K. Hyrc et al., IONIZED INTRACELLULAR CALCIUM-CONCENTRATION PREDICTS EXCITOTOXIC NEURONAL DEATH - OBSERVATIONS WITH LOW-AFFINITY FLUORESCENT CALCIUM INDICATORS, The Journal of neuroscience, 17(17), 1997, pp. 6669-6677
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.