Convergent Control of NREM Sleep and Anesthesia by Prefrontal Layer 5 Extratelencephalic Neurons

Convergent Control of NREM Sleep and Anesthesia by Prefrontal Layer 5 Extratelencephalic Neurons | 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 Convergent Control of NREM Sleep and Anesthesia by Prefrontal Layer 5 Extratelencephalic Neurons Alex Proekt, Andrzej Wasilczuk, Daiki Takekawa, Joseph Cichon, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7861434/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract The cortical mechanisms that actively suppress consciousness remain poorly understood. Here, we identify a specific subset of prefrontal cortical (PFC) neurons preferentially active under anesthesia while most neurons are suppressed. Chemogenetic activation of these excitatory neurons enhances anesthetic potency and deepens NREM sleep, whereas their inhibition blunts anesthetic effects. We identify these NREM and Anesthesia Promoting (NAP) neurons as PFC Layer 5 extratelencephalic (L5 ET) neurons. Remarkably, NAPs have sparse cortical projections and predominately communicate with subcortical nuclei including anterior and reticular thalamic nuclei, hypothalamus, and claustrum. We identify a transgenic mouse line that labels L5 ET neurons and verify that PFC L5 ET neurons are uniquely activated under anesthesia. Furthermore, we show that activation of PFC L5 ET neurons promotes deep NREM sleep. These findings identify a unique excitatory PFC circuit that promotes both naturally-occurring and drug-induced unconsciousness, with implications for both sleep regulation and anesthetic action. Biological sciences/Neuroscience/Circadian rhythms and sleep/Non-REM sleep Biological sciences/Neuroscience/Neural circuits Biological sciences/Neuroscience/Cellular neuroscience Full Text Additional Declarations There is NO Competing Interest. Supplementary Files supfigure1.png Supplemental Figure 1 supfigure2.png Supplemental Figure 2 supfigure3.png Supplemental Figure 3 supfigure4.png Supplemental Figure 4 supfigure5.png Supplemental Figure 5 supfigure6.png Supplemental Figure 6 supfigure7.png Supplemental Figure 7 supfigure8.png Supplemental Figure 8 InputOutputTable.pdf Supplemental Table 1 Cite Share Download PDF Status: Under Review 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-7861434","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":547121481,"identity":"9c4e43bd-1048-48b7-a029-0ba84048cbd7","order_by":0,"name":"Alex Proekt","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsklEQVRIiWNgGAWjYJACxgYGGxDVeIAELQlpYIokLYfBDOK0mEsfPvhw5o/zdmvbDwNtqbGJJqjFsi8t2XBDwu3kbWcSgVqOpeU2ENJicIbHTPIBUIvZAaAWxobDRGkx//kg4Vyy2fmHxGsxY9yQcMDO7Aaxtlj2sCVLzkhLTjC7AbQlgRi/mPMwH/zYY2Nnb3Y+/eGDDzU2RDgMSieCVSYQUo6sxZ4YxaNgFIyCUTBCAQCjYEgvmUiGoAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-9272-5337","institution":"University of Pennsylvania","correspondingAuthor":true,"prefix":"","firstName":"Alex","middleName":"","lastName":"Proekt","suffix":""},{"id":547121482,"identity":"f924c96b-d386-4361-bc50-e2fa5cd348a9","order_by":1,"name":"Andrzej Wasilczuk","email":"","orcid":"https://orcid.org/0000-0002-1492-2319","institution":"University of Pennsylvania","correspondingAuthor":false,"prefix":"","firstName":"Andrzej","middleName":"","lastName":"Wasilczuk","suffix":""},{"id":547121483,"identity":"29c212e4-1192-432f-afb5-94724b2b314a","order_by":2,"name":"Daiki Takekawa","email":"","orcid":"https://orcid.org/0000-0003-4005-9536","institution":"Hirosaki University","correspondingAuthor":false,"prefix":"","firstName":"Daiki","middleName":"","lastName":"Takekawa","suffix":""},{"id":547121484,"identity":"08a8a47e-6d2f-4b81-beff-12d8937a22cb","order_by":3,"name":"Joseph Cichon","email":"","orcid":"https://orcid.org/0000-0003-4318-9707","institution":"University of Pennsylvania","correspondingAuthor":false,"prefix":"","firstName":"Joseph","middleName":"","lastName":"Cichon","suffix":""},{"id":547121485,"identity":"63e9cfe9-d3ff-4027-a5c3-a9ca1926fe99","order_by":4,"name":"Xiaoyan Zhang","email":"","orcid":"","institution":"University of Pennsylvania","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyan","middleName":"","lastName":"Zhang","suffix":""},{"id":547121486,"identity":"caa29403-2e13-4a32-86fd-fde4896718c9","order_by":5,"name":"Ethan Blackwood","email":"","orcid":"https://orcid.org/0000-0002-3049-0640","institution":"University