Functional significance of brain glycogen in sustaining glutamatergic neurotransmission

The involvement of brain glycogen in sustaining neuronal activity has previously been demonstrated. However, to what extent energy derived from glycogen is consumed by astrocytes themselves or is transferred to the neurons in the form of lactate for oxidative metabolism to proceed is at present unclear. The significance of glycogen in fueling glutamate uptake into astrocytes was specifically addressed in cultured astrocytes. Moreover, the objective was to elucidate whether glycogen derived energy is important for maintaining glutamatergic neurotransmission, induced by repetitive exposure to NMDA in co-cultures of cerebellar neurons and astrocytes. In the astrocytes it was shown that uptake of the glutamate analogue d -[³H]aspartate was impaired when glycogen degradation was inhibited irrespective of the presence of glucose, signifying that energy derived from glycogen degradation is important for the astrocytic compartment. By inhibiting glycogen degradation in co-cultures it was evident that glycogen provides energy to sustain glutamatergic neurotransmission, i.e. release and uptake of glutamate. The relocation of glycogen derived lactate to the neuronal compartment was investigated by employing d -lactate, a competitive substrate for the monocarboxylate transporters. Neurotransmitter release was affected by the presence of d -lactate indicating that glycogen derived energy is important not only in the astrocytic but also in the neuronal compartment.     

Astrocytic energetics during excitatory neurotransmission: What are contributions of glutamate oxidation and glycolysis?

Astrocytic energetics of excitatory neurotransmission is controversial due to discrepant findings in different experimental systems in vitro and in vivo. The energy requirements of glutamate uptake are believed by some researchers to be satisfied by glycolysis coupled with shuttling of lactate to neurons for oxidation. However, astrocytes increase glycogenolysis and oxidative metabolism during sensory stimulation in vivo, indicating that other sources of energy are used by astrocytes during brain activation. Furthermore, glutamate uptake into cultured astrocytes stimulates glutamate oxidation and oxygen consumption, and glutamate maintains respiration as well as glucose. The neurotransmitter pool of glutamate is associated with the faster component of total glutamate turnover in vivo, and use of neurotransmitter glutamate to fuel its own uptake by oxidation-competent perisynaptic processes has two advantages, substrate is supplied concomitant with demand, and glutamate spares glucose for use by neurons and astrocytes. Some, but not all, perisynaptic processes of astrocytes in adult rodent brain contain mitochondria, and oxidation of only a small fraction of the neurotransmitter glutamate taken up into these structures would be sufficient to supply the ATP required for sodium extrusion and conversion of glutamate to glutamine. Glycolysis would, however, be required in perisynaptic processes lacking oxidative capacity. Three lines of evidence indicate that critical cornerstones of the astrocyte-to-neuron lactate shuttle model are not established and normal brain does not need lactate as supplemental fuel: (i) rapid onset of hemodynamic responses to activation delivers oxygen and glucose in excess of demand, (ii) total glucose utilization greatly exceeds glucose oxidation in awake rodents during activation, indicating that the lactate generated is released, not locally oxidized, and (iii) glutamate-induced glycolysis is not a robust phenotype of all astrocyte cultures. Various metabolic pathways, including glutamate oxidation and glycolysis with lactate release, contribute to cellular energy demands of excitatory neurotransmission.     

星形胶质细胞功能障碍在抑郁症发病中的作用研究进展

星型胶质细胞在突触形成、神经元代谢、神经递质传递等方面起重要作用,其退行性病变可引起突触蛋白水平降低、神经元体积减小及神经递质传递异常,进而引起神经精神性疾病的发生。抑郁症患者前额叶皮层、海马、杏仁核以及前扣带回等多个脑区均有星型胶质细胞密度减低,提示星形胶质细胞与抑郁症发病密切相关。研究表明,能量和营养支持、谷氨酸(glutamate,Glu)转运和代谢、N-甲基-D-天(门)冬氨酸(N-methyl-D-aspartate,NMDA)受体活性调节以及炎症反应异常等星形胶质细胞功能障碍参与抑郁症的发生。本文就星形胶质细胞功能障碍在抑郁症发病机理中的作用进行综述。     

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.     

