{"paper_id":"2dd0959f-ed8b-4d20-9839-10ddcf384eaa","body_text":"The medial orbitofrontal cortex mediates neuropathic pain and anxiodepressive-like behaviors via two distinct pathways | 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 Research Article The medial orbitofrontal cortex mediates neuropathic pain and anxiodepressive-like behaviors via two distinct pathways Zhixiao Li, Tianen Si, Liangliang Gao, Yijie Meng, Xinyu Su, Liang Tao, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8000273/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Mar, 2026 Read the published version in The Journal of Headache and Pain → Version 1 posted 10 You are reading this latest preprint version Abstract Background Chronic neuropathic pain is frequently accompanied by anxiety and depression, yet the cortical circuit mechanisms underlying this comorbidity remain unclear. The medial orbitofrontal cortex (mOFC) is a key cortical region involved in emotional regulation and valuation, and clinical imaging studies have reported altered mOFC activity in patients with chronic pain. However, it remains unclear how mOFC neuronal activity contributes to both the pain hypersensitivity and anxiodepressive-like behaviors associated with neuropathic pain. This study aimed to determine the pathway-specific roles of mOFC glutamatergic neurons in chronic pain and comorbid affective disturbances. Methods Neuropathic pain was induced by chronic constriction injury (CCI) of the sciatic nerve. Pain sensitivity and anxiodepressive-like behaviors were assessed using von Frey, Hargreaves, open field, elevated plus maze, tail suspension, and forced swim tests. Electrophysiological recording and fiber photometry were used to monitor neuronal activity. Neuronal projection tracing identified projection patterns of mOFC glutamatergic neurons to mediodorsal thalamus (MD) and basolateral amygdala (BLA). Chemogenetic and optogenetic manipulations were applied to selectively modulate the mOFC CaMKIIα -MD and mOFC CaMKIIα -BLA pathways. Results The mOFC glutamatergic neurons are activated in neuropathic pain mice accompanied by anxiodepressive-like phenotypes. Chemogenetic and optogenetic inhibition of mOFC glutamatergic neurons attenuates both pain hypersensitivity and anxiodepressive-like behaviors after nerve injury. Retrograde labeling result revealed two non-overlapping mOFC CaMKIIα neurons projecting to the MD and BLA, respectively. Selective inhibition of mOFC CaMKIIα -MD pathway leads to amelioration of CCI-induced pain hypersensitivity. While selective inhibition of mOFC CaMKIIα -BLA pathway leads to amelioration of CCI-induced anxiodepressive-like behaviors. Conclusions The present study has revealed that the critical involvement of mOFC glutamatergic neurons in the comorbidity of chronic pain and affective disturbances. Inhibition of mOFC glutamatergic neurons attenuates both pain hypersensitivity and anxiodepressive-like behaviors after nerve injury. The discrete mOFC CaMKIIα -MD and mOFC CaMKIIα -BLA pathway independently control the sensory and affective components of neuropathic pain. neuropathic pain medial orbitofrontal cortex (mOFC) anxiety and depression basolateral amygdala (BLA) mediodorsal thalamus (MD) Full Text Additional Declarations No competing interests reported. Supplementary Files Supplementaryinformation.docx Cite Share Download PDF Status: Published Journal Publication published 18 Mar, 2026 Read the published version in The Journal of Headache and Pain → Version 1 posted Editorial decision: Revision requested 15 Dec, 2025 Reviews received at journal 20 Nov, 2025 Reviews received at journal 19 Nov, 2025 Reviewers agreed at journal 12 Nov, 2025 Reviewers agreed at journal 12 Nov, 2025 Reviewers agreed at journal 10 Nov, 2025 Reviewers invited by journal 10 Nov, 2025 Editor assigned by journal 04 Nov, 2025 Submission checks completed at journal 04 Nov, 2025 First submitted to journal 31 Oct, 2025 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. 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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-8000273\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":543903767,\"identity\":\"8e07fe32-ff05-4996-9279-0db9bcf74058\",\"order_by\":0,\"name\":\"Zhixiao Li\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Zhengzhou University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Zhixiao\",\"middleName\":\"\",\"lastName\":\"Li\",\"suffix\":\"\"},{\"id\":543903768,\"identity\":\"b7e99624-096d-4476-94c0-b64400b1b279\",\"order_by\":1,\"name\":\"Tianen 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pathways\",\"fulltext\":[],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":false,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"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\":\"info@researchsquare.com\",\"identity\":\"the-journal-of-headache-and-pain\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"tjhp\",\"sideBox\":\"Learn more about [The Journal of Headache and Pain](https://thejournalofheadacheandpain.biomedcentral.com/)\",\"snPcode\":\"10194\",\"submissionUrl\":\"https://submission.nature.com/new-submission/10194/3\",\"title\":\"The Journal of Headache and Pain\",\"twitterHandle\":\"@BioMedCentral\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC/SO AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"neuropathic pain, medial orbitofrontal cortex (mOFC), anxiety and depression, basolateral amygdala (BLA), mediodorsal thalamus (MD)\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-8000273/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-8000273/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003ch2\\u003eBackground\\u003c/h2\\u003e\\u003cp\\u003eChronic neuropathic pain is frequently accompanied by anxiety and depression, yet the cortical circuit mechanisms underlying this comorbidity remain unclear. The medial orbitofrontal cortex (mOFC) is a key cortical region involved in emotional regulation and valuation, and clinical imaging studies have reported altered mOFC activity in patients with chronic pain. However, it remains unclear how mOFC neuronal activity contributes to both the pain hypersensitivity and anxiodepressive-like behaviors associated with neuropathic pain. This study aimed to determine the pathway-specific roles of mOFC glutamatergic neurons in chronic pain and comorbid affective disturbances.