Astrocytes and stroke: networking for survival?

Astrocytes are now known to be involved in the most integrated functions of the central nervous system. These functions are not only necessary for the normally working brain but are also critically involved in many pathological conditions, including stroke. Astrocytes may contribute to damage by propagating spreading depression or by sending proapoptotic signals to otherwise healthy tissue via gap junction channels. Astrocytes may also inhibit regeneration by participating in formation of the glial scar. On the other hand, astrocytes are important in neuronal antioxidant defense and secrete growth factors, which probably provide neuroprotection in the acute phase, as well as promoting neurogenesis and regeneration in the chronic phase after injury. A detailed understanding of the astrocytic response, as well as the timing and location of the changes, is necessary to develop effective treatment strategies for stroke patients.     

Electrochemical monitoring of brain ascorbic acid changes associated with hypoxia,spreading depression,and seizure activity

In vivo electrochemistry has been a valuable tool in detecting real time neurochemical changes in extracellular fluid. Absolute selectivity has been difficult to achieve previously, but we report here a carbon fiber electrode and measurement technique which is specific for one oxidizable species: ascorbic acid. Ascorbic acid is highly concentrated in extra- as well as intracellular brain spaces, and appears to undergo dynamic changes in response to a variety of physiological and pathophysiological circumstances. Recent studies have implicated glutamatergic mechanisms which give rise to extracellular changes in brain ascorbate, and we confirm and extend these observations. Preliminary studies, directed towards examining ascorbic acid as an index and/or result of hypoxia, spreading depression, and seizure activity, have been undertaken and the results are reported herein.Special issue dedicated to Dr. Frederick E. Samson.     

Spreading depression-induced preconditioning in the mouse cortex: differential changes in the protein expression of ionotropic nicotinic acetylcholine and glutamate receptors

Preconditioning of the cerebral cortex was induced in mice by repeated cortical spreading depression (CSD), and the major ionotropic glutamate (GluRs) and nicotinic acetylcholine receptor (nAChRs) subunits were compared by quantitative immunoblotting between sham- and preconditioned cortex, 24 h after treatment. A 30% reduction in alpha-amino-3-hydroxy-5-methyl-4-iso- xazolepropionate (AMPA) GluR1 and 2 subunit immunoreactivities was observed in the preconditioned cortex (p < 0.03), but there was no significant change in the NMDA receptor subunits, NR1, NR2A and NR2B. A 12-15-fold increase in alpha7 nAChR subunit expression following in vivo CSD (p < 0.001) was by far the most remarkable change associated with preconditioning. In contrast, the alpha4 nAChR subunit was not altered. These data point to the alpha7 nAChR as a potential new target for neuroprotection because preconditioning increases consistently the tolerance of the brain to acute insults such as ischaemia. These data complement recent studies implicating alpha7 nAChR overexpression in the amelioration of chronic neuropathologies, notably Alzheimer's disease (AD).     

Evidence for increased expression of the vesicular glutamate transporter, VGLUT1, by a course of antidepressant treatment

The therapeutic effect of a course of antidepressant treatment is believed to involve a cascade of neuroadaptive changes in gene expression leading to increased neural plasticity. Because glutamate is linked to mechanisms of neural plasticity, this transmitter may play a role in these changes. This study investigated the effect of antidepressant treatment on expression of the vesicular glutamate transporters, VGLUT1-3 in brain regions of the rat. Repeated treatment with fluoxetine, paroxetine or desipramine increased VGLUT1 mRNA abundance in frontal, orbital, cingulate and parietal cortices, and regions of the hippocampus. Immunoautoradiography analysis showed that repeated antidepressant drug treatment increased VGLUT1 protein expression. Repeated electroconvulsive shock (ECS) also increased VGLUT1 mRNA abundance in regions of the cortex and hippocampus compared to sham controls. The antidepressant drugs and ECS did not alter VGLUT1 mRNA abundance after acute administration, and no change was detected after repeated treatment with the antipsychotic agents, haloperidol and chlorpromazine. In contrast to VGLUT1, the different antidepressant treatments did not commonly increase the expression of VGLUT2 or VGLUT3 mRNA. These data suggest that a course of antidepressant drug or ECS treatment increases expression of VGLUT1, a key gene involved in the regulation of glutamate secretion.     

