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

<div><p>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.</p> </div

Relationship between FMP responses, membrane potential and the external K⁺ concentration.

<p>(<b>A</b>) Mean values of the relative changes of the FMP fluorescence (ΔF/F₀) shown in Fig. 2F were transformed and fitted with eq. 9. For RT/F = 25.43 mV (at 22°C), the best fit to the data indicates that the apparent charge of FMP (z′) has a value of −0.71. Changes of membrane potential (ΔE) are given with respect to −60 mV. (<b>B</b>) Mean values of the data shown in Fig. 2D were transformed using the K⁺ concentration of 4 mM as reference (K_r). The fit of the data with eq. 11 indicates that z′ = −0.64. (<b>C</b>) Graph showing relationship between the extracellular KCl concentration and ΔF/F₀ for different neuronal cell types. ΔF/F₀ was measured in experiments similar to those shown in Fig. 2C, in which high K⁺ pulses were applied from a reference K⁺ concentration of either 4 mM or 5.4 mM (solution BS). Hippocampal neurons (HIPPs), n = 17; DRGs, n = 12; RGCs, n = 15. A sigmoidal relationship between the KCl concentration and ΔF/F₀ is apparent. (<b>D</b>) Mean values of the data shown in (C) were transformed and fitted with eq. 11, where the fraction K_x/K_r represents the change of the external K⁺ concentration (K_x) with respect to a reference (K_r) concentration that was either 4 mM or 5.4 mM. Calculated z′ values are: −0.71 (DRGs); −0.72 (HIPPs); −0.62 (RGCs).</p

FMP responses during stimulation of neurons with various neurotransmitters.

<p>(<b>A</b>) FMP signals elicited by activation of ionotropic and metabotropic glutamate receptors. Retinal ganglion cell (RGCs) were treated with 25 µM glutamate (i), 100 µM NMDA (ii) and 100 µM AMPA (iii). Septal neurons were exposed to 200 µM (S)-3,5-DHPG (iv). (<b>B</b>) Cortical neurons were treated with 100 µM acetylcholine (ACH) resulting in rapid oscillations of the FMP fluorescence. (<b>C</b>) The spontaneous oscillations of the FMP fluorescence observed in some hippocampal neurons were silenced upon addition of 10 µM GABA and 0.5 µM diazepam (i). Addition of 10 µM GABA in the presence (i) and absence (ii) of diazepam slightly enhanced the FMP fluorescence in most hippocampal neurons. (<b>D</b>) Dorsal root ganglion neurons (DRGs) were treated with 250 µM (-)-menthol to activate cold receptors, followed by 0.6 µM (E)-capsaicin to activate heat receptors. Experiments were performed in HBSS. Shown are representative FMP signals of individual neurons depicted in colour.</p

Monitoring of spontaneous neuronal activity with FMP.

<p>(<b>A</b>) FMP fluorescence in hippocampal neurons. The image shows the basal FMP fluorescence (530 nm excitation; 605 nm emission). Regions of interest on the neuronal soma (ns) and along neurites (n1–n3) are marked. Scale bars, 20 µm. (<b>B</b>) Time courses of ΔF/F₀ recorded on the neuronal soma and neurites as indicated in A. The signals were calibrated with eq. 9. z′ = −0.72. RT/F = 25.43 mV (22°C). Images were sampled at a rate of 20 frames/s. (<b>C</b>) Single frames of neuronal activity recorded at the indicated time points. Images of relative FMP fluorescence (ΔF/F₀) were computed pixel-by-pixel.</p

FMP dye responses to changes in cell membrane potential.

