Neuronal Stimulation Triggers Neuronal Glycolysis and Not Lactate Uptake

Abstract

Proper brain function requires a substantial energy supply, up to 20% of whole-body energy in humans, and brain activation produces large dynamic variations in energy demand. While local increases in cerebral blood flow are well known, the cellular responses to energy demand are controversial. During brain excitation, glycolysis of glucose to lactate temporarily exceeds the rate of mitochondrial fuel oxidation; although the increased energy demand occurs mainly within neurons, some have suggested this glycolysis occurs mainly in astrocytes, which then shuttle lactate to neurons as their primary fuel. Using metabolic biosensors in acute hippocampal slices and brains of awake mice, we find that neuronal metabolic responses to stimulation do not depend on astrocytic stimulation by glutamate release, nor do they require neuronal uptake of lactate; instead they reflect increased direct glucose consumption by neurons. Neuronal glycolysis temporarily outstrips oxidative metabolism, and provides a rapid response to increased energy demand.

Keywords: astrocyte-neuron lactate shuttle; brain metabolism; glycolysis.

Figure 1. NADH_CYT transients with synaptic stimulation

(A) Filmstrip of metabolic transients in dentate granule neurons. A sequence of acquisitions shows the brief increase in RCaMP lifetime reflecting an augmented Ca²⁺ concentration after synaptic stimulation. The peak in the Peredox lifetime elevation occurs approximately 1 min later, reflecting changes in the cytosolic NADH/NAD⁺ ratio (NADH_CYT) toward a more reduced state. Images are pseudocolored according to sensor lifetimes. The color scale bar to the right shows the range of lifetime values for both RCaMP and Peredox sensors. For the latter, the color scale is accompanied by an axis converting lifetimes to calibrated NADH/NAD⁺ ratios. (B) Individual traces from a cell in the filmstrip illustrate the time course of synaptically stimulated transients in Peredox (top) and RCaMP1h lifetimes (bottom). (C) Average transient increases in Peredox lifetime after synaptic stimulation of 60 brief pulses delivered at 20 Hz. Traces represent the mean ± SEM (events=447, neurons=166, slices=27, mice=14; SEM calculated using number of neurons). (D) The magnitude of the NADH_CYT increase correlates with the amplitude of the neuronal Ca²⁺ spike in a wide range of Ca²⁺ responses elicited by synaptic stimulation. The scatter plot summarizes data resulting of events elicited with different stimulation strengths (events=554, neurons=166, slices=27, mice=14). (E) Glycolysis and lactate utilization enhance neuronal NADH production. The cartoon summarizes the main pathways leading to an increased cytosolic NADH/NAD⁺ ratio in neurons. After passive uptake via GLUT transporters, glucose is phosphorylated and enters glycolysis, where NADH is produced by the enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Lactate import occurs through monocarboxylate transporters (MCT) and is then converted to pyruvate by the enzyme lactate dehydrogenase (LDH), also reducing NAD⁺ to NADH.

Figure 2. Glutamate release is neither sufficient nor necessary for NADH_CYT transients

