The GluN2B subunit of NMDA receptors represents a perspective therapeutic target in various CNS pathologies, including epilepsy. Because of its predominant expression in the immature brain, selective GluN2B antagonists are expected to be more effective early in postnatal development. The aim of this study was to identify age-dependent differences in the anticonvulsant activity of the GluN2B-selective antagonist Ro 25-6981 and assess the safety of this drug for the developing brain. Anticonvulsant activity of Ro 25-6981 (1, 3, and 10 mg/kg) was tested in a pentylenetetrazol (PTZ) model in infantile (12-day-old, P12) and juvenile (25-day-old, P25) rats. Ro 25-6981 (1 or 3 mg/kg/day) was administered from P7 till P11 to assess safety for the developing brain. Animals were then tested repeatedly in a battery of behavioral tests focusing on sensorimotor development, cognition, and emotionality till adulthood. Effects of early exposure to Ro 25-6981 on later seizure susceptibility were tested in the PTZ model. Ro 25-6981 was effective against PTZ-induced seizures in infantile rats, specifically suppressing the tonic phase of the generalized tonic-clonic seizures, but it failed in juveniles. Neither sensorimotor development nor cognitive abilities and emotionality were affected by early-life exposure to Ro 25-6981. Treatment cessation did not affect later seizure susceptibility. Our data are in line with the maturational gradient of the GluN2B-subunit of NMDA receptors and demonstrate developmental differences in the anti-seizure activity of the GluN2B-selective antagonist and its safety for the developing brain.

Department of Developmental Epileptology Institute of Physiology Czech Academy of Sciences 14220 Prague Czech Republic

Department of Rehabilitation and Sport Medicine 2nd Medical Faculty Charles University 15006 Prague Czech Republic

Zobrazit více v PubMed

Hernan A.E., Holmes G.L. Antiepileptic drug treatment strategies in neonatal epilepsy. Prog. Brain Res. 2016;226:179–193. doi: 10.1016/bs.pbr.2016.03.011. PubMed DOI

Meador K.J. Neurodevelopmental effects of antiepileptic drugs. Curr. Neurol. Neurosci. Rep. 2002;2:373–378. doi: 10.1007/s11910-002-0013-6. PubMed DOI

Kim J.S., Kondratyev A., Tomita Y., Gale K. Neurodevelopmental impact of antiepileptic drugs and seizures in the immature brain. Epilepsia. 2007;48((Suppl. 5)):19–26. doi: 10.1111/j.1528-1167.2007.01285.x. PubMed DOI

Marsh E.D., Brooks-Kayal A.R., Porter B.E. Seizures and antiepileptic drugs: Does exposure alter normal brain development? Epilepsia. 2006;47:1999–2010. doi: 10.1111/j.1528-1167.2006.00894.x. PubMed DOI

Holmes G.L. Drug Treatment of Epilepsy Neuropsychiatric Comorbidities in Children. Paediatr. Drugs. 2021;23:55–73. doi: 10.1007/s40272-020-00428-w. PubMed DOI PMC

Wasterlain C.G., Gloss D.S., Niquet J., Wasterlain A.S. Epileptogenesis in the developing brain. Handb. Clin. Neurol. 2013;111:427–439. doi: 10.1016/b978-0-444-52891-9.00046-4. PubMed DOI

Cull-Candy S., Brickley S., Farrant M. NMDA receptor subunits: Diversity, development and disease. Curr. Opin. Neurobiol. 2001;11:327–335. doi: 10.1016/S0959-4388(00)00215-4. PubMed DOI

Monyer H., Burnashev N., Laurie D.J., Sakmann B., Seeburg P.H. Developmental and regional expression in the rat brain and functional properties of four NMDA receptors. Neuron. 1994;12:529–540. doi: 10.1016/0896-6273(94)90210-0. PubMed DOI

Szczurowska E., Mareš P. NMDA and AMPA receptors: Development and status epilepticus. Physiol. Res. 2013;62((Suppl. 1)):S21–S38. doi: 10.33549/physiolres.932662. PubMed DOI

