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2.  Distribution of temperature changes and neurovascular coupling in rat brain following 3,4-methylenedioxymethamphetamine (MDMA,‘ecstasy’) exposure 
NMR in biomedicine  2015;28(10):1257-1266.
(+/−)3,4-methylenedioxymethamphetamine (MDMA, ‘ecstasy’) is an abused psychostimulant producing strong monoaminergic stimulation and whole-body hyperthermia. MDMA-induced thermogenesis involves activation of uncoupling proteins (UCP), primarily a type specific to skeletal muscle (UCP-3) and which is absent in brain, although other UCP types are expressed in brain (e.g., thalamus) and might contribute to thermogenesis. Since neuroimaging of brain temperature could provide insights of MDMA action, we measured spatial distributions of systemically-administered MDMA-induced temperature changes and dynamics in rat cortex and subcortex using a novel magnetic resonance method, Biosensor Imaging of Redundant Deviation of Shifts (BIRDS), with an exogenous temperature-sensitive probe (thulium ion and macrocyclic chelate 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetramethyl-1,4,7,10-tetraacetate (DOTMA4−)). The MDMA-induced temperature rise in cortex was greater than in subcortex (1.6±0.4°C vs. 1.3±0.4°C) and occurred more rapidly (2.0±0.2°C/h vs. 1.5±0.2°C/h). MDMA-induced temperature changes and dynamics in cortex and body were correlated, although body temperature exceeded cortex before and after MDMA. Temperature, neuronal activity, and blood flow (CBF) were measured simultaneously in cortex and subcortex (i.e., thalamus) to investigate possible differences of MDMA-induced warming across brain regions. MDMA-induced warming correlated with increases in neuronal activity and blood flow in cortex, suggesting that the normal neurovascular response to increased neural activity was maintained. In contrast to cortex, a biphasic relationship was seen in subcortex (i.e., thalamus), with a decline in CBF as temperature and neural activity rose, transitioning to a rise in CBF for temperature >37°C, suggesting that MDMA affected CBF and neurovascular coupling differently in subcortical regions. Considering that MDMA effects on CBF and heat dissipation (as well as potential heat generation) may vary regionally, neuroprotection may require different cooling strategies.
PMCID: PMC4573923  PMID: 26286889
BIRDS; blood flow; ecstasy; heat; oxidative metabolism; TmDOTMA−
3.  Effects of GAT1 Inhibition by Tiagabine on Brain Glutamate and GABA Metabolism in the Anesthetized Rat In Vivo 
Journal of neuroscience research  2015;93(7):1101-1108.
GABA clearance from the extracellular space after release from neurons involves reuptake into terminals and astrocytes through GABA transporters. The relative flows through these two pathways for GABA released from neurons remains unclear. In this study we determined the effect of tiagabine, a selective inhibitor of neuronal GABA transporter-1 on the rates of glutamate and GABA metabolism, and GABA re-synthesis via the GABA-glutamine cycle. Halothane anesthetized rats were administered tiagabine (30 mg/kg, i.p.), and 45 min later received an intravenous infusion of either [1,6-13C2]glucose (in vivo) or [2-13C]acetate (ex vivo). Non-treated rats served as controls. Metabolites and 13C enrichments were measured with 1H-[13C]-NMR spectroscopy and referenced to their corresponding end-point values measured in extracts from in situ frozen brain. Metabolic flux estimates of GABAergic and glutamatergic neurons were determined by fitting a metabolic model to the 13C turnover data measured in vivo during [1,6-13C2]glucose infusion. Tiagabine-treated rats were indistinguishable (P>0.05) from control in tissue amino acid levels or 13C enrichments from [2-13C]acetate. Tiagabine reduced average rates of glucose oxidation and neurotransmitter cycling in both glutamatergic neurons (↓18%, CMRglc(ox)Glu: control, 0.27±0.05 vs. tiagabine, 0.22±0.04 μmol/g/min; ↓11%, Vcyc(Glu-Gln): control, 0.23±0.05 vs. tiagabine, 0.21±0.04 μmol/g/min) and GABAergic neurons (↓18-25%, CMRglc(ox)GABA: control, 0.09±0.02 vs. tiagabine, 0.07±0.03 μmol/g/min; Vcyc(GABA-Gln): control, 0.08±0.02 vs. tiagabine, 0.07±0.03 μmol/g/min), but the changes in glutamatergic and GABAergic fluxes were not significant (P>0.10). The results suggest that any reduction in GABA metabolism by tiagabine might be an indirect response to reduced glutamatergic drive rather than direct compensatory effects.