High-Glucose and S100B Stimulate Glutamate Uptake in C6 Glioma Cells

Diabetes mellitus is a disease associated with several changes in the central nervous system, including oxidative stress and abnormal glutamatergic neurotransmission, and the astrocytes play an essential role in these alterations. In vitro studies of astroglial function have been performed using cultures of primary astrocytes or C6 glioma cells. Herein, we investigated glutamate uptake, glutamine synthetase and S100B secretion in C6 glioma cells cultured in a high-glucose environment, as well as some parameters of oxidative stress and damage. C6 glioma cells, cultured in 12 mM glucose medium, exhibited signals of oxidative and nitrosative stress similar to those found in diabetes mellitus and other models of diabetic disease (decrease in glutathione, elevated NO, DNA damage). Interestingly, we found an increase in glutamate uptake and S100B secretion, and a decrease in glutamine synthetase, which might be linked to the altered glutamatergic communication in diabetes mellitus. Moreover, glutamate uptake in C6 glioma cells, like primary astrocytes, was stimulated by extracellular S100B. Aminoguanidine partially prevented the glial alterations induced by the 12 mM glucose medium. Together, these data emphasize the relevance of astroglia in diabetes mellitus, as well as the importance of glial parameters in the evaluation of diabetic disease progression and treatment.     

Astrocytes and energy metabolism

Astrocytes are glial cells, which play a significant role in a number of processes, including the brain energy metabolism. Their anatomical position between blood vessels and neurons make them an interface for effective glucose uptake from blood. After entering astrocytes, glucose can be involved in different metabolic pathways, e.g. in glycogen production. Glycogen in the brain is localized mainly in astrocytes and is an important energy source in hypoxic conditions and normal brain functioning. The portion of glucose metabolized into glycogen molecules in astrocytes is as high as 40%. It is thought that the release of gliotransmitters (such as glutamate, neuroactive peptides and ATP) into the extracellular space by regulated exocytosis supports a significant part of communication between astrocytes and neurons. On the other hand, neurotransmitter action on astrocytes has a significant role in brain energy metabolism. Therefore, understanding the astrocytes energy metabolism may help understanding neuron-astrocyte interactions.     

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.     

Neuron-astrocyte interactions in the regulation of brain energy metabolism: a focus on NMR spectroscopy

An adequate and timely production of ATP by brain cells is of cardinal importance to support the major energetic cost of the rapid processing of information via synaptic and action potentials. Recently, evidence has been accumulated to support the view that the regulation of brain energy metabolism is under the control of an intimate dialogue between astrocytes and neurons. In vitro studies on cultured astrocytes and in vivo studies on rodents have provided evidence that glutamate and Na(+) uptake in astrocytes is a key triggering signal regulating glucose use in the brain. With the advent of NMR spectroscopy, it has been possible to provide experimental evidence to show that energy consumption is mainly devoted to glutamatergic neurotransmission and that glutamate-glutamine cycling is coupled in a approximately 1 : 1 molar stoichiometry to glucose oxidation, at least in the cerebral cortex. This improved understanding of neuron-astrocyte metabolic interactions offers the potential for developing novel therapeutic strategies for many neurological disorders that include a metabolic deficit.     

