Kevin L. Behar

Cerebral intracellular pH by 31P nuclear magnetic resonance spectroscopy

We determined cerebral intracellular pH in living rabbits and rats under physiologic conditions, using phosphorus NMR spectroscopy and new titration curves thought to be appropriate for brain. Mean values for the two species were, respectively, 7.14 ± 0.04 (SD) and 7.13 ± 0.03. These are toward the alkaline end of the range of values obtained by other methods, as values reported by other NMR workers also tend to be.

Neuronal-glial glucose oxidation and glutamatergic-GABAergic function

Prior 13C magnetic resonance spectroscopy (MRS) experiments, which simultaneously measured in vivo rates of total glutamate-glutamine cycling (Vcyc(tot)) and neuronal glucose oxidation (CMRglc(ox), N), revealed a linear relationship between these fluxes above isoelectricity, with a slope of ~1. In vitro glial culture studies examining glutamate uptake indicated that glutamate, which is cotransported with Na+, stimulated glial uptake of glucose and release of lactate. These in vivo and in vitro results were consolidated into a model: recycling of one molecule of neurotransmitter between glia and neurons was associated with oxidation of one glucose molecule in neurons; however, the glucose was taken up only by glia and all the lactate (pyruvate) generated by glial glycolysis was transferred to neurons for oxidation. The model was consistent with the 1:1 relationship between ΔCMRglc(ox), N and ΔVcyc(tot) measured by 13C MRS. However, the model could not specify the energetics of glia and γ-amino butyric acid (GABA) neurons because quantitative values for these pathways were not available. Here, we review recent 13C and 14C tracer studies that enable us to include these fluxes in a more comprehensive model. The revised model shows that glia produce at least 8% of total oxidative ATP and GABAergic neurons generate ~18% of total oxidative ATP in neurons. Neurons produce at least 88% of total oxidative ATP, and take up ~26% of the total glucose oxidized. Glial lactate (pyruvate) still makes the major contribution to neuronal oxidation, but ~30% less than predicted by the prior model. The relationship observed between ΔCMRglc(ox), N and ΔVcyc(tot) is determined by glial glycolytic ATP as before. Quantitative aspects of the model, which can be tested by experimentation, are discussed.

Glial pathology in an animal model of depression: reversal of stress-induced cellular, metabolic and behavioral deficits by the glutamate-modulating drug riluzole.

Growing evidence indicates that glia pathology and amino-acid neurotransmitter system abnormalities contribute to the pathophysiology and possibly the pathogenesis of major depressive disorder. This study investigates changes in glial function occurring in the rat prefrontal cortex (PFC) after chronic unpredictable stress (CUS), a rodent model of depression. Furthermore, we analyzed the effects of riluzole, a Food and Drug Administration-approved drug for the treatment of amyotrophic laterosclerosis, known to modulate glutamate release and facilate glutamate uptake, on CUS-induced glial dysfunction and depressive-like behaviors. We provide the first experimental evidence that chronic stress impairs cortical glial function. Animals exposed to CUS and showing behavioral deficits in sucrose preference and active avoidance exhibited significant decreases in 13C-acetate metabolism reflecting glial cell metabolism, and glial fibrillary associated protein (GFAP) mRNA expression in the PFC. The cellular, metabolic and behavioral alterations induced by CUS were reversed and/or blocked by chronic treatment with the glutamate-modulating drug riluzole. The beneficial effects of riluzole on CUS-induced anhedonia and helplessness demonstrate the antidepressant action of riluzole in rodents. Riluzole treatment also reversed CUS-induced reductions in glial metabolism and GFAP mRNA expression. Our results are consistent with recent open-label clinical trials showing the drugs effect in mood and anxiety disorders. This study provides further validation of hypothesis that glial dysfunction and disrupted amino-acid neurotransmission contribute to the pathophysiology of depression and that modulation of glutamate metabolism, uptake and/or release represent viable targets for antidepressant drug development.

Cerebral energetics and spiking frequency: The neurophysiological basis of fMRI

Functional MRI (fMRI) is widely assumed to measure neuronal activity, but no satisfactory mechanism for this linkage has been identified. Here we derived the changes in the energetic component from the blood oxygenation level-dependent (BOLD) fMRI signal and related it to changes in the neuronal spiking frequency in the activated voxels. Extracellular recordings were used to measure changes in cerebral spiking frequency (Δν/ν) of a neuronal ensemble during forepaw stimulation in the α-chloralose anesthetized rat. Under the same conditions localized changes in brain energy metabolism (ΔCMRO2/CMRO2) were obtained from BOLD fMRI data in conjunction with measured changes in cerebral blood flow (ΔCBF/CBF), cerebral blood volume (ΔCBV/CBV), and transverse relaxation rates of tissue water (T\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}_{2}^{*}\end{equation*}\end{document} and T2) by MRI methods at 7T. On stimulation from two different depths of anesthesia ΔCMRO2/CMRO2 ≈ Δν/ν. Previous 13C magnetic resonance spectroscopy studies, under similar conditions, had shown that ΔCMRO2/CMRO2 was proportional to changes in glutamatergic neurotransmitter flux (ΔVcyc/Vcyc). These combined results show that ΔCMRO2/CMRO2 ≈ ΔVcyc/Vcyc ≈ Δν/ν, thereby relating the energetic basis of brain activity to neuronal spiking frequency and neurotransmitter flux. Because ΔCMRO2/CMRO2 had the same high spatial and temporal resolutions of the fMRI signal, these results show how BOLD imaging, when converted to ΔCMRO2/CMRO2, responds to localized changes in neuronal spike frequency.

