Abstract
Brain is one of the most energy-demanding organs. Energy in the form of ATP is produced in brain cells predominantly in oxidative phosphorylation coupled to mitochondrial respiration. Any alteration of the mitochondrial metabolism or prolonged ischemic or anoxic conditions can lead to serious neurological conditions, including neurodegenerative disorders. Assessment of mitochondrial metabolism is important for understanding physiological and pathological processes in the brain. Bioenergetics in central nervous system is dependent on multiple parameters including neuron–glia interactions and considering this, in vivo or ex vivo, the measurements of mitochondrial metabolism should also be complimenting the experiments on isolated mitochondria or cell cultures. To assess the mitochondrial function, there are several key bioenergetic parameters which indicate mitochondrial health. One of the major characteristics of mitochondria is the mitochondrial membrane potential (ΔΨm) which is used as a proton motive force for ATP production and generated by activity of the electron transport chain. Major donor of electrons for the mitochondrial respiratory chain is NADH. Here we demonstrate how to measure mitochondrial NADH/NAD(P)H autofluorescence and ΔΨm in acute brain slices in a time-dependent manner and provide information for the identification of NADH redox index, mitochondrial NADH pool, and the rate of NADH production in the Krebs cycle. Additionally, non-mitochondrial NADH/NADPH autofluorescence can signify the level of activity of the pentose phosphate pathway.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Abramov AY, Angelova PR (2019) Mitochondrial dysfunction and energy deprivation in the mechanism of neurodegeneration. Turkish J Biochem 44(6):723–729
Chance B (1954) Spectrophotometry of intracellular respiratory pigments. Science 120(3124):767–775
Chance B, Schoener B, Oshino R et al (1979) Oxidation-reduction ratio studies of mitochondria in freeze-trapped samples. NADH and flavoprotein fluorescence signals. J Biol Chem 254(11):4764–4771
Chance B, Thorell B (1959) Fluorescence measurements of mitochondrial pyridine nucleotide in aerobiosis and anaerobiosis. Nature 184:931–934
Bartolome F, Abramov AY (2015) Measurement of mitochondrial NADH and FAD autofluorescence in live cells. Methods Mol Biol 1264:263–270
Zamzami N, Marchetti P, Castedo M et al (1995) Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death. J Exp Med 182(2):367–377
Gasser UE, Hatten ME (1990) Neuron-glia interactions of rat hippocampal cells in vitro: glial-guided neuronal migration and neuronal regulation of glial differentiation. J Neurosci 10(4):1276–1285
Angelova PR, Abramov AY (2014) Interaction of neurons and astrocytes underlies the mechanism of Abeta-induced neurotoxicity. Biochem Soc Trans 42(5):1286–1290
Angelova PR, Barilani M, Lovejoy C et al (2018) Mitochondrial dysfunction in parkinsonian mesenchymal stem cells impairs differentiation. Redox Biol 14:474–484
Arber C, Angelova PR, Wiethoff S et al (2017) iPSC-derived neuronal models of PANK2-associated neurodegeneration reveal mitochondrial dysfunction contributing to early disease. PLoS One 12(9):e0184104
Kinghorn KJ, Castillo-Quan JI, Bartolome F et al (2015) Loss of PLA2G6 leads to elevated mitochondrial lipid peroxidation and mitochondrial dysfunction. Brain 138(Pt 7):1801–1816
Ludtmann MHR, Angelova PR, Horrocks MH et al (2018) α-Synuclein oligomers interact with ATP synthase and open the permeability transition pore in Parkinson’s disease. Nat Commun 9(1):2293
Tufi R, Gandhi S, de Castro IP et al (2014) Enhancing nucleotide metabolism protects against mitochondrial dysfunction and neurodegeneration in a PINK1 model of Parkinson’s disease. Nat Cell Biol 16(2):157–166
Abeti R, Parkinson MH, Hargreaves IP et al (2016) Mitochondrial energy imbalance and lipid peroxidation cause cell death in Friedreich’s ataxia. Cell Death Dis 7:e2237
Kovac S, Domijan AM, Walker MC et al (2012) Prolonged seizure activity impairs mitochondrial bioenergetics and induces cell death. J Cell Sci 125(Pt 7):1796–1806
Kovac S, Preza E, Houlden H et al (2019) Impaired bioenergetics in mutant mitochondrial DNA determines cell fate during seizure-like activity. Mol Neurobiol 56(1):321–334
Angelova P, Muller W (2006) Oxidative modulation of the transient potassium current IA by intracellular arachidonic acid in rat CA1 pyramidal neurons. Eur J Neurosci 23(9):2375–2384
Egorov AV, Angelova PR, Heinemann U et al (2003) Ca2+−independent muscarinic excitation of rat medial entorhinal cortex layer V neurons. Eur J Neurosci 18(12):3343–3351
Acknowledgments
This study was supported by the Russian Federation Government grant No. 075-15-2019-1877. VD kindly acknowledges the personal support from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No. 839888.
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Vinokurov, A.Y., Dremin, V.V., Piavchenko, G.A., Stelmashchuk, O.A., Angelova, P.R., Abramov, A.Y. (2021). Assessment of Mitochondrial Membrane Potential and NADH Redox State in Acute Brain Slices. In: Weissig, V., Edeas, M. (eds) Mitochondrial Medicine . Methods in Molecular Biology, vol 2276. Springer, New York, NY. https://doi.org/10.1007/978-1-0716-1266-8_14
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1266-8_14
Published:
Publisher Name: Springer, New York, NY
Print ISBN: 978-1-0716-1265-1
Online ISBN: 978-1-0716-1266-8
eBook Packages: Springer Protocols