{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T19:48:48Z","timestamp":1789156128461,"version":"build-2803163510"},"reference-count":38,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2023,2,21]],"date-time":"2023-02-21T00:00:00Z","timestamp":1676937600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Ministry of Science, Technological Development and Innovation of the Republic of Serbia","award":["451-03-68\/2022-14\/200017"],"award-info":[{"award-number":["451-03-68\/2022-14\/200017"]}]},{"name":"Ministry of Science, Technological Development and Innovation of the Republic of Serbia","award":["451-03-47\/2023-01\/200178"],"award-info":[{"award-number":["451-03-47\/2023-01\/200178"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceuticals"],"abstract":"<jats:p>Alprazolam (ALP), a benzodiazepine (BDZ) used to treat anxiety, panic, and sleep disorders, is one of the most prescribed psychotropic drugs worldwide. The side effects associated with long-term (mis)use of ALP have become a major challenge in pharmacotherapy, emphasizing the unmet need to further investigate their underlying molecular mechanisms. Prolonged BDZ exposure may induce adaptive changes in the function of several receptors, including the primary target, gammaaminobutyric acid receptor type A (GABAAR), but also other neurotransmitter receptors such as glutamatergic. The present study investigated the potential effects of prolonged ALP treatment on components of glutamatergic neurotransmission, with special emphasis on N-Methyl-D-aspartate receptor (NMDAR) in the hippocampus of adult male Wistar rats. The study revealed behavioral changes consistent with potential onset of tolerance and involvement of the glutamatergic system in its development. Specifically, an increase in NMDAR subunits (NR1, NR2A, NR2B), a decrease in vesicular glutamate transporter 1 (vGlut1), and differential modulation of excitatory amino acid transporters 1 and 2 (EAAT1\/2, in vivo and in vitro) were observed, alongside a decrease in \u03b11-containing GABAAR following the treatment. By describing the development of compensatory actions in the glutamatergic system, the present study provides valuable information on neuroadaptive mechanisms following prolonged ALP intake.<\/jats:p>","DOI":"10.3390\/ph16030331","type":"journal-article","created":{"date-parts":[[2023,2,22]],"date-time":"2023-02-22T02:31:55Z","timestamp":1677033115000},"page":"331","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":16,"title":["Prolonged Alprazolam Treatment Alters Components of Glutamatergic Neurotransmission in the Hippocampus of Male Wistar Rats\u2014The Neuroadaptive Changes following Long-Term Benzodiazepine (Mis)Use"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1635-9766","authenticated-orcid":false,"given":"Marina","family":"Zaric Kontic","sequence":"first","affiliation":[{"name":"Department of Molecular Biology and Endocrinology, VIN\u010cA Institute of Nuclear Sciences\u2014National Institute of the Republic of Serbia, University of Belgrade, 11351 Belgrade, Serbia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4855-6131","authenticated-orcid":false,"given":"Milorad","family":"Dragic","sequence":"additional","affiliation":[{"name":"Laboratory for Neurobiology, Department of General Physiology and Biophysics, Faculty of Biology, University of Belgrade, 11158 Belgrade, Serbia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jelena","family":"Martinovic","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology and Endocrinology, VIN\u010cA Institute of Nuclear Sciences\u2014National Institute of the Republic of Serbia, University of Belgrade, 11351 Belgrade, Serbia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9385-5002","authenticated-orcid":false,"given":"Katarina","family":"Mihajlovic","sequence":"additional","affiliation":[{"name":"Laboratory for Neurobiology, Department of General Physiology and Biophysics, Faculty of Biology, University of Belgrade, 11158 Belgrade, Serbia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zeljka","family":"Brkic","sequence":"additional","affiliation":[{"name":"Department of Biomedicine, Aarhus University, 8000 Aarhus, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Natasa","family":"Mitrovic","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology and Endocrinology, VIN\u010cA Institute of Nuclear Sciences\u2014National Institute of the Republic of Serbia, University of Belgrade, 11351 Belgrade, Serbia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4476-7871","authenticated-orcid":false,"given":"Ivana","family":"Grkovic","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology and Endocrinology, VIN\u010cA Institute of Nuclear Sciences\u2014National Institute of the Republic of Serbia, University of Belgrade, 11351 Belgrade, Serbia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,2,21]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"4","DOI":"10.1097\/ADM.0000000000000350","article-title":"A Review of Alprazolam Use, Misuse, and Withdrawal","volume":"12","author":"Hamby","year":"2018","journal-title":"J. Addict. Med."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"404","DOI":"10.1093\/fampra\/cmt010","article-title":"Tolerance to benzodiazepines among long-term users in primary care","volume":"30","author":"Willems","year":"2013","journal-title":"Fam. Pract."