\\u003c/p\\u003e\\u003ch2\\u003eMethods\\u003c/h2\\u003e\\u003cp\\u003eNeuropathic pain was induced by chronic constriction injury (CCI) of the sciatic nerve. Pain sensitivity and anxiodepressive-like behaviors were assessed using von Frey, Hargreaves, open field, elevated plus maze, tail suspension, and forced swim tests. Electrophysiological recording and fiber photometry were used to monitor neuronal activity. Neuronal projection tracing identified projection patterns of mOFC glutamatergic neurons to mediodorsal thalamus (MD) and basolateral amygdala (BLA). Chemogenetic and optogenetic manipulations were applied to selectively modulate the mOFC\\u003csup\\u003eCaMKIIα\\u003c/sup\\u003e-MD and mOFC\\u003csup\\u003eCaMKIIα\\u003c/sup\\u003e-BLA pathways.\\u003c/p\\u003e\\u003ch2\\u003eResults\\u003c/h2\\u003e\\u003cp\\u003eThe mOFC glutamatergic neurons are activated in neuropathic pain mice accompanied by anxiodepressive-like phenotypes. Chemogenetic and optogenetic inhibition of mOFC glutamatergic neurons attenuates both pain hypersensitivity and anxiodepressive-like behaviors after nerve injury. Retrograde labeling result revealed two non-overlapping mOFC\\u003csup\\u003eCaMKIIα\\u003c/sup\\u003e neurons projecting to the MD and BLA, respectively. Selective inhibition of mOFC\\u003csup\\u003eCaMKIIα\\u003c/sup\\u003e-MD pathway leads to amelioration of CCI-induced pain hypersensitivity. While selective inhibition of mOFC\\u003csup\\u003eCaMKIIα\\u003c/sup\\u003e-BLA pathway leads to amelioration of CCI-induced anxiodepressive-like behaviors.\\u003c/p\\u003e\\u003ch2\\u003eConclusions\\u003c/h2\\u003e\\u003cp\\u003eThe present study has revealed that the critical involvement of mOFC glutamatergic neurons in the comorbidity of chronic pain and affective disturbances. Inhibition of mOFC glutamatergic neurons attenuates both pain hypersensitivity and anxiodepressive-like behaviors after nerve injury. The discrete mOFC\\u003csup\\u003eCaMKIIα\\u003c/sup\\u003e-MD and mOFC\\u003csup\\u003eCaMKIIα\\u003c/sup\\u003e-BLA pathway independently control the sensory and affective components of neuropathic pain.\\u003c/p\\u003e\",\"manuscriptTitle\":\"The medial orbitofrontal cortex mediates neuropathic pain and anxiodepressive-like behaviors via two distinct pathways\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-11-19 17:46:42\",\"doi\":\"10.21203/rs.3.rs-8000273/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2025-12-15T07:24:52+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-11-20T14:15:34+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-11-20T02:57:35+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"76106375957458687674086396800287273735\",\"date\":\"2025-11-12T11:42:32+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"136057641328479201140752017281476932351\",\"date\":\"2025-11-12T10:28:19+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"82316334186021654341134831735080087693\",\"date\":\"2025-11-11T01:37:25+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2025-11-10T11:34:41+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2025-11-04T14:37:22+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2025-11-04T13:08:09+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"The Journal of Headache and Pain\",\"date\":\"2025-10-31T16:19:27+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"the-journal-of-headache-and-pain\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"tjhp\",\"sideBox\":\"Learn more about [The Journal of Headache and Pain](https://thejournalofheadacheandpain.biomedcentral.com/)\",\"snPcode\":\"10194\",\"submissionUrl\":\"https://submission.nature.com/new-submission/10194/3\",\"title\":\"The Journal of Headache and Pain\",\"twitterHandle\":\"@BioMedCentral\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC/SO AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"609af329-faa1-4e38-917b-27d8be0da280\",\"owner\":[],\"postedDate\":\"November 19th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2026-03-23T16:10:01+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-8000273\",\"link\":\"https://doi.org/10.1186/s10194-026-02335-w\",\"journal\":{\"identity\":\"the-journal-of-headache-and-pain\",\"isVorOnly\":false,\"title\":\"The Journal of Headache and Pain\"},\"publishedOn\":\"2026-03-18 15:59:34\",\"publishedOnDateReadable\":\"March 18th, 2026\"},\"versionCreatedAt\":\"2025-11-19 17:46:42\",\"video\":\"\",\"vorDoi\":\"10.1186/s10194-026-02335-w\",\"vorDoiUrl\":\"https://doi.org/10.1186/s10194-026-02335-w\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-8000273\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-8000273\",\"identity\":\"rs-8000273\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}