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Mitochondrial Calcium Waves by Electrical Stimulation in Cultured Hippocampal Neurons

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Abstract

Mitochondria are critical to cellular Ca2+ homeostasis via the sequestering of cytosolic Ca2+ in the mitochondrial matrix. Mitochondrial Ca2+ buffering regulates neuronal activity and neuronal death by shaping cytosolic and presynaptic Ca2+ or controlling energy metabolism. Dysfunction in mitochondrial Ca2+ buffering has been implicated in psychological and neurological disorders. Ca2+ wave propagation refers to the spreading of Ca2+ for buffering and maintaining the associated rise in Ca2+ concentration. We investigated mitochondrial Ca2+ waves in hippocampal neurons using genetically encoded Ca2+ indicators. Neurons transfected with mito-GCaMP5G, mito-RCaMP1h, and CEPIA3mt exhibited evidence of mitochondrial Ca2+ waves with electrical stimulation. These waves were observed with 200 action potentials at 40 Hz or 20 Hz but not with lower frequencies or fewer action potentials. The application of inhibitors of mitochondrial calcium uniporter and oxidative phosphorylation suppressed mitochondrial Ca2+ waves. However, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors and N-methyl-d-aspartate receptor blockade had no effect on mitochondrial Ca2+ wave were propagation. The Ca2+ waves were not observed in endoplasmic reticula, presynaptic terminals, or cytosol in association with electrical stimulation of 200 action potentials at 40 Hz. These results offer novel insights into the mechanisms underlying mitochondrial Ca2+ buffering and the molecular basis of mitochondrial Ca2+ waves in neurons in response to electrical stimulation.

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Data Availability

The data that support the findings of the study are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank Drs. Franck Polleux (mito-GCaMP5G, mito-RCaMP1h, and vGlut1-GCaMP5G), Timothy Ryan (ER-GCaMP6-150), Masamitsu Iino (CEPIA3mt), Justin Taraska (VAMP2-pHuji), and Michael Davidson (DsRed2) for providing plasmids.

Funding

This work was supported by the Korean government (MSIT) (No. 2020R1C1C1008852) and the Korean Ministry of Environment under the ‘Environmental Health R&D Program’ (2021003310005).

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Author notes

  1. Yunkyung Eom and Sung Rae Kim are contributed equally to this work.

Authors and Affiliations

  1. College of Pharmacy, Chung-Ang University, Seoul, 06974, Republic of Korea

    Yunkyung Eom, Sung Rae Kim, Yeong-Kyeong Kim & Sung Hoon Lee

  2. Brain Research Core Facilities of Korea Brain Research Institute (KBRI), Daegu, 41068, Republic of Korea

    Sung Rae Kim

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  1. Yunkyung Eom
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Contributions

EK and SRK: Conceptualization, investigation, review, editing. YK: Investigation. SHL: Conceptualization, writing, supervision.

Corresponding author

Correspondence to Sung Hoon Lee.

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All procedures with animals were performed according to the guidelines of the Animal Care and Use Committee at Chung-Ang University (Approval No. 2017–00093) and according to the National Institutes of Health Guidelines for Laboratory Animal Care.

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Supplementary file1 Supplementary Movie 1 (Relate to Figure 1 and Supplementary Figure 2). Mitochondrial Ca2+ waves after electrical stimulation. Pseudocolor time-lapse imaging of mito-GCaMP5G–expressing neurons in association with various levels of electrical stimulation. Depiction of ROIs along mito-GCaMP5G–expressing neuronal processes (red, green, and blue). (A) 200 APs at 40 Hz. Sequential increases in mitochondrial Ca2+ from red to blue (AVI 402 KB)

Supplementary file2 Supplementary Movie 1 (Relate to Figure 1 and Supplementary Figure 2). Mitochondrial Ca2+ waves after electrical stimulation. Pseudocolor time-lapse imaging of mito-GCaMP5G–expressing neurons in association with various levels of electrical stimulation. Depiction of ROIs along mito-GCaMP5G–expressing neuronal processes (red, green, and blue). (B) Similar to A with a higher temporal resolution (5 Hz) (AVI 180 KB)

