Anant B. Patel

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.

Glutamatergic Neurotransmission and Neuronal Glucose Oxidation Are Coupled During Intense Neuronal Activation

13C nuclear magnetic resonance (NMR) experiments have previously shown that glutamatergic neurotransmitter flux (Vcycle(Glu/Gln)) changes proportionately with neuronal glucose oxidation (CMRglc(ox)N) in the nonactivated cortex of anesthetized rats. Positron Emission Tomography measurements of glucose and oxygen uptake during sensory stimulation had shown that the incremental glucose utilization is greater than oxygen leading to the suggestion that the energy required for stimulated neuronal activity arises from nonoxidative glucose metabolism. In this study, the authors used spatially localized 1H-observed, 13C-edited NMR spectroscopy during an infusion of [1,6–13C2]glucose to assess the relationship between changes in Vcycle(Glu/Gln) and glucose utilization (CMRglc(ox)N and CMRglc(nonox)) during the intense cortical activity associated with bicuculline-induced seizures. Metabolic fluxes were determined by model-based analysis of the 13C-enrichment time courses of glutamate-C4 and glutamine-C4 (CMRglc(ox)N, Vcycle(Glu/Gln)) and lactate-C3 (CMRglc(nonox)). The exchange rate between α-ketoglutarate and glutamate was found to be significantly faster than TCA cycle flux both for control (41 μmol·g−1·min−1; 95% CI, 5 to 109 μmol·g−1·min−1) and during seizures (21 μmol·g−1·min−1; 95% CI, 4.4 to 51.8 μmol·g−1·min−1). During seizures, total glucose utilization (CMRglc(ox+nonox)) increased substantially (466% between 0 and 6 minutes; 277% between 6 and 55 minutes). Glucose oxidation (CMRglc(ox)N) also increased (214%; from 0.26 ± 0.02 to 0.57 ± 0.07 μmol·g−1·min−1) but to a lesser degree, resulting in a large increase in cortical lactate concentration. Vcycle(Glu/Gln) increased 233% (from 0.22 ± 0.04 to 0.52 ± 0.07 μmol·g−1·min−1), which was similar to the increase in glucose oxidation. The value of Vcycle(Glu/Gln) and CMRglc(ox)N obtained here lie on the line predicted in a previous study. These results indicate that neuronal glucose oxidation and not total glucose utilization is coupled to the glutamate/glutamine cycle during intense cortical activation.

Direct evidence for activity-dependent glucose phosphorylation in neurons with implications for the astrocyte-to-neuron lactate shuttle

Significance A near one-to-one relationship had previously been observed between increments in the fluxes of the glutamate−glutamine neurotransmitter cycle and neuronal glucose oxidation in the tricarboxylic acid (TCA) cycle. This flux relationship was consistent with a hypothesized mechanism involving glycolytic ATP in astrocytes and astrocyte-to-neuron lactate shuttling. Here, 2-fluoro-2-deoxy-d-glucose was used to evaluate the glucose flux through glycolysis and the TCA cycle in nerve terminals isolated from the brains of rats under baseline and high-activity conditions. In a direct contradiction of this hypothesis, the results show that nerve terminals metabolize significant amounts of glucose. Previous 13C magnetic resonance spectroscopy experiments have shown that over a wide range of neuronal activity, approximately one molecule of glucose is oxidized for every molecule of glutamate released by neurons and recycled through astrocytic glutamine. The measured kinetics were shown to agree with the stoichiometry of a hypothetical astrocyte-to-neuron lactate shuttle model, which predicted negligible functional neuronal uptake of glucose. To test this model, we measured the uptake and phosphorylation of glucose in nerve terminals isolated from rats infused with the glucose analog, 2-fluoro-2-deoxy-d-glucose (FDG) in vivo. The concentrations of phosphorylated FDG (FDG6P), normalized with respect to known neuronal metabolites, were compared in nerve terminals, homogenate, and cortex of anesthetized rats with and without bicuculline-induced seizures. The increase in FDG6P in nerve terminals agreed well with the increase in cortical neuronal glucose oxidation measured previously under the same conditions in vivo, indicating that direct uptake and oxidation of glucose in nerve terminals is substantial under resting and activated conditions. These results suggest that neuronal glucose-derived pyruvate is the major oxidative fuel for activated neurons, not lactate-derived from astrocytes, contradicting predictions of the original astrocyte-to-neuron lactate shuttle model under the range of study conditions.

