Regional distribution of glutamate and aspartate in adult and old human brain

In previous studies on rat brain we found that the observed heterogeneity of the regional distribution of amino acids was much greater when small well-defined anatomical structures were assayed. We therefore reinvestigated the distribution of glutamate and aspartate in 50 discrete areas from adult and old human brain. The concentration of glutamate in the area of highest level was 4.5 and 4.7 times as high as in the area of lowest level in adult and old brain respectively; for aspartate these values were 3.0 and 6.6. Several changes in old brain were noted. The human pattern differed from that in rat.     

Distribution and stress-induced increase of glutamate and aspartate levels in discrete brain nuclei of rats

Concentrations of glutamate and aspartate have been measured in 45 microdissected brain areas and nuclei in rat. Both amino acids are ubiquitously present and distributed unevenly in the central nervous system. Very high glutamate levels were found in the cerebellum and the insular cortex, high levels in neocortical and limbic cortical areas, and in the nuclei of the medial hypothalamus. Aspartate is distributed rather uniformly with the highest concentration in the hypothalamic arcuate nucleus and the lowest in the midbrain central gray matter and the cerebellum. Acute formalin (pain) stress elevated glutamate and aspartate levels in the cortical areas and substantia nigra significantly, but had minor or no effects on other brain nuclei. Increased locomotor and behavioral activities due to a high dose of amphetamine resulted in a 2-5-fold increase of glutamate and aspartate concentrations, particularly in the biogenic amine-containing brain nuclei.     

Proline, glutamate and glutamine metabolism in mouse brain synaptosomes

In nerve terminals, glutamate (Glu) may serve as precursor of the inhibitory neurotransmitter, GABA, and the putative excitatory transmitter, aspartate (Asp), in addition to exerting its own excitatory neurotransmitter role in brain. Glu carbon can originate from glucose through glycolysis and the Krebs cycle, from glutamine (Gln) subsequent to uptake, and from proline (Pro) and ornithine (Orn). Orn, but not Glu, is an effective precursor in nerve terminals of Pro, a putative inhibitory neurotransmitter. [3H]Arg can be converted in mouse brain nerve terminals to Orn, which in turn gives rise to Glu, Pro and GABA. In the present study, the conversion subsequent to uptake of labeled Glu, Gln and Pro to other amino acids was studied in unfractionated and subfractionated synaptosomal particles which layered, respectively, on 1.0 M, 1.2 M, 1.3 M and 1.5 M sucrose after centrifugation in a discontinuous gradient (fractions 1-4, respectively). Fraction 1 contained small synaptosomal fragments with vesicles and almost no mitochondria. Fractions 2 and 3 showed numerous normal-appearing mitochondria-containing synaptosomes, and fraction 4 contained large synaptosomes and more free mitochondria than the other fractions. Glu was readily taken up in all fractions and converted to Asp, Gln and GABA, the greatest formation of Asp from Glu occurring in fractions 2 and 3 and of Gln in fraction 4. In contrast, Gln was taken up poorly in fraction 1 and not metabolized, converted extensively to Glu and GABA in fractions 2-4, giving rise only to very small amounts of Asp in fractions 2 and 3. Although Pro was taken up to the greatest extent in fraction 2, it was by far most readily converted to Glu, Gln and GABA in fraction 1, showing only small amounts of Asp formation in fractions 1-3 and none in 4. There was no significant production of Pro from Glu or Gln or of Arg and Orn from any of the 3 precursors studied. The above results suggest that Glu, Gln and Pro may be taken up largely in different classes of synaptosomes which are distributed among the centrifugally separated fractions and which possess differing transport and metabolic characteristics. Determination of glutamate decarboxylase activity (GAD) indicated that GABA-forming nerve terminals were present in all synaptosomal fractions studied. Amino acid determinations by HPLC in the subfractionated synaptosomes showed a similar distribution for Glu, Asp and GABA contents, peaking in fraction 2, and an inverse relationship of the latter 3 with Arg contents.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nervous tissue thiamine metabolism in vivo. I. Transport of thiamine and thiamine monophosphate from plasma to different brain regions of the rat

