Hyperactivity of the Amygdala Mediates Depressive-Like Phenotypes and Decreased Serotonin Release

Hyperactivity of the Amygdala Mediates Depressive-Like Phenotypes and Decreased Serotonin Release | 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 Hyperactivity of the Amygdala Mediates Depressive-Like Phenotypes and Decreased Serotonin Release Jufang He, kyuhee KIM, YuanYing Lai, Qianqian Gao, Huajie Wang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7251588/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 Clinical and preclinical studies have consistently demonstrated a correlation between hyperactivity of the amygdala and the onset of depression. However, the underlying mechanisms influencing serotonin levels a critical neurotransmitter implicated in depression and a primary target for selective serotonin reuptake inhibitors (SSRIs) remain inadequately understood. In this study, we employed a restrained inescapable shock (RIS) model to investigate these mechanisms in mice. The RIS paradigm elicited depressive-like phenotypes, increased c-Fos expression in the amygdala, diminished serotonin levels, and elevated corticosterone concentrations in serum. Notably, chemogenetic inhibition of the amygdala mitigated depressive symptoms, reduced neuronal activity in this region, and restored serotonin levels. Anatomical analyses revealed a strong connectivity between the central amygdala (CeA) and the dorsal raphe nucleus (DRN). Fiber photometry recordings indicated that serotonergic neuronal activity in the DRN decreased in response to aversive stimuli, accompanied by amygdala activation following RIS, with no notable alterations in DRN GABAergic activity. These findings suggest that chronic stress may exacerbate amygdala hyperactivity, which subsequently inhibits serotonin release in the brain, potentially intensifying depressive states. Therefore, targeting amygdala hyperactivity may represent a novel therapeutic strategy for the management of stress-related depressive and anxiety disorders. Biological sciences/Neuroscience/Molecular neuroscience Health sciences/Diseases/Psychiatric disorders/Depression Amygdala Depression Serotonin Restrained tail shock stress model Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Full Text Additional Declarations The authors have declared there is NO conflict of interest to disclose Supplementary Files Supplementary1.pdf Supplementary 1. Acute foot shock and foot shock with laser activation of CeA induced change in DRN neuronal activity. A. Showing calcium traces during FS (aversive stimuli) and FS paired with CeA activation in mice. B. Average change in DRN activity before and during FS and FS paired with CeA activation in mice. RM Two-way ANOVA, Interaction: ns P < 0.39, F (1, 10) = 0.79. Row Factor: ns P < 0.55, F (1, 10) = 0.38. Column Factor: ****P < 0.0001, F (1, 10) = 97.17 Baseline vs FS: ***P < 0.0002, t = 6.34, df = 10. Baseline vs FS-Laser: ****P < 0.0001, t = 7.60, df = 10. Adjustment: Bonferroni. N = 6 mice. C. Showing the average changes between FS and FS paired with CeA activation in mice. RM Two-way ANOVA, Interaction: ns P < 0.54, F (1, 10) = 0.40. Row Factor: ****P < 0.0001, F (1, 10) = 49.93. Column Factor: ns P 0.9999, t = 0.085, df = 10. FS vs FS-Laser: ns P > 0.9999, t = 0.98, df = 10. Adjustment: Bonferroni. N = 6 mice Supplementary2.pdf Supplementary 2. Acute foot shock or foot shock with laser activation of CeA induces a change in the subtype of DRN neuronal activity. A. Schematical representation of the experimental design for GCaMP6s virus injection. B. Showing calcium traces during FS (aversive stimuli) and FS paired with CeA activation in mDlx-GCaMP6s expression mice. C. Average change in DRN activity before and during FS and FS paired with CeA activation. RM Two-way ANOVA, Interaction: ns P < 0.43, F (1, 8) = 0.67. Row Factor: ns P < 0.62, F (1, 8) = 0.25 Column Factor: ***P < 0.0002, F (1, 8) = 42.71. Baseline vs FS: **P < 0.0075, t = 4.04, df = 8. Baseline