Measurement of neuro-energetics and neurotransmission in the rat olfactory bulb using 1 H and 1 H-[13 C] NMR spectroscopy.

The olfactory bulb (OB) plays a fundamental role in the sense of smell and has been implicated in several pathologies, including Alzheimer's disease. Despite its importance, high metabolic activity and unique laminar architecture, the OB is not frequently studied using MRS methods, likely due to the small size and challenging location. Here we present a detailed metabolic characterization of OB metabolism, in terms of both static metabolite concentrations using 1 H MRS and metabolic fluxes associated with neuro-energetics and neurotransmission by tracing the dynamic 13 C flow from intravenously administered [1,6-13 C2 ]-glucose, [2-13 C]-glucose and [2-13 C]-acetate to downstream metabolites, including [4-13 C]-glutamate, [4-13 C]-glutamine and [2-13 C]-GABA. The unique laminar architecture and associated metabolism of the OB, distinctly different from that of the cerebral cortex, is characterized by elevated GABA and glutamine levels, as well as increased GABAergic and astroglial energy metabolism and neurotransmission. The results show that, despite the technical challenges, high-quality 1 H and 1 H-[13 C] MR spectra can be obtained from the rat OB in vivo. The derived metabolite concentrations and metabolic rates demonstrate a unique metabolic profile for the OB. The metabolic model provides a solid basis for future OB studies on functional activation or pathological conditions.

Cell-type specific modulation of NMDA receptors triggers antidepressant actions.

Both the NMDA receptor (NMDAR) positive allosteric modulator (PAM), and antagonist, can exert rapid antidepressant effects as shown in several animal and human studies. However, how this bidirectional modulation of NMDARs causes similar antidepressant effects remains unknown. Notably, the initial cellular trigger, specific cell-type(s), and subunit(s) of NMDARs mediating the antidepressant-like effects of a PAM or an antagonist have not been identified. Here, we used electrophysiology, microdialysis, and NMR spectroscopy to evaluate the effect of a NMDAR PAM (rapastinel) or NMDAR antagonist, ketamine on NMDAR function and disinhibition-mediated glutamate release. Further, we used cell-type specific knockdown (KD), pharmacological, and behavioral approaches to dissect the cell-type specific role of GluN2B, GluN2A, and dopamine receptor subunits in the actions of NMDAR PAM vs. antagonists. We demonstrate that rapastinel directly enhances NMDAR activity on principal glutamatergic neurons in medial prefrontal cortex (mPFC) without any effect on glutamate efflux, while ketamine blocks NMDAR on GABA interneurons to cause glutamate efflux and indirect activation of excitatory synapses. Behavioral studies using cell-type-specific KD in mPFC demonstrate that NMDAR-GluN2B KD on Camk2a- but not Gad1-expressing neurons blocks the antidepressant effects of rapastinel. In contrast, GluN2B KD on Gad1- but not Camk2a-expressing neurons blocks the actions of ketamine. The results also demonstrate that Drd1-expressing pyramidal neurons in mPFC mediate the rapid antidepressant actions of ketamine and rapastinel. Together, these results demonstrate unique initial cellular triggers as well as converging effects on Drd1-pyramidal cell signaling that underlie the antidepressant actions of NMDAR-positive modulation vs. NMDAR blockade.

Glutaminase activity in GLS1 Het mouse brain compared to putative pharmacological inhibition by ebselen using ex vivo MRS.

Glutaminase mediates the recycling of neurotransmitter glutamate, supporting most excitatory neurotransmission in the mammalian central nervous system. A constitutive heterozygous reduction in GLS1 engenders in mice a model of schizophrenia resilience and associated increases in Gln, reductions in Glu and activity-dependent attenuation of excitatory synaptic transmission. Hippocampal brain slices from GLS1 heterozygous mice metabolize less Gln to Glu. Whether glutaminase activity is diminished in the intact brain in GLS1 heterozygous mice has not been assessed, nor the regional impact. Moreover, it is not known whether pharmacological inhibition would mimic the genetic reduction. We addressed this using magnetic resonance spectroscopy to assess amino acid content and 13C-acetate loading to assess glutaminase activity, in multiple brain regions. Glutaminase activity was reduced significantly in the hippocampus of GLS1 heterozygous mice, while acute treatment with the putative glutaminase inhibitor ebselen did not impact glutaminase activity, but did significantly increase GABA. This approach identifies a molecular imaging strategy for testing target engagement by comparing genetic and pharmacological inhibition, across brain regions.

The effects of ketamine on prefrontal glutamate neurotransmission in healthy and depressed subjects.

