Regional glucose metabolism and glutamatergic neurotransmission in rat brainin vivo

Abstract

, 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...

Modeling of Brain Metabolism and Pyruvate Compartmentation Using 13C NMR in Vivo: Caution Required

“…Although the model currently allows the use of [1,[6][7][8][9][10][11][12][13] C 2 ]glucose only as enriched substrate, it can be extended to simultaneously analyze mixtures of substrates with appropriately different labeling patterns. This will help determine the extent to which different substrates are used in different compartments of the brain.…”

“…[10][11][12][13] In this study common assumptions involving the metabolism involved are those found in Figure 1 and outlined in the Materials and Methods section. All flux and substrate enrichment parameters were free parameters, determined only by fitting to the measured data.…”

“…The time courses of 13 C multiplets used in this study were presented in a report on the metabolism of [1,[6][7][8][9][10][11][12][13] C 2 ]glucose in the rat brain. 4 The measurements from glutamine and glutamate were analyzed.…”

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Two variants of a widely used two-compartment model were prepared for fitting the time course of [1,[6][7][8][9][10][11][12][13] C 2 ]glucose metabolism in rat brain. Features common to most models were included, but in one model the enrichment of the substrates entering the glia and neuronal citric acid cycles was allowed to differ.

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1H‐[13C] NMR spectroscopy of the rat brain during infusion of [2‐13C] acetate at 14.1 T

“…To increase the sensitivity, indirect detection of 13 C through the more sensitive 1 H nuclei is an alternative, albeit at the expense of a lower spectral resolution, such as the partial separation of labeling in Glu and Gln (23,25), which limits the achievable dynamic information and the reliability of the two-compartment metabolic modeling (20). For instance, Pfeuffer et al (23) proposed a modified stimulated echo acquisition mode localization for performing adiabatic carbon editing and decoupling, and demonstrated the measurement of GluC4 and GlnC4 time courses with glucose infusion at 9.4 T. A proton-observed carbon-edited NMR sequence (7) based on three-dimensional image-selected in vivo spectroscopy localization was performed with glucose infusion at 7 T in rat brain, and the reported time courses were limited to GluC4, GlnC4, and GluC3 þ GlnC3 (27).…”