Yasuzo Tsukada

Effects of ammonia on the anaplerotic pathway and amino acid metabolism in the brain: an ex vivo 13C NMR spectroscopic study of rats after administering [2-13C]] glucose with or without ammonium acetate.

The 13C-label incorporation into glutamate, glutamine, aspartate and gamma-aminobutyric acid (GABA) from [2-13C] glucose was measured by 13C nuclear magnetic resonance (NMR) spectroscopy to directly examine the effects of ammonia on the activity of pyruvate carboxylase (i.e., the anaplerotic pathway) and the amino acid metabolism in the rat brain in vivo. Rats were sacrificed by exposure to microwaves at 7.5, 15, 30, and 60 min after an i.v. injection of [2-13C] glucose with or without ammonium acetate. After the injection of ammonium acetate, the brain contents of glutamate, aspartate and GABA had decreased, however, the percentage of 13C enrichment of C3 of glutamine, glutamate and GABA, and C2 and C3 of aspartate had increased. The 13C entered the TCA cycle via pyruvate carboxylase from [2-13C] glucose, labeling the C2 or C3 positions of aspartate, the C2 or C3 positions of glutamate and glutamine, and the C3 or C4 positions of GABA first and second turns of the tricarboxylic acid (TCA) cycle. The C4/C3 labeling ratio in GABA was lower than the analogous ratio in glutamate (C2/C3) and higher than that of glutamine (C2/C3). The order of these ratios (glutamate > GABA > glutamine) was not altered by the injection of ammonium acetate. These findings directly indicate that ammonia increases the anaplerotic pathway and that the 13C-skeletons entered glial glutamine through the anaplerotic pathway flow from glia to neuron. A fraction of the glutamine is used in the direct synthesis of GABA via glutamate, whereas the remaining fraction of glutamine passed through the neuronal TCA cycle before synthesizing GABA.

Human brain glucose metabolism mapping using multislice 2D 1H‐13C correlation HSQC spectroscopy

A method for multivolume 2D 1H‐13C correlation spectroscopy, multislice heteronuclear single quantum coherence (HSQC), is proposed. This permits human brain metabolism from glucose to amino acids to be followed using a 2‐T whole‐body scanner. The modifications from the conventional HSQC are that the 180°(13C) and 180°(1H) pulses are separated in time in the preparation period and that the 180°(13C) pulse is applied at 1/(4JCH) before the 90°(1H) polarization transfer (PT) pulse. The preparation (echo) time can be set longer than 1/(2JCH) so that, even in a whole‐body system, slice‐selective pulses and gradients can be applied. Another modification is that the 90°(1H) reverse PT pulses after the creation of 2IzSz are used as multislice pulses. The time‐course of glutamate C4 could be followed with 15‐min temporal resolution from the HSQC spectra obtained from the brains of volunteers after the oral administration of glucose C1, and the maximum S/N was 3. Magn Reson Med 43:525–533, 2000.

Molecular cloning and characterization of avian N-methyl-D-aspartate receptor type1 (NMDA-R1) gene

Birds have several advantages in the study of memory formation, as imprinting and passive avoidance behaviors in chick are often used as model systems. However, the primary structure of the bird N-methyl-d-aspartate (NMDA) responsive glutamate receptor, which is assumed to play a critical role in memory formation, has not been determined. In this report we describe the cDNA cloning of a subunit of NMDA receptors (NMDA-R1) from duck and analysis of its structure and distribution in the brain. The N-terminal 898 amino acids of the NMDA-R1 were well conserved between duck and mammals, but the homology was completely lost in the C-terminus. In situ hybridization showed that the duck NMDA-R1 gene was expressed throughout the brain as it is in mammals.

