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Imbalance of excitatory and inhibitory neurotransmission is implicated in a wide range of psychiatric and neurologic disorders. Here we tested the hypothesis that insertion of a methyl group on the stereogenic alpha carbon of L-Glu or L-Gln would impact the γ-aminobutyric acid (GABA) shunt and the glutamate-glutamine cycle. (S)-2-methylglutamate, or (S)-2MeGlu, was efficiently transported into brain and synaptosomes where it was released by membrane depolarization in a manner equivalent to endogenous L-Glu. (R)-2MeGlu was transported less efficiently into brain and synaptosomes but was not released by membrane depolarization. Each enantiomer of 2MeGlu had limited activity across a panel of over 30 glutamate and GABA receptors. While neither enantiomer of 2MeGlu was metabolized along the GABA shunt, (S)-2MeGlu was selectively converted to (S)-2-methylglutamine, or (S)-2MeGln, which was subsequently slowly hydrolyzed back to (S)-2MeGlu in brain. rac-2MeGln was also transported into brain, with similar efficiency as (S)-2MeGlu. A battery of behavioral tests in young adult wild type mice showed safety with up to single 900 mg/kg dose of (R)-2MeGlu, (S)-2MeGlu, or rac-2MeGln, suppressed locomotor activity with single ≥ 100 mg/kg dose of (R)-2MeGlu or (S)-2MeGlu. No effect on anxiety or hippocampus-dependent learning was evident. Enantiomers of 2MeGlu and 2MeGln show promise as potential pharmacologic agents and imaging probes for cells that produce or transport L-Gln.

N-(2- 18 F-fluoropropionyl)-l-glutamate ( 18 F-FPGLU), a new N-substituted 18 F-labeling l-glutamate, is a potential amino acid tracer for oncology PET imaging with good tumor-to-background contrast in several tumor-bearing mice. Herein, we evaluated the potential value of 18 F-FPGLU for PET imaging of glioma in orthotopic glioma-bearing SD rats. A series of competitive inhibition experiments with various types of inhibitors were conducted with C6 cells to investigate the transport mechanism of 18 F-FPGLU in glioma. Establishment of orthotopic rat C6 glioma-bearing SD rats models was confirmed by MRI. Then PET imaging of 18 F-FPGLU was performed on the orthotopic C6 glioma-bearing SD rats and compared with that of 18 F-FDG. After the rats sacrificed, the whole brain was collected and immunofluorescence staining of glial fibrillary acidic protein (GFAP) and matrix metalloproteinase 2 (MMP2) were processed. Na + -dependent system X AG - and Na + -independent system X C - are the mainly transporters of 18 F-FPGLU in C6 cells. N-methyl-d-aspartate (NMDA) receptor, which is associated with the invasiveness and proliferation of glioma cells, is also involved in the uptake of 18 F-FPGLU. High uptake and retention of 18 F-FPGLU was observerd in orthotopic glioma with good visualization and the tumor/background ratio reached 2.35 at 60 min post-injection, which was significantly higher than that of 18 F-FDG (1.72) in small-animal PET images. High expression of MMP-2 and GFAP was observed in the immunofluorescence staining of glioma xerography slices. 18 F-FPGLU seems to be a better potential PET tracer than 18 F-FDG for brain glioma imaging with good visualization and ability to assess the tumor activity., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background/aim: The effects of coadministration of melatonin and theanine (Mel/Thea) on pentylenetetrazole (PTZ)-induced seizures and brain tissue oxidative damage were investigated in ovariectomized (OVX) and sham-operated rats., Materials and Methods: The rats were divided into the following groups: 1) sham, 2) ovariectomized (OVX), 3) sham-PTZ, 4) OVX- PTZ, 5) sham-Mel/Thea-PTZ, and 6) OVX-Mel/Thea-PTZ. Groups 1-4 received saline, while groups 5 and 6 received a combination of Mel/Thea for 6 weeks. All animals except for those in groups 1 and 2 received a single injection of PTZ., Results: The OVX-PTZ group had higher generalized tonic-clonic seizure (GTCS) latency compared to the sham-PTZ group. Administration of Mel/Thea increased minimal clonic seizure and GTCS latencies in both the sham-Mel/Thea-PTZ and OVX-Mel/Thea-PTZ groups compared to the controls. Additionally, PTZ exposure increased malondialdehyde levels and reduced thiol concentrations in brain tissues of both the sham-PTZ and OVX-PTZ groups. Mel/Thea pretreatment resulted in MDA reduction and thiol increase in brain tissues., Conclusion: The results of this study demonstrated the antioxidant and anticonvulsant activities of Mel/Thea despite the presence or absence of ovarian hormones.

