Intercellular metabolic compartmentation in the brain: past, present and future

The first indication of 'metabolic compartmentation' in brain was the demonstration that glutamine after intracisternal [14C]glutamate administration is formed from a compartment of the glutamate pool that comprises at most one-fifth of the total glutamate content in the brain. This pool, which was designated 'the small compartment,' is now known to be made up predominantly or exclusively of astrocytes, which accumulate glutamate avidly and express glutamine synthetase activity, whereas this enzyme is absent from neurons, which eventually were established to constitute 'the large compartment.' During the following decades, the metabolic compartment concept was refined, aided by emerging studies of energy metabolism and glutamate uptake in cellularly homogenous preparations and by the histochemical observations that the two key enzymes glutamine synthetase and pyruvate carboxylase are active in astrocytes but absent in neurons. It is, however, only during the last few years that nuclear magnetic resonance (NMR) spectroscopy, assisted by previously obtained knowledge of metabolic pathways, has allowed accurate determination in the human brain in situ of actual metabolic fluxes through the neuronal tricarboxylic acid (TCA) cycle, the glial, presumably mainly astrocytic, TCA cycle, pyruvate carboxylation, and the 'glutamate-glutamine cycle,' connecting neuronal and astrocytic metabolism. Astrocytes account for 20% of oxidative metabolism of glucose in the human brain cortex and accumulate the bulk of neuronally released transmitter glutamate, part of which is rapidly converted to glutamine and returned to neurons in the glutamate-glutamine cycle. However, one-third of released transmitter glutamate is replaced by de novo synthesis of glutamate from glucose in astrocytes, suggesting that at steady state a corresponding amount of glutamate is oxidatively degraded. Net degradation of glutamate may not always equal its net production from glucose and enhanced glutamatergic activity, occurring during different types of cerebral stimulation, including the establishment of memory, may be associated with increased de novo synthesis of glutamate. This process may contribute to a larger increase in glucose utilization rate than in rate of oxygen consumption during brain activation. The energy yield in astrocytes from glutamate formation is strongly dependent upon the fate of the generated glutamate.     

Role of glutamine and neuronal glutamate uptake in glutamate homeostasis and synthesis during vesicular release in cultured glutamatergic neurons

Glutamate exists in a vesicular as well as a cytoplasmic pool and is metabolically closely related to the tricarboxylic acid (TCA) cycle. Glutamate released during neuronal activity is most likely to a large extent accumulated by astrocytes surrounding the synapse. A compensatory flux from astrocytes to neurons of suitable precursors is obligatory as neurons are incapable of performing a net synthesis of glutamate from glucose. Glutamine appears to play a major role in this context. Employing cultured cerebellar granule cells, as a model system for glutamatergic neurons, details of the biosynthetic machinery have been investigated during depolarizing conditions inducing vesicular release. [U-13C]Glucose and [U-13C]glutamine were used as labeled precursors for monitoring metabolic pathways by nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) technologies. To characterize release mechanisms and influence of glutamate transporters on maintenance of homeostasis in the glutamatergic synapse, a quantification was performed by HPLC analysis of the amounts of glutamate and aspartate released in response to depolarization by potassium (55 mM) in the absence and presence of DL-threo-beta-benzyloxyaspartate (TBOA) and in response to L-trans-pyrrolidine-2,4-dicarboxylate (t-2,4-PDC), a substrate for the glutamate transporter. Based on labeling patterns of glutamate the biosynthesis of the intracellular pool of glutamate from glutamine was found to involve the TCA cycle to a considerable extent (approximately 50%). Due to the mitochondrial localization of PAG this is unlikely only to reflect amino acid exchange via the cytosolic aspartate aminotransferase reaction. The involvement of the TCA cycle was significantly lower in the synthesis of the released vesicular pool of glutamate. However, in the presence of TBOA, inhibiting glutamate uptake, the difference between the intracellular and the vesicular pool with regard to the extent of involvement of the TCA cycle in glutamate synthesis from glutamine was eliminated. Surprisingly, the intracellular pool of glutamate was decreased after repetitive release from the vesicular pool in the presence of TBOA indicating that neuronal reuptake of released glutamate is involved in the maintenance of the neurotransmitter pool and that 0.5 mM glutamine exogenously supplied is inadequate to sustain this pool.     

