Research of treatment options addressing the cognitive deficit associated with neurodegenerative disorders is of particular importance. Application of trimethyltin (TMT) to rats represents a promising model replicating multiple relevant features of such disorders. N-methyl-D-aspartate (NMDA) receptor antagonists and gamma-aminobutyric acid type A (GABAA) receptor potentiators have been reported to alleviate the TMT-induced cognitive deficit. These compounds may provide synergistic interactions in other models. The aim of this study was to investigate, whether co-application of NMDA receptor antagonist dizocilpine (MK-801) and GABAA receptor potentiator midazolam would be associated with an improved effect on the TMT-induced model of cognitive deficit. Wistar rats injected with TMT were repeatedly (12 days) treated with MK-801, midazolam, or both. Subsequently, cognitive performance was assessed. Finally, after a 17-day drug-free period, hippocampal neurodegeneration (neuronal density in CA2/3 subfield in the dorsal hippocampus, dentate gyrus morphometry) were analyzed. All three protective treatments induced similar degree of therapeutic effect in Morris water maze. The results of histological analyses were suggestive of minor protective effect of the combined treatment (MK-801 and midazolam), while these compounds alone were largely ineffective at this time point. Therefore, in terms of mitigation of cognitive deficit, the combined treatment was not associated with improved effect.

2nd Faculty of Medicine Charles University 5 Uvalu 84 150 06 Prague 5 Czech Republic

Institute of Physiology of the Czech Academy of Sciences Videnska 1083 142 20 Prague 4 Czech Republic

National Institute of Mental Health Topolova 748 250 67 Klecany Czech Republic

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Nava-Mesa M.O., Jiménez-Díaz L., Yajeya J., Navarro-Lopez J.D. GABAergic neurotransmission and new strategies of neuromodulation to compensate synaptic dysfunction in early stages of Alzheimer’s disease. Front. Cell. Neurosci. 2014;8:167. doi: 10.3389/fncel.2014.00167. PubMed DOI PMC

Wilkinson D., Andersen H.F. Analysis of the Effect of Memantine in Reducing the Worsening of Clinical Symptoms in Patients with Moderate to Severe Alzheimer’s Disease. Dement. Geriatr. Cogn. Disord. 2007;24:138–145. doi: 10.1159/000105162. PubMed DOI

Martinez-Coria H., Green K.N., Billings L.M., Kitazawa M., Albrecht M., Rammes G., Parsons C.G., Gupta S., Banerjee P., LaFerla F.M. Memantine Improves Cognition and Reduces Alzheimer’s-Like Neuropathology in Transgenic Mice. Am. J. Pathol. 2010;176:870–880. doi: 10.2353/ajpath.2010.090452. PubMed DOI PMC

Companys-Alemany J., Turcu A.L., Bellver-Sanchis A., Loza M.I., Brea J.M., Canudas A.M., Leiva R., Vázquez S., Pallàs M., Griñán-Ferré C. A Novel NMDA Receptor Antagonist Protects against Cognitive Decline Presented by Senescent Mice. Pharmaceutics. 2020;12:284. doi: 10.3390/pharmaceutics12030284. PubMed DOI PMC

Ponce-Lopez T., Liy-Salmeron G., Hong E., Meneses A. Lithium, phenserine, memantine and pioglitazone reverse memory deficit and restore phospho-GSK3β decreased in hippocampus in intracerebroventricular streptozotocin induced memory deficit model. Brain Res. 2011;1426:73–85. doi: 10.1016/j.brainres.2011.09.056. PubMed DOI

Minkeviciene R., Banerjee P., Tanila H. Memantine Improves Spatial Learning in a Transgenic Mouse Model of Alzheimer’s Disease. J. Pharmacol. Exp. Ther. 2004;311:677–682. doi: 10.1124/jpet.104.071027. PubMed DOI

