Prior 13C magnetic resonance spectroscopy (MRS) experiments, which simultaneously measured in vivo rates of total glutamate-glutamine cycling (V(cyc(tot))) and neuronal glucose oxidation (CMR(glc(ox), N)), revealed a linear relationship between these fluxes above isoelectricity, with a slope of approximately 1. In vitro glial culture studies examining glutamate uptake indicated that glutamate, which is cotransported with Na+, stimulated glial uptake of glucose and release of lactate. These in vivo and in vitro results were consolidated into a model: recycling of one molecule of neurotransmitter between glia and neurons was associated with oxidation of one glucose molecule in neurons; however, the glucose was taken up only by glia and all the lactate (pyruvate) generated by glial glycolysis was transferred to neurons for oxidation. The model was consistent with the 1:1 relationship between DeltaCMR(glc(ox), N) and DeltaV(cyc(tot)) measured by 13C MRS. However, the model could not specify the energetics of glia and gamma-amino butyric acid (GABA) neurons because quantitative values for these pathways were not available. Here, we review recent 13C and 14C tracer studies that enable us to include these fluxes in a more comprehensive model. The revised model shows that glia produce at least 8% of total oxidative ATP and GABAergic neurons generate approximately 18% of total oxidative ATP in neurons. Neurons produce at least 88% of total oxidative ATP, and take up approximately 26% of the total glucose oxidized. Glial lactate (pyruvate) still makes the major contribution to neuronal oxidation, but approximately 30% less than predicted by the prior model. The relationship observed between DeltaCMR(glc(ox), N) and DeltaV(cyc(tot)) is determined by glial glycolytic ATP as before. Quantitative aspects of the model, which can be tested by experimentation, are discussed.

Previous studies have shown that the glutamate͞glutamine (Glu͞ Gln) neurotransmitter cycle and neuronal glucose oxidation are proportional (1:1), with increasing neuronal activity above isoelectricity. GABA, a product of Glu metabolism, is synthesized from astroglial Gln and contributes to total Glu͞Gln neurotransmitter cycling, although the fraction contributed by GABA is unknown. In the present study, we used 13 C NMR spectroscopy together with i.v. infusions of [1,6-13 C2]glucose and [2-13 C]acetate to separately determine rates of Glu͞Gln and GABA͞Gln cycling and their respective tricarboxylic acid cycles in the rat cortex under conditions of halothane anesthesia and pentobarbital-induced isoelectricity. Under 1% halothane anesthesia, GABA͞Gln cycle flux comprised 23% of total (Glu plus GABA) neurotransmitter cycling and 18% of total neuronal tricarboxylic acid cycle flux. In isoelectric cortex, glucose oxidation was reduced >3-fold in glutamatergic and GABAergic neurons, and neurotransmitter cycling was below detection. Hence, in both cell types, the primary energetic costs are associated with neurotransmission, which increase together as cortical activity is increased. The contribution of GABAergic neurons and inhibition to cortical energy metabolism has broad implications for the interpretation of functional imaging signals.GABAergic neurons ͉ glutamatergic neurons ͉ magnetic resonance spectroscopy ͉ neuronal-glial interactions G lutamate (Glu) and GABA are the major excitatory and inhibitory neurotransmitters in the mature cerebral cortex and together account for Ϸ90% of total cortical synapses (1). Excitatory synapses outnumber inhibitory synapses Ϸ5:1 (2), suggesting that excitation plays an energetically dominant role in the cortex. The energetic cost of GABAergic neurotransmission remains an open question, however, because current methods used to assess cortical activity are based on changes in local blood f low or metabolism (e.g., glucose or oxygen consumption), which cannot differentiate glutamatergic from GABAergic neurons. Thus, the interpretation of changes in cortical activity in terms of the energetics of excitation and inhibition requires other methods that are sensitive to the synthesis of these neurotransmitters.Glutamatergic neurotransmitter cycling flux and energy consumption have been reported for rat and human cortex by using 13 C NMR spectroscopy during the i.v. infusion of 13 C-labeled glucose (3-10). These studies show that the Glu͞glutamine (Gln) cycling flux (V cyc ) is substantial, from Ϸ30% to 42% of neuronal tricarboxylic acid (TCA) cycle flux in anesthetized rats (V TCAn ) (4, 5, 8) to Ϸ38-50% of V TCAn in resting awake rat and human cortex (7, 9, 10). In the cortex of anesthetized rats, changes in V cyc and V TCAn are proportional (Ϸ1:1) over a large cortical activity range above isoelectricity (4,8).The determination of V cyc from Gln and Glu 13 C turnover, using [1-13 C]glucose as tracer, includes contributions from GABA as well as Glu (4,5,11). This is because Gln is a com...