Glutamate-induced exocytosis of glutamate from astrocytes

Recent studies indicate that astrocytes can play a much more active role in neuronal circuits than previously believed, by releasing neurotransmitters such as glutamate and ATP. Here we report that local application of glutamate or glutamine synthetase inhibitors induces astrocytic release of glutamate, which activates a slowly decaying transient inward current (SIC) in CA1 pyramidal neurons and a transient inward current in astrocytes in hippocampal slices. The occurrence of SICs was accompanied by an appearance of large vesicles around the puffing pipette. The frequency of SICs was positively correlated with [glutamate]o. EM imaging of anti-glial fibrillary acid protein-labeled astrocytes showed glutamate-induced large astrocytic vesicles. Imaging of FM 1-43 fluorescence using two-photon laser scanning microscopy detected glutamate-induced formation and fusion of large vesicles identified as FM 1-43-negative structures. Fusion of large vesicles, monitored by collapse of vesicles with a high intensity FM 1-43 stain in the vesicular membrane, coincided with SICs. Glutamate induced two types of large vesicles with high and low intravesicular [Ca2+]. The high [Ca2+] vesicle plays a major role in astrocytic release of glutamate. Vesicular fusion was blocked by infusing the Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, or the SNARE blocker, tetanus toxin, suggesting Ca2+- and SNARE-dependent fusion. Infusion of the vesicular glutamate transport inhibitor, Rose Bengal, reduced astrocytic glutamate release, suggesting the involvement of vesicular glutamate transports in vesicular transport of glutamate. Our results demonstrate that local [glutamate]o increases induce formation and exocytotic fusion of glutamate-containing large astrocytic vesicles. These large vesicles could play important roles in the feedback control of neuronal circuits and epileptic seizures.     

Neuronal-astrocytic and cytosolic-mitochondrial metabolite trafficking during brain activation, hyperammonemia and energy deprivation

A novel concept is described, according to which both neurons and astrocytes are capable of metabolizing glucose all the way to CO(2) and water, but in addition interact metabolically in a process generating glutamate from glucose, and subsequently, metabolizing excess glutamate to CO(2) and water Hertz, L., Dringen, R., Schousboe, A., Robinson, S.R., 1999. Astrocytes: Glutamate producers for neurons (Journal of Neuroscience Research 57, 417-428). The proposed metabolic degradation of glucose via glutamate serves the purpose of adjusting transmitter pools of glutamate to the demands for glutamatergic transmission, and it must account for a major fraction of glucose utilization. Evidence in favor of this concept is presented and a multitude of in vivo data are interpreted in the context of metabolic trafficking between neurons and astrocytes. In addition, intracellular trafficking occurs between cytosol and mitochondria during synthesis of transmitter glutamate, partly explaining a robust quantitative correlation between glutamine synthesis, as a measure of release of transmitter glutamate, and glucose utilization, reported by several authors. Both intracellular and intercellular metabolic trafficking may be affected during pathological conditions, as evidenced by effects of hyperammonemia (mimicking hepatic encephalopathy) and energy deprivation (mimicking stroke). It is suggested that neuronal-astrocytic interactions may also be impaired during degenerative dementing diseases.     

The role of glia in neuronal recovery following anoxia: In vitro evidence of neuronal adaptation

We investigated the effects of 3h of anoxia on metabolism of neurons and astrocytes, using a robust cell-based model system that mimics closely the living tissue milieu, i.e., in 3D neural aggregates cultured in bioreactors. Cells were incubated simultaneously with [1-(13)C]glucose and [1,2-(13)C]acetate; and, the gliotoxin fluorocitrate (FC) was used for glial tricarboxylic acid (TCA) cycle inhibition to assess the role of astrocytes for neuronal metabolism after oxygen deprivation. Results show that culture viability was not compromised by exposure to anoxia with and without FC. Interaction between astrocytes and glutamatergic neurons was altered due to anoxia: labeling in glutamine from [1-(13)C]glucose was decreased, whereas that in glutamate from [1,2-(13)C]acetate was increased. In contrast, GABA labeling was not affected by anoxia. It was shown that anoxia did not affect astrocytic capacity to synthesize glutamine in the reoxygenation period. The selective action of FC on astrocytes was confirmed. However, the presence of small amounts of glutamate and GABA labeled from acetate indicated residual activity of the glial TCA cycle. Although major metabolic changes were found due to FC-treatment, the intracellular pool of GABA was kept unchanged. Overall, our data clearly confirm that the glutamate-glutamine cycle depends on astrocytic TCA cycle activity and that mitochondrial impairment of astrocytes will ultimately stop metabolic trafficking between astrocytes and glutamatergic neurons. Additionally, our data suggest a metabolic independence of GABAergic neurons from astrocytes even after situations of complete oxygen depletion.     