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"594","DOI":"10.3390\/neurolint13040059","article-title":"Benzodiazepines: Uses, Dangers, and Clinical Considerations","volume":"13","author":"Edinoff","year":"2021","journal-title":"Neurol. Int."},{"key":"ref_4","first-page":"1","article-title":"Drugs Most Frequently Involved in Drug Overdose Deaths: United States, 2011\u20132016","volume":"67","author":"Hedegaard","year":"2018","journal-title":"Natl. Vital Stat. Rep."},{"key":"ref_5","unstructured":"George, T.T., and Tripp, J. (2022). StatPearls, StatPearls Publishing."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"587","DOI":"10.1038\/75761","article-title":"Sedative but not anxiolytic properties of benzodiazepines are mediated by the GABA(A) receptor alpha1 subtype","volume":"3","author":"McKernan","year":"2000","journal-title":"Nat. Neurosci."},{"key":"ref_7","first-page":"416864","article-title":"Mechanisms Underlying Tolerance after Long-Term Benzodiazepine Use: A Future for Subtype-Selective GABA(A) Receptor Modulators?","volume":"2012","author":"Vinkers","year":"2012","journal-title":"Adv. Pharmacol. Sci."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"3483","DOI":"10.1073\/pnas.051628698","article-title":"Glutamic acid decarboxylase and glutamate receptor changes during tolerance and dependence to benzodiazepines","volume":"98","author":"Izzo","year":"2001","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"6889","DOI":"10.1073\/pnas.90.14.6889","article-title":"Diazepam dependence prevented by glutamate antagonists","volume":"90","author":"Steppuhn","year":"1993","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_10","first-page":"595","article-title":"Chronic benzodiazepine administration alters hippocampal CA1 neuron excitability: NMDA receptor function and expression","volume":"43","author":"Cox","year":"2002","journal-title":"Neuropharmacology"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1016\/S0163-7258(03)00029-9","article-title":"Neuroadaptive processes in GABAergic and glutamatergic systems in benzodiazepine dependence","volume":"98","author":"Allison","year":"2003","journal-title":"Pharmacol. Ther."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"108602","DOI":"10.1016\/j.neuropharm.2021.108602","article-title":"Glutamate transporters: Critical components of glutamatergic transmission","volume":"192","author":"Ortega","year":"2021","journal-title":"Neuropharmacology"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"702","DOI":"10.1038\/269702a0","article-title":"High densities of benzodiazepine receptors in human cortical areas","volume":"269","author":"Braestrup","year":"1977","journal-title":"Nature"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"602","DOI":"10.1523\/JNEUROSCI.08-02-00615.1988","article-title":"Localization of the GABAA receptor in the rat brain with a monoclonal antibody to the 57,000 Mr peptide of the GABAA receptor\/benzodiazepine receptor\/Cl- channel complex","volume":"8","author":"Vitorica","year":"1988","journal-title":"J. Neurosci. Off. J. Soc. Neurosci."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"732","DOI":"10.1038\/266732a0","article-title":"Benzodiazepine receptors in rat brain","volume":"266","author":"Squires","year":"1977","journal-title":"Nature"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1016\/j.ejphar.2009.10.014","article-title":"Hippocampal NMDA receptors and anxiety: At the interface between cognition and emotion","volume":"626","author":"Barkus","year":"2010","journal-title":"Eur. J. Pharmacol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"670","DOI":"10.1016\/j.neuron.2018.01.016","article-title":"Anxiety Cells in a Hippocampal-Hypothalamic Circuit","volume":"97","author":"Jimenez","year":"2018","journal-title":"Neuron"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"7","DOI":"10.1016\/j.neuron.2009.11.031","article-title":"Are the dorsal and ventral hippocampus functionally distinct structures?","volume":"65","author":"Fanselow","year":"2010","journal-title":"Neuron"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"17513","DOI":"10.1073\/pnas.0602841103","article-title":"Redefining the concept of reactive astrocytes as cells that remain within their unique domains upon reaction to injury","volume":"103","author":"Wilhelmsson","year":"2006","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Malik, A.R., and Willnow, T.E. (2019). Excitatory Amino Acid Transporters in Physiology and Disorders of the Central Nervous System. Int. J. Mol. Sci., 20.","DOI":"10.3390\/ijms20225671"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"113","DOI":"10.1016\/0149-7634(85)90037-5","article-title":"Tolerance to the behavioral actions of benzodiazepines","volume":"9","author":"File","year":"1985","journal-title":"Neurosci. Biobehav. Rev."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"361","DOI":"10.1016\/0165-6147(88)90255-6","article-title":"Tolerance to the anticonvulsant effect of benzodiazepines","volume":"9","author":"Haigh","year":"1988","journal-title":"Trends Pharmacol. Sci."