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1.  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.
doi:10.1016/j.neuroimage.2013.04.099
PMCID: PMC3700380  PMID: 23648961
cognitive dementia; thalamocortical responses; neurovascular coupling; aging; energy metabolism; neuroimaging
2.  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.
doi:10.1007/s11064-012-0898-7
PMCID: PMC3702378  PMID: 23104556
3.  Roles of Glutamine Synthetase Inhibition in Epilepsy 
Neurochemical research  2012;37(11):2339-2350.
Glutamine synthetase (GS, E.C. 6.3.1.2) 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.
doi:10.1007/s11064-012-0766-5
PMCID: PMC3731630  PMID: 22488332
Astrocytes; Glutamate; Hippocampus; Seizures; Temporal lobe epilepsy
4.  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.
doi:10.1021/ac102285c
PMCID: PMC3711472  PMID: 21142125
rat brain extract; spectral fitting; 1H NMR
5.  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.
doi:10.1002/nbm.1761
PMCID: PMC3694136  PMID: 21919099
NMR spectroscopy; direct 13C detection; indirect 1H-[13C] detection; broadband decoupling; RF power deposition; metabolism; metabolic modeling
6.  1H -[13C]-NMR Spectroscopy Measures of Ketamine’s Effect on Amino Acid Neurotransmitter Metabolism 
Biological psychiatry  2011;71(11):1022-1025.
Background
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.
Methods
[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.
Results
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.
Conclusion
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.
doi:10.1016/j.biopsych.2011.11.006
PMCID: PMC3660962  PMID: 22169441
NMDA; glutamate/glutamine cycle; ketamine; magnetic resonance spectroscopy; prefrontal cortex; GABA
7.  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.
doi:10.1002/nbm.1772
PMCID: PMC3651027  PMID: 21882281
8.  Intravenous ethanol infusion decreases human cortical GABA and NAA as measured with 1H-MRS at 4T 
Biological Psychiatry  2011;71(3):239-246.
Background
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).
Methods
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.
Results
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).
Conclusions
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.
doi:10.1016/j.biopsych.2011.06.026
PMCID: PMC3227760  PMID: 21855054
1H MRS; GABA; alcohol; NAA; glutamate; brain
9.  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.
doi:10.1038/jcbfm.2011.91
PMCID: PMC3323194  PMID: 21731032
energy metabolism; kinetic modeling; MR metabolite; MR spectroscopy; neuronal–glial interaction
10.  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.
doi:10.1038/jcbfm.2010.2
PMCID: PMC2879471  PMID: 20125180
brain acetate transport and utilization; fatty acids; glutamate; glutamine; nuclear magnetic resonance spectroscopy; neuron-glia trafficking
11.  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.
doi:10.1523/JNEUROSCI.2040-10.2010
PMCID: PMC2996729  PMID: 20962220
Human; Brain metabolism; Lactate transport; NMR; In vivo 13C Spectroscopy; reversible Michaelis-Menten
12.  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.
doi:10.1111/j.1471-4159.2009.06428.x
PMCID: PMC2843425  PMID: 19818103
GABA; 1H-[13C] NMR; glutamate; cerebral metabolism; rat
13.  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.
doi:10.1038/jcbfm.2009.197
PMCID: PMC2949111  PMID: 19794401
aging; human brain; metabolism; mitochondria; 13C MRS; 1H MRS
14.  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.
doi:10.1038/jcbfm.2010.2
PMCID: PMC2879471  PMID: 20125180
brain acetate transport and utilization; fatty acids; glutamate; glutamine; neuron-glia trafficking; nuclear magnetic resonance spectroscopy
15.  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
16.  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.
doi:10.1007/s00232-009-9155-7
PMCID: PMC2929962  PMID: 19242745
NH3 permeability; Surface pH measurement; Xenopus oocytes; AmtB
17.  Recurrent Antecedent Hypoglycemia Alters Neuronal Oxidative Metabolism In Vivo 
Diabetes  2009;58(6):1266-1274.
OBJECTIVE
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.
RESEARCH DESIGN AND METHODS
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.
RESULTS
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.