A Tribute to Mary C. McKenna: Glutamate as Energy Substrate and Neurotransmitter—Functional Interaction Between Neurons and Astrocytes

Glutamate metabolism in the brain is extremely complex not only involving a large variety of enzymes but also a tight partnership between neurons and astrocytes, the latter cells being in control of de novo synthesis of glutamate. This review provides an account of the processes involved, i.e. pyruvate carboxylation and recycling as well as the glutamate–glutamine cycle, focusing on the many seminal contributions from Dr. Mary McKenna. The ramification of the astrocytic end feet allowing contact and control of hundreds of thousands of synapses at the same time obviously puts these cells in a prominent position to regulate neural activity. Additionally, the astrocytes take active part in the neurotransmission processes by releasing a variety of gliotransmitters including glutamate. Hence, the term “the tripartite synapse”, in which there is an active and dynamic interplay between the pre- and post-synaptic neurons and the ensheathing astrocytes, has been coined. The studies of Mary McKenna and her colleagues over several decades have been of paramount importance for the elucidation of compartmentation in astrocytes and synaptic terminals and the intricate metabolic processes underlying the glutamatergic neurotransmission process.     

Role of the Intracellular Nucleoside Transporter ENT3 in Transmitter and High K+ Stimulation of Astrocytic ATP Release Investigated Using siRNA Against ENT3

This study investigates the role of the intracellular adenosine transporter equilibrative nucleoside transporter 3 (ENT3) in stimulated release of the gliotransmitter adenosine triphosphate (ATP) from astrocytes. Within the past 20 years, our understanding of the importance of astrocytic handling of adenosine, its phosphorylation to ATP, and release of astrocytic ATP as an important transmitter has become greatly expanded. A recent demonstration that the mainly intracellular nucleoside transporter ENT3 shows much higher expression in freshly isolated astrocytes than in a corresponding neuronal preparation leads to the suggestion that it was important for the synthesis of gliotransmitter ATP from adenosine. This would be consistent with a previously noted delay in transmitter release of ATP in astrocytes but not in neurons. The present study has confirmed and quantitated stimulated ATP release in response to glutamate, adenosine, or an elevated K⁺ concentration from well-differentiated astrocyte cultures, measured by a luciferin–luciferase reaction. It showed that the stimulated ATP release was abolished by downregulation of ENT3 with small interfering RNA (siRNA), regardless of the stimulus. The concept that transmitter ATP in mature astrocytes is synthesized directly from adenosine prior to release is supported by the postnatal development of the expression of the vesicular transporter SLC17A9 in astrocytes. In neurons, this transporter carries ATP into synaptic vesicles, but in astrocytes, its expression is pronounced only in immature cells and shows a rapid decline during the first 3 postnatal weeks so that it has almost disappeared at the end of the third week in well-differentiated astrocytes, where its role has probably been taken over by ENT3.     

