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 fluorometric method 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.