{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,13]],"date-time":"2025-11-13T13:03:43Z","timestamp":1763039023934,"version":"3.45.0"},"reference-count":108,"publisher":"MDPI AG","issue":"22","license":[{"start":{"date-parts":[[2025,11,13]],"date-time":"2025-11-13T00:00:00Z","timestamp":1762992000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Concurrent consumption of ethanol (EtOH) and herbal preparations containing Withania somnifera (WS, ashwagandha) is increasingly common, but the neurobehavioral and molecular consequences of such interactions remain poorly characterized. This study investigated how three purified withanolides\u2014withanolide A (WITA), withanone (WIN), and withaferin A (WTFA)\u2014modulate the effects of acute EtOH exposure in zebrafish (Danio rerio) larvae. Locomotor behavior was quantified under EtOH concentrations ranging from 0 to 4.0%, and the expression of four GABAA receptor subunit genes (gabra1, gabra2, gabrd, gabrg2) was analyzed by qPCR. EtOH alone induced a biphasic locomotor response, with stimulation at low-to-moderate doses and suppression at higher doses. WITA and WIN modulated this pattern in a dose-dependent manner, preserving or enhancing hyperactivity, while WTFA consistently potentiated locomotor suppression. mRNA profile analysis revealed subunit-specific changes, including downregulation of gabra1 and gabra2, compound-dependent regulation of gabrd, and complex gabrg2 responses. These results demonstrate that individual withanolides distinctly shape behavioral and molecular outcomes of EtOH exposure, suggesting specific interactions at the level of inhibitory neurotransmission. The findings provide mechanistic insight into the combined effects of WS-derived compounds and EtOH and highlight the importance of considering such interactions in both experimental and applied contexts.<\/jats:p>","DOI":"10.3390\/ijms262210991","type":"journal-article","created":{"date-parts":[[2025,11,13]],"date-time":"2025-11-13T12:57:13Z","timestamp":1763038633000},"page":"10991","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Ethanol\u2013Withanolides Interactions: Compound-Specific Effects on Zebrafish Larvae Locomotor Behavior and GABAA Receptor Subunit Expression"],"prefix":"10.3390","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2873-4746","authenticated-orcid":false,"given":"Kamila","family":"Czora-Poczwardowska","sequence":"first","affiliation":[{"name":"Department of Pharmacology, Poznan University of Medical Sciences, 60-806 Poznan, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2389-0689","authenticated-orcid":false,"given":"Rados\u0142aw","family":"Kujawski","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, Poznan University of Medical Sciences, 60-806 Poznan, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-7639-4841","authenticated-orcid":false,"given":"Weronika","family":"Jarczak","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, Poznan University of Medical Sciences, 60-806 Poznan, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-3394-5336","authenticated-orcid":false,"given":"Emilia","family":"Cicha","sequence":"additional","affiliation":[{"name":"Animal Facility, Poznan University of Medical Sciences, 60-806 Poznan, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1252-6523","authenticated-orcid":false,"given":"Przemys\u0142aw","family":"Miko\u0142ajczak","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, Poznan University of Medical Sciences, 60-806 Poznan, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3295-2789","authenticated-orcid":false,"given":"Micha\u0142","family":"Szulc","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, Poznan University of Medical Sciences, 60-806 Poznan, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,11,13]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Song, F., and Walker, M.P. (2023). Sleep, alcohol, and caffeine in financial traders. PLoS ONE, 18.","DOI":"10.1371\/journal.pone.0291675"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Fern\u00e1ndez-Sol\u00e0, J. (2020). The effects of ethanol on the heart: Alcoholic cardiomyopathy. Nutrients, 12.","DOI":"10.3390\/nu12020572"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1097\/HCR.0000000000000042","article-title":"An expanding knowledge of the mechanisms and effects of alcohol consumption on cardiovascular disease","volume":"34","author":"Matsumoto","year":"2014","journal-title":"J. Cardiopulm. Rehabil. Prev."