A possible role of alanine for ammonia transfer between astrocytes and glutamatergic neurons

The metabolism of [U-(13)C]lactate (1 mM) in the presence of unlabeled glucose (2.5 mM) was investigated in glutamatergic cerebellar granule cells, cerebellar astrocytes, and corresponding co-cultures. It was evident that lactate is primarily a neuronal substrate and that lactate produced glycolytically from glucose in astrocytes serves as a substrate in neurons. Alanine was highly enriched with (13)C in the neurons, whereas this was not the case in the astrocytes. Moreover, the cellular content and the amount of alanine released into the medium were higher in neurons than astrocytes. On incubation of the different cell types in medium containing alanine (1 mM), the astrocytes exhibited the highest level of accumulation. Altogether, these results indicate a preferential synthesis and release of alanine in glutamatergic neurons and uptake in cerebellar astrocytes. A new functional role of alanine may be suggested as a carrier of nitrogen from glutamatergic neurons to astrocytes, a transport that may operate to provide ammonia for glutamine synthesis in astrocytes and dispose of ammonia generated by the glutaminase reaction in glutamatergic neurons. Hence, a model of a glutamate-glutamine/lactate-alanine shuttle is presented. To elucidate if this hypothesis is compatible with the pattern of alanine metabolism observed in the astrocytes and neurons from cerebellum, the cells were incubated in a medium containing [(15)N]alanine (1 mM) and [5-(15)N]glutamine (0.5 mM), respectively. Additionally, neurons were incubated with [U-(13)C]glutamine to estimate the magnitude of glutamine conversion to glutamate. Alanine was labeled from [5-(15)N]glutamine to 3.3% and [U-(13)C]glutamate generated from [U-(13)C]glutamine was labeled to 16%. In spite of the modest labeling in alanine, it is clear that nitrogen from ammonia is transferred to alanine via transamination with glutamate formed by reductive amination of alpha-ketoglutarate. With regard to the astrocytic part of the shuttle, glutamine was labeled to 22% in one nitrogen atom whereas 3.2% was labeled in two when astrocytes were incubated in [(15)N]alanine. Moreover, in co-cultures, [U-(13)C]alanine labeled glutamate and glutamine equally, whereas [U-(13)C]lactate preferentially labeled glutamate. Altogether, these results support the role proposed above of alanine as a possible ammonia nitrogen carrier between glutamatergic neurons and surrounding astrocytes and they show that lactate is preferentially metabolized in neurons and alanine in astrocytes.     

Astrocyte and Neuron Intone Through Glutamate

The unexpected finding of astrocytes to release glutamate as gliotransmitter challenges the traditional concepts on astrocyte being “passive” in CNS communications. Glutamate is the major excitatory transmitter in transferring information between neurons, but is now also known to activate astrocyte through transporters and receptors. Together with the sensitive swelling response, astrocytes could respond directly to glutamate and neuronal activity. Other new functions of astrocytes include modulation of synaptic plasticity and cerebral blood flow (CBF). The classic glutamate deplenishment through glutamine synthesis and CO₂ production does not account for the total glutamate internalized into astrocytes. This leads us to speculate there are many hidden functions of glutamate in neurons and astrocytes waiting to be discovered. In this review, we attempted to reexamine some of these new and older functions of glutamate and to reevaluate the roles of glutamate intoning these two cell types. Special issue article in honor of Dr. Anna Maria Giuffrida-Stella.     

Distinct changes in neuronal and astrocytic amino acid neurotransmitter metabolism in mice with reduced numbers of synaptic vesicles

The relations between glutamate and GABA concentrations and synaptic vesicle density in nerve terminals were examined in an animal model with 40–50% reduction in synaptic vesicle numbers caused by inactivation of the genes encoding synapsin I and II. Concentrations and synthesis of amino acids were measured in extracts from cerebrum and a crude synaptosomal fraction by HPLC and ¹³C nuclear magnetic resonance spectroscopy (NMRS), respectively. Analysis of cerebrum extracts, comprising both neurotransmitter and metabolic pools, showed decreased concentration of GABA, increased concentration of glutamine and unchanged concentration of glutamate in synapsin I and II double knockout (DKO) mice. In contrast, both glutamate and GABA concentrations were decreased in crude synaptosomes isolated from synapsin DKO mice, suggesting that the large metabolic pool of glutamate in the cerebral extracts may overshadow minor changes in the transmitter pool. ¹³C NMRS studies showed that the changes in amino acid concentrations in the synapsin DKO mice were caused by decreased synthesis of GABA (20–24%) in cerebral neurons and increased synthesis of glutamine (36%) in astrocytes. In a crude synaptosomal fraction, the glutamate synthesis was reduced (24%), but this reduction could not be detected in cerebrum extracts. We suggest that lack of synaptic vesicles causes down-regulation of neuronal GABA and glutamate synthesis, with a concomitant increase in astrocytic synthesis of glutamine, in order to maintain normal neurotransmitter concentrations in the nerve terminal cytosol.     

