A genetically defined midbrain-pontine circuit gates vocal communication

A genetically defined midbrain-pontine circuit gates vocal communication | 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 Biological Sciences - Article A genetically defined midbrain-pontine circuit gates vocal communication Xian Zhang, Qingliang Liao, Ruiming Xu, Yang Qiao, Wenting Huang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8634654/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Vocal communication is selectively expressed in social and emotional contexts (1-3), suggesting the presence of neural substrates that gate the conversion of internal states into vocal motor output. Although the periaqueductal gray (PAG) is known to be essential for vocalization (4-6), the genetic identity and circuit logic of the neurons that initiate and shape vocal signals remain unclear. Here, we identify a genetically defined population of somatostatin-expressing neurons in the lateral and ventrolateral PAG (l/vlPAG-SST) that functions as a premotor gate for ultrasonic vocalizations in mice. In vivo calcium imaging shows that l/vlPAG-SST neurons are selectively recruited around vocal onset. Manipulating l/vlPAG-SST neuron activity bidirectionally regulates vocal output: activation prolongs call duration by coordinating respiratory, laryngeal, and orofacial motor programs, whereas silencing suppresses courtship vocalizations. Projection-specific manipulations further demonstrate that descending glutamatergic, but not neuropeptidergic, output from l/vlPAG-SST neurons to the dorsal pontine tegmentum is sufficient to drive vocal production. Together, these findings define a genetically specified midbrain–pontine circuit that gates vocal communication and provides prospective access for linking internal conditions to vocal expressions. Biological sciences/Neuroscience/Neural circuits Biological sciences/Neuroscience/Social behaviour Biological sciences/Neuroscience/Motivation Full Text Additional Declarations There is NO Competing Interest. Supplementary Files Video4Optogeneticsstimulationflatrespiratoryairflow.mp4 Supplementary Video 4. Photoactivation of l/vlPAG-SST neurons induces flat-expiration airflow. Video3Twophotonimaging.mp4 Supplementary Video 2. Fiber photometry recordings of l/vlPAG-SST neuronal activity during courtship USVs (4× speed). Video6Optogeneticsinhibition.mp4 Supplementary Video 6. Photoinhibition of l/vlPAG-SST neurons abolishes ongoing courtship USVs. Video7TeLCinhibition.mp4 Supplementary Video 7. Synaptic silencing of l/vlPAG-SST neurons abolishes courtship USVs (5× speed). Video5Optogeneticsstimulationvocalcord.mp4 Supplementary Video 5. Photoactivation of l/vlPAG-SST neurons drives frequency-dependent vocal-cord adduction. Video2Photometry.mp4 Supplementary Video 3. Freely moving two-photon calcium imaging of l/vlPAG-SST neurons during courtship USVs. Video8OptogeneticsstimulationofPAGdPnTgpathway.mp4 Supplementary Video 8. Photoactivation of l/vlPAG-SST axon terminals in dPnTg evokes USVs. Video1Optogeneticsstimulation.mp4 Supplementary Video 1. Photoactivation of l/vlPAG-SST neurons with different frequencies triggers USVs. Cite Share Download PDF Status: Posted 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-8634654","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Biological Sciences - Article","associatedPublications":[],"authors":[{"id":577628133,"identity":"d6ccaee0-b294-40d9-8303-51e5fe1e21bf","order_by":0,"name":"Xian Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABE0lEQVRIiWNgGAWjYDACZiBmbAAS7D3MCFEeorTwnAExDYjQwgDTIpFDpBaD48zPHv7cYZcnH/n2sDHPnz8M8v4HGB+8bWOQN8ehRbKZzdxA8kxyseHtvORk3jYDBsMDB5gN57YxGO5swK6Fn5nBTMKwjTlx4+wc48O8DQb1Gxsb2KR52xgSDA5g18LGzP5NIrGtPnHjzDPGh3n+AG1pZmD/jU8LPzOPmcTBtsOJ8yV4jJN52AwY5NmA5uDTItnMUybZ2HY8cQNPjjHQC8YMBjyMzZJzzkkYbsChxeD88W2SP9uqE+e3nzGWePNHjkG+//DBD2/KbORx2YLQewDOgEQTAfVAIN+AzhgFo2AUjIJRAAUAf3ZS8bzSm0UAAAAASUVORK5CYII=","orcid":"https://orcid.org/0009-0004-3416-3948","institution":"The Hong Kong University of Science and Technology (Guangzhou)","correspondingAuthor":true,"prefix":"","firstName":"Xian","middleName":"","lastName":"Zhang","suffix":""},{"id":577628134,"identity":"cf552229-5bd8-4f5c-b880-5daf736538cd","order_by":1,"name":"Qingliang