The glutamate transporter, GLAST, participates in a macromolecular complex that supports glutamate metabolism

GLAST is the predominant glutamate transporter in the cerebellum and contributes substantially to glutamate transport in forebrain. This astroglial glutamate transporter quickly binds and clears synaptically released glutamate and is principally responsible for ensuring that synaptic glutamate concentrations remain low. This process is associated with a significant energetic cost. Compartmentalization of GLAST with mitochondria and proteins involved in energy metabolism could provide energetic support for glutamate transport. Therefore, we performed immunoprecipitation and co-localization experiments to determine if GLAST might co-compartmentalize with proteins involved in energy metabolism. GLAST was immunoprecipitated from rat cerebellum and subunits of the Na(+)/K(+) ATPase, glycolytic enzymes, and mitochondrial proteins were detected. GLAST co-localized with mitochondria in cerebellar tissue. GLAST also co-localized with mitochondria in fine processes of astrocytes in organotypic hippocampal slice cultures. From these data, we hypothesized that mitochondria participate in a macromolecular complex with GLAST to support oxidative metabolism of transported glutamate. To determine the functional metabolic role of this complex, we measured CO(2) production from radiolabeled glutamate in cultured astrocytes and compared it to overall glutamate uptake. Within 15min, 9% of transported glutamate was converted to CO(2). This CO(2) production was blocked by inhibitors of glutamate transport and glutamate dehydrogenase, but not by an inhibitor of glutamine synthetase. Our data support a model in which GLAST exists in a macromolecular complex that allows transported glutamate to be metabolized in mitochondria to support energy production.     

Inhibition of Gap Junction Elevates Glutamate Uptake in Cultured Astrocytes

Glutamate uptake is a main function of astrocytes to keep extracellular glutamate levels low and protect neurons against glutamate-induced excitotoxicity. On the other hand, astrocyte networks formed by gap junctions, which are consisted with connexins and connecting neighboring cells, are reported to play a critical role in maintaining the homeostasis in the brain. In the present study, we examined the effects of gap junction inhibitors on the glutamate uptake activity in cultured rat cortical astrocytes. At first, we confirmed the effects of gap junction inhibitors, 1-octanol and carbenoxolone, on cell–cell communication by the scrape-loading assay using a fluorescent dye Lucifer yellow. Both of 1-octanol and carbenoxolone treatments for 20 min in cultured astrocytes significantly suppressed the cell–cell communication assessed as the distance of dye-spreading. 1-octanol and carbenoxolone increased the glutamate uptake by astrocytes and glutamate aspartate transporter (GLAST) expression on the cell membrane. These results suggest that gap junction inhibitors increase the glutamate uptake activity through the increase of GLAST proteins located on the cell membrane. The regulation of gap junction in astrocytes might protect neurons against glutamate-induced excitotoxicity.     

Exposure to high glutamate concentration activates aerobic glycolysis but inhibits ATP‐linked respiration in cultured cortical astrocytes

Astrocytes play a key role in removing the synaptically released glutamate from the extracellular space and maintaining the glutamate below neurotoxic level in the brain. However, high concentration of glutamate leads to toxicity in astrocytes, and the underlying mechanisms are unclear. The purpose of this study was to investigate whether energy metabolism disorder, especially impairment of mitochondrial respiration, is involved in the glutamate‐induced gliotoxicity. Exposure to 10‐mM glutamate for 48 h stimulated glycolysis and respiration in astrocytes. However, the increased oxygen consumption was used for proton leak and non‐mitochondrial respiration, but not for oxidative phosphorylation and ATP generation. When the exposure time extended to 72 h, glycolysis was still activated for ATP generation, but the mitochondrial ATP‐linked respiration of astrocytes was reduced. The glutamate‐induced astrocyte damage can be mimicked by the non‐metabolized substrate d ‐aspartate but reversed by the non‐selective glutamate transporter inhibitor TBOA. In addition, the glutamate toxicity can be partially reversed by vitamin E. These findings demonstrate that changes of bioenergetic profile occur in cultured cortical astrocytes exposed to high concentration of glutamate and highlight the role of mitochondria respiration in glutamate‐induced gliotoxicity in cortical astrocytes. Copyright © 2014 John Wiley & Sons, Ltd.     