Activity-dependent regulation of vesicular glutamate and GABA transporters: a means to scale quantal size

The functional balance of glutamatergic and GABAergic signaling in neuronal cortical circuits is under homeostatic control. That is, prolonged alterations of global network activity leads to opposite changes in quantal amplitude at glutamatergic and GABAergic synapses. Such scaling of excitatory and inhibitory transmission within cortical circuits serves to restore and maintain a constant spontaneous firing rate of pyramidal neurons. Our recent work shows that this includes alterations in the levels of expression of vesicular glutamate (VGLUT1 and VGLUT2) and GABA (VIAAT) transporters. Other vesicle markers, such as synaptophysin or synapsin, are not regulated in this way. Endogenous regulation at the level of mRNA and synaptic protein controls the number of transporters per vesicle and hence, the level of vesicle filling with transmitter. Bidirectional and opposite activity-dependent regulation of VGLUT1 and VIAAT expression would serve to adjust the balance of glutamate and GABA release and therefore the level of postsynaptic receptor saturation. In some excitatory neurons and synapses, co-expression of VGLUT1 and VGLUT2 occurs. Bidirectional and opposite changes in the levels of two excitatory vesicular transporters would enable individual neocortical neurons to scale up or scale down the level of vesicular glutamate storage, and thus, the amount available for release at individual synapses. Regulated vesicular transmitter storage and release via selective changes in the level of expression of vesicular glutamate and GABA transporters indicates that homeostatic plasticity of synaptic strength at cortical synapses includes presynaptic elements.     

Redefining chemical anatomy of glutamatergic neurotransmission

With the recent identification of the two isoforms of vesicular glutamate transporters VGLUT1 and VGLUT2 and of the presumed neuronal glutamine transporter SAT1 novel tools have been made available to unequivocally define the anatomy of glutamatergic pathways on the cellular and synaptic level. Using highly specific antisera and cRNA probes two distinct glutamatergic pathways expressing either VGLUT1 or VGLUT2 could be detected throughout the central nervous system. Areas where VGLUT1 predominated included the cerebral and cerebellar cortex and the hippocampus. VGLUT2 was mainly expressed in the thalamus, hypothalamus and brain stem. VGLUT1 and VGLUT2 synapses exhibited distinct region- and pathway-specific relationships with each other and with other classical transmitter and peptidergic systems. The glutamine transporter SAT1 was expressed in CNS neurons and in ependymal cells. Neuronal SAT1 expression comprised virtually all glutamatergic neurons but also specific subsets of cholinergic, GABAergic and aminergic neurons in the CNS. In addition to widespread expression of VGLUT1 and VGLUT2 in the CNS, peripheral tissues such as sensory neurons and pancreatic islet cells differentially expressed VGLUT isoforms and SAT1.
Our results suggest pathway-specific functional duality in the regulation of vesicular glutamate release at excitatory synapses and provide evidence for glutamine transport and metabolism in excitatory glutamatergic and diverse nonglutamatergic neurons as well.     

The metabolic role of isoleucine in detoxification of ammonia in cultured mouse neurons and astrocytes