McDonald J.W., Silverstein F.S., Johnston M.V. Neuroprotective effects of MK-801, TCP, PCP and CPP against N-methyl-d-aspartate induced neurotoxicity in an in vivo perinatal rat model. Brain Res. 1989;490:33–40. doi: 10.1016/0006-8993(89)90427-7. PubMed DOI

Schauwecker P.E. Neuroprotection by glutamate receptor antagonists against seizure-induced excitotoxic cell death in the aging brain. Exp. Neurol. 2010;224:207–218. doi: 10.1016/j.expneurol.2010.03.013. PubMed DOI PMC

Chen H.-S.V., Lipton S.A. The chemical biology of clinically tolerated NMDA receptor antagonists. J. Neurochem. 2006;97:1611–1626. doi: 10.1111/j.1471-4159.2006.03991.x. PubMed DOI

Muir K.W., Lees K.R. Clinical Experience With Excitatory Amino Acid Antagonist Drugs. Stroke. 1995;26:503–513. doi: 10.1161/01.STR.26.3.503. PubMed DOI

Pilipenko V., Narbute K., Pupure J., Rumaks J., Jansone B., Klusa V. Neuroprotective action of diazepam at very low and moderate doses in Alzheimer’s disease model rats. Neuropharmacol. 2019;144:319–326. doi: 10.1016/j.neuropharm.2018.11.003. PubMed DOI

Delorey T.M., Olsens R.W. y-Aminobutyric Acid A Receptor Structure and Function. J. Biol. Chem. 1992;267:16747–16750. doi: 10.1016/S0021-9258(18)41841-8. PubMed DOI

Ito H., Watanabe Y., Isshiki A., Uchino H. Neuroprotective properties of propofol and midazolam, but not pentobarbital, on neuronal damage induced by forebrain ischemia, based on the GABAA receptors. Acta Anaesthesiol. Scand. 1999;43:153–162. doi: 10.1034/j.1399-6576.1999.430206.x. PubMed DOI

Harman F., Hasturk A.E., Yaman M., Arca T., Kilinc K., Sargon M.F., Kaptanoglu E. Neuroprotective effects of propofol, thiopental, etomidate, and midazolam in fetal rat brain in ischemia-reperfusion model. Child’s Nerv. Syst. 2012;28:1055–1062. doi: 10.1007/s00381-012-1782-0. PubMed DOI

Shibuta S., Varathan S., Mashimo T. Ketamine and thiopental sodium: Individual and combined neuroprotective effects on cortical cultures exposed to NMDA or nitric oxide. Br. J. Anaesth. 2006;97:517–524. doi: 10.1093/bja/ael192. PubMed DOI

Sarnowska A., Beręsewicz M., Zabłocka B., Domańska-Janik K. Diazepam neuroprotection in excitotoxic and oxidative stress involves a mitochondrial mechanism additional to the GABAAR and hypothermic effects. Neurochem. Int. 2009;55:164–173. doi: 10.1016/j.neuint.2009.01.024. PubMed DOI

Liu J.-Y., Guo F., Wu H.-L., Wang Y., Liu J.-S. Midazolam anesthesia protects neuronal cells from oxidative stress-induced death via activation of the JNK-ERK pathway. Mol. Med. Rep. 2016;15:169–179. doi: 10.3892/mmr.2016.6031. PubMed DOI PMC

Lanctôt K.L., Herrmaan N., Mazzotta P., Khan L.R., Ingber N. GABAergic function in Alzheimer’s disease: Evidence for dysfunction and potential as a therapeutic target for the treatment of behavioral and psychological symptoms of dementia. Can. J. Psychiatry. 2004;49:439–453. doi: 10.1177/070674370404900705. PubMed DOI

Robertson D.G., Gray R.H., De Laiglesia F.A. Quantitative Assessment of Trimethyltin Induced Pathology of the Hippocampus. Toxicol. Pathol. 1987;15:7–17. doi: 10.1177/019262338701500102. PubMed DOI