谷氨酸、NMDA 和吗啡对培养大鼠星形胶质细胞内钙振荡的影响

目的:研究谷氨酸、NMDA、吗啡对原代培养的大鼠星形胶质细胞的胞内钙信号的影响及受体作用机制.方法:利用Leica AF6000活细胞工作站,检测谷氨酸、NMDA、吗啡分别灌流前后Fura-2/AM加载的星形胶质细胞内钙信号的动态变化,进一步观 察分别阻断代谢性谷氨酸受体5 (mGluR5)、NMDA受体(NMDA receptor,NMDAR)和阿片μ受体对诱导的胞内钙振荡的影响.结果:谷氨酸、NMDA、吗啡均可明显升高胞内游离钙的浓度([Ca2+]i),而将其相应受体拮抗后,星形胶质细胞[Ca2+]i升高的现象可以被显著抑制.结论:离体培养的星形胶质细胞膜上存在mGluR5、NMDAR和阿片μ受体,这些受体的激活可以升高星影胶质细胞的[Ca2+]i,且这些受体依赖的[Ca2+]i的调控机制可能是星形胶质细胞与神经元交互作用的重要途径之一.     

The Discovery of Human of GLUD2 Glutamate Dehydrogenase and Its Implications for Cell Function in Health and Disease

While the evolutionary changes that led to traits unique to humans remain unclear, there is increasing evidence that enrichment of the human genome through DNA duplication processes may have contributed to traits such as bipedal locomotion, higher cognitive abilities and language. Among the genes that arose through duplication in primates during the period of increased brain development was GLUD2, which encodes the hGDH2 isoform of glutamate dehydrogenase expressed in neural and other tissues. Glutamate dehydrogenase GDH is an enzyme central to the metabolism of glutamate, the main excitatory neurotransmitter in mammalian brain involved in a multitude of CNS functions, including cognitive processes. In nerve tissue GDH is expressed in astrocytes that wrap excitatory synapses, where it is thought to play a role in the metabolic fate of glutamate removed from the synaptic cleft during excitatory transmission. Expression of GDH rises sharply during postnatal brain development, coinciding with nerve terminal sprouting and synaptogenesis. Compared to the original hGDH1 (encoded by the GLUD1 gene), which is potently inhibited by GTP generated by the Krebs cycle, hGDH2 can function independently of this energy switch. In addition, hGDH2 can operate efficiently in the relatively acidic environment that prevails in astrocytes following glutamate uptake. This adaptation is thought to provide a biological advantage by enabling enhanced enzyme catalysis under intense excitatory neurotransmission. While the novel protein may help astrocytes to handle increased loads of transmitter glutamate, dissociation of hGDH2 from GTP control may render humans vulnerable to deregulation of this enzyme’s function. Here we will retrace the cloning and characterization of the novel GLUD2 gene and the potential implications of this discovery in the understanding of mechanisms that permitted the brain and other organs that express hGDH2 to fine-tune their functions in order to meet new challenging demands. In addition, the potential role of gain-of-function of hGDH2 variants in human neurodegenerative processes will be considered.     

Effect of ammonia on GABA uptake and release in cultured astrocytes

While the pathogenesis of hepatic encephalopathy (HE) is unclear, there is evidence of enhanced GABAergic neurotransmission in this condition. Ammonia is believed to play a major pathogenetic role in HE. To determine whether ammonia might contribute to abnormalities in GABAergic neurotransmission, its effects on GABA uptake and release were studied in cultured astrocytes, cells that appear to be targets of ammonia neurotoxicity. Acutely, ammonium chloride (5 mM) inhibited GABA uptake by 30%, and by 50-60% after 4-day treatment. GABA uptake inhibition was associated with a predominant decrease in Vmax; the Km was also decreased. Ammonia also enhanced GABA release after 4-day treatment, although such release was initially inhibited. These effects of ammonia (inhibition of GABA uptake and enhanced GABA release) may elevate extracellular levels of GABA and contribute to a dysfunction of GABAergic neurotransmission in HE and other hyperammonemic states.     