Repeated Restraint Stress Alters Hippocampal Glutamate Uptake and Release in the Rat

Glutamatergic mechanisms are thought to be involved in stress-induced changes of brain function, especially in the hippocampus. We hypothesized that alterations caused by the hormonal changes associated with chronic and acute stress may affect glutamate uptake and release from hippocampal synaptosomes in Wistar rats. It was found that [3H]glutamate uptake and release by hippocampal nerve endings, when measured 24 h after 1 h of acute restraint, presented no significant difference. The exposure to repeated restraint stress for 40 days increased neuronal presynaptic [3H]glutamate uptake as well as basal and K+-stimulated glutamate release when measured 24 h after the last stress session. Chronic treatment also caused a significant decrease in [3H]glutamate binding to hippocampal membranes. We suggest that changes in the glutamatergic system are likely to take part in the mechanisms involved in nervous system plasticity following repeated stress exposure.     

The synthesis of glutamate and the control of glutamate dehydrogenase in pea mitochondria

The synthesis of glutamate from α-oxoglutarate and NH₄⁺ by pea seedling mitochondria has been demonstrated under certain defined but non-physiological conditions. Malate acts as a hydrogen donor for the synthesis of glutamate but isocitrate is more effective, whilst succinate, in the presence or absence of ATP, is a poor donor of hydrogen. Glutamate dehydrogenase has been purified from pea mitochondria and from the cytosol. The similarities between the two preparations are interpreted to mean that the soluble glutamate dehydrogenase is released from the mitochondria during isolation. The kinetics of the mitochondrial enzyme and the effect of various metabolites on its activity have been examined. The results are discussed in relation to the proposed role of this enzyme and it is suggested that the ratio NADH-NAD⁺ may play a role in the control of glutamate metabolism.     

Hypoxia regulates glutamate metabolism and membrane transport in rat PC12 cells

We investigated the effect of hypoxia on glutamate metabolism and uptake in rat pheochromocytoma (PC12) cells. Various key enzymes relevant to glutamate production, metabolism and transport were coordinately regulated by hypoxia. PC12 cells express two glutamate-metabolizing enzymes, glutamine synthetase (GS) and glutamate decarboxylase (GAD), as well as the glutamate-producing enzyme, phosphate-activated glutaminase (PAG). Exposure to hypoxia (1% O(2)) for 6 h or longer increased expression of GS mRNA and protein and enhanced GS enzymatic activity. In contrast, hypoxia caused a significant decrease in expression of PAG mRNA and protein, and also decreased PAG activity. In addition, hypoxia led to an increase in GAD65 and GAD67 protein levels and GAD enzymatic activity. PC12 cells express three Na(+)-dependent glutamate transporters; EAAC1, GLT-1 and GLAST. Hypoxia increased EAAC1 and GLT-1 protein levels, but had no effect on GLAST. Chronic hypoxia significantly enhanced the Na(+)-dependent component of glutamate transport. Furthermore, chronic hypoxia decreased cellular content of glutamate, but increased that of glutamine. Taken together, the hypoxia-induced changes in enzymes related to glutamate metabolism and transport are consistent with a decrease in the extracellular concentration of glutamate. This may have a role in protecting PC12 cells from the cytotoxic effects of glutamate during chronic hypoxia.     

Effects of Dehydroevodiamine Exposure on Glutamate Release and Uptake in the Cultured Cerebellar Cells

Dehydroevodiamine has been reported to have neuroprotective and antiamnesic effects. This study examined the effects of dehydroevodiamine on glutamate release and uptake in cultured cerebellar cells. Chronic dehydroevodiamine exposure decreased the viability of granule cells. The basal and N-methyl-D-aspartate (NMDA)-induced release of glutamate from granule cells were decreased (26 and 14%) by dehydroevodiamine. The NMDA-induced release of glutamate was concentration-dependently inhibited in the granule cells. The basal and NMDA-induced releases of glutamate in chronically dehydroevodiamine-preexposed granule cells were unaffected by dehydroevodiamine. Glutamate uptake in the glial cells incubated without and with cAMP was inhibited (31% and 8%, respectively) by dehydroevodiamine. In the chronically dehydroevodiamine-preexposed glial cells, glutamate uptake was increased (8%) in the cAMP-coexposed glial cells by dehydroevodiamine but was unaffected in the naive cells. In addition, dehydroevodiamine potentiated (from 20% to 34%) the inhibition of L-pyrollidine-2,4-dicarboxylic acid (PDC) on glutamate uptake in naive glial cells, but this inhibition was reduced (from 41% to 26%) in cAMP-coexposed glial cells. These results suggest that dehydroevodiamine inhibits glutamate uptake and release. Furthermore, the results suggest that the characteristics of glutamate release and uptake in granule and glial cells may be altered by chronic exposure to dehydroevodiamine.     