Whittington D.L., Woodruff M.L., Baisden R.H. The time-course of trimethyltin-induced fiber and terminal degeneration in hippocampus. Neurotoxicol. Teratol. 1989;11:21–33. doi: 10.1016/0892-0362(89)90081-0. PubMed DOI

Balaban C., Callaghan J., Billingsle M. Trimethyltin-induced neuronal damage in the rat brain: Comparative studies using silver degeneration stains, immunocytochemistry and immunoassay for neuronotypic and gliotypic proteins. Neuroscience. 1988;26:337–361. doi: 10.1016/0306-4522(88)90150-9. PubMed DOI

Brown A.W., Aldridge W.N., Street B.W., Verschoyle R.D. The behavioral and neuropathologic sequelae of intoxication by trimethyltin compounds in the rat. Am. J. Pathol. 1979;97:59–82. PubMed PMC

Earley B., Burke M., Leonard B.E. Behavioural, biochemical and histological effects of trimethyltin (TMT) induced brain damage in the rat. Neurochem. Int. 1992;21:351–366. doi: 10.1016/0197-0186(92)90186-U. PubMed DOI

Ishida N., Akaike M., Tsutsumi S., Kanai H., Masui A., Sadamatsu M., Kuroda Y., Watanabe Y., McEwen B.S., Kato N. Trimethyltin syndrome as a hippocampal degeneration model: Temporal changes and neurochemical features of seizure susceptibility and learning impairment. Neuroscience. 1997;81:1183–1191. doi: 10.1016/S0306-4522(97)00220-0. PubMed DOI

Kaur S., Nehru B. Alteration in Glutathione Homeostasis and Oxidative Stress During the Sequelae of Trimethyltin Syndrome in Rat Brain. Biol. Trace Element Res. 2013;153:299–308. doi: 10.1007/s12011-013-9676-x. PubMed DOI

Lalkovicova M., Burda J., Nemethova M., Burda R., Danielisova V., Maria L., Jozef B., Miroslava N., Rastislav B., Viera D. Postconditioning Effectively Prevents Trimethyltin Induced Neuronal Damage in the Rat Brain. Folia Biol. 2016;64:97–103. doi: 10.3409/fb64_2.97. PubMed DOI

Scallet A.C., Pothuluri N., Rountree R.L., Matthews J.C. Quantitating silver-stained neurodegeneration: The neurotoxicity of trimethlytin (TMT) in aged rats. J. Neurosci. Methods. 2000;98:69–76. doi: 10.1016/S0165-0270(00)00191-6. PubMed DOI

Brabeck C., Michetti F., Geloso M.C., Corvino V., Goezalan F., Meyermann R., Schluesener H.J. Expression of EMAP-II by Activated Monocytes/Microglial Cells in Different Regions of the Rat Hippocampus after Trimethyltin-Induced Brain Damage. Exp. Neurol. 2002;177:341–346. doi: 10.1006/exnr.2002.7985. PubMed DOI

Misiti F., Orsini F., Clementi M.E., Lattanzi W., Giardina B., Michetti F. Mitochondrial oxygen consumption inhibition importance for TMT-dependent cell death in undifferentiated PC12 cells. Neurochem. Int. 2008;52:1092–1099. doi: 10.1016/j.neuint.2007.11.008. PubMed DOI

Dawson R., Patterson T.A., Eppler B. Endogenous excitatory amino acid release from brain slices and astrocyte cultures evoked by trimethyltin and other neurotoxic agents. Neurochem. Res. 1995;20:847–858. doi: 10.1007/BF00969697. PubMed DOI

Aschner M., Gannon M., Kimelberg H. Interactions of trimethyl tin (TMT) with rat primary astrocyte cultures: Altered uptake and efflux of rubidium,l-glutamate andD-aspartate. Brain Res. 1992;582:181–185. doi: 10.1016/0006-8993(92)90131-R. PubMed DOI

