title: Membrane potential measurements of isolated neurons using a voltage-sensitive dye.
abstract: 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.
authors.affiliation: Department of Neuro-Oncology, University Clinic Heidelberg, Heidelberg, Germany.
authors.forename: Richard
authors.initials: R
authors.lastname: Fairless
authors.forename: Andreas
authors.initials: A
authors.lastname: Beck
authors.forename: Mykola
authors.initials: M
authors.lastname: Kravchenko
authors.forename: Sarah K
authors.initials: SK
authors.lastname: Williams
authors.forename: Ulrich
authors.initials: U
authors.lastname: Wissenbach
authors.forename: Ricarda
authors.initials: R
authors.lastname: Diem
authors.forename: Adolfo
authors.initials: A
authors.lastname: Cavalié
mesh_terms: D000818:Animals; D002460:Cell Line; D058948:Cell Tracking; D005456:Fluorescent Dyes / Q000378:metabolism*; D006624:Hippocampus; D006801:Humans; D007477:Ions / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D018377:Neurotransmitter Agents / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D051381:Rats; D012165:Retinal Ganglion Cells / Q000502:physiology
chemical_list: D005456:Fluorescent Dyes; D007477:Ions; D018377:Neurotransmitter Agents; D011188:Potassium