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Kamal, H., Tan, G.C., Ibrahim, S.F., Shaikh, M.F., Mohamed, I.N., Mohamed, R.M.P., Hamid, A.A., Ugusman, A., and Kumar, J. (2020). Alcohol use disorder, neurodegeneration, Alzheimer\u2019s and Parkinson\u2019s disease: Interplay between oxidative stress, neuroimmune response and excitotoxicity. Front. Cell. Neurosci., 14.","DOI":"10.3389\/fncel.2020.00282"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"390","DOI":"10.3934\/Neuroscience.2021021","article-title":"Effect of alcohol on the central nervous system to develop neurological disorder: Pathophysiological and lifestyle modulation can be potential therapeutic options for alcohol-induced neurotoxication","volume":"8","author":"Pervin","year":"2021","journal-title":"AIMS Neurosci."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"4965","DOI":"10.7314\/APJCP.2013.14.9.4965","article-title":"Alcohol Consumption and Risk of Cancer: A Systematic Literature Review","volume":"14","author":"Bergmann","year":"2013","journal-title":"Asian Pac. J. Cancer Prev."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Begun, A.L., and Murray, M.M. (2020). Introduction to psychoactive substances. The Routledge Handbook of Social Work and Addictive Behaviors, Routledge.","DOI":"10.4324\/9780429203121"},{"key":"ref_8","unstructured":"World Health Organization (2024). Global Status Report on Alcohol and Health and Treatment of Substance Use Disorders, WHO."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"S51","DOI":"10.1590\/1516-4446-2013-S105","article-title":"Use of psychoactive substances by adolescents: Current panorama","volume":"35","author":"Lopes","year":"2013","journal-title":"Rev. Bras. Psychiatry"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"36","DOI":"10.1186\/s13011-021-00373-y","article-title":"Use of alcohol, tobacco, cannabis, and other substances during the first wave of the SARS-CoV-2 pandemic in Europe: A survey on 36,000 European substance users","volume":"16","author":"Manthey","year":"2021","journal-title":"Subst. Abuse Treat. Prev. Policy"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"34","DOI":"10.1111\/add.13056","article-title":"The drug situation in Europe: An overview of data available on illicit drugs and new psychoactive substances from European monitoring in 2015","volume":"111","author":"Mounteney","year":"2016","journal-title":"Addiction"},{"key":"ref_12","unstructured":"(2025, March 31). European Monitoring Centre for Drugs and Drug Addiction. Health Responses to New Psychoactive Substances. Available online: https:\/\/www.emcdda.europa.eu\/publications\/pods\/health-responses-for-new-psychoactive-substances_en."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"231","DOI":"10.1016\/0147-7552(77)90025-0","article-title":"Biphasic action of ethanol","volume":"1","author":"Pohorecky","year":"1977","journal-title":"Biobehav. Rev."},{"key":"ref_14","first-page":"185","article-title":"Neurobiology of alcohol dependence: Focus on motivational mechanisms","volume":"31","author":"Gilpin","year":"2008","journal-title":"Alcohol Res. Health"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"998","DOI":"10.1111\/j.1530-0277.1998.tb03695.x","article-title":"Effects of moderate alcohol consumption on the central nervous system","volume":"22","author":"Eckardt","year":"1998","journal-title":"Alcohol. Clin. Exp. Res."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.ejim.2022.08.013","article-title":"Identification and management of acute alcohol intoxication","volume":"108","author":"Mirijello","year":"2023","journal-title":"Eur. J. Intern. Med."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1515","DOI":"10.1016\/j.bcp.2004.07.031","article-title":"A role for GABA mechanisms in the motivational effects of alcohol","volume":"68","author":"Koob","year":"2004","journal-title":"Biochem. Pharmacol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"2053","DOI":"10.1073\/pnas.0437926100","article-title":"Ethanol increases GABAergic transmission at both pre- and postsynaptic sites in rat central amygdala neurons","volume":"100","author":"Roberto","year":"2003","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"217","DOI":"10.1038\/npp.2009.110","article-title":"Neurocircuitry of addiction","volume":"35","author":"Koob","year":"2010","journal-title":"Neuropsychopharmacology"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"111","DOI":"10.1016\/j.physbeh.2015.04.040","article-title":"The neurobiology of offensive aggression: Revealing a modular view","volume":"146","author":"Olivier","year":"2015","journal-title":"Physiol. Behav."