Koczyk D. How does trimethyltin affect the brain: Facts and hypotheses. Acta Neurobiol. Exp. 1996;56:587–596. PubMed

Little A., Miller D., Li S., Kashon M., O’Callaghan J., Little R. Trimethyltin-induced neurotoxicity: Gene expression pathway analysis, q-RT-PCR and immunoblotting reveal early effects associated with hippocampal damage and gliosis. Neurotoxicol. Teratol. 2012;34:72–82. doi: 10.1016/j.ntt.2011.09.012. PubMed DOI

Nilsberth C., Kostyszyn B., Luthman J. Changes in APP, PS1 and other factors related to Alzheimer’s disease pathophysiology after trimethyltin-induced brain lesion in the rat. Neurotox. Res. 2002;4:625–636. doi: 10.1080/1029842021000045471. PubMed DOI

Geloso M.C., Corvino V., Michetti F. Trimethyltin-induced hippocampal degeneration as a tool to investigate neurodegenerative processes. Neurochem. Int. 2011;58:729–738. doi: 10.1016/j.neuint.2011.03.009. PubMed DOI

Corvino V., Marchese E., Michetti F., Geloso M.C. Neuroprotective Strategies in Hippocampal Neurodegeneration Induced by the Neurotoxicant Trimethyltin. Neurochem. Res. 2013;38:240–253. doi: 10.1007/s11064-012-0932-9. PubMed DOI

Earley B., Burke M., Leonard B., Gouret C., Junien J. A comparison of the psychopharmacological profiles of phencyclidine, ketamine and (+) SKF 10,047 in the trimethyltin rat model. Neuropharmacology. 1990;29:695–703. doi: 10.1016/0028-3908(90)90121-7. PubMed DOI

O’Connell A., Earley B., Leonard B.E. Effects of the GABA agonist THIP (gaboxadol) on trimethyltin-induced behavioural neurotoxicity in the rat. Med. Sci. Res. 1994;22:201–202.

Shuto M., Seko K., Kuramoto N., Sugiyama C., Kawada K., Yoneyama M., Nagashima R., Ogita K. Activation of c-Jun N-Terminal Kinase Cascades Is Involved in Part of the Neuronal Degeneration Induced by Trimethyltin in Cortical Neurons of Mice. J. Pharmacol. Sci. 2009;109:60–70. doi: 10.1254/jphs.08211FP. PubMed DOI

Gunasekar P., Li L., Prabhakaran K., Eybl V., Borowitz J.L., Isom G.E. Mechanisms of the Apoptotic and Necrotic Actions of Trimethyltin in Cerebellar Granule Cells. Toxicol. Sci. 2001;64:83–89. doi: 10.1093/toxsci/64.1.83. PubMed DOI

Zimmer L., Woolley D., Chang L. Does phenobarbital protect against trimethyltin-induced neuropathology of limbic structures? Life Sci. 1985;36:851–858. doi: 10.1016/0024-3205(85)90209-7. PubMed DOI

Kabir T., Uddin S., Al Mamun A., Jeandet P., Aleya L., Mansouri R.A., Ashraf G.M., Mathew B., Bin-Jumah M.N., Abdel-Daim M.M. Combination Drug Therapy for the Management of Alzheimer’s Disease. Int. J. Mol. Sci. 2020;21:3272. doi: 10.3390/ijms21093272. PubMed DOI PMC

Martin B.S., Kapur J. A combination of ketamine and diazepam synergistically controls refractory status epilepticus induced by cholinergic stimulation. Epilepsia. 2007;49:248–255. doi: 10.1111/j.1528-1167.2007.01384.x. PubMed DOI PMC

Niquet J., Baldwin R., Norman K., Suchomelova L., Lumley L., Wasterlain C.G. Midazolam-ketamine dual therapy stops cholinergic status epilepticus and reduces Morris water maze deficits. Epilepsia. 2016;57:1406–1415. doi: 10.1111/epi.13480. PubMed DOI PMC

