Detection of de- and hyperpolarization of mitochondria of cultured astrocytes and neurons by the cationic fluorescent dye rhodamine 123 - PubMed (original) (raw)

Detection of de- and hyperpolarization of mitochondria of cultured astrocytes and neurons by the cationic fluorescent dye rhodamine 123

Stefan Kahlert et al. J Neurosci Methods. 2008.

Abstract

The mitochondrial potential is an essential regulator in cellular physiology and detection of this parameter in living cells is still under discussion. Here we present a protocol which allows the use of rhodamine 123 as a probe for quantifying the mitochondrial potential. To avoid dequenching artefacts the detection area is limited to the area above the nucleus. In co-cultured rat hippocampal astrocytes and neurons, we analysed the mitochondrial accumulation of the cationic fluorescent dye rhodamine 123 (Rh123). Application of the uncoupler carbonyl cyanide 4-(trifluoro-methoxy)phenylhydrazone (FCCP, 4 micromol/L) together with the ATP-synthase inhibitor oligomycin (Oli, 10 micromol/L) induced an immediate fluorescence increase of Rh123-loaded mitochondria. This effect is due to the well-known fluorescence dequenching caused by the reduction in concentration of Rh123 in the mitochondria after depolarization. However, above the nucleus an increase in fluorescence was registered. Due to the absence of mitochondria in the area above the nucleus this fluorescence increase is most likely caused by the Rh123 release from mitochondria. Pre-treatment of cells with antimycin A abolished the response to FCCP/Oli. Furthermore, a 10-min exposure to 50 mmol/L K+, which causes a plasma membrane depolarization in neurons, did not significantly change the FCCP/Oli-mediated Rh123 release measured above the nucleus of neurons. However, application of 100 micromol/L glutamate enhanced the effect of FCCP/Oli both in astrocytes and neurons. This enhancement is interpreted as an increase in mitochondrial potential above the control potential. Thus, this Rh123 method described here allows a cell type-specific determination of changes of mitochondrial polarization.

PubMed Disclaimer

Cited by

Improved mitochondrial function in brain aging and Alzheimer disease - the new mechanism of action of the old metabolic enhancer piracetam.
Leuner K, Kurz C, Guidetti G, Orgogozo JM, Müller WE. Leuner K, et al. Front Neurosci. 2010 Sep 7;4:44. doi: 10.3389/fnins.2010.00044. eCollection 2010. Front Neurosci. 2010. PMID: 20877425 Free PMC article.
Physiological Concentration of Exogenous Lactate Reduces Antimycin A Triggered Oxidative Stress in Intestinal Epithelial Cell Line IPEC-1 and IPEC-J2 In Vitro.
Kahlert S, Junnikkala S, Renner L, Hynönen U, Hartig R, Nossol C, Barta-Böszörményi A, Dänicke S, Souffrant WB, Palva A, Rothkötter HJ, Kluess J. Kahlert S, et al. PLoS One. 2016 Apr 7;11(4):e0153135. doi: 10.1371/journal.pone.0153135. eCollection 2016. PLoS One. 2016. PMID: 27054581 Free PMC article.
Inhibition of the Mitochondrial Glutamate Carrier SLC25A22 in Astrocytes Leads to Intracellular Glutamate Accumulation.
Goubert E, Mircheva Y, Lasorsa FM, Melon C, Profilo E, Sutera J, Becq H, Palmieri F, Palmieri L, Aniksztejn L, Molinari F. Goubert E, et al. Front Cell Neurosci. 2017 May 31;11:149. doi: 10.3389/fncel.2017.00149. eCollection 2017. Front Cell Neurosci. 2017. PMID: 28620281 Free PMC article.
A series of carbazole cationic compounds with large two-photon absorption cross sections for imaging mitochondria in living cells with two-photon fluorescence microscopy.
Liu X, Sun Y, Zhang Y, Zhao N, Zhao H, Wang G, Yu X, Liu H. Liu X, et al. J Fluoresc. 2011 Mar;21(2):497-506. doi: 10.1007/s10895-010-0736-8. Epub 2010 Oct 16. J Fluoresc. 2011. PMID: 20953825
Glutamate transport decreases mitochondrial pH and modulates oxidative metabolism in astrocytes.
Azarias G, Perreten H, Lengacher S, Poburko D, Demaurex N, Magistretti PJ, Chatton JY. Azarias G, et al. J Neurosci. 2011 Mar 9;31(10):3550-9. doi: 10.1523/JNEUROSCI.4378-10.2011. J Neurosci. 2011. PMID: 21389211 Free PMC article.

Detection of de- and hyperpolarization of mitochondria of cultured astrocytes and neurons by the cationic fluorescent dye rhodamine 123 - PubMed (original) (raw)