<p>(<b>A</b>) Reversibility of the FMP signal (ΔF/F₀) assayed in DRG neurons as the cell membrane was depolarised and repolarised by changing the K⁺ concentration in BS. Bold red line represents mean response, n = 16; individual cell responses are shown as light red lines. (<b>B</b>) Time courses of FMP signals during high K⁺ pulses (left panel) and voltage-clamp pulses (right panel). HEK cells were exposed to the indicated K⁺ concentrations (left panel); mean (black) and single cell examples (grey) are shown. The membrane potential of HEK cells was clamped to the indicated potentials (right panel) using the perforated patch clamp technique and the FMP fluorescence was measured using a photometry system. The exponential fittings (blue) show that similar time constants were achieved for the rise and decay of FMP signals, when HEK cells were depolarised and repolarised using high K⁺ and voltage-clamp pulses. (<b>C</b>) FMP signals observed in HEK cells exposed to increasing K⁺ concentrations in BS. Mean (black) and single cell (grey) signals from example experiments are shown. K⁺ step protocols are depicted above graphs. (<b>D</b>) Relationship between ΔF/F₀ and the KCl concentration in BS from experiments performed with HEK cells as illustrated in (C), n: 21–30. The reference K⁺ concentration was 4 mM and the data was fitted with an empirical sigmoidal function (grey line). (<b>E</b>) FMP signals elicited in HEK cells by voltage-clamp pulses. Photometric measurements and voltage clamp were performed as in (B). The voltage-clamp pulse protocol is shown above the FMP signal. (<b>F</b>) Relationship between ΔF/F₀ and membrane potential (V_m) obtained in combined voltage clamp and photometry experiments. The resting membrane potential was −60 mV. The grey line is a data fit with an empirical sigmoidal function; n = 5. Data is presented as mean ± SEM.</p

Calibration of FMP signals measured with a single cell imaging system.

<p>Changes of membrane potential (ΔE) induced by high K⁺ steps were calculated with eq. 9. RT/F = 25.43 mV (22°C). ΔF/F₀ values were from Fig. 2C–D and Fig. 4C. (<b>A</b>) FMP signals measured in HEK (black). z′ = −0.64. (<b>B</b>) FMP signals from the indicated neuronal cell types (red). z′: −0.71 (DRGs); −0.72 (HIPPs); −0.62 (RGCs). For comparison, the plot ΔE vs. ΔF/F₀ (grey) obtained using a photometric system on voltage-clamped HEK cells (Fig. 2 F) is superimposed on the graphs.</p

Intracellular localization and spectral properties of the FMP dye in neurons.

<p>(<b>A</b>) Phase contrast image of hippocampal neurons on top of astrocytic feeder layer. Scale bars, 20 μm. (<b>B</b>) Confocal-like optical sections of the neurons shown in (A) 15 min after labelling with FMP in Hanks' balanced salt solution (HBSS). Strong fluorescence of neurons (left panel) and low background of astrocytic feeder layer (right panel) show preferential labelling of neurons with intracellular localization of the FMP dye. (<b>C</b>) Epifluorescence images showing increase in FMP emission intensity upon depolarizing dorsal root ganglion (DRG) neurons by increasing KCl from a resting concentration of 5.4 mM (5.4 K⁺) to 25 mM (25 K⁺) in the basic solution (BS). Images of resting DRGs and intermediate images as fluorescence intensity increases are shown. roi, region of interest; lp, line profile; a. u., arbitrary units. (<b>D</b>) Cross-section line profiles of FMP fluorescence in a DRG neuron as indicated in (C). The example illustrates the homogenous increase in cytosolic fluorescence during transition of neurones from resting (5.4 K⁺). (<b>E</b>) Excitation spectra of the FMP dye in DRG neurons exposed to 5.4 K⁺ (left) and 25 K⁺ (right). Average spectra of neurons (bold red, n = 17) and astrocytic feeder cells (bold black, n = 36) are shown with examples of individual cells (light red and grey) for excitation wavelengths between 400 and 545 nm. (<b>F</b>) Relative changes of FMP fluorescence intensity calculated as ΔF/F₀ from the spectra shown in (E). Average values of neurons (red circles) and astrocytic feeder cells (grey circles) are plotted with individual cell traces (light red and grey lines). Data is presented as mean ± SEM. The relative changes of the FMP fluorescence measured at 530 nm excitation displayed a high signal-to-noise ratio (F) independently of individual cell fluorescence intensities (E).</p

Ionic contribution to membrane potential changes detected by the FMP dye.

