TY - JOUR
T1 - Fluorescent detection of Ca2+-permeable AMPA/kainate receptor activation in murine neocortical neurons
AU - Dravid, Shashank M.
AU - Murray, Thomas F.
N1 - Funding Information:
This work was supported by NIH grant GM63554 to TFM.
PY - 2003/11/20
Y1 - 2003/11/20
N2 - 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.
AB - 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.
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U2 - 10.1016/j.neulet.2003.07.017
DO - 10.1016/j.neulet.2003.07.017
M3 - Article
C2 - 14623127
AN - SCOPUS:0242499507
VL - 351
SP - 145
EP - 148
JO - Neuroscience Letters
JF - Neuroscience Letters
SN - 0304-3940
IS - 3
ER -