Gallagher M.J., Huang H., Pritchett D.B., Lynch D.R. Interactions between ifenprodil and the NR2B subunit of the N-methyl-D-aspartate receptor. J. Biol. Chem. 1996;271:9603–9611. doi: 10.1074/jbc.271.16.9603. PubMed DOI

Williams K. Ifenprodil discriminates subtypes of the N-methyl-D-aspartate receptor: Selectivity and mechanisms at recombinant heteromeric receptors. Mol. Pharmacol. 1993;44:851–859. PubMed

McAllister K.H. N-methyl-D-aspartate receptor antagonists and channel blockers have different effects upon a spinal seizure model in mice. Eur. J. Pharmacol. 1992;211:105–108. doi: 10.1016/0014-2999(92)90269-A. PubMed DOI

Wang X.M., Bausch S.B. Effects of distinct classes of N-methyl-D-aspartate receptor antagonists on seizures, axonal sprouting and neuronal loss in vitro: Suppression by NR2B-selective antagonists. Neuropharmacology. 2004;47:1008–1020. doi: 10.1016/j.neuropharm.2004.07.036. PubMed DOI

Yen W., Williamson J., Bertram E.H., Kapur J. A comparison of three NMDA receptor antagonists in the treatment of prolonged status epilepticus. Epilepsy Res. 2004;59:43–50. doi: 10.1016/j.eplepsyres.2004.03.004. PubMed DOI PMC

Yourick D.L., Repasi R.T., Rittase W.B., Staten L.D., Meyerhoff J.L. Ifenprodil and arcaine alter amygdala-kindling development. Eur. J. Pharmacol. 1999;371:147–152. doi: 10.1016/S0014-2999(99)00183-1. PubMed DOI

Mareš P. Age and activation determines the anticonvulsant effect of ifenprodil in rats. Naunyn Schmiedebergs Arch. Pharmacol. 2014;387:753–761. doi: 10.1007/s00210-014-0987-z. PubMed DOI

Mareš P., Mikulecká A. Different effects of two N-methyl-D-aspartate receptor antagonists on seizures, spontaneous behavior, and motor performance in immature rats. Epilepsy Behav. 2009;14:32–39. doi: 10.1016/j.yebeh.2008.08.013. PubMed DOI

Mareš P., Tsenov G., Kubová H. Anticonvulsant Action of GluN2A-Preferring Antagonist PEAQX in Developing Rats. Pharmaceutics. 2021;13:415. doi: 10.3390/pharmaceutics13030415. PubMed DOI PMC

Fischer G., Mutel V., Trube G., Malherbe P., Kew J.N., Mohacsi E., Heitz M.P., Kemp J.A. Ro 25-6981, a highly potent and selective blocker of N-methyl-D-aspartate receptors containing the NR2B subunit. Characterization in vitro. J. Pharmacol. Exp. Ther. 1997;283:1285–1292. PubMed

Lynch D.R., Shim S.S., Seifert K.M., Kurapathi S., Mutel V., Gallagher M.J., Guttmann R.P. Pharmacological characterization of interactions of RO 25-6981 with the NR2B (epsilon2) subunit. Eur. J. Pharmacol. 2001;416:185–195. doi: 10.1016/S0014-2999(01)00868-8. PubMed DOI

Dong F., Yao R., Yu H., Liu Y. Neuroprotection of Ro 25-6981 Against Ischemia/Reperfusion-Induced Brain Injury via Inhibition of Autophagy. Cell. Mol. Neurobiol. 2017;37:743–752. doi: 10.1007/s10571-016-0409-5. PubMed DOI PMC

Yellepeddi V.K., Zhudeva M.Y., Movahedi F., Vo A., Phan J., Kirsh R.D., Rawlins D.B., Talbot J.N. Biopharmaceutical Characterization and Oral Efficacy of a New Rapid Acting Antidepressant Ro 25-6981. J. Pharm. Sci. 2018;107:2472–2478. doi: 10.1016/j.xphs.2018.05.005. PubMed DOI

