{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,4,11]],"date-time":"2026-04-11T04:42:50Z","timestamp":1775882570860,"version":"3.50.1"},"posted":{"date-parts":[[2025,1,7]]},"group-title":"Neuroscience","reference-count":42,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,1,7]],"date-time":"2025-01-07T00:00:00Z","timestamp":1736208000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,1,7]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>Stress is central to many neuropsychiatric conditions, including alcohol use disorder (AUD). Stress influences the initiation and continued use of alcohol, the progression to AUD, and relapse. Identifying the neurocircuits activated during stress, and individual variability in these responses is critical for developing new treatment targets for AUD, particularly to mitigate stress-induced relapse. Using a longitudinal approach, this study examined the relationship between sub-chronic stress exposure and negative affect during protracted abstinence following chronic ethanol exposure. Sub-chronic restraint stress heightened negative affect-like behavior in protracted abstinence. Interestingly, this was driven by a subset of \u201cstress-susceptible\u201d female mice. We examined the mid-insula, a hub in the brain\u2019s salience network, as a driver of this effect, given its role in emotional regulation and links to alcohol craving, consumption, and abstinence-induced negative affect. Mid-insula GCaMP fiber photometry revealed that GCaMP activity during stress exposure was positively correlated with activity during the novelty-suppressed feeding test (NSFT) two weeks into abstinence. A distinct subset of mice exhibited increasing activity during the consummatory phase, implicating the mid-insula as a neural basis for heightened negative affect in abstinence. Chemogenetic inhibition of mid-insula neurons projecting to the dorsal BNST during stress disrupted the emergence of stress susceptibility, highlighting this circuit as a key determinant of susceptibility to abstinence-induced negative affect. These outcomes were female-specific, addressing a critical gap in understanding AUD risk in women. Furthermore, female mice exhibited higher struggling behavior during stress than males. However, this effect was blocked by chemogenetic inhibition of the insula-BNST pathway during stress. By linking pre-alcohol stress response with abstinence outcomes, this work positions the insula-BNST pathway as a potential AUD circuit activity biomarker and therapeutic target.<\/jats:p>","DOI":"10.1101\/2025.01.07.631325","type":"posted-content","created":{"date-parts":[[2025,1,8]],"date-time":"2025-01-08T02:40:44Z","timestamp":1736304044000},"source":"Crossref","is-referenced-by-count":0,"title":["A stress-activated mid-insula to BNST pathway regulates susceptibility to abstinence-induced negative affect in female mice"],"prefix":"10.64898","author":[{"given":"Benjamin M.","family":"Williams","sequence":"first","affiliation":[{"name":"Department of Translational Neuroscience, Wake Forest University School of Medicine, Winston-Salem, NC, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jincy R.","family":"Little","sequence":"additional","affiliation":[{"name":"Department of Translational Neuroscience, Wake Forest University School of Medicine, Winston-Salem, NC, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nathaniel S.","family":"O\u2019Connell","sequence":"additional","affiliation":[{"name":"Department of Biostatistics and Data Science, Wake Forest University School of Medicine, Winston-Salem, NC, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3941-7677","authenticated-orcid":false,"given":"Samuel W.","family":"Centanni","sequence":"additional","affiliation":[{"name":"Department of Translational Neuroscience, Wake Forest University School of Medicine, Winston-Salem, NC, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2026041020432793000_2025.01.07.631325v1.1","doi-asserted-by":"publisher","DOI":"10.35946\/arcr.v42.1.12"},{"key":"2026041020432793000_2025.01.07.631325v1.2","doi-asserted-by":"publisher","DOI":"10.1176\/appi.ajp.2020.20091375"},{"key":"2026041020432793000_2025.01.07.631325v1.3","doi-asserted-by":"crossref","unstructured":"Sinha R . Stress and substance use disorders: risk, relapse, and treatment outcomes. The Journal of clinical investigation. 