PMCID: PMC4441585  PMID: 25663257
Glutamate-GABA-glutamine cycle; GABA reuptake; GAT-1 transporter; 13C turnover; NMR spectroscopy
4.  Characterization of cerebral glutamine uptake from blood in the mouse brain: implications for metabolic modeling of 13C NMR data 
13C Nuclear Magnetic Resonance (NMR) studies of rodent and human brain using [1-13C]/[1,6-13C2]glucose as labeled substrate have consistently found a lower enrichment (∼25% to 30%) of glutamine-C4 compared with glutamate-C4 at isotopic steady state. The source of this isotope dilution has not been established experimentally but may potentially arise either from blood/brain exchange of glutamine or from metabolism of unlabeled substrates in astrocytes, where glutamine synthesis occurs. In this study, the contribution of the former was evaluated ex vivo using 1H-[13C]-NMR spectroscopy together with intravenous infusion of [U-13C5]glutamine for 3, 15, 30, and 60 minutes in mice. 13C labeling of brain glutamine was found to be saturated at plasma glutamine levels >1.0 mmol/L. Fitting a blood–astrocyte–neuron metabolic model to the 13C enrichment time courses of glutamate and glutamine yielded the value of glutamine influx, VGln(in), 0.036±0.002 μmol/g per minute for plasma glutamine of 1.8 mmol/L. For physiologic plasma glutamine level (∼0.6 mmol/L), VGln(in) would be ∼0.010 μmol/g per minute, which corresponds to ∼6% of the glutamine synthesis rate and rises to ∼11% for saturating blood glutamine concentrations. Thus, glutamine influx from blood contributes at most ∼20% to the dilution of astroglial glutamine-C4 consistently seen in metabolic studies using [1-13C]glucose.
PMCID: PMC4269725  PMID: 25074745
glutamate; neurotransmitter; nuclear magnetic resonance spectroscopy
5.  The contribution of ketone bodies to basal and activity-dependent neuronal oxidation in vivo 
The capacity of ketone bodies to replace glucose in support of neuronal function is unresolved. Here, we determined the contributions of glucose and ketone bodies to neocortical oxidative metabolism over a large range of brain activity in rats fasted 36 hours and infused intravenously with [2,4-13C2]-D-β-hydroxybutyrate (BHB). Three animal groups and conditions were studied: awake ex vivo, pentobarbital-induced isoelectricity ex vivo, and halothane-anesthetized in vivo, the latter data reanalyzed from a recent study. Rates of neuronal acetyl-CoA oxidation from ketone bodies (VacCoA-kbN) and pyruvate (VpdhN), and the glutamate-glutamine cycle (Vcyc) were determined by metabolic modeling of 13C label trapped in major brain amino acid pools. VacCoA-kbN increased gradually with increasing activity, as compared with the steeper change in tricarboxylic acid (TCA) cycle rate (VtcaN), supporting a decreasing percentage of neuronal ketone oxidation: ∼100% (isoelectricity), 56% (halothane anesthesia), 36% (awake) with the BHB plasma levels achieved in our experiments (6 to 13 mM). In awake animals ketone oxidation reached saturation for blood levels >17 mM, accounting for 62% of neuronal substrate oxidation, the remainder (38%) provided by glucose. We conclude that ketone bodies present at sufficient concentration to saturate metabolism provides full support of basal (housekeeping) energy needs and up to approximately half of the activity-dependent oxidative needs of neurons.
PMCID: PMC4083391  PMID: 24780902
13C isotopes; glucose utilization; glutamate/glutamine cycle; ketone body utilization; neuroenergetics; nuclear magnetic resonance spectroscopy
6.  Detection of cerebral NAD+ by in vivo 1H NMR spectroscopy 
NMR in biomedicine  2014;27(7):802-809.
Nicotinamide adenine dinucleotide (NAD+) plays a central role in cellular metabolism both as coenzyme for electron-transfer enzymes as well as a substrate for a wide range of metabolic pathways. In the current study NAD+ was detected on rat brain in vivo at 11.7 T by 3D localized 1H MRS of the NAD+ nicotinamide protons in the 8.7 – 9.5 ppm spectral region. Avoiding water perturbation was critical to the detection of NAD+ as strong, possibly indirect cross-relaxation between NAD+ and water would lead to a several-fold reduction of the NAD+ intensity in the presence of water suppression. Water perturbation was minimized through the use of LASER localization in combination with frequency-selective excitation. The NAD+ concentration in the rat cerebral cortex was determined at 296 ± 28 μmol/L, which is in good agreement with recently published 31P NMR-based results as well as results from brain extracts in vitro (355 ± 34 μmol/L). The T1 relaxation time constants of the NAD+ nicotinamide protons as measured by inversion recovery were 280 ± 65 ms and 1136 ± 122 ms in the absence and presence of water inversion, respectively. This confirms the strong interaction between NAD+ nicotinamide and water protons as observed during water suppression. The T2 relaxation time constants of the NAD+ nicotinamide protons were determined at 60 ± 13 ms after confounding effects of scalar coupling evolution were taken into account. The simplicity of the MR sequence together with the robustness of NAD+ signal detection and quantification makes the presented method a convenient choice for studies on NAD+ metabolism and function. As the method does not critically rely on magnetic field homogeneity and spectral resolution it should find immediate applications in rodents and humans even at lower magnetic fields.
