10mM). Telenzepine did not change the effect of the muscarine on rheobase (\u003cstrong\u003eA2\u003c/strong\u003e), Rin\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eA3\u003c/strong\u003e), AP height and AHP depth (\u003cstrong\u003eA4\u003c/strong\u003e). \u003cstrong\u003eA5-A9. \u003c/strong\u003eStatistical results listed for the RMP, Rheobase, AP height AHP depth and Rin of 5-HT neurons recorded in control (black), 20mM muscarine (red) and 10mM telenzepine (green). \u003cstrong\u003eB. \u003c/strong\u003eM3 receptor was involved the modulation of muscarine on 5-HT neurons. \u003cstrong\u003eB1.\u003c/strong\u003eMuscarine-mediated RMP depolarization were blocked by 4-DAMP, M3 receptor antagonist. 4-DAMP removed the muscarine-induced decrease in rheobase (\u003cstrong\u003eB2\u003c/strong\u003e), Rin (\u003cstrong\u003eB3\u003c/strong\u003e), AP height, and AHP depth (\u003cstrong\u003eB4\u003c/strong\u003e). \u003cstrong\u003eB5-B9. \u003c/strong\u003eStatistical results showed for the RMP, Rheobase, AP height AHP depth and Rin of 5-HT neurons recorded in control (black), 20mM muscarine (red) and 5 mM 4-DAMP (green), respectively. \u003cstrong\u003eC. \u003c/strong\u003eM5 receptor was not involved in muscarinic modulation of 5-HT neurons. \u003cstrong\u003eC1.\u003c/strong\u003eMuscarine-mediated depolarization persisted following application of 10 mM VU 6008667, M5 inhibitor. UV 6008667 did not alter the effect of muscarine on rheobase (\u003cstrong\u003eC2\u003c/strong\u003e), input resistance\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eC3\u003c/strong\u003e), AP height and AHP depth (\u003cstrong\u003eC4\u003c/strong\u003e). \u003cstrong\u003eC5-C9. \u003c/strong\u003eStatistical results for the RMP, Rheobase, AP height AHP depth and Rin of 5-HT neurons recorded in control (black), 20 mM muscarine (red) and 10mM UV 6008667 (green), respectively. Mus: muscarine; Tel: Telenzepine; UV: UV 6008667. Error bars represented SD; paired t-test performed; ∗:P \u0026lt; 0.05, ∗∗: P \u0026lt; 0.01, ∗∗∗: P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6559559/v1/89cbdebf2a9ca9b88a3e8643.png"},{"id":82054303,"identity":"2ed096d5-0443-4d60-9b21-e76340f47ef9","added_by":"auto","created_at":"2025-05-06 10:21:17","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":846486,"visible":true,"origin":"","legend":"\u003cp\u003eThe\u003cstrong\u003e \u003c/strong\u003emAChR M2 and M4 receptors mediated the muscarine-induced inhibitory effect on medullar 5-HT neurons. \u003cstrong\u003eA. \u003c/strong\u003eM2 receptor was involved the modulation of muscarine on 5-HT neurons. \u003cstrong\u003eA1.\u003c/strong\u003e Muscarine-mediated RMP hyperpolarization were blocked by methoctramine, M2 receptor antagonist. Methoctramine (5mM) blocked the effect of muscarine-induced increase in rheobase (\u003cstrong\u003eA2\u003c/strong\u003e) and decrease in Rin (\u003cstrong\u003eA3\u003c/strong\u003e), AP height, and AHP depth (\u003cstrong\u003eA4\u003c/strong\u003e). \u003cstrong\u003eA5-A9. \u003c/strong\u003eStatistical results summarized for the RMP, Rheobase, AP height AHP depth and Rin of 5-HT neurons recorded in control (black), 20mMmuscarine (blue) and 5mM methoctramine (orange). \u003cstrong\u003eB. \u003c/strong\u003eM4 receptor mediated muscarinic modulation of 5-HT neurons. \u003cstrong\u003eB1.\u003c/strong\u003e Muscarine-induced RMP hyperpolarization were blocked by 10mM tropicamide, M4 receptor antagonist. Tropicamide blocked the effect of muscarine-induced increase in rheobase (\u003cstrong\u003eB2\u003c/strong\u003e), and decrease in Rin (\u003cstrong\u003eB3\u003c/strong\u003e), AP height, AHP depth (\u003cstrong\u003eB4\u003c/strong\u003e). \u003cstrong\u003eB5-B9. \u003c/strong\u003eStatistical results shown for the RMP, Rheobase, AP height AHP depth and Rin of 5-HT neurons in control (black), 20mM muscarine (blue) and 10mM tropicamide (green), respectively. \u003cstrong\u003eC-D.