3D localized 1H-13C heteronuclear single-quantum coherence correlation spectroscopy in vivo

A method for spatially three‐dimensional (3D) localized two‐dimensional (2D) 1H‐13C correlation spectroscopy, localized HSQC, is proposed. This method has the following special feature in the preparation period. The 180°(13C) and 180°(1H) pulses are separated in time, and the 180°(13C) pulse is applied at 1/(4 1JCH) before the 90°(1H) polarization transfer pulse. The preparation (echo) period 2τ can then be set substantially longer than 1/(2 1JCH), so that even in a whole‐body system, slice‐selective 90°(1H) pulses and gradient pulses can be applied in that period. The localization capabilities of this method were confirmed in a phantom experiment. The 3D localized 2D 1H‐13C correlation spectra from a monkey brain in vivo were obtained after [1‐13C]glucose injection, and amino acid metabolism was detected; that is, [4‐13C]glutamate appeared immediately after the injection, followed by the appearance of [2‐13C]glutamate, [3‐13C]glutamate, and [4‐13C]glutamine. Magn Reson Med 43:200–210, 2000.

In vivo Investigation of Glutamate–Glutamine Metabolism in Hyperammonemic Monkey Brain Using 13C-Magnetic Resonance Spectroscopy

To investigate the metabolism of glutamate and glutamine in living monkey brain, a system of in vivo 13C magnetic resonance spectroscopy (MRS) using 1H-decoupled 13C spectroscopy combined with monitoring temperature changes in the brain by MR phase mapping was developed. Serial 13C-NMR spectra of the amino acids glutamate and glutamine were acquired non-invasively over 4 h from anesthetized monkey brain after the intravenous administration of [1-13C]glucose (0.5–1.0 g/kg). In the acute hyperammonemic state induced by the administration of ammonium acetate (77 mg/kg bolus), it was observed that 13C incorporation into glutamine-4 was clearly accelerated, without changes of 13C incorporation into glutamate-4. During hyperammonemia, it was shown directly by [2-13C]glucose administration that the anaplerotic pathway for the TCA cycle was also augmented, contributing to the formation of glutamine in the astroglia.

Neurotransmitter Release from the Medial Hyperstriatum Ventrale of the Chick Forebrain Accompanying Filial Imprinting Behavior, Measured by In Vivo Microdialysis

The imprinting behavior of chicks was quantified as a preference score (correct response ratio) achieved in a running wheel apparatus. A total of 249 chicks were exposed to an imprinting stimulus and tested for stimulus-approaching behavior. The chicks were then classified as good learners (imprinted), poor learners (non-imprinted) and a gray-zone group, those were 46%, 31% and 23% of the total chicks respectively. Using the classified chicks, the acetylcholine (ACh) and glutamate releases from the medial hyperstriatum ventrale (MHV) of the chick forebrains were determined by in vivo microdialysis. The non-imprinted chicks were used as yoked controls. Increases of ACh and glutamate released were observed in the imprinted chicks during exposure to the imprinting stimulus, whereas there were no changes in the release of these neurotransmitters in the non-imprinted chicks during the imprinting exposure. These results might be indicated that cholinergic and glutamatergic synapses which are newly formed as functioning synapses with imprinting stimulus in the MHV are involved in the performance of imprinting behavior.

Molecular cloning of the kainate-binding protein and calmodulin genes which are induced by an imprinting stimulus in ducklings

For the formation of imprinting in birds, protein synthesis is known to be essential in the medial hyperstriatum ventrale (MHV) of the forebrain after presentation of an imprinting stimulus. We have searched for the genes whose expressions are increased in ducklings MHV during formation of imprinting, and identified kainate-binding protein and calmodulin genes. This may reflect the formation of glutamatergic pathways in MHV.

Temperature monitoring of internal body heating induced by decoupling pulses in animal 13C-MRS experiments

A temperature monitoring method to promote safety with regard to tissue heating induced by RF irradiation during MRI procedures, especially carbon‐13 magnetic resonance spectroscopy (13C‐MRS), is proposed. The method is based on the temperature dependence of the water proton chemical shift (−0.01 ppm/°C) combined with phase mapping. Using this method, temperature changes were measured in rats (n = 4) employing practical 1H‐decoupled 13C‐MRS pulse sequences for 1D projections (TR = 1000 ms, acquisition time = 15 ms, matrix = 256, spatial resolution = 0.2 mm) and 2D images (TR = 1500 ms, acquisition time = 840 ms, matrix = 128 × 32, spatial resolution = 0.8 × 1.5 mm). Measurement error was 0.18°C (SD) for 1D acquisition and 0.39°C (SD) for 2D acquisition, demonstrating the feasibility of this temperature mapping method. Further studies should be conducted in human subjects to monitor patient safety and to optimize the pulse sequences employed. Magn Reson Med 43:796–803, 2000.