Simultaneous determination of the rates of the TCA cycle, glucose utilization, α-ketoglutarate/glutamate exchange, and glutamine synthesis in human brain by NMR

13C isotopic tracer data previously obtained by 13C nuclear magnetic resonance in the human brain in vivo were analyzed using a mathematical model to determine metabolic rates in a region of the human neocortex. The tricarboxylic acid (TCA) cycle rate was 0.73 ± 0.19 μmol min−1 g−1 (mean ± SD; n = 4). The standard deviation reflects primarily intersubject variation, since individual uncertainties were low. The rate of α-ketoglutarate/glutamate exchange was 57 ± 26 μmol min−1 g−1 (n = 3), which is much greater than the TCA cycle rate; the high rate indicates that α-ketoglutarate and glutamate are in rapid exchange and can be treated as a single combined kinetic pool. The rate of synthesis of glutamine from glutamate was 0.47 μmol min−1 g−1 (n = 4), with 95% confidence limits of 0.139 and 3.094 μmol min−1 g−1; individual uncertainties were biased heavily toward high synthesis rates. From the TCA cycle rate the brain oxygen consumption was estimated to be 2.14 ± 0.48 μmol min−1 g−1 (5.07 ± 1.14 ml 100 g−1 min−1; n = 4), and the rate of brain glucose consumption was calculated to be 0.37 ± 0.08 μmol min−1 g−1 (n = 4). The sensitivity of the model to the assumptions made was evaluated, and the calculated values were found to be unchanged as long as the assumptions remained near reported physiological values.

NMR Determination of the TCA Cycle Rate and α-Ketoglutarate/Glutamate Exchange Rate in Rat Brain:

A mathematical model of cerebral glucose metabolism was developed to analyze the isotopic labeling of carbon atoms C4 and C3 of glutamate following an intravenous infusion of [1-13C]glucose. The model consists of a series of coupled metabolic pools representing glucose, glycolytic intermediates, tricarboxylic acid (TCA) cycle intermediates, glutamate, aspartate, and glutamine. Based on the rate of 13C isotopic labeling of glutamate C4 measured in a previous study, the TCA cycle rate in rat brain was determined to be 1.58 ± 0.41 μmol min−1 g−1 (mean ± SD, n = 5). Analysis of the difference between the rates of isotopic enrichment of glutamate C4 and C3 permitted the rate of exchange between α-ketoglutarate (α-KG) and glutamate to be assessed in vivo. In rat brain, the exchange rate between α-KG and glutamate is between 89 ± 35 and 126 ± 22 times faster than the TCA cycle rate (mean ± SD, n = 4). The sensitivity of the calculated value of the TCA cycle rate to other metabolic fluxes and to concentrations of glycolytic and TCA cycle intermediates was tested and found to be small.

The flux from glucose to glutamate in the rat brain in vivo as determined by 1H-observed, 13C-edited NMR spectroscopy.

The rate of incorporation of carbon from [1-13C]glucose into the [4-CH2] and [3-CH2] of cerebral glutamate was measured in the rat brain in vivo by 1H-observed, 13C-edited (POCE) nuclear magnetic resonance (NMR) spectroscopy. Spectra were acquired every 98 s during a 60-min infusion of [1-13C]glucose. Complete time courses were obtained from six animals. The measured intensity of the unresolved [4-13CH2] resonances of glutamate and glutamine increased exponentially during the infusion and attained a steady state in ∼20 min with a first-order rate constant of 0.130 ± 0.010 min−1 (t1/2 = 5.3 ± 0.5 min). The appearance of the [3-13CH2] resonance in the POCE difference spectrum lagged behind that of the [4-13CH2] resonance and had not reached steady state at the end of the 60-min infusion (t1/2 = 26.6 ± 4.1 min). The increase observed in 13C-labeled glutamate represented isotopic enrichment and was not due to a change in the total glutamate concentration. The glucose infusion did not affect the levels of high-energy phosphates or intracellular pH as determined by 31P NMR spectroscopy. Since glucose carbon is incorporated into glutamate by rapid exchange with the tricarboxylic acid (TCA) cycle intermediate α-ketoglutarate, the rate of glutamate labeling provided an estimate of TCA cycle flux. We have determined the flux of carbon through the TCA cycle to be ≈1.4 μmol g−1 min−1. These experiments demonstrate the feasibility of measuring metabolic fluxes in vivo using 13C-labeled glucose and the technique of 1H-observed, 13C-decoupled NMR spectroscopy.

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.

High magnetic field water and metabolite proton T1 and T2 relaxation in rat brain in vivo

Comprehensive and quantitative measurements of T1 and T2 relaxation times of water, metabolites, and macromolecules in rat brain under similar experimental conditions at three high magnetic field strengths (4.0 T, 9.4 T, and 11.7 T) are presented. Water relaxation showed a highly significant increase (T1) and decrease (T2) with increasing field strength for all nine analyzed brain structures. Similar but less pronounced effects were observed for all metabolites. Macromolecules displayed field‐independent T2 relaxation and a strong increase of T1 with field strength. Among other features, these data show that while spectral resolution continues to increase with field strength, the absolute signal‐to‐noise ratio (SNR) in T1/T2‐based anatomical MRI quickly levels off beyond ∼7 T and may actually decrease at higher magnetic fields. Magn Reson Med, 2006.

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 h infusion study, 13C incorporation in the amino acid pools of glutamate and glutamine were measured with a 5 min 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 that (1) in the physiological range, plasma lactate unidirectional transport (Vin) and concentration in tissue increase close to linearly with the lactate concentration in plasma; (2) 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 supraphysiological plasma lactate concentrations when the transporters are saturated; (3) the half-saturation constant KT is 5.1 ± 2.7 mmol/L and VMAX is 0.40 ± 0.13 μmol · g−1 · min−1 (68% confidence interval); and (4) the majority of plasma lactate is metabolized in neurons similar to glucose.