Effects of estrogen treatment on glutamate uptake in cultured human astrocytes derived from cortex of Alzheimer's disease patients

Estrogen is thought to play a protective role against neurodegeneration through a variety of mechanisms including the activation of growth factors, the control of synaptic plasticity, and the reduction of response to various insults, such as iron and glutamate. Increasing evidence indicates an increased level of extracellular glutamate and a down-regulation of glutamate transporters in Alzheimer's disease (AD). In this study, we show that glutamate uptake in astrocytes derived from Alzheimer's patients is significantly lower than that from non-demented controls. Estrogen treatment increases glutamate uptake in a dose-dependent pattern. Two glutamate transporters, GLT-1 and GLAST, are expressed in the astrocytes. Up-regulation of the glutamate transporters is induced by estrogen treatment in AD astrocytes only. Our data suggest that the action of estrogen on glutamate uptake by astrocytes might contribute to its potential neuroprotective role in AD.     

Inhibition of astroglial glutamate transport by polyunsaturated fatty acids: Evidence for a signalling role of docosahexaenoic acid

Brain cells are especially rich in polyunsaturated fatty acids (PUFA), mainly the n-3 PUFA docosahexaenoic acid (DHA) and the n-6 PUFA arachidonic acid (AA). They are released from membranes by PLA2 during neurotransmission, and may regulate glutamate uptake by astroglia, involved in controlling glutamatergic transmission. AA has been shown to inhibit glutamate transport in several model systems, but the contribution of DHA is less clear and has not been evaluated in astrocytes. Because the high DHA content of brain membranes is essential for brain function, we investigated the role of DHA in the regulation of astroglial glutamate transport.We evaluated the actions of DHA and AA using cultured rat astrocytes and suspensions of rat brain membranes (P1 fractions). DHA reduced d-[³H]aspartate uptake by cultured astrocytes and cortical membrane suspensions, while AA did not. This also occurred in astrocytes enriched with α-tocopherol, indicating that it was not due to peroxidation products. The reduction of d-[³H]aspartate uptake by DHA did not involve any change in the concentrations of membrane-associated astroglial glutamate transporters (GLAST and GLT-1), suggesting that DHA reduced the activity of the transporters. In contrast with the inhibition induced by free-DHA, we found no effect of membrane-bound DHA on d-[³H]aspartate uptake. Indeed, the uptake was similar in astrocytes with varying amount of DHA in their membrane (induced by long-term supplementation with DHA or AA). Therefore, DHA reduces glutamate uptake through a signal-like effect but not through changes in the PUFA composition of the astrocyte membranes. Also, reactive astrocytes, induced by a medium supplement (G5), were insensitive to DHA. This suggests that DHA regulates synaptic glutamate under basal condition but does not impair glutamate scavenging under reactive conditions.These results indicate that DHA slows astroglial glutamate transport via a specific signal-like effect, and may thus be a physiological synaptic regulator.     

3-hydroxyglutaric acid enhances glutamate uptake into astrocytes from cerebral cortex of young rats

A predominantly neurological presentation is common in patients with glutaric acidemia type I (GA-I). 3-hydroxyglutaric acid (3-OHGA), which accumulates in affected patients, has recently been demonstrated to play a central role in the neuropathogenesis of this disease. In the present study, we investigated the in vitro effects of 3-OHGA at concentrations ranging from 10 to 1000 microM on various parameters of the glutamatergic system, such as the basal and potassium-induced release of [3H]glutamate by synaptosomes, as well as on Na+-dependent [3H]glutamate uptake by synaptosomes and astrocytes and Na+-independent [3H]glutamate uptake by synaptic vesicles from cerebral cortex of 30-day-old Wistar rats. First, we observed that exposure of cultured astrocytes to 3-OHGA for 20 h did not reduce their viability. Furthermore, 3-OHGA significantly increased Na+-dependent [3H]glutamate uptake by astrocytes by up to 80% in a dose-dependent manner at doses as low as 30 microM. This effect was not dependent on the presence of the metabolite during the uptake assay, since it occurred even when 3-OHGA was withdrawn from the medium after cultured cells had been exposed to the acid for approximately 1 h. All other parameters investigated were not influenced by this organic acid, indicating a selective action of 3-OHGA on astrocyte transporters. Although the exact mechanisms involved in 3-OHGA-stimulatory effect on astrocyte glutamate uptake are unknown, the present findings contribute to the understanding of the pathophysiology of GA-I, suggesting that astrocytes may protect neurons against excitotoxic damage caused by 3-OHGA by increasing glutamate uptake and therefore reducing the concentration of this excitatory neurotransmitter in the synaptic cleft.     

