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. 2009 Oct;111(2):522-36.
doi: 10.1111/j.1471-4159.2009.06333.x. Epub 2009 Aug 13.

Astrocytes are poised for lactate trafficking and release from activated brain and for supply of glucose to neurons

Gautam K Gandhi  1 , Nancy F Cruz, Kelly K Ball, Gerald A Dienel
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Astrocytes are poised for lactate trafficking and release from activated brain and for supply of glucose to neurons

Gautam K Gandhi et al. J Neurochem. .
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Abstract

Brain is a highly-oxidative organ, but during activation, glycolytic flux is preferentially up-regulated even though oxygen supply is adequate. The biochemical and cellular basis of metabolic changes during brain activation and the fate of lactate produced within brain are important, unresolved issues central to understanding brain function, brain images, and spectroscopic data. Because in vivo brain imaging studies reveal rapid efflux of labeled glucose metabolites during activation, lactate trafficking among astrocytes and between astrocytes and neurons was examined after devising specific, real-time, sensitive enzymatic fluorescent assays to measure lactate and glucose levels in single cells in adult rat brain slices. Astrocytes have a 2- to 4-fold faster and higher capacity for lactate uptake from extracellular fluid and for lactate dispersal via the astrocytic syncytium compared to neuronal lactate uptake from extracellular fluid or shuttling of lactate to neurons from neighboring astrocytes. Astrocytes can also supply glucose to neurons as well as glucose can be taken up by neurons from extracellular fluid. Astrocytic networks can provide neuronal fuel and quickly remove lactate from activated glycolytic domains, and the lactate can be dispersed widely throughout the syncytium to endfeet along the vasculature for release to blood or other brain regions via perivascular fluid flow.