Measurement of amino acid metabolism derived from [1-13C]glucose in the rat brain using13C magnetic resonance spectroscopy

To clarify the unique characteristics of amino acid metabolism derived from glucose in the central nervous system (CNS), we injected [1-13C]glucose intraperitoneally to the rat, and extracted the free amino acids from several kinds of tissues and measured the amount of incorporation of13C derived from [1-13C]glucose into each amino acid using13C-magnetic resonance spectroscopy (NMR). In the adult rat brain, the intensities of resonances from13C-amino acids were observed in the following order: glutamate, glutamine, aspartate, γ-aminobutyrate (GABA) and alanine. There seemed no regional difference on this labeling pattern in the brain. However, only in the striatum and thalamus, the intensities of resonances from [2-13C]GABA were larger than that from [2,3-13C]aspartate. In the other tissues, such as heart, kidney, liver, spleen, muscle, lung and small intestine, the resonances from GABA were not detected and every intensity of resonances from13C-amino acids, except13C-alanine, was much smaller than those in the brain and spinal cord. In the serum,13C-amino acid was not detected at all. When the rats were decapitated, in the brain, the resonances from [1-13C]glucose greatly reduced and the intensities of resonances from [3-13C]lactate, [3-13C]alanine, [2, 3, 4-13C]GABA and [2-13C]glutamine became larger as compared with those in the case that the rats were sacrificed with microwave. In other tissues, the resonances from [1-13C]glucose were clearly detected even after the decapitation. In the glioma induced by nitrosoethylurea in the spinal cord, the large resonances from glutamine and alanine were observed; however, the intensities of resonances from glutamate were considerably reduced and the resonances from GABA and aspartate were not detected. These results show that the pattern of13C label incorporation into amino acids is unique in the central nervous tissues and also suggest that the metabolic compartmentalization could exist in the CNS through the metabolic trafficking between neurons and astroglia.

Changes in the rates of the tricarboxylic acid (TCA) cycle and glutamine synthesis in the monkey brain with hemiparkinsonism induced by intracarotid infusion of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP): studies by non-invasive 13C-magnetic resonance spectroscopy.

The tricarboxylic acid cycle rate (Vtca) and the rate of glutamine synthesis (Vgln) in the pre- and post-MPTP-treated cynomolgus monkey (Macaca fascicularis) brain were measured non-invasively using a 2 Tesla 13C-magnetic resonance spectroscopy (13C-MRS; multislice 1H-13C correlation heteronuclear single quantum coherence spectroscopy) system. Before the infusion of 1-methyl-4-phenyl-1,2,3,6-tertahydropyridine (MPTP) into three monkeys, spectra were obtained by 13C-MRS from each monkey under anesthesia after the bolus injection of [1-13C] glucose (99% atom excess, 0.28 g/kg) followed by the continuous infusion of [1-13C] glucose (99% atom excess, 0.72 g/kg) into the saphenous vein for 3 h. The average values of Vtca were 0.475+/-0.077 (mean+/-S.D.) and 0.472+/-0.073 micromol/g/min, and the average values of Vgln were 0.042+/-0.007 and 0.041+/-0.008 mumol/g/min on the left and on the right hemisphere, respectively. Three monkeys were induced hemiparkinsonism by intracarotid (left) infusion of MPTP (0.6 mg/kg) and then were employed in 13C-MRS studies for 2 (5, 14 days), 3 (3, 8, 71 days) or 4 (5, 11, 27, 78 days) times, respectively, after the MPTP treatment. The average ratios of Vtca and Vgln on the left hemisphere to those on the right hemisphere in pre- and post-MPTP-treated monkeys were 0.837+/-0.085 and 1.373+/-0.132, respectively. These results of non-invasive 13C-MRS analysis of the MPTP primate model of Parkinsons disease indicate that the loss of the dopaminergic innervation from the caudate putamen may modulate the overall glucose metabolism to glutamate and glutamine in the ipsilateral cerebrum.