2024;134(16).","DOI":"10.1172\/JCI172883"},{"key":"2026041020432793000_2025.01.07.631325v1.4","doi-asserted-by":"publisher","DOI":"10.1111\/adb.12395"},{"key":"2026041020432793000_2025.01.07.631325v1.5","doi-asserted-by":"publisher","DOI":"10.2174\/0929867323666161202092908"},{"key":"2026041020432793000_2025.01.07.631325v1.6","doi-asserted-by":"publisher","DOI":"10.1038\/s41583-021-00513-0"},{"key":"2026041020432793000_2025.01.07.631325v1.7","doi-asserted-by":"publisher","DOI":"10.1037\/0022-3514.65.5.1010"},{"key":"2026041020432793000_2025.01.07.631325v1.8","doi-asserted-by":"publisher","DOI":"10.1017\/S0033291701003877"},{"key":"2026041020432793000_2025.01.07.631325v1.9","doi-asserted-by":"crossref","unstructured":"Ibrahim C , Rubin-Kahana DS , Pushparaj A , Musiol M , Blumberger DM , Daskalakis ZJ , et al. The Insula: A Brain Stimulation Target for the Treatment of Addiction. Frontiers in pharmacology. 2019;10:720.","DOI":"10.3389\/fphar.2019.00720"},{"key":"2026041020432793000_2025.01.07.631325v1.10","doi-asserted-by":"publisher","DOI":"10.1007\/s00429-010-0262-0"},{"key":"2026041020432793000_2025.01.07.631325v1.11","doi-asserted-by":"crossref","unstructured":"Centanni SW , Janes AC , Haggerty DL , Atwood B , Hopf FW . Better living through understanding the insula: Why subregions can make all the difference. Neuropharmacology. 2021;198:108765.","DOI":"10.1016\/j.neuropharm.2021.108765"},{"key":"2026041020432793000_2025.01.07.631325v1.12","doi-asserted-by":"publisher","DOI":"10.1016\/j.tins.2017.02.002"},{"key":"2026041020432793000_2025.01.07.631325v1.13","doi-asserted-by":"crossref","unstructured":"Centanni SW , Janes AC , Haggerty DL , Atwood B , Hopf FW . Better living through understanding the insula: Why subregions can make all the difference. Neuropharmacology. 2021:108765.","DOI":"10.1016\/j.neuropharm.2021.108765"},{"key":"2026041020432793000_2025.01.07.631325v1.14","doi-asserted-by":"publisher","DOI":"10.1038\/nrn2555"},{"key":"2026041020432793000_2025.01.07.631325v1.15","doi-asserted-by":"publisher","DOI":"10.1111\/add.12437"},{"key":"2026041020432793000_2025.01.07.631325v1.16","doi-asserted-by":"publisher","DOI":"10.1038\/sj.npp.1300295"},{"key":"2026041020432793000_2025.01.07.631325v1.17","doi-asserted-by":"publisher","DOI":"10.1016\/j.biopsych.2023.08.024"},{"key":"2026041020432793000_2025.01.07.631325v1.18","doi-asserted-by":"crossref","unstructured":"Gorka SM , Radoman M , Jimmy J , Kreutzer KA , Manzler C , Culp S . Behavioral and brain reactivity to uncertain stress prospectively predicts binge drinking in youth. Neuropsychopharmacology: official publication of the American College of Neuropsychopharmacology. 2023.","DOI":"10.1038\/s41386-023-01571-x"},{"key":"2026041020432793000_2025.01.07.631325v1.19","doi-asserted-by":"publisher","DOI":"10.1038\/s41386-018-0257-8"},{"key":"2026041020432793000_2025.01.07.631325v1.20","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-021-23674-z"},{"key":"2026041020432793000_2025.01.07.631325v1.21","doi-asserted-by":"crossref","unstructured":"Gehrlach DA , Weiand C , Gaitanos TN , Cho E , Klein AS , Hennrich AA , et al. A whole-brain connectivity map of mouse insular cortex. Elife. 2020;9.","DOI":"10.7554\/eLife.55585"},{"issue":"1","key":"2026041020432793000_2025.01.07.631325v1.22","first-page":"63","article-title":"Neurobiological mechanisms of addiction: focus on corticotropin-releasing factor","volume":"11","year":"2010","journal-title":"Curr Opin Investig Drugs"},{"key":"2026041020432793000_2025.01.07.631325v1.23","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.3432-05.2005"},{"key":"2026041020432793000_2025.01.07.631325v1.24","doi-asserted-by":"publisher","DOI":"10.1002\/hbm.25314"},{"key":"2026041020432793000_2025.01.07.631325v1.25","doi-asserted-by":"crossref","unstructured":"Flook EA , Feola B , Avery SN , Winder DG , Woodward ND , Heckers S , et al. BNST-insula structural connectivity in humans. NeuroImage. 