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1145","DOI":"10.1016\/S0278-5846(01)00166-X","article-title":"Behavioral analysis indicates benzodiazepine-tolerance mediated by the benzodiazepine binding-site at the GABA(A)-receptor","volume":"25","author":"Schmitt","year":"2001","journal-title":"Prog. Neuro-Psychopharmacol. Biol. Psychiatry"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"92","DOI":"10.1016\/j.phrs.2015.12.030","article-title":"Activation-induced regulation of GABAA receptors: Is there a link with the molecular basis of benzodiazepine tolerance?","volume":"109","author":"Gravielle","year":"2016","journal-title":"Pharmacol. Res."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"108985","DOI":"10.1016\/j.drugalcdep.2021.108985","article-title":"Tolerance and dependence following chronic alprazolam treatment in rhesus monkeys: Role of GABA(A) receptor subtypes","volume":"228","author":"Duke","year":"2021","journal-title":"Drug Alcohol Depend."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Barker, J.S., and Hines, R.M. (2020). Regulation of GABAA Receptor Subunit Expression in Substance Use Disorders. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21124445"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1016\/j.pscychresns.2010.08.003","article-title":"The acute and late CNS glutamine response to benzodiazepine challenge: A pilot pharmacokinetic study using proton magnetic resonance spectroscopy","volume":"184","author":"Henry","year":"2010","journal-title":"Psychiatry Res."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"163","DOI":"10.1007\/s00213-002-1373-y","article-title":"Adaptive changes in the rat hippocampal glutamatergic neurotransmission are observed during long-term treatment with lorazepam","volume":"166","author":"Bonavita","year":"2003","journal-title":"Psychopharmacology"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"119","DOI":"10.1016\/S0166-4328(03)00072-X","article-title":"NMDA-NR1 and -NR2B subunits mRNA expression in the hippocampus of rats tolerant to Diazepam","volume":"144","author":"Perez","year":"2003","journal-title":"Behav. Brain Res."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"309","DOI":"10.1007\/PL00005172","article-title":"Hypersusceptibility to DMCM-induced seizures during diazepam withdrawal in mice: Evidence for upregulation of NMDA receptors","volume":"357","author":"Tsuda","year":"1998","journal-title":"Naunyn-Schmiedeberg\u2019Pharmacol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"823","DOI":"10.1016\/0091-3057(94)90282-8","article-title":"Dizocilpine prevents the development of tolerance to the sedative effects of diazepam in rats","volume":"47","author":"File","year":"1994","journal-title":"Pharmacol. Biochem. Behav."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1011","DOI":"10.1016\/0028-3908(93)90066-C","article-title":"Kindling to the benzodiazepine receptor inverse agonist, FG 7142: Evidence for involvement of NMDA, but not non-NMDA, glutamatergic receptors","volume":"32","author":"Stephens","year":"1993","journal-title":"Neuropharmacology"},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Briley, M., and File, S.E. (1991). New Concepts in Anxiety, Macmillan Education.","DOI":"10.1007\/978-1-349-11847-2"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"321","DOI":"10.1016\/S0197-0186(01)00087-0","article-title":"Benzodiazepines differently modulate EAAT1\/GLAST and EAAT2\/GLT1 glutamate transporters expressed in CHO cells","volume":"40","author":"Palmada","year":"2002","journal-title":"Neurochem. Int."},{"key":"ref_35","first-page":"624","article-title":"The Laboratory Rat: Relating Its Age With Human\u2019s","volume":"4","author":"Sengupta","year":"2013","journal-title":"Int. J. Prev. Med."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"100","DOI":"10.1016\/j.ygcen.2016.06.018","article-title":"Regional and sex-related differences in modulating effects of female sex steroids on ecto-5\u2032-nucleotidase expression in the rat cerebral cortex and hippocampus","volume":"235","author":"Mitrovic","year":"2016","journal-title":"Gen. Comp. Endocrinol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"206","DOI":"10.1016\/0003-2697(78)90586-9","article-title":"A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples","volume":"87","author":"Markwell","year":"1978","journal-title":"Anal. Biochem."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1792","DOI":"10.1007\/s12035-020-02273-x","article-title":"Trimethyltin Increases Intracellular Ca(2+) Via L-Type Voltage-Gated Calcium Channels and Promotes Inflammatory Phenotype in Rat Astrocytes In Vitro","volume":"58","author":"Dragic","year":"2021","journal-title":"Mol. Neurobiol."}],"container-title":["Pharmaceuticals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1424-8247\/16\/3\/331\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T18:38:33Z","timestamp":1760121513000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1424-8247\/16\/3\/331"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,2,21]]},"references-count":38,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2023,3]]}},"alternative-id":["ph16030331"],"URL":"https:\/\/doi.org\/10.3390\/ph16030331","relation":{},"ISSN":["1424-8247"],"issn-type":[{"value":"1424-8247","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,2,21]]}}}