Strategies for data analyses in a high resolution 1H NMR based metabolomics study of a mouse model of Batten disease

Using an NMR based approach, employing both solution state and high resolution magic angle spinning (HR MAS) ¹H NMR spectroscopy, in conjunction with an array of statistical methods, we report cerebral metabolic deficits in a mouse model of Batten disease (Cln3 null mutant mice). Batten disease is the most common progressive neurodegenerative disorder of childhood and is caused by mutations in the Cln3 gene. In particular, brain tissue from Cln3 mice was characterised by increased concentrations of glutamine, myo-inositol, scyllo-inositol, aspartate and lactate, alongside decreased concentrations of N-acetyl-l-aspartate (NAA), N-acetyl-l-glutamate (NAG), γ-amino butyric acid (GABA), glutamate and creatine. Accompanying changes in lipid deposition were also detected in intact cortical tissue by HR MAS ¹H NMR spectroscopy. To realise the true potential of metabolomic datasets necessitates a comprehensive analysis of the data, such that useful biological information can be extracted and used to generate hypotheses which can be further tested and refined. We found that using a combination of univariate and multivariate analyses, a maximal number of metabolic deficits were successfully identified. In particular the complementary nature of the statistical approaches allowed the definition of changes which were relative, absolute or simply a change in variance, allowing a greater understanding of the disease processes detected.     

A metabolomic comparison of mouse models of the Neuronal Ceroid Lipofuscinoses

The Neuronal Ceroid Lipofuscinoses (NCL) are a group of fatal inherited neurodegenerative diseases in humans distinguished by a common clinical pathology, characterized by the accumulation of storage body material in cells and gross brain atrophy. In this study, metabolic changes in three NCL mouse models were examined looking for pathways correlated with neurodegeneration. Two mouse models; motor neuron degeneration (mnd) mouse and a variant model of late infantile NCL, termed the neuronal ceroid lipofuscinosis (nclf) mouse were investigated experimentally. Both models exhibit a characteristic accumulation of autofluorescent lipopigment in neuronal and non neuronal cells. The NMR profiles derived from extracts of the cortex and cerebellum from mnd and nclf mice were distinguished according to disease/wildtype status. In particular, a perturbation in glutamine and glutamate metabolism, and a decrease in γ-amino butyric acid (GABA) in the cerebellum and cortices of mnd (adolescent mice) and nclf mice relative to wildtype at all ages were detected. Our results were compared to the Cln3 mouse model of NCL. The metabolism of mnd mice resembled older (6?month) Cln3 mice, where the disease is relatively advanced, while the metabolism of nclf mice was more akin to younger (1-2?months) Cln3 mice, where the disease is in its early stages of progression. Overall, our results allowed the identification of metabolic traits common to all NCL subtypes for the three animal models.     

MRL/lpr mice have alterations in brain metabolism as shown with [1H-13C] NMR spectroscopy

Cerebral glucose metabolism and cerebral blood flow are altered in patients with lupus who have neuropsychiatric manifestations. However, the dynamics of changes in glucose metabolism remain unclear. The present study was undertaken using 1H and 13C nuclear magnetic resonance (NMR) spectroscopy to determine the rates of incorporation of glucose into amino acids and lactate via cell-specific pathways in mice with lupus. In the well-established MRL/lpr lupus mouse model, 24-week-old mice had a significant increase of 30-80% (P<0.001) in total brain glutamine, glutamate and lactate concentrations, while alanine, aspartate, N-acetyl aspartate (NAA) and gamma-aminobutyric acid (GABA) remained unchanged as compared to the congenic MRL+/+control mice. Although succinate concentration was increased in lupus brain, it did not reach statistical significance. Furthermore, 13C isotopomer analysis showed a selective increase of de novo synthesis of lactate from [1-(13)C] glucose through glycolysis resulting in 1.5-fold increased fractional 13C enrichments in lactate in MRL/lpr mice. [4-(13)C] Glutamate, which is synthesized mainly by the neuronal pyruvate dehydogenase, was selectively increased, while [2-(13)C] and [3-(13)C] GABA synthesis were decreased by 25% compared to controls. In accordance with the total concentrations, aspartate synthesis remained unaltered in brains of lupus mice, while alanine synthesis was elevated, indicating increased utilization of alanine. Creatine was unchanged in MRL/lpr mice as compared to controls. An interesting finding was a significant increase (158%, P<0.005) in choline concentration in MRL/lpr mice while the myo-inositol concentration remained the same in both groups. Furthermore a significant increase in total brain water content was observed, indicative of possible edema. In conclusion, the cumulative effect of increased brain lactate synthesis, altered glucose metabolism and intracellular glutamine accumulation could be an important mechanism causing brain pathology in SLE. The alteration in metabolites could alter downstream pathways and cause neurological dysfunction. Future NMR spectroscopic studies using stable isotopes and real-time measurements of metabolic rates, along with levels of metabolites in plasma and cerebrospinal fluid, could be valuable in the elucidation of the cerebral metabolic consequences of systemic lupus erythematosis (SLE) in humans.     