vs FS-Laser: **P < 0.0016, t = 5.20, df = 8. Adjustment: Bonferroni. N = 5 mice. D. Showing the average changes between FS and FS paired with CeA activation. RM Two-way ANOVA, Interaction: ns P < 0.17, F (1, 8) = 018. Row Factor: ***P < 0.0004, F (1, 8) = 32.79. Column Factor: ns P < 0.49, F (1, 8) = 051. Baseline (FS) vs Baseline (FS-Laser): nsP < 0.99, t = 0.53, df = 8. FS vs FS-Laser: ns P < 0.32, t = 1.55, df = 8. Adjustment: Bonferroni. N = 5 mice. E. Schematical representation of the experimental design for GCaMP6s virus injection. F. Showing calcium traces during FS (aversive stimuli) and FS paired with CeA activation in TPH2-GCaMP6s expression mice. G. Average change in DRN activity before and during FS and FS paired with CeA activation. RM Two-way ANOVA, Interaction: nsP < 0.44, F (1, 10) = 0.63. Row Factor: ns P < 0.40, F (1, 10) = 0.75. Column Factor: **P < 0.0024, F (1, 10) = 16.30. Baseline vs FS: ns P < 0.089, t = 2.29, df = 10. Baseline vs FS-Laser: *P < 0.013, t = 3.42, df = 10. Adjustment: Bonferroni. N = 6 mice. H. Showing the average changes between FS and FS paired with CeA activation. RM Two-way ANOVA, Interaction: ns P < 0.44, F (1, 10) = 0.62. Row Factor: **P < 0.0015, F (1, 10) = 18.55. Column Factor: ns P < 0.44, F (1, 10) = 0.65. Baseline (FS) vs Baseline (FS-Laser): nsP < 0.99, t = 0.013, df = 10. FS vs FS-Laser: ns 782 P < 0.57, t = 1.133, df = 10. Adjustment: Bonferroni. N = 6 mice StatisticalTable20250728KIM.pdf 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-7251588","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":500718667,"identity":"55cea5a1-8426-447b-9329-83e558859f6d","order_by":0,"name":"Jufang He","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYBACxgbGB0DqAAM/kAkROsDABmPi0MJsAFYm2cDY2ECUFgYGqBaDAzBrCGlhnpHM+Lng1x054+OH2x/ztjHI8d1IYHs4A5/DZiQzS8/se2ZsdiaxsRmoxVjyRgK74Qa8WvIPSPP2HE7cdoMRrCVxA9AWyQcEbPkN1FK/eQZESz0xWtikeX4cTjCQgGhJMABpweuwnsds1rwNhw1nAP0yc845CcOZZx62SeLzvmF7MvNtnj+H5fnbjz/48KbMRp7vePIxyR58WhpAVrVBOEw8DBIgbgMeDQwM8mDyD9SVP/CqHQWjYBSMgpEKAKL2VjkPTcKfAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-4288-5957","institution":"City University of Hong Kong","correspondingAuthor":true,"prefix":"","firstName":"Jufang","middleName":"","lastName":"He","suffix":""},{"id":500718668,"identity":"0870d0b3-01e0-424e-9724-fb5832d1863a","order_by":1,"name":"kyuhee KIM","email":"","orcid":"","institution":"City University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"kyuhee","middleName":"","lastName":"KIM","suffix":""},{"id":500718669,"identity":"2b7c0e7d-f56d-4380-9655-be06baf4a6e9","order_by":2,"name":"YuanYing Lai","email":"","orcid":"","institution":"City University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"YuanYing","middleName":"","lastName":"Lai","suffix":""},{"id":500718670,"identity":"d1a98383-e56f-4b7a-b6be-1508b1f1e143","order_by":3,"name":"Qianqian Gao","email":"","orcid":"","institution":"City University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"Qianqian","middleName":"","lastName":"Gao","suffix":""},{"id":500718671,"identity":"614f68ab-8e34-4761-88d2-9bb664864eac","order_by":4,"name":"Huajie Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Huajie","middleName":"","lastName":"Wang","suffix":""},{"id":500718672,"identity":"90277b12-c87f-4c38-904f-94e1445bf809","order_by":5,"name":"Kwok Tsz Ying","email":"","orcid":"","institution":"City University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"Kwok","middleName":"Tsz","lastName":"Ying","suffix":""},{"id":500718673,"identity":"bd92b031-2e8b-4fe2-9f98-40ed134cae9c","order_by":6,"name":"Muhammad ASIM","email":"","orcid":"https://orcid.org/0000-0002-6912-4461","institution":"Stanford University","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"","lastName":"ASIM","suffix":""}],"badges":[],"createdAt":"2025-07-30 10:10:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7251588/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7251588/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89983642,"identity":"f53482cc-8d15-4a45-a317-46e053464c61","added_by":"auto","created_at":"2025-08-27 