Graphical abstract
The NAD+ nicotinamide H2, H4 and H6 resonances were detected and quantified in rat brain in vivo at 11.7 T. Minimizing water perturbation by frequency-selective excitation (B) was critical for NAD+ detection as cross-relaxation between NAD+ and water would lead to NAD+ signal destruction in the presence of water suppression (A). Detailed NAD+ T1 and T2 measurements further characterized the in vivo NAD+ signal.
PMCID: PMC4459131  PMID: 24831866
NAD+; 1H MRS; brain; T1; T2; water
7.  Quantification of High-Resolution 1H-[13C] NMR Spectra from Rat Brain Extracts 
Analytical Chemistry  2014;86(10):5032-5038.
NMR spectroscopy in combination with 13C-labeled substrate infusion is a unique technique to obtain information about dynamic metabolic fluxes noninvasively in vivo. In many cases, the in vivo information content obtained during dynamic 13C studies in rodents can be enhanced by high-resolution 1H-[13C] NMR spectroscopy on brain extracts. Previously, it has been shown that 1H NMR spectra from rat brain extracts can be accurately quantified with a spectral fitting routine utilizing simulated basis sets using complete prior knowledge of chemical shifts and scalar couplings. The introduction of 13C label into the various metabolites presents complications that demand modifications of the spectral fitting routine. As different multiplets within a given molecule accumulate various amounts of 13C label, the fixed amplitude relationship between multiplets typical for 1H NMR spectra must be abandoned. In addition, 13C isotope effects lead to spectral multiplet patterns that become dependent on the amount of 13C label accumulation, thereby preventing the use of a common basis set. Here a modified spectral fitting routine is presented that accommodates variable 13C label accumulation and 13C isotope effects. Spectral fitting results are quantitatively compared to manual integration on column-separated samples in which spectral overlap is minimized.
PMCID: PMC4033633  PMID: 24773047
8.  Functional MRI and neural responses in a rat model of Alzheimer’s disease 
NeuroImage  2013;79:404-411.
Based on the hypothesis that brain plaques and tangles can affect cortical functions in Alzheimer's disease (AD) and thus modify functional activity, we investigated functional responses in an AD rat model (called the Samaritan Alzheimer’s rat achieved by ventricular infusion of amyloid peptide) and age-matched healthy control. High-field functional magnetic resonance imaging (fMRI) and extracellular neural activity measurements were applied to characterize sensory-evoked responses. Electrical stimulation of the forepaw led to BOLD and neural responses in the contralateral somatosensory cortex and thalamus. In AD brain we noted much smaller BOLD activation patterns in the somatosensory cortex (i.e., about 50% less activated voxels compared to normal brain). While magnitudes of BOLD and neural responses in the cerebral cortex were markedly attenuated in AD rats compared to normal rats (by about 50%), the dynamic coupling between the BOLD and neural responses in the cerebral cortex, as assessed by transfer function analysis, remained unaltered between the groups. However thalamic BOLD and neural responses were unaltered in AD brain compared to controls. Thus cortical responses in the AD model were indeed diminished compared to controls, but the thalamic responses in the AD and control rats were quite similar. Therefore these results suggest that Alzheimer’s disease may affect cortical function more than subcortical function, which may have implications for interpreting altered human brain functional responses in fMRI studies of Alzheimer’s disease.
PMCID: PMC3700380  PMID: 23648961
cognitive dementia; thalamocortical responses; neurovascular coupling; aging; energy metabolism; neuroimaging
9.  Is there in vivo evidence for amino acid shuttles carrying ammonia from neurons to astrocytes? 
Neurochemical research  2012;37(11):2597-2612.
