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Volume 17, Number 17,
Issue of September 1, 1997
pp. 6669-6677
Copyright ©1997 Society for Neuroscience
Ionized Intracellular Calcium Concentration Predicts Excitotoxic
Neuronal Death: Observations with Low-Affinity Fluorescent Calcium
Indicators
Received April 18, 1997; revised June 2, 1997; accepted June 12, 1997.
Krzysztof Hyrc1,
Shawn
D. Handran1,
Steven M. Rothman1, 2, and
Mark P. Goldberg1
1 Center for the Study of Nervous System Injury and
Departments of Neurology and Anatomy and Neurobiology, Washington
University School of Medicine, St. Louis, Missouri 63110, and
2 Department of Pediatric Neurology, St. Louis Children's
Hospital, St. Louis, Missouri 63110
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 µM) or AMPA (450 µM). 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 ~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 µM
during lethal NMDA exposure, but
[Ca2+]i levels were <1.5
µM 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 µM. 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.
Key words:
AMPA;
calcium;
excitotoxicity;
fura-2;
glutamate;
kainate;
NMDA;
videomicroscopy
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