Theanine (r-glutamylethylamide) is one of the major amino acid components in green tea. Recent studies suggest that theanine affects neurotransmission, especially inhibitory neurotransmission. In this study, we investigated whether theanine affects brain development in infant rats, because inhibitory neurotransmission is required for mature brain function. Mother rats were fed theanine ad libitum after confinement. The body weight gain rate of infants was not different from control infants. We detected theanine in the infant serum and measured neurotransmitter concentration and nerve growth factor (NGF) mRNA level in the infant rat brain. Some neurotransmitters, including dopamine, serotonin, glycine and GABA concentration, increased in the infant brain and NGF mRNA level increased in the cerebral cortex and hippocampus. However, these differences were lost by the end of nerve maturity. These results suggest that theanine enhanced synthesis of nerve growth factor and neurotransmitters during a nerve maturing period and promoted central nerve system maturation (CNS). Thus, theanine accelerated maturation. In conclusion, theanine may assist in healthy brain function development.

Pemetrexed disodium is a novel antifolate that exhibits potent inhibitory effects on multiple enzymes in folate metabolism. Phase II/III clinical trials have shown that pemetrexed is effective against various solid tumors. Like methotrexate, pemetrexed may be useful in treatment of primary and secondary brain tumors. In this study, we examined the central nervous system (CNS) distribution of pemetrexed and the interaction with an organic anion transport inhibitor indomethacin. Male Wistar rats were administered pemetrexed by either single intravenous bolus or constant intravenous infusion. Unbound pemetrexed in blood and brain was measured by simultaneous arterial blood and frontal cortex microdialysis sampling. In the i.v. bolus experiments, indomethacin was administered by i.v. bolus (10 mg/kg) followed by i.v. infusion (0.1 mg/kg/h) in a crossover manner. In the infusion experiments, the same dose of indomethacin was administered after a steady state was reached for pemetrexed. CNS distributional kinetics was analyzed by compartmental and noncompartmental methods. Both bolus and infusion studies showed that pemetrexed has a limited CNS distribution. The mean area under concentration-time curve (AUC)(brain)/AUC(plasma) ratio of unbound pemetrexed was 0.078 +/- 0.038 in the i.v. bolus study. The pemetrexed steady-state brain-to-plasma unbound concentration ratio after i.v. infusion was 0.106 +/- 0.054. The distributional clearance into the brain was approximately 10% of the clearance out of the brain in both the compartmental and noncompartmental analyses. Indomethacin had no effect on either the brain-to-plasma AUC ratio or the steady-state brain-to-plasma concentration ratio. The distribution of pemetrexed into the brain is limited, and an efflux clearance process, such as an efflux transporter, may be involved.

The excitotoxicity of the neurotransmitter glutamate has been shown to be connected with many acute and chronic diseases of the CNS. High affinity sodium-dependent glutamate transporters play a key role in maintaining adequate levels of extracellular glutamate. In the present study, we used slices of striatum, hippocampus and cortex from rat brain to describe the in vitro profile of glutamate uptake during development and ageing, and its sensitivity to guanosine. In all structures, glutamate uptake was higher in immature animals. There was a maximum decrease in glutamate uptake in striatum and hippocampus in 15-month-old rats, which later increased, while in cortex there was a significant decrease in rats aged 60 days old. The effect of guanosine seems to be age and structure dependent since the increase in basal glutamate uptake was only seen in slices of cortex from 10-day-old animals.