Liao","email":"","orcid":"","institution":"The Hong Kong University of Science and Technology (Guangzhou)","correspondingAuthor":false,"prefix":"","firstName":"Qingliang","middleName":"","lastName":"Liao","suffix":""},{"id":577628135,"identity":"c7b811da-7fc2-435f-a640-d36035849f0a","order_by":2,"name":"Ruiming Xu","email":"","orcid":"https://orcid.org/0009-0003-7830-9868","institution":"The Hong Kong University of Science and Technology (Guangzhou)","correspondingAuthor":false,"prefix":"","firstName":"Ruiming","middleName":"","lastName":"Xu","suffix":""},{"id":577628137,"identity":"bc96f3db-58be-45e4-af6d-08d2f317b269","order_by":3,"name":"Yang Qiao","email":"","orcid":"","institution":"The Hong Kong University of Science and Technology (Guangzhou)","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Qiao","suffix":""},{"id":577628138,"identity":"b73da8d9-48e0-4d99-b4f8-3eb36ea9a403","order_by":4,"name":"Wenting Huang","email":"","orcid":"","institution":"The Hong Kong University of Science and Technology (Guangzhou)","correspondingAuthor":false,"prefix":"","firstName":"Wenting","middleName":"","lastName":"Huang","suffix":""}],"badges":[],"createdAt":"2026-01-19 03:35:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8634654/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8634654/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102963589,"identity":"65ca0dbb-c9ad-473e-832b-474199f1d5af","added_by":"auto","created_at":"2026-02-19 04:19:12","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8830958,"visible":true,"origin":"","legend":"Article File","description":"","filename":"PAGSSTMousevocalizationLiaoetal.combinedfiles.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8634654/v1_covered_ba34115f-c80c-4fad-a3e5-e330c9c17ed4.pdf"},{"id":101230003,"identity":"e82d7cd9-10f8-4d8a-8c9d-d1690784ad11","added_by":"auto","created_at":"2026-01-27 13:27:45","extension":"mp4","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":11580815,"visible":true,"origin":"","legend":"Supplementary Video 4. Photoactivation of l/vlPAG-SST neurons induces flat-expiration airflow.","description":"","filename":"Video4Optogeneticsstimulationflatrespiratoryairflow.mp4","url":"https://assets-eu.researchsquare.com/files/rs-8634654/v1/931b1c63a43b9e0fba5f94f7.mp4"},{"id":101296841,"identity":"c727fad4-ddca-4c3d-940d-960f60f4816b","added_by":"auto","created_at":"2026-01-28 09:21:36","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":8087139,"visible":true,"origin":"","legend":"Supplementary Video 2. Fiber photometry recordings of l/vlPAG-SST neuronal activity during courtship USVs (4\u0026#x00D7; speed).","description":"","filename":"Video3Twophotonimaging.mp4","url":"https://assets-eu.researchsquare.com/files/rs-8634654/v1/5cabda226438115090592344.mp4"},{"id":101230000,"identity":"c0f3ed74-fd23-41bf-a1ab-71cf43e92d40","added_by":"auto","created_at":"2026-01-27 13:27:44","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":13496469,"visible":true,"origin":"","legend":"Supplementary Video 6. Photoinhibition of l/vlPAG-SST neurons abolishes ongoing courtship USVs.","description":"","filename":"Video6Optogeneticsinhibition.mp4","url":"https://assets-eu.researchsquare.com/files/rs-8634654/v1/9363f2042ea594f88c231dfe.mp4"},{"id":101230005,"identity":"541aac86-76e4-49db-b8e9-4c04f4b277d6","added_by":"auto","created_at":"2026-01-27 13:27:46","extension":"mp4","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":10671218,"visible":true,"origin":"","legend":"Supplementary Video 7. Synaptic silencing of l/vlPAG-SST neurons abolishes courtship USVs (5\u0026#x00D7; speed).","description":"","filename":"Video7TeLCinhibition.mp4","url":"https://assets-eu.researchsquare.com/files/rs-8634654/v1/d27301a4e244c565be28e567.mp4"},{"id":101229997,"identity":"b63706fa-14f8-4366-9135-efb07aaf6a44","added_by":"auto","created_at":"2026-01-27 13:27:43","extension":"mp4","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":13833390,"visible":true,"origin":"","legend":"Supplementary Video 5. Photoactivation of l/vlPAG-SST neurons drives frequency-dependent vocal-cord