The transport of thiamine (T) and thiamine monophosphate (TMP) across the blood-brain barrier was measured in vivo in the rat. Different doses of [¹⁴C]T (15–550 nmol) and [¹⁴]TMP (11–110 nmol) were injected into the femoral vein. The content of T and its phosphoesters in blood and brain tissue (cerebellum, pons, medulla and cerebral cortex) 20 s after the injection was determined radiometrically after electrophoretic separation. Blood flow and blood volume in the same regions of the brain was also determined. Both T and TMP entered rapidly the cerebral tissue, where they were found chemically unmodified.The cerebral tissue extracted less than 7% of plasma T. At physiological plasma T concentrations, the rate of transport ranged from 0.43 to 0.65 nmol·g⁻¹·h⁻¹ with only minor differences among the various regions. T was transported into the nervous tissue by two separate mechanisms: one saturable, that at physiological plasma T levels accounted for 95% (cerebellum) to 91% (cerebral cortex) of the total T taken up, and one non-saturable, that was most efficient in the cerebral cortex. The K_m (half-saturation constant) of the former transport mechanism ranged from 1.95 to 2.75 nmol·ml⁻¹ in the 4 areas investigated. V_max (maximal transport rate) values ranged from 6 to 9 nmol·g⁻¹·h⁻¹, the highest value being found in the cerebellum. The overall transport rate of TMP was on average 5–10 times as low as that of T and also showed a saturable and a non-saturable component. Both components were slower than those observed for T.     

Inhibitors of glutamate transport modulate distinct patterns in brain metabolism

High affinity uptake of glutamate plays a major role in the termination of excitatory neurotransmission. Identification of the ramifications of transporter function is essential to understand the diseases in which defective excitatory amino acid transporters (EAAT) have been implicated. In this work we incubated Guinea pig cortical tissue slices with [3-(13)C]pyruvate and major currently available glutamate uptake inhibitors and studied the resultant metabolic sequelae by (13)C and (1)H NMR spectroscopy using a multivariate statistical approach. Perturbation of glutamate uptake produced significant effects on metabolic flux through the Krebs cycle, and on glutamate/glutamine cycling rates, with this effect accounting for 76% of the variation in the total data set. The effects of all inhibitors were separable from each other along three major principal components. The competitive inhibitor L-CCG III ((2S,1'S,2'R)-2-carboxycyclopropyl)glycine) differed most from the other inhibitors, showing negative weightings on both the first and second principal components, whereas the EAAT2-specific inhibitor dihydrokainate (DHK) showed metabolic patterns similar to that of anti-endo-3,4-methanopyrolidine dicarboxylate but separate from those of DL-threo-beta-benzyloxyaspartate (TBOA) and L-trans-pyrrolidine-2,4-dicarboxylate (L-tPDC). This indicates that different inhibition mechanisms or different colocalisation of the separate transporter subtypes with glutamate receptors can produce significantly different metabolic and functional outcomes for the brain.     

Alterations in cerebral energy metabolism induced by traumatic brain injury

Energy metabolism of the brain is unique, possessing high aerobic metabolism with no significant capacity for anaerobic glycolysis and limited tissue stores of glucose. A steady supply of oxygen and glucose is essential in order to maintain cerebral function and integrity. Extensive research in experimental and human head injury has been conducted regarding the delivery of oxygen and outcome. This research has provided evidence which indicates that in addition to the availability of oxygen and glucose, other factors, such as perturbation of mitochondrial energy transducing processes which also follow head trauma, play significant roles. In this paper, the salient findings from biochemical studies of experimental and clinical brain injury are summarized and indicate that the mitochondrial respiratory chain-linked oxidative phosphorylation and calcium transport are compromised by trauma-induced brain injury and support the idea that oxidative stress and perturbation of cellular calcium homeostasis play significant roles in traumatic brain injury.     

