title: Kainate-induced calcium overload of cortical neurons in vitro: Dependence on expression of AMPAR GluA2-subunit and down-regulation by subnanomolar ouabain.
abstract: Whereas kainate (KA)-induced neurodegeneration has been intensively investigated, the contribution of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) in neuronal Ca2+ overload ([Ca2+]i) is still controversial. Using Ca2+ imaging and patch-clamp techniques, we found different types of Ca2+ entry in cultured rat cortical neurons. The presence of Ca2+ in the extracellular solution was required to generate the [Ca2+]i responses to 30 μM N-methyl-d-aspartate (NMDA) or KA. The dynamics of NMDA-induced [Ca2+]i responses were fast, while KA-induced responses developed slower reaching high [Ca2+]i. Ifenprodil, a specific inhibitor of the GluN2B subunit of NMDARs, reduced NMDA-induced [Ca2+]i responses suggesting expression of GluN1/GluN2B receptors. Using IEM-1460, a selective blocker of Ca(2+)-permeable GluA2-subunit lacking AMPARs, we found three neuronal responses to KA: (i) IEM-1460 resistant neurons which are similar to pyramidal neurons expressing Ca(2+)-impermeable GluA2-rich AMPARs; (ii) Neurons exhibiting nearly complete block of both KA-induced currents and [Ca2+]i signals by IEM-1460 may represent interneurons expressing GluA2-lacking AMPARs and (iii) neurons with moderate sensitivity to IEM-1460. Ouabain at 1 nM prevented the neuronal Ca2+ overload induced by KA. The data suggest, that cultured rat cortical neurons maintain functional phenotypes of the adult brain cortex, and demonstrate the key contribution of the Na/K-ATPase in neuroprotection against KA excitotoxicity.
authors.affiliation: Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, Saint-Petersburg, Russian Federation.
authors.forename: Polina A
authors.initials: PA
authors.lastname: Abushik
authors.forename: Dmitry A
authors.initials: DA
authors.lastname: Sibarov
authors.forename: Misty J
authors.initials: MJ
authors.lastname: Eaton
authors.forename: Serguei N
authors.initials: SN
authors.lastname: Skatchkov
authors.forename: Sergei M
authors.initials: SM
authors.lastname: Antonov
mesh_terms: D000218:Adamantane / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000378:metabolism* / Q000473:pathology; D004195:Disease Models, Animal; D015536:Down-Regulation / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D066298:In Vitro Techniques; D007608:Kainic Acid / Q000009:adverse effects / Q000494:pharmacology*; D009410:Nerve Degeneration / Q000139:chemically induced / Q000378:metabolism / Q000473:pathology; D009474:Neurons / Q000378:metabolism* / Q000473:pathology; D010042:Ouabain / Q000494:pharmacology*; D018408:Patch-Clamp Techniques; D010880:Piperidines / Q000494:pharmacology; D011247:Pregnancy; D051381:Rats; D017208:Rats, Wistar; D018091:Receptors, AMPA / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; C110702:IEM 1460; D010880:Piperidines; D018091:Receptors, AMPA; D010042:Ouabain; C104722:glutamate receptor ionotropic, AMPA 2; D000218:Adamantane; C010739:ifenprodil; D007608:Kainic Acid; D002118:Calcium
keywords: Calcium; Cortical neurons; Excitotoxicity; Glutamate receptors; Ouabain; Subunit selective antagonists; Whole-cell currents