In vitro studies showed deltamethrin (1 x 10(-7)-1 x 10(-4) mol/L) could inhibit 3H-DL-glutamate uptake in rat cerebral cortex and hippocampus synaptosome by 7.2%-21.5% and 10.4%-25.1%, respectively, with a dose-response pattern. But only in high-dose permethrin (1 x 10(-4) mol/L) can significantly reduce 3H-DL-glutamate uptake in rat hippocampus synaptosome. In addition, deltamethrin also can reduce 3H-DL-glutamate uptake in cerebellum and striatum of rats by 16.5% and 31.8%, respectively. It is suggested that both deltamethrin and permethrin, especially the former, can disturb the function of brain tissue in high-affinity-glutamate uptake with their alpha-cyano-group. So pyrethroids could play an important role in cerebral stimulation in mammals.

The transplantation of fetal dopamine neurons has previously been shown to ameliorate locomotor deficits in laboratory animals with nigrostriatal dopamine lesions. These studies have also demonstrated a reinstatement of striatal dopamine synthesis and release associated with the graft-to-host fiber innervation. More recent studies in the intact striatum indicate that striatal dopamine release can be regulated by corticostriatal glutamatergic projections. In the present study we hypothesized that secretion of dopamine by grafted neurons may also be regulated by similar mechanisms. To test this hypothesis we have measured dopamine release in the striatum of rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal pathway and grafts containing dopamine neurons. Intrastriatal dopamine levels were determined using methods of in vivo microdialysis. The administration of dihydrokainic acid, an inhibitor of glutamate uptake, resulted in the release of striatal dopamine in grafted and normal rats. In contrast, dopamine was not detectable in animals with 6-OHDA lesions alone. Additional studies were conducted to determine the type of glutamate receptor mediating the release of striatal dopamine. Kainic acid administration into the perfusate of the dialysis probe resulted in the release of striatal dopamine in a dose-dependent fashion in both grafted and normal rats. N-methyl-D-aspartate had no effect on dopamine release except at abnormally high concentrations. These results demonstrate that dopamine neurons which are transplanted into the striatum of rats with 6-OHDA lesions become functionally incorporated into the neural circuitry of the host brain, and that the release of dopamine by grafted neurons can be regulated by afferent fibers from the host brain.(ABSTRACT TRUNCATED AT 250 WORDS)

In search of factors mitigating the final outcome of ischemic and epileptic brain damage, we tested a novel dibenzoxazepine derivative (BY-1949), as the compound has been shown to be effective under these two conditions. First, using rat brain, we assessed whether or not BY-1949 affects the Na+,K(+)-ATPase activity. Although in vitro applications of either BY-1949 or its three major metabolites did not cause any apparent effects, both acute and chronic oral administrations of the compound (10 mg/kg) invariably increased the Na+,K(+)-ATPase activity in the synaptosomal plasma membranes by increasing Vmax values. Second, it was shown by this study that the drug treatment caused marked increases in the uptake of both glutamic acid and gamma-aminobutyric acid into the synaptosomes. These results suggest that the activity against ischemic/epileptic brain damage by BY-1949 is explicable, at least partly, in terms of improvement of ionic derangements across the neural membranes via Na+,K(+)-ATPase activation.

A new miniaturized triple-barreled cannula system has been developed for the push-pull perfusion of a circumscribed area of brain tissue and/or simultaneous microinjection of a drug or other chemical substance at the same site. A three-lumen, microbore glass capillary tube is drawn to a length of 10-15 mm with a tip diameter of 150-190 microM and connected by lengths of PE tubing to a multichannel peristaltic pump. An artificial CSF is then perfused through either one or two tubes at a rate of 5.0-10.0 microliters/min at a selected site. Thus, a pharmacological receptor agonist and/or antagonist, for example, can be delivered simultaneously to the tissue quantitatively to achieve steady-state levels over time. Alternatively, a single volume of a given drug can be micro-injected at the perfusion site during the course of a push-pull perfusion, simply by replacing one push channel with an injection tube. In both cases, aliquots of CSF perfusate from the pull channel(s) are collected on ice for assay by HPLC or other procedures. Because of the diminutive size of the triple-bore cannula system, it is now possible to perfuse an individual nucleus in the hypothalamus or lower brainstem not only with minimal cytopathological damage but also with a punctate dispersion of the perfusion medium. The large number of possible in vivo manipulations which can be undertaken using this cannula system is illustrated experimentally by recovery studies of [14C]glutamate and 45Ca from the nucleus tractus solitarius and hypothalamus of the rat.