\u003c/strong\u003eThe coupling effects of M2 and M4 receptors on 5-HT neurons. \u003cstrong\u003eC1. \u003c/strong\u003eMuscarine\u003cstrong\u003e-\u003c/strong\u003einduced hyperpolarization was not blocked by M2 antagonist methoctramine, but was completely removed by M4 antagonist tropicamide.\u003cstrong\u003e C2. \u003c/strong\u003eThe RMP, Rheobase, AP height, AHP depth and Rin of three 5-HT neurons were recorded in control (black), 20mM muscarine (blue), 5mM methoctramine (orange) and 10mM tropicamide (green), respectively.\u003cstrong\u003eD1. \u003c/strong\u003eMuscarine\u003cstrong\u003e-\u003c/strong\u003einduced hyperpolarization was not blocked by M4 antagonist tropicamide, but was blocked by M2 antagonist methoctramine. \u003cstrong\u003eD2. \u003c/strong\u003eThe RMP, Rheobase, AP height, AHP depth and Rin of two 5-HT neurons were recorded in control (black), 20mM muscarine (blue), 10mM tropicamide (green) and 5mM methoctramine (orange), respectively. Mus: muscarine; Meth: methoctramine; Trop:tropicamide; Error bars represented SD; paired t-test performed; ∗: P \u0026lt; 0.05, ∗∗: P \u0026lt; 0.01, ∗∗∗: P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6559559/v1/3544a5413096da718e7d6306.png"},{"id":82054299,"identity":"284e9dcd-9c82-4e60-b137-1d5b64231506","added_by":"auto","created_at":"2025-05-06 10:21:17","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2302313,"visible":true,"origin":"","legend":"\u003cp\u003eImages from immunofluorescence and retrograde labeling experiments \u003cstrong\u003eA. \u003c/strong\u003eLocalization of M2 (\u003cstrong\u003eA1\u003c/strong\u003e), M3 (\u003cstrong\u003eA2\u003c/strong\u003e), and M4 (\u003cstrong\u003eA3\u003c/strong\u003e) receptors in the medulla. The first column contained images of a representative transverse medulla section, demonstrating the position of the view within the black box. The next three columns contained images of 5-HT neurons (green), receptors (red), and the merged images (yellow). \u003cstrong\u003eB. \u003c/strong\u003eInjection of a retrograde tracer, dextran tetramethylrhodamine (TMR), into the ventral medulla. Distribution of cholinergic neuronal clusters in sagittal brainstem sections of transgenic mice (Chat-IRES-EYFP), with green fluorescence indicating cholinergic neurons (\u003cstrong\u003eB1\u003c/strong\u003e). Distribution of tetramethylrhodamine in the sagittal brainstem (\u003cstrong\u003eB2\u003c/strong\u003e). \u003cstrong\u003eC.\u003c/strong\u003e Examples of fluorescence staining of cholinergic neurons (green) with retrograde labeling (red) in the pedunculopontine nucleus (PPN). Noncholinergic neurons of the cuneiform nucleus (CnF) were also fluorescently labeled.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6559559/v1/c4f61fcaaed4268f6b19471c.png"},{"id":82055877,"identity":"f9ada617-b024-4e55-b90b-b3d39e2700b5","added_by":"auto","created_at":"2025-05-06 10:29:17","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":355856,"visible":true,"origin":"","legend":"\u003cp\u003eThe cholinergic role of brainstem in generating fictive locomotion \u003cstrong\u003eA. \u003c/strong\u003eSchematic representation of the whole spinal cord ENG recording with glass electrodes from the lumbar segment L5 (both right and left). Vaseline (black line) was used to divide the medulla (yellow area) and spinal cord (grey area) in the cervical region. 5-HT and NMDA were applied to the spinal cord area and muscarine were applied to medulla area. Ventral root recordings were recorded before and after bath application of methoctramine (\u003cstrong\u003eB\u003c/strong\u003e), 4-DAMP (\u003cstrong\u003eC\u003c/strong\u003e), and tropicamide (\u003cstrong\u003eD\u003c/strong\u003e) in the medulla, respectively.\u003cstrong\u003e B2\u003c/strong\u003e, \u003cstrong\u003eC2\u003c/strong\u003e\u0026amp;\u003cstrong\u003eD2\u003c/strong\u003e. Polar plots represented the fictive locomotion pre- and post-drug application.