Golam M. I. Chowdhury

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.

Mild prenatal stress enhances learning performance in the non-adopted rat offspring.

The present study was designed to investigate whether mild stress during pregnancy affects offspring behaviors, including learning performance. Prenatal stress was induced by short-lasting, mild restraint stress, which had previously been shown to facilitate the morphological development of fetal brain neurons. Adult offspring whose dams had been restrained in a small cage for 30min daily from gestation day 15 to 17 showed enhanced active avoidance and radial maze learning performance. In addition, the prenatally stressed rats showed weaker emotional responses than unstressed control, as indicated by decreases both in ambulation upon initial exposure to an open field and in Fos expression in the amygdala induced by physical stress. The observed effects of prenatal stress on learning performance and emotional behavior were attenuated by foster rearing by unstressed dams. Fos expression in the hypothalamic paraventricular nucleus following physical stress and corticosterone secretion during physical and psychological stress did not differ between the prenatally stressed and unstressed control rats. From these results we suggest that mild prenatal stress facilitates learning performance in the adult offspring. The enhancement of learning performance appears to be accompanied by reduced emotionality, but not by any apparent alterations in hypothalamic-pituitary-adrenal responses. In addition, the observation of differential behaviors in the adopted and non-adopted animals supports the notion that the postnatal environment modifies the behavioral effects of prenatal stress.

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.

1H-[13C]-Nuclear Magnetic Resonance Spectroscopy Measures of Ketamine's Effect on Amino Acid Neurotransmitter Metabolism

Ketamine has recently gained significant attention owing to its psychotomimetic and more recently discovered rapid antidepressant-like properties. ¹H-[¹³C]-nuclear magnetic resonance studies were employed to explore potential physiological processes underlying these unique effects. [1-¹³C]glucose and [2-¹³C]acetate-nuclear magnetic resonance ex vivo studies were performed on the medial prefrontal cortex (mPFC) and hippocampus of rats acutely treated with 30 mg/kg or 80 mg/kg ketamine and compared with saline-treated animals to determine the effects of ketamine on amino acid neurotransmitter cycling and glial metabolism. A subanesthetic, but not anesthetic, dose of ketamine significantly increased the percentage of ¹³C-enrichments of glutamate, γ-aminobutyric acid, and glutamine in the mPFC of rats. Subanesthetic doses of ketamine increased 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 subanesthetic doses of ketamine, are related to the drugs acute effects on cognition, perception, and mood.

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.

Transiently increased glutamate cycling in rat PFC is associated with rapid onset of antidepressant-like effects.

Several drugs have recently been reported to induce rapid antidepressant effects in clinical trials and rodent models. Although the cellular mechanisms involved remain unclear, reports suggest that increased glutamate transmission contributes to these effects. Here, we demonstrate that the antidepressant-like efficacy of three unique drugs, with reported rapid onset antidepressant properties, is coupled with a rapid transient rise in glutamate cycling in the medial prefronal cortex (mPFC) of awake rats as measured by ex vivo 1H-[13C]-nuclear magnetic resonance spectroscopy. Rats were acutely pretreated by intraperitoneal injection with a single dose of ketamine (1, 3, 10, 30 and 80 mg kg−1), Ro 25-6981 (1, 3 and 10 mg kg−1), scopolamine (5, 25 and 100 μg kg−1) or vehicle (controls). At fixed times after drug injection, animals received an intravenous infusion of [1,6-13C2]glucose for 8 min to enrich the amino-acid pools of the brain with 13C, followed by rapid euthanasia. The mPFC was dissected, extracted with ethanol and metabolite 13C enrichments were measured. We found a clear dose-dependent effect of ketamine and Ro 25-6981 on behavior and the percentage of 13C enrichment of glutamate, glutamine and GABA (γ-aminobutyric acid). Further, we also found an effect of scopolamine on both cycling and behavior. These studies demonstrate that three pharmacologically distinct classes of drugs, clinically related through their reported rapid antidepressant actions, share the common ability to rapidly stimulate glutamate cycling at doses pertinent for their antidepressant-like efficacy. We conclude that increased cycling precedes the antidepressant action at behaviorally effective doses and suggest that the rapid change in cycling could be used to predict efficacy of novel agents or identify doses with antidepressant activity.