We have investigated factors which may affect accumulated glutamate levels in synaptic vesicles and glutamate efflux. Agents which dissipate the electrochemical proton gradient resulted in a rapid reduction of steady-state vesicular glutamate levels, which was prevented by N-ethylmaleimide. Glutamate efflux was found to occur even in the presence of an electrochemical proton gradient, but was effectively inhibited by N-ethylmaleimide. These results suggest that accumulated glutamate levels in synaptic vesicles are not maintained unless glutamate is taken up continuously by an active transport mechanism, and they could provide an explanation for the lack of convincing evidence for the enrichment of endogenous glutamate in isolated synaptic vesicles.

Quisqualate, a glutamate analogue, displaced L-[3H]glutamate binding in a biphasic manner, corresponding to "high-affinity" and "low-affinity" binding sites. High-affinity quisqualate sites were termed "quisqualate-sensitive L-[3H]glutamate" binding sites. Quisqualate-sensitive L-[3H]glutamate binding was regionally distributed, with the highest levels present in the cerebellar molecular layer. This binding was stimulated by millimolar concentrations of chloride and calcium. The stimulatory effects of calcium required the presence of chloride ions, whereas chloride's stimulatory effects did not require calcium. All of the L-[3H]glutamate binding stimulated by chloride/calcium was quisqualate sensitive and only weakly displaced by N-methyl-D-aspartate, L-aspartate, or kainate. At high concentrations (1 mM), the anion blockers 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid both reduced, by 41 and 43%, respectively, the stimulatory effects of chloride. At concentrations of 100 microM, kynurenate, L-aspartate, (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and L-2-amino-4-phosphonobutyric acid (L-APB) failed to displace quisqualate-sensitive L-[3H]glutamate binding in the cerebellar molecular layer. In the presence of KSCN, however, 100 microM AMPA displaced 44% of binding. Quisqualate-sensitive L-[3H]glutamate binding was not sensitive to freezing, and, in contrast to other chloride- and calcium-dependent L-[3H]glutamate binding sites that have been reported, quisqualate-sensitive binding observed by autoradiography was enhanced at 4 degrees C compared with 37 degrees C. Quisqualate-sensitive L-[3H]glutamate binding likely represents binding to the subclass of postsynaptic neuronal glutamate receptors known as quisqualate receptors, rather than binding to previously described APB receptors, chloride-driven sequestration into vesicles, or binding to astrocytic membrane binding sites.

To permit a molecular characterization of neurotransmitter transporter proteins, we have studied uptake activities induced in Xenopus laevis oocytes after injection of adult rat forebrain, cerebellum, brainstem, and spinal cord poly(A)+ RNA. L-Glutamate uptake could be observed as early as 24 hr after injection, was linearly related to the quantity of mRNA injected, and could be induced after injection of as little as 1 ng of cerebellar mRNA. Transport of radiolabeled L-glutamate, gamma-aminobutyric acid, glycine, dopamine, serotonin, and choline could be measured in single microinjected oocytes with a regional profile consistent with the anatomical distribution of particular neurotransmitter synthesizing soma. Forebrain L-glutamate and dopamine uptake, as well as cerebellar L-glutamate transport, were found to be Na+-dependent. Cerebellar mRNA-induced L-glutamate transport was both time and temperature-dependent, was saturable by substrate, suggesting a single activity with an apparent transport Km of 14.2 microM and a Vmax of 15.2 pmol/hr per oocyte, and was sensitive to inhibitors of brain L-glutamate transport. Thus, the oocyte L-glutamate transport induced by injection of adult rat cerebellar mRNA appears essentially identical to the high-affinity, Na+-dependent L-glutamate uptake found in brain slices and nerve terminals. Experiments with size-fractionated cerebellar mRNA reveal single, comigrating peaks for cerebellar L-glutamate and gamma-aminobutyric acid transport, with peak activity obtained in fractions of approximately 2.7 kilobases, suggesting the presence of single or similarly sized mRNAs encoding each of these activities.