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"321","DOI":"10.1146\/annurev-pharmtox-010611-134625","article-title":"Addiction circuitry in the human brain","volume":"52","author":"Volkow","year":"2012","journal-title":"Annu. Rev. Pharmacol. Toxicol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"27100","DOI":"10.1016\/S0021-9258(18)47131-1","article-title":"Distribution, prevalence, and drug binding profile of gamma-aminobutyric acid type A receptor subtypes differing in the beta-subunit variant","volume":"269","author":"Benke","year":"1994","journal-title":"J. Biol. Chem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"243","DOI":"10.1124\/pr.108.00505","article-title":"International Union of Pharmacology. LXX. Subtypes of gamma-aminobutyric acid(A) receptors: Classification on the basis of subunit composition, pharmacology, and function. Update","volume":"60","author":"Olsen","year":"2008","journal-title":"Pharmacol. Rev."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"7769","DOI":"10.1073\/pnas.0400220101","article-title":"Positioning of the alpha-subunit isoforms confers a functional signature to gamma-aminobutyric acid type A receptors","volume":"101","author":"Minier","year":"2004","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"12367","DOI":"10.1523\/JNEUROSCI.2786-07.2007","article-title":"Mechanisms of reversible GABAA receptor plasticity after ethanol intoxication","volume":"27","author":"Liang","year":"2007","journal-title":"J. Neurosci."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"8379","DOI":"10.1523\/JNEUROSCI.2040-04.2004","article-title":"Low ethanol concentrations selectively augment the tonic inhibition mediated by delta subunit-containing GABAA receptors in hippocampal neurons","volume":"24","author":"Wei","year":"2004","journal-title":"J. Neurosci."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"15218","DOI":"10.1073\/pnas.2435171100","article-title":"Ethanol enhances \u03b14\u03b23\u03b4 and \u03b16\u03b23\u03b4 \u03b3-aminobutyric acid type A receptors at low concentrations known to affect humans","volume":"100","author":"Wallner","year":"2003","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"529","DOI":"10.1007\/s00213-009-1562-z","article-title":"The role of GABA(A) receptors in the acute and chronic effects of ethanol: A decade of progress","volume":"205","author":"Kumar","year":"2009","journal-title":"Psychopharmacology"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"126","DOI":"10.1046\/j.1471-4159.1997.69010126.x","article-title":"Bidirectional alterations of GABA(A) receptor subunit peptide levels in rat cortex during chronic ethanol consumption and withdrawal","volume":"69","author":"Devaud","year":"1997","journal-title":"J. Neurochem."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1016\/j.alcohol.2019.08.005","article-title":"Correlations between subunits of GABAA and NMDA receptors after chronic alcohol treatment or withdrawal, and the effect of taurine in the hippocampus of rats","volume":"82","author":"Hansen","year":"2020","journal-title":"Alcohol"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1016\/j.pbb.2008.03.007","article-title":"The role of GABA(A) receptors in the development of alcoholism","volume":"90","author":"Enoch","year":"2008","journal-title":"Pharmacol. Biochem. Behav."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1245","DOI":"10.1038\/npp.2008.171","article-title":"GABRG1 and GABRA2 as independent predictors for alcoholism in two populations","volume":"34","author":"Enoch","year":"2009","journal-title":"Neuropsychopharmacology"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"2445","DOI":"10.1111\/acer.13215","article-title":"Effects of Gabra2 Point Mutations on Alcohol Intake: Increased Binge-Like and Blunted Chronic Drinking by Mice","volume":"40","author":"Newman","year":"2016","journal-title":"Alcohol. Clin. Exp. Res."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Chandrasekar, R. (2013). Alcohol and NMDA receptor: Current research and future direction. Front. Mol. Neurosci., 6.","DOI":"10.3389\/fnmol.2013.00014"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"377","DOI":"10.1016\/S0197-0186(02)00046-3","article-title":"Ethanol sensitivity of NMDA receptors","volume":"41","author":"Allgaier","year":"2002","journal-title":"Neurochem. Int."