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Updated: Jan 8, 2026
Measurement of Total Calcium in Neurons by Electron Probe X-ray Microanalysis
Published on: November 20, 2013
Effects of Isoflurane Inhibition of Mitochondrial Complex I on Calcium Removal in Mouse Neuronal Cultures
Sangwook Jung1, Jan-Marino Ramirez2, Margaret M Sedensky3
1Sangwook Jung, Ph.D.: Norcliffe Foundation Center for Integrative Brain Research, Seattle Children's Research Institute, Seattle, Washington.
Background:
One mechanism proposed for anesthetic-induced neurotoxicity (AIN) is elevated neuronal calcium, leading to mitochondrial damage and caspase activation. Increased cytosolic calcium could arise from increased entry or decreased removal. The relative importance of these distinct mechanisms is unknown. Isoflurane inhibits mitochondrial complex I and reduces adenosine triphosphate (ATP) at presynaptic terminals leading to synaptic quiescence. The authors hypothesized that mitochondrial inhibition initiates calcium dysregulation in mouse wild-type and mitochondrial mutant neurons, leading to AIN.
Methods:
Presynaptic calcium levels were monitored using VGlut1-GCaMP5 or an endoplasmic reticulum-specific GCaMP6 during electrical stimulations of neuronal cultures. Cultures were stimulated in the presence of isoflurane and blockers or activators of calcium removal. Mitochondrial damage was monitored using MitoView (Biotium, USA). Cleaved caspase induction assessed AIN.
Results:
In the absence of isoflurane, neuronal stimulation transiently increased presynaptic calcium levels. Isoflurane increased the half-life for calcium decay in wild-type cultures (time [s], unexposed, 14 [10]; exposed, 160 [77]; P = 0.001). Maintaining ATP levels rescued the isoflurane-induced defective removal of calcium (time [s], 30 mM glucose, 16 [14]; n = 8; P = 0.001). Activation of sarcoplasmic endoplasmic reticulum calcium adenosine triphosphatase (SERCA) alleviated the isoflurane-induced defective removal of calcium (time [s], no SERCA activator, 159 [78]; SERCA activator, 36 [18]; P = 0.002). Similar results were seen for mutant cultures exposed to lower, but equipotent, concentrations of isoflurane. Isoflurane induced a SERCA-dependent decrease in uptake of MitoView and an increase in cleaved caspase in wild-type cultures.
Conclusions:
Isoflurane causes a failure of SERCA-dependent calcium removal by inhibition of mitochondrial production of ATP. The increase in intracellular calcium leads to early signs of cellular toxicity.
Insights
Anesthetic-induced neurotoxicity (AIN) involves elevated neuronal calcium. Isoflurane impairs calcium removal by inhibiting mitochondrial ATP production, leading to cellular damage.
Area of Science:
- Neuroscience
- Cellular Biology
- Anesthesiology
Background:
- Anesthetic-induced neurotoxicity (AIN) is linked to elevated neuronal calcium, mitochondrial damage, and caspase activation.
- Isoflurane inhibits mitochondrial complex I, reducing ATP and causing synaptic quiescence.
- The precise mechanisms of calcium dysregulation in AIN remain unclear.
Purpose of the Study:
- To investigate the role of mitochondrial inhibition in isoflurane-induced calcium dysregulation and neurotoxicity.
- To determine whether impaired calcium removal or increased calcium entry is the primary driver of AIN.
- To examine the impact of ATP levels and SERCA activity on isoflurane's effects on neuronal calcium.
Main Methods:
- Neuronal cultures (wildtype and mitochondrial mutants) were stimulated electrically in the presence of isoflurane.
- Presynaptic and endoplasmic reticulum calcium levels were monitored using genetically encoded calcium indicators (GCaMP5, GCaMP6).
- Mitochondrial damage (MitoViewTM uptake) and caspase activation were assessed to evaluate neurotoxicity.
Main Results:
- Isoflurane significantly prolonged calcium decay half-life, indicating impaired calcium removal.
- Restoring ATP levels or activating SERCA (sarcoplasmic endoplasmic reticulum calcium ATPase) rescued the defective calcium removal.
- Isoflurane induced mitochondrial damage and caspase activation in a SERCA-dependent manner.
Conclusions:
- Isoflurane causes impaired calcium removal by inhibiting mitochondrial ATP production, leading to SERCA dysfunction.
- Elevated intracellular calcium resulting from impaired removal contributes to early cellular toxicity.
- This study elucidates a key mechanism of anesthetic-induced neurotoxicity involving mitochondrial-ER calcium handling.