06:34:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":845501,"visible":true,"origin":"","legend":"\u003cp\u003eRestrained inescapable shock triggers depressive-like behavior in mice. \u0026nbsp;A. An Experimental design to induce depressive-like behavior in rodents. B. Sucrose \u0026nbsp;preference percentage among control, stress, and 6 weeks after stress group. One-way ANOVA, \u0026nbsp;Treatment (column): **P \u0026lt; 0.0023, F (2, 26) = 7.721. Control vs. Stress: *P\u0026lt; 0.0157, t = 3.049, \u0026nbsp;df = 26. Control vs. 6weeks after stress: **P \u0026lt; 0.0030, t = 3.712, df = 26. Stress Vs. 6weeks after stress: ns P \u0026gt; 0.9999, t = 0.6817, df = 26. Adjustment: Bonferroni. N = \u0026nbsp;9-10 mice. C. Immobility time (sec) during TST. One-way ANOVA, Treatment (column): \u0026nbsp;****P \u0026lt; 0.0001, F (2, 27) = 96.53. Control vs. Stress: ****P\u0026lt; 0.0001, t = 9.310, df = 27. \u0026nbsp;Control vs. 6weeks after stress: ****P \u0026lt; 0.0001, t = 13.59, df = 27. Stress Vs. 6weeks after \u0026nbsp;stress: ***P \u0026lt;0.0006, t = 4.277, df = 27. Adjustment: Bonferroni. \u0026nbsp;N = 10 mice. D. Trajectory map during OFT. E. Time spent in center (sec) during OFT. Oneway ANOVA, Treatment (column): *P \u0026lt; 0.0464, F (2, 27) = 3.448. Control vs. Stress: ns P\u0026lt; \u0026nbsp;0.3883, t = 1.564, df = 27. Control vs. 6weeks after stress: *P \u0026lt; 0.0439, t = 2.609, df = 27. Stress Vs. 6weeks after stress: ns P \u0026lt;0.9163, t = 1.045, df = 27. Adjustment: Bonferroni. \u0026nbsp;N = 10 mice. F. Total distance explored (cm) during OFT. One-way ANOVA, Treatment (column): nsP \u0026lt; 0.5752, F (2, 27) = 0.5646. Control vs. Stress: ns P\u0026lt; 0.9883, t = 0.9932, df = 27. \u0026nbsp;P \u0026gt; 0.9999, t = 0.1695, df = 27. Stress Vs. 6weeks after stress: ns P \u0026gt; 0.9999, t = 0.8237, df = 27. Adjustment: Bonferroni. N = 10 mice. G. c-Fos expression \u0026nbsp;control (Left), stress (Middle), and 6 weeks after stress (Right) group in BLA. H. Measured c Fos expression levels in BLA. One-way ANOVA, Treatment(column): *P \u0026lt; 0.0251, F (2, 17) = 4.611. Control vs. Stress: ns P\u0026lt; 0.3795, t = 1.607, df = 17. Control vs. 6weeks after stress: *P \u0026lt; 0.0226, t = 3.032, df = 17. Stress Vs. 6weeks after stress: ns P \u0026gt; 0.4650, t = 1.488, df = 17. \u0026nbsp;Adjustment: Bonferroni. N = 3 mice (6-7 slices). I. Showing the corticosterone level changes \u0026nbsp;between control and depressive-like behavior mice. Unpaired t-test, *P \u0026lt; 0.0421, t = 2.188, df \u0026nbsp;= 18, N = 10 mice. J. Showing the 5-ht level changes between control and depressive-like \u0026nbsp;behavior mice. Unpaired t-test, **P \u0026lt; 0.0012, t = 3.856, df = 18, N = 10 mice\u003c/p\u003e","description":"","filename":"Binder21.png","url":"https://assets-eu.researchsquare.com/files/rs-7251588/v1/0ab94b354572fcd99ad6b369.png"},{"id":89983640,"identity":"199672dd-d46b-40fc-822e-345e949751e6","added_by":"auto","created_at":"2025-08-27 06:34:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":656384,"visible":true,"origin":"","legend":"\u003cp\u003eChemogenetic inhibition of the amygdala alleviates depressive-like behaviors in RIS mice. A. Schematical representation of experimental design. B. hM4Di-mCherry virus expression for chemogenetic inhibition of BLA. C. Sucrose preference percentage among control, stress, and hM4Di group. One-way ANOVA, Treatment (column): ***P \u0026lt; 0.0003, F (2, 34) = 10.52. Control vs. Stress: ***P\u0026lt; 0.0006, t = 4.189, df = 34. Control vs. Treatment: ns P \u0026gt; 0.9999, t = 0.5268, df = 34. Stress Vs. Treatment: **P \u0026lt; 0.0026, t = 3.652, df = 34. Adjustment: Bonferroni. N = 13-14 mice. D. Immobility time (sec) during TST. One-way ANOVA, Treatment (column): ****P \u0026lt; 0.0001, F (2, 37) = 12.04. Control vs. Stress: **P\u0026lt; 