A Metabolomic Study of Brain Tissues from Aged Mice with Low Expression of the Vesicular Monoamine Transporter 2 (VMAT2) Gene

The vesicular monoamine transporter 2 (VMAT2) sequesters monoamines into synaptic vesicles in preparation for neurotransmission. Samples of cerebellum, cortex, hippocampus, substantia nigra and striatum from VMAT2-deficient mice were compared to age-matched control mice. Multivariate statistical analyses of ¹H NMR spectral profiles separated VMAT2-deficient mice from controls for all five brain regions. Although the data show that metabolic alterations are region- and age-specific, in general, analyses indicated decreases in the concentrations of taurine and creatine/phosphocreatine and increases in glutamate and N-acetyl aspartate in VMAT2-deficient mouse brain tissues. This study demonstrates the efficacy of metabolomics as a functional genomics phenotyping tool for mouse models of neurological disorders, and indicates that mild reductions in the expression of VMAT2 affect normal brain metabolism. Special issue article in honor of Dr. Frode Fonnum.     

Regional and developmental variations in metabolite concentration in the rat brain and eye: A study using1H NMR spectroscopy and high performance liquid chromatography

Regional and developmental changes in metabolite concentrations were measured by¹H NMR spectroscopy and HPLC of perchloric acid extracts from rat brain and eye. The highest concentrations of N-acetylaspartate were found in grey matter as opposed to white matter with concentration increasing with age from neonate to adult, while the related compound N-acetylaspartylglutamate was highest in adult optic nerve. Creatine and choline-containing compounds were present in all regions throughout development, with higher levels of creatine found in grey matter compared to other regions. Choline-containing compounds were present at the highest concentrations in the eye at all ages examined, and tended to decrease in concentration to minimum values in adulthood in all regions. The presence of hypotaurine in corpus callosum and optic nerve was consistent with the metabolic profiles of O-2A progenitor cells and oligodendrocytes, which are cells composing these tissues. The neurotransmitters glutamate and GABA reached their highest concentrations in the olfactory bulb (higher than in adult cortex). Special issue dedicated to Dr. Herman Bachelard.     

Early changes in gene expression in two models of Batten disease

Infantile and juvenile neuronal ceroid lipofuscinosis (NCLs) are progressive neurodegenerative disorders of childhood with distinct ages of clinical onset, but with a similar pathological outcome. Infantile and juvenile NCL are inherited in an autosomal recessive manner due to mutations in the CLN1 and CLN3 genes, respectively. Recently developed Cln1- and Cln3-knockout mouse models share similarities in pathology with the respective human disease. Using oligonucleotide arrays we identified reproducible changes in gene expression in the brains of both 10-week-old Cln1- and Cln3-knockout mice as compared to wild-type controls, and confirmed changes in levels of several of the cognate proteins by immunoblotting. Despite the similarities in pathology, the two mutations affect the expression of different, non-overlapping sets of genes. The possible significance of these changes and the pathological mechanisms underlying NCL diseases are discussed.     

Distinct changes in neuronal and astrocytic amino acid neurotransmitter metabolism in mice with reduced numbers of synaptic vesicles

The relations between glutamate and GABA concentrations and synaptic vesicle density in nerve terminals were examined in an animal model with 40–50% reduction in synaptic vesicle numbers caused by inactivation of the genes encoding synapsin I and II. Concentrations and synthesis of amino acids were measured in extracts from cerebrum and a crude synaptosomal fraction by HPLC and ¹³C nuclear magnetic resonance spectroscopy (NMRS), respectively. Analysis of cerebrum extracts, comprising both neurotransmitter and metabolic pools, showed decreased concentration of GABA, increased concentration of glutamine and unchanged concentration of glutamate in synapsin I and II double knockout (DKO) mice. In contrast, both glutamate and GABA concentrations were decreased in crude synaptosomes isolated from synapsin DKO mice, suggesting that the large metabolic pool of glutamate in the cerebral extracts may overshadow minor changes in the transmitter pool. ¹³C NMRS studies showed that the changes in amino acid concentrations in the synapsin DKO mice were caused by decreased synthesis of GABA (20–24%) in cerebral neurons and increased synthesis of glutamine (36%) in astrocytes. In a crude synaptosomal fraction, the glutamate synthesis was reduced (24%), but this reduction could not be detected in cerebrum extracts. We suggest that lack of synaptic vesicles causes down-regulation of neuronal GABA and glutamate synthesis, with a concomitant increase in astrocytic synthesis of glutamine, in order to maintain normal neurotransmitter concentrations in the nerve terminal cytosol.     