GLT-1 down-regulation induced by clozapine in rat frontal cortex is associated with synaptophysin up-regulation

In rat frontal cortex, extracellular levels of glutamate are raised by the anti-psychotic drug clozapine. We have recently shown that a significant reduction in the levels of the glutamate transporter GLT-1 may be one of the mechanisms responsible for this elevation. Here we studied whether GLT-1 down-regulation induced by chronic clozapine treatment is associated with changes in the expression of synaptophysin, synaptosome-associated protein of 25 kDa (SNAP-25) and vesicular glutamate transporter 1 (VGLUT1), three major presynaptic proteins involved in neurotransmitter release. Quantitative high-resolution confocal microscopy studies in vivo showed that GLT-1 down-regulation is closely associated with a significant increase in synaptophysin, but not SNAP-25 and VGLUT1, expression. This was confirmed in vitro studies, and in western blotting studies of synaptophysin, SNAP-25 and VGLUT1. In addition, our results show that, following clozapine treatment, synaptophysin expression increases in the very cortical regions in which GLT-1 expression is down-regulated. These findings suggest that part of the effects of clozapine may be exerted via an action on the presynaptic machinery involved in neurotransmitter release.     

Changes in the Glutamate Release and Uptake of Cerebellar Cells in Perinatally Nicotine-Exposed Rat Pups

Cerebellar granule and glial cells were cultured from 7 day-old rat pups after pre- and post-natal nicotine treatment. Ten days later, the basal release of glutamate in the granule cells prepared from the pre- and post-natally nicotine-exposed pups was higher and lower than the controls, respectively. The N-methyl-D-aspartate-induced release of glutamate was higher in the granule cells of post-natal nicotine exposed rats. However, the nicotine-induced glutamate release was either unchanged or was lower in the granule cells of all nicotine-treated pups. The basal glutamate uptake was higher in the glial cells from those exposed pre-natally and lower in the continuously nicotine-exposed pups. The sensitivities of L-trans-pyrrolidine-2,4-dicarboxylic acid on glutamate uptake were higher in all nicotine treated groups. There was a higher number of specific [³H]dizocilpine binding sites in the pre- or continuously nicotine-exposed group. These results suggest that the cerebellar cell properties are altered after perinatal nicotine exposure and that the development of an excitatory amino acid system might be affected differently depending on the nicotine exposure time.     

谷氨酸转运体与疼痛

中枢神经系统谷氨酸生理浓度主要依赖神经细胞和神经胶质细胞上谷氨酸转运体维持,谷氨酸转运体的功能紊乱会导致谷氨酸的累积。谷氨酸转运体在吗啡镇痛及耐受中扮演一定的角色,并在神经病理性痛中发挥重要作用。谷氨酸转运体可能作为治疗疼痛的一个潜在的药物靶点。     

Metabotropic Glutamate Receptors Modulate Ischemia-Induced GABA Release in Mouse Hippocampal Slices

The involvement of glutamate receptors in GABA release in ischemia was investigated in hippocampal slices from adult (3-month-old) and developing (7-day-old) mice. For in vitro ischemia, the slices were superfused in glucose-free media under nitrogen. Ionotropic glutamate receptor agonists failed to affect the ischemia-induced basal GABA release at either age. The K(+)-stimulated release in the immature hippocampus was potentiated by N-methyl-D-aspartate receptors, whereas in adults this release was reduced by both kainate and 2-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate receptor activation. The group I metabotropic receptor agonist (1+/-)-1-aminocyclopentane-trans-1,3-dicarboxylate enhanced the basal ischemic GABA release in a receptor-mediated manner in adults, this being concordant with the positive modulation of GABAergic neurotransmission by group I metabotropic glutamate receptors. (1 +/-)-1-Aminocyclopentane-trans-1,3-dicarboxylate and (S)-3,5-dihydroxyphenylglycine also enhanced the K(+)-stimulated release in the developing hippocampus in a receptor-mediated manner. Because group I receptors generally increase neuronal excitability, the enhanced GABA release may attenuate hyperexcitation or strengthen inhibition, being thus neuroprotective, particularly under ischemic conditions. Group III metabotropic glutamate receptors were not at all involved in ischemic GABA release in the immature mice, but in adults their activation by O-phospho-L-serine potentiated the basal release and reduced the K(+)-stimulated release. These opposite effects were abolished by the antagonist (RS)-2-cyclopropyl-4-phosphonophenylglycine. Metabotropic glutamate receptors, namely group I and III receptors, are able to modify the release of GABA from hippocampal slices under ischemic conditions, both positive and negative effects being discernible, depending on the age and type of receptor activated.     