(A) Typical electrode placement for synaptic stimulation. The picture shows an infra-red differential interference contrast image of a horizontal slice from the hippocampus, where the stimulating electrode (s.e.) is placed in the molecular layer (m.l.) and an extracellular recording electrode (r.e.) is inserted in the layer of dentate granule neurons (DGNs, following the arrow direction). (B) Inhibition of ionotropic glutamate receptors abolishes the metabolic transients. A representative trace of a synaptically stimulated DGN (using a train of 60 pulses at 20 Hz) shows that the Peredox signal disappears after blocking synaptic transmission with a mixture of 5 μM NBQX and 25 μM D-AP5 in the ACSF. The complete blockade of synaptic transmission is corroborated by the absence of the typical RCaMP1h signal after stimulation. The effect of synaptic blockers was partially reversed after 5 min of washout in regular ACSF, and both Ca²⁺ and NADH transients were completely recovered after 10 min in ACSF. (C) Dataset for the effect of ionotropic glutamate receptor block. Data were compared by a paired Student’s t-test (neurons=46, slices=6, mice=3). (D) Typical electrode placement for antidromic (axonal) stimulation. In this experimental paradigm, the stimulating electrode (s.e.) is placed in the the hilus (hil) and an extracellular recording electrode (r.e.) is inserted in the layer of dentate granule neurons (DGNs, following the arrow direction). (E) Average transient increases in Peredox lifetime after antidromic stimulation of 100 brief pulses delivered at 50 Hz. Traces represent the mean ± SEM (events=179, neurons=84, slices=18, mice=11; SEM calculated using number of neurons). The lower panel shows the putative average Ca²⁺ spike in the cells, as a proxy for neuronal excitation. (F) Elevation in the NADH/NAD⁺ ratio is directly correlated with the neuronal Ca²⁺ increase after electrical antidromic stimulation in DGNs, similarly to synaptic stimulation. Scatter plots of changes in Peredox lifetime versus variations in RCaMP lifetime for events recorded after antidromic stimulation (open circles; events=205, neurons=87, slices=19, mice=12), overlap with data collected using synaptic stimulation (dark squares).

Figure 3. Neuronal lactate import is not required for the NADH_CYT transients

(A) Monocarboxylate transporter (MCT) inhibition abolishes NADH transients induced by the application of external lactate but enhances the NADH_CYT rise after synaptic stimulation. The MCT inhibitor AR-C155858 (2 μM, 5 min) prevents the increase in the NADH/NAD⁺ ratio in response to external lactate (10 mM). However, the endogenous NADH transients in response to synaptic stimulation (60 pulses at 20 Hz) were bigger in the presence of the MCT inhibitor (MCT inhib). (B) Lactate-induced changes in Peredox lifetime were significantly reduced in the presence of the MCT inhibitor. Data were compared by a paired Student’s t-test (neurons=36, slices=5, mice=5). (C) Scatter plots of changes in Peredox lifetime versus changes in RCaMP lifetime, show that acute (5 min) or long-lasting (>10 min; as in A) exposure to the MCT inhibitor increases the size of NADH_CYT transients for similar Ca²⁺ transients, making the slope of the positive correlation steeper. All comparisons were made using a paired Student’s t-test. Control data points are open symbols while symbols for treatments are filled, using circles or squares for acute (neurons=39, slices=7, mice=5) and long-lasting (neurons=36, slices=5, mice=5) application of the MCT inhibitor respectively. The dashed lines and surrounding shaded zones show the linear fit and 95% confidence interval for the (unpaired) data with (black) and without (gray) MCT inhibitor; the lower dashed line shows the prediction of the ANLS that the stimulation-induced transients should be reduced comparably to the lactate-induced transients in B. (D) Lactate dehydrogenase (LDH) inhibition also decreases the NADH_CYT transients induced by the application of external lactate and increases the NADH/NAD⁺ ratio after synaptic stimulation. (E) The LDH inhibitor GSK-2837808A (2 μM, 30 min) diminishes the change of the cytosolic NADH/NAD⁺ ratio in response to external lactate (10 mM). Data were compared by a Wilcoxon signed-rank test (neurons=54, slices=5, mice=5). (F) Scatter plots of changes in Peredox lifetime versus changes in RCaMP lifetime, show that sustained (>30 min; as in A) exposure to the LDH inhibitor increases the size of NADH_CYT transients for similar Ca²⁺ transients, making the slope of the positive correlation steeper. Control data points are open circles (events=179, neurons=54, slices=5, mice=5) while black squares correspond to LDH inhibition (events=54, neurons=54, slices=5, mice=5). The dashed lines and surrounding shaded zones show the linear fit and 95% confidence interval for the (unpaired) data with (black) and without (gray) LDH inhibitor; the lower dashed line shows the prediction of the ANLS that the stimulation-induced transients should be reduced comparably to the lactate-induced transients in E. (G) Left. Representative traces of changes in CFP/YFP fluorescence emission ratio for Laconic before and after the application of the MCT inhibitor (2 μM, 5 min). Right. The relative increase in the CFP/YFP ratio was unaffected by MCT inhibition (neurons=41, slices=5, mice=4; Wilcoxon Signed Rank Test, p>0.05).