Effect of 2-Amino-7-Phosphonoheptanoic Acid on Regional Brain Amino Acid Levels in Fed and Fasted Rodents

Abstract: 2-Amino-7-phosphonoheptanoic acid, an antagonist of excitation caused by dicarboxylic amino acids with a selective action on N -methyl-d-aspartate receptors, has been administered in an anticonvulsant dose (1 mmol/kg i.p.) to fed or fasted rats and mice. The drug impaired motor activity in fasted mice. Glucose and amino acids were determined in dissected regions of brain fixed by microwave irradiation. Glucose content was low in the brains of fasted rats and mice but was restored to normal (fed) concentration 45 min after the administration of 2-amino-7-phosphonoheptanoic acid in fasted mice. In fed animals, 2-amino-7-phosphonoheptanoic acid did not change brain aspartate concentration. In fasted animals, aspartate concentration was raised in most brain regions. In fasted rats and mice, 2-amino-7-phosphonoheptanoic acid significantly increased glutamine in rat cortex and mouse striatum, decreased glutamate content in rat striatum, and decreased aspartate concentration in all regions except mouse cortex and striatum. GABA levels were significantly decreased in rat striatum and hippocampus. These changes are consistent with an increased synaptic release of glutamate and aspartate following blockage of their post-synaptic action at selected sites.     

Sex-differences and stress: effects on regional high and low affinity [3H]GABA binding

Sex-differences are observed in the GABAergic neurotransmitter system both at rest and following acute stress, yet the brain regions and functional implications of these differences are unknown. We examined sex-differences in the number of low- and high-affinity [3H]GABA binding sites in various brain regions of male and female mice and the effect of stress on such sex-differences. Male (n=6) and female (n=6) QS mice were exposed to a brief swim stress (3 min at 32+/-1 degrees C) either individually or with cage-mates whilst control males (n=6) and females (n=6) remained undisturbed in the home cage. Using quantitative receptor autoradiography, sections of mouse brain were labelled with either 30 or 1000 nM [3H]GABA to label high or low affinity binding sites, respectively. Results indicated that males had more low affinity [3H]GABA binding sites in various forebrain cortical regions but less high affinity binding sites in many of these regions compared with females. Forced swim stress-induced rapid changes in forebrain GABA binding sites in females and group stressed males, suggesting a mechanism for rapid GABAergic adaptations. However the number of functional binding sites for GABA in certain forebrain regions was altered by stress in opposite directions in males and females, such that baseline sex-differences were removed following stress. These results exemplify sex-differences in brain chemical function and stress responses, and are of potential importance for understanding sex-differences in response to GABAergic compounds and disorders with sex and stress as predisposing factors.     

Metabolic characterization of the R6/2 transgenic mouse model of Huntington's disease by high-resolution MAS 1H NMR spectroscopy

The metabolic consequences of Huntington's disease in the R6/2 mouse model were investigated using NMR spectroscopy and pattern recognition to characterize selected brain regions, muscle, blood, and urine. Global increases in relative brain concentrations of osmolytes, creatine, glutamine, and lactate, and decreases in acetate and N-acetylaspartate were found together with striatal-specific lower concentrations of GABA and choline. Clear differentiation of R6/2 and wild-type mice was also obtained for urine and blood metabolite profiles that may have applicability for monitoring HD in human populations.     