Shakarjian M.P., Ali M.S., Velíšková J., Stanton P.K., Heck D.E., Velíšek L. Combined diazepam and MK-801 therapy provides synergistic protection from tetramethylenedisulfotetramine-induced tonic–clonic seizures and lethality in mice. NeuroToxicology. 2015;48:100–108. doi: 10.1016/j.neuro.2015.03.007. PubMed DOI PMC

Ellison G. The N-methyl-d-aspartate antagonists phencyclidine, ketamine and dizocilpine as both behavioral and anatomical models of the dementias. Brain Res. Rev. 1995;20:250–267. doi: 10.1016/0165-0173(94)00014-G. PubMed DOI

Kanto J.H. Midazolam: The First Water-soluble Benzodiazepine; Pharmacology, Pharmacokinetics and Efficacy in Insomnia and Anesthesia. Pharmacother. J. Hum. Pharmacol. Drug Ther. 1985;5:138–155. doi: 10.1002/j.1875-9114.1985.tb03411.x. PubMed DOI

Morris R.G.M., Garrud P., Rawlins J.N.P., O’Keefe J. Place navigation impaired in rats with hippocampal lesions. Nat. Cell Biol. 1982;297:681–683. doi: 10.1038/297681a0. PubMed DOI

Kochli D.E., Thompson E.C., Fricke E.A., Postle A.F., Quinn J.J. The amygdala is critical for trace, delay, and contextual fear conditioning. Learn. Mem. 2015;22:92–100. doi: 10.1101/lm.034918.114. PubMed DOI PMC

Maren S., Fanselow M.S. Electrolytic Lesions of the Fimbria/Fornix, Dorsal Hippocampus, or Entorhinal Cortex Produce Anterograde Deficits in Contextual Fear Conditioning in Rats. Neurobiol. Learn. Mem. 1997;67:142–149. doi: 10.1006/nlme.1996.3752. PubMed DOI

Bahník Š. Carousel Maze Manager (Version 0.4.0) [Software] [(accessed on 21 April 2015)];2014 Available online: https://github.com/bahniks/CM_Manager_0_4_0.

Vorhees C.V., Williams M.T. Morris water maze: Procedures for assessing spatial and related forms of learning and memory. Nat. Protoc. 2006;1:848–858. doi: 10.1038/nprot.2006.116. PubMed DOI PMC

Whiting M.D., Kokiko-Cochran O.N. Assessment of cognitive function in the water maze task: Maximizing data collection and analysis in animal models of brain injury. In: Kobeissy F., editor. Injury Models of the Central Nervous System: Methods and Protocols, Methods in Molecular Biology. Volume 1462. Springer Science + Business Media; New York, NY, USA: 2016. pp. 553–571. PubMed

Mátéffyová A., Otáhal J., Tsenov G., Mareš P., Kubová H. Intrahippocampal injection of endothelin-1 in immature rats results in neuronal death, development of epilepsy and behavioral abnormalities later in life. Eur. J. Neurosci. 2006;24:351–360. doi: 10.1111/j.1460-9568.2006.04910.x. PubMed DOI

Rustay N., Browman K., Curzon P. Methods of Behavior Analysis in Neuroscience. 2nd ed. CRC Press/Taylor & Francis; Boca Raton, FL, USA: 2008. Cued and Contextual Fear Conditioning for Rodents; pp. 19–37. PubMed

Krsek P., Mikulecká A., Druga R., Kubová H., Hliňák Z., Suchomelová L., Mareš P. Long-term behavioral and morphological consequences of nonconvulsive status epilepticus in rats. Epilepsy Behav. 2004;5:180–191. doi: 10.1016/j.yebeh.2003.11.032. PubMed DOI

Paxinos G., Watson C. The Rat Brain in Stereotaxic Coordinates. 6th ed. Academic Press; London, UK: 2007.

