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

Agonist stimulated Co(2+) uptake is used to identify neurons expressing Ca(2+)-permeable AMPA/kaniate receptors (Ca-A/K receptors). Based on the selective permeability of Co(2+) through these receptors we have developed a fluorometric method that utilizes the fluorescence laser imaging plate reader (FLIPR). We used the dye calcein whose fluorescence is stoichiometrically quenched by Co(2+), while being only minimally affected by variations in intracellular Ca(2+). Application of AMPA in the presence of cyclothiazide led to a concentration-dependent increase in Co(2+) uptake in the neocortical neurons. Similar concentration-dependent increments in Co(2+) 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 Co(2+) influx. The fluorometric method described affords a rapid, high throughput and quantitative procedure for investigation of Ca-A/K receptors in intact neurons.