CONCLUSIONS
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.
doi:10.2337/db08-1664
PMCID: PMC2682668  PMID: 19276443
18.  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.
doi:10.1038/jcbfm.2008.102
PMCID: PMC2613170  PMID: 18766194
brain glucose metabolism; glutamate–glutamine neurotransmitter cycling; magnetic resonance spectroscopy; 13C isotopes
19.  Neurovascular and Neurometabolic Couplings in Dynamic Calibrated fMRI: Transient Oxidative Neuroenergetics for Block-Design and Event-Related Paradigms 
Functional magnetic resonance imaging (fMRI) with blood-oxygenation level dependent (BOLD) contrast is an important tool for mapping brain activity. Interest in quantitative fMRI has renewed awareness in importance of oxidative neuroenergetics, as reflected by cerebral metabolic rate of oxygen consumption(CMRO2), for supporting brain function. Relationships between BOLD signal and the underlying neurophysiological parameters have been elucidated to allow determination of dynamic changes inCMRO2 by “calibrated fMRI,” which require multi-modal measurements of BOLD signal along with cerebral blood flow (CBF) and volume (CBV). But how doCMRO2 changes, steady-state or transient, derived from calibrated fMRI compare with neural activity recordings of local field potential (LFP) and/or multi-unit activity (MUA)? Here we discuss recent findings primarily from animal studies which allow high magnetic fields studies for superior BOLD sensitivity as well as multi-modal CBV and CBF measurements in conjunction with LFP and MUA recordings from activated sites. A key observation is that while relationships between neural activity and sensory stimulus features range from linear to non-linear, associations between hyperemic components (BOLD, CBF, CBV) and neural activity (LFP, MUA) are almost always linear. More importantly, the results demonstrate good agreement between the changes inCMRO2 and independent measures of LFP or MUA. The tight neurovascular and neurometabolic couplings, observed from steady-state conditions to events separated by <200 ms, suggest rapid oxygen equilibration between blood and tissue pools and thus calibrated fMRI at high magnetic fields can provide high spatiotemporal mapping ofCMRO2 changes.
doi:10.3389/fnene.2010.00018
PMCID: PMC2936934  PMID: 20838476
glia; glucose; glutamate; mitochondria; neuron; oxygen transport
20.  High Resolution NMR Spectroscopy of Rat Brain In Vivo Through Indirect Zero-Quantum-Coherence Detection 
The time evolution of zero-quantum-coherences (ZQCs) is insensitive to magnetic field inhomogeneity. Using a 2D indirect ZQC detection method it is shown that high-resolution 1H NMR spectra can be obtained from rat brain in vivo at 11.74 T that are immune to magnetic field inhomogeneity. Simulations based on the density matrix formalism, as well as in vitro measurements are used to demonstrate the features of 2D ZQC NMR spectra. Unique spectral information which is normally not directly available from regular 1H NMR spectra can be extracted and used for compound identification or improved prior knowledge during spectral fitting.
doi:10.1016/j.jmr.2007.06.001
PMCID: PMC2788487  PMID: 17587617
zero-quantum-coherences; indirect detection; rat brain
21.  Effects of Continuous Hypoxia on Energy Metabolism in Cultured Cerebro-Cortical Neurons 
Brain research  2008;1229:147-154.
Mechanisms underlying hypoxia-induced neuronal adaptation have not been fully elucidated. In the present study we investigated glucose metabolism and the activities of glycolytic and TCA cycle enzymes in cerebro-cortical neurons exposed to hypoxia (3 days in 1% of O2) or normoxia (room air). Hypoxia led to increased activities of LDH (251%), PK (90%), and HK (24%) and decreased activities of CS (15%) and GDH (34%). Neurons were incubated with [1-13C]glucose for 45 and 120 min under normoxic or hypoxic (120 min only) conditions and 13C enrichment determined in the medium and cell extract using 1H-{13C}-NMR. In hypoxia-treated neurons [3-13C]lactate release into the medium was 428% greater than in normoxia-treated controls (45-min normoxic incubation) and total flux through lactate was increased by 425%. In contrast glucose oxidation was reduced significantly in hypoxia-treated neurons, even when expressed relative to total cellular protein, which correlated with the reduced activities of the measured mitochondrial enzymes. The results suggest that surviving neurons adapt to prolonged hypoxia by up-regulation of glycolysis and down-regulation of oxidative energy metabolism, similar to certain other cell types. The factors leading to adaptation and survival for some neurons but not others remains to be determined.
