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Activation of T and natural killer (NK) cells leads to the tyrosine phosphorylation of pp36 and to its association with several signaling molecules, including phospholipase Cgamma-1 and Grb2. Microsequencing of peptides derived from purified rat pp36 protein led to the cloning, in rat and man, of cDNA encoding a T- and NK cell-specific protein with several putative Src homology 2 domain-binding motifs. A rabbit antiserum directed against a peptide sequence from the cloned rat molecule recognized tyrosine phosphorylated pp36 from pervanadate-treated rat thymocytes. When expressed in 293T human fibroblast cells and tyrosine-phosphorylated, pp36 associated with phospholipase Cgamma-1 and Grb2. Studies with GST-Grb2 fusion proteins demonstrated that the association was specific for the Src homology 2 domain of Grb-2. Molecular cloning of the gene encoding pp36 should facilitate studies examining the role of this adaptor protein in proximal signaling events during T and NK cell activation.

The cystine/glutamate antiporter system x c - has been identified as the major source of extracellular glutamate in several brain regions as well as a modulator of neuroinflammation, and genetic deletion of its specific subunit xCT (xCT -/- ) is protective in mouse models for age-related neurological disorders. However, the previously observed oxidative shift in the plasma cystine/cysteine ratio of adult xCT -/- mice led to the hypothesis that system x c - deletion would negatively affect life- and healthspan. Still, till now the role of system x c - in physiological aging remains unexplored. We therefore studied the effect of xCT deletion on the aging process of mice, with a particular focus on the immune system, hippocampal function, and cognitive aging. We observed that male xCT -/- mice have an extended lifespan, despite an even more increased plasma cystine/cysteine ratio in aged compared to adult mice. This oxidative shift does not negatively impact the general health status of the mice. On the contrary, the age-related priming of the innate immune system, that manifested as increased LPS-induced cytokine levels and hypothermia in xCT +/+ mice, was attenuated in xCT -/- mice. While this was associated with only a very moderate shift towards a more anti-inflammatory state of the aged hippocampus, we observed changes in the hippocampal metabolome that were associated with a preserved hippocampal function and the retention of hippocampus-dependent memory in male aged xCT -/- mice. Targeting system x c - is thus not only a promising strategy to prevent cognitive decline, but also to promote healthy aging., (© 2022. The Author(s).)

In contrast to water soluble enzymes which can be purified and studied while in solution, studies of solute carrier (transporter) proteins require both that the protein of interest is situated in a phospholipid membrane and that this membrane forms a closed compartment. An additional challenge to the study of transporter proteins has been that the transport depends on the transmembrane electrochemical gradients. Baruch I. Kanner understood this early on and first developed techniques for studying plasma membrane vesicles. This advanced the field in that the experimenter could control the electrochemical gradients. Kanner, however, did not stop there, but started to solubilize the membranes so that the transporter proteins were taken out of their natural environment. In order to study them, Kanner then had to find a way to reconstitute them (reinsert them into phospholipid membranes). The scope of the present review is both to describe the reconstitution method in full detail as that has never been done, and also to reveal the scientific impact that this method has had. Kanner's later work is not reviewed here although that also deserves a review because it too has had a huge impact., (© 2021. The Author(s).)

Objective: The astroglial enzyme glutamine synthetase (GS) is deficient in small loci in the brain in adult patients with different types of focal epilepsy; however, the role of this deficiency in the pathogenesis of epilepsy has been difficult to assess due to a lack of sufficiently sensitive and specific animal models. The aim of this study was to develop an in vivo approach for precise and specific deletions of the GS gene in the postnatal brain., Methods: We stereotaxically injected various adeno-associated virus (AAV)-Cre recombinase constructs into the hippocampal formation and neocortex in 22-70-week-old GS flox/flox mice to knock out the GS gene in a specific and focal manner. The mice were subjected to seizure threshold determination, continuous video-electroencephalographic recordings, advanced in vivo neuroimaging, and immunocytochemistry for GS., Results: The construct AAV8-glial fibrillary acidic protein-green fluorescent protein-Cre eliminated GS in >99% of astrocytes in the injection center with a gradual return to full GS expression toward the periphery. Such focal GS deletion reduced seizure threshold, caused spontaneous recurrent seizures, and diminished functional connectivity., Significance: These results suggest that small loci of GS deficiency in the postnatal brain are sufficient to cause epilepsy and impaired functional connectivity. Additionally, given the high specificity and precise spatial resolution of our GS knockdown approach, we anticipate that this model will be extremely useful for rigorous in vivo and ex vivo studies of astroglial GS function at the brain-region and single-cell levels., (© 2021 International League Against Epilepsy.)

Objective: To test the hypothesis that glutamate and GABA are linked to the formation of epilepsy networks and the triggering of spontaneous seizures, we examined seizure initiation/propagation characteristics and neurotransmitter levels during epileptogenesis in a translationally relevant rodent model of mesial temporal lobe epilepsy., Methods: The glutamine synthetase (GS) inhibitor methionine sulfoximine was infused into one of the hippocampi in laboratory rats to create a seizure focus. Long-term video-intracranial EEG recordings and brain microdialysis combined with mass spectrometry were used to examine seizure initiation, seizure propagation, and extracellular brain levels of glutamate and GABA., Results: All seizures (n = 78 seizures, n = 3 rats) appeared first in the GS-inhibited hippocampus of all animals, followed by propagation to the contralateral hippocampus. Propagation time decreased significantly from 11.65 seconds early in epileptogenesis (weeks 1-2) to 6.82 seconds late in epileptogenesis (weeks 3-4, paired t test, p = 0.025). Baseline extracellular glutamate levels were 11.6-fold higher in the hippocampus of seizure propagation (7.3 µM) vs the hippocampus of seizure onset (0.63 µM, analysis of variance/Fisher least significant difference, p = 0.01), even though the concentrations of the major glutamate transporter proteins excitatory amino acid transporter subtypes 1 and 2 and xCT were unchanged between the brain regions. Finally, extracellular GABA in the seizure focus decreased significantly from baseline several hours before a spontaneous seizure (paired t test/false discovery rate)., Conclusion: The changes in glutamate and GABA suggest novel and potentially important roles of the amino acids in epilepsy network formation and in the initiation and propagation of spontaneous seizures., (© 2021 American Academy of Neurology.)

