Glycolysis and oxidative phosphorylation in neurons and astrocytes during network activity in hippocampal slices

Abstract

Network activation triggers a significant energy metabolism increase in both neurons and astrocytes. Questions of the primary neuronal energy substrate (e.g., glucose vs. lactate) as well as the relative contributions of glycolysis and oxidative phosphorylation and their cellular origin (neurons vs. astrocytes) are still a matter of debates. Using simultaneous measurements of electrophysiological and metabolic parameters during synaptic stimulation in hippocampal slices from mature mice, we show that neurons and astrocytes use both glycolysis and oxidative phosphorylation to meet their energy demands. Supplementation or replacement of glucose in artificial cerebrospinal fluid (ACSF) with pyruvate or lactate strongly modifies parameters related to network activity-triggered energy metabolism. These effects are not induced by changes in ATP content, pH(i), [Ca(2+)](i) or accumulation of reactive oxygen species. Our results suggest that during network activation, a significant fraction of NAD(P)H response (its overshoot phase) corresponds to glycolysis and the changes in cytosolic NAD(P)H and mitochondrial FAD are coupled. Our data do not support the hypothesis of a preferential utilization of astrocyte-released lactate by neurons during network activation in slices--instead, we show that during such activity glucose is an effective energy substrate for both neurons and astrocytes.

Figure 1

NAD(P)H transient waveshape is similar throughout the depth of the slice. (A) Example of recording electrodes' configuration and regions of interest for fluorescence measurements in a slice (photo on the left); original NAD(P)H transients and the corresponding oxygen transients measured at different depths (distance from the upper slice surface) of the same slice. Signals were induced by a 10- Hz, 10-second stimulation of Schaffer collaterals. Note that the variable location of the stimulation electrode due to its moving into the slice explains the difference in response values. (B) The dependence of NAD(P)H oxidation/overshoot amplitudes ratio on the depth in slice. Linear regression (straight line) shows no correlation between these two parameters (R²=0.013).

Figure 2

Neuronal and astrocytic metabolic signaling induced by synaptic stimulation. (A) NAD(P)H and oxygen transients induced by a 10-Hz, 10-second stimulation of Schaffer collaterals. Red, signals recorded in artificial cerebrospinal fluid (ACSF); blue, signals recorded after the addition of a cocktail of blockers consisting of NBQX (10 μmol/L), AP-5 (40 μmol/L), gabazine (10 μmol/L), and E4CPG (500 μmol/L). The inset (A) shows local field potentials (LFPs) within the stimulation train. Note multiple population spikes induced by short-term synaptic plasticity. (B) Summary of all similar experiments. The NAD(P)H oxidation dip and overshoot amplitudes were used in analysis. Oxygen consumption was estimated as an integral of pO₂ transient below the baseline. Responses are normalized to the amplitudes in ‘cocktail-free' ACSF. **P<0.01, ***P<0.001.

Figure 3

Neuronal and astrocytic metabolic signaling induced by the glutamate puff train stimulation. (A) Electrodes' configuration and regions of interest (ROIs) for fluorescence measurements in a slice. Original NAD(P)H transients in each ROI in response to a 10-ms, 10-Hz, 1-second glutamate (10 mmol/L) puff train stimulation. In blue, signals recorded in standard artificial cerebrospinal fluid (ACSF), in yellow—after addition a cocktail of blockers consisting of NBQX (10 μmol/L), AP-5 (40 μmol/L), gabazine (10 μmol/L), E4CPG (500 μmol/L), and TTX (1 μmol/L). Reference ROI (#1) is not shown. (B) NAD(P)H and oxygen transients induced by a 10-ms, 10-Hz, 5-second glutamate (10 mmol/L) puff stimulation in ACSF (red), ACSF+cocktail+TTX (blue) and ACSF+cocktail+TTX+TFB-TBOA (green). The inset shows field recordings during the stimulation train. (C) Summary of similar experiments. Responses are normalized to the amplitudes in ‘cocktail-free' ACSF. (D) Averaged transients of NAD(P)H and FAD responses (n=4) to the glutamate puffs train in ACSF (red) and ACSF+cocktail+TTX (blue). **P<0.01, ***P<0.001.

Figure 4

NADH fluorescence depends on the glucose and pyruvate content in artificial cerebrospinal fluid (ACSF). (A) Example of a long-lasting NAD(P)H fluorescence recording in a slice of P44 mouse. Black trace shows raw data from one region of interest (ROI) (right Y axis). The initial fragment of baseline recording (15-minute duration) was used to fit the baseline drift with an exponential decay function. This function was extrapolated to the entire experiment duration and applied as F₀(t) to the calculation of ΔF(t)/F₀(t) (red trace). Black arrows indicate stimulations of Shaffer collaterals (10 Hz, 10 seconds). (B, C) NAD(P)H responses to stimulations at different glucose concentrations in ACSF shown in panel A (a–c) are shown. (D) Summary of four similar experiments. NAD(P)H overshoot amplitude decreased with a decrease in glucose concentration (top graph, black bars) whereas NAD(P)H oxidation dip was insensitive to such change (lower graph, black bars). Gray bars show data scaled to the responses measured in 10 mmol/L glucose. (E, F) NAD(P)H responses to stimulations shown in panel A (c–e) are shown. (G) Summary of four similar experiments. Pyruvate modified both phases of NADH transients (top and lower graphs black bars). Gray bars show data scaled to the initial responses measured in 10 mmol/L glucose (see text for details). *P<0.05, **P<0.01, ***P<0.001.

Figure 5

Overshoot in NAD(P)H transients mainly reflects the glycolysis-generated NAD(P)H. (A) Replacement of glucose (5 mmol/L, red) with pyruvate (10 mmol/L, blue) in artificial cerebrospinal fluid (ACSF) results in robust changes in the NAD(P)H response to synaptic stimulation and in the increased oxygen consumption. (B) Summary of similar experiments. Responses are normalized to the amplitudes in glucose-ACSF. (C) Similar recordings as in (A) but with the glutamate (10 mmol/L) puff (10 ms) train stimulation (10 Hz, 1 second). (D) Summary of experiments as in panel D. (E) Summary of similar to (D) experiments but with cocktail+TTX added to ACSF to reveal the astrocytic fraction of responses. Cocktail of blockers consists of NBQX (10 μmol/L), AP-5 (40 μmol/L), gabazine (10 μmol/L), and E4CPG (500 μmol/L). *P<0.05, **P<0.01, ***P<0.001.

Figure 6

Coupling between cytosolic and mitochondrial redox states. (A) NAD(P)H and FAD transients in response to synaptic stimulation in 5 mmol/L glucose-artificial cerebrospinal fluid (ACSF) (red) and 10 mmol/L pyruvate-ACSF (blue). Black bar indicates stimulation. (B) Summary of similar experiments. Responses are normalized to the amplitudes in glucose-ACSF. (C) Averaged (n=6) transients recorded consequently in 5 mmol/L glucose-ACSF (red), 10 mmol/L pyruvate-ACSF (blue), and 10 mmol/L lactate-ACSF (green). In each experiment, signals were normalized to the overshoot (undershoot) amplitude in glucose-ACSF. (D) Histograms of changes (maximal values) in neuronal pH_i after replacement of glucose in ACSF with pyruvate or lactate. ***P<0.001.