Boyce S., Wyatt A., Webb J.K., O’Donnell R., Mason G., Rigby M., Sirinathsinghji D., Hill R.G., Rupniak N.M. Selective NMDA NR2B antagonists induce antinociception without motor dysfunction: Correlation with restricted localisation of NR2B subunit in dorsal horn. Neuropharmacology. 1999;38:611–623. doi: 10.1016/S0028-3908(98)00218-4. PubMed DOI

Velíšek L., Kubová H., Pohl M., Staňková L., Mareš P., Schickerová R. Pentylenetetrazol-induced seizures in rats: An ontogenetic study. Naunyn Schmiedebergs Arch. Pharmacol. 1992;346:588–591. doi: 10.1007/BF00169017. PubMed DOI

de Casrilevitz M., Engelhardt E., Esberard C.A. Maturation of convulsogenic activity induced by leptazol in the albino rat. Br. J. Pharmacol. 1971;42:31–42. doi: 10.1111/j.1476-5381.1971.tb07084.x. PubMed DOI PMC

Brožičková C., Mikulecká A., Otahál J. Effect of 7-Nitroindazole, a Neuronal Nitric Oxide Synthase Inhibitor, on Behavioral and Physiological Parameters. Physiol. Res. 2014;63:637–648. doi: 10.33549/physiolres.932781. PubMed DOI

Mikulecká A., Mareš P. NMDA receptor antagonists impair motor performance in immature rats. Psychopharmacology. 2002;162:364–372. doi: 10.1007/s00213-002-1122-2. PubMed DOI

Hrncić D., Rasić-Marković A., Susić V., Djurić D., Stanojlović O. Influence of NR2B-selective NMDA antagonist on lindane-induced seizures in rats. Pharmacology. 2009;84:234–239. doi: 10.1159/000238055. PubMed DOI

Burket J.A., Cannon W.R., Jacome L.F., Deutsch S.I. MK-801, a noncompetitive NMDA receptor antagonist, elicits circling behavior in the genetically inbred Balb/c mouse strain. Brain Res. Bull. 2010;83:337–339. doi: 10.1016/j.brainresbull.2010.08.014. PubMed DOI

Tsuda M., Suzuki T., Misawa M. Age-related decrease in the antiseizure effect of ifenprodil against pentylenetetrazole in mice. Brain Res. Dev. Brain Res. 1997;104:201–204. doi: 10.1016/S0165-3806(97)00140-5. PubMed DOI

Szczurowska E., Mareš P. Different action of a specific NR2B/NMDA antagonist Ro 25-6981 on cortical evoked potentials and epileptic afterdischarges in immature rats. Brain Res. Bull. 2015;111:1–8. doi: 10.1016/j.brainresbull.2014.11.001. PubMed DOI

Kolarová A., Ringer R., Tauber M.G., Leib S.L. Blockade of NMDA receptor subtype NR2B prevents seizures but not apoptosis of dentate gyrus neurons in bacterial meningitis in infant rats. BMC Neurosci. 2003;4:21. doi: 10.1186/1471-2202-4-21. PubMed DOI PMC

Loftis J.M., Janowsky A. The N-methyl-D-aspartate receptor subunit NR2B: Localization, functional properties, regulation, and clinical implications. Pharmacol. Ther. 2003;97:55–85. doi: 10.1016/S0163-7258(02)00302-9. PubMed DOI

Klinger W. Developmental pharmacology and toxicology: Biotransformation of drugs and other xenobiotics during postnatal development. Eur. J. Drug Metab. Pharm. 2005;30:3–17. doi: 10.1007/BF03226403. PubMed DOI

Kato R., Takanaka A. Effect of phenobarbital on electron transport system, oxidation and reduction of drugs in liver microsomes of rats of different age. J. Biochem. 1968;63:406–408. PubMed

Schmitt G., Parrott N., Prinssen E., Barrow P. The great barrier belief: The blood-brain barrier and considerations for juvenile toxicity studies. Reprod. Toxicol. 2017;72:129–135. doi: 10.1016/j.reprotox.2017.06.043. PubMed DOI