Altered sensitivity of cerebellar granule cells to glutamate receptor overactivation in the Cln3(Δex7/8)-knock-in mouse model of juvenile neuronal ceroid lipofuscinosis

The juvenile onset form of neuronal ceroid lipofuscinoses (JNCL) is a recessively inherited lysosomal storage disorder characterized by progressive neurodegeneration. JNCL results from mutations in the CLN3 gene that encodes a lysosomal membrane protein with unknown function. Utilizing a Cln3-knock-out mouse model of JNCL that was created on the 129S6/SvEv genetic background, we have previously demonstrated that CLN3-deficient cerebellar granule cells (CGCs) have a selectively increased sensitivity to AMPA-type glutamate receptor-mediated toxicity. Our recent findings that CGCs from 129S6/SvEv and C57BL/6J wild type (WT) mice have significant differences in glutamate receptor expression and in excitotoxic vulnerability indicated that the genetic background possibly have a strong influence on how glutamate receptor function is dysregulated in CLN3-deficient neurons. Indeed, here we show that in the Cln3(Δex7/8)-knock-in mouse model, that is on the C57BL/6J genetic background, mimics the most frequent mutation observed in JNCL patients and considered a null mutant, the sensitivity of CGCs to both AMPA- and NMDA-type glutamate receptor overactivations is altered. Cultured wild type and Cln3(Δex7/8) CGCs were equally sensitive to AMPA toxicity after 2 or 3 weeks in vitro, whereas the subunit-selective AMPA receptor agonist, CPW-399, induced significantly more cell death in mature, 3-week-old Cln3(Δex7/8) cultures. NMDA receptor-mediated toxicity changed during in vitro development: Cln3(Δex7/8) CGCs were less sensitive to high concentration of NMDA after 2 weeks in culture but became more vulnerable than their WT counterparts after 3 weeks in vitro. Abnormally altered glutamate receptor function in the cerebellum may result in motor deficits, and we confirmed that 7-week-old Cln3(Δex7/8) mice, similarly to Cln3-knock-out mice, have a motor coordination deficit as measured by an accelerating rotarod. Our results demonstrate altered glutamate receptor function in Cln3(Δex7/8) neurons and suggest that both AMPA and NMDA receptors are potential therapeutic targets in JNCL.     

Quantification of the GABA Shunt and the Importance of the GABA Shunt Versus the 2-Oxoglutarate Dehydrogenase Pathway in GABAergic Neurons

Abstract: We investigated the activity of the cerebral GABA shunt relative to the overall cerebral tricarboxylic acid (TCA) cycle and the importance of the GABA shunt versus 2-oxoglutarate dehydrogenase for the conversion of 2-oxoglutarate into succinate in GABAergic neurons. Awake mice were dosed with [1-¹³C]glucose, and brain extracts were analyzed by ¹³C NMR spectroscopy. The percent enrichments of GABA C-2 and glutamate C-4 were the same: 5.0 ± 1.6 and 5.1 ± 0.2%, respectively (mean ± SD). This, together with previous data, indicates that the flux through the GABA shunt relative to the overall cerebral TCA cycle flux equals the GABA/glutamate pool size ratio, which in the mouse is 17%. It has previously been shown that under the experimental conditions used in this study, the ¹³C labeling of aspartate from [1-¹³C]glucose specifically reflects the metabolic activity of GABAergic neurons. In the present study, the reduction in the formation of [¹³C]aspartate during inhibition of the GABA shunt by γ-vinyl-GABA indicated that not more than half the flux from 2-oxoglutarate to succinate in GABAergic neurons goes via the GABA shunt. Therefore, because fluxes through the GABA shunt and 2-oxoglutarate dehydrogenase in GABAergic neurons are approximately the same, the TCA cycle activity of GABAergic neurons could account for one-third of the overall cerebral TCA cycle activity in the mouse. Treatment with γ-vinyl-GABA, which increased GABA levels dramatically, caused changes in the ¹³C labeling of glutamate and glutamine, which indicated a reduction in the transfer of glutamate from neurons to glia, implying reduced glutamatergic neurotransmission. In the most severely affected animals these alterations were associated with convulsions.     

13C Nuclear Magnetic Resonance Evidence for γ-Aminobutyric Acid Formation via Pyruvate Carboxylase in Rat Brain: A Metabolic Basis for Compartmentation0