Proper glutamatergic neurotransmission requires a balance between glutamate release and removal. The removal is mainly catalyzed by the glutamate transporters EAAT1-3, while the glutamate-cystine exchanger (system x c - with specific subunit xCT) represents one of the release mechanisms. Previous studies of the spinal cord have focused on the cellular distribution of EAAT1-3 with special reference to the dorsal horn, but have not provided quantitative data and have not systematically compared multiple segments. Here we have studied the distribution of EAAT1-3 and xCT in sections of multiple spinal cord segments using knockout tissue as negative controls. EAAT2 and EAAT3 were evenly expressed in all gray matter areas at all segmental levels, albeit with slightly higher levels in laminae 1-4 (dorsal horn). Somewhat higher levels of EAAT2 were also seen in lamina 9 (ventral horn), while EAAT3 was also detected in the lateral spinal nucleus. EAAT1 was concentrated in laminae 1-3, lamina 10, the intermediolateral nucleus and the sacral parasympathetic nucleus, while xCT was concentrated in laminae 1-3, lamina 10 and the leptomeninges. The levels of these four transporters were low in white matter, which represents 42% of the spinal cord volume. Quantitative immunoblotting revealed that the average level of EAAT1 in the whole spinal cord was 0.6 ± 0.1% of that in the cerebellum, while the levels of EAAT2, EAAT3 and xCT were, respectively, 41.6 ± 12%, 39.8 ± 7.6%, and 30.8 ± 4.3% of the levels in the hippocampus (mean values ± SEM). Conclusions: Because the hippocampal tissue content of EAAT2 protein is two orders of magnitude higher than the content of the EAAT3, it follows that most of the gray matter in the spinal cord depends almost exclusively on EAAT2 for glutamate removal, while the lamina involved in the processing of autonomic and nociceptive information rely on a complex system of transporters., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Elevated blood ammonia (hyperammonemia) is believed to be a major contributor to the neurological sequelae following severe liver disease. Ammonia is cleared via two main mechanisms, the urea cycle pathway and the glutamine synthetase reaction. Recent studies of genetically modified animals confirm the importance of the urea cycle, but also suggest that the glutamine synthetase reaction is more important than previously recognized. While the liver clears about two-thirds of the body's ammonia via the combined action of the urea cycle and glutamine synthetase, extrahepatic tissues do not express all the components required for performing a complete urea cycle and therefore depend on the glutamine synthetase reaction for ammonia clearance. The brain is particularly vulnerable to the effects of hyperammonemia, which include impaired extracellular potassium buffering and brain edema. Moreover, the glutamine synthetase reaction is intimately linked to the metabolism of the excitatory and inhibitory neurotransmitters glutamate and gamma aminobutyric acid (GABA), implicating a key role for this enzyme in neurotransmission. This review discusses the emerging roles of glutamine synthetase in brain pathophysiology, particularly aspects related to ammonia homeostasis and hepatic encephalopathy., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objectives: The cystine/glutamate antiporter (system xc-) is believed to contribute to nonvesicular glutamate release from glial cells in various brain areas. Although recent investigations implicate system xc- in mood disorders, unambiguous evidence has not yet been established. Therefore, we evaluated the possible role of system xc- in the depressive state. Methods: We conducted a protein expression analysis of the specific subunit of system xc- (xCT) in brain regions of the corticosterone mouse model, Flinders Sensitive Line rat model and post-mortem tissue of depressed patients. We next subjected system xc- deficient mice to the corticosterone model and analysed their behaviour in several tests. Lastly, we subjected additional cohorts of xCT-deficient and wild-type mice to N -acetylcysteine treatment to unveil whether the previously reported antidepressant-like effects are dependent upon system xc-. Results: We did not detect any changes in xCT expression levels in the animal models or patients compared to proper controls. Furthermore, loss of system xc- had no effect on depression- and anxiety-like behaviour. Finally, the antidepressant-like effects of N- acetylcysteine are not mediated via system xc-. Conclusions: xCT protein expression is not altered in the depressed brain and system xc- deficiency does not affect depression-associated behaviour in the corticosterone mouse model.

The excitatory amino acid transporter type 2 (EAAT2) represents the major mechanism for removal of extracellular glutamate. In the hippocampus, there is some EAAT2 in axon-terminals, whereas most of the protein is found in astroglia. The functional importance of the neuronal EAAT2 is unknown, and it is debated whether EAAT2-expressing nerve terminals are present in other parts of the brain. Here we selectively deleted the EAAT2 gene in neurons (by crossing EAAT2-flox mice with synapsin 1-Cre mice in the C57B6 background). To reduce interference from astroglial EAAT2, we measured glutamate accumulation in crude tissue homogenates. EAAT2 proteins levels were measured by immunoblotting. Although synapsin 1-Cre mediated gene deletion only reduced the forebrain tissue content of EAAT2 protein to 95.5 ± 3.4% of wild-type (littermate) controls, the glutamate accumulation in homogenates of neocortex, hippocampus, striatum and thalamus were nevertheless diminished to, respectively, 54 ± 4, 46 ± 3, 46 ± 2 and 65 ± 7% of controls (average ± SEM, n = 3 pairs of littermates). GABA uptake was unaffected. After injection of U- 13 C-glucose, lack of neuronal EAAT2 resulted in higher 13 C-labeling of glutamine and GABA in the hippocampus suggesting that neuronal EAAT2 is partly short-circuiting the glutamate-glutamine cycle in wild-type mice. Crossing synapsin 1-Cre mice with Ai9 reporter mice revealed that Cre-mediated excision occurred efficiently in hippocampus CA3, but less efficiently in other regions and hardly at all in the cerebellum. Conclusions: (1) EAAT2 is expressed in nerve terminals in multiple brain regions. (2) The uptake catalyzed by neuronal EAAT2 plays a role in glutamate metabolism, at least in the hippocampus. (3) Synapsin 1-Cre does not delete floxed genes in all neurons, and the contribution of neuronal EAAT2 is therefore likely to be larger than revealed in the present study., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Glutamate-ammonia ligase (glutamine synthetase; Glul) is enriched in astrocytes and serves as the primary enzyme for ammonia detoxification and glutamate inactivation in the brain. Loss of astroglial Glul is reported in hippocampi of epileptic patients, but the mechanism by which Glul deficiency might cause disease remains elusive. Here we created a novel mouse model by selectively deleting Glul in the hippocampus and neocortex. The Glul deficient mice were born without any apparent malformations and behaved unremarkably until postnatal week three. There were reductions in tissue levels of aspartate, glutamate, glutamine and GABA and in mRNA encoding glutamate receptor subunits GRIA1 and GRIN2A as well as in the glutamate transporter proteins EAAT1 and EAAT2. Adult Glul-deficient mice developed progressive neurodegeneration and spontaneous seizures which increased in frequency with age. Importantly, progressive astrogliosis occurred before neurodegeneration and was first noted in astrocytes along cerebral blood vessels. The responses to CO 2 -provocation were attenuated at four weeks of age and dilated microvessels were observed histologically in sclerotic areas of cKO. Thus, the abnormal glutamate metabolism observed in this model appeared to cause epilepsy by first inducing gliopathy and disrupting the neurovascular coupling., (Copyright © 2018. Published by Elsevier Ltd.)

The communication between the immune and central nervous system (CNS) is affected in many neurological disorders. Peripheral injections of the endotoxin lipopolysaccharide (LPS) are widely used to study this communication: an LPS challenge leads to a biphasic syndrome that starts with acute sickness and is followed by persistent brain inflammation and chronic behavioral alterations such as depressive-like symptoms. In vitro, the response to LPS treatment has been shown to involve enhanced expression of system x c - . This cystine-glutamate antiporter, with xCT as specific subunit, represents the main glial provider of extracellular glutamate in mouse hippocampus. Here we injected male xCT knockout and wildtype mice with a single intraperitoneal dose of 5 mg/kg LPS. LPS-injection increased hippocampal xCT expression but did not alter the mainly astroglial localization of the xCT protein. Peripheral and central inflammation (as defined by cytokine levels and morphological activation of microglia) as well as LPS-induced sickness and depressive-like behavior were significantly attenuated in xCT-deficient mice compared with wildtype mice. Our study is the first to demonstrate the involvement of system x c - in peripheral and central inflammation in vivo and the potential therapeutic relevance of its inhibition in brain disorders characterized by peripheral and central inflammation, such as depression., (© 2018 Wiley Periodicals, Inc.)