2020;210.","DOI":"10.1016\/j.neuroimage.2020.116555"},{"key":"2026041020432793000_2025.01.07.631325v1.26","doi-asserted-by":"crossref","unstructured":"Marino RAM , Girven KS , Figueiredo A , Navarrete J , Doty C , Sparta DR . Binge ethanol drinking associated with sex-dependent plasticity of neurons in the insula that project to the bed nucleus of the stria terminalis. Neuropharmacology. 2021;196:108695.","DOI":"10.1016\/j.neuropharm.2021.108695"},{"key":"2026041020432793000_2025.01.07.631325v1.27","unstructured":"Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington (DC); 2003."},{"key":"2026041020432793000_2025.01.07.631325v1.28","doi-asserted-by":"publisher","DOI":"10.1038\/npp.2016.3"},{"key":"2026041020432793000_2025.01.07.631325v1.29","doi-asserted-by":"crossref","unstructured":"Holleran KM , Wilson HH , Fetterly TL , Bluett RJ , Centanni SW , Gilfarb RA , et al. Ketamine and MAG Lipase Inhibitor-Dependent Reversal of Evolving Depressive-Like Behavior During Forced Abstinence From Alcohol Drinking. Neuropsychopharmacology. 2016.","DOI":"10.1038\/npp.2016.3"},{"key":"2026041020432793000_2025.01.07.631325v1.30","doi-asserted-by":"crossref","unstructured":"Olsen CM , Winder DG . Operant sensation seeking in the mouse. J Vis Exp. 2010(45).","DOI":"10.3791\/2292-v"},{"key":"2026041020432793000_2025.01.07.631325v1.31","doi-asserted-by":"publisher","DOI":"10.1111\/ejn.12195"},{"issue":"1","key":"2026041020432793000_2025.01.07.631325v1.32","doi-asserted-by":"crossref","first-page":"114","DOI":"10.1111\/acer.14742","article-title":"The impact of intermittent exercise on mouse ethanol drinking and abstinence-associated affective behavior and physiology","volume":"46","year":"2022","journal-title":"Alcoholism, clinical and experimental research"},{"key":"2026041020432793000_2025.01.07.631325v1.33","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbr.2008.03.030"},{"key":"2026041020432793000_2025.01.07.631325v1.34","doi-asserted-by":"publisher","DOI":"10.1111\/j.1460-9568.2005.03916.x"},{"key":"2026041020432793000_2025.01.07.631325v1.35","doi-asserted-by":"publisher","DOI":"10.1016\/j.alcohol.2024.07.004"},{"key":"2026041020432793000_2025.01.07.631325v1.36","doi-asserted-by":"publisher","DOI":"10.1016\/j.neuropharm.2011.02.017"},{"key":"2026041020432793000_2025.01.07.631325v1.37","doi-asserted-by":"publisher","DOI":"10.1007\/s00213-011-2443-9"},{"key":"2026041020432793000_2025.01.07.631325v1.38","doi-asserted-by":"publisher","DOI":"10.1037\/adb0000436"},{"key":"2026041020432793000_2025.01.07.631325v1.39","doi-asserted-by":"publisher","DOI":"10.1080\/10673220902899680"},{"key":"2026041020432793000_2025.01.07.631325v1.40","doi-asserted-by":"publisher","DOI":"10.35946\/arcr.v40.3.05"},{"key":"2026041020432793000_2025.01.07.631325v1.41","doi-asserted-by":"crossref","unstructured":"Jaramillo AA , Williford KM , Marshall C , Winder DG , Centanni SW . BNST transient activity associates with approach behavior in a stressful environment and is modulated by the parabrachial nucleus. Neurobiology of Stress. 2020;13:100247.","DOI":"10.1016\/j.ynstr.2020.100247"},{"key":"2026041020432793000_2025.01.07.631325v1.42","doi-asserted-by":"crossref","unstructured":"Starski P , Morningstar MD , Katner SN , Frasier RM , De Oliveira Sergio T , Wean S , et al. Neural Activity in the Anterior Insula at Drinking Onset and Licking Relates to Compulsion-Like Alcohol Consumption. The Journal of neuroscience: the official journal of the Society for Neuroscience. 2024;44(9).","DOI":"10.1523\/JNEUROSCI.1490-23.2023"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2025.01.07.631325","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,4,11]],"date-time":"2026-04-11T03:43:32Z","timestamp":1775879012000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2025.01.07.631325"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,1,7]]},"references-count":42,"URL":"https:\/\/doi.org\/10.1101\/2025.01.07.631325","relation":{},"subject":[],"published":{"date-parts":[[2025,1,7]]},"subtype":"preprint"}}