The compartmentation of amino acid metabolism is an active and important area of brain research. 13C labeling and 13C nuclear magnetic resonance (NMR) are powerful tools for studying metabolic pathways, because information about the metabolic histories of metabolites can be determined from the appearance and position of the label in products. We have used 13C labeling and 13C NMR in order to investigate the metabolic history of gamma-aminobutyric acid (GABA) and glutamate in rat brain. [1-13C]Glucose was infused into anesthetized rats and the 13C labeling patterns in GABA and glutamate examined in brain tissue extracts obtained at various times after infusion of the label. Five minutes after infusion, most of the 13C label in glutamate appeared at the C4 position; at later times, label was also present at C2 and C3. This 13C labeling pattern occurs when [1-13C]glucose is metabolized to pyruvate by glycolysis and enters the pool of tricarboxylic acid (TCA) intermediates via pyruvate dehydrogenase. The label exchanges into glutamate from the TCA cycle pool through glutamate transaminases or dehydrogenase. After 30 min of infusion, approximately 10% of the total 13C in brain extracts appeared in GABA, primarily (greater than 80%) at the amino carbon (C4), indicating that the GABA detected is labeled through pyruvate carboxylase. The different labeling patterns observed for glutamate and GABA show that the large detectable glutamate pool does not serve as the precursor to GABA. Our NMR data support previous experiments suggesting compartmentation of metabolism in brain, and further demonstrate that GABA is formed from a pool of TCA cycle intermediates derived from an anaplerotic pathway involving pyruvate carboxylase.     

Metabonomic alterations in hippocampus, temporal and prefrontal cortex with age in rats

The metabolic changes in hippocampus, temporal cortex and prefrontal cortex in SD rats along with aging were explored using a metabonomic approach, which based on high resolution “magic angle spinning” ¹H NMR spectroscopy. The metabolite profiles were analyzed by partial least squares-discriminant analysis, and the results showed that the metabolites of the above three brain regions in old rats were dramatically different from that in the adult and young rats. The old rats showed increased myo-inositol and lactate in all of the three brain regions, and decreased N-acetylaspartate in temporal and frontal cortex, Glutamate–GABA level became imbalance in temporal cortex of old rats. In addition, compared with the adult female rats, male rats had higher levels of N-acetylaspartate, taurine, and creatine in temporal or frontal cortex. The age-related metabolic changes may indicate the early functional alterations of neural cells in these brain regions, especially the temporal cortex. The gender-related metabolic changes suggest the significance of the hormonal regulation in brain metabolism. Our work highlights the potential of metabolic profiling to enhance our understanding of biological mechanisms of brain aging.     

Loss of Ahi1 Impairs Neurotransmitter Release and Causes Depressive Behaviors in Mice

Major depression is becoming one of the most prevalent forms of psychiatric disorders. However, the mechanisms of major depression are still not well-understood. Most antidepressants are only effective in some patients and produce some serious side effects. Animal models of depression are therefore essential to unravel the mechanisms of depression and to develop novel therapeutic strategies. Our previous studies showed that Abelson helper integration site-1 (Ahi1) deficiency causes depression-like behaviors in mice. In this study, we characterized the biochemical and behavioral changes in Ahi1 knockout (KO) mice. In Ahi1 KO mice, neurotransmitters including serotonin and dopamine were significantly decreased in different brain regions. However, glutamate and GABA levels were not affected by Ahi1 deficiency. The antidepressant imipramine attenuated depressive behaviors and partially restored brain serotonin level in Ahi1 KO mice. Our findings suggest that Ahi1 KO mice can be used for studying the mechanisms of depression and screening therapeutic targets.     

Metabonomic Characterization of the 3-Nitropropionic Acid Rat Model of Huntington’s Disease

3-Nitropropionic acid (3-NP)-induced neurotoxicity can be used as a model for the genetic neurodegenerative disorder Huntington’s disease (HD). A metabolic profiling strategy was adopted to explore the biochemical consequences of 3-NP administered to rats in specific brain regions. ¹H NMR spectroscopy was used to characterize the metabolite composition of several brain regions following 3-NP-intoxication. Dose-dependent increases in succinate levels were observed in all neuroanatomical regions, resulting from the 3-NP-induced inhibition of succinate dehydrogenase. Global decreases in taurine and GABA were observed in the majority of brain regions, whereas altered lipid profiles were observed only in the globus pallidus and dorsal striatum. Depleted phosphatidylcholine and elevated glycerol levels, which are indicative of apoptosis, were also observed in the frontal cortex of the 3-NP model. Many of the metabolic anomalies are consistent with those reported in HD. The 3-NP-induced model of HD provides a means of monitoring potential mechanisms of pathology and therapeutic response for drug interventions, which can be efficiently assessed using metabolic profiling strategies.     