The cystine-glutamate exchanger (xCT) promotes glutathione synthesis by catalyzing cystine uptake and glutamate release. The released glutamate may modulate normal neural signaling and contribute to excitotoxicity in pathological situations. Uncertainty, however, remains as neither the expression levels nor the distribution of xCT have been unambiguously determined. In fact, xCT has been reported in astrocytes, neurons, oligodendrocytes and microglia, but most of the information derives from cell cultures. Here, we show by immunohistochemistry and by Western blotting that xCT is widely expressed in the central nervous system of both sexes. The labeling specificity was validated using tissue from xCT knockout mice as controls. Astrocytes were selectively labeled, but showed greatly varying labeling intensities. This astroglial heterogeneity resulted in an astrocyte domain-like labeling pattern. Strong xCT labeling was also found in the leptomeninges, along some blood vessels, in selected circumventricular organs and in a subpopulation of tanycytes residing the lateral walls of the ventral third ventricle. Neurons, oligodendrocytes and resting microglia, as well as reactive microglia induced by glutamine synthetase deficiency, were unlabeled. The concentration of xCT protein in hippocampus was compared with that of the EAAT3 glutamate transporter by immunoblotting using a chimeric xCT-EAAT3 protein to normalize xCT and EAAT3 labeling intensities. The immunoblots suggested an xCT/EAAT3 ratio close to one (0.75 ± 0.07; average ± SEM; n = 4) in adult C57BL6 mice., Conclusions: xCT is present in select blood/brain/CSF interface areas and in an astrocyte subpopulation, in sufficient quantities to support the notion that system xc- provides physiologically relevant transport activity., (© 2018 Wiley Periodicals, Inc.)

Glutamate transport activities have been identified not only in the brain, but also in the liver, kidney, and intestine. Although glutamate transporter distributions in the central nervous system are fairly well known, there are still uncertainties with respect to the distribution of these transporters in peripheral organs. Quantitative information is mostly lacking, and few of the studies have included genetically modified animals as specificity controls. The present study provides validated qualitative and semi-quantitative data on the excitatory amino acid transporter (EAAT)1-3 subtypes in the mouse liver, kidney, and intestine. In agreement with the current view, we found high EAAT3 protein levels in the brush borders of both the distal small intestine and the renal proximal tubules. Neither EAAT1 nor EAAT2 was detected at significant levels in murine kidney or intestine. In contrast, the liver only expressed EAAT2 (but 2 C-terminal splice variants). EAAT2 was detected in the plasma membranes of perivenous hepatocytes. These cells also expressed glutamine synthetase. Conditional deletion of hepatic EAAT2 did neither lead to overt neurological disturbances nor development of fatty liver.

Immunocytochemistry and Western blotting are still major methods for protein localization, but they rely on the specificity of the antibodies. Validation of antibody specificity remains challenging mostly because ideal negative controls are often unavailable. Further, immunochemical labeling patterns are also influenced by a number of other factors such as postmortem changes, fixation procedures and blocking agents as well as the general assay conditions (e.g., buffers, temperature, etc.). Western blotting similarly depends on tissue collection and sample preparation as well as the electrophoretic separation, transfer to blotting membranes and the immunochemical probing of immobilized molecules. Publication of inaccurate information on protein distribution has downstream consequences for other researchers because the interpretation of physiological and pharmacological observations depends on information on where ion channels, receptors, enzymes or transporters are located. Despite numerous reports, some of which are strongly worded, erroneous localization data are being published. Here we describe the extent of the problem and illustrate the nature of the pitfalls with examples from studies of neurotransmitter transporters. We explain the importance of supplementing immunochemical observations with other measurements (e.g., mRNA levels and distribution, protein activity, mass spectrometry, electrophysiological recordings, etc.) and why quantitative considerations are integral parts of the quality control. Further, we propose a practical strategy for researchers who plan to embark on a localization study. We also share our thoughts about guidelines for quality control. GLIA 2016;64:2045-2064., (© 2016 Wiley Periodicals, Inc.)

Little is known about olfactory glands' regulation despite their presumed importance for normal functioning of the cilia of olfactory neurons. The aim of this study was to establish an assay for olfactory gland activation by using large-scale quantitative electron microscopy (EM). In addition we wanted to test the hypothesis that cholinergic drugs activate the olfactory glands, by using our newly established EM assay. In total, over 70 000 secretory gland vesicles were quantified in over 3000 cells. Olfactory gland cell size (40.8 µm 2 ± 2.0 SD), vesicle diameter (812 nm ± 57 SD) and vesicles per cell (21.6 ± 4.2 SD) were also quantified. The vesicle percentage of the cell area varied between 24% and 30%. In a blinded study we found no significant effects of cholinergic agents on parameters of vesicle number or vesicle diameter. Unexpectedly, pilocarpine treatment increased olfactory gland size, probably by inducing cell swelling. In conclusion, we have established a quantitative EM assay for olfactory gland activation and provided new data on basic olfactory gland cell characteristics. By using the EM assay, olfactory glands are shown not to be activated by cholinergic agents, which indicates an alternative regulation pathway or constitutive secretion from olfactory glands., (© The Author 2016. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved.For permissions, please e-mail: journals.permissions@oup.com.)