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Ekor, M. (2014). The growing use of herbal medicines: Issues relating to adverse reactions and challenges in monitoring safety. Front. Pharmacol., 4.","DOI":"10.3389\/fphar.2013.00177"},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Dhara, A.K., and Mandal, S.C. (2023). Market overview of herbal medicines for lifestyle diseases. Role of Herbal Medicines: Management of Lifestyle Diseases, Springer Nature.","DOI":"10.1007\/978-981-99-7703-1"},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Lippert, A., and Renner, B. (2022). Herb-Drug Interaction in Inflammatory Diseases: Review of Phytomedicine and Herbal Supplements. J. Clin. Med., 11.","DOI":"10.3390\/jcm11061567"},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Sivertsen, K., Lukic, M., and Kristoffersen, A.E. (2018). Gender specific association between the use of complementary and alternative medicine (CAM) and alcohol consumption and injuries caused by drinking in the sixth Troms\u00f8 study. BMC Complement. Altern. Med., 18.","DOI":"10.1186\/s12906-018-2301-y"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"45","DOI":"10.16899\/jcm.1607686","article-title":"Assessment of the use of herbal supplements and attitudes of adults taking medication","volume":"15","author":"Karakaya","year":"2025","journal-title":"J. Contemp. Med."},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Bashir, A., Nabi, M., Tabassum, N., Afzal, S., and Ayoub, M. (2023). An updated review on phytochemistry and molecular targets of Withania somnifera (L.) Dunal (Ashwagandha). Front. Pharmacol., 14.","DOI":"10.3389\/fphar.2023.1049334"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"112876","DOI":"10.1016\/j.jep.2020.112876","article-title":"Ashwagandha in brain disorders: A review of recent developments","volume":"257","author":"Zahiruddin","year":"2020","journal-title":"J. Ethnopharmacol."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Mikulska, P., Malinowska, M., Ignacyk, M., Szustowski, P., Nowak, J., Pesta, K., Szel\u0105g, M., Szklanny, D., Judasz, E., and Kaczmarek, G. (2023). Ashwagandha (Withania somnifera)\u2014Current Research on the Health-Promoting Activities: A Narrative Review. Pharmaceutics, 15.","DOI":"10.3390\/pharmaceutics15041057"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1067","DOI":"10.1080\/17512433.2022.2121699","article-title":"Potential clinical applications of Ashwagandha (Withania somnifera) in medicine and neuropsychiatry","volume":"15","author":"Andrade","year":"2022","journal-title":"Expert Rev. Clin. Pharmacol."},{"key":"ref_45","first-page":"40","article-title":"Alcohol and medication interactions","volume":"23","author":"Weathermon","year":"1999","journal-title":"Alcohol Res. Health"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"478","DOI":"10.1111\/acer.13594","article-title":"Alcohol and Opioid Use, Co-Use, and Chronic Pain in the Context of the Opioid Epidemic: A Critical Review","volume":"42","author":"Witkiewitz","year":"2018","journal-title":"Alcohol. Clin. Exp. Res."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"80","DOI":"10.1038\/s41572-022-00406-1","article-title":"Hazardous drinking and alcohol use disorders","volume":"8","author":"MacKillop","year":"2022","journal-title":"Nat. Rev. Dis. Primers"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"809653","DOI":"10.1155\/2012\/809653","article-title":"Complementary medicine, exercise, meditation, diet, and lifestyle modification for anxiety disorders: A review of current evidence","volume":"2012","author":"Sarris","year":"2012","journal-title":"Evid. Based Complement. Altern. Med."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"719","DOI":"10.1517\/17425255.3.5.719","article-title":"Drug interactions involving ethanol and alcoholic beverages","volume":"3","author":"Jang","year":"2007","journal-title":"Expert Opin. Drug Metab. Toxicol."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"18","DOI":"10.1016\/j.tox.2017.04.001","article-title":"The induction of cytochrome P450 2E1 by ethanol leads to the loss of synaptic proteins via PPAR\u03b1 down-regulation","volume":"385","author":"Na","year":"2017","journal-title":"Toxicology"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"1115","DOI":"10.1007\/s40262-014-0190-x","article-title":"Pharmacokinetic and pharmacodynamic drug interactions with ethanol (alcohol)","volume":"53","author":"Chan","year":"2014","journal-title":"Clin. Pharmacokinet."