The high in vivo flux of the glutamate/glutamine cycle puts a strong demand on the return of ammonia released by phosphate activated glutaminase from the neurons to the astrocytes in order to maintain nitrogen balance. In this paper we review several amino acid shuttles that have been proposed for balancing the nitrogen flows between neurons and astrocytes in the glutamate/glutamine cycle. All of these cycles depend on the directionality of glutamate dehydrogenase, catalyzing reductive glutamate synthesis (forward reaction) in the neuron in order to capture the ammonia released by phosphate activated glutaminase, while catalyzing oxidative deamination of glutamate (reverse reaction) in the astrocytes to release ammonia for glutamine synthesis. Reanalysis of results from in vivo experiments using 13N and 15N labeled ammonia and leucine in rats suggests that the maximum flux of the alanine/lactate or branched chain amino acid/branched chain amino acid transaminase shuttles between neurons and astrocytes are approximately 3-5 times lower than would be required to account for the ammonia transfer from neurons to astrocytes needed for glutamine synthesis (amide nitrogen) to sustain the glutamate/glutamine cycle. However, in the rat brain both the total ammonia fixation rate by glutamate dehydrogenase and the total branched chain amino acid transaminase activity are sufficient to support a branched chain amino acid/branched chain keto acid shuttle, as proposed by Hutson and coworkers, which would support the de novo synthesis of glutamine in the astrocyte to replace the ∼ 20% of neurotransmitter glutamate that is oxidized. A higher fraction of the nitrogen needs of total glutamate neurotransmitter cycling could be supported by hybrid cycles in which glutamate and tricarboxylic acid cycle intermediates act as a nitrogen shuttle. A limitation of all in vivo studies in animals conducted to date is that none have shown transfer of nitrogen for glutamine amide synthesis, either as free ammonia or via an amino acid from the neurons to the astrocytes. Future work will be needed, perhaps using methods for selectively labeling nitrogen in neurons, to conclusively establish the rate of amino acid nitrogen shuttles in vivo and their coupling to the glutamate/glutamine cycle.
PMCID: PMC3702378  PMID: 23104556
10.  Roles of Glutamine Synthetase Inhibition in Epilepsy 
Neurochemical research  2012;37(11):2339-2350.
Glutamine synthetase (GS, E.C. is a ubiquitous and highly compartmentalized enzyme that is critically involved in several metabolic pathways in the brain, including the glutamine-glutamate-GABA cycle and detoxification of ammonia. GS is normally localized to the cytoplasm of most astrocytes, with elevated concentrations of the enzyme being present in perivascular endfeet and in processes close to excitatory synapses. Interestingly, an increasing number of studies have indicated that the expression, distribution, or activity of brain GS is altered in several brain disorders, including Alzheimer’s disease, schizophrenia, depression, suicidality, and mesial temporal lobe epilepsy (MTLE). Although the metabolic and functional sequelae of brain GS perturbations are not fully understood, it is likely that a deficiency in brain GS will have a significant biological impact due to the critical metabolic role of the enzyme. Furthermore, it is possible that restoration of GS in astrocytes lacking the enzyme could constitute a novel and highly specific therapy for these disorders. The goals of this review are to summarize key features of mammalian GS under normal conditions, and discuss the consequences of GS deficiency in brain disorders, specifically MTLE.
PMCID: PMC3731630  PMID: 22488332
Astrocytes; Glutamate; Hippocampus; Seizures; Temporal lobe epilepsy
11.  Quantification of High-Resolution 1H NMR Spectra from Rat Brain Extracts 
Analytical chemistry  2010;83(1):216-224.
Extracting quantitative information about absolute concentrations from high-resolution 1H NMR spectra of complex mixtures such as brain extracts remains challenging. Partial overlap of resonances complicates integration, whereas simple line fitting algorithms cannot accommodate the spectral complexity of coupled spin-systems. Here it is shown that high-resolution 1H NMR spectra of rat brain extracts from 11 distinct brain regions can be reproducibly quantified using a basis set of 29 compounds. The basis set is simulated with the density matrix formalism using complete prior knowledge of chemical shifts and scalar couplings. A crucial aspect to obtain reproducible results was the inclusion of a line shape distortion common among all 73 resonances of the 29 compounds. All metabolites could be quantified with <10% and <3% inter- and intrasubject variation, respectively.
PMCID: PMC3711472  PMID: 21142125
rat brain extract; spectral fitting; 1H NMR
12.  State-of-the-Art Direct 13C and Indirect 1H-[13C] NMR Spectroscopy In Vivo 
NMR in biomedicine  2011;24(8):958-972.