Cerebral hyperammonemia is a hallmark of hepatic encephalopathy, a debilitating condition arising secondary to liver disease. Pyruvate oxidation including tricarboxylic acid (TCA) cycle metabolism has been suggested to be inhibited by hyperammonemia at the pyruvate and -ketoglutarate dehydrogenase steps. Catabolism of the branched-chain amino acid isoleucine provides both acetyl-CoA and succinyl-CoA, thus by-passing both the pyruvate dehydrogenase and the -ketoglutarate dehydrogenase steps. Potentially, this will enable the TCA cycle to work in the face of ammonium-induced inhibition. In addition, this will provide the -ketoglutarate carbon skeleton for glutamate and glutamine synthesis by glutamate dehydrogenase and glutamine synthetase (astrocytes only), respectively, both reactions fixing ammonium. Cultured cerebellar neurons (primarily glutamatergic) or astrocytes were incubated in the presence of either [U-¹³C]glucose (2.5 mM) and isoleucine (1 mM) or [U-¹³C]isoleucine and glucose. Cell cultures were treated with an acute ammonium chloride load of 2 (astrocytes) or 5 mM (neurons and astrocytes) and incorporation of ¹³C-label into glutamate, aspartate, glutamine and alanine was determined employing mass spectrometry. Labeling from [U-¹³C]glucose in glutamate and aspartate increased as a result of ammonium-treatment in both neurons and astrocytes, suggesting that the TCA cycle was not inhibited. Labeling in alanine increased in neurons but not in astrocytes, indicating elevated glycolysis in neurons. For both neurons and astrocytes, labeling from [U-¹³C]isoleucine entered glutamate and aspartate albeit to a lower extent than from [U-¹³C]glucose. Labeling in glutamate and aspartate from [U-¹³C]isoleucine was decreased by ammonium treatment in neurons but not in astrocytes, the former probably reflecting increased metabolism of unlabeled glucose. In astrocytes, ammonia treatment resulted in glutamine production and release to the medium, partially supported by catabolism of [U-¹³C]isoleucine. In conclusion, i) neuronal and astrocytic TCA cycle metabolism was not inhibited by ammonium and ii) isoleucine may provide the carbon skeleton for synthesis of glutamate/glutamine in the detoxification of ammonium.     

Present status and significance of the glutamine cycle in neural tissues

Evidence derived from various types of neurochemical experiments indicates that in the CNS of vertebrates there is a net flux of glutamate and GABA from neurons to astroglia and a metabolic conversion of these amino acids to glutamine. This glutamine is apparently released into the interstitial fluid and is in part taken up neurons and converted back into glutamate and GABA. This process, which is frequently referred to as “the glutamine cycle”, probably reflects the involvement of astrocytes in maintaining very low extracellular levels of glutamate and GABA, and the role of glutamine as a metabolic precursor of the transmitter pools of glutamate and GABA. The synthesis and release of glutamine by astrocytes may also reflect the role of these cells in ammonia detoxification. The quantitative importance of glutamine as a precursor of the neurotransmitter pools of glutamate and GABA has yet to be established. Other potential metabolic precursors such as α-ketoglutarate have not yet been evaluated adequately.     

From glutamate co-release to vesicular synergy: vesicular glutamate transporters

Recent data indicate that 'classical' neurotransmitters can also act as co-transmitters. This notion has been strengthened by the demonstration that three vesicular glutamate transporters (vesicular glutamate transporter 1 (VGLUT1), VGLUT2 and VGLUT3) are present in central monoamine, acetylcholine and GABA neurons, as well as in primarily glutamatergic neurons. Thus, intriguing questions are raised about the morphological and functional organization of neuronal systems endowed with such a dual signalling capacity. In addition to glutamate co-release, vesicular synergy - a process leading to enhanced packaging of the 'primary' transmitter - is increasingly recognized as a major property of the glutamatergic co-phenotype. The behavioural relevance of this co-phenotype is presently the focus of considerable interest.     

The glutamate/GABA-glutamine cycle: aspects of transport, neurotransmitter homeostasis and ammonia transfer

Neurons are metabolically handicapped in the sense that they are not able to perform de novo synthesis of neurotransmitter glutamate and gamma-aminobutyric acid (GABA) from glucose. A metabolite shuttle known as the glutamate/GABA-glutamine cycle describes the release of neurotransmitter glutamate or GABA from neurons and subsequent uptake into astrocytes. In return, astrocytes release glutamine to be taken up into neurons for use as neurotransmitter precursor. In this review, the basic properties of the glutamate/GABA-glutamine cycle will be discussed, including aspects of transport and metabolism. Discussions of stoichiometry, the relative role of glutamate vs. GABA and pathological conditions affecting the glutamate/GABA-glutamine cycling are presented. Furthermore, a section is devoted to the accompanying ammonia homeostasis of the glutamate/GABA-glutamine cycle, examining the possible means of intercellular transfer of ammonia produced in neurons (when glutamine is deamidated to glutamate) and utilized in astrocytes (for amidation of glutamate) when the glutamate/GABA-glutamine cycle is operating. A main objective of this review is to endorse the view that the glutamate/GABA-glutamine cycle must be seen as a bi-directional transfer of not only carbon units but also nitrogen units.     