The ability of ketamine administration to activate prefrontal glutamate neurotransmission is thought to be a key mechanism contributing to its transient psychotomimetic effects and its delayed and sustained antidepressant effects. Rodent studies employing carbon-13 magnetic resonance spectroscopy (13C MRS) methods have shown ketamine and other N-methyl-D-aspartate (NMDA) receptor antagonists to transiently increase measures reflecting glutamate-glutamine cycling and glutamate neurotransmission in the frontal cortex. However, there are not yet direct measures of glutamate neurotransmission in vivo in humans to support these hypotheses. The current first-level pilot study employed a novel prefrontal 13C MRS approach similar to that used in the rodent studies for direct measurement of ketamine effects on glutamate-glutamine cycling. Twenty-one participants (14 healthy and 7 depressed) completed two 13C MRS scans during infusion of normal saline or subanesthetic doses of ketamine. Compared to placebo, ketamine increased prefrontal glutamate-glutamine cycling, as indicated by a 13% increase in 13C glutamine enrichment (t = 2.4, p = 0.02). We found no evidence of ketamine effects on oxidative energy production, as reflected by 13C glutamate enrichment. During ketamine infusion, the ratio of 13C glutamate/glutamine enrichments, a putative measure of neurotransmission strength, was correlated with the Clinician-Administered Dissociative States Scale (r = -0.54, p = 0.048). These findings provide the most direct evidence in humans to date that ketamine increases glutamate release in the prefrontal cortex, a mechanism previously linked to schizophrenia pathophysiology and implicated in the induction of rapid antidepressant effects.

Impaired Glutamatergic Neurotransmission in the Ventromedial Hypothalamus May Contribute to Defective Counterregulation in Recurrently Hypoglycemic Rats.

The objectives of this study were to understand the role of glutamatergic neurotransmission in the ventromedial hypothalamus (VMH) in response to hypoglycemia and to elucidate the effects of recurrent hypoglycemia (RH) on this neurotransmitter. We 1) measured changes in interstitial VMH glutamate levels by using microdialysis and biosensors, 2) identified the receptors that mediate glutamate's stimulatory effects on the counterregulatory responses, 3) quantified glutamate metabolic enzyme levels in the VMH, 4) examined astrocytic glutamate reuptake mechanisms, and 5) used 1H-[13C]-nuclear magnetic resonance (NMR) spectroscopy to evaluate the effects of RH on neuronal glutamate metabolism. We demonstrated that glutamate acts through kainic acid receptors in the VMH to augment counterregulatory responses. Biosensors showed that the normal transient rise in glutamate levels in response to hypoglycemia is absent in RH animals. More importantly, RH reduced extracellular glutamate concentrations partly as a result of decreased glutaminase expression. Decreased glutamate was also associated with reduced astrocytic glutamate transport in the VMH. NMR analysis revealed a decrease in [4-13C]glutamate but unaltered [4-13C]glutamine concentrations in the VMH of RH animals. The data suggest that glutamate release is important for proper activation of the counterregulatory response to hypoglycemia and that impairment of glutamate metabolic and resynthetic pathways with RH may contribute to counterregulatory failure.

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-(13)C2]-D-ß-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 (V(acCoA-kbN)) and pyruvate (V(pdhN)), and the glutamate-glutamine cycle (V(cyc)) were determined by metabolic modeling of (13)C label trapped in major brain amino acid pools. V(acCoA-kbN) increased gradually with increasing activity, as compared with the steeper change in tricarboxylic acid (TCA) cycle rate (V(tcaN)), 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.

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

Previous (13)C 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.

Quantification of high-resolution ¹H-[¹³C] NMR spectra from rat brain extracts.

NMR spectroscopy in combination with (13)C-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 (13)C studies in rodents can be enhanced by high-resolution (1)H-[(13)C] NMR spectroscopy on brain extracts. Previously, it has been shown that (1)H 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 (13)C 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 (13)C label, the fixed amplitude relationship between multiplets typical for (1)H NMR spectra must be abandoned. In addition, (13)C isotope effects lead to spectral multiplet patterns that become dependent on the amount of (13)C label accumulation, thereby preventing the use of a common basis set. Here a modified spectral fitting routine is presented that accommodates variable (13)C label accumulation and (13)C isotope effects. Spectral fitting results are quantitatively compared to manual integration on column-separated samples in which spectral overlap is minimized.

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 drug's acute effects on cognition, perception, and mood.

Quantification of high-resolution (1)H 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.

As one of the most widespread drugs of abuse, nicotine has long been known to impact the brain, particularly with respect to addiction. However, the regional effects of nicotine on the concentrations and kinetics of amino acid neurotransmitters and some energetically related neurochemicals have been little studied. In this investigation, acute effects of nicotine were measured by (1)H-observed/(13)C-edited nuclear magnetic resonance spectroscopy method in extracts obtained from nicotine-naïve, freely moving rats given 0.7 mg/kg nicotine or saline, with [1-(13)C] glucose to track metabolism. Nicotine was observed to exert significant effects on the concentrations of N-acetylaspartate and GABA, particularly in the striatum. Nicotine decreased brain glucose oxidation, glutamate-glutamine neurotransmitter cycling, and GABA synthesis regionally, including in the parietal and occipital cortices and the striatum. The olfactory bulb showed kinetics that differed markedly from those observed in the rest of the brain. Independently of nicotine, the concentration of glutamate was found to be correlated significantly with levels of N-acetylaspartate and GABA, suggesting a potential interplay of energetics and excitatory and inhibitory neurotransmission. In summary, the study revealed that the neurochemicals were most affected in the cortex and striatum of the rat brain after acute nicotine treatment.