Evidence is presented that glutamate binding to rat brain synaptic plasma membranes measured in Cl(-)- and Na+-free but Ca2+-supplemented buffers is partly due to uptake of glutamate into resealed membrane vesicles. An intravesicular volume of 7.9 microliter/mg protein was measured. Ca2+-induced glutamate binding to synaptic plasma membranes was found to be sensitive to low temperatures as well as to an increase in osmolarity and was abolished by short pulses of ultrasonication. None of several glutamate receptor agonists tested discriminated between basal and Ca2+-induced binding, but 4 out of 5 glutamate uptake inhibitors did. The Ca2+-induced increase in glutamate binding was the same irrespective of whether calcium acetate, calcium sulfate or calcium gluconate in either Tris-acetate of Tris-citrate buffer was used.

The relationship between the ontogeny of the vesicular glutamate uptake system and synaptogenesis in rats was investigated. For this purpose we have developed a simplified procedure for the preparation of crude synaptic vesicles which are sufficiently pure to demonstrate a highly ATP-dependent glutamate uptake. ATP-dependent glutamate uptake into synaptic vesicles was found to increase dramatically starting on postnatal day 10 and reaching a maximum on day 30 (76 +/- 40 and 657 +/- 40 pmol/mg protein/10 min, respectively), correlating well with the active period of synaptogenesis. Stimulation of uptake by chloride also developed in parallel with the vesicular glutamate uptake. In contrast, combined non-ATP-dependent uptake and non-specific binding remained constant (21 +/- 6 pmol/mg protein/10 min). This development of vesicular glutamate uptake during the period of synaptogenesis supports the notion that synaptic vesicles play an important role in glutamate synaptic transmission.

A chloride-dependent transport process for glutamate has been identified in partially purified rat brain synaptosomes. This process shares many characteristics with the chloride-dependent sequestration process for glutamate in brain sonicates, which was previously thought to represent a quisqualate receptor, such as sensitivity to specific inhibitors and regulation by anions. Increasing the concentrations of chloride led to an increase in the apparent Vmax without affecting the KT. Synaptosomes preincubated with [3H]-L-glutamate exhibit an efflux of the radiolabel, which was stimulated by a substrate for the carrier in the incubating medium, indicating the bidirectional nature of the transport. The chloride-dependent transfer process is restricted to the brain, and regional and developmental profiles clearly distinguish it from the sodium-dependent high-affinity uptake process for glutamate. Nevertheless, the effects of excitotoxic lesions strongly suggest a neuronal localization of the chloride-dependent transport.

Joro spider toxin (JSTX), a specific blocker of glutamate receptors, was found to exert a prominent suppressive action on the Na+-dependent binding of L-glutamate to synaptic membranes and on glutamate uptake by synaptosomes in a dose-dependent manner. In contrast, the synthesized 2,4-dihydroxyphenylacetylasparagine (2,4-DHPA-ASN), a common moiety of spider toxins, which has been shown to exhibit almost the same activity as intact JSTX with respect to the inhibition of Na+-independent glutamate binding to its synaptic membrane receptors, shows lower potency in inhibiting Na+-dependent binding and uptake of L-glutamate. From these findings, it is clear that JSTX has the ability to inhibit not only L-glutamate binding to its synaptic membrane receptors but also L-glutamate uptake by synaptosomes, and that polyamines linked to 2,4-DHPA-ASN in the molecule of spider toxins may participate in the inhibition of L-glutamate uptake.