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Updated: Aug 13, 2026
Measurement of Total Calcium in Neurons by Electron Probe X-ray Microanalysis
Published on: November 20, 2013
Mitochondrial permeability transition and calcium dynamics in striatal neurons upon intense NMDA receptor activation
Conrad C Alano1, Gisela Beutner, Robert T Dirksen
1Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, New York 14642, USA.
Abstract:
Deregulation of the intracellular Ca2+ homeostasis by NMDA receptor activation leads to neuronal cell death. Induction of the mitochondrial permeability transition pore (MPT) by Ca2+ is a critical event in mediating cell death. In this study, we used fluorescent Ca2+ indicators to investigate the effect of high concentrations of NMDA on cytosolic and mitochondrial Ca2+ concentrations ([Ca2+]c and [Ca2+]m, respectively) in cultured striatal neurons. Exposure to NMDA resulted in an immediate, sustained increase in [Ca2+]c followed by a secondary increase in [Ca2+]c. This second increase of [Ca2+]c was prevented by pretreatment with N-methyl-valine-4-cyclosporin (NMV-Cys). Exposure of neurons to NMDA also resulted in an increase in [Ca2+]m that was followed by a precipitous decrease in the rhod-2 signal. This decrease followed the time frame of the secondary increase in [Ca2+]c. Preincubation of the neurons with NMV-Cys prevented the decrease in rhod-2 fluorescence. These dynamic changes in the rhod-2 signal and [Ca2+]m in response to NMDA were confirmed by using confocal microscopy. The presented results indicate that MPT can be detected in living neurons using fluorescent Ca2+ indicators, which would allow the study of the physiological role of MPT in cell death.
Insights
NMDA receptor activation disrupts calcium (Ca2+) homeostasis, leading to neuronal death. Cyclosporin derivative NMV-Cys prevents mitochondrial permeability transition pore (MPT) opening, suggesting a role in neuroprotection.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- NMDA receptor activation can cause neuronal cell death by disrupting intracellular calcium homeostasis.
- Mitochondrial permeability transition pore (MPT) induction by calcium is a key event in cell death pathways.
Purpose of the Study:
- To investigate the effects of high NMDA concentrations on cytosolic and mitochondrial calcium levels in cultured striatal neurons.
- To determine if NMDA-induced mitochondrial calcium changes are linked to MPT.
- To assess the neuroprotective potential of N-methyl-valine-4-cyclosporin (NMV-Cys) against NMDA-induced mitochondrial dysfunction.
Main Methods:
- Utilized fluorescent Ca2+ indicators to monitor cytosolic ([Ca2+]c) and mitochondrial ([Ca2+]m) calcium concentrations in real-time.
- Employed confocal microscopy to visualize dynamic changes in mitochondrial Ca2+ signals.
- Administered NMDA and NMV-Cys to cultured striatal neurons to observe their effects on Ca2+ homeostasis and MPT.
Main Results:
- NMDA exposure caused an immediate, sustained increase in [Ca2+]c, followed by a secondary rise.
- NMDA induced an increase in [Ca2+]m, preceding a significant decrease in mitochondrial Ca2+ signal (rhod-2 fluorescence).
- NMV-Cys pretreatment abolished the secondary [Ca2+]c increase and prevented the decrease in mitochondrial Ca2+ signal, indicating MPT inhibition.
Conclusions:
- MPT can be detected in living neurons using fluorescent Ca2+ indicators.
- NMDA-induced neuronal cell death involves MPT, which can be inhibited by NMV-Cys.
- Fluorescent Ca2+ indicators offer a valuable tool for studying the physiological role of MPT in neuronal cell death.
