Tomoyuki Kanamatsu

Glutamatergic Neurotransmission and Neuronal Glucose Oxidation Are Coupled During Intense Neuronal Activation

13C nuclear magnetic resonance (NMR) experiments have previously shown that glutamatergic neurotransmitter flux (Vcycle(Glu/Gln)) changes proportionately with neuronal glucose oxidation (CMRglc(ox)N) in the nonactivated cortex of anesthetized rats. Positron Emission Tomography measurements of glucose and oxygen uptake during sensory stimulation had shown that the incremental glucose utilization is greater than oxygen leading to the suggestion that the energy required for stimulated neuronal activity arises from nonoxidative glucose metabolism. In this study, the authors used spatially localized 1H-observed, 13C-edited NMR spectroscopy during an infusion of [1,6–13C2]glucose to assess the relationship between changes in Vcycle(Glu/Gln) and glucose utilization (CMRglc(ox)N and CMRglc(nonox)) during the intense cortical activity associated with bicuculline-induced seizures. Metabolic fluxes were determined by model-based analysis of the 13C-enrichment time courses of glutamate-C4 and glutamine-C4 (CMRglc(ox)N, Vcycle(Glu/Gln)) and lactate-C3 (CMRglc(nonox)). The exchange rate between α-ketoglutarate and glutamate was found to be significantly faster than TCA cycle flux both for control (41 μmol·g−1·min−1; 95% CI, 5 to 109 μmol·g−1·min−1) and during seizures (21 μmol·g−1·min−1; 95% CI, 4.4 to 51.8 μmol·g−1·min−1). During seizures, total glucose utilization (CMRglc(ox+nonox)) increased substantially (466% between 0 and 6 minutes; 277% between 6 and 55 minutes). Glucose oxidation (CMRglc(ox)N) also increased (214%; from 0.26 ± 0.02 to 0.57 ± 0.07 μmol·g−1·min−1) but to a lesser degree, resulting in a large increase in cortical lactate concentration. Vcycle(Glu/Gln) increased 233% (from 0.22 ± 0.04 to 0.52 ± 0.07 μmol·g−1·min−1), which was similar to the increase in glucose oxidation. The value of Vcycle(Glu/Gln) and CMRglc(ox)N obtained here lie on the line predicted in a previous study. These results indicate that neuronal glucose oxidation and not total glucose utilization is coupled to the glutamate/glutamine cycle during intense cortical activation.

A comparison of 13C NMR measurements of the rates of glutamine synthesis and the tricarboxylic acid cycle during oral and intravenous administration of [1-13C]glucose

13C-labeled glucose is increasingly used in conjunction with magnetic resonance spectroscopy to measure rates of metabolic pathways in the brain in vivo. Most studies of human subjects have used intravenous infusions to administer the labeled compounds, but the procedure is cumbersome and can be uncomfortable for patients with neurological or psychiatric disorders. It may be possible to improve the practicality of the method by administering the glucose orally instead of intravenously. This report describes the performance and comparison of the oral and intravenous protocols in the same subjects. The conclusion is that oral administration does yield the same result as intravenous administration but with lower precision. That sensitivity of the oral protocol may be improved by several ways that are available today.

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.

Demonstration of a Neural Circuit Critical for Imprinting Behavior in Chicks

Imprinting behavior in birds is elicited by visual and/or auditory cues. It has been demonstrated previously that visual cues are recognized and processed in the visual Wulst (VW), and imprinting memory is stored in the intermediate medial mesopallium (IMM) of the telencephalon. Alteration of neural responses in these two regions according to imprinting has been reported, yet direct evidence of the neural circuit linking these two regions is lacking. Thus, it remains unclear how memory is formed and expressed in this circuit. Here, we present anatomical as well as physiological evidence of the neural circuit connecting the VW and IMM and show that imprinting training during the critical period strengthens and refines this circuit. A functional connection established by imprint training resulted in an imprinting behavior. After the closure of the critical period, training could not activate this circuit nor induce the imprinting behavior. Glutamatergic neurons in the ventroposterior region of the VW, the core region of the hyperpallium densocellulare (HDCo), sent their axons to the periventricular part of the HD, just dorsal and afferent to the IMM. We found that the HDCo is important in imprinting behavior. The refinement and/or enhancement of this neural circuit are attributed to increased activity of HDCo cells, and the activity depended on NR2B-containing NMDA receptors. These findings show a neural connection in the telencephalon in Aves and demonstrate that NR2B function is indispensable for the plasticity of HDCo cells, which are key mediators of imprinting.

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.

Imprinting modulates processing of visual information in the visual wulst of chicks

BackgroundImprinting behavior is one form of learning and memory in precocial birds. With the aim of elucidating of the neural basis for visual imprinting, we focused on visual information processing.ResultsA lesion in the visual wulst, which is similar functionally to the mammalian visual cortex, caused anterograde amnesia in visual imprinting behavior. Since the color of an object was one of the important cues for imprinting, we investigated color information processing in the visual wulst. Intrinsic optical signals from the visual wulst were detected in the early posthatch period and the peak regions of responses to red, green, and blue were spatially organized from the caudal to the nasal regions in dark-reared chicks. This spatial representation of color recognition showed plastic changes, and the response pattern along the antero-posterior axis of the visual wulst altered according to the color the chick was imprinted to.ConclusionThese results indicate that the thalamofugal pathway is critical for learning the imprinting stimulus and that the visual wulst shows learning-related plasticity and may relay processed visual information to indicate the color of the imprint stimulus to the memory storage region, e.g., the intermediate medial mesopallium.

FK-506 extended the therapeutic time window for thrombolysis without increasing the risk of hemorrhagic transformation in an embolic rat stroke model.

FK-506 confers a neuroprotective effect and is thought to extend the time window for thrombolytic treatment of cerebral ischemia. These effects have not been assessed in an embolic stroke model. In addition, clinical studies have raised concern that FK-506 may increase the risk of hemorrhagic transformation by damaging vascular endothelial cells. We investigated whether combined administration of recombinant tissue plasminogen activator (rt-PA) and FK-506 would extend the therapeutic time window without increasing the hemorrhagic transformation in a rat embolic stroke model. Male Sprague-Dawley rats (n=66) were subjected to embolic infarction and assigned into eight groups. Six of the groups were treated with or without FK-506 (0.3 mg/kg) administration at 60 min after embolization, together with and all six groups received systemic rt-PA administration (10 mg/kg) at 60, 90, or 120 min. Two permanent ischemia groups were administered saline either with or without FK-506. Infarct and hemorrhagic volume were assessed at 24 h after embolization. Diffusion-weighted and perfusion-weighted magnetic resonance imaging (MRI) were performed in the groups administered rt-PA at 90 min and a vehicle control group to assess whether FK-506 influenced the effectiveness of MRI in revealing ischemic lesion. FK-506 extended the therapeutic time window for systemic thrombolysis compared to rt-PA alone without increasing the risk for hemorrhage. Combined therapy with FK-506 salvaged some of the MRI, revealing ischemic lesions destined to infarction in the animals treated by rt-PA alone. Single low dose of FK-506 alone did not ameliorate the embolic infarction, but it did prove effective in extending the therapeutic time windows for thrombolysis without increasing the risk of hemorrhagic transformation.

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.