Soares R.V., Do T.M., Mabondzo A., Pons G., Chhun S. Ontogeny of ABC and SLC transporters in the microvessels of developing rat brain. Fundam. Clin. Pharmacol. 2016;30:107–116. doi: 10.1111/fcp.12175. PubMed DOI

Burket J.A., Mastropaolo J., Rosse R.B., Katz E.U., Deutsch S.I. NMDA NR2B subtype-selective receptor antagonists fail to antagonize electrically-precipitated seizures and elicit popping in mice. Eur. Neuropsychopharmacol. 2010;20:207–210. doi: 10.1016/j.euroneuro.2009.11.008. PubMed DOI

Browning R.A., Nelson D.K. Modification of electroshock and pentylenetetrazol seizure patterns in rats after precollicular transections. Exp. Neurol. 1986;93:546–556. doi: 10.1016/0014-4886(86)90174-3. PubMed DOI

Mareš P., Kubová H. What is the role of neurotransmitter systems in cortical seizures? Physiol. Res. 2008;57((Suppl. 3)):S111–S120. doi: 10.33549/physiolres.931605. PubMed DOI

Löscher W. Animal Models of Seizures and Epilepsy: Past, Present, and Future Role for the Discovery of Antiseizure Drugs. Neurochem. Res. 2017;42:1873–1888. doi: 10.1007/s11064-017-2222-z. PubMed DOI

Kubová H., Mareš P. Partial agonist of benzodiazepine receptors Ro 19-8022 elicits withdrawal symptoms after short-term administration in immature rats. Physiol. Res. 2012;61:319–323. doi: 10.33549/physiolres.932343. PubMed DOI

Chazot P.L. The NMDA receptor NR2B subunit: A valid therapeutic target for multiple CNS pathologies. Curr. Med. Chem. 2004;11:389–396. doi: 10.2174/0929867043456061. PubMed DOI

Kumar A. NMDA Receptor Function During Senescence: Implication on Cognitive Performance. Front. Neurosci. Switz. 2015;9 doi: 10.3389/fnins.2015.00473. PubMed DOI PMC

Dalton G.L., Wu D.C., Wang Y.T., Floresco S.B., Phillips A.G. NMDA GluN2A and GluN2B receptors play separate roles in the induction of LTP and LTD in the amygdala and in the acquisition and extinction of conditioned fear. Neuropharmacology. 2012;62:797–806. doi: 10.1016/j.neuropharm.2011.09.001. PubMed DOI

Radiske A., Gonzalez M.C., Noga D.A., Rossato J.I., Bevilaqua L.R.M., Cammarota M. GluN2B and GluN2A-containing NMDAR are differentially involved in extinction memory destabilization and restabilization during reconsolidation. Sci. Rep. 2021;11:186. doi: 10.1038/s41598-020-80674-7. PubMed DOI PMC

Kutsuwada T., Sakimura K., Manabe T., Takayama C., Katakura N., Kushiya E., Natsume R., Watanabe M., Inoue Y., Yagi T., et al. Impairment of suckling response, trigeminal neuronal pattern formation, and hippocampal LTD in NMDA receptor epsilon 2 subunit mutant mice. Neuron. 1996;16:333–344. doi: 10.1016/S0896-6273(00)80051-3. PubMed DOI

Bhardwaj S.K., Forcelli P.A., Palchik G., Gale K., Srivastava L.K., Kondratyev A. Neonatal exposure to phenobarbital potentiates schizophrenia-like behavioral outcomes in the rat. Neuropharmacology. 2012;62:2337–2345. doi: 10.1016/j.neuropharm.2012.02.001. PubMed DOI PMC

Mikulecká A., Šubrt M., Pařízková M., Mareš P., Kubová H. Consequences of early postnatal benzodiazepines exposure in rats. II. Social behavior. Front. Behav. Neurosci. 2014;8:169. doi: 10.3389/fnbeh.2014.00169. PubMed DOI PMC

Mikulecká A., Šubrt M., Stuchlík A., Kubová H. Consequences of early postnatal benzodiazepines exposure in rats. I. Cognitive-like behavior. Front. Behav. Neurosci. 2014;8:101. doi: 10.3389/fnbeh.2014.00101. PubMed DOI PMC

