Calcium green-5N, a novel fluorescent probe for monitoring high intracellular free Ca2+ concentrations associated with glutamate excitotoxicity in cultured rat brain neurons

We describe here the properties of calcium green-5N (CG5N), a novel Ca(2+)-sensitive fluorescent probe. CG5N binds Ca2+ with an affinity of 4.29 +/- 0.67 microM. Ca2+ binding is associated with a 14.7-fold increase in fluorescence. Mg2+ binds to CG5N with an affinity of 5 mM but produces a very small change in fluorescence. Using CG5N [Ca2+]i was recorded in single cultured neurons from fetal rat forebrain. Glutamate together with glycine increased [Ca2+]i by 1.17 +/- 0.15 microM. Pretreating cells with 5 mM potassium cyanide significantly potentiated the response to glutamate, resulting in a mean increase of 8.8 +/- 2 microM. These results demonstrate that CG5N is a useful indicator for measuring very large changes in [Ca2+]i in neurons.