\u003cstrong\u003e \u003c/strong\u003eChanges in frequency of step cycles\u003cstrong\u003e (B3\u003c/strong\u003e, \u003cstrong\u003eC3\u003c/strong\u003e \u0026amp; \u003cstrong\u003eD3) \u003c/strong\u003eand amplitude of ENG (\u003cstrong\u003eB4\u003c/strong\u003e, \u003cstrong\u003eC4 \u0026amp; D4) \u003c/strong\u003ebefore and after drug administration during fictive locomotion.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6559559/v1/f04c1d050aa29fb21c20b41f.png"},{"id":82054297,"identity":"049e8227-81a3-4675-b097-beaf1794e4a0","added_by":"auto","created_at":"2025-05-06 10:21:17","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":501876,"visible":true,"origin":"","legend":"\u003cp\u003eModeling study of rhythmic generation with network of central pattern generator (CPG). \u003cstrong\u003eA.\u003c/strong\u003e CPG model with rhythmic generation. \u003cstrong\u003eA1. \u003c/strong\u003eThe CPG model was composed of left and right half-center pools, coupled by reciprocal inhibition of commissural interneuron pools (CIN), and driven by cholinergic inputs from the medullar 5-HT neuron pool\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003eA2.\u003c/strong\u003e Rhythmic activities of left and right half-centers were generated by excitatory (M3 receptor) and inhibitory (M2\u0026amp;M4) synaptic inputs to 5-HT neuron pool. g\u003csub\u003esye_Exc\u003c/sub\u003e=0.2uS and g\u003csub\u003esye_Inh\u003c/sub\u003e=0.2uS were set as control values. Polar plots described coordination of locomotion. The step frequency was measured as 0.3±0.03Hz. \u003cstrong\u003eB.\u003c/strong\u003e Effect of reducing synaptic conductance on rhythmic activities. \u003cstrong\u003eB1. \u003c/strong\u003eReducing excitatory synaptic conductance gsye\u003csub\u003e_Exc\u003c/sub\u003e to 25% decreased rhythmic frequency to 0.16±0.06 Hz and disrupted locomotion. \u003cstrong\u003eB2.\u003c/strong\u003e Reducing inhibitory synaptic conductance g\u003csub\u003esye_Inh\u003c/sub\u003e to 25% increased the rhythmic frequency to 0.38±0.03 Hz with preserved gait stability. \u003cstrong\u003eC.\u003c/strong\u003e Effect of increasing synaptic conductance on rhythmic activities. \u003cstrong\u003eC1. \u003c/strong\u003eIncreasing g\u003csub\u003esye_Exc\u003c/sub\u003e to 200% increased the rhythmic frequency to 0.33±0.04 Hz with stable gait. \u003cstrong\u003eC2.\u003c/strong\u003e Increasing g\u003csub\u003esye_Inh\u003c/sub\u003e to 200% reduced rhythmic frequency to 0.23±0.02 Hz with stable gait. Error bars represented SD; paired t-test performed; ∗: P \u0026lt; 0.05; ∗∗: P \u0026lt; 0.01. Dash lines in rhythmic bursting represented boundaries within which the recordings were used to calculate frequency of step cycle and polar plots.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6559559/v1/0ad0cf1664ec270798b11b39.png"},{"id":82056514,"identity":"51d0619c-3444-47ad-88da-16e4926f6b90","added_by":"auto","created_at":"2025-05-06 10:37:17","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":395260,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional roles of cholinergic pathway from MLR to spinal cord in generating locomotion. Release of acetylcholine from MLR to medullar area activates M2\u0026amp;M4 receptors, reduces excitability of medullar 5-HT neurons, and slows down locomotor frequency. Acetylcholine from MLR activates M3 receptors, increases excitability of medullar 5-HT neurons, and accelerates locomotor frequency.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-6559559/v1/7aabbfce0f4f39fa69347e02.png"},{"id":98578891,"identity":"f3fd8bdf-d786-49ae-ad73-2ad80cdf29b2","added_by":"auto","created_at":"2025-12-19 08:06:48","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5061359,"visible":true,"origin":"","legend":"","description":"","filename":"Activationofmuscarinicreceptorsinmedullary5HTneurons20250430.