Neither normal brain function nor the pathological processes involved in neurological diseases can be adequately understood without knowledge of the release, uptake and metabolism of glutamate. The reason for this is that glutamate (a) is the most abundant amino acid in the brain, (b) is at the cross-roads between several metabolic pathways, and (c) serves as the major excitatory neurotransmitter. In fact most brain cells express glutamate receptors and are thereby influenced by extracellular glutamate. In agreement, brain cells have powerful uptake systems that constantly remove glutamate from the extracellular fluid and thereby limit receptor activation. It has been clear since the 1970s that both astrocytes and neurons express glutamate transporters. However the relative contribution of neuronal and glial transporters to the total glutamate uptake activity, however, as well as their functional importance, has been hotly debated ever since. The present short review provides (a) an overview of what we know about neuronal glutamate uptake as well as an historical description of how we got there, and (b) a hypothesis reconciling apparently contradicting observations thereby possibly resolving the paradox., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Developmental cortical malformations (DCMs) are linked with severe epilepsy and are caused by both genetic and environmental insults. DCMs include several neurological diseases, such as focal cortical dysplasia, polymicrogyria, schizencephaly, and others. Human studies have implicated astrocyte reactivity and dysfunction in the pathophysiology of DCMs, but their specific role is unknown. As astrocytes powerfully regulate glutamate neurotransmission, and glutamate levels are known to be increased in human epileptic foci, understanding the role of astrocytes in the pathological sequelae of DCMs is extremely important. Additionally, recent studies examining astrocyte glutamate uptake in DCMs have reported conflicting results, adding confusion to the field. In this study we utilized the freeze lesion (FL) model of DCM, which is known to induce reactive astrocytosis and cause significant changes in astrocyte morphology, proliferation, and distribution. Using whole-cell patch clamp recording from astrocytes, we recorded both UV-uncaging and synaptically evoked glutamate transporter currents (TCs), widely accepted assays of functional glutamate transport by astrocytes. With this approach, we set out to test the hypothesis that astrocyte membrane properties and glutamate transport were disrupted in this model of DCM. Though we found that the developmental maturation of astrocyte membrane resistance was disrupted by FL, glutamate uptake by individual astrocytes was robust throughout FL development. Interestingly, using an immunolabeling approach, we observed spatial and developmental differences in excitatory amino acid transporter (EAAT) expression in FL cortex. Spatially specific differences in EAAT2 (GLT-1) and EAAT1 (GLAST) expression suggest that the relative contribution of each EAAT to astrocytic glutamate uptake may be altered in FL cortex. Lastly, we carefully analyzed the amplitudes and onset times of both synaptically- and UV uncaging-evoked TCs. We found that in the FL cortex, synaptically-evoked, but not UV uncaging-evoked TCs, were larger in amplitude. Additionally, we found that the amount of electrical stimulation required to evoke a synaptic TC was significantly reduced in the FL cortex. Both of these findings are consistent with increased excitatory input to the FL cortex, but not with changes in how individual astrocytes remove glutamate. Taken together, our results demonstrate that the maturation of astrocyte membrane resistance, local distribution of glutamate transporters, and glutamatergic input to the cortex are altered in the FL model, but that single-cell astrocytic glutamate uptake is robust., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The cystine/glutamate antiporter or system Xc- exchanges cystine for glutamate, thereby supporting intracellular glutathione synthesis and nonvesicular glutamate release. The role of system Xc- in neurological disorders can be dual and remains a matter of debate. One important reason for the contradictory findings that have been reported to date is the use of nonspecific anti-xCT (the specific subunit of system Xc-) antibodies. Often studies rely on the predicted molecular weight of 55.5 kDa to identify xCT on Western blots. However, using brain extracts from xCT knockout (xCT(-/-)) mice as negative controls, we show that xCT migrates as a 35-kDa protein. Misinterpretation of immunoblots leads to incorrect assessment of antibody specificity and thereby to erroneous data interpretation. Here we have verified the specificity of most commonly used commercial and some in-house-developed anti-xCT antibodies by comparing their immunoreactivity in brain tissue of xCT(+/+) and xCT(-/-) mice by Western blotting and immunohistochemistry. The Western blot screening results demonstrate that antibody specificity not only differs between batches produced by immunizing different rabbits with the same antigen but also between bleedings of the same rabbit. Moreover, distinct immunohistochemical protocols have been tested for all the anti-xCT antibodies that were specific on Western blots in order to obtain a specific immunolabeling. Only one of our in-house-developed antibodies could reveal specific xCT labeling and exclusively on acetone-postfixed cryosections. Using this approach, we observed xCT protein expression throughout the mouse forebrain, including cortex, striatum, hippocampus, midbrain, thalamus, and amygdala, with greatest expression in regions facing the cerebrospinal fluid and meninges., (© 2015 Wiley Periodicals, Inc.)

Epilepsy is a complex, multifactorial disease characterized by spontaneous recurrent seizures and an increased incidence of comorbid conditions such as anxiety, depression, cognitive dysfunction, and sudden unexpected death. About 70 million people worldwide are estimated to suffer from epilepsy, and up to one-third of all people with epilepsy are expected to be refractory to current medications. Development of more effective and specific antiepileptic interventions is therefore requisite. Perturbations in the brain's glutamate-glutamine cycle, such as increased extracellular levels of glutamate, loss of astroglial glutamine synthetase, and changes in glutaminase and glutamate dehydrogenase, are frequently encountered in patients with epilepsy. Hence, manipulations of discrete glutamate-glutamine cycle components may represent novel approaches to treat the disease. The goal of his review is to discuss some of the glutamate-glutamine cycle components that are altered in epilepsy, particularly neurotransmitters and metabolites, enzymes, amino acid transporters, and glutamate receptors. We will also review approaches that potentially could be used in humans to target the glutamate-glutamine cycle. Examples of such approaches are treatment with glutamate receptor blockers, glutamate scavenging, dietary intervention, and hypothermia.

Glutamate uptake by astrocytes controls the time course of glutamate in the extracellular space and affects neurotransmission, synaptogenesis, and circuit development. Astrocytic glutamate uptake has been shown to undergo post-natal maturation in the hippocampus, but has been largely unexplored in other brain regions. Notably, glutamate uptake has never been examined in the developing neocortex. In these studies, we investigated the development of astrocytic glutamate transport, intrinsic membrane properties, and control of neuronal NMDA receptor activation in the developing neocortex. Using astrocytic and neuronal electrophysiology, immunofluorescence, and Western blot analysis we show that: (1) glutamate uptake in the neonatal neocortex is slow relative to neonatal hippocampus; (2) astrocytes in the neonatal neocortex undergo a significant maturation of intrinsic membrane properties; (3) slow glutamate uptake is accompanied by lower expression of both GLT-1 and GLAST; (4) glutamate uptake is less dependent on GLT-1 in neonatal neocortex than in neonatal hippocampus; and (5) the slow glutamate uptake we report in the neonatal neocortex corresponds to minimal astrocytic control of neuronal NMDA receptor activation. Taken together, our results clearly show fundamental differences between astrocytic maturation in the developing neocortex and hippocampus, and corresponding changes in how astrocytes control glutamate signaling., (© 2015 Wiley Periodicals, Inc.)

GLT-1 (EAAT2; slc1a2) is the major glutamate transporter in the brain, and is predominantly expressed in astrocytes, but at lower levels also in excitatory terminals. We generated a conditional GLT-1 knock-out mouse to uncover cell-type-specific functional roles of GLT-1. Inactivation of the GLT-1 gene was achieved in either neurons or astrocytes by expression of synapsin-Cre or inducible human GFAP-CreERT2. Elimination of GLT-1 from astrocytes resulted in loss of ∼80% of GLT-1 protein and of glutamate uptake activity that could be solubilized and reconstituted in liposomes. This loss was accompanied by excess mortality, lower body weight, and seizures suggesting that astrocytic GLT-1 is of major importance. However, there was only a small (15%) reduction that did not reach significance of glutamate uptake into crude forebrain synaptosomes. In contrast, when GLT-1 was deleted in neurons, both the GLT-1 protein and glutamate uptake activity that could be solubilized and reconstituted in liposomes were virtually unaffected. These mice showed normal survival, weight gain, and no seizures. However, the synaptosomal glutamate uptake capacity (Vmax) was reduced significantly (40%). In conclusion, astrocytic GLT-1 performs critical functions required for normal weight gain, resistance to epilepsy, and survival. However, the contribution of astrocytic GLT-1 to glutamate uptake into synaptosomes is less than expected, and the contribution of neuronal GLT-1 to synaptosomal glutamate uptake is greater than expected based on their relative protein expression. These results have important implications for the interpretation of the many previous studies assessing glutamate uptake capacity by measuring synaptosomal uptake., (Copyright © 2015 the authors 0270-6474/15/355187-15$15.00/0.)

