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

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

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

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

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

Low glucose-induced LC3-II and p62 accumulation is principally due to a defect of autophagosome/lysosome fusion.

<p>661W cells were cultured as mentioned in material & methods, and then incubated at low (1 mM) or high (25 mM) glucose for 48 h in absence or in presence of fusion inhibitors (10 µg/ml Pepstatin/E64 and 75 µM Chloroquine) during the last 4 h. <b>A)</b> Representative western blot analysis of LC3-II protein expression and quantification. Results are expressed as mean ± SEM of 4 experiments, *p<0.03 (1 mM <i>vs.</i> 25 mM without inhibitors). <b>B)</b> Transfection of 661W cells with a lentivirus expressing the GFP-LC3 chimeric protein and incubation for 48 hrs at 1 mM (a-d) and 25 mM (e-h) glucose in absence (a and e) or in presence of 50 nM Bafilomycin (b and f), 10 µg/ml Pepstatine/E64 (c and g) and 75 µM Chloroquine (d and h) for the last 4 h. <b>C)</b> Transfection of 661W cells with a lentivirus vector expressing the mRFP-GFP-LC3 chimeric protein and incubation for 48 h at 1 mM (a–f) and 25 mM (g–l) glucose in absence (a–c and g–i) or in presence (d–f and j–l) of 75 µM chloroquine during the last 4 h.</p

3-MA chemical inhibition of autophagy increases low glucose-induced cell death and caspase 3 activity.

<p>661W cells were cultured as mentioned in material & methods, and then incubated at low (1 mM) or high (25 mM) glucose for different periods of time (8 or 48 h). <b>A)</b> TUNEL assay was performed in absence (a, d) or in presence (b, c and e, f) of 600 µM 3-MA, at 1 mM (a, b, c) or 25 mM (d, e, f) glucose concentrations, for different periods of time as indicated to the left. White arrows show TUNEL positive cells. Quantification of TUNEL positive cells was performed in three different experiments, *p<0.05; #p<0.0001 and **p<0.002 <b>B)</b> Measures of Caspase 3 activity, results are expressed as mean ± SEM of 3 experiments (n = 13), *p<0.04 and #p<0.0002, and immunostaining of cleaved Caspase 3 in 661W cells incubated at 1 mM (a and b) or 25 mM (c and d) glucose concentrations in absence (a and c) or presence of 600 µM 3-MA (b and d).</p

Low glucose induces a decrease in LAMP2 expression.

<p>661W cells were cultured as mentioned in material & methods, and then incubated at low (1 mM) or high (25 mM) glucose for diverse periods of time. <b>A)</b> Quantification of <i>Lamp2</i> mRNA by qPCR analysis, RL8 gene was used to normalize gene expression. Results are expressed as mean ± SEM of 3 experiments, * p<0.02 and # p<0.0008. <b>B)</b> Western blot analysis of LAMP2a protein expression. Results are expressed as mean ± SEM of 3 experiments, * p<0.03 and #p<0.001. <b>C)</b> Immunofluorescence staining of LAMP2a (green) in 661W cells cultured at 1 mM and 25 mM glucose concentration. <b>D)</b> Western blot analysis and quantification of p62 in 661W cells, western blot is representative of three experiments performed in triplicate and results are expressed as mean ± SEM, ** p<0.0011. <b>E)</b> Western blot and quantification of p62 expression in retinal explants, cultured at 1 mM or 25 mM glucose. Western blot is representative of 2–3 experiments and results are expressed as mean ± SEM of 5 different retinas for each condition, * p<0.002.</p