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 Ca²⁺-permeable GluA2-subunit lacking AMPARs, we found three neuronal responses to KA: (i) IEM-1460 resistant neurons which are similar to pyramidal neurons expressing Ca²⁺-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.