},{"key":"ref_52","unstructured":"(2025, April 01). Horizon Databook: The World\u2019s Largest Portal for Market Reports & Statistics Global Ashwagandha Supplements Market Size & Outlook. Available online: https:\/\/www.grandviewresearch.com\/horizon\/outlook\/ashwagandha-supplements-market-size\/global."},{"key":"ref_53","doi-asserted-by":"crossref","unstructured":"Pandit, S., Srivastav, A.K., Sur, T.K., Chaudhuri, S., Wang, Y., and Biswas, T.K. (2024). Effects of Withania somnifera Extract in Chronically Stressed Adults: A Randomized Controlled Trial. Nutrients, 16.","DOI":"10.3390\/nu16091293"},{"key":"ref_54","doi-asserted-by":"crossref","unstructured":"Wici\u0144ski, M., Fajkiel-Madajczyk, A., Kurant, Z., Kurant, D., Gryczka, K., Falkowski, M., Wi\u015bniewska, M., S\u0142upski, M., Ohla, J., and Zabrzy\u0144ski, J. (2023). Can Ashwagandha Benefit the Endocrine System?\u2014A Review. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms242216513"},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"1043","DOI":"10.1142\/S0192415X13500705","article-title":"GABA-mimetic actions of Withania somnifera on substantia gelatinosa neurons of the trigeminal subnucleus caudalis in mice","volume":"41","author":"Yin","year":"2013","journal-title":"Am. J. Chin. Med."},{"key":"ref_56","first-page":"278","article-title":"Sleep-promoting activity of amylase-treated Ashwagandha (Withania somnifera L. Dunal) root extract via GABA receptors","volume":"31","author":"Park","year":"2023","journal-title":"J. Food Drug Anal."},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"105867","DOI":"10.1016\/j.neuint.2024.105867","article-title":"Neuroprotective effects of Withania somnifera on ischemic stroke are mediated via anti-inflammatory response and modulation of neurotransmitter levels","volume":"180","author":"Sood","year":"2024","journal-title":"Neurochem. Int."},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"466","DOI":"10.1055\/a-1438-2816","article-title":"Withanolides from Withania somnifera Ameliorate Neutrophil Infiltration in Endotoxin-Induced Peritonitis by Regulating Oxidative Stress and Inflammatory Cytokines","volume":"88","author":"Balkrishna","year":"2022","journal-title":"Planta Med."},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"4445","DOI":"10.1007\/s00018-015-2012-1","article-title":"Pharmacologic overview of Withania somnifera, the Indian Ginseng","volume":"72","author":"Dar","year":"2015","journal-title":"Cell. Mol. Life Sci."},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"119224","DOI":"10.1016\/j.jep.2024.119224","article-title":"Ashwagandha (Withania somnifera (L.) dunal) root extract containing withanolide a alleviates depression-like behavior in mice by enhancing the brain-derived neurotrophic factor pathway under unexpected chronic mild stress","volume":"340","author":"Kim","year":"2025","journal-title":"J. Ethnopharmacol."},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"745","DOI":"10.1016\/j.phymed.2013.10.021","article-title":"Withania somnifera root extract prolongs analgesia and suppresses hyperalgesia in mice treated with morphine","volume":"21","author":"Marchese","year":"2014","journal-title":"Phytomedicine"},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"1915","DOI":"10.1007\/s11064-012-0810-5","article-title":"Oxidative stress induced NMDA receptor alteration leads to spatial memory deficits in temporal lobe epilepsy: Ameliorative effects of Withania somnifera and Withanolide A","volume":"37","author":"Soman","year":"2012","journal-title":"Neurochem. Res."},{"key":"ref_63","doi-asserted-by":"crossref","unstructured":"Paul, S., Chakraborty, S., Anand, U., Dey, S., Nandy, S., Ghorai, M., Saha, S.C., Patil, M.T., Kandimalla, R., and Pro\u0107k\u00f3w, J. (2021). Withania somnifera (L.) Dunal (Ashwagandha): A comprehensive review on ethnopharmacology, pharmacotherapeutics, biomedicinal and toxicological aspects. Biomed. Pharmacother., 143.","DOI":"10.1016\/j.biopha.2021.112175"},{"key":"ref_64","first-page":"1501","article-title":"Withania somnifera L.: Insights into the phytochemical profile, therapeutic potential, clinical trials, and future prospective","volume":"23","author":"Saleem","year":"2020","journal-title":"Iran. J. Basic Med. Sci."},{"key":"ref_65","doi-asserted-by":"crossref","first-page":"1191","DOI":"10.1177\/0269881115600132","article-title":"Withania somnifera Dunal (Indian ginseng) impairs acquisition and expression of ethanol-elicited conditioned place preference and conditioned place aversion","volume":"29","author":"Spina","year":"2015","journal-title":"J. Psychopharmacol."