Figure 4. NADH_CYT transients most likely arise from neuronal glycolysis

(A) Treatment with the GAPDH inhibitor iodoacetic acid (IAA) reduces the NADH_CYT transients in response to antidromic stimulation. Representative trace of NADH_CYT transients elicited by antidromic stimulation (same conditions as Fig. 2), before and after IAA treatment. The bars indicate the times of application of both lactate (2 mM) and IAA (0.5 mM). IAA inhibits glycolysis irreversibly; the temperature was lowered (shaded zone) before and during IAA application to minimize damage to the slice’s health. (Temperature affects sensor lifetime directly, which is why there are large changes in the sensors’ response during the temperature changes.) After this treatment, the cells were stimulated again, leading to a much smaller NADH_CYT transient than before IAA treatment. (B) IAA decreased the ability of neurons to produce NADH in response to excitation, as observed in the reduction of the Δ(Peredox LT)/Δ(RCaMP LT) ratio (neurons=18, slices=5, mice=5). This ratio normalizes the NADH_CYT change to the size of the Ca²⁺ transients, as they are correlated (Figs. 1, 2) and the Ca²⁺ transients are somewhat affected by IAA treatment. Figure S4 shows additional controls and analysis.

Figure 5. Stimulation induces transient dips of neuronal intracellular glucose, consistent with increased neuronal glycolysis

(A) Representative trace of glucose dips elicited by synaptic stimulation of 60 pulses at 50 Hz. Glucose transporter inhibition by 0.2 μM Cytochalasin B (10 min) increased the size of the glucose dips. Figure S5 shows the properties of the SweetieTS sensor. (B) The structural analog Cytochalasin D (0.2 μM, 10 min) does not affect the glucose dips. This experiment serves as a negative control for the possible effects of CytoB on the cytoskeleton. The effect sizes for CytoB (neurons=22, slices=4, mice=4) and CytoD (neurons=17, slices=4, mice=3) were compared using an unpaired Student’s t-test. (C) Representative recording of glucose dips in the continuous presence of 0.2 μM CytoB (to enhance the glucose dips), before and after the application of MCT inhibitor (2 μM). (D) The acute application of 2 μM MCT inhibitor (5 min) reduced the glucose dips. Comparisons were done using the ratio between two consecutive control pulses and the ratio between the dip in the presence of MCT inhibitor and the previous control pulse (neurons=28, slices=7, mice=4). The nonparametric Wilcoxon signed-rank test was used because at least one of the distributions was not Gaussian.

Figure 6. NADH_CYT transients occur independently of the mitochondrial malate-aspartate shuttle

