IONIZED INTRACELLULAR CALCIUM-CONCENTRATION PREDICTS EXCITOTOXIC NEURONAL DEATH - OBSERVATIONS WITH LOW-AFFINITY FLUORESCENT CALCIUM INDICATORS

Cytosolic calcium ([Ca2+](i)) is an important mediator of neuronal sig nal transduction, partic pating in diverse biochemical reactions that elicit changes in synaptic efficacy, metabolic rate, and gene transcri ption. Excessive [Ca2+](i) also has been implicated as a cause of acut e neuronal injury, although measurement of [Ca2+](i) in living neurons by fluorescent calcium indicators has not consistently demonstrated a correlation between [Ca2+](i) and the likelihood of neuronal death af ter a variety of potentially lethal insults. Using fluorescence videom icroscopy and microinjected calcium indicators, we measured [Ca2+](i) in cultured cortical neurons during intense activation with either NMD A (300 mu M) or AMPA (450 mu M). At these concentrations NMDA killed > 80% of the cultured neurons by the next day, whereas neuronal death fr om AMPA was <20%, Using the conventional calcium indicator, fura-2/AM, we estimated [Ca2+](i) elevations to be similar to 300-400 nM during exposure to either glutamate agonist. In contrast, indicators with low er affinity for calcium, benzothiazole coumarin (ETC), and fura-2/dext ran reported [Ca2+](i) levels >5 mu M during lethal NMDA exposure, but [Ca2+](i) levels were <1.5 mu M during nonlethal activation of AMPA r eceptors or voltage-gated calcium channels. Fura-2 reported [Ca2+](i) responses during brief exposure to glutamate, NMDA, AMPA, kainate, and elevated extracellular K+ between 0.5 and 1 mu M. With the use of ETC , only NMDA and glutamate exposures resulted in micromolar [Ca2+](i) l evels, Neurotoxic glutamate receptor activation is associated with sus tained, micromolar [Ca2+](i) elevation. The widely used calcium indica tor fura-2 selectively underestimates [Ca2+](i), depending on the rout e of entry, even at levels that appear to be within its range of detec tion.