},{"key":"ref_66","doi-asserted-by":"crossref","unstructured":"Haque, I.M., Mishra, A., Kalra, B.S., and Chawla, S. (2021). Role of Standardized Plant Extracts in Controlling Alcohol Withdrawal Syndrome\u2014An Experimental Study. Brain Sci., 11.","DOI":"10.3390\/brainsci11070919"},{"key":"ref_67","doi-asserted-by":"crossref","unstructured":"Bassareo, V., Talani, G., Frau, R., Porru, S., Rosas, M., Kasture, S.B., Peana, A.T., Loi, E., Sanna, E., and Acquas, E. (2019). Inhibition of Morphine- and Ethanol-Mediated Stimulation of Mesolimbic Dopamine Neurons by Withania somnifera. Front. Neurosci., 13.","DOI":"10.3389\/fnins.2019.00545"},{"key":"ref_68","doi-asserted-by":"crossref","first-page":"795","DOI":"10.1007\/s00213-022-06069-w","article-title":"Effects of docosanyl ferulate, a constituent of Withania somnifera, on ethanol- and morphine-elicited conditioned place preference and ERK phosphorylation in the accumbens shell of CD1 mice","volume":"239","author":"Maccioni","year":"2022","journal-title":"Psychopharmacology"},{"key":"ref_69","doi-asserted-by":"crossref","first-page":"264","DOI":"10.1016\/j.jep.2015.05.058","article-title":"Direct evidence for GABAergic activity of Withania somnifera on mammalian ionotropic GABAA and GABA\u03c1 receptors","volume":"171","author":"Candelario","year":"2015","journal-title":"J. Ethnopharmacol."},{"key":"ref_70","doi-asserted-by":"crossref","first-page":"77","DOI":"10.33069\/cim.2024.0010","article-title":"Molecular Docking and Dynamics Studies of Withania somnifera Derived Compounds as GABA-A Receptor Modulators for Insomnia","volume":"6","author":"Alam","year":"2024","journal-title":"Chronobiol. Med."},{"key":"ref_71","doi-asserted-by":"crossref","first-page":"300","DOI":"10.1016\/j.neuroscience.2016.07.018","article-title":"Characterization of the \u03b3-aminobutyric acid signaling system in the zebrafish (Danio rerio Hamilton) central nervous system by reverse transcription-quantitative polymerase chain reaction","volume":"343","author":"Cocco","year":"2017","journal-title":"Neuroscience"},{"key":"ref_72","doi-asserted-by":"crossref","unstructured":"Monesson-Olson, B., McClain, J.J., Case, A.E., Dorman, H.E., Turkewitz, D.R., Steiner, A.B., and Downes, G.B. (2018). Expression of the eight GABAA receptor \u03b1 subunits in the developing zebrafish central nervous system. PLoS ONE, 13.","DOI":"10.1101\/244590"},{"key":"ref_73","doi-asserted-by":"crossref","unstructured":"Sadamitsu, K., Shigemitsu, L., Suzuki, M., Ito, D., Kashima, M., and Hirata, H. (2021). Characterization of zebrafish GABAA receptor subunits. Sci. Rep., 11.","DOI":"10.1038\/s41598-021-84646-3"},{"key":"ref_74","doi-asserted-by":"crossref","first-page":"310","DOI":"10.1038\/s12276-021-00571-5","article-title":"Zebrafish as an animal model for biomedical research","volume":"53","author":"Choi","year":"2021","journal-title":"Exp. Mol. Med."},{"key":"ref_75","doi-asserted-by":"crossref","first-page":"204","DOI":"10.1016\/j.bbr.2013.05.065","article-title":"Time-course of behavioural changes induced by ethanol in zebrafish (Danio rerio)","volume":"252","author":"Tran","year":"2013","journal-title":"Behav. Brain Res."},{"key":"ref_76","doi-asserted-by":"crossref","first-page":"425","DOI":"10.1016\/j.ntt.2012.03.002","article-title":"Locomotor activity assay in zebrafish larvae: Influence of age, strain and ethanol","volume":"34","author":"Slieker","year":"2012","journal-title":"Neurotoxicol. Teratol."},{"key":"ref_77","doi-asserted-by":"crossref","first-page":"727","DOI":"10.1016\/j.alcohol.2015.08.003","article-title":"Influences of acute ethanol exposure on locomotor activities of zebrafish larvae under different illumination","volume":"49","author":"Guo","year":"2015","journal-title":"Alcohol"},{"key":"ref_78","doi-asserted-by":"crossref","unstructured":"Hillman, C., Kearn, J., and Parker, M.O. (2024). A unified approach to investigating 4 dpf zebrafish larval behaviour through a standardised light\/dark assay. Prog. Neuropsychopharmacol. Biol. Psychiatry, 134.","DOI":"10.1016\/j.pnpbp.2024.111084"},{"key":"ref_79","doi-asserted-by":"crossref","first-page":"647","DOI":"10.1016\/j.pbb.2004.01.003","article-title":"Acute effects of alcohol on larval zebrafish: A genetic system for large-scale screening","volume":"77","author":"Lockwood","year":"2004","journal-title":"Pharmacol. Biochem. Behav."