(A) Inhibition of the malate-aspartate shuttle (MAS) by aminooxyacetate (AOA) increases the cytosolic NADH/NAD⁺ ratio. The right panel shows representative traces of Peredox lifetime in two neurons, which increases after the application of 10 mM AOA in the ACSF. In the control cell, Peredox LT remains high in the continuous presence of 10 mM AOA, whereas the exposure of the second cell to 1.5 mM pyruvate (Pyr) decreased the Peredox LT to baseline levels, even in the presence of the shuttle inhibitor. The right panel summarizes the effect of AOA and pyruvate (as well as their combination) on Peredox lifetime (neurons=40, slices=5, mice=5). Values are significantly different from each other with a p<0.01, except for the conditions 1.5Pyr and 1.5Pyr+10 AOA. (B) Representative traces of activity-induced changes in Peredox lifetime of a neuron before and after AOA application, both obtained in the presence of 1.5 mM pyruvate in the ACSF. Antidromic stimulation was delivered in the presence of picrotoxin (100 μM) in addition to the ionotropic glutamate receptor blockers. This demonstrates that neuronal activation still produces a rise in the cytosolic NADH/NAD⁺ ratio, in spite of MAS inhibition. (C) The size of the NADH_CYT transient is marginally affected by MAS blockade. Left. The repetitive exposure to pyruvate increases the NADH_CYT transients around 11% in slices that were not treated with AOA (neurons=33, slices=3, mice=3). By contrast, the transients obtained in the simultaneous presence of AOA and pyruvate are ~8% smaller than the initial transients in the presence of pyruvate alone (neurons=40, slices=5, mice=5; Wilcoxon matched-pairs signed rank test, *p<0.05, **p<0.01). Right. For comparisons, we took into account the hysteresis effect by dividing the Peredox LT/RCaMP LT ratio at the final condition (second exposure to pyruvate in controls or AOA plus pyruvate in treated neurons) by the initial pulse in the presence of pyruvate alone. NADH_CYT transients in the presence of AOA plus pyruvate, are approximately 19% smaller than expected (Mann-Whitney test, **p<0.01).

Figure 7. NADH_CYT transients in vivowith sensory stimulation

(A) Representative pseudo-colored lifetime image of neurons expressing RCaMP1h (left panels) and nuclear-localized Peredox (right panels) in layers II/III of primary somatosensory cortex of an awake mouse. Scale bar = 10 μm. The images were acquired 0.33 minutes before (top panels), during (middle panels), and 1 minute after (bottom panels) whisker stimulation. Stimulation resulted in a brief increase in RCaMP lifetime in the responsive neuron, reflecting an augmented Ca²⁺ concentration (middle left panel). This was followed approximately 1 minute later by an increase in Peredox lifetime for the responsive neuron, reflecting changes in the cytosolic NADH/NAD⁺ ratio (NADH_CYT) toward a more reduced state (bottom right panel). The color scale bar to the right shows the range of lifetime values for both RCaMP1h and Peredox sensors. (B) Average transient increases, within neuronal regions of interest, in cytosolic NADH/NAD⁺ ratio (top panel) and Ca²⁺ concentration (bottom panel) following whisker stimulation (red arrow) consisting of 10 seconds of mechanical stimulation at 5 Hz. Traces represent the mean ± SEM; n=19 neurons, N= 9 mice (3 males, 6 females). (C) Elevation in the NADH/NAD⁺ ratio is directly correlated with the increase in intracellular Ca²⁺ following whisker stimulation. (D) Comparison of lactate-induced Δ(Peredox LT) (50mM lactate) before and after MCT inhibition (50 μM AR-C155858) in vivo using a paired Student’s t-test. n= 59 neurons, N= 6 mice (3 females, 3 males). (E) Representative experiment on the effect of MCT inhibition on neuronal NADH_CYT transients elicited by whisker stimulation. In vivo application of MCT inhibitor AR-C155858 (50μM, 20 minutes) did not reduce the endogenous NADH transients in response to whisker stimulation (10 sec of mechanical stimulation at 5 Hz). (F) The scatter plot of Δ(Peredox LT)/Δ(RCaMP LT) shows that in vivo exposure to MCT inhibitor does not reduce the size of the NADH/NAD⁺ transients and the slope of the relationship. The ratio of stimulation-induced Δ(Peredox LT)/Δ(RCaMP LT) in the absence of MCT inhibitor was 0.13 with a 95%CI: 0.10–0.16. In the presence of MCT inhibitor, the ratio of stimulation-induced Δ(Peredox LT)/Δ(RCaMP LT) was 0.15 with a 95%CI: 0.11–0.20. n=4 neurons, N=3 mice (2 females, 1 male). The dashed line shows the prediction of the ANLS that the stimulation-induced transients should be reduced comparably to the lactate-induced transients in D (based on adjusting the slope for the linear fit of the control measurements).