Carbon-13 NMR spectroscopy in combination with 13C-labeled substrate infusion is a powerful technique to measure a large number of metabolic fluxes non-invasively in vivo. It has been used to quantify glycogen synthesis rates, establish quantitative relationships between energy metabolism and neurotransmission and evaluate the importance of different substrates. All measurements can, in principle, be performed through direct 13C NMR detection or via indirect 1H-[13C] NMR detection of the protons attached to 13C nuclei. The choice for detection scheme and pulse sequence depends on the magnetic field strength, whereas substrate selection depends on the metabolic pathways that are studied. 13C NMR spectroscopy remains a challenging technique that requires several non-standard hardware modifications, infusion of 13C-labeled substrates and sophisticated processing and metabolic modeling. Here the various aspects of direct 13C and indirect 1H-[13C] NMR are reviewed with the aim of providing a practical guide.
PMCID: PMC3694136  PMID: 21919099
NMR spectroscopy; direct 13C detection; indirect 1H-[13C] detection; broadband decoupling; RF power deposition; metabolism; metabolic modeling
13.  1H -[13C]-NMR Spectroscopy Measures of Ketamine’s Effect on Amino Acid Neurotransmitter Metabolism 
Biological psychiatry  2011;71(11):1022-1025.
Ketamine has recently gained significant attention owing to its psychotomimetic and more recently discovered rapid antidepressant-like properties. 1H-[13C]-NMR studies were employed to explore potential physiological processes underlying these unique effects.
[1-13C]glucose and [2-13C]acetate-NMR ex vivo studies were performed on the mPFC and hippocampus of rats acutely treated with 30mg/kg or 80mg/kg ketamine and compared to saline treated animals to determine the effects of ketamine on amino acid neurotransmitter cycling and glial metabolism.
A sub-anesthetic, but not anesthetic, dose of ketamine significantly increased the percentage 13C-enrichments of Glutamate, GABA, and Glutamine in the mPFC of rats.
Sub-anesthetic doses of ketamine increase mPFC amino acid neurotransmitter cycling as well as neuronal and glial energy metabolism. These data add to previous reports suggesting increased mPFC levels of glutamate release, following the administration of sub-anesthetic doses of ketamine, are related to the drug’s acute effects on cognition, perception and mood.
PMCID: PMC3660962  PMID: 22169441
NMDA; glutamate/glutamine cycle; ketamine; magnetic resonance spectroscopy; prefrontal cortex; GABA
14.  13C MRS studies of neuroenergetics and neurotransmitter cycling in humans 
NMR in biomedicine  2011;24(8):943-957.
In the last 25 years 13C MRS has been established as the only non invasive method for measuring glutamate neurotransmission and cell specific neuroenergetics. Although technically and experimentally challenging 13C MRS has already provided important new information on the relationship between neuroenergetics and neuronal function, 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 paper the current state of 13C MRS as it is applied to study 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 13C MRS studies in animals are discussed from the standpoint of validation of MRS measurements of neuroenergetics and neurotransmitter cycling and where they have helped identify key questions to address in human research. Controversies concerning the relation of neuroenergetics and neurotransmitter cycling and factors impacting accurate determination of fluxes through mathematical modeling are addressed. We further touch upon different 13C labeled substrates used to study brain metabolism, before reviewing a number of human brain diseases studied using 13C MRS. Future technological developments are discussed that will help to overcome limitations of 13C MRS with special attention on recent developments in hyperpolarized 13C MRS.
PMCID: PMC3651027  PMID: 21882281
15.  Intravenous ethanol infusion decreases human cortical GABA and NAA as measured with 1H-MRS at 4T 
Biological Psychiatry  2011;71(3):239-246.
Ethanol modulates glutamate and GABA function. However, little is known about the acute pharmacologic effects of ethanol on levels of GABA, glutamate, and other metabolites measurable in the human cortex in vivo using 1H magnetic resonance spectroscopy (MRS).
Eleven healthy social drinkers received two intravenous ethanol infusions that raised breath alcohol levels to a clamped plateau of 60 mg/dL over 60–70 minutes. The first infusion established tolerability of the procedure, and the second procedure, conducted 15±12 days later, was performed during 1H MRS of occipital GABA, glutamate, and other metabolites.
The time course of brain ethanol approximated that of breath ethanol, but venous ethanol lagged by about 7 minutes. GABA fell 13±8% after 5 minutes of the ethanol infusion and remained reduced (p=0.003) throughout the measurement. The combination of N-acetylaspartate and N-acetylaspartyl glutamate (summed as NAA) fell steadily during the infusion by 8±3% (p=0.0036).
Ethanol reduced cortical GABA and NAA levels in humans. Reductions in GABA levels are consistent with facilitation of GABAA receptor function by ethanol. The gradual decline in NAA levels suggests inhibition of neural or metabolic activity in the brain.