Alterations of noradrenaline and serotonin uptake and metabolism in chronic cobalt-induced epilepsy in the rat

The high affinity uptake of noradrenaline and serotonin, and the concentrations of these monoamines and their metabolites, have been measured in the perifocal cortical area at various stages of the evolution of cobalt-induced epilepsy in the rat. Noradrenaline uptake was maximally reduced at days 8-10 after cortical cobalt application, a time corresponding to the onset of epileptic discharges; it remained diminished during the spiking activity period of the focus (days 14-20) and was back to normal values at day 40, at which time the epileptic syndrome had disappeared. Serotonin uptake was also diminished at days 8-10 but to a lesser extent than was noradrenaline uptake. In the homotopic cerebral cortex contralateral to cobalt application, noradrenaline uptake was reduced at day 10 only and to a lesser extent than in the perifocal area, whereas serotonin uptake was unaffected. Kinetic analysis of the cobalt-induced monoamine uptake alterations at day 10 revealed a diminution of the maximal velocity with no change in the Km. Noradrenaline and dihydroxyphenylethyleneglycol concentrations in the perifocal area were also maximally reduced at days 8-10 but were unaffected at day 2 and day 40 post cobalt application. A reduction of serotonin levels in the perifocal area was observed only at days 8-10 while 5-hydroxyindoleacetic acid remained unaffected throughout the time period studied. The levels of these monoamines and their metabolites were unchanged in the homotopic contralateral cortex 2-40 days after cobalt application. These results indicate that cortical cobalt application induces alterations of the biochemical indices of the density of noradrenaline-containing terminals that closely parallel the evolution of the epileptic syndrome. These data further emphasize the important role of the cortical noradrenergic system in cobalt-induced epilepsy.     

Alterations in brain metabolism during the first year of HIV infection

Migration of both uninfected and infected monocytes into the brain during acute HIV infection likely initiates metabolic changes that can be observed with magnetic resonance spectroscopy (MRS). Herein, we measured changes in brain metabolism during the first year of HIV infection and examined the relationship of these metabolite levels to CD16+ monocyte populations measured in the blood. MRS was performed on nine HIV+ subjects identified during acute HIV infection and nine seronegative control subjects. HIV+ subjects were examined within 90 days of an indeterminate Western blot, then again 2 and 6 months later, during early infection. Blood samples were collected for plasma viral RNA and monocyte subset quantification. HIV+ subjects were identified with acute viral ailment and did not display severe cognitive deficits such as dementia or minor cognitive motor disorder. Changes in lipid membrane metabolism (choline levels) in the frontal cortex and white matter were observed during the initial year of HIV infection. Greater numbers of CD16+ monocytes were associated with lower N-acetylaspartate levels and higher choline levels in the brain. These results suggest that HIV infection induces metabolic changes in the brain early during infection and that these changes may be related to monocyte dynamics in the periphery.     

Alterations in cerebral oxygen metabolism after traumatic brain injury in children

Traumatic brain injury (TBI) is the most common cause of acquired disability in children. Metabolic defects, and in particular mitochondrial dysfunction, are important contributors to brain injury after TBI. Studies of metabolic dysfunction are limited, but magnetic resonance methods suitable for use in children are overcoming this limitation. We performed noninvasive measurements of cerebral blood flow and oxygen metabolic index (OMI) to assess metabolic dysfunction in children with severe TBI. Cerebral blood flow is variable after TBI but hypoperfusion and low OMI are predominant, supporting metabolic dysfunction. This finding is consistent with preclinical and adult clinical studies of brain metabolism and mitochondrial dysfunction after TBI.     

Regulation of human glutamate dehydrogenases: implications for glutamate, ammonia and energy metabolism in brain.

Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of glutamate to alpha-ketoglutarate using NAD or NADP as cofactors. In mammalian brain, GDH is located predominantly in astrocytes, where it is probably involved in the metabolism of transmitter glutamate. The exact mechanisms that regulate glutamate fluxes through this pathway, however, have not been fully understood. In the human, GDH exists in heat-resistant and heat-labile isoforms, encoded by the GLUD1 (housekeeping) and GLUD2 (nerve tissue-specific) genes, respectively. These forms differ in their catalytic and allosteric properties. Kinetic studies showed that the K(m) value for glutamate for the nerve tissue GDH is within the range of glutamate levels in astrocytes (2.43 mM), whereas for the housekeeping enzyme, this value is significantly higher (7.64 mM; P < 0.01). The allosteric activators ADP (0.1-1.0 mM) and L-leucine (1.0-10.0 mM) induce a concentration-dependent enzyme stimulation that is proportionally greater for the nerve tissue-specific GDH (up to 1,600%) than for the housekeeping enzyme (up to 150%). When used together at lower concentrations, ADP (10-50 mM) and L-leucine (75-200 microM) act synergistically in stimulating GDH activity. GTP exerts a powerful inhibitory effect (IC(50) = 0.20 mM) on the housekeeping GDH; in contrast, the nerve tissue isoenzyme is resistant to GTP inhibition. Thus, although the housekeeping GDH is regulated primarily by GTP, the nerve tissue GDH activity depends largely on available ADP or L-leucine levels. Conditions associated with enhanced hydrolysis of ATP to ADP (e.g., intense glutamatergic transmission) are likely to activate nerve tissue-specific GDH leading to an increased glutamate flux through this pathway.     

