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 micro- injected calcium indicators, we measured [Ca2+], 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) levers >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+] levels. Neurotoxic glutamate receptor activation s 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.
|Original language||English (US)|
|Number of pages||9|
|Journal||Journal of Neuroscience|
|State||Published - Sep 11 1997|
- Fura- 2
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