13C MRS studies of neuroenergetics and neurotransmitter cycling in humans

Abstract

In the last 25 years, (13)C MRS has been established as the only noninvasive method for the measurement of glutamate neurotransmission and cell-specific neuroenergetics. Although technically and experimentally challenging, (13)C MRS has already provided important new information on the relationship between neuroenergetics and neuronal function, the energy cost of brain function, the high neuronal activity in the resting brain state and how neuroenergetics and neurotransmitter cycling are altered in neurological and psychiatric disease. In this article, the current state of (13)C MRS as it is applied to the study of neuroenergetics and neurotransmitter cycling in humans is reviewed. The focus is predominantly on recent findings in humans regarding metabolic pathways, applications to clinical research and the technical status of the method. Results from in vivo (13)C MRS studies in animals are discussed from the standpoint of the validation of MRS measurements of neuroenergetics and neurotransmitter cycling, and where they have helped to identify key questions to address in human research. Controversies concerning the relationship between neuroenergetics and neurotransmitter cycling and factors having an impact on the accurate determination of fluxes through mathematical modeling are addressed. We further touch upon different (13)C-labeled substrates used to study brain metabolism, before reviewing a number of human brain diseases investigated using (13)C MRS. Future technological developments are discussed that will help to overcome the limitations of (13)C MRS, with special attention given to recent developments in hyperpolarized (13)C MRS.

Figure 1. Diagram of the glutamate/glutamine cycle

The figure above shows a schematic of metabolic pathways within glutamatergic neurons and surrounding astroglial cells. Glucose and lactate will enter both the glial (V_TCAa) and neuronal TCA cycles (V_TCAn) via pyruvate dehydrogenase (V_pdh), beta-hydroxybutyrate is directly incorporated into the neuronal and astroglial TCA cycles while acetate is near exclusively incorporated into the glial TCA cycle. Neuronal glutamate (Glu) that is released via neurotransmission will be taken up by astroglial cells and converted by glutamine synthetase to glutamine at a rate proportional to the glutamate/glutamine cycle. The synthesis of glutamine is believed to be exclusively within astroglia and other glial cells. In addition to neurotransmitter cycling glutamine may be synthesized de novo starting with the pyruvate carboxylase (PC) reaction (V_PC). Glutamine synthesized via PC can replace neurotransmitter glutamate oxidized in the astrocyte or elsewhere (and be recycled back to the neuron) or leave the brain (V_efflux) to remove ammonia and maintain nitrogen balance (5,17,26). To measure the rates of these pathways ¹³C labeled substrates are used and the flow of ¹³C isotope into glutamate and glutamine measured using ¹³C MRS. For detailed descriptions of how these pathways are tracked using ¹³C MRS and isotopically labeled substrates and rates then calculated by metabolic modeling see (–26,44,45,47,143).

Figure 2

Localized ¹³C MR spectra acquired at 4 Tesla from the midline occipito-parietal lobe of a volunteer infused with ¹³C-labeled glucose, lactate or acetate. Upper spectrum: acquired during the last 18 min of a 2-hour [1-¹³C] glucose infusion. Middle spectrum: acquired during the last 18 min of a 2-hour [3-¹³C] lactate infusion ([Lac]_Plasma ~1.5 mmol/L and ¹³C fractional enrichment, ~30%). Lower spectrum: acquired during the last 18 min of a 2-hour [2-¹³C] acetate infusion. Spectra are scaled to NAA C3 to exhibit the differences in ¹³C fractional enrichment reached for glutamate (Glu) and glutamine (Gln) and aspartate. The highest fractional enrichment is attained with glucose as label precursor. For glucose or lactate as precursor the majority of labeling appears in glutamate C4, consistent with the majority of brain metabolism of these substrates occurring in the neurons which contain the majority of glutamate under normal conditions (25). In contrast label from [2-¹³C] acetate is highly enriched in glutamine C4, consistent with the localization of acetate metabolism in the astrocyte TCA cycle as shown in figure 1 resulting in preferential labeling of glutamine C4. Abbreviations Glu: Glutamate, Gln: Glutamine, Asp: Aspartate, NAA: N-Acetyl aspartate, GABA: Gamma-Aminobutyric Acid.

Figure 3. Approximately 1:1 relationship between the neuronal TCA cycle (0.5 * V_TCAn) and the glutamate/glutamine cycle (V_cyc) with increasing electrical activity in the rat cerebral cortex

The plot shows the mean values of 0.5 * V_TCAn (equivalent to CMR_glc(ox)N in Sibson et al. (28)) plotted versus V_cyc reported from 11 published studies at activity levels ranging from awake to isoelectricity (,,,,,,,–153). Regression analysis yields a slope of 0.89 (R² = 0.92) and an intercept of 0.5 * V_TCAn of 0.09 at isoelectricity (V_cyc ~0), values similar to those found in the original 1998 study by Sibson et al. (28). In the case of reference (39) for both anesthetized and awake state, values of V_TCAn were calculated from the time constants reported for glutamate turnover during a glucose infusion. The ratio of glutamate to glutamine steady state fractional enrichment during [2-¹³C] acetate infusion was used to calculate V_cyc using the equation described in Lebon et al. (25).

Figure 4. Comparison of V_TCAn versus glutamate and NAA concentrations in the midline occipital parietal lobe of healthy elderly subjects

The results show a strong correlation between the rate of the neuronal TCA cycle and the concentrations of glutamate (Glu) and NAA, both of which have been associated with cellular dysfunction and chronically reduced mitochondrial activity in other studies. Pearson correlation coefficients appear in upper left corners of a and b Closed circles, values measured for the individual elderly subjects (n=7). Open circles, average values for the respective metabolite concentrations from a young cohort (n=7). Fluxes and metabolite concentrations are expressed as µmol.g⁻¹.min⁻¹ and µmol.g⁻¹ respectively.

Figure 5. Comparison of steady state ¹³C MRS spectra during [2-¹³C] acetate infusion from a type 1 diabetic with a healthy control subject

Brain ¹³C MRS spectra were averaged over the final 45 min of a hypoglycemic period during infusion of [2-¹³C] acetate. The diabetic subject (top spectrum) had significantly greater labeling in glutamate (Glu) and glutamine (Gln) C4 than the control (bottom spectrum). The acetate C2 signal was also greater in the diabetic subject. Other resonances labeled in the figure include N-acetylaspartate (NAA) C4, GABA C2, and Glu C3.

Figure 6. ¹³C MRS time course spectra of glutamate, glutamine, and aspartate turnover detected in the occipital lobe during intravenous infusion of [2-¹³C] glucose

Lorentz–Gauss transformation (LB = 3 Hz, GB = −0.3) was applied. The time-averaged decoupling power was 1.46 W. Each spectrum corresponds to 8.5-min of signal averaging (128 scans). Glu C5 (182.0 ppm) and C1 (175.4 ppm), Gln C5 (178.5 ppm) and C1 (174.9 ppm), Asp C4 (178.3 ppm) and C1 (175.0 ppm), as well as NAA C5 (174.3 ppm) were detected. No baseline corrections were made. Glu, glutamate; Gln, glutamine; Asp, aspartate; NAA, N-acetylaspartate. Figure adapted from Li et al. (141).