<p>(<b>A</b>) HEK cells over-expressing the Na⁺-selective channel TRPM8 were co-labelled with both FMP and SBFI, and treated with 500 µM menthol in HBSS. Cells were monitored for changes in membrane potential and intracellular Na⁺ using FMP and SBFI dyes, respectively (left panels). Shown are FMP (ΔF/F₀) and SBFI (F₃₄₀/F₃₈₀) signals of individual cells. Upon overlaying average FMP (black) and SBFI (green) signals, similar responses are seen (right panel). (<b>B</b>) FMP signals induced by the Na⁺ ionophore SQI-Pr. Two different concentrations of SQI-Pr (5 µM and 10 µM) were tested. Mock applications (0 µM) were performed as controls. (<b>C</b>) FMP responses to high K⁺ steps in the absence (0 µM) and presence (2 µM) of the K⁺ ionophore valinomycin (VAL). Experiments in (B) and (C) were carried out with HEK cells in BS. Average traces are shown in bold and individual traces are in grey; arrows indicate the time point of mock, SQI-Pr and valinomycin applications.</p

Administration of anti-mouse TNFR1 on the day of immunization ameliorated EAE.

<p>Anti-mouse TNFR1 was injected intra-peritoneally in C57BL/6 mice, on the day of disease induction, at a dosage of 100 µg (equivalent to 5 mg/kg). Mice were subsequently monitored on a daily basis until 21 days after the onset of clinical symptoms (EAE day 21). Antibody treatment resulted in a reduced EAE severity compared to mice receiving control IgG (<b>A, B</b>). Furthermore, mice injected with anti-TNFR1 also showed a significant delay in the onset of spinal cord symptoms in comparison to mice receiving control IgG (<b>C</b>). (A) Results from one representative experiment out of four shown (control IgG n = 4; anti TNFR1 n = 6), (B, C) results from four combined experiments (control IgG n = 16, anti-TNFR1 n = 19). * P<0.05, **P<0.01.</p

Anti-TNFR1 treatment on the day of immunization resulted in a significant reduction in demyelination and neuronal loss and a mild reduction in inflammatory infiltration.

<p>Spinal cord histopathology was performed at day 21 of EAE, following treatment with anti-mouse TNFR1 on the day of immunization. Representative images are shown from control IgG treated mice, with an EAE score of 2.0 (<b>B, E, H</b>, and <b>K</b>) and from anti-TNFR1-treated animals, with an EAE score of 1.0 (<b>C, F, I</b> and <b>L</b>). The level of spinal cord demyelination was assessed using sections stained with LFB (<b>A–C</b>). Mice treated prophylactically with anti-TNFR1 had significantly reduced levels of demyelination compared to control-treated mice (<b>A–C</b>). Immunohistochemistry with an anti-CD3 antibody was used to detect T cells and showed a decrease, although not significant, in the number of T cells within the spinal cord of anti-TNFR1 treated mice, in comparison to control animals (<b>D–F</b>). Immunohistochemistry with an antibody to Mac-3 was used to detect activated microglia and macrophages and demonstrated a decrease in the number of positive cells in anti-TNFR1 treated mice, although again this was not significant (<b>G–I</b>). <b>J-L</b>: Immunohistochemistry with an anti-NeuN antibody was used to detect neuronal cell bodies, which were quantified within the spinal cord grey matter. Anti-TNFR1 treated mice had significantly elevated numbers of surviving neuronal cell bodies. Scale bars in <b>B, C, E, F, H, I, K, L</b>: 200 µm.</p