0.021, t = 3.692, df = 37. Control vs. Treatment: ns P \u0026gt; 0.9999, t = 0.9030, df = 37. Stress Vs. Treatment: ***P \u0026lt;0.0001, t = 4.612, df = 37. Adjustment: Bonferroni. N = 13-14 mice. E. Trajectory map during OFT. F. Time spent in center (sec) during OFT. One-way ANOVA, Treatment (column): ***P \u0026lt; 0.0003, F (2, 37) = 10.12. Control vs. Stress: **P\u0026lt; 0.0020, t = 3.721, df = 37. Control vs. Treatment: ns 638 P \u0026gt; 0.9999, t = 0.2835, df = 37. Stress Vs. Treatment ***P \u0026lt; 0.0009, t = 4.010, df = 37. Adjustment: Bonferroni. N = 13-14 mice. G. Total distance explored(cm) during OFT. One-way ANOVA, Treatment (column): ns P \u0026lt; 0.4281, F (2, 37) = 0.6486. Control vs. Stress: ns P \u0026gt; 0.9999, t = 0.5915, df = 37. Control vs. Treatment: nsP \u0026gt; 0.9999, t = 0.3233, df = 37. Stress Vs. Treatment ns P \u0026lt; 0.9999, t = 0.9207, df = 37. Adjustment: Bonferroni. N = 13-14 mice. H. c-Fos expression among control, stress, and hM4Di group in BLA. I. Measured c-fos expression levels in BLA. Unpaired t-test, *P \u0026lt; 0.0317, t = 2.459, df = 11, N = 3 mice (6-7 slices). J. Showing the corticosterone level changes among control, stress, and hM4Di groups. One-way ANOVA, Treatment (column): nsP \u0026lt; 0.0530, F (2, 25) = 3.311. Control vs. Stress: ns P \u0026lt; 0.0646, t = 2.452, df = 25. Control vs. Treatment: nsP \u0026gt; 0.9999, t = 0.4684, df = 25. Stress Vs. Treatment: ns P \u0026gt; 0.2313, t = 1.844, df = 25. Adjustment: Bonferroni. N = 8-10 mice. K. The 5-HT level changes control, stress, and the hM4Di group. One-way ANOVA, Treatment (column): **P \u0026lt; 0.0057, F (2, 25) = 6.410. Control vs. Stress: *P \u0026lt; 0.0137, t = 3.116, df = 25. Control vs. Treatment: ns P \u0026gt; 0.9999, t = 0.08747, df = 25. Stress Vs. Treatment: *P \u0026lt; 0.0171, t = 3.025, df = 25. Adjustment: Bonferroni, N = 8-10 mice\u003c/p\u003e","description":"","filename":"Binder22.png","url":"https://assets-eu.researchsquare.com/files/rs-7251588/v1/bbb43f6829498237c1b2922b.png"},{"id":89983641,"identity":"1b8b58de-08bd-48f7-ae0b-d715219751b7","added_by":"auto","created_at":"2025-08-27 06:34:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1486007,"visible":true,"origin":"","legend":"\u003cp\u003eAnatomical confirmation of the amygdala connectivity with dorsal raphe nucleus using a retrograde virus. A. Schematical representation of the experimental design for AAV-retro virus injection in DRN. B. EGFP expression in CeA and fibers in DRN. C. tdTtomato expression in DRN Ai 14 mice, and tdTomato regrade cells in CeA. D. Schematical representation of the experimental design for Cre-dependant mCherry expression in CeA-DRN pathway. E. mCherry expression in DRN. F. Co-localization with 5-HT antibody. G. Co-localization with GAD67 positive- cells. H. Measurement of Co-localization with 5-HT and GAD-67 in DRN. Unpaired t-test, *P \u0026lt; 0.0105, t = 2.953, df = 14, N = 3 mice(8slices). I. Percentage of co-localized cells with 5HT and GAD 67 in DRN.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Binder23.png","url":"https://assets-eu.researchsquare.com/files/rs-7251588/v1/31e8f3efcd5141661510b3de.png"},{"id":89983643,"identity":"7ba18e18-2e22-465a-be93-86b9f1ab000d","added_by":"auto","created_at":"2025-08-27 06:34:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":373382,"visible":true,"origin":"","legend":"\u003cp\u003eDRN neuronal activities in response to Foot shock and Foot shock paired with CeA activation. A. An Experimental Design. B. Schematical representation of experimental design for Fiber photometry. C. ChrimsonR virus expression in CeA and GCaMP6s virus expression in the DRN. D. Schematical representation of the experimental design for foot shock. E. Showing calcium traces during FS in experimental mice with restrained inescapable shock. F. Average change in DRN activity before and during FS in experimental mice with restrained inescapable shock. RM Two-way ANOVA, Interaction: nsP \u0026lt; 0.19, F (1, 10) = 2.018. Row Factor: ns P \u0026lt; 0.93, F (1, 10) = 0.008. Column Factor: ****P \u0026lt; 0.0001, F (1, 10) = 109.7. Baseline vs FS (Pre-Stress-Day1): ***P \u0026lt; 0.0002, t = 6.402, df = 10. Baseline vs FS (Post Stress-Day5): ****P \u0026lt; 0.0001, t = 8.411, df = 10. Adjustment: Bonferroni. N = 6 mice. G. Showing the average changes during FS in experimental mice with restrained inescapable shock. RM Two-way ANOVA, Interaction: nsP \u0026lt; 0.31, F (1, 10) = 1.104. Row Factor ****P \u0026lt; 0.0001, F (1, 10) = P \u0026lt; 0.91, F (1, 10) = 0.013. Baseline (Pre-Stress-Day1) vs Baseline (Post Stress-Day5): ns P \u0026gt; 0.86, t = 0.82, df = 10. FS (Pre-Stress-Day1) vs FS (Post Stress Day5): ns P \u0026lt; 0.99, t = 0.66, df = 10. Adjustment: Bonferroni. N = 6 mice. H. Schematical representation of the experimental design for foot shock paired with CeA activation. I. Showing calcium traces during FS paired with CeA activation in experimental mice with restrained inescapable shock. J. Average change in DRN activity before and during FS paired with CeA activation in experimental mice with restrained inescapable shock. RM Two-way ANOVA, Interaction: nsP \u0026lt; 0.27, F (1, 10) = 1.36. Row Factor: ns P \u0026lt; 0.19, F (1, 10) = 1.93. Column Factor: ****P \u0026lt; 0.0001, F (1, 10) = 52.72. Baseline vs FS-Laser (Pre-Stress-Day1): **P \u0026lt; 0.0031, t = 4.31, df = 10. Baseline vs FS-Laser (Post Stress-Day5): ***P \u0026lt; 0.0003, t = 5.96, df = 10. Adjustment: Bonferroni. N = 6 mice. K. Showing the average changes during FS paired with CeA activation in experimental mice with restrained inescapable shock. RM Two-way ANOVA, Interaction: ns P \u0026lt; 0.15, F (1, 10) = 2.33. Row Factor: ***P \u0026lt; 0.0002, F (1, 10) = 33.05. Column Factor: nsP \u0026lt; 0.076, F (1, 10) = 3.90. Baseline (Pre-Stress-Day1) vs Baseline (Post Stress-Day5): ns P \u0026lt; 0.99, t = 0.32, df = 10. FS-Laser (Pre-Stress-Day1) vs FS-Laser (Post Stress-Day5): nsP \u0026lt; 0.066, t = 2.48, df = 10. Adjustment: Bonferroni, N = 6 mice.\u003c/p\u003e","description":"","filename":"Binder24.png","url":"https://assets-eu.researchsquare.com/files/rs-7251588/v1/0c4d9c4bb60ebb6708aff826.png"},{"id":89983637,"identity":"192117ed-adf6-4dbf-a810-9c1291f03386","added_by":"auto","created_at":"2025-08-27 06:34:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":314641,"visible":true,"origin":"","legend":"\u003cp\u003eRIS induced a change in DRN GABAergic neuronal activity. \u0026nbsp;A. An experimental design. B. Schematical representation of the experimental design for \u0026nbsp;GCaMP6s virus injection. C. ChrimsonR virus expression in CeA and GCaMP6s virus \u0026nbsp;expression in the DRN. D. Showing calcium traces during FS in mDlx-GCaMP6s virus \u0026nbsp;expression mice with restrained inescapable shock. E. Average change in DRN activity before \u0026nbsp;and during FS in mDlx-GCaMP6s virus expression mice with restrained inescapable shock. RM Two-way ANOVA, Interaction: nsP \u0026lt; 0.91, F (1, 8) = 0.015. Row Factor: ns P \u0026lt; 0.91, F (1, \u0026nbsp;8) = 0.012. Column Factor: ***P \u0026lt; 0.0004, F (1, 8) = 34.40. Baseline vs FS (Pre-Stress-Day1): \u0026nbsp;**P \u0026lt; 0.0057, t = 4.23, df = 8. Baseline vs FS (Post Stress-Day5): **P \u0026lt; 0.0073, t = 4.061, df \u0026nbsp;= 8. Adjustment: Bonferroni. N = 5 mice. F. Showing the average changes between FS in \u0026nbsp;mDlx-GCaMP6s virus expression mice with restrained inescapable shock. RM Two-way ANOVA, Interaction: ns P \u0026lt; 0.85, F (1, 8) = 0.039. Row Factor: ****P \u0026lt; 0.0015 F (1, 8) = 22.12. Column Factor: ns P \u0026lt; 0.87, F (1, 8) = 0.030. Baseline (Pre-Stress-Day1) vs Baseline (Post Stress-Day5): ns P \u0026gt; 0.9999, t = 0.016, df = 8. FS (Pre-Stress-Day1) vs FS (Post Stress Day5): ns P \u0026gt; 0.9999, t = 0.26, df = 8. Adjustment: Bonferroni. N = 5 mice. G. Showing calcium traces during FS paired with CeA activation in mDlx-GCaMP6svirus