Glutamate transport decreases mitochondrial pH and modulates oxidative metabolism in astrocytes

Abstract

During synaptic activity, the clearance of neuronally released glutamate leads to an intracellular sodium concentration increase in astrocytes that is associated with significant metabolic cost. The proximity of mitochondria at glutamate uptake sites in astrocytes raises the question of the ability of mitochondria to respond to these energy demands. We used dynamic fluorescence imaging to investigate the impact of glutamatergic transmission on mitochondria in intact astrocytes. Neuronal release of glutamate induced an intracellular acidification in astrocytes, via glutamate transporters, that spread over the mitochondrial matrix. The glutamate-induced mitochondrial matrix acidification exceeded cytosolic acidification and abrogated cytosol-to-mitochondrial matrix pH gradient. By decoupling glutamate uptake from cellular acidification, we found that glutamate induced a pH-mediated decrease in mitochondrial metabolism that surpasses the Ca(2+)-mediated stimulatory effects. These findings suggest a model in which excitatory neurotransmission dynamically regulates astrocyte energy metabolism by limiting the contribution of mitochondria to the metabolic response, thereby increasing the local oxygen availability and preventing excessive mitochondrial reactive oxygen species production.

Figure 1.

Glutamate evokes mitochondrial acidification in astrocytes. a, Images of cortical astrocytes expressing MitoSypHer loaded with the mitochondrial-selective fluorescent marker MitoTracker Red. Scale bar, 10 μm. b₁, Calibration curve of MitoSypHer fluorescence excitation ratio versus pH. b₂, Glutamate (200 μm) superfusion on intact astrocytes acidified the mitochondrial matrix. A typical trace from a single astrocyte is shown, along with pseudocolor intensity modulated fluorescence ratio images of cells before (ctrl) and during (glut) glutamate application. Scale bar, 20 μm. c, Amplitude of glutamate-induced intracellular acidifications induced by glutamate in the cytosol (n = 23, 172 cells), MIMS (n = 9, 26 cells), and mitochondrial matrix (n = 46, 168 cells). Data are shown as means ± SEM. d, Scheme for the localization of mitochondrial pH sensors (MIMS–EYFP and MitoSypHer). IMM, Inner mitochondrial membrane; OMM, outer mitochondrial membrane.

Figure 2.

Glutamate evokes a concentration-dependent mitochondrial matrix acidification mediated by glutamate transporters. a, Concentration dependence of the amplitude (open circles) and initial slope (filled triangles) of glutamate-induced mitochondrial matrix acidification (n = 24, 80 cells). b, TBOA reversibly inhibited a 200 μm glutamate-evoked mitochondrial matrix acidification. Amplitude of glutamate-induced mitochondrial matrix acidification before during and after TBOA (n = 5, 30 cells). See also supplemental Figure S2 (available at www.jneurosci.org as supplemental material). c, Ca²⁺ (top curves) and pH_mit (bottom curves) responses were simultaneously monitored using fura-2 in MitoSypHer-transfected astrocytes. The applications of the mGluR agonist (t-ACPD, 100 μm) and glutamate transporter substrate D-asp (200 μm) are indicated in the graph. Traces are shown as mean signals with error bars (SEM) displayed every third time point for graphical clarity (n = 5, 8 cells).

Figure 3.

Glutamate-induced cellular acidification abrogates the cytosol-to-mitochondrial matrix pH gradient. a, Representative images of MitoSypHer-transfected astrocytes loaded with the cytosolic pH-sensitive red dye SNARF-1. Scale bar, 20 μm. b, Spectral selectivity of both probes by measuring emission spectra in nucleus (Nuc) and cytosol (Cyt) of transfected (Tr) and untransfected (UnTr) astrocytes labeled with SNARF-1. Both fluorophores were excited at 488 nm, and emission fluorescence was split into three channels, for MitoSypHer (Ch.1) and SNARF-1 (Ch.2 and Ch.3), respectively. c, d, Glutamate transporter-mediated acidification abrogates cytosol-to-mitochondrial matrix pH gradient. Original traces (c) and averaged data (d) of cytosolic and mitochondrial matrix pH during agonist-induced acidification. Both glutamate (Glu) and d-aspartate (D-Asp) caused a collapse of cytosol-to-mitochondrial matrix pH gradient (n = 7, 11 cells). Kai, Kainate. Error bars indicate SEM.