Computational Flux Balance Analysis Predicts that Stimulation of Energy Metabolism in Astrocytes and their Metabolic Interactions with Neurons Depend on Uptake of K<Superscript>+</Superscript> Rather than Glutamate

Brain activity involves essential functional and metabolic interactions between neurons and astrocytes. The importance of astrocytic functions to neuronal signaling is supported by many experiments reporting high rates of energy consumption and oxidative metabolism in these glial cells. In the brain, almost all energy is consumed by the Na⁺/K⁺ ATPase, which hydrolyzes 1 ATP to move 3 Na⁺ outside and 2 K⁺ inside the cells. Astrocytes are commonly thought to be primarily involved in transmitter glutamate cycling, a mechanism that however only accounts for few % of brain energy utilization. In order to examine the participation of astrocytic energy metabolism in brain ion homeostasis, here we attempted to devise a simple stoichiometric relation linking glutamatergic neurotransmission to Na⁺ and K⁺ ionic currents. To this end, we took into account ion pumps and voltage/ligand-gated channels using the stoichiometry derived from available energy budget for neocortical signaling and incorporated this stoichiometric relation into a computational metabolic model of neuron-astrocyte interactions. We aimed at reproducing the experimental observations about rates of metabolic pathways obtained by ¹³C-NMR spectroscopy in rodent brain. When simulated data matched experiments as well as biophysical calculations, the stoichiometry for voltage/ligand-gated Na⁺ and K⁺ fluxes generated by neuronal activity was close to a 1:1 relationship, and specifically 63/58 Na⁺/K⁺ ions per glutamate released. We found that astrocytes are stimulated by the extracellular K⁺ exiting neurons in excess of the 3/2 Na⁺/K⁺ ratio underlying Na⁺/K⁺ ATPase-catalyzed reaction. Analysis of correlations between neuronal and astrocytic processes indicated that astrocytic K⁺ uptake, but not astrocytic Na⁺-coupled glutamate uptake, is instrumental for the establishment of neuron-astrocytic metabolic partnership. Our results emphasize the importance of K⁺ in stimulating the activation of astrocytes, which is relevant to the understanding of brain activity and energy metabolism at the cellular level.     

Alanine metabolism, transport, and cycling in the brain

Brain glutamate/glutamine cycling is incomplete without return of ammonia to glial cells. Previous studies suggest that alanine is an important carrier for ammonia transfer. In this study, we investigated alanine transport and metabolism in Guinea pig brain cortical tissue slices and prisms, in primary cultures of neurons and astrocytes, and in synaptosomes. Alanine uptake into astrocytes was largely mediated by system L isoform LAT2, whereas alanine uptake into neurons was mediated by Na⁺-dependent transporters with properties similar to system B⁰ isoform B⁰AT2. To investigate the role of alanine transport in metabolism, its uptake was inhibited in cortical tissue slices under depolarizing conditions using the system L transport inhibitors 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid and cycloleucine (1-aminocyclopentanecarboxylic acid; cLeu). The results indicated that alanine cycling occurs subsequent to glutamate/glutamine cycling and that a significant proportion of cycling occurs via amino acid transport system L. Our results show that system L isoform LAT2 is critical for alanine uptake into astrocytes. However, alanine does not provide any significant carbon for energy or neurotransmitter metabolism under the conditions studied.