expression mice with \u0026nbsp;restrained inescapable shock. H. Average change in DRN activity before and during FS paired \u0026nbsp;with CeA activation in mDlx-GCaMP6s virus expression mice with restrained inescapable shock. RM Two-way ANOVA, Interaction: nsP \u0026lt; 0.79, F (1, 8) = 0.07. Row Factor: ns P \u0026lt; 0.59, F (1, 8) = 0.30. Column Factor: ***P \u0026lt; 0.0005, F (1, 8) = 31.30. Baseline vs FS-Laser (Pre Stress Day1): *P \u0026lt; 0.011, t = 3.77, df =8. Baseline vs FS-Laser (Post Stress-Day5): **P \u0026lt; \u0026nbsp;0.0065, t = 4.15, df = 8. Adjustment: Bonferroni. N = 5 mice. I. The average changes during \u0026nbsp;FS paired with CeA activation in mDlx-GCaMP6s virus expression mice with restrained inescapable shock. RM Two-way ANOVA, Interaction: ns P \u0026lt; 0.64, F (1, 8) = 0.63. Row Factor: ***P \u0026lt; 0.002, F (1, 8) = 2081. Column Factor: ns P \u0026lt; 0.39, F (1, 8) = 0.81. Baseline (Pre-Stress Day1) vs Baseline (Post Stress-Day5): ns P \u0026gt; 0.9999, t = 0.29, df = 8. FS-Laser (Pre-Stress Day1) vs FS Laser (Post Stress-Day5): ns P \u0026gt; 0.71, t = 0.98, df = 8. Adjustment: Bonferroni, N = 5 mice\u003c/p\u003e","description":"","filename":"Binder25.png","url":"https://assets-eu.researchsquare.com/files/rs-7251588/v1/24ff2b07364e7a468847e647.png"},{"id":89983650,"identity":"2a11b612-55a3-4efa-84a5-94eb38ecbbb0","added_by":"auto","created_at":"2025-08-27 06:34:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":325706,"visible":true,"origin":"","legend":"\u003cp\u003eRIS induced a change in DRN serotonin neuronal activity. A. An experimental design. B. Schematical representation of the experimental design for GCaMP6s virus injection. C. ChrimsonR virus expression in CeA and GCAMP6s virus expression in the DRN. D. Showing calcium traces during FS in TPH2-GCaMP6s expression mice with restrained inescapable shock. E. Average change in DRN activity before and during FS in TPH2-GCaMP6s expression mice with restrained inescapable shock. RM Two-way ANOVA, Interaction: nsP \u0026lt; 0.26, F (1, 10) = 1.44. Row Factor: ns P \u0026lt; 0.20, F (1, 10) = 1.83. Column Factor: **P \u0026lt; 0.0033, F (1, 10) = 14.69. Baseline vs FS (Pre-Stress-Day1): *P \u0026lt; 0.0103, t = 3.56, df = 10. Baseline vs FS (Post Stress-Day5): ns P \u0026lt; 0.18, t = 1.86, df = 10. Adjustment: Bonferroni. N = 6 mice. F. Showing the average changes between FS in TPH2 GCaMP6s expression mice with restrained inescapable shock. RM Two-way ANOVA Interaction: **P \u0026lt; 0.006, F (1, 10) = 12.44. Row Factor: *P \u0026lt; 0.02, F (1, 10) = 7.86 Column Factor: **P \u0026lt; 0.003, F (1, 10) = 15.49. Baseline (Pre-Stress Day1) vs Baseline (Post Stress Day5): ns P \u0026gt; 0.9999, t = 0.29, df = 10. FS (Pre-Stress-Day1) vs FS (Post Stress-Day5): ***P \u0026lt; 0.0007, t = 5.28, df = 10. Adjustment: Bonferroni. N = 6 mice. G. Showing calcium traces during FS paired with CeA activation in TPH2-GCaMP6s expression mice with restrained inescapable shock. H. Average change in DRN activity before and during FS paired with CeA activation in TPH2-GCaMP6s expression mice with restrained inescapable shock. RM Two- way ANOVA, Interaction: nsP \u0026lt; 0.35, F (1, 10) = 0.94. Row Factor: ns 739 P \u0026lt; 0.17, F (1, 10) = 2.13 Column Factor: **P \u0026lt; 0.0044, F (1, 10) = 13.38. Baseline vs FS-Laser (Pre-Stress-Day1): *P \u0026lt; 0.016, t = 3.28, df = 10. Baseline vs FS-Laser (Post Stress-Day5): ns P \u0026lt; 0.17, t = 1.89, df = 10. Adjustment: Bonferroni. N = 6 mice. I. The average changes during FS paired with CeA activation in TPH2-GCaMP6s expression mice with restrained inescapable shock were shown. RM Two-way ANOVA, Interaction: ns P \u0026lt; 0.12, F (1, 10) = 0.122. Row Factor: *P \u0026lt; 0.0106, F (1, 10) = 9.82. Column Factor: ns P \u0026lt; 0.0609, F (1, 10) = 4.46. Baseline (Pre-Stress-Day1) vs Baseline (Post Stress-Day5): ns P \u0026lt; 0.99, t = 0.29, df = 10. FS-Laser (Pre-Stress-Day1) vs FS Laser (Post Stress-Day5): *P \u0026lt; 0.046, t = 2.69, df = 10. Adjustment: Bonferroni. N = 6 mice.