Glutamine uptake by System A transporters maintains neurotransmitter GABA synthesis and inhibitory synaptic transmission

GABA synthesis is necessary to maintain synaptic vesicle filling, and key proteins in its biosynthetic pathways may play a role in regulating inhibitory synaptic stability and strength. GABAergic neurons require a source of precursor glutamate, possibly from glutamine, although it is controversial whether glutamine contributes to the synaptic pool of GABA. Here we report that inhibition of System A glutamine transporters with alpha-(methyl-amino) isobutyric acid rapidly reduced the amplitude of inhibitory post-synaptic currents and miniature inhibitory post-synaptic currents (mIPSCs) recorded in rat hippocampal area cornu ammonis 1 (CA1) pyramidal neurons, indicating that synaptic vesicle content of GABA was reduced. After inhibiting astrocytic glutamine synthesis by either blocking glutamate transporters or the glutamine synthetic enzyme, the effect of alpha-(methyl-amino) isobutyric acid on mIPSC amplitudes was abolished. Exogenous glutamine did not affect mIPSC amplitudes, suggesting that the neuronal transporters are normally saturated. Our findings demonstrate that a constitutive supply of glutamine is provided by astrocytes to inhibitory neurons to maintain vesicle filling. Therefore, glutamine transporters, like those for glutamate, are potential regulators of inhibitory synaptic strength. However, in contrast to glutamate, extracellular glutamine levels are normally high. Therefore, we propose a supportive role for glutamine, even under resting conditions, to maintain GABA vesicle filling.     

Role of glycogenolysis in stimulation of ATP release from cultured mouse astrocytes by transmitters and high K+ concentrations

This study investigates the role of glycogenolysis in stimulated release of ATP as a transmitter from astrocytes. Within the last 20 years our understanding of brain glycogenolysis has changed from it being a relatively uninteresting process to being a driving force for essential brain functions like production of transmitter glutamate and homoeostasis of potassium ions (K⁺) after their release from excited neurons. Simultaneously, the importance of astrocytic handling of adenosine, its phosphorylation to ATP and release of some astrocytic ATP, located in vesicles, as an important transmitter has also become to be realized. Among the procedures stimulating Ca²⁺-dependent release of vesicular ATP are exposure to such transmitters as glutamate and adenosine, which raise intra-astrocytic Ca²⁺ concentration, or increase of extracellular K⁺ to a depolarizing level that opens astrocytic L-channels for Ca²⁺ and thereby also increase intra-astrocytic Ca²⁺ concentration, a prerequisite for glycogenolysis. The present study has confirmed and quantitated stimulated ATP release from well differentiated astrocyte cultures by glutamate, adenosine or elevated extracellular K⁺ concentrations, measured by a luciferin/luciferase reaction. It has also shown that this release is virtually abolished by an inhibitor of glycogenolysis as well as by inhibitors of transmitter-mediated signaling or of L-channel opening by elevated K⁺ concentrations.     

中枢神经系统谷氨酸转运体的研究进展

在中枢神经系统,谷氨酸转运体在谷氨酸一谷氨酰胺循环中发挥着重要作用。谷氨酸转运体有高亲和力转运体,即兴奋性氨基酸转运体（excitatory amino acid transporters,EAATs）和低亲和力转运体,即囊泡谷氨酸转运体（vesicular glutamate transporters,VGLUTs）两种类型。其中,VGLUTs的功能是特异地将突触囊泡外的谷氨酸转运进入突触囊泡内,它包括三个成员,分别是VGLUT1、VGLUT2和VGLUT3。一方面,VGLUT1和VGLUT2标记了所有的谷氨酸能神经元,是谷氦酸能神经元和它们轴突末端高度特异的标志;另一方面,VGLUT1标志着皮质一皮质投射,而VGLUT2则标志着丘脑一皮层投射,VGLUT3则位于抑制性突触末端。