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.
Dravid
Shashank M
SM
Department of Physiology and Pharmacology, The University of Georgia, College of Veterinary Medicine, Room 2223, Athens, GA 30602, USA .
Murray
Thomas F
TF
D000818:Animals; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods; D019579:Neocortex / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D009474:Neurons / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D018091:Receptors, AMPA / Q000032:analysis / Q000378:metabolism*; D018092:Kainic Acid Receptors / Q000032:analysis / Q000378:metabolism*; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000378:metabolism / Q000494:pharmacology
D002118:Calcium; D018091:Receptors, AMPA; D018092:Kainic Acid Receptors; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid