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| TR |
TITLE |
AUTHORS |
KEYWORDS |
MATERIALS & METHODS
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MICROSCOPIC
TECHNIQUES |
SPECIES |
MORPHOLOGY |
CELL
LINE |
| 604 |
Ionized
Intracellular Calcium Concentration Predicts Excitotoxic
Neuronal Death: Observations with Low-Affinity Fluorescent
Calcium Indicators |
Krzysztof
Hyrc, Shawn D. Handran, Steven M. Rothman, and Mark P. Goldberg |
AMPA,
calcium, excitotoxicity, fura-2, glutamate, kainate, NMDA, videomicroscopy |
Cells were plated (0.5 hemisphere
per dish) onto glass-bottom 35 mm dishes (Mattek, Ashland, MA) previously coated
with a mixture of poly-D-lysine (0.5 mg/ml) and laminin (4 mg/ml).
|
videomicroscopy |
mouse |
neoronal |
neocortical
neurons |
| Abstract |
| |
Cytosolic calcium ([Ca21]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 [Ca21]i also
has been implicated as a cause of acute neuronal injury, although measurement of [Ca21]i in
living neurons by fluorescent calcium indicators has not consistently demonstrated a correlation
between [Ca21]i and the likelihood of neuronal death after a variety of potentially lethal insults.
Using fluorescence videomicroscopy and microinjected calcium indicators, we measured [Ca21]i
in cultured cortical neurons during intense activation with either NMDA (300 mM) or AMPA (450
mM). 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
[Ca21]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 [Ca21]i levels .5 mM during lethal NMDA exposure, but [Ca21]i levels were ,1.5 mM during
nonlethal activation of AMPA receptors or voltage-gated calcium channels. Fura-2 reported [Ca21]i
responses during brief exposure to glutamate, NMDA, AMPA, kainate, and elevated extracellular
K1 between 0.5 and 1 mM. With the use of BTC, only NMDA and glutamate exposures resulted in
micromolar [Ca21]i levels. Neurotoxic glutamate receptor activation is associated with sustained,
micromolar [Ca21]i elevation. The widely used calcium indicator fura-2 selectively underestimates
[Ca21]i , depending on the route of entry, even at levels that appear to be within its range
of detection.. |
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