},{"key":"ref_80","doi-asserted-by":"crossref","first-page":"84","DOI":"10.1016\/j.ntt.2009.04.066","article-title":"Acute neuroactive drug exposures alter locomotor activity in larval zebrafish","volume":"32","author":"Irons","year":"2010","journal-title":"Neurotoxicol. Teratol."},{"key":"ref_81","doi-asserted-by":"crossref","first-page":"52","DOI":"10.1016\/j.neuro.2008.09.011","article-title":"Locomotion in larval zebrafish: Influence of time of day, lighting and ethanol","volume":"30","author":"MacPhail","year":"2009","journal-title":"Neurotoxicology"},{"key":"ref_82","doi-asserted-by":"crossref","unstructured":"Basnet, R.M., Zizioli, D., Taweedet, S., Finazzi, D., and Memo, M. (2019). Zebrafish larvae as a behavioral model in neuropharmacology. Biomedicines, 7.","DOI":"10.3390\/biomedicines7010023"},{"key":"ref_83","doi-asserted-by":"crossref","first-page":"1749","DOI":"10.1523\/JNEUROSCI.4702-05.2006","article-title":"Chronic intermittent ethanol-induced switch of ethanol actions from extrasynaptic to synaptic hippocampal GABAA receptors","volume":"26","author":"Liang","year":"2006","journal-title":"J. Neurosci."},{"key":"ref_84","doi-asserted-by":"crossref","first-page":"705","DOI":"10.1086\/383283","article-title":"Variations in GABRA2, encoding the \u03b12 subunit of the GABAA receptor, are associated with alcohol dependence and with brain oscillations","volume":"74","author":"Edenberg","year":"2004","journal-title":"Am. J. Hum. Genet."},{"key":"ref_85","first-page":"1708","article-title":"GABAA-Receptor \u03b4 Subunit Knockout Mice Have Multiple Defects in Behavioral Responses to Ethanol","volume":"25","author":"Mihalek","year":"2001","journal-title":"Alcohol Clin. Exp. Res."},{"key":"ref_86","first-page":"040782","article-title":"Targeted knockout of GABA-A receptor gamma 2 subunit provokes transient light-induced reflex seizures in zebrafish larvae","volume":"12","author":"Liao","year":"2019","journal-title":"Dis. Model. Mech."},{"key":"ref_87","doi-asserted-by":"crossref","first-page":"791","DOI":"10.1111\/acer.14000","article-title":"GABAA Receptor Subtypes and the Abuse-Related Effects of Ethanol in Rhesus Monkeys: Experiments with Selective Positive Allosteric Modulators","volume":"43","author":"Berro","year":"2019","journal-title":"Alcohol. Clin. Exp. Res."},{"key":"ref_88","doi-asserted-by":"crossref","first-page":"728","DOI":"10.1038\/nrn920","article-title":"Excitatory actions of gaba during development: The nature of the nurture","volume":"3","year":"2002","journal-title":"Nat. Rev. Neurosci."},{"key":"ref_89","doi-asserted-by":"crossref","first-page":"1588","DOI":"10.1523\/JNEUROSCI.3791-07.2008","article-title":"Neurogenic role of the depolarizing chloride gradient revealed by global overexpression of KCC2 from the onset of development","volume":"28","author":"Reynolds","year":"2008","journal-title":"J. Neurosci."},{"key":"ref_90","doi-asserted-by":"crossref","unstructured":"Zhang, R., Zhang, S., and Du, J. (2013). KCC2-dependent subcellular E(Cl) difference of ON-OFF retinal ganglion cells in larval zebrafish. Front. Neural Circuits, 7.","DOI":"10.3389\/fncir.2013.00103"},{"key":"ref_91","doi-asserted-by":"crossref","first-page":"48","DOI":"10.1002\/cne.25411","article-title":"In vivo tracking of KCC2b expression during early brain development","volume":"531","author":"Jones","year":"2023","journal-title":"J. Comp. Neurol."},{"key":"ref_92","doi-asserted-by":"crossref","first-page":"223","DOI":"10.1016\/j.bbr.2009.10.008","article-title":"Zebrafish antipredatory responses: A future for translational research?","volume":"207","author":"Gerlai","year":"2010","journal-title":"Behav. Brain Res."},{"key":"ref_93","doi-asserted-by":"crossref","first-page":"287","DOI":"10.1007\/s00213-021-05990-w","article-title":"The zebrafish (Danio rerio) anxiety test battery: Comparison of behavioral responses in the novel tank diving and light-dark tasks following exposure to anxiogenic and anxiolytic compounds","volume":"239","author":"Fontana","year":"2022","journal-title":"Psychopharmacology"},{"key":"ref_94","doi-asserted-by":"crossref","first-page":"12757","DOI":"10.1523\/JNEUROSCI.3340-09.2009","article-title":"Extrasynaptic GABAA receptors: Form, pharmacology, and function","volume":"29","author":"Belelli","year":"2009","journal-title":"J. Neurosci."