Figure 4.

Independence of cellular Na⁺ and pH responses to glutamate. a, Glutamate-evoked cellular Na⁺ responses without concomitant pH changes. After a first control pulse of glutamate application, glutamate was superfused together with 6 mm TREA, the concentration found to compensate glutamate-induced cytosolic acidification. pH measurements in the cytosol (pH_cyt; n = 4, 32 cells) and mitochondrial matrix (pH_mit; n = 6, 20 cells) and Na⁺ measurements in the cytosol (Na_cyt⁺; n = 4, 30 cells) and mitochondria (Na_mit⁺; n = 4, 32 cells) were taken after 1.5 min of stimulation. Data are presented as means ± SEM. b, Cytosolic pH measurement during a pulse of 200 μm glutamate (solid line) and during glutamate plus TREA application (dotted line). In the example shown, 10.7 mm TREA effectively prevented the acidification during glutamate application, as measured after 5 min of glutamate application.

Figure 5.

Glutamate induces a pH-mediated decrease of oxygen consumption rate. Glycolysis was inhibited using 2-DG (10 mm) to promote mitochondrial activity, and glucose (5 mm) and pyruvate (5 mm) were given as substrates. a, Time course of oxygen consumption rate in intact astrocytes in the absence and presence of glutamate (top; n = 7). When glutamate-induced cellular acidification was compensated by coadministration of TREA (20 mm), glutamate no longer significantly decreased oxygen consumption rate (bottom; n = 6). As a control, mitochondrial respiration was inhibited using oligomycin (5 μm) and then increased using FCCP (2 μm) at the end of each experiment. b, Average data of glutamate-induced decrease in oxygen consumption rate with increasing concentrations of TREA. Data were normalized to oxygen consumption rate before glutamate addition for each experiment (n = 42).

Figure 6.

Glutamate induced a pH-mediated alteration of mROS production rate. Glycolysis was inhibited using 2-DG (10 mm) to promote mitochondrial activity, and pyruvate (5 mm) was given as mitochondrial substrate. The mROS production rate during 2-DG plus pyruvate phase was considered as the reference rate of 100%. a, Time course of mROS production rate during stimulation with glutamate, coadministration of glutamate and TREA (10.7 mm), glutamate and MCPG (1 mm), or glutamate in the presence of the ROS scavenger MnTMPyP (50 μm). At the end of each experiment, antimycin A (Ant. A) was used as a positive control of mROS detection. Traces are shown as mean signals with error bars (SEM) displayed every 10th time point for graphical clarity. b, Mean values of mROS production rate after 5 min of stimulation. Conditions: MCPG (n = 5, 30 cells); TBOA (n = 3, 12 cells); TREA (n = 6, 34 cells); glutamate (n = 6, 34 cells); glutamate + MCPG (n = 5, 30 cells); glutamate + MnTMPyP (n = 3, 26 cells); glutamate + TBOA (n = 6, 36 cells); glutamate + TBOA + MCPG (n = 6, 45 cells); glutamate + TREA (n = 6, 30 cells). Statistical analysis was done for each group by comparison with the MitoSOX slope before stimulation. All data shown are means ± SEM.

Figure 7.

Neuronal release of glutamate triggers glutamate transporter-mediated mitochondrial matrix acidification in astrocytes. Images depicting fluorescent MitoSypHer expression in astrocytes (a₁) surrounding neurons visible under DIC (a₂). Scale bar, 20 μm. b₁, MitoSypHer fluorescence excitation ratio monitored in astrocytes during a 20 s stimulation of neurons using NMDA (10 μm). Representative trace (b₁) and individual data (b₂) of mitochondrial matrix pH of astrocytes at baseline and after NMDA stimulation. c, Amplitudes of NMDA-evoked astrocyte mitochondrial matrix acidification in the absence or presence of TBOA and neurons. NMDA (n = 9, 16 cells); NMDA + TBOA (n = 5, 7 cells); NMDA without neurons (n = 4, 11 cells). * p < 0.05 and ** p < 0.01 using unpaired Student's t test.