Furuie H., Yamada K., Ichitani Y. Differential effects of N-methyl-D-aspartate receptor blockade during the second and third postnatal weeks on spatial working and reference memory in adult rats. Brain Res. 2019;1721:146339. doi: 10.1016/j.brainres.2019.146339. PubMed DOI

McLamb R.L., Williams L.R., Nanry K.P., Wilson W.A., Tilson H.A. MK-801 impedes the acquisition of a spatial memory task in rats. Pharmacol. Biochem. Behav. 1990;37:41–45. doi: 10.1016/0091-3057(90)90038-J. PubMed DOI

Clancy B., Darlington R.B., Finlay B.L. Translating developmental time across mammalian species. Neuroscience. 2001;105:7–17. doi: 10.1016/S0306-4522(01)00171-3. PubMed DOI

Workman A.D., Charvet C.J., Clancy B., Darlington R.B., Finlay B.L. Modeling transformations of neurodevelopmental sequences across mammalian species. J. Neurosci. 2013;33:7368–7383. doi: 10.1523/JNEUROSCI.5746-12.2013. PubMed DOI PMC

Ikonomidou C., Bosch F., Miksa M., Bittigau P., Vöckler J., Dikranian K., Tenkova T.I., Stefovska V., Turski L., Olney J.W. Blockade of NMDA receptors and apoptotic neurodegeneration in the developing brain. Science. 1999;283:70–74. doi: 10.1126/science.283.5398.70. PubMed DOI

Kawabe K. Effects of chronic forced-swim stress on behavioral properties in rats with neonatal repeated MK-801 treatment. Pharmacol. Biochem. Behav. 2017;159:48–54. doi: 10.1016/j.pbb.2017.06.009. PubMed DOI

Uehara T., Sumiyoshi T., Seo T., Itoh H., Matsuoka T., Suzuki M., Kurachi M. Long-term effects of neonatal MK-801 treatment on prepulse inhibition in young adult rats. Psychopharmacology. 2009;206:623–630. doi: 10.1007/s00213-009-1527-2. PubMed DOI

Lima-Ojeda J.M., Vogt M.A., Pfeiffer N., Dormann C., Köhr G., Sprengel R., Gass P., Inta D. Pharmacological blockade of GluN2B-containing NMDA receptors induces antidepressant-like effects lacking psychotomimetic action and neurotoxicity in the perinatal and adult rodent brain. Prog. Neuropsychopharmacol. Biol. Psychiatry. 2013;45:28–33. doi: 10.1016/j.pnpbp.2013.04.017. PubMed DOI

Mysliveček J., Hassmannová J. Step-down passive avoidance in the rat ontogeny. Acta Neurobiol. Exp. 1991;51:89–96. PubMed

Prut L., Belzung C. The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: A review. Eur. J. Pharmacol. 2003;463:3–33. doi: 10.1016/S0014-2999(03)01272-X. PubMed DOI

Crawley J.N. Behavioral phenotyping strategies for mutant mice. Neuron. 2008;57:809–818. doi: 10.1016/j.neuron.2008.03.001. PubMed DOI

Altman J., Sudarshan K. Postnatal development of locomotion in the laboratory rat. Anim. Behav. 1975;23:896–920. doi: 10.1016/0003-3472(75)90114-1. PubMed DOI

Geisler H.C., Westerga J., Gramsbergen A. Development of posture in the rat. Acta Neurobiol. Exp. 1993;53:517–523. PubMed

Gramsbergen A. Posture and locomotion in the rat: Independent or interdependent development? Neurosci. Biobehav. Rev. 1998;22:547–553. doi: 10.1016/S0149-7634(97)00043-2. PubMed DOI

Jänicke B., Schulze G., Coper H. Motor performance achievements in rats of different ages. Exp. Gerontol. 1983;18:393–407. doi: 10.1016/0531-5565(83)90018-9. PubMed DOI

Epilepsy Research in the Institute of Physiology of the Czech Academy of Sciences in Prague

The outcome of early life status epilepticus-lessons from laboratory animals