Dopamine neurons in culture express VGLUT2 explaining their capacity to release glutamate at synapses in addition to dopamine

Dopamine neurons have been suggested to use glutamate as a cotransmitter. To identify the basis of such a phenotype, we have examined the expression of the three recently identified vesicular glutamate transporters (VGLUT1-3) in postnatal rat dopamine neurons in culture. We found that the majority of isolated dopamine neurons express VGLUT2, but not VGLUT1 or 3. In comparison, serotonin neurons express only VGLUT3. Single-cell RT-PCR experiments confirmed the presence of VGLUT2 mRNA in dopamine neurons. Arguing for phenotypic heterogeneity among axon terminals, we find that only a proportion of terminals established by dopamine neurons are VGLUT2-positive. Taken together, our results provide a basis for the ability of dopamine neurons to release glutamate as a cotransmitter. A detailed analysis of the conditions under which DA neurons gain or loose a glutamatergic phenotype may provide novel insight into pathophysiological processes that underlie diseases such as schizophrenia, Parkinson's disease and drug dependence.     

NAD(H)-dependent glutamate dehydrogenase is essential for the survival of Arabidopsis thaliana during dark-induced carbon starvation

Interconversion between glutamate and 2-oxoglutarate, which can be catalysed by glutamate dehydrogenase (GDH), is a key reaction in plant carbon (C) and nitrogen (N) metabolism. However, the physiological role of plant GDH has been a controversial issue for several decades. To elucidate the function of GDH, the expression of GDH in various tissues of Arabidopsis thaliana was studied. Results suggested that the expression of two Arabidopsis GDH genes was differently regulated depending on the organ/tissue types and cellular C availability. Moreover, Arabidopsis mutants defective in GDH genes were identified and characterized. The two isolated mutants, gdh1-2 and gdh2-1, were crossed to make a double knockout mutant, gdh1-2/gdh2-1, which contained negligible levels of NAD(H)-dependent GDH activity. Phenotypic analysis on these mutants revealed an increased susceptibility of gdh1-2/gdh2-1 plants to C-deficient conditions. This conditional phenotype of the double knockout mutant supports the catabolic role of GDH and its role in fuelling the TCA cycle during C starvation. The reduced rate of glutamate catabolism in the gdh2-1 and gdh1-2/gdh2-1 plants was also evident by the growth retardation of these mutants when glutamate was supplied as the alternative N source. Furthermore, amino acid profiles during prolonged dark conditions were significantly different between WT and the gdh mutant plants. For instance, glutamate levels increased in WT plants but decreased in gdh1-2/gdh2-1 plants, and aberrant accumulation of several amino acids was detected in the gdh1-2/gdh2-1 plants. These results suggest that GDH plays a central role in amino acid breakdown under C-deficient conditions.     

Enzymes of glutamine metabolism in testa-pericarp and endosperm of developing wheat grain

Glutamate dehydrogenase, glutamine synthetase, glutamate synthase, glutamate puruvate transaminase and glutamate oxaloacetate transaminase have been assayed in developing testa-pericarp and endosperm of two wheat varieties, namely Shera (11.6% protein) and C-306 (9.8% protein). On per organ basis, activities of all the enzymes studied, except glutamine synthetase, increased during development. Glutamine synthetase activity decreased during development in the testa-pericarp, whereas, no glutamine synthetase activity could be detected in endosperm of either variety at any stage of development. Compared to testa-pericarp, endosperm had higher activities of glutamate synthase and glutamate pyruvate transaminase. On the whole, enzyme activities in Shera were higher, as compared to C-306. Developmental patterns and relative levels of enzyme activities in the two varieties were more or less the same, when expressed on dry weight basis or as specific activities. The results suggest that ammonia assimilation in developing wheat grain takes place by the glutamate dehydrogenase pathway in the endosperm; and both by the glutamate dehydrogenase and glutamine synthetase—glutamate synthase pathways in the testa-pericarp.     