adduction.","description":"","filename":"Video5Optogeneticsstimulationvocalcord.mp4","url":"https://assets-eu.researchsquare.com/files/rs-8634654/v1/3119dbf47b5e921332196ed7.mp4"},{"id":101229999,"identity":"028ed09e-6cb4-482f-b1b5-86308150d4f9","added_by":"auto","created_at":"2026-01-27 13:27:44","extension":"mp4","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":19218415,"visible":true,"origin":"","legend":"Supplementary Video 3. Freely moving two-photon calcium imaging of l/vlPAG-SST neurons during courtship USVs.","description":"","filename":"Video2Photometry.mp4","url":"https://assets-eu.researchsquare.com/files/rs-8634654/v1/af3da4bda7c6e17cc7851a6c.mp4"},{"id":101230002,"identity":"d148425a-6d2d-45b7-a3dc-eb150c192119","added_by":"auto","created_at":"2026-01-27 13:27:44","extension":"mp4","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":15577605,"visible":true,"origin":"","legend":"Supplementary Video 8. Photoactivation of l/vlPAG-SST axon terminals in dPnTg evokes USVs.","description":"","filename":"Video8OptogeneticsstimulationofPAGdPnTgpathway.mp4","url":"https://assets-eu.researchsquare.com/files/rs-8634654/v1/8175eccfa648d6b6ba47ed20.mp4"},{"id":101229998,"identity":"efc76c16-7cf6-45a7-b4d6-294c95d4770d","added_by":"auto","created_at":"2026-01-27 13:27:44","extension":"mp4","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":19434600,"visible":true,"origin":"","legend":"Supplementary Video 1. Photoactivation of l/vlPAG-SST neurons with different frequencies triggers USVs.","description":"","filename":"Video1Optogeneticsstimulation.mp4","url":"https://assets-eu.researchsquare.com/files/rs-8634654/v1/8d99a56669c1731860172f64.mp4"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"A genetically defined midbrain-pontine circuit gates vocal communication","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8634654/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8634654/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Vocal communication is selectively expressed in social and emotional contexts (1-3), suggesting the presence of neural substrates that gate the conversion of internal states into vocal motor output. Although the periaqueductal gray (PAG) is known to be essential for vocalization (4-6), the genetic identity and circuit logic of the neurons that initiate and shape vocal signals remain unclear. Here, we identify a genetically defined population of somatostatin-expressing neurons in the lateral and ventrolateral PAG (l/vlPAG-SST) that functions as a premotor gate for ultrasonic vocalizations in mice. In vivo calcium imaging shows that l/vlPAG-SST neurons are selectively recruited around vocal onset. Manipulating l/vlPAG-SST neuron activity bidirectionally regulates vocal output: activation prolongs call duration by coordinating respiratory, laryngeal, and orofacial motor programs, whereas silencing suppresses courtship vocalizations. Projection-specific manipulations further demonstrate that descending glutamatergic, but not neuropeptidergic, output from l/vlPAG-SST neurons to the dorsal pontine tegmentum is sufficient to drive vocal production. Together, these findings define a genetically specified midbrain–pontine circuit that gates vocal communication and provides prospective access for linking internal conditions to vocal expressions.","manuscriptTitle":"A genetically defined midbrain-pontine circuit gates vocal communication","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-27 13:27:16","doi":"10.21203/rs.3.rs-8634654/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"60783955-1ad2-416c-a28f-64bebcc1ff91","owner":[],"postedDate":"January 27th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":61455275,"name":"Biological sciences/Neuroscience/Neural circuits"},{"id":61455276,"name":"Biological sciences/Neuroscience/Social behaviour"},{"id":61455277,"name":"Biological sciences/Neuroscience/Motivation"}],"tags":[],"updatedAt":"2026-05-13T09:32:45+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-27 13:27:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8634654","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8634654","identity":"rs-8634654","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}