Supplementary file3 Supplementary Movie 1 (Relate to Figure 1 and Supplementary Figure 2). Mitochondrial Ca2+ waves after electrical stimulation. Pseudocolor time-lapse imaging of mito-GCaMP5G–expressing neurons in association with various levels of electrical stimulation. Depiction of ROIs along mito-GCaMP5G–expressing neuronal processes (red, green, and blue). (C) 200 APs at 20 Hz. Sequential increases in mitochondrial Ca2+ from red to blue (AVI 206 KB)

Supplementary file4 Supplementary Movie 1 (Relate to Figure 1 and Supplementary Figure 2). Mitochondrial Ca2+ waves after electrical stimulation. Pseudocolor time-lapse imaging of mito-GCaMP5G–expressing neurons in association with various levels of electrical stimulation. Depiction of ROIs along mito-GCaMP5G–expressing neuronal processes (red, green, and blue). (D) 200 APs at 10 Hz. Simultaneous mitochondrial Ca2+ increases from red to blue (AVI 178 KB)

Supplementary file5 Supplementary Movie 1 (Relate to Figure 1 and Supplementary Figure 2). Mitochondrial Ca2+ waves after electrical stimulation. Pseudocolor time-lapse imaging of mito-GCaMP5G–expressing neurons in association with various levels of electrical stimulation. Depiction of ROIs along mito-GCaMP5G–expressing neuronal processes (red, green, and blue). (E) 40 APs at 40 Hz. Simultaneous mitochondrial Ca2+ increases from red to blue. (AVI 340 KB)

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Supplementary file6 Supplementary Figure 1. Co-localization of GECIs of mitochondria with MitoTracker in hippocampal neurons. (A) Representative image of MitoTracker (red) and mito-GCaMP5G expression (green). (B) Representative image of mito-RCaMP1h expression (red) and MitoTracker (green). (C) Representative image of MitoTracker (red) and CEPIA3mt expression (green). (TIF 1938 KB)

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Supplementary file7 Supplementary Figure 2. Mitochondrial Ca2+ waves with electrical stimulation at a higher temporal resolution. (A and B) Similar to Fig. 1A, B but with higher temporal resolution (5 Hz) (TIF 3540 KB)

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Supplementary file8 Supplementary Figure 3 (Related to Figure 5). Effect of AMPAR and NMDAR inhibition on mitochondrial Ca2+ waves. Application of electrical stimulation in mito-GCaMP5G transfected neurons in the presence of CNQX and APV. (A) Representative images of ROIs showing significant mitochondrial Ca2+ increases in response to electrical stimulation (left) and quantification of positive ROIs (right). Blockade of AMPAR and NMDAR reduced the number of ROIs. n = 8 independent cultures for Veh, n = 5 independent cultures for CNQX and APV treatment. Data are presented as the mean ± SEM. *p < 0.05 compared with Veh. Mann-Whitney U test. (B) Averaged normalized traces for ROIs showing mitochondrial Ca2+ increase in response to electrical stimulation with or without CNQX and APV. The average peak amplitude of mitochondrial Ca2+ increased in response to electrical stimulation (200 APs at 40 Hz). n = 664 responses from 8 independent cultures for Veh, n = 282 responses from 5 independent cultures for CNQX and APV treatment. Data are presented as the mean ± SEM. *p < 0.05 and ***p < 0.001 compared to Veh. Mann-Whitney U test. (TIF 3907 KB)

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Supplementary file9 Supplementary Figure 4. Intracellular Ca2+ changes associated with electrical stimulation. (A) Representative whole images of DsRed2-expressing and Fluo-4-AM–labeled neurons (left). The dashed box (white) was magnified, and the individual and merged images are shown (right). Depiction of ROIs along a single DsRed2-expressing neuronal process (crosses). (B) Normalized traces of ROIs from the Fluo-4-AM of panel A (left) and magnification of the dashed box (right). Simultaneous intracellular Ca2+ increases in all ROIs (TIF 3681 KB)

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Eom, Y., Kim, S.R., Kim, YK. et al. Mitochondrial Calcium Waves by Electrical Stimulation in Cultured Hippocampal Neurons. Mol Neurobiol 61, 3477–3489 (2024). https://doi.org/10.1007/s12035-023-03795-w

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