PMCID: PMC3227760  PMID: 21855054
1H MRS; GABA; alcohol; NAA; glutamate; brain
16.  Cortical substrate oxidation during hyperketonemia in the fasted anesthetized rat in vivo 
Ketone bodies are important alternate brain fuels, but their capacity to replace glucose and support neural function is unclear. In this study, the contributions of ketone bodies and glucose to cerebral cortical metabolism were measured in vivo in halothane-anesthetized rats fasted for 36 hours (n=6) and receiving intravenous [2,4-13C2]--β-hydroxybutyrate (BHB). Time courses of 13C-enriched brain amino acids (glutamate-C4, glutamine-C4, and glutamate and glutamine-C3) were measured at 9.4 Tesla using spatially localized 1H-[13C]-nuclear magnetic resonance spectroscopy. Metabolic rates were estimated by fitting a constrained, two-compartment (neuron–astrocyte) metabolic model to the 13C time-course data. We found that ketone body oxidation was substantial, accounting for 40% of total substrate oxidation (glucose plus ketone bodies) by neurons and astrocytes. -β-Hydroxybutyrate was oxidized to a greater extent in neurons than in astrocytes (∼70:30), and followed a pattern closely similar to the metabolism of [1-13C]glucose reported in previous studies. Total neuronal tricarboxylic acid cycle (TCA) flux in hyperketonemic rats was similar to values reported for normal (nonketotic) anesthetized rats infused with [1-13C]glucose, but neuronal glucose oxidation was 40% to 50% lower, indicating that ketone bodies had compensated for the reduction in glucose use.
PMCID: PMC3323194  PMID: 21731032
energy metabolism; kinetic modeling; MR metabolite; MR spectroscopy; neuronal–glial interaction
17.  Evaluation of cerebral acetate transport and metabolic rates in the rat brain in vivo using 1H-[13C]-NMR 
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 for 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]-Nuclear Magnetic Resonance 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 to 5 mmol/L. Analysis assuming reversible, symmetric Michaelis–Menten kinetics for transport yielded 27±2mmol/L and 1.3±0.3 μmol/g/min for Kt and Tmax, respectively, and for utilization, 0.17±0.24 mmol/L 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 tricarboxylic acid cycle fluxes were 0.37±0.03 μmol/g/min and 1.41±0.11 μmol/g/min, respectively; astrocytes thus comprised ∼21%±3% of total oxidative metabolism.
PMCID: PMC2879471  PMID: 20125180
brain acetate transport and utilization; fatty acids; glutamate; glutamine; nuclear magnetic resonance spectroscopy; neuron-glia trafficking
18.  The Contribution of Blood Lactate to Brain Energy Metabolism in Humans Measured by Dynamic 13C Nuclear Magnetic Resonance Spectroscopy 
To determine whether plasma lactate can be a significant fuel for human brain energy metabolism infusions of [3-13C]lactate and 1H-13C polarization transfer spectroscopy were used to detect the entry and utilization of lactate. During the 2-hour infusion study, 13C incorporation in the amino acid pools of glutamate and glutamine were measured with a 5 minutes time-resolution. With a plasma concentration ([Lac]P) being in the 0.8–2.8 mmol/L range, the tissue lactate concentration ([Lac]B) was assessed as well as the fractional contribution of lactate to brain energy metabolism (CMRlac). From the measured relationship between unidirectional lactate influx (Vin) and plasma and brain lactate concentrations lactate transport constants were calculated using a reversible Michaelis-Menten model. The results show (i) that in the physiological range plasma lactate unidirectional transport (Vin) and concentration in tissue increases close to linearly with the lactate concentration in plasma, (ii) the maximum potential contribution of plasma lactate to brain metabolism is 10% under basal plasma lactate conditions of ~ 1.0 mmol/L and as much as 60% at supra-physiological plasma lactate concentrations when the transporters are saturated, (iii) the half-saturation constant KT is 5.1±2.7 mmol/L and VMAX is 0.40±0.13 μmol/g/min (68% confidence interval), (iv) the majority of plasma lactate is metabolized in neurons similar to glucose.
PMCID: PMC2996729  PMID: 20962220
Human; Brain metabolism; Lactate transport; NMR; In vivo 13C Spectroscopy; reversible Michaelis-Menten
19.  In Vivo Neurochemical Profiling of Rat Brain by 1H-[13C] NMR Spectroscopy. Cerebral Energetics and Glutamatergic/GABAergic Neurotransmission 
Journal of neurochemistry  2009;112(1):24-33.