Summary:13 C nuclear magnetic resonance (NMR) experiments have previously shown that glutamatergic neurotransmitter flux (V cycle(Glu/Gln) ) changes proportionately with neuronal glucose oxidation (CMR glc(ox)N ) in the nonactivated cortex of anesthetized rats. Positron Emission Tomography measurements of glucose and oxygen uptake during sensory stimulation had shown that the incremental glucose utilization is greater than oxygen leading to the suggestion that the energy required for stimulated neuronal activity arises from nonoxidative glucose metabolism. In this study, the authors used spatially localized 1 H-observed, 13 C-edited NMR spectroscopy during an infusion of [1,6-13 C 2 ]glucose to assess the relationship between changes in V cycle(Glu/Gln) and glucose utilization (CMR glc(ox)N and CMR glc(nonox) ) during the intense cortical activity associated with bicuculline-induced seizures. Metabolic fluxes were determined by model-based analysis of the 13 C-enrichment time courses of glutamate-C4 and glutamine-C4 (CMR glc(ox)N , V cycle(Glu/Gln) ) and lactate-C3 (CMR glc(nonox) ). The exchange rate between ␣-ketoglutarate and glutamate was found to be significantly faster than TCA cycle flux both for control (41 mol·g ), which was similar to the increase in glucose oxidation. The value of V c y c l e ( G l u / G l n ) and CMR glc(ox)N obtained here lie on the line predicted in a previous study. These results indicate that neuronal glucose oxidation and not total glucose utilization is coupled to the glutamate/glutamine cycle during intense cortical activation.

Previous 13C magnetic resonance spectroscopy experiments have shown that over a wide range of neuronal activity, approximately one molecule of glucose is oxidized for every molecule of glutamate released by neurons and recycled through astrocytic glutamine. The measured kinetics were shown to agree with the stoichiometry of a hypothetical astrocyte-to-neuron lactate shuttle model, which predicted negligible functional neuronal uptake of glucose. To test this model, we measured the uptake and phosphorylation of glucose in nerve terminals isolated from rats infused with the glucose analog, 2-fluoro-2-deoxy-D-glucose (FDG) in vivo. The concentrations of phosphorylated FDG (FDG 6P ), normalized with respect to known neuronal metabolites, were compared in nerve terminals, homogenate, and cortex of anesthetized rats with and without bicuculline-induced seizures. The increase in FDG 6P in nerve terminals agreed well with the increase in cortical neuronal glucose oxidation measured previously under the same conditions in vivo, indicating that direct uptake and oxidation of glucose in nerve terminals is substantial under resting and activated conditions. These results suggest that neuronal glucose-derived pyruvate is the major oxidative fuel for activated neurons, not lactate-derived from astrocytes, contradicting predictions of the original astrocyte-to-neuron lactate shuttle model under the range of study conditions. neuroenergetics | glutamate−glutamine cycle | neuronal glucose phosphorylation | synaptoneurosomes | 2-fluorodeoxyglucose M etabolic and neurophysiological research has experimentally related brain energy consumption, in the form of glucose oxidation, to the brain work supporting neuronal firing. Carbon-13 magnetic resonance spectroscopy (MRS) measurements (1, 2) of the associated fluxes in cerebral cortex of anesthetized rats over a range of electrical activity revealed, surprisingly, a near 1:1 relationship (in molar equivalent units) between increments in the glutamate−glutamine neurotransmitter cycle and neuronal glucose oxidation. Subsequent studies of rat and human cerebral cortex have been consistent with this finding (3, 4). The near 1:1 flux relation was consistent with a cellular/ molecular model, originally proposed by Pellerin and Magistretti (5), and subsequently expanded to include the glutamate/glutamine cycle (1, 6). Evidence for the astrocyte-to-neuron lactate shuttle (ANLS) model is summarized in ref. 7. In this model (Fig. 1A), glutamate released from neurons is taken up by astrocytes and converted to glutamine using ATP derived from glycolysis. Lactate produced by this process is transferred to neurons where oxidation occurs. This ANLS model predicts a 1:1 relationship between increments in astrocytic glutamate uptake and glycolysis. Glycolytically derived ATP might provide for more rapid clearance of glutamate from the synaptic cleft into astrocyte processes devoid of mitochondria (8).The ANLS hypothesis has been challenged on biochemical, in vivo, in situ, and in vitro experimental a...