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6559559/v1_covered_cd6a70a9-9b5e-4684-9371-2013d319fccf.pdf"},{"id":82054292,"identity":"9f433afb-584a-4107-89e0-194e49864008","added_by":"auto","created_at":"2025-05-06 10:21:17","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":50839,"visible":true,"origin":"","legend":"Supplementary Tables","description":"","filename":"SupplementaryTablesnewversion.docx","url":"https://assets-eu.researchsquare.com/files/rs-6559559/v1/9db16432e227df3038cb3d51.docx"},{"id":82056513,"identity":"668d42bf-1fce-4578-8516-85869720be67","added_by":"auto","created_at":"2025-05-06 10:37:17","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1803061,"visible":true,"origin":"","legend":"Supplementary Figures","description":"","filename":"SupplementaryFigureswithLegends.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6559559/v1/774597b3db05bb7c542adf11.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Activation of Muscarinic Receptors Mediated Varying Cholinergic Effects on Medullar 5-HT Neurons and Locomotor Activities in ePet-EYFP Mice","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Motor Control, Locomotion, Medulla, Neuromodulation, Serotonergic Neurons","lastPublishedDoi":"10.21203/rs.3.rs-6559559/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6559559/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSerotonergic neurons (5-HT) in medulla play an essential role in initiating locomotion. These neurons receive cholinergic input and display varying response to acetylcholine (ACh). However, the mechanism behind remains unknown. Using ePet-EYFP transgenic mice (P3\u0026ndash;P6) and multiple approaches, we investigated mechanisms underlying cholinergic modulation of medullar 5-HT neurons and generating locomotion. Our results included: (1) Significant morphological difference in 5-HT neurons was shown between parapyramidal region (PPR) and midline raphe nuclei (MRN). (2) ACh induced triphasic effects (excitatory, inhibitory, and neutral) on 5-HT neurons, which could be duplicated by muscarine, with PPR neurons more excitable and MRN neurons less sensitive to ACh. (3) ACh enhanced excitability via mAChR-M3 receptors, while M2\u0026amp;M4 receptors mediated inhibitory effects. (4) Blocking M3 pathway reduced gait frequency and disrupted locomotion, whereas antagonizing M2\u0026amp;M4 pathways increased gait frequency. This study unveiled interactions between medullary cholinergic and serotonergic systems in modulating neuronal excitability and generating locomotion.\u003c/p\u003e","manuscriptTitle":"Activation of Muscarinic Receptors Mediated Varying Cholinergic Effects on Medullar 5-HT Neurons and Locomotor Activities in ePet-EYFP Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-06 10:21:12","doi":"10.21203/rs.3.rs-6559559/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"275bdf1f-baa3-4bbc-8bbe-241a2da14214","owner":[],"postedDate":"May 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":48002203,"name":"Biological sciences/Neuroscience/Neuronal physiology/Intrinsic excitability"},{"id":48002204,"name":"Biological sciences/Neuroscience/Motor control/Spinal cord"},{"id":48002205,"name":"Biological sciences/Neuroscience/Motor control/Central pattern generators"},{"id":48002206,"name":"Biological sciences/Neuroscience/Neuronal physiology/Inhibition\u0026#x2013;excitation balance"}],"tags":[],"updatedAt":"2025-12-19T08:06:30+00:00","versionOfRecord":{"articleIdentity":"rs-6559559","link":"https://doi.org/10.1038/s42003-025-09217-y","journal":{"identity":"communications-biology","isVorOnly":false,"title":"Communications Biology"},"publishedOn":"2025-11-25 05:00:00","publishedOnDateReadable":"November 25th, 2025"},"versionCreatedAt":"2025-05-06 10:21:12","video":"","vorDoi":"10.1038/s42003-025-09217-y","vorDoiUrl":"https://doi.org/10.1038/s42003-025-09217-y","workflowStages":[]},"version":"v1","identity":"rs-6559559","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6559559","identity":"rs-6559559","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}