Intracellular characterization of the K⁺ indicator APG-1.

<p>(<b>A</b>) Fluorescence image of primary astrocytes loaded using APG-1 AM. Scale bar 50 µm. (<b>B</b>) <i>In situ</i> excitation and emission spectra measured by fluorescence microscopy. Intracellular spectra were ∼10 nm red-shifted compared with measurement in cuvettes. (<b>C</b>) Representative experimental trace depicting the <i>in situ</i> calibration procedure. At the time indicated by the arrow, the cell membrane was permeabilized for K⁺ using valinomycin and nigericin while the Na⁺/K⁺ ATPase was inhibited by ouabain. Solutions of different [K⁺] were then sequentially applied until stable fluorescence plateaus were obtained. (<b>D</b>) Calibration curve obtained by plotting the fluorescence plateau values measured for each known [K⁺].</p

Spectrofluorimetric characterization of the K⁺ indicator APG-1.

<p>(<b>A</b>) Emission spectra recorded in the presence of different [K⁺] in intracellular-like solutions following excitation at 515 nm. Emission maximum was ∼540 nm. (<b>B</b>) Fluorescence emission plotted as a function of [K⁺] showing a monotonic relationship of APG-1 fluorescence with increasing [K⁺] (circles). The same analysis was performed on APG-2, a related indicator with identical spectral properties (diamonds) but lower Kd for K⁺. The plots show that APG-2 fluorescence becomes saturated at [K⁺]>80 mM, which is not the case with APG-1. (<b>C</b>) Na⁺ dependency of APG-1 fluorescence measured in intracellular-like solution containing 135 mM K⁺ (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109243#pone.0109243.s001" target="_blank">Fig. S1</a>). (<b>D</b>) pH dependency of APG-1 fluorescence measured in intracellular-like solution containing 135 mM K⁺. The pH of each solution was adjusted using NMDG. This pH analysis was repeated three times. Data are presented as means ± SEM of triplicate measurements.</p

Intracellular K⁺ is modulated by [K⁺]_o level changes.

<p>(<b>A</b>) Representative single-cell [K⁺]_i trace during bath application of solutions with different K⁺ concentrations in the range 3 to 10 mM, as are found during physiological and pathological conditions. (<b>B</b>) Relationship between steady-state [K⁺]_i (measured on plateau levels) and externally applied [K⁺]_o (n = 120 cells from 12 exp). The graph indicates a steady increase in [K⁺]_i in the [K⁺]_o range 3–10 mM (plain circles), which yielded a slope of 1.04±0.06 (r = 0.82). A higher [K⁺]_o of 15 mM (open circle) failed to further increase [K⁺]_i. (<b>C</b>) Intracellular K⁺ is influenced by localized K⁺-gluconate puff applications. Representative [K⁺]_i traces (average values of 7 cells each) during puff application (black arrows) of K⁺ gluconate in close proximity to the pipette (upper trace) and at>90 µm distance (lower trace). Insets: magnification of the trace after single extracellular applications of K⁺. Average amplitude (<b>D</b>) and duration of [K⁺]_i rise (<b>E</b>) induced by K⁺ puffs (black bar) compared with responses observed in the presence of 200 µM Ba²⁺ (white bar) or 20 µM carbenoxolone (CBX, grey bar) (n = 62 cells, 5 exp). No significant changes in amplitudes were found, whereas the response duration was significantly prolonged by CBX and reduced by Ba²⁺.</p

D-lactate effects on neuronal activity.

<p>(a) Sample trace of calcium transients in control or 5 mM D-lactate containing solution. (b) D-lactate substantially decreased calcium transient frequency. (c) The concentration-response analysis yielded an apparent IC₅₀ of 4.6±1.2 mM (n = 127 cells; 21exp).</p

Effects of L-lactate on calcium spiking frequency.

<p>(a) Original traces of calcium transients in control or 5 mM L-lactate containing solution. (b) Calcium spiking frequency for principal glutamatergic neurons and GABAergic interneurons are shown as percent of activity measured during control solution. Data are obtained from 49 principal cells and 35 interneurons from 13 experiments.</p

Concentration dependency of L-lactate effects.

<p>The decrease in calcium spiking frequency was concentration dependent. Apparent IC₅₀ values obtained by nonlinear curve fitting yielded 4.2±1.9 mM for principal neurons (n = 175 cells, 56 exp) and 4.2±2.8 mM for GABAergic neurons (n = 83 cells, 35 exp).</p

HCA1 receptor involvement in the lactate sensitivity.

<p>(a) Confocal images showing immunostaining for NeuN (green), HCA1 (red) and the merged image in mouse primary cortical neurons. Scale bar, 20 µm. (b) Representative Western blot showing that HCA1 is expressed in mouse primary cortical neuronal cultures. Each track represents one independent cultured dish of mouse primary cortical neurons (c) Comparison of lactate effect on calcium spiking frequency in cells incubated or not with pertussis toxin (PTX). PTX incubation strongly reduced the effects of lactate on neuronal activity. Data are obtained from 8 experiments and 61 cells for non-treated group and 8 experiments and 62 cells for PTX treated group.</p

Intracellular pH effects of lactate isomers on cortical neurons.

<p>Intracellular pH measured using BCECF and calibrated <i>in situ</i> in cortical neurons. (a) Original pH trace during sequences of L- and D-lactate application. (b) Summary of acidification (pH amplitude) measured during L- and D-lactate application. (n = 39 cells; 7exp).</p

Neuronal activity monitored with calcium imaging.

<p>Comparison between simultaneous intracellular calcium imaging sampled at a frame rate of 10 Hz and whole-cell patch clamp recordings. A representative experiment out of 15 is shown with the upper trace representing calcium transients (arbitrary fluorescence units, AFU) and lower trace action potentials recorded in current-clamp configuration from the same neuron. The tick marks above the calcium trace indicate the occurrence of action potentials detected in the same cell using patch-clamp recordings.</p

Energy metabolite dependency of calcium spiking frequency.

<p>Calcium spikes frequency shown as percent of activity measured during control solution. (a) Effects of pyruvate on calcium spiking frequency (n = 188 cells, 24 exp). Glucose (5 mM) was present throughout the experiments. (b) Effects of glucose concentration on spiking frequency (n = 68 cells, 10 exp).</p