Epilepsy is one of the most common chronic neurologic diseases, yet approximately one-third of affected patients do not respond to anticonvulsive drugs that target neurons or neuronal circuits. Reactive astrocytes are commonly found in putative epileptic foci and have been hypothesized to be disease contributors because they lose essential homeostatic capabilities. However, since brain pathology induces astrocytes to become reactive, it is difficult to distinguish whether astrogliosis is a cause or a consequence of epileptogenesis. We now present a mouse model of genetically induced, widespread chronic astrogliosis after conditional deletion of β1-integrin (Itgβ1). In these mice, astrogliosis occurs in the absence of other pathologies and without BBB breach or significant inflammation. Electroencephalography with simultaneous video recording revealed that these mice develop spontaneous seizures during the first six postnatal weeks of life and brain slices show neuronal hyperexcitability. This was not observed in mice with neuronal-targeted β1-integrin deletion, supporting the hypothesis that astrogliosis is sufficient to induce epileptic seizures. Whole-cell patch-clamp recordings from astrocytes further suggest that the heightened excitability was associated with impaired astrocytic glutamate uptake. Moreover, the relative expression of the cation-chloride cotransporters (CCC) NKCC1 (Slc12a2) and KCC2 (Slc12a5), which are responsible for establishing the neuronal Cl(-) gradient that governs GABAergic inhibition were altered and the NKCC1 inhibitor bumetanide eliminated seizures in a subgroup of mice. These data suggest that a shift in the relative expression of neuronal NKCC1 and KCC2, similar to that observed in immature neurons during development, may contribute to astrogliosis-associated seizures., (Copyright © 2015 the authors 0270-6474/15/353330-16$15.00/0.)

The oxidation resistance gene 1 (OXR1) prevents oxidative stress-induced cell death by an unknown pathway. Here, depletion of human OXR1 (hOXR1) sensitized several human cell lines to hydrogen peroxide-induced oxidative stress, reduced mtDNA integrity, and increased apoptosis. In contrast, depletion of hOXR1 in cells lacking mtDNA showed no significant change in ROS or viability, suggesting that OXR1 prevents intracellular hydrogen peroxide-induced increase in oxidative stress levels to avoid a vicious cycle of increased oxidative mtDNA damage and ROS formation. Furthermore, expression of p21 and the antioxidant genes GPX2 and HO-1 was reduced in hOXR1-depleted cells. In sum, these data reveal that human OXR1 upregulates the expression of antioxidant genes via the p21 signaling pathway to suppress hydrogen peroxide-induced oxidative stress and maintain mtDNA integrity., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

The EAAT2 glutamate transporter, accounts for >90% of hippocampal glutamate uptake. Although EAAT2 is predominantly expressed in astrocytes, ∼10% of EAAT2 molecules are found in axon terminals. Despite the lower level of EAAT2 expression in glutamatergic terminals, when hippocampal slices are incubated with low concentration of d-aspartate (an EAAT2 substrate), axon terminals accumulate d-aspartate as quickly as astroglia. This implies an unexplained mismatch between the distribution of EAAT2 protein and of EAAT2-mediated transport activity. One hypothesis is that (1) heteroexchange of internal substrate with external substrate is considerably faster than net uptake and (2) terminals favor heteroexchange because of high levels of internal glutamate. However, it is currently unknown whether heteroexchange and uptake have similar or different rates. To address this issue, we used a reconstituted system to compare the relative rates of the two processes in rat and mice. Net uptake was sensitive to changes in the membrane potential and was stimulated by external permeable anions in agreement with the existence of an uncoupled anion conductance. By using the latter, we also demonstrate that the rate of heteroexchange also depends on the membrane potential. Additionally, our data further suggest the presence of a sodium leak in EAAT2. By incorporating the new findings in our previous model of glutamate uptake by EAAT2, we predict that the voltage sensitivity of exchange is caused by the voltage-dependent third Na(+) binding. Further, both our experiments and simulations suggest that the relative rates of net uptake and heteroexchange are comparable in EAAT2., (Copyright © 2014 the authors 0270-6474/14/3413472-14$15.00/0.)

Glutamate is the most abundant free amino acid in the brain and is at the crossroad between multiple metabolic pathways. Considering this, it was a surprise to discover that glutamate has excitatory effects on nerve cells, and that it can excite cells to their death in a process now referred to as "excitotoxicity". This effect is due to glutamate receptors present on the surface of brain cells. Powerful uptake systems (glutamate transporters) prevent excessive activation of these receptors by continuously removing glutamate from the extracellular fluid in the brain. Further, the blood-brain barrier shields the brain from glutamate in the blood. The highest concentrations of glutamate are found in synaptic vesicles in nerve terminals from where it can be released by exocytosis. In fact, glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. It took, however, a long time to realize that. The present review provides a brief historical description, gives a short overview of glutamate as a transmitter in the healthy brain, and comments on the so-called glutamate-glutamine cycle. The glutamate transporters responsible for the glutamate removal are described in some detail.

The physiological roles of the betaine/GABA transporter (BGT1; slc6a12) are still being debated. BGT1 is a member of the solute carrier family 6 (the neurotransmitter, sodium symporter transporter family) and mediates cellular uptake of betaine and GABA in a sodium- and chloride-dependent process. Most of the studies of BGT1 concern its function and regulation in the kidney medulla where its role is best understood. The conditions here are hostile due to hyperosmolarity and significant concentrations of NH4Cl and urea. To withstand the hyperosmolarity, cells trigger osmotic adaptation, involving concentration of a transcriptional factor TonEBP/NFAT5 in the nucleus, and accumulate betaine and other osmolytes. Data from renal cells in culture, primarily MDCK, revealed that transcriptional regulation of BGT1 by TonEBP/NFAT5 is relatively slow. To allow more acute control of the abundance of BGT1 protein in the plasma membrane, there is also post-translation regulation of BGT1 protein trafficking which is dependent on intracellular calcium and ATP. Further, betaine may be important in liver metabolism as a methyl donor. In fact, in the mouse the liver is the organ with the highest content of BGT1. Hepatocytes express high levels of both BGT1 and the only enzyme that can metabolize betaine, namely betaine:homocysteine -S-methyltransferase (BHMT1). The BHMT1 enzyme removes a methyl group from betaine and transfers it to homocysteine, a potential risk factor for cardiovascular disease. Finally, BGT1 has been proposed to play a role in controlling brain excitability and thereby represents a target for anticonvulsive drug development. The latter hypothesis is controversial due to very low expression levels of BGT1 relative to other GABA transporters in brain, and also the primary location of BGT1 at the surface of the brain in the leptomeninges. These issues are discussed in detail.