The simultaneous quantification of excitatory and inhibitory neurotransmission and the associated energy metabolism is crucial for a proper understanding of brain function. While the detection of glutamatergic neurotransmission in vivo by 13C NMR spectroscopy is now relatively routine, the detection of GABAergic neurotransmission in vivo has remained elusive due to the low GABA concentration and spectral overlap. Using 1H-[13C] NMR spectroscopy at high magnetic field in combination with robust spectral modeling and the use of different substrates, [U-13C6]-glucose and [2-13C]-acetate, it is shown that GABAergic, as well as glutamatergic neurotransmitter fluxes can be detected non-invasively in rat brain in vivo.
PMCID: PMC2843425  PMID: 19818103
GABA; 1H-[13C] NMR; glutamate; cerebral metabolism; rat
20.  Altered brain mitochondrial metabolism in healthy aging as assessed by in vivo magnetic resonance spectroscopy 
A decline in brain function is a characteristic feature of healthy aging; however, little is known about the biologic basis of this phenomenon. To determine whether there are alterations in brain mitochondrial metabolism associated with healthy aging, we combined 13C/1H magnetic resonance spectroscopy with infusions of [1-13C]glucose and [2-13C]acetate to quantitatively characterize rates of neuronal and astroglial tricarboxylic acid cycles, as well as neuroglial glutamate–glutamine cycling, in healthy elderly and young volunteers. Compared with young subjects, neuronal mitochondrial metabolism and glutamate–glutamine cycle flux was ∼30% lower in elderly subjects. The reduction in individual subjects correlated strongly with reductions in N-acetylaspartate and glutamate concentrations consistent with chronic reductions in brain mitochondrial function. In elderly subjects infused with [2-13C]acetate labeling of glutamine, C4 and C3 differed from that of the young subjects, indicating age-related changes in glial mitochondrial metabolism. Taken together, these studies show that healthy aging is associated with reduced neuronal mitochondrial metabolism and altered glial mitochondrial metabolism, which may in part be responsible for declines in brain function.
PMCID: PMC2949111  PMID: 19794401
aging; human brain; metabolism; mitochondria; 13C MRS; 1H MRS
21.  Evaluation of Cerebral Acetate Transport and Metabolic Rates in the Rat Brain In Vivo using 1H-[13C] NMR 
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.
PMCID: PMC2879471  PMID: 20125180
brain acetate transport and utilization; fatty acids; glutamate; glutamine; neuron-glia trafficking; nuclear magnetic resonance spectroscopy
22.  Astroglial Contribution to Brain Energy Metabolism in Humans Revealed by 13C Nuclear Magnetic Resonance Spectroscopy: Elucidation of the Dominant Pathway for Neurotransmitter Glutamate Repletion and Measurement of Astrocytic Oxidative Metabolism 
Increasing evidence supports a crucial role for glial metabolism in maintaining proper synaptic function and in the etiology of neurological disease. However, the study of glial metabolism in humans has been hampered by the lack of noninvasive methods. To specifically measure the contribution of astroglia to brain energy metabolism in humans, we used a novel noninvasive nuclear magnetic resonance spectroscopic approach. We measured carbon 13 incorporation into brain glutamate and glutamine in eight volunteers during an intravenous infusion of [2-13C] acetate, which has been shown in animal models to be metabolized specifically in astroglia. Mathematical modeling of the three established pathways for neurotransmitter glutamate repletion indicates that the glutamate/glutamine neurotransmitter cycle between astroglia and neurons (0.32 ± 0.07 μmol · gm−1 · min−1) is the major pathway for neuronal glutamate repletion and that the astroglial TCA cycle flux (0.14 ± 0.06 μmol · gm−1 · min−1) accounts for ~14% of brain oxygen consumption. Up to 30% of the glutamine transferred to the neurons by the cycle may derive from replacement of oxidized glutamate by anaplerosis. The further application of this approach could potentially enlighten the role of astroglia in supporting brain glutamatergic activity and in neurological and psychiatric disease.
PMCID: PMC2995528  PMID: 11880482
human; brain; astrocyte; glutamate/glutamine cycle; TCA cycle; NMR; acetate
23.  Concentration-Dependent Effects on Intracellular and Surface pH of Exposing Xenopus oocytes to Solutions Containing NH3/NH4+ 
The Journal of membrane biology  2009;228(1):15-31.