doi:10.1016/j.brainres.2008.06.074
PMCID: PMC2658263  PMID: 18621040
continuous hypoxia; glycolysis; TCA cycle; metabolic flux; NMR
22.  Chronic riluzole treatment increases glucose metabolism in rat prefrontal cortex and hippocampus 
Riluzole is believed to modulate glutamatergic function by reducing glutamate release and facilitating astroglial uptake. We measured the incorporation of 13C during a 10 min infusion of [1-13C]glucose into metabolites in prefrontal-cortex and hippocampus of urethane-anesthetized rats treated with riluzole (21d, 4mg/kg, i.p. daily) or saline. Total and 13C-concentrations of metabolites were determined in extracts using 1H-[13C]NMR. In prefrontal-cortex (P<0.05) and hippocampus (P<0.01) riluzole increased 13C-labeling over saline in to glutamate-C4 (112% and 130%), GABA-C2 (142% and 171%) and glutamine-C4 (118% and 233%) without affecting total metabolite levels (P>0.2). Our findings indicate that chronic riluzole enhances brain glutamate labeling from [1-13C]glucose.
doi:10.1038/jcbfm.2008.78
PMCID: PMC2739056  PMID: 18628780
glutamate; GABA; glutamine; TCA cycle; riluzole; nuclear magnetic resonance
23.  Natural Abundance 17O NMR Spectroscopy of Rat Brain In Vivo 
Oxygen is an abundant element that is present in almost all biologically relevant molecules. NMR observation of oxygen has been relatively limited since the NMR-active isotope, oxygen-17, is only present at a 0.037% natural abundance. Furthermore, as a spin 5/2 nucleus oxygen-17 has a moderately strong quadrupole moment which leads to fairly broad resonances (T2* = 1 - 4 ms). However, the similarly short T1 relaxation constants allow substantial signal averaging, whereas the large chemical shift range (> 300 ppm) improves the spectral resolution of 17O NMR. Here it is shown that high-quality, natural abundance 17O NMR spectra can be obtained from rat brain in vivo at 11.74 T. The chemical shifts and line widths of more than 20 oxygen-containing metabolites are established and the sensitivity and potential for 17O-enriched NMR studies are estimated.
doi:10.1016/j.jmr.2008.04.019
PMCID: PMC2587261  PMID: 18456525
17O NMR; natural abundance; rat brain; high magnetic field
24.  A Neuronal Glutamate Transporter Contributes to Neurotransmitter GABA Synthesis and Epilepsy 
The predominant neuronal glutamate transporter, EAAC1 (for excitatory amino acid carrier-1), is localized to the dendrites and somata of many neurons. Rare presynaptic localization is restricted to GABA terminals. Because glutamate is a precursor for GABA synthesis, we hypothesized that EAAC1 may play a role in regulating GABA synthesis and, thus, could cause epilepsy in rats when inactivated. Reduced expression of EAAC1 by antisense treatment led to behavioral abnormalities, including staring–freezing episodes and electrographic (EEG) seizures. Extracellular hippocampal and thalamocortical slice recordings showed excessive excitability in antisense-treated rats. Patch-clamp recordings of miniature IPSCs (mIPSCs) conducted in CA1 pyramidal neurons in slices from EAAC1 antisense-treated animals demonstrated a significant decrease in mIPSC amplitude, indicating decreased tonic inhibition. There was a 50% loss of hippocampal GABA levels associated with knockdown of EAAC1, and newly synthesized GABA from extracellular glutamate was significantly impaired by reduction of EAAC1 expression. EAAC1 may participate in normal GABA neurosynthesis and limbic hyperexcitability, whereas epilepsy can result from a disruption of the interaction between EAAC1 and GABA metabolism.
PMCID: PMC2483507  PMID: 12151515
EAAC1; transport; antisense; GABA; metabolism; epilepsy

Results 1-24 (24)