Differential regulation of GLAST immunoreactivity and activity by protein kinase C: evidence for modification of amino and carboxyl termini

Many neurotransmitter transporters, including the GLT-1 and EAAC1 subtypes of the glutamate transporter, are regulated by protein kinase C (PKC) and these effects are associated with changes in cell surface expression. In the present study, the effects of PKC activation on the glutamate aspartate transporter (GLAST) subtype of glutamate transporter were examined in primary astrocyte cultures. Acute (30 min) exposure to the phorbol 12-myristate 13-acetate (PMA) increased (approximately 20%) transport activity but had the opposite effect on both total and cell surface immunoreactivity. Chronic treatment (6 or 24 h) with PMA had no effect on transport activity but caused an even larger decrease in total and cell surface immunoreactivity. This loss of immunoreactivity was observed using antibodies directed against three different cytoplasmic epitopes, and was blocked by the PKC antagonist, bisindolylmaleimide II. We provide biochemical and pharmacological evidence that the activity observed after treatment with PMA is mediated by GLAST. Two different flag-tagged variants of the human homolog of GLAST were introduced into astrocytes using lentiviral vectors. Although treatment with PMA caused a loss of transporter immunoreactivity, flag immunoreactivity did not change in amount or size. Together, these studies suggest that activation of PKC acutely up-regulates GLAST activity, but also results in modification of several different intracellular epitopes so that they are no longer recognized by anti-GLAST antibodies. We found that exposure of primary cultures of neurons/astrocytes to transient hypoxia/glucose deprivation also caused a loss of GLAST immunoreactivity that was attenuated by the PKC antagonist, bisindolylmaleimide II, suggesting that some acute insults previously thought to cause a loss of GLAST protein may mimic the phenomenon observed in the present study.     

Group I and II metabotropic glutamate receptors alter brain cortical metabolic and glutamate/glutamine cycle activity: a 13C NMR spectroscopy and metabolomic study

Metabotropic glutamate receptors (mGluR) modulate neuronal function. Here, we tested the effect on metabolism of a range of Group I and II mGluR ligands in Guinea pig brain cortical tissue slices, applying 13C NMR spectroscopy and metabolomic analysis using multivariate statistics. The effects of Group I agonists (S)-3,5-dihydroxyphenylglycine (DHPG) and (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) depended upon concentration and were mostly stimulatory, increasing both net metabolic flux through the Krebs cycle and glutamate/glutamine cycle activity. Only the higher (50 microm) concentrations of CHPG had the opposite effect. The Group I antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA), consistent with its neuroprotective role, caused significant decreases in metabolism. With principal components analysis of the metabolic profiles generated by these ligands, the effects could be separated by two principal components. Agonists at Group II mGluR [(2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV) and 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate (APDC)] generally stimulated metabolism, including glutamate/glutamine cycling, although this varied with concentration. The antagonist (2S)-alpha-ethylglutamic acid (EGLU) stimulated astrocyte metabolism with minimal impact on glutamate/glutamine cycling. (RS)-1-Aminophosphoindan-1-carboxylic acid (APICA) decreased metabolism at 5 microm but had a stimulatory effect at 50 microm. All ligand effects were separated from control and from each other using two principal components. The ramifications of these findings are discussed.     

Uptake and release for glutamine and glutamate in a crude synaptosomal fraction from rat brain

[14C]Glutamine uptake in a crude synaptosomal (P2) fraction, (representing the sum of [¹⁴C]glutamine accumulated and [¹⁴C]glutamate formed by hydrolysis), is distinct from glutamate uptake. Glutamine uptake is Na⁺-independent and unaffected by the Na⁺–K⁺-ATPase inhibitor ouabain, whereas glutamate uptake is Na⁺-dependent and inhibited by ouabain. The uptake of both glutamine and glutamate is unaffected by the gamma-glutamyltransferase inhibitor, Acivicin. This indicates that glutamine uptake is not mediated by a carrier, as distinct from that of glutamate, and also not linked to gamma-glutamyl-transferase. Na⁺ affects the distribution of glutamine-derived glutamate by increasing the synaptosomal content and reducing that of the medium. When glutamate release from synaptosomes preloaded with [¹⁴C]glutamate is measured by superfusion technique in order to prevent reuptake, Na⁺ has been found to inhibit release in a non-depolarizing medium (Ringer buffer with no Ca²⁺) of the [¹⁴C]glutamate as well as of endogenous glutamate. The specific activity of the [¹⁴C]glutamine-derived glutamate in the incubation medium is much higher than that in the synaptosomes, indicating that there exists a readily releasable pool of newly formed glutamate in addition to another pool. The latter glutamate pool is partially reduced by Na⁺.Special Issue Dedicated to Dr. Abel Lajtha.