Glutamine is the major precursor for GABA synthesis in rat neocortex in vivo following acute GABA-transaminase inhibition

The objective of the present study was to assess the degree to which astrocytic glutamine provides carbon for net synthesis of GABA in the rat neocortex in vivo. Isotopic labeling of GABA and glutamate from astrocytic glutamine was followed in halothane anesthetized and ventilated rats during an intravenous infusion of [2-(13)C]glucose. A net increase in GABA was achieved by administration of the GABA-transaminase inhibitor, gabaculine to suppress catabolism of GABA and recycling of (13)C label. (13)C Percentage enrichments of GABA, glutamate and glutamine were assessed in tissue extracts using (13)C-edited (1)H nuclear magnetic resonance at 8.4 T. GABA levels increased 2.6 micromol/g at 2 h and 6.1 micromol/g at 5 h after gabaculine, whereas glutamate and glutamine decreased in toto by 5.6 micromol/g at 2 h and 3.1 micromol/g at 5 h. Selective enrichment of glutamine, glutamate, and GABA C3s over other carbon positions was observed consistent with a precursor role for astrocytic glutamine. Between 1 h (control) and 3 h (gabaculine-treated) of [2-(13)C]glucose infusion, (13)C percentage enrichment increased in glutamine C3 (from 3.2+/-0.5 to 7.0+/-0.9%), glutamate C3 (from 1.8+/-0.5 to 3.4+/-0.9%), and GABA C3 (from 2.7+/-1.6 to 4.8+/-0.4%). The measured incremental [3-(13)C]GABA concentration (0.15 micromol/g) was close to the predicted value (0.13 micromol/g) that would be expected if the increase in GABA were produced entirely from glutamine compared to glutamate (0.07 micromol/g) based on the average precursor enrichments between 1 and 3 h. We conclude that glutamine is the major source of GABA carbon in the rat neocortex produced acutely following GABA-T inhibition by gabaculine in vivo.

Evidence that GAD65 mediates increased GABA synthesis during intense neuronal activity in vivo

In this study we tested the hypothesis that the 65‐kDa isoform of glutamate decarboxylase (GAD65) mediates activity‐dependent GABA synthesis as invoked by seizures in anesthetized rats. GABA synthesis was measured following acute GABA‐transaminase inhibition by gabaculine using spatially localized 1H NMR spectroscopy before and after bicuculline‐induced seizures. Experiments were conducted with animals pre‐treated with vigabatrin 24 h earlier in order to reduce GAD67 protein and also with non‐treated controls. GAD isoform content was quantified by immunoblotting. GABA was higher in vigabatrin‐treated rats compared to non‐treated controls. In vigabatrin‐treated animals, GABA synthesis was 28% lower compared to controls [p < 0.05; vigabatrin‐treated, 0.043 ± 0.011 µmol/(g min); non‐treated, 0.060 ± 0.014 µmol/(g min)] and GAD67 was 60% lower. No difference between groups was observed for GAD65. Seizures increased GABA synthesis in both control [174%; control, 0.060 ± 0.014 µmol/(g min) vs. seizures, 0.105 ± 0.043 µmol/(g min)] and vigabatrin‐treated rats [214%; control, 0.043 ± 0.011 µmol/(g min); seizures, 0.092 ± 0.018 µmol/(g min)]. GAD67 could account for at least half of basal GABA synthesis but only 20% of the two‐fold increase observed in vigabatrin‐treated rats during seizures. The seizure‐induced activation of GAD65 in control cortex occurs concomitantly with a 2.3‐fold increase in inorganic phosphate, known to be a potent activator of apoGAD65in vitro. Our results are consistent with a major role for GAD65 in activity‐dependent GABA synthesis.

Decrease in GABA synthesis rate in rat cortex following GABA-transaminase inhibition correlates with the decrease in GAD67 protein

gamma-Aminobutyric acid (GABA) synthesis in the brain is mediated by two major isoforms of glutamic acid decarboxylase, GAD(65) and GAD(67). The contribution of these isoforms to GABA synthesis flux (V(GAD)) is not known quantitatively. In the present study we compared V(GAD) in cortex of control and vigabatrin-treated rats under alpha-chloralose/70% nitrous oxide anesthesia, with total GAD activity and GAD isoform composition (GAD(65) and GAD(67)) measured by enzymatic assay and quantitative immunoblotting. V(GAD) was determined by re-analysis of 13C NMR data obtained ex vivo and in vivo during infusions of [1-13C]glucose using an extension of a model of glutamate-glutamine cycling that included a discrete GABAergic neuronal compartment with relevant interconnecting fluxes. V(GAD) was significantly lower in vigabatrin-treated rats (0.030-0.05 micromol/min per g, P<0.003) compared to the non-treated control group (0.10-0.15 micromol/min per g). The 67-70% decrease in V(GAD) was associated with a 13% decrease in total GAD activity (P=0.01) and a selective 44+/-15% decrease in GAD(67) protein (from 0.63+/-0.10 to 0.35+/-0.08 microg protein/mg tissue, P<0.05); GAD(65) protein was unchanged. The reduction in GAD(67) protein could account for a maximum of approximately 65% of the decrease in V(GAD) in vigabatrin-treated animals suggesting that inhibition of GAD(65) must have also occurred in these experiments, although product inhibition of GAD(67) by increased GABA could play a role. GAD(67) could account for 56-85% of cortical GABA synthesis flux under basal conditions and the entire flux after vigabatrin treatment.

