Fluorescent detection of Ca2+-permeable AMPA/kainate receptor activation in murine neocortical neurons

Agonist stimulated Co2+ uptake is used to identify neurons expressing Ca2+-permeable AMPA/kaniate receptors (Ca-A/K receptors). Based on the selective permeability of Co2+ through these receptors we have developed a fluorometricmethod that utilizes the fluorescence laser imaging plate reader (FLIPR). We used the dye calcein whose fluorescence is stoichiometrically quenched by Co2+, while being only minimally affected by variations in intracellular Ca2+. Application of AMPA in the presence of cyclothiazide led to a concentration-dependent increase in Co2+ uptake in the neocortical neurons. Similar concentration-dependent increments in Co2+ uptake were observed with kainate treatment. 2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX), an AMPA/kainate receptor antagonist. blocked the AMPA-induced Co2+ influx. The fluorometric method described affords a rapid, high throughput and quantitative procedure for investigation of Ca-A/K receptors in intact neurons. (C) 2003 Elsevier Ireland Ltd. All rights reserved.