Figures

Figure 1. Lactate oxidase system to assay lactate trafficking among astrocytes and neurons
(a) Left panel: Two enzymes, lactate oxidase and horseradish peroxidase, are used to oxidize lactate and generate resorufin, a fluorescent molecule. Right panel: The stoichiometry of the reaction carried out by horseradish peroxidase (Amplex red plus hydrogen peroxide) and the coupled reaction (Amplex red plus lactate) is 1:1. The inset shows the 1:1 relationship between fluorescence of known quantities of resorufin and oxidation of known amounts of H2O2. Plotted values are representative of 6 independent assays. (b) Schematic of three types of lactate transfer assays: lactate uptake from a point source in extracellular fluid into neurons and astrocytes (scheme 1, blue arrows), lactate shuttling from astrocytes to neurons (scheme 2, green arrows), and lactate trafficking among gap junction-coupled astrocytes (scheme 3, red arrows).
Figure 2. Single cell responses to uptake extracellular lactate
Neurons (a) or astrocytes (c) were identified under differential interference contrast (DIC) in slices of inferior colliculus from adult rat brain, and a single cell was impaled with a micropipette containing the enzymatic reaction mixture plus a gap junction-permeable fluorescent dye, Lucifer yellow VS (LYVS). Dye spread labeled the impaled neuron (b) and astrocyte (d), plus gap junction-coupled astrocytes (e) and their perivascular structures (arrow), i.e., astrocytic endfeet. Then a second micropipette containing L-lactate was placed in extracellular fluid about 2 µm from the membrane of the cell containing the enzymatic reporter system (Fig 1b, scheme 1). Lactate uptake into neurons (f, g) and astrocytes (h, i) generated a fluorescent response that increased with time, more in astrocytes (i) than neurons (g). Scale bars = 10 µm and apply to all panels.
Figure 3. Standardization of the lactate assay in neurons and astrocytes in slices of inferior colliculus from adult rat brain
(a) Impaling a single cell with a micropipette containing known concentrations of resorufin gives a stable fluorescence response with repeated sampling with 100 ms exposures at 30 s intervals (n=3/group). (b) The linear ranges of standard curves were identified by diffusing known concentrations of resorufin into single neurons and astrocytes (n = 5/data point); the equation of the linear regression line is y = 28.8x + 14.1. The inset shows the non-linear fluorescence response of higher resorufin concentrations. (c) Area labeled by co-diffusion of Lucifer yellow plus resorufin into single astrocytes (n = 5/group; paired t test). (d) Net increase in fluorescence (ΔF) due to lactate uptake from extracellular fluid into astrocytes and neurons located at the indicated distances from the tip of the micropipette containing L-lactate (10 mmol/L). P values for indicated comparisons were determined by ANOVA and Tukey’s test (n=5 for astrocytes and neurons at 2 µm, n=4 for astrocytes at 50 µm). Values are means and vertical bars represent ±1SD; if not visible, SDs are smaller than the symbol.
Figure 4. Lactate uptake into astrocytes and neurons from extracellular space
Time courses of lactate uptake (ΔF) from a micropipette placed about 2 µm from the membrane of astrocytes (a) and neurons (b) in slices of adult rat inferior colliculus (see Fig. 1b, Scheme 1, blue arrows). Initial lactate uptake rates (ΔF/s, c, d) were calculated as the slope during the first 60 s (a, b), and net lactate uptake (ΔF, e, f) was calculated as the mean of the values measured at 240–300 s (a, b). Values at zero lactate (a–f) are endogenous responses to impaling the reporter cell and placing a buffer-containing pipette outside the cell; similar values were obtained by only placing the reporter pipette into the cell (data not shown; n= 5/group for astrocytes and neurons). Values are means and vertical bars are 1 SD. For astrocytes: n = 5, 15, 8, 5, and 5 for buffer, 2,10, 20, and 40 mmol/L lactate, respectively. For neurons: n = 5, 10, 5, and 5 for buffer, 2,10, and 20 mmol/L lactate, respectively. P values for comparisons (unpaired, two-tailed t test) between corresponding values for astrocytes and neurons at each lactate concentration are shown on the neuron graphs. The goodness of fit (r2) and slope (all slopes were different from zero, P < 0.006) are indicated for the linear regression lines for astrocytic (solid lines) and neuronal (dashed lines) lactate initial uptake rates (c, d) and net lactate uptake (e, f). Significant differences in the respective slopes of the astrocytic and neuronal regression lines were identified with the unpaired, two-tailed t test and are indicated in neuron graphs (d, f).
Figure 5. Lactate trafficking among gap junction-coupled astrocytes and from astrocytes to neurons
Concentration-dependent temporal profiles of lactate shuttling from (a) one astrocyte to another astrocyte (Fig. 1b, Scheme 3, red arrows) or (b) an astrocyte to a neuron (Fig. 1b, Scheme 2, green arrows) in slices of adult rat inferior colliculus were assayed by inserting a lactate-containing pipette into an astrocyte located 50 ± 5 µm from the enzyme-containing reporter cell. Initial lactate uptake rates (c, ΔF/s) and net lactate uptake (d, ΔF) were calculated from the 0–60 and 240–300 s intervals of the time courses. Lines connect the points in panels a–c. P values (unpaired, two-tailed t test) are indicated for comparisons between neuronsv and astrocytes at each lactate concentration and for the slopes of the linear regression lines in panel d for astrocytic (solid lines) and neuronal (dashed lines) net lactate uptake. Zero lactate denotes the endogenous response to impaling the reporter cell and placing a pipette outside the cell; similar control values were obtained by only placing the reporter pipette into the cell, with no second pipette (not shown, n= 5/group for astrocytes and neurons).Values are means; vertical bars are 1SD. For astrocytes: n = 5, 8, 7, and 7, for buffer, 2, 5, and 10, lactate, respectively. For neurons: n = 5, 8, 7, and 6 for buffer, 2, 5, and 10, lactate, respectively. Astrocyte-to-astrocyte lactate (10 mmol/L) transfer was gap junction-dependent; ΔF = 866 ± 511 (n = 7) and 230 ± 140 (n=5), in the absence and presence of octanol (gap junction inhibitor), respectively.
Figure 6. Glucose delivery to neurons from astrocytes
Neuronal glucose uptake from a micropipette containing 20 mmol glucose/L and located 2 µm from the neuronal membrane (a) or from an astrocyte impaled with a micropipette containing 20 mmol glucose/L and located 30–50 µm from the neuron (b) in slices of adult rat inferior colliculus. Coupled glucose oxidase-horseradish peroxidase assays were carried out in the absence or presence of cytochalasin B (10 µmol/L in the perfusion fluid) to block glucose transporters. Initial glucose uptake rates (c) and net transfer (d) were calculated from values over the 0–60s and 240–300s intervals (a, b). P values for indicated comparisons were determined with the unpaired, two-tailed t test (n=5/group).
Figure 7. Dye and metabolite trafficking via gap junction-coupled astrocytes
Astrocytes in the adult rat inferior colliculus are highly coupled by gap junctions, and diffusion of Lucifer yellow VS into a single astrocyte for 5 min labels thousands of cells (a, scale bar = 100 µm) and their perivascular endfeet (b, scale bar = 25 µm). (c) A model emphasizing diffusion and transporter-mediated pathways for (i) uptake of glucose from blood into brain and distribution within tissue and (ii) clearance of lactate from glycolytic domains via intracellular and extracellular routes during brain activation. For clarity, the tortuosity of extracellular space, cell-cell connections, and more distant non-activated cells are not portrayed. Glucose and lactate fluxes in interstitial fluid and within cells follow their local concentration gradients; hypothetical lactate gradients during brain activation are illustrated as gray-scales in intracellular and extracellular fluid and from brain to blood and thicker lines through transporters. In normal activated brain lactate is generated intracellularly and will be dispersed by diffusion and carrier-mediated co-transport with H+, which is influenced by local pH. Because Caesar et al. (2008) reported that metabolic responses (increased blood flow, CMRO2, CMRglc, extracellular lactate level) to stimulation in the cerebellum were blocked by postsynaptic AMPA receptor blockers, lactate gradients in postsynaptic neuronal domains are emphasized; recent modeling studies (Simpson et al., 2007; Mangia et al., 2009b) also support predominant neuronal glucose utilization and neuronal lactate generation, but astrocytic glycolytic activity is also likely. Because astrocytes have a faster and greater capacity for uptake and lactate trafficking than neurons (Fig. 4, Fig. 5), lactate dispersal among thousands of astrocytes via gap junctions (a) and to distant perivascular fluid (b) is likely to facilitate rapid lactate release from activated tissue domains by diffusion and transport among cells, ultimately to blood (c). Lactate in interstitial fluid is also expected to diffuse down its extracellular concentration gradient to perivascular space and blood (c) Relative rates of lactate transport (table in c) were calculated for each monocarboxylic acid transporter (MCT) isoform, assuming Michaelis-Menten kinetics and using Km values from Table 1 of Manning Fox et al. (2000). MCT2 is located mainly in neurons whereas MCT1 and MCT4 are mainly in astrocytes (Bergersen, 2007). Abbreviations: Glc, glucose; Lac, lactate; MCT, monocarboxylic acid transporter; GLUT, glucose transporter.
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