Glutamatergic and GABAergic Neurotransmitter Cycling and Energy Metabolism in Rat Cerebral Cortex during Postnatal Development

The contribution of glutamatergic and γ-aminobutyric acid (GABA)ergic neurons to oxidative energy metabolism and neurotransmission in the developing brain is not known. Glutamatergic and GABAergic fluxes were assessed in neocortex of postnatal day 10 (P10) and 30 (P30) urethane-anesthetized rats infused intravenously with [1,6-13C2]glucose for different time intervals (time course) or with [2-13C]acetate for 2 to 3 h (steady state). Amino acid levels and 13C enrichments were determined in tissue extracts ex vivo using 1H-[13C]-NMR spectroscopy. Metabolic fluxes were estimated from the best fits of a three-compartment metabolic model (glutamatergic neurons, GABAergic neurons, and astroglia) to the 13C-enrichment time courses of amino acids from [1,6-13C2]glucose, constrained by the ratios of neurotransmitter cycling (Vcyc)-to-tricarboxylic acid (TCA) cycle flux (VTCAn) calculated from the steady-state [2-13C]acetate enrichment data. From P10 to P30 increases in total neuronal (glutamate plus GABA) TCA cycle flux (3×; 0.24±0.05 versus 0.71 ± 0.07 μmol per g per min, P < 0.0001) and total neurotransmitter cycling flux (3.1 to 5×; 0.07 to 0.11 (± 0.03) versus 0.34 ± 0.03 μmol per g per min, P < 0.0001) were approximately proportional. Incremental changes in total cycling (δ Vcyc(tot)) and neuronal TCA cycle flux (δ VTCAn(tot)) between P10 and P30 were 0.23 to 0.27 and 0.47 μmol per g per min, respectively, similar to the ∼ 1:2 relationship previously reported for adult cortex. For the individual neurons, increases in VTCAn and Vcyc were similar in magnitude (glutamatergic neurons, 2.7× versus 2.8 to 4.6×; GABAergic neurons, ∼ 5× versus ∼7×), although GABAergic flux changes were larger. The findings show that glutamate and GABA neurons undergo large and approximately proportional increases in neurotransmitter cycling and oxidative energy metabolism during this major postnatal growth spurt.

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.

Dysfunctional Glutamatergic and γ-Aminobutyric Acidergic Activities in Prefrontal Cortex of Mice in Social Defeat Model of Depression

BACKGROUND Depression is a complex neuropsychiatric syndrome that is often very severe and life threatening. In spite of the remarkable progress in understanding the neural biology, the etiopathophysiology of depression is still elusive. In this study, we have investigated molecular mechanisms in the prefrontal cortex of mice showing depression-like phenotype induced by chronic defeat stress. METHODS Depression-like phenotype was induced in C57BL/6 mice by subjecting them to a 10-day social defeat paradigm. The metabolic activity of excitatory (glutamatergic) and inhibitory (γ-aminobutyric acid [GABA]ergic) neurons of the prefrontal cortex was measured by (1)H-[(13)C]-nuclear magnetic resonance spectroscopy together with infusion of [1,6-(13)C2]glucose. In addition, the expression level of genes associated with glutamatergic and GABAergic pathways was monitored by quantitative polymerase chain reaction. RESULTS Mice showing depression-like phenotype exhibit significant reduction in the levels of glutamate, glutamine, N-acetyl aspartate, and taurine in the prefrontal cortex. Most importantly, findings of reduced (13)C labeling of glutamate-C4, glutamate-C3, and GABA-C2 from [1,6-(13)C2]glucose indicate decreased glutamatergic and GABAergic neuronal metabolism and neurotransmitter cycling in the depressed mice. The reduced glutamine-C4 labeling suggests decreased neurotransmitter cycling in depression. Quantitative polymerase chain reaction analysis revealed reduced transcripts of Gad1 and Eaat2 genes, which code for enzymes involved in the synthesis of GABA and the clearance of glutamate from synapses, respectively. CONCLUSIONS These data indicate that the activities of glutamatergic and GABAergic neurons are reduced in mice showing a depression-like phenotype, which is supported by molecular data for the expression of genes involved in glutamate and GABA pathways.

Synthesis, spectroscopic, electrochemical and antibacterial studies of new Ru(II) 1,10-phenanthroline complexes containing aryldiazopentane-2,4-dione as co-ligand

Mononuclear and dinuclear Ru(II) complexes of 1,10-phenanthroline containing aryldiazopentane-2,4-diones (L1H–L3H2) as co-ligands were prepared and characterised using IR, 1H NMR, UV/Vis spectra in addition to their elemental analysis and FAB mass spectral data. A representative ligand L2H2 and its complex [Ru2L2(phen)4]2+ (phen=1,10-phenanthroline) were characterised also by 1H–1H COSY, TOCSY and 1H–13C HMBC spectral data. Electrochemical behaviour of the complexes was studied in acetonitrile solution and showed irreversible RuII/RuIII redox couples. Luminescence and UV/Vis spectral properties of the complexes in the presence and absence of buffered solutions of calf thymus DNA were also compared. Antibacterial activity of the complexes has been evaluated against Pseudomonas aeruginosa.