Objectives: Loss of glutamate from cardiomyocytes during ischaemia may aggravate ischaemia-reperfusion injury in open heart surgery. This may be due to reversal of excitatory amino acid transporters (EAATs). However, the expression of such transporters in cardiomyocytes is ambiguous and quantitative data are lacking. Our objective was to study whether EAATs were expressed in the rat heart and to study whether blocking of transporter operation during cardiac ischaemia could be beneficial., Methods: We used TaqMan real-time PCR and immunoisolation followed by western blotting to unequivocally identify EAAT subtypes in rat hearts. We used a novel high-affinity non-transportable competitive inhibitor, named LL-TBOA [(2S,3S)-3-(3-(6-(6-(2-(2-(2-(2-(2-aminoethoxy)ethoxy)-ethoxy)ethoxy) acetamido)hexanamido)- hexanamido)-5-(4-(trifluoromethyl)benzamido)benzyloxy) aspartic acid], to block EAAT-mediated transport during global ischaemia and reperfusion of isolated rat hearts., Results: Rat hearts expressed EAAT subtypes 1 and 3, while subtypes 2 and 4 were not detected. Hearts were isolated and perfused with 1.6 µM LL-TBOA for 5 min before 30 min of induced global ischaemia and 60 min of reperfusion (n = 8). Control hearts were perfused either with the solvent dimethylsulfoxide 3.5 mM (n = 7) or with no pretreatment (n = 8). Infarct size was evaluated by triphenyl tetrazolium chloride (TTC) staining. LL-TBOA reduced infarct size from 33 ± 14 to 20 ± 5% (mean ± SD) (P = 0.015). Dimethylsulfoxide alone had no effect (35 ± 2%). Reperfusion arrhythmias were reduced by LL-TBOA (P = 0.009), but not by dimethylsulfoxide alone., Conclusion: Rat hearts express EAAT1 and EAAT3, but the mRNA levels are, respectively, ∼ 25 and 200 times lower than in the brain. Addition of LL-TBOA has a beneficial effect against ischaemia-reperfusion injury.

Glutamate is the major excitatory neurotransmitter, and is inactivated by cellular uptake catalyzed mostly by the glutamate transporter subtypes GLT-1 (EAAT2) and GLAST (EAAT1). Astrocytes express both GLT-1 and GLAST, while axon terminals in the neocortex only express GLT-1. To evaluate the role of GLT-1 in glutamate homeostasis, we injected GLT-1 knockout (KO) mice and wild-type littermates with [1-(13)C]glucose and [1,2-(13)C]acetate 15 min before euthanization. Metabolite levels were analyzed in extracts from neocortex and cerebellum and (13)C labeling in neocortex. Whereas the cerebellum in GLT-1-deficient mice had normal levels of glutamate, glutamine, and (13)C labeling of metabolites, glutamate level was decreased but labeling from [1-(13)C] glucose was unchanged in the neocortex. The contribution from pyruvate carboxylation toward labeling of these metabolites was unchanged. Labeling from [1,2-(13)C] acetate, originating in astrocytes, was decreased in glutamate and glutamine in the neocortex indicating reduced mitochondrial metabolism in astrocytes. The decreased amount of glutamate in the cortex indicates that glutamine transport into neurons is not sufficient to replenish glutamate lost because of neurotransmission and that GLT-1 plays a role in glutamate homeostasis in the cortex. Glutamate is the major excitatory neurotransmitter, and is inactivated by uptake via GLT-1 (EAAT2) and GLAST (EAAT1) transporters, while axon terminals in the neocortex only express GLT-1. To evaluate the role of GLT-1 in glutamate homeostasis, we used [1-(13)C]glucose and [1,2-(13)C]acetate injection and NMR spectroscopy. The results indicate that glutamine transport into neurons is not sufficient to replenish glutamate lost because of neurotransmission and that GLT-1 plays a role in glutamate homeostasis in the neocortex., (© 2013 International Society for Neurochemistry.)

Islet function is incompletely understood in part because key steps in glutamate handling remain undetermined. The glutamate (excitatory amino acid) transporter 2 (EAAT2; Slc1a2) has been hypothesized to (a) provide islet cells with glutamate, (b) protect islet cells against high extracellular glutamate concentrations, (c) mediate glutamate release, or (d) control the pH inside insulin secretory granules. Here we floxed the EAAT2 gene to produce the first conditional EAAT2 knock-out mice. Crossing with Nestin-cyclization recombinase (Cre) eliminated EAAT2 from the brain, resulting in epilepsy and premature death, confirming the importance of EAAT2 for brain function and validating the genetic construction. Crossing with insulin-Cre lines (RIP-Cre and IPF1-Cre) to obtain pancreas-selective deletion did not appear to affect survival, growth, glucose tolerance, or β-cell number. We found (using TaqMan RT-PCR, immunoblotting, immunocytochemistry, and proteome analysis) that the EAAT2 levels were too low to support any of the four hypothesized functions. The proteome analysis detected more than 7,000 islet proteins of which more than 100 were transporters. Although mitochondrial glutamate transporters and transporters for neutral amino acids were present at high levels, all other transporters with known ability to transport glutamate were strikingly absent. Glutamate-metabolizing enzymes were abundant. The level of glutamine synthetase was 2 orders of magnitude higher than that of glutaminase. Taken together this suggests that the uptake of glutamate by islets from the extracellular fluid is insignificant and that glutamate is intracellularly produced. Glutamine synthetase may be more important for islets than assumed previously.

The mammalian genome contains four genes encoding GABA transporters (GAT1, slc6a1; GAT2, slc6a13; GAT3, slc6a11; BGT1, slc6a12) and five glutamate transporter genes (EAAT1, slc1a3; EAAT2, slc1a2; EAAT3, slc1a1; EAAT4, slc1a6; EAAT5, slc1a7). These transporters keep the extracellular levels of GABA and excitatory amino acids low and provide amino acids for metabolic purposes. The various transporters have different properties both with respect to their transport functions and with respect to their ability to act as ion channels. Further, they are differentially regulated. To understand the physiological roles of the individual transporter subtypes, it is necessary to obtain information on their distributions and expression levels. Quantitative data are important as the functional capacity is limited by the number of transporter molecules. The most important and most abundant transporters for removal of transmitter glutamate in the brain are EAAT2 (GLT-1) and EAAT1 (GLAST), while GAT1 and GAT3 are the major GABA transporters in the brain. EAAT3 (EAAC1) does not appear to play a role in signal transduction, but plays other roles. Due to their high uncoupled anion conductance, EAAT4 and EAAT5 seem to be acting more like inhibitory glutamate receptors than as glutamate transporters. GAT2 and BGT1 are primarily expressed in the liver and kidney, but are also found in the leptomeninges, while the levels in brain tissue proper are too low to have any impact on GABA removal, at least in normal young adult mice. The present review will provide summary of what is currently known and will also discuss some methodological pitfalls.

Glutamate transporters (GLT-1, GLAST, EAAC1) limit the actions of excitatory amino acids. Because a disturbed transporter operation can cause or aggravate neurological diseases, transporters are of considerable neuropathological interest. Human samples, however, are seldom obtained fresh. Here, we used mice brains to study how fast glutamate transporters are degraded after death. Immunoblots showed that terminal GLT-1 epitopes (within residues 1-26 and 518-573) had mostly disappeared after 24 hr. GLAST termini (1-25 and 522-543) degraded slightly slower. In contrast, epitopes within central parts of GLT-1 (493-508) and the EAAC1 C-terminus (510-523) were readily detectable after 72 hr. The decline in immunoreactivity of the GLT-1 and GLAST termini was also seen in tissue sections, but proteolysis did not happen synchronously in all cells. At 24 hr, scattered cells remained strongly immunopositive, while the majority of cells were completely immunonegative. GLAST and GLT-1 co-localized in neocortical tissue, but at 12 hr, many GLAST-positive cells had lost the GLT-1 termini. The uneven disappearance of labeling was not observed with the antibodies to GLT-1 residues 493-508. The immunoreactivity to this epitope correlated better with the reported glutamate uptake activity. Thus, postmortem delay may affect epitopes differently, possibly causing erroneous conclusions about relative expression levels.

