Ionized intracellular calcium concentration predicts excitotoxic neuronal death: Observations with low-affinity fluorescent calcium indicators

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Abstract

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 languageEnglish (US)
Pages (from-to)6669-6677
Number of pages9
JournalJournal of Neuroscience
Volume17
Issue number17
StatePublished - 1997

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N-Methylaspartate
alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid
Calcium
Fura-2
Neurons
Glutamic Acid
Video Microscopy
AMPA Receptors
Kainic Acid
Glutamate Receptors
Calcium Channels
Signal Transduction
Fluorescence
Wounds and Injuries
Genes

Keywords

  • AMPA
  • Calcium
  • excitotoxicity
  • Fura- 2
  • Glutamate
  • Kainate
  • NMDA
  • Videomicroscopy

ASJC Scopus subject areas

  • Neuroscience(all)

Cite this

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title = "Ionized intracellular calcium concentration predicts excitotoxic neuronal death: Observations with low-affinity fluorescent calcium indicators",
abstract = "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.",
keywords = "AMPA, Calcium, excitotoxicity, Fura- 2, Glutamate, Kainate, NMDA, Videomicroscopy",
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volume = "17",
pages = "6669--6677",
journal = "Journal of Neuroscience",
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TY - JOUR

T1 - Ionized intracellular calcium concentration predicts excitotoxic neuronal death

T2 - Observations with low-affinity fluorescent calcium indicators

AU - Goldberg, Mark P.

PY - 1997

Y1 - 1997

N2 - 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.

AB - 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.

KW - AMPA

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KW - excitotoxicity

KW - Fura- 2

KW - Glutamate

KW - Kainate

KW - NMDA

KW - Videomicroscopy

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