Methylmercury inhibits glutamate uptake by synaptic vesicles from rat brain

Methylmercury (MeHg) is an environmental contaminant that continues to cause risk to human health. The toxic effects of MeHg on the CNS implicate the involvement of glutamatergic system. In this study, we evaluated the effects of MeHg on [3H]glutamate uptake by synaptic vesicles. MeHg inhibited [3H]glutamate uptake in a concentration dependent manner. Since glutamate uptake by synaptic vesicles is driven by an electrochemical gradient, formed across the vesicle membrane by a bafilomycin A(1)-sensitive H+-ATPase, we further investigated the effect of MeHg on activity of this enzyme.MeHg inhibited the H+-ATPase activity and also dissipated the proton gradient (DeltapH), indicating that MeHg decreased [3H]glutamate uptake involving the H+-ATPase activity. Until now, the toxic effects of MeHg on CNS were attributed mainly to an impairment of glial glutamate transporters. These findings contribute for the understanding of the neurotoxicity by MeHg, pointing to the involvement of vesicular glutamate.     

Postnatal changes in the activity of glutamate dehydrogenase and aspartate aminotransferase in the rat nervous system with special reference to the glutamate transmitter metabolism

The activities of aspartate aminotransferase (AAT) and glutamate dehydrogenase (GIDH), the major glutamate metabolizing enzymes, were studied in hippocampal formation, cerebellar cortex, dorsal root ganglia, superior cervical ganglia and liver as a function of postnatal development. At birth, in all these nervous tissues the enzyme activities were quite low and showed similar levels (AAT 7-15 U/g wet weight; 0.18-0.23 U/mg protein; GIDH 3.4-13 U/g wet weight; 0.07-0.18 U/mg protein). Based on protein, AAT activity increased during the postnatal period studied 5.8 and 3.8 times in the hippocampal formation and cerebellar cortex, respectively, while the respective GIDH rise was 5.2 and 2.3 times. During postnatal maturation, enzyme activities in dorsal root ganglia showed only minor changes. In superior cervical ganglia, AAT and GIDH were remarkably constant. In liver the enzyme activities changed during postnatal development, but the activity curve profile was quite distinct from those obtained for brain regions. The steep rise of AAT and GIDH activities in brain regions is discussed as being a consequence of the maturation of preferably glutamatergic structures. Glutamatergic transmission processes obviously do not take place in superior cervical ganglia and dorsal root ganglia, and certainly not in liver. The present results suggest a quantitatively significant participation of glutamate transmitter metabolism in proportion to the whole glutamate metabolism of the CNS.     

Imaging brain deoxyglucose uptake and metabolism by glucoCEST MRI

2-Deoxy-D-glucose (2DG) is a known surrogate molecule that is useful for inferring glucose uptake and metabolism. Although ¹³C-labeled 2DG can be detected by nuclear magnetic resonance (NMR), its low sensitivity for detection prohibits imaging to be performed. Using chemical exchange saturation transfer (CEST) as a signal-amplification mechanism, 2DG and the phosphorylated 2DG-6-phosphate (2DG6P) can be indirectly detected in ¹H magnetic resonance imaging (MRI). We showed that the CEST signal changed with 2DG concentration, and was reduced by suppressing cerebral metabolism with increased general anesthetic. The signal changes were not affected by cerebral or plasma pH, and were not correlated with altered cerebral blood flow as demonstrated by hypercapnia; neither were they related to the extracellular glucose amounts as compared with injection of D- and L-glucose. In vivo ³¹P NMR revealed similar changes in 2DG6P concentration, suggesting that the CEST signal reflected the rate of glucose assimilation. This method provides a new way to use widely available MRI techniques to image deoxyglucose/glucose uptake and metabolism in vivo without the need for isotopic labeling of the molecules.