of Pennsylvania","correspondingAuthor":false,"prefix":"","firstName":"Ethan","middleName":"","lastName":"Blackwood","suffix":""},{"id":547121487,"identity":"11cab401-541b-4eaf-a349-84fbfea73cd2","order_by":6,"name":"Jeffrey Parra-Munevar","email":"","orcid":"https://orcid.org/0009-0008-4606-3724","institution":"University of Washingtion","correspondingAuthor":false,"prefix":"","firstName":"Jeffrey","middleName":"","lastName":"Parra-Munevar","suffix":""},{"id":547121488,"identity":"83c5db46-4a6e-4fc2-84ed-e3493171065a","order_by":7,"name":"Mean-Hwan Kim","email":"","orcid":"","institution":"DGIST","correspondingAuthor":false,"prefix":"","firstName":"Mean-Hwan","middleName":"","lastName":"Kim","suffix":""},{"id":547121489,"identity":"71472e07-f364-4c04-8a2d-71f47260e3f3","order_by":8,"name":"William Spain","email":"","orcid":"","institution":"University of Washington","correspondingAuthor":false,"prefix":"","firstName":"William","middleName":"","lastName":"Spain","suffix":""},{"id":547121490,"identity":"9b0badec-e4d9-4c41-ad7b-aa02183f121a","order_by":9,"name":"Aaron Krom","email":"","orcid":"https://orcid.org/0000-0002-0266-4957","institution":"Hadassah-Hebrew University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Aaron","middleName":"","lastName":"Krom","suffix":""},{"id":547121491,"identity":"36b0c7e1-1889-438c-82d8-33c2ea9c9f7f","order_by":10,"name":"Max Kelz","email":"","orcid":"https://orcid.org/0000-0002-2803-6078","institution":"University of Pennsylvania","correspondingAuthor":false,"prefix":"","firstName":"Max","middleName":"","lastName":"Kelz","suffix":""},{"id":547121492,"identity":"7dae0841-4a87-46e9-8071-1ea4d77a0b79","order_by":11,"name":"Nikolai DEMBROW","email":"","orcid":"https://orcid.org/0000-0002-7699-6591","institution":"University of Washington","correspondingAuthor":false,"prefix":"","firstName":"Nikolai","middleName":"","lastName":"DEMBROW","suffix":""}],"badges":[],"createdAt":"2025-10-14 19:00:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7861434/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7861434/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":96368819,"identity":"eb55f60f-9022-4fba-9832-d69dbdd71cd1","added_by":"auto","created_at":"2025-11-20 10:19:18","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2611557,"visible":true,"origin":"","legend":"Article File","description":"","filename":"NNA93048WasilczuketalPFCsleep.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7861434/v1_covered_8b233bfa-27c7-42e6-bb07-4cbf463ecac2.pdf"},{"id":96262957,"identity":"eecfda45-9333-4fab-8bbe-f3bd12bb3113","added_by":"auto","created_at":"2025-11-19 08:18:34","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":74488,"visible":true,"origin":"","legend":"Supplemental Figure 1","description":"","filename":"supfigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7861434/v1/30e998ee1d74566d4cd76249.png"},{"id":96262964,"identity":"90476574-9f07-4fd8-aa82-54935de9da51","added_by":"auto","created_at":"2025-11-19 08:18:34","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":220817,"visible":true,"origin":"","legend":"Supplemental Figure 2","description":"","filename":"supfigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7861434/v1/07503a9996a7ebecf1d5379d.png"},{"id":96363867,"identity":"4c5a0fc5-8c82-4024-872d-3322cb5e78a0","added_by":"auto","created_at":"2025-11-20 10:08:13","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":953328,"visible":true,"origin":"","legend":"Supplemental Figure 3","description":"","filename":"supfigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7861434/v1/24cd77f06e501fb7cb9148a8.png"},{"id":96262961,"identity":"e477cfa9-2819-4123-958f-e0dd04e4c5e8","added_by":"auto","created_at":"2025-11-19 08:18:34","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":228387,"visible":true,"origin":"","legend":"Supplemental Figure 4","description":"","filename":"supfigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7861434/v1/74d7f2a3fa211af671d17631.png"},{"id":96363176,"identity":"620ddc36-1f71-4c6d-92db-5a4bbfd70461","added_by":"auto","created_at":"2025-11-20 10:05:14","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":242543,"visible":true,"origin":"","legend":"Supplemental Figure 