Membrane Potential Measurements of Isolated Neurons Using a Voltage-Sensitive Dye

被引:27
作者
Fairless, Richard [1 ,2 ]
Beck, Andreas [3 ]
Kravchenko, Mykola [3 ]
Williams, Sarah K. [1 ,2 ]
Wissenbach, Ulrich [3 ]
Diem, Ricarda [1 ,2 ]
Cavalie, Adolfo [3 ]
机构
[1] Univ Clin Heidelberg, Dept Neurooncol, Heidelberg, Germany
[2] Univ Saarland, Dept Neurol, Homburg, Germany
[3] Univ Saarland, Dept Pharmacol & Toxicol, Homburg, Germany
来源
PLOS ONE | 2013年 / 8卷 / 03期
关键词
TRP CHANNELS; FLUORESCENCE; IDENTIFICATION; VALIDATION; INDICATORS; FLIPR; GABA;
D O I
10.1371/journal.pone.0058260
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
The ability to monitor changes in membrane potential is a useful tool for studying neuronal function, but there are only limited options available at present. Here, we have investigated the potential of a commercially available FLIPR membrane potential (FMP) dye, developed originally for high throughput screening using a plate reader, for imaging the membrane potential of cultured cells using an epifluorescence-based single cell imaging system. We found that the properties of the FMP dye make it highly suitable for such imaging since 1) its fluorescence displayed a high signal-to-noise ratio, 2) robust signals meant only minimal exposure times of around 5 ms were necessary, and 3) bidirectional changes in fluorescence were detectable resulting from hyper- or depolarising conditions, reaching equilibrium with a time constant of 4-8 s. Measurements were possible independently of whether membrane potential changes were induced by voltage clamping, or manipulating the ionic distribution of either Na+ or K+. Since FMP behaves as a charged molecule which accumulates in the cytosol, equations based on the Boltzmann distribution were developed determining that the apparent charge of FMP which represents a measure of the voltage sensitivity of the dye, is between -0.62 and -0.72. Finally, we demonstrated that FMP is suitable for use in a variety of neuronal cell types and detects membrane potential changes arising from spontaneous firing of action potentials and through stimulation with a variety of excitatory and inhibitory neurotransmitters.
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页数:14
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