Cerebral pyruvate carboxylase flux is unaltered during bicuculline-seizures.

Glutamine synthesis in the astroglia reflects the sum of neurotransmitter cycling (glutamate and γ‐aminobutyric acid [GABA]) and de novo synthesis (anaplerosis), the latter catalyzed by pyruvate carboxylase. Previous studies have shown that the glutamate plus GABA cycling flux is correlated strongly with neuronal activity; however, the relationship between pyruvate carboxylase flux and neuronal activity is not known. In this study, pyruvate carboxylase flux was assessed during intravenous infusion of [2‐13C]glucose using localized 1H‐[13C] NMR spectroscopy at 7 Tesla in vivo in halothane‐ anesthetized and ventilated adult Wistar rats during 85 min of bicuculline‐induced seizures (1 mg/kg, intravenously) and in nontreated controls. During seizures, concentrations of lactate, alanine, glutamine, GABA, and succinate increased whereas glutamate and aspartate decreased such that the decrease in glutamate plus aspartate equaled the increase in glutamine plus GABA. Pyruvate carboxylase flux was assessed by the sum of [2‐13C] and [3‐13C] of glutamine and glutamate (Glx2+3) labeling during [2‐13C]glucose infusion. During seizures the initial rate of Glx2+3 synthesis (0.069 ± 0.013 μmol/g/min) was not significantly different (P = 0.68) from that of the controls (0.059 ± 0.010 μmol/g/min), indicating that anaplerotic flow through pyruvate carboxylase was unaltered. Intense neuronal activation of seizures did not seem to increase anaplerosis through pyruvate carboxylase, despite the substantial increase in neuronal activity and glutamate/glutamine cycling shown in a previous study (Patel et al., 2004b ).

Altered cerebral glucose and acetate metabolism in succinic semialdehyde dehydrogenase-deficient mice: evidence for glial dysfunction and reduced glutamate/glutamine cycling

Succinic semialdehyde dehydrogenase (SSADH) catalyzes the NADP‐dependent oxidation of succinic semialdehyde to succinate, the final step of the GABA shunt pathway. SSADH deficiency in humans is associated with excessive elevation of GABA and γ‐hydroxybutyrate (GHB). Recent studies of SSADH‐null mice show that elevated GABA and GHB are accompanied by reduced glutamine, a known precursor of the neurotransmitters glutamate and GABA. In this study, cerebral metabolism was investigated in urethane‐anesthetized SSADH‐null and wild‐type 17‐day‐old mice by intraperitoneal infusion of [1,6‐13C2]glucose or [2‐13C]acetate for different periods. Cortical extracts were prepared and measured using high‐resolution 1H‐[13C] NMR spectroscopy. Compared with wild‐type, levels of GABA, GHB, aspartate, and alanine were significantly higher in SSADH‐null cortex, whereas glutamate, glutamine, and taurine were lower. 13C Labeling from [1,6‐13C2]glucose, which is metabolized in neurons and glia, was significantly lower (expressed as μmol of 13C incorporated per gram of brain tissue) for glutamate‐(C4,C3), glutamine‐C4, succinate‐(C3/2), and aspartate‐C3 in SSADH‐null cortex, whereas Ala‐C3 was higher and GABA‐C2 unchanged. 13C Labeling from [2‐13C]acetate, a glial substrate, was lower mainly in glutamine‐C4 and glutamate‐(C4,C3). GHB was labeled by both substrates in SSADH‐null mice consistent with GABA as precursor. Our findings indicate that SSADH deficiency is associated with major alterations in glutamate and glutamine metabolism in glia and neurons with surprisingly lesser effects on GABA synthesis.

Quantification of High-Resolution 1H-[13C] NMR Spectra from Rat Brain Extracts

Extracting quantitative information about absolute concentrations from high-resolution (1)H 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 (1)H 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.

Regional metabolite levels and turnover in the awake rat brain under the influence of nicotine

J. Neurochem. (2010) 113, 1447–1458.