Latini L., Geloso M.C., Corvino V., Giannetti S., Florenzano F., Viscomi M.T., Michetti F., Molinari M. Trimethyltin intoxication up-regulates nitric oxide synthase in neurons and purinergic ionotropic receptor 2 in astrocytes in the hippocampus. J. Neurosci. Res. 2009;88:500–509. doi: 10.1002/jnr.22238. PubMed DOI

Florian C., Roullet P. Hippocampal CA3-region is crucial for acquisition and memory consolidation in Morris water maze task in mice. Behav. Brain Res. 2004;154:365–374. doi: 10.1016/j.bbr.2004.03.003. PubMed DOI

Hunsaker M.R., Rosenberg J.S., Kesner R.P. The role of the dentate gyrus, CA3a,b, and CA3c for detecting spatial and environmental novelty. Hippocampus. 2008;18:1064–1073. doi: 10.1002/hipo.20464. PubMed DOI

West M.J., Slomianka L., Gundersen H.J.G. Unbiased stereological estimation of the total number of neurons in the subdivisions of the rat hippocampus using the optical fractionator. Anat. Rec. Adv. Integr. Anat. Evol. Biol. 1991;231:482–497. doi: 10.1002/ar.1092310411. PubMed DOI

Dyer R.S., Deshields T.L., Wonderlin W.F. Trimethyltin-induced changes in gross morphology of the hippocampus. Neurobehav. Toxicol. Teratol. 1982;4:141–147. PubMed

Meera P., Wallner M., Otis T.S. Molecular basis for the high THIP/gaboxadol sensitivity of extrasynaptic GABAA receptors. J. Neurophysiol. 2011;106:2057–2064. doi: 10.1152/jn.00450.2011. PubMed DOI PMC

Takahashi H. Automated Measurement of Freezing Time to Contextual and Auditory Cues in Fear Conditioning as a Simple Screening Method to Assess Learning and Memory Abilities in Rats. J. Toxicol. Sci. 2004;29:53–61. doi: 10.2131/jts.29.53. PubMed DOI

Gill R., Brazell C., Woodruff G.N., Kemp J.A. The neuroprotective action of dizocilpine (MK-801) in the rat middle cerebral artery occlusion model of focal ischaemia. Br. J. Pharmacol. 1991;103:2030–2036. doi: 10.1111/j.1476-5381.1991.tb12371.x. PubMed DOI PMC

Mazzone G.L., Nistri A. Modulation of extrasynaptic GABAergic receptor activity influences glutamate release and neuronal survival following excitotoxic damage to mouse spinal cord neurons. Neurochem. Int. 2019;128:175–185. doi: 10.1016/j.neuint.2019.04.018. PubMed DOI

Nelson R.M., Green A.R., Lambert D.G., Hainsworth A.H. On the regulation of ischaemia-induced glutamate efflux from rat cortex by GABA;in vitrostudies with GABA, clomethiazole and pentobarbitone. Br. J. Pharmacol. 2000;130:1124–1130. doi: 10.1038/sj.bjp.0703398. PubMed DOI PMC

Krüger K., Diepgrond V., Ahnefeld M., Wackerbeck C., Madeja M., Binding N., Musshoff U. Blockade of glutamatergic and GABAergic receptor channels by trimethyltin chloride. Br. J. Pharmacol. 2005;144:283–292. doi: 10.1038/sj.bjp.0706083. PubMed DOI PMC

Chang L.W. Neuropathology of trimethyltin: A proposed pathogenetic mechanism. Fundam. Appl. Toxicol. 1986;6:217–232. doi: 10.1016/0272-0590(86)90235-6. PubMed DOI

Chang L.W., Dyer R.S. Early effects of trimethyltin on the dentate gyrus basket cells: A morphological study. J. Toxicol. Environ. Health Part A. 1985;16:641–653. doi: 10.1080/15287398509530770. PubMed DOI