Lactate modulates the activity of primary cortical neurons through a receptor-mediated pathway

Abstract

Lactate is increasingly described as an energy substrate of the brain. Beside this still debated metabolic role, lactate may have other effects on brain cells. Here, we describe lactate as a neuromodulator, able to influence the activity of cortical neurons. Neuronal excitability of mouse primary neurons was monitored by calcium imaging. When applied in conjunction with glucose, lactate induced a decrease in the spontaneous calcium spiking frequency of neurons. The effect was reversible and concentration dependent (IC50 ∼4.2 mM). To test whether lactate effects are dependent on energy metabolism, we applied the closely related substrate pyruvate (5 mM) or switched to different glucose concentrations (0.5 or 10 mM). None of these conditions reproduced the effect of lactate. Recently, a Gi protein-coupled receptor for lactate called HCA1 has been introduced. To test if this receptor is implicated in the observed lactate sensitivity, we incubated cells with pertussis toxin (PTX) an inhibitor of Gi-protein. PTX prevented the decrease of neuronal activity by L-lactate. Moreover 3,5-dyhydroxybenzoic acid, a specific agonist of the HCA1 receptor, mimicked the action of lactate. This study indicates that lactate operates a negative feedback on neuronal activity by a receptor-mediated mechanism, independent from its intracellular metabolism.

Figure 1. Neuronal activity monitored with calcium imaging.

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.

Figure 2. Effects of L-lactate on calcium spiking frequency.

(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.

Figure 3. Concentration dependency of L-lactate effects.

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).

Figure 4. Energy metabolite dependency of calcium spiking frequency.

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).

Figure 5. D-lactate effects on neuronal activity.

(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).

Figure 6. Intracellular pH effects of lactate isomers on cortical neurons.

Intracellular pH measured using BCECF and calibrated in situ 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).

Figure 7. HCA1 receptor involvement in the lactate sensitivity.

(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.

Figure 8. Reduction by 3,5-DHBA and 3-HBA of the calcium spiking frequency.

Calcium spiking frequency shown as percent of activity measured during control solution. (a) Effects of 3,5-DHBA on calcium spiking frequency (n = 155 from 22 experiments). (b) Effects of 3-HBA on spiking frequency (n = 10 from 79 experiments).