The GABA transporters (GAT1, GAT2, GAT3, and BGT1) have mostly been discussed in relation to their potential roles in controlling the action of transmitter GABA in the nervous system. We have generated the first mice lacking the GAT2 (slc6a13) gene. Deletion of GAT2 (both mRNA and protein) neither affected growth, fertility, nor life span under nonchallenging rearing conditions. Immunocytochemistry showed that the GAT2 protein was predominantly expressed in the plasma membranes of periportal hepatocytes and in the basolateral membranes of proximal tubules in the renal cortex. This was validated by processing tissue from wild-type and knockout mice in parallel. Deletion of GAT2 reduced liver taurine levels by 50%, without affecting the expression of the taurine transporter TAUT. These results suggest an important role for GAT2 in taurine uptake from portal blood into liver. In support of this notion, GAT2-transfected HEK293 cells transported [(3)H]taurine. Furthermore, most of the uptake of [(3)H]GABA by cultured rat hepatocytes was due to GAT2, and this uptake was inhibited by taurine. GAT2 was not detected in brain parenchyma proper, excluding a role in GABA inactivation. It was, however, expressed in the leptomeninges and in a subpopulation of brain blood vessels. Deletion of GAT2 increased brain taurine levels by 20%, suggesting a taurine-exporting role for GAT2 in the brain.

The extracellular levels of excitatory amino acids are kept low by the action of the glutamate transporters. Glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) are the most abundant subtypes and are essential for the functioning of the mammalian CNS, but the contribution of the EAAC1 subtype in the clearance of synaptic glutamate has remained controversial, because the density of this transporter in different tissues has not been determined. We used purified EAAC1 protein as a standard during immunoblotting to measure the concentration of EAAC1 in different CNS regions. The highest EAAC1 levels were found in the young adult rat hippocampus. Here, the concentration of EAAC1 was ∼0.013 mg/g tissue (∼130 molecules μm⁻³), 100 times lower than that of GLT-1. Unlike GLT-1 expression, which increases in parallel with circuit formation, only minor changes in the concentration of EAAC1 were observed from E18 to adulthood. In hippocampal slices, photolysis of MNI-D-aspartate (4-methoxy-7-nitroindolinyl-D-aspartate) failed to elicit EAAC1-mediated transporter currents in CA1 pyramidal neurons, and D-aspartate uptake was not detected electron microscopically in spines. Using EAAC1 knock-out mice as negative controls to establish antibody specificity, we show that these relatively small amounts of EAAC1 protein are widely distributed in somata and dendrites of all hippocampal neurons. These findings raise new questions about how so few transporters can influence the activation of NMDA receptors at excitatory synapses.

The biomedical research community relies directly or indirectly on immunocytochemical data. Unfortunately, validation of labeling specificity is difficult. A common specificity test is the preadsorption test. This test was intended for testing crude antisera but is now frequently used to validate monoclonal and affinity purified polyclonal antibodies. Here, the authors assess the power of this test. Nine affinity purified antibodies to different epitopes on 3 proteins (EAAT3, slc1a1; EAAT2, slc1a2; BGT1, slc6a12) were tested on samples (tissue sections and Western blots with or without fixation). The selected antibodies displayed some degree of cross-reactivity as defined by labeling of samples from knockout mice. The authors show that antigen preadsorption blocked all labeling of both wild-type and knockout samples, implying that preadsorption also blocked binding to cross-reactive epitopes. They show how this can give an illusion of specificity and illustrate sensitivity-specificity relationships, the importance of good negative controls, that fixation can create new epitopes, and that cross-reacting epitopes present in sections may not be present on Western blots and vice versa. In conclusion, they argue against uncritical use of the preadsorption test and, in doing so, address a number of other issues related to immunocytochemistry specificity testing.

The Na(+)- and Cl(-)-dependent GABA-betaine transporter (BGT1) has received attention mostly as a protector against osmolarity changes in the kidney and as a potential controller of the neurotransmitter GABA in the brain. Nevertheless, the cellular distribution of BGT1, and its physiological importance, is not fully understood. Here we have quantified mRNA levels using TaqMan real-time PCR, produced a number of BGT1 antibodies, and used these to study BGT1 distribution in mice. BGT1 (protein and mRNA) is predominantly expressed in the liver (sinusoidal hepatocyte plasma membranes) and not in the endothelium. BGT1 is also present in the renal medulla, where it localizes to the basolateral membranes of collecting ducts (particularly at the papilla tip) and the thick ascending limbs of Henle. There is some BGT1 in the leptomeninges, but brain parenchyma, brain blood vessels, ependymal cells, the renal cortex, and the intestine are virtually BGT1 deficient in 1- to 3-mo-old mice. Labeling specificity was assured by processing tissue from BGT1-deficient littermates in parallel as negative controls. Addition of 2.5% sodium chloride to the drinking water for 48 h induced a two- to threefold upregulation of BGT1, tonicity-responsive enhancer binding protein, and sodium-myo-inositol cotransporter 1 (slc5a3) in the renal medulla, but not in the brain and barely in the liver. BGT1-deficient and wild-type mice appeared to tolerate the salt treatment equally well, possibly because betaine is one of several osmolytes. In conclusion, this study suggests that BGT1 plays its main role in the liver, thereby complementing other betaine-transporting carrier proteins (e.g., slc6a20) that are predominantly expressed in the small intestine or kidney rather than the liver.

The sodium-dependent inorganic phosphate transporter NaPi-IIa is expressed in the kidney. Here, the authors used a polyclonal antiserum raised against NaPi-IIa- and NaPi-IIa-deficient mice to characterize its expression in nervous tissue. Western blots showed that a NaPi-IIa immunoreactive band (~90 kDa) was only present in wild-type kidney membranes and not in kidney knockout or wild-type brain membranes. In the water-soluble fraction of wild-type and knockout brains, another band (~50 kDa) was observed; this band was not detected in the kidney. Light and electron microscopic immunohistochemistry using the NaPi-IIa antibodies showed immunolabeling of kidney tubules in wild-type but not knockout mice. In the brain, labeling of presynaptic nerve terminals was present also in NaPi-IIa-deficient mice. This labeling pattern was also produced by the NaPi-IIa preimmune serum. The authors conclude that the polyclonal antiserum is specific toward NaPi-IIa in the kidney, but in the brain, immunolabeling is caused by a cross-reaction of the antiserum with an unknown cytosolic protein that is not present in the kidney. This tissue-specific cross-reactivity highlights a potential pitfall when validating antibody specificity using knockout mouse-derived tissue other than the specific tissue of interest and underlines the utility of specificity testing using preimmune sera.