Others have shown that exposing oocytes to high levels of NH3/NH4+ (10–20 mM) causes a paradoxical fall in intracellular pH (pHi), whereas low levels (e.g., 0.5 mM) cause little pHi change. Here we monitored pHi and extra-cellular surface pH (pHS) while exposing oocytes to 5 or 0.5 mM NH3/NH4+. We confirm that 5 mM NH3/NH4+ causes a paradoxical pHi fall (−ΔpHi ≅ 0.2), but also observe an abrupt pHS fall (−ΔpHS ≅ 0.2)—indicative of NH3 influx—followed by a slow decay. Reducing [NH3/NH4+] to 0.5 mM minimizes pHi changes but maintains pHS changes at a reduced magnitude. Expressing AmtB (bacterial Rh homologue) exaggerates −ΔpHS at both NH3/NH4+ levels. During removal of 0.5 or 5 mM NH3/NH4+, failure of pHS to markedly overshoot bulk extracellular pH implies little NH3 efflux and, thus, little free cytosolic NH3/NH4+. A new analysis of the effects of NH3 vs. NH4+ fluxes on pHS and pHi indicates that (a) NH3 rather than NH4+ fluxes dominate pHi and pHS changes and (b) oocytes dispose of most incoming NH3. NMR studies of oocytes exposed to 15N-labeled NH3/NH4+ show no significant formation of glutamine but substantial NH3/NH4+ accumulation in what is likely an acid intracellular compartment. In conclusion, parallel measurements of pHi and pHS demonstrate that NH3 flows across the plasma membrane and provide new insights into how a protein molecule in the plasma membrane—AmtB—enhances the flux of a gas across a biological membrane.
PMCID: PMC2929962  PMID: 19242745
NH3 permeability; Surface pH measurement; Xenopus oocytes; AmtB
24.  Recurrent Antecedent Hypoglycemia Alters Neuronal Oxidative Metabolism In Vivo 
Diabetes  2009;58(6):1266-1274.
The objective of this study was to characterize the changes in brain metabolism caused by antecedent recurrent hypoglycemia under euglycemic and hypoglycemic conditions in a rat model and to test the hypothesis that recurrent hypoglycemia changes the brain's capacity to utilize different energy substrates.
Rats exposed to recurrent insulin-induced hypoglycemia for 3 days (3dRH rats) and untreated controls were subject to the following protocols: [2-13C]acetate infusion under euglycemic conditions (n = 8), [1-13C]glucose and unlabeled acetate coinfusion under euglycemic conditions (n = 8), and [2-13C]acetate infusion during a hyperinsulinemic-hypoglycemic clamp (n = 8). In vivo nuclear magnetic resonance spectroscopy was used to monitor the rise of13C-labeling in brain metabolites for the calculation of brain metabolic fluxes using a neuron-astrocyte model.
At euglycemia, antecedent recurrent hypoglycemia increased whole-brain glucose metabolism by 43 ± 4% (P < 0.01 vs. controls), largely due to higher glucose utilization in neurons. Although acetate metabolism remained the same, control and 3dRH animals showed a distinctly different response to acute hypoglycemia: controls decreased pyruvate dehydrogenase (PDH) flux in astrocytes by 64 ± 20% (P = 0.01), whereas it increased by 37 ± 3% in neurons (P = 0.01). The 3dRH animals decreased PDH flux in both compartments (−75 ± 20% in astrocytes, P < 0.001, and −36 ± 4% in neurons, P = 0.005). Thus, acute hypoglycemia reduced total brain tricarboxylic acid cycle activity in 3dRH animals (−37 ± 4%, P = 0.001), but not in controls.
Our findings suggest that after antecedent hypoglycemia, glucose utilization is increased at euglycemia and decreased after acute hypoglycemia, which was not the case in controls. These findings may help to identify better methods of preserving brain function and reducing injury during acute hypoglycemia.
PMCID: PMC2682668  PMID: 19276443
25.  Determination of the glutamate–glutamine cycling flux using two-compartment dynamic metabolic modeling is sensitive to astroglial dilution 
Over the last decade 13C magnetic resonance spectroscopy (13C MRS) combined with the infusion of [1-13C]glucose has been used to measure the cerebral rate of the glutamate–glutamine cycle (Vcyc). However, the effect of the astroglial label dilution pathways on the accuracy and precision of the 13C MRS measurement of Vcyc has not been evaluated or realized. In this report, we use the numerical Monte Carlo method to study the effect of astroglial dilution on the reliability of extracting Vcyc using the neuronal–astroglial two-compartment metabolic model and [1-13C]glucose infusion. The results show that omission of the astroglial dilution flux leads to a large loss in the sensitivity of the glutamine turnover curve to Vcyc. When the measured isotopic dilution of cerebral glutamine is accounted for in the analysis, the value of Vcyc can be precisely and accurately determined.
PMCID: PMC2613170  PMID: 18766194
brain glucose metabolism; glutamate–glutamine neurotransmitter cycling; magnetic resonance spectroscopy; 13C isotopes

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