In situ 3D magnetic resonance metabolic imaging of microwave-irradiated rodent brain: a new tool for metabolomics research.

The rapid elevation in rat brain temperature achieveable with focused beam microwave irradiation (FBMI) leads to a permanent inactivation of enzymes, thereby minimizing enzyme-dependent post-mortem metabolic changes. An additional characteristic of FBMI is that the NMR properties of the tissue are close to those of the in vivo condition and remain so for at least 12 h. These features create an opportunity to develop magnetic resonance spectroscopy and imaging on microwave-irradiated samples into a technique with a resolution, coverage and sensitivity superior to any experiment performed directly in vivo. Furthermore, when combined with pre-FBMI infusion of (13)C-labeled substrates, like [1-(13)C]-glucose, the technique can generate maps of metabolic fluxes, like the tricarboxylic acid and glutamate-glutamine neurotransmitter cycle fluxes at an unprecedented spatial resolution.

Effects of continuous hypoxia on energy metabolism in cultured cerebro-cortical neurons.

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 (194%), PK (90%), and HK (24%) and decreased activities of CS (15%) and GDH (34%). Neurons were incubated with [1-(13)C]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-(13)C]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 remain to be determined.

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 (13)C labeling in metabolites in prefrontal cortex and hippocampus during a 10 mins infusion of [1-(13)C]glucose in urethane anesthetized rats treated with riluzole (21 days, 4 mg/kg per day, i.p.) or saline. Total and (13)C concentrations of metabolites were determined in extracts using (1)H-[(13)C] NMR spectroscopy. In prefrontal cortex (P<0.05) and hippocampus (P<0.05) riluzole increased (13)C labeling over saline in glutamate-C4 (to 112% and 130%), GABA-C2 (to 142% and 171%), and glutamine-C4 (to 118% and 233%) without affecting total metabolite levels (P>0.2). Our findings indicate that contrary to expectation chronic riluzole enhanced glucose oxidative metabolism and glutamate/glutamine cycling.

Glutamatergic and GABAergic neurotransmitter cycling and energy metabolism in rat cerebral cortex during postnatal development.

The contribution of glutamatergic and gamma-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-(13)C(2)]glucose for different time intervals (time course) or with [2-(13)C]acetate for 2 to 3 h (steady state). Amino acid levels and (13)C enrichments were determined in tissue extracts ex vivo using (1)H-[(13)C]-NMR spectroscopy. Metabolic fluxes were estimated from the best fits of a three-compartment metabolic model (glutamatergic neurons, GABAergic neurons, and astroglia) to the (13)C-enrichment time courses of amino acids from [1,6-(13)C(2)]glucose, constrained by the ratios of neurotransmitter cycling (V(cyc))-to-tricarboxylic acid (TCA) cycle flux (V(TCAn)) calculated from the steady-state [2-(13)C]acetate enrichment data. From P10 to P30 increases in total neuronal (glutamate plus GABA) TCA cycle flux (3 x ; 0.24+/-0.05 versus 0.71+/-0.07 micromol per g per min, P<0.0001) and total neurotransmitter cycling flux (3.1 to 5 x ; 0.07 to 0.11 (+/-0.03) versus 0.34+/-0.03 micromol per g per min, P<0.0001) were approximately proportional. Incremental changes in total cycling (DeltaV(cyc(tot))) and neuronal TCA cycle flux (DeltaV(TCAn(tot))) between P10 and P30 were 0.23 to 0.27 and 0.47 micromol per g per min, respectively, similar to the approximately 1:2 relationship previously reported for adult cortex. For the individual neurons, increases in V(TCAn) and V(cyc) were similar in magnitude (glutamatergic neurons, 2.7 x versus 2.8 to 4.6 x ; GABAergic neurons, approximately 5 x versus approximately 7 x), 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.

Cerebral pyruvate carboxylase flux is unaltered during bicuculline-seizures.

Glutamine synthesis in the astroglia reflects the sum of neurotransmitter cycling (glutamate and gamma-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-(13)C]glucose using localized (1)H-[(13)C] 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-(13)C] and [3-(13)C] of glutamine and glutamate (Glx(2+3)) labeling during [2-(13)C]glucose infusion. During seizures the initial rate of Glx(2+3) synthesis (0.069 +/- 0.013 micromol/g/min) was not significantly different (P = 0.68) from that of the controls (0.059 +/- 0.010 micromol/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).