ABSTRACT:13 C NMR spectroscopy in combination with the infusion of 13 C-labeled precursors is currently the only technique that is capable of quantitatively studying energy metabolism, neurotransmission and other metabolic pathways non-invasively in vivo. H-[13 C]-NMR spectroscopy is a high-sensitivity alternative to direct 13 C NMR spectroscopy. The development of improved NMR methods for water suppression, spatial localization, broadband decoupling, shimming and signal quantification, together with the availability of high magnetic field strengths, has made 1 H-[13 C]-NMR spectroscopy the method of choice for the detection of metabolism at a high spatial and/or temporal resolution. H-[13 C]-NMR spectroscopy can now be used to discriminate glutamatergic (excitatory) and GABAergic (inhibitory) neuronal activity. The improved sensitivity allows the detection of metabolism in different tissues (e.g. gray and white matter) and potentially even in smaller structures, like cortical layers. Finally, 1 H-[ 13 C]-NMR spectroscopy allows the detection of energy metabolism and neurotransmission during functional activation, thereby further strengthening our understanding of the neurochemical basis of brain function.

Acetate is a well-known astrocyte-specific substrate that has been used extensively to probe astrocytic function in vitro and in vivo. Analysis of amino acid turnover curves from 13C-acetate has been limited mainly to estimation of first-order rate constants from exponential fitting or calculation of relative rates from steady-state 13C enrichments. In this study we used 1H-[13C]-NMR spectroscopy with intravenous infusion of [2-13C]acetate-Na+ in vivo to measure the cerebral kinetics of acetate transport and utilization in anesthetized rats. Kinetics were assessed using a two-compartment (neuron/astrocyte) analysis of the 13C turnover curves of glutamate-C4 and glutamine-C4 from [2-13C]acetate-Na+, brain acetate levels, and the dependence of steady state glutamine-C4 enrichment on blood acetate levels. The steady-state enrichment of glutamine-C4 increased with blood acetate concentration until 90% of plateau for plasma acetate of 4–5 mM. Analysis_assuming reversible, symmetric Michaelis-Menten kinetics for transport yielded 27±2 mM and 1.3±0.3 µmol/g/min for Kt and Tmax, respectively, and for utilization, 0.17±0.24 mM and 0.14±0.02 µmol/g/min for KM_util and Vmax_util, respectively. The distribution space for acetate was only 0.32±0.12 mL/g, indicative of a large excluded volume. The astrocytic and neuronal TCA cycle fluxes were 0.37±0.03 µmol/g/min and 1.41±0.11 µmol/g, respectively; astrocytes thus comprised ~21±3% of total oxidative metabolism.