5","description":"","filename":"supfigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7861434/v1/ffd62088bbeec9e7502b228f.png"},{"id":96262958,"identity":"6c27c3c9-6075-44cf-b1d6-5a8c7e790c9d","added_by":"auto","created_at":"2025-11-19 08:18:34","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":65137,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental Figure 6\u003c/p\u003e","description":"","filename":"supfigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7861434/v1/f7fde3e7e1978a9a189a9958.png"},{"id":96363544,"identity":"52e78650-48a2-4bd3-b0b8-4e1dd7580988","added_by":"auto","created_at":"2025-11-20 10:07:17","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":46388,"visible":true,"origin":"","legend":"Supplemental Figure 7","description":"","filename":"supfigure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7861434/v1/959e61175de244dc80cac294.png"},{"id":96262960,"identity":"c189527e-1739-48cf-b2cb-cfb9bb6d55dd","added_by":"auto","created_at":"2025-11-19 08:18:34","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":540885,"visible":true,"origin":"","legend":"Supplemental Figure 8","description":"","filename":"supfigure8.png","url":"https://assets-eu.researchsquare.com/files/rs-7861434/v1/f886030bfc5805cb9d30d3fd.png"},{"id":96362812,"identity":"1c595a93-a9b8-44a6-a4df-e0b5277f7a48","added_by":"auto","created_at":"2025-11-20 09:56:29","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":249328,"visible":true,"origin":"","legend":"Supplemental Table 1","description":"","filename":"InputOutputTable.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7861434/v1/19ee3615a48bfbc73676c768.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Convergent Control of NREM Sleep and Anesthesia by Prefrontal Layer 5 Extratelencephalic Neurons","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"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":"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":"","lastPublishedDoi":"10.21203/rs.3.rs-7861434/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7861434/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The cortical mechanisms that actively suppress consciousness remain poorly understood. Here, we identify a specific subset of prefrontal cortical (PFC) neurons preferentially active under anesthesia while most neurons are suppressed. Chemogenetic activation of these excitatory neurons enhances anesthetic potency and deepens NREM sleep, whereas their inhibition blunts anesthetic effects. We identify these NREM and Anesthesia Promoting (NAP) neurons as PFC Layer 5 extratelencephalic (L5 ET) neurons. Remarkably, NAPs have sparse cortical projections and predominately communicate with subcortical nuclei including anterior and reticular thalamic nuclei, hypothalamus, and claustrum. We identify a transgenic mouse line that labels L5 ET neurons and verify that PFC L5 ET neurons are uniquely activated under anesthesia. Furthermore, we show that activation of PFC L5 ET neurons promotes deep NREM sleep. These findings identify a unique excitatory PFC circuit that promotes both naturally-occurring and drug-induced unconsciousness, with implications for both sleep regulation and anesthetic action.","manuscriptTitle":"Convergent Control of NREM Sleep and Anesthesia by Prefrontal Layer 5 Extratelencephalic Neurons","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-19 08:18:30","doi":"10.21203/rs.3.rs-7861434/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-neuroscience","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"neuro","sideBox":"Learn more about [Nature Neuroscience](http://www.nature.com/neuro/)","snPcode":"","submissionUrl":"","title":"Nature Neuroscience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"950f3f62-a769-4009-8e22-5b93ee3d4112","owner":[],"postedDate":"November 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":58208473,"name":"Biological sciences/Neuroscience/Circadian rhythms and sleep/Non-REM sleep"},{"id":58208474,"name":"Biological sciences/Neuroscience/Neural circuits"},{"id":58208475,"name":"Biological sciences/Neuroscience/Cellular neuroscience"}],"tags":[],"updatedAt":"2026-01-09T12:47:14+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-19 08:18:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7861434","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7861434","identity":"rs-7861434","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}