},{"key":"ref_95","doi-asserted-by":"crossref","first-page":"4","DOI":"10.1111\/j.1742-7843.2012.00879.x","article-title":"Acute effects of ethanol on glutamate receptors","volume":"111","author":"Korpi","year":"2012","journal-title":"Basic Clin. Pharmacol. Toxicol."},{"key":"ref_96","doi-asserted-by":"crossref","unstructured":"Modi, S.J., Tiwari, A., Ghule, C., Pawar, S., Saste, G., Jagtap, S., Singh, R., Deshmukh, A., Girme, A., and Hingorani, L. (2022). Pharmacokinetic Study of Withanosides and Withanolides from Withania somnifera Using Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry (UHPLC-MS\/MS). Molecules, 27.","DOI":"10.3390\/molecules27051476"},{"key":"ref_97","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1177\/0023677219869037","article-title":"Zebrafish: Housing and husbandry recommendations","volume":"54","author":"Midtlyng","year":"2020","journal-title":"Lab. Anim."},{"key":"ref_98","unstructured":"Westerfield, M. (2000). The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Danio rerio), University of Oregon Press. [4th ed.]."},{"key":"ref_99","doi-asserted-by":"crossref","first-page":"134490","DOI":"10.1016\/j.scitotenv.2019.134490","article-title":"DMSO effects larval zebrafish (Danio rerio) behavior, with additive and interaction effects when combined with positive controls","volume":"709","author":"Christou","year":"2020","journal-title":"Sci. Total Environ."},{"key":"ref_100","doi-asserted-by":"crossref","unstructured":"Ingebretson, J.J., and Masino, M.A. (2013). Quantification of locomotor activity in larval zebrafish: Considerations for the design of high-throughput behavioral studies. Front. Neural Circuits, 7.","DOI":"10.3389\/fncir.2013.00109"},{"key":"ref_101","doi-asserted-by":"crossref","unstructured":"Jarema, K.A., Hunter, D.L., Hill, B.N., Olin, J.K., Britton, K.N., Waalkes, M.R., and Padilla, S. (2022). Developmental Neurotoxicity and Behavioral Screening in Larval Zebrafish with a Comparison to Other Published Results. Toxics, 10.","DOI":"10.3390\/toxics10050256"},{"key":"ref_102","doi-asserted-by":"crossref","first-page":"2526","DOI":"10.1242\/jeb.003939","article-title":"Modulation of locomotor activity in larval zebrafish during light adaptation","volume":"210","author":"Burgess","year":"2007","journal-title":"J. Exp. Biol."},{"key":"ref_103","doi-asserted-by":"crossref","first-page":"156","DOI":"10.1016\/0003-2697(87)90021-2","article-title":"Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction","volume":"162","author":"Chomczynski","year":"1987","journal-title":"Anal. Biochem."},{"key":"ref_104","doi-asserted-by":"crossref","unstructured":"Houseright, R.A., Rosowski, E.E., Lam, P.-Y., Tauzin, S.J.M., Mulvaney, O., Dewey, C.N., and Huttenlocher, A. (2020). Cell type specific gene expression profiling reveals a role for complement component C3 in neutrophil responses to tissue damage. Sci. Rep., 10.","DOI":"10.1038\/s41598-020-72750-9"},{"key":"ref_105","doi-asserted-by":"crossref","first-page":"402","DOI":"10.1006\/meth.2001.1262","article-title":"Analysis of relative gene expression data using real-time quantitative PCR and the 2\u2212\u0394\u0394Ct Method","volume":"25","author":"Livak","year":"2001","journal-title":"Methods"},{"key":"ref_106","doi-asserted-by":"crossref","first-page":"597","DOI":"10.1677\/jme.1.01755","article-title":"Quantitative real-time RT-PCR\u2014A perspective","volume":"34","author":"Bustin","year":"2005","journal-title":"J. Mol. Endocrinol."},{"key":"ref_107","doi-asserted-by":"crossref","first-page":"1537","DOI":"10.1373\/clinchem.2016.268953","article-title":"MIQE: A step toward more robust and reproducible quantitative PCR","volume":"63","author":"Bustin","year":"2017","journal-title":"Clin. Chem."},{"key":"ref_108","doi-asserted-by":"crossref","first-page":"384","DOI":"10.1111\/j.1745-7270.2007.00283.x","article-title":"Validation of zebrafish (Danio rerio) reference genes for quantitative real-time RT-PCR normalization","volume":"39","author":"Tang","year":"2007","journal-title":"Acta Biochim. Biophys. Sin."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/22\/10991\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,11,13]],"date-time":"2025-11-13T12:59:40Z","timestamp":1763038780000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/22\/10991"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,11,13]]},"references-count":108,"journal-issue":{"issue":"22","published-online":{"date-parts":[[2025,11]]}},"alternative-id":["ijms262210991"],"URL":"https:\/\/doi.org\/10.3390\/ijms262210991","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,11,13]]}}}