A Regional Study of 4-Aminobutyrate Metabolism and Amino Acid Levels in Rat Brain Following Chronic Oral Administration of Ethanolamine O-Sulphate

Abstract: Ethanolamine O-sulphate (EOS) dissolved in the drinking water (5mg-ml⁻¹) was administered ad libitum to rats for 26 days. At the end of this period, glutamate decarboxylase (GAD) and GABA-transaminase (GABA-T) activities, 4-aminobutyrate (GABA) concentration, and the levels of six other amino acids were measured in various brain regions. Significant inhibition of GABA-T accompanied by significant increases in GABA content were observed throughout the brain, although the magnitudes of these effects varied according to region. GAD activity was significantly reduced in most brain regions, although this effect was apparently not related to cofactor availability or the direct actions of EOS or increased GABA concentration. Glutamine levels were significantly reduced to approximately 72% of control values in all brain regions. Aspartate levels were significantly reduced to approximately 84% of control values in all regions except the striatum and cerebellum. Minor changes in other amino acid levels were also detected. These neurochemical changes which accompanied the primary effect of EOS on GABA-T are discussed in terms of indirect secondary metabolic changes rather than nonspecific enzyme inhibition by EOS.     

Acute and short term effects of ethanol on the metabolism of glutamic acid and GABA in rat brain

Although alcoholic intoxication is attributed to its pharmacological effects on the cell membranes in brain, the rapid metabolic utilisation of the same alters the metabolism of brain affecting the metabolism of glutamate and GABA which have varied metabolic roles besides serving a major proportion of synaptic activity. A study on the effects of ethanol, both acute and short-term, on glutamate (glu) and GABA metabolism in various regions of rat brain was carried out. Increased activities of glutamic acid decarboxylase (GAD) and aspartic acid aminotransferase (AST) in all brain regions, but decreased activity of glutamic acid dehydrogenase (GDH) in cerebral cortex (CC) and cerebellum (CB) following ethanol administration in brain was observed. Differential effects of ethanol were also obtained on the contents of glu and aspartate (asp), which were increased in CC, CB, and brain stem (BS) regions, as opposed to GABA content, which, although found to increase in acute toxicity, showed a decrease in all of the above brain regions in short-term toxicity. It is concluded that the above changes in glu, asp and GABA represent the consequences of metabolic utilization of alcohol in the brain, probably more a state of cerebral excitation than depression, and the changes may be a compensatory phenomenon.     

Pentylenetetrazole decreases metabolic glutamate turnover in rat brain

Seizures were induced in rats by intraperitoneal injection of pentylenetetrazole (PTZ, 70 mg/kg), followed, 30 min later, by injection of [1-13C]glucose and [1,2-13C]acetate. Analyses of extracts from cortex, subcortex and cerebellum were performed using 13C magnetic resonance spectroscopy and HPLC. It could be shown that PTZ affected different brain regions differently. The total amounts of glutamate, glutamine, GABA, aspartate and taurine were decreased in the cerebellum and unchanged in the other brain regions. GABAergic neurones in the cortex and subcortex were not affected, whereas those in the cerebellum showed a pronounced decrease of GABA synthesis. However, glutamatergic neurones in all brain regions showed a decrease in glutamate labelling and in addition a decreased turnover in cerebellum. It could be shown that this decrease was in the metabolic pool of glutamate whereas release of glutamate was unaffected since glutamine labelling from glutamate was unchanged. Aspartate turnover was also decreased in all brain regions. Changes in astrocyte metabolism were not detected, indicating that PTZ had no effect on astrocyte metabolism in the early postictal stage.     

Nondestructive Detection of Glutamate by 1H Nuclear Magnetic Resonance Spectroscopy in Cortical Brain Slices from the Guinea Pig: Evidence for Changes in Detectability During Severe Anoxic Insults

31P and 1H nuclear magnetic resonance spectroscopy (NMR) was used to study the metabolism of intact superfused cortical brain slices during normoxia and anoxia. Attention was focused on quantification of 1H NMR-detected glutamate by a water-suppressed spin-echo method, using N-acetyl aspartate as an internal concentration reference. To quantify the 1H NMR signals, the spin-spin relaxation times and saturation effects were estimated for given metabolites. In addition, absolute concentrations of metabolites were determined by biochemical methods from acid extracts of the preparations after NMR experiments. Under aerobic conditions, 1H NMR detected 79% of the glutamate determined biochemically from the brain slice extracts. During anoxia in the absence of glucose when a severe energetic failure was evident, both 1H NMR and biochemical assays gave closely matching levels for glutamate. We conclude that in the brain cortex 21% of glutamate is located in an intracellular compartment in which this amino acid does not contribute to the 1H NMR signal. However, during severe anoxia an intracellular reorganisation occurs increasing the detectability of this amino acid neurotransmitter by NMR.