, and is indicative of a glutamate (Glu)͞glutamine (Gln) neurotransmitter cycling flux between glutamatergic neurons and surrounding astroglia. Although 13 C NMR spectroscopy offers a high spectral resolution (1), it suffers from an inherently low sensitivity, thereby limiting the detection to relatively large volumes. As an alternative to direct 13 C NMR spectroscopy, the protons bound to 13 C atoms can be detected by proton-observed, carbon-edited 1 H-[ C]Glx (Glx ϭ Glu ϩ Gln) is measured in volumes that span the cerebral cortex, corpus callosum, hippocampus, and thalamus. In combination with quantitative tissue segmentation by T 1 relaxation mapping and multicompartment metabolic modeling, the rates of the neuronal TCA cycle and the Glu͞Gln neurotransmitter cycle can be calculated in pure cerebral gray matter and white matter. MethodsAnimal Preparation. Six male Sprague-Dawley rats (140 Ϯ 12 g, mean Ϯ SD) were studied in accordance with the guidelines established by the Yale Animal Care and Use Committee. After an overnight fast (12-16 h), the animals were tracheotomized and ventilated with a mixture of 70% nitrous oxide and 28.5% oxygen under 1.5% halothane anesthesia. A femoral artery was cannulated for monitoring of blood gases (pO 2 and pCO 2 ), pH, and blood pressure. Physiological variables were maintained within normal limits by small adjustments in ventilation [pCO 2 ϭ 33-42 mmHg; pO 2 Ͼ 120 mmHg; pH ϭ 7.30-7.58; blood pressure ϭ 95-110 mmHg (1 mmHg ϭ 133 Pa)]. A femoral vein was cannulated for infusion of [1,[6][7][8][9][10][11][12][13] C 2 ]glucose. After all of the surgeries were completed, anesthesia was maintained by 0.3-0.8% halothane in combination with 70% nitrous oxide. During NMR experiments animals were restrained in a head holder, and additional immobilization was achieved with D-tubocurarine chloride (0.5 mg͞kg every 40 min, i.p.). The core body temperature was measured with a rectal thermosensor and was maintained at 37 Ϯ 1°C by means of a heated water pad. The animals were infused with [1,6-13 C 2 ]glucose (Cambridge Isotope Laboratories, Cambridge, MA) according to a protocol described in ref.13. Blood samples were taken before infusion and every 25 min after the start of infusion. The plasma glucose fractional 13 C enrichments were measured by GC-MS.In Vivo 1 H NMR Spectroscopy. Experiments were performed on a 7.05-T Bruker magnet and console (Billerica, MA) equipped with a 12-cm-diameter actively shielded gradient coil insert (190 mT͞m in 200 s). Radiofrequency pulse transmission and NMR signal reception for protons (300.3 MHz) were performed with a 14-mm-diameter surface coil. Radiofrequency pulse transmission on carbon-13 (75.5 MHz) was achieved with two orthogonal 21-mm-diameter surface coils driven in quadrature. The magnetic field homogeneity over the volume-of-interest was optimized with the FASTMAP algorithm (14), which resulted in signal linewidths of 10-13 Hz for water and 8-9 Hz for metabolites in a volume of 100 l. (15), an adiabatic sequence employing frequency-selectiv...

Succinic semialdehyde dehydrogenase (SSADH) catalyzes the NADP-dependent oxidation of succinic semialdehyde to succinate, the final step of the GABA shunt pathway. SSADH deficiency in humans is associated with excessive elevation of GABA and c-hydroxybutyrate (GHB). Recent studies of SSADH-null mice show that elevated GABA and GHB are accompanied by reduced glutamine, a known precursor of the neurotransmitters glutamate and GABA. In this study, cerebral metabolism was investigated in urethane-anesthetized SSADH-null and wild-type 17-day-old mice by intraperitoneal infusion of [1,[6][7][8][9][10][11][12][13] C incorporated per gram of brain tissue) for glutamate-(C4,C3), glutamine-C4, succinate-(C3/2), and aspartate-C3 in SSADH-null cortex, whereas Ala-C3 was higher and GABA-C2 unchanged.13 C Labeling from [2-13 C]acetate, a glial substrate, was lower mainly in glutamine-C4 and glutamate-(C4,C3). GHB was labeled by both substrates in SSADH-null mice consistent with GABA as precursor. Our findings indicate that SSADH deficiency is associated with major alterations in glutamate and glutamine metabolism in glia and neurons with surprisingly lesser effects on GABA synthesis.