\u003c/p\u003e","description":"","filename":"Binder26.png","url":"https://assets-eu.researchsquare.com/files/rs-7251588/v1/d2ca6064515f8f02cbd096b9.png"},{"id":105752211,"identity":"ad5cc3f8-7b93-420a-bb9c-27a0e5147f93","added_by":"auto","created_at":"2026-03-30 15:55:52","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1563113,"visible":true,"origin":"","legend":"Article File","description":"","filename":"CeADRNManuscriptFileTranslationalPsychiatry.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7251588/v1_covered_5b6f2999-56f3-4a49-9901-d85034a45371.pdf"},{"id":89985279,"identity":"d5a329f8-dc75-41ce-aed6-3d7ea3b09692","added_by":"auto","created_at":"2025-08-27 06:42:08","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":160974,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary 1. Acute foot shock and foot shock with laser activation of CeA induced change in DRN neuronal activity. A. Showing calcium traces during FS (aversive stimuli) and FS paired with CeA activation in mice. B. Average change in DRN activity before and during FS and FS paired with CeA activation in mice. RM Two-way ANOVA, Interaction: ns P \u0026lt; 0.39, F (1, 10) = 0.79. Row Factor: ns P \u0026lt; 0.55, F (1, 10) = 0.38. Column Factor: ****P \u0026lt; 0.0001, F (1, 10) = 97.17 Baseline vs FS: ***P \u0026lt; 0.0002, t = 6.34, df = 10. Baseline vs FS-Laser: ****P \u0026lt; 0.0001, t = 7.60, df = 10. Adjustment: Bonferroni. N = 6 mice. C. Showing the average changes between FS and FS paired with CeA activation in mice. RM Two-way ANOVA, Interaction: ns P \u0026lt; 0.54, F (1, 10) = 0.40. Row Factor: ****P \u0026lt; 0.0001, F (1, 10) = 49.93. Column Factor: ns P \u0026lt; 0.47, F (1, 10) = 0.57. Baseline (FS) vs Baseline (FS-Laser): ns P \u0026gt; 0.9999, t = 0.085, df = 10. FS vs FS-Laser: ns P \u0026gt; 0.9999, t = 0.98, df = 10. Adjustment: Bonferroni. N = 6 mice\u003c/p\u003e","description":"","filename":"Supplementary1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7251588/v1/f1181890eed10535d57038e4.pdf"},{"id":89983648,"identity":"2e5c65c6-43b0-4818-a147-c7ef801518d2","added_by":"auto","created_at":"2025-08-27 06:34:09","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":320079,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary 2. Acute foot shock or foot shock with laser activation of CeA induces a change in the subtype of DRN neuronal activity. A. Schematical representation of the experimental design for GCaMP6s virus injection. B. Showing calcium traces during FS (aversive stimuli) and FS paired with CeA activation in mDlx-GCaMP6s expression mice. C. Average change in DRN activity before and during FS and FS paired with CeA activation. RM Two-way ANOVA, Interaction: ns P \u0026lt; 0.43, F (1, 8) = 0.67. Row Factor: ns P \u0026lt; 0.62, F (1, 8) = 0.25 Column Factor: ***P \u0026lt; 0.0002, F (1, 8) = 42.71. Baseline vs FS: **P \u0026lt; 0.0075, t = 4.04, df = 8. Baseline vs FS-Laser: **P \u0026lt; 0.0016, t = 5.20, df = 8. Adjustment: Bonferroni. N = 5 mice. D. Showing the average changes between FS and FS paired with CeA activation. RM Two-way ANOVA, Interaction: ns P \u0026lt; 0.17, F (1, 8) = 018. Row Factor: ***P \u0026lt; 0.0004, F (1, 8) = 32.79. Column Factor: ns P \u0026lt; 0.49, F (1, 8) = 051. Baseline (FS) vs Baseline (FS-Laser): nsP \u0026lt; 0.99, t = 0.53, df = 8. FS vs FS-Laser: ns P \u0026lt; 0.32, t = 1.55, df = 8. Adjustment: Bonferroni. N = 5 mice. E. Schematical representation of the experimental design for GCaMP6s virus injection. F. Showing calcium traces during FS (aversive stimuli) and FS paired with CeA activation in TPH2-GCaMP6s expression mice. G. Average change in DRN activity before and during FS and FS paired with CeA activation. RM Two-way ANOVA, Interaction: nsP \u0026lt; 0.44, F (1, 10) = 0.63. Row Factor: ns P \u0026lt; 0.40, F (1, 10) = 0.75. Column Factor: **P \u0026lt; 0.0024, F (1, 10) = 16.30. Baseline vs FS: ns P \u0026lt; 0.089, t = 2.29, df = 10. Baseline vs FS-Laser: *P \u0026lt; 0.013, t = 3.42, df = 10. Adjustment: Bonferroni. N = 6 mice. H. Showing the average changes between FS and FS paired with CeA activation. RM Two-way ANOVA, Interaction: ns P \u0026lt; 0.44, F (1, 10) = 0.62. Row Factor: **P \u0026lt; 0.0015, F (1, 10) = 18.55. Column Factor: ns P \u0026lt; 0.44, F (1, 10) = 0.65. Baseline (FS) vs Baseline (FS-Laser): nsP \u0026lt; 0.99, t = 0.013, df = 10. FS vs FS-Laser: ns 782 P \u0026lt; 0.57, t = 1.133, df = 10. Adjustment: Bonferroni. N = 6 mice\u003c/p\u003e","description":"","filename":"Supplementary2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7251588/v1/d6cd893a4cc35af885420331.pdf"},{"id":89983651,"identity":"1330d27c-348f-4b2c-b2ae-0436cceceb50","added_by":"auto","created_at":"2025-08-27 06:34:09","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":119713,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"StatisticalTable20250728KIM.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7251588/v1/a34a97100e6ce252f28222cc.pdf"}],"financialInterests":"The authors have declared there is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose","formattedTitle":"\u003cp\u003eHyperactivity of the Amygdala Mediates Depressive-Like Phenotypes and Decreased Serotonin Release\u003c/p\u003e","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"Amygdala, Depression, Serotonin, Restrained tail shock stress model","lastPublishedDoi":"10.21203/rs.3.rs-7251588/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7251588/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eClinical and preclinical studies have consistently demonstrated a correlation between hyperactivity of the amygdala and the onset of depression. However, the underlying mechanisms influencing serotonin levels a critical neurotransmitter implicated in depression and a primary target for selective serotonin reuptake inhibitors (SSRIs) remain inadequately understood. In this study, we employed a restrained inescapable shock (RIS) model to investigate these mechanisms in mice. The RIS paradigm elicited depressive-like phenotypes, increased c-Fos expression in the amygdala, diminished serotonin levels, and elevated corticosterone concentrations in serum. Notably, chemogenetic inhibition of the amygdala mitigated depressive symptoms, reduced neuronal activity in this region, and restored serotonin levels. Anatomical analyses revealed a strong connectivity between the central amygdala (CeA) and the dorsal raphe nucleus (DRN). Fiber photometry recordings indicated that serotonergic neuronal activity in the DRN decreased in response to aversive stimuli, accompanied by amygdala activation following RIS, with no notable alterations in DRN GABAergic activity. These findings suggest that chronic stress may exacerbate amygdala hyperactivity, which subsequently inhibits serotonin release in the brain, potentially intensifying depressive states. Therefore, targeting amygdala hyperactivity may represent a novel therapeutic strategy for the management of stress-related depressive and anxiety disorders.\u003c/p\u003e","manuscriptTitle":"Hyperactivity of the Amygdala Mediates Depressive-Like Phenotypes and Decreased Serotonin Release","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-27 06:34:03","doi":"10.21203/rs.3.rs-7251588/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":"cfeaa8ed-045f-49b9-ad7c-9bfa96771e7e","owner":[],"postedDate":"August 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":53761712,"name":"Biological sciences/Neuroscience/Molecular neuroscience"},{"id":53761713,"name":"Health sciences/Diseases/Psychiatric disorders/Depression"}],"tags":[],"updatedAt":"2026-03-30T15:16:41+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-27 06:34:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7251588","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7251588","identity":"rs-7251588","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}