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 signal transduction, participating in diverse biochemical reactions that elicit changes in synaptic efficacy, metabolic rate, and gene transcription. Excessive [Ca2+]i also has been implicated as a cause of acute 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 after a variety of potentially lethal insults. Using fluorescence videomicroscopy and microinjected calcium indicators, we measured [Ca2+]i in cultured cortical neurons during intense activation with either NMDA (300 microM) or AMPA (450 microM). At these concentrations NMDA killed >80% of the cultured neurons by the next day, whereas neuronal death from AMPA was <20%. Using the conventional calcium indicator, fura-2/AM, we estimated [Ca2+]i elevations to be approximately 300-400 nM during exposure to either glutamate agonist. In contrast, indicators with lower affinity for calcium, benzothiazole coumarin (BTC), and fura-2/dextran reported [Ca2+]i levels >5 microM during lethal NMDA exposure, but [Ca2+]i levels were <1.5 microM during nonlethal activation of AMPA receptors 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 microM. With the use of BTC, only NMDA and glutamate exposures resulted in micromolar [Ca2+]i levels. Neurotoxic glutamate receptor activation is associated with sustained, micromolar [Ca2+]i elevation. The widely used calcium indicator fura-2 selectively underestimates [Ca2+]i, depending on the route of entry, even at levels that appear to be within its range of detection.
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Center for the Study of Nervous System Injury, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Handran
S D
SD
Rothman
S M
SM
Goldberg
M P
MP
D000818:Animals; D052160:Benzothiazoles; D002118:Calcium / Q000378:metabolism*; D016923:Cell Death; D003374:Coumarins; D003911:Dextrans; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D005456:Fluorescent Dyes; D005544:Forecasting; D016257:Fura-2 / Q000031:analogs & derivatives; D007425:Intracellular Membranes / Q000378:metabolism*; D007477:Ions; D051379:Mice; D016202:N-Methylaspartate / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects*; D009498:Neurotoxins / Q000494:pharmacology*; D009994:Osmolar Concentration; D013844:Thiazoles; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000494:pharmacology
D052160:Benzothiazoles; D003374:Coumarins; D003911:Dextrans; D018690:Excitatory Amino Acid Agonists; D005456:Fluorescent Dyes; D007477:Ions; D009498:Neurotoxins; D013844:Thiazoles; C104515:benzothiazole coumarin; C049925:fura-2-am; D016202:N-Methylaspartate; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D002118:Calcium; D016257:Fura-2