High-resolution detection of ¹³C multiplets from the conscious mouse brain by ex vivo NMR spectroscopy

Abstract

Glucose readily supplies the brain with the majority of carbon needed to sustain neurotransmitter production and utilization. The rate of brain glucose metabolism can be computed using (13)C nuclear magnetic resonance (NMR) spectroscopy by detecting changes in (13)C contents of products generated by cerebral metabolism. As previously observed, scalar coupling between adjacent (13)C carbons (multiplets) can provide additional information to (13)C contents for the computation of metabolic rates. Most NMR studies have been conducted in large animals (often under anesthesia) because the mass of the target organ is a limiting factor for NMR. Yet, despite the challengingly small size of the mouse brain, NMR studies are highly desirable because the mouse constitutes a common animal model for human neurological disorders. We have developed a method for the ex vivo resolution of NMR multiplets arising from the brain of an awake mouse after the infusion of [1,6-(13)C(2)]glucose. NMR spectra obtained by this method display favorable signal-to-noise ratios. With this infusion protocol, the (13)C multiplets of glutamate, glutamine, GABA and aspartate achieved steady state after 150 min. The method enables the accurate resolution of multiplets over time in the awake mouse brain. We anticipate that this method can be broadly applicable to compute brain fluxes in normal and transgenic mouse models of neurological disorders.

Copyright © 2011 Elsevier B.V. All rights reserved.

Figures

FIGURE 1. Evolution of glutamate labeling in the mouse brain TCA cycle

Schematic of the key reactions of the TCA cycle and the consequent 13C-labeling and spectral pattern of brain glutamate (GLU) labeled in position 4 after the administration of [1,6-13C2]glucose. The intermediates citrate (CIT), α-ketoglutarate (α-KG), succinate (SUC) and oxaloacetate (OAA), are numbered from bottom (carbon 1) to top. This model assumes that the only source of 13C for the TCA cycle is acetyl-CoA and that 100% of the acetyl-CoA is [2-13C]acetyl-CoA. The resulting labeling pattern of glutamate via TCA cycle intermediate metabolism and the predicted 13C NMR spectrum of glutamate arising from each “turn” of the cycle (number to the left) are shown. Note that carbons 4 and 5 of glutamate are derived directly from acetyl-CoA and carbons 1, 2 and 3 originate from oxaloacetate.

FIGURE 2. 13C-NMR spectrum of the mouse forebrain at 75 min of infusion

Insets display the multiplets of some isotopomers detected in the 13C spectrum. GLU: glutamate, GLN: glutamine, ASP: aspartate, NAA: n-acetylaspartate, TAU: taurine. C#: carbon labeled in position #. Sx: singlet, Dxx: doublet, T: triplet, Q: quartet.

FIGURE 3. Evolution of 13C multiplets of GABA C2, glutamate C3, C4 and glutamine C4 over time

The largest resonance achievable for each isotopomer is reached at 150 min of 13C-glucose infusion initiation.

FIGURE 4. Time course of 13C multiplets of glutamate, glutamine, GABA and aspartate in all carbon positions as detected by 13C NMR spectroscopy

The multiplet fractional amount is calculated by the area of each multiplet relative to the total resonance area of the isotopomer as described in the methods section. Overall, the fractional amount of each multiplet in each isotopomer reaches steady state at 150 min with the present infusion protocol, implying brain metabolic equilibrium. Open square: singlet; C2: open circle: doublet12, closed circle: doublet23, diamond: quartet; C3: open circle: doublet, closed square: triplet. C4: open circle: doublet34. For aspartate C3: open circle: doublet23, closed circle: doublet34.