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian brain. Once released, it is removed from the extracellular space by cellular uptake catalyzed by GABA transporter proteins. Four GABA transporters (GAT1, GAT2, GAT3 and BGT1) have been identified. Inhibition of the GAT1 by the clinically available anti-epileptic drug tiagabine has been an effective strategy for the treatment of some patients with partial seizures. Recently, the investigational drug EF1502, which inhibits both GAT1 and BGT1, was found to exert an anti-convulsant action synergistic to that of tiagabine, supposedly due to inhibition of BGT1. The present study addresses the role of BGT1 in seizure control and the effect of EF1502 by developing and exploring a new mouse line lacking exons 3-5 of the BGT1 (slc6a12) gene. The deletion of this sequence abolishes the expression of BGT1 mRNA. However, homozygous BGT1-deficient mice have normal development and show seizure susceptibility indistinguishable from that in wild-type mice in a variety of seizure threshold models including: corneal kindling, the minimal clonic and minimal tonic extension seizure threshold tests, the 6Hz seizure threshold test, and the i.v. pentylenetetrazol threshold test. We confirm that BGT1 mRNA is present in the brain, but find that the levels are several hundred times lower than those of GAT1 mRNA; possibly explaining the apparent lack of phenotype. In conclusion, the present results do not support a role for BGT1 in the control of seizure susceptibility and cannot provide a mechanistic understanding of the synergism that has been previously reported with tiagabine and EF1502., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The unravelling of the polarized distribution of AQP4 in perivascular astrocytic endfeet has revitalized the interest in the role of astrocytes in controlling water and ion exchange at the brain-blood interface. The importance of the endfeet is based on the premise that they constitute a complete coverage of the vessel wall. Despite a number of studies based on different microscopic techniques this question has yet to be resolved. We have made an electron microscopic 3D reconstruction of perivascular endfeet in CA1 (stratum moleculare) of rat hippocampus. The endfeet interdigitate and overlap, leaving no slits between them. Only in a few sites do processes--tentatively classified as processes of microglia--extend through the perivascular glial sheath to establish direct contact with the endothelial basal lamina. In contrast to the endfoot covering of the endothelial tube, the endfoot covering of the pericyte is incomplete, allowing neuropil elements to touch the basal lamina that enwraps this type of cell. The 3D reconstruction also revealed large bundles of mitochondria in the endfoot processes that came in close apposition to the perivascular endfoot membrane. Our data support the idea that in pathophysiological conditions, the perivascular astrocytic covering may control the exchange of water and solutes between blood and brain and that free diffusion is limited to narrow clefts between overlapping endfeet., (Copyright 2010 Wiley-Liss, Inc.)

The neurotransmitter glutamate is inactivated by cellular uptake; mostly catalyzed by the glutamate transporter GLT1 (slc1a2, excitatory amino acid transporter [EAAT2]) subtype which is expressed at high levels in brain astrocytes and at lower levels in neurons. Three coulombs-terminal variants of GLT1 exist (GLT1a, GLT1b and GLT1c). Their cellular distributions are currently being debated (that of GLT1b in particular). Here we have made antibodies to the variants and produced pure preparations of the individual variant proteins. The immunoreactivities of each variant per amount of protein were compared to that of total GLT1 immunoisolated from Wistar rat brains. At eight weeks of age GLT1a, GLT1b and GLT1c represented, respectively 90%+/-1%, 6+/-1% and 1%+/-0.5% (mean+/-SEM) of total hippocampal GLT1. The levels of all three variants were low at birth and increased towards adulthood, but GLT1a increased relatively more than the other two. At postnatal day 14 the levels of GLT1b and GLT1c relative to total GLT1 were, respectively, 1.7+/-0.1 and 2.5+/-0.1 times higher than at eight weeks. In tissue sections, antibodies to GLT1a gave stronger labeling than antibodies to GLT1b, but the distributions of GLT1a and GLT1b were similar in that both were predominantly expressed in astroglia, cell bodies as well as their finest ramifications. GLT1b was not detected in nerve terminals in normal brain tissue. The findings illustrate the need for quantitative measurements and support the notion that the importance of the variants may not be due to the transporter molecules themselves, but rather that their expression represents the activities of different regulatory pathways.

Human studies of intrasex variability have shown that males are intellectually more variable. Here we have performed retrospective statistical analysis of human intrasex variability in several different properties and performances that are unrelated or indirectly related to intelligence: (a) birth weights of nearly 48,000 babies (Medical Birth Registry of Norway); (b) adult weight, height, body mass index and blood parameters of more than 2,700 adults aged 18-90 (NORIP); (c) physical performance in the 60 meter dash event of 575 junior high school students; and (d) psychological performance reflected by the results of more than 222,000 undergraduate university examination grades (LIST). For all characteristics, the data were analyzed using cumulative distribution functions and the resultant intrasex variability for males was compared with that for females. The principal finding is that human intrasex variability is significantly higher in males, and consequently constitutes a fundamental sex difference.

The relative distribution of the excitatory amino acid transporter 2 (EAAT2) between synaptic terminals and astroglia, and the importance of EAAT2 for the uptake into terminals is still unresolved. Here we have used antibodies to glutaraldehyde-fixed d-aspartate to identify electron microscopically the sites of d-aspartate accumulation in hippocampal slices. About 3/4 of all terminals in the stratum radiatum CA1 accumulated d-aspartate-immunoreactivity by an active dihydrokainate-sensitive mechanism which was absent in EAAT2 glutamate transporter knockout mice. These terminals were responsible for more than half of all d-aspartate uptake of external substrate in the slices. This is unexpected as EAAT2-immunoreactivity observed in intact brain tissue is mainly associated with astroglia. However, when examining synaptosomes and slice preparations where the extracellular space is larger than in perfusion fixed tissue, it was confirmed that most EAAT2 is in astroglia (about 80%). Neither d-aspartate uptake nor EAAT2 protein was detected in dendritic spines. About 6% of the EAAT2-immunoreactivity was detected in the plasma membrane of synaptic terminals (both within and outside of the synaptic cleft). Most of the remaining immunoreactivity (8%) was found in axons where it was distributed in a plasma membrane surface area several times larger than that of astroglia. This explains why the densities of neuronal EAAT2 are low despite high levels of mRNA in CA3 pyramidal cell bodies, but not why EAAT2 in terminals account for more than half of the uptake of exogenous substrate by hippocampal slice preparations. This and the relative amount of terminal versus glial uptake in the intact brain remain to be discovered.

The excitatory amino acid transporters (EAATs) underlie the so-called "high affinity" uptake of glutamate, which is well characterized. In contrast, the "low-affinity" uptake of glutamate remains poorly defined, and it has been discussed whether it may represent a mere in vitro artifact. Here we have visualized "low-affinity" excitatory amino acid uptake sites by incubating rat hippocampal slices with the glutamate analogue D-aspartate in the presence of PMB-TBOA, which blocks the EAATs. After fixation of the slices, D-aspartate taken up into the tissue was localized with the use of light microscopic immunoperoxidase and electron microscopic immunogold methods, exploiting highly specific antibodies against D-aspartate. PMB-TBOA blocked uptake of both low and high exogenous D-aspartate concentrations (0.01-1.0 mM) into nerve terminals, as well as the uptake of 0.01 mM D-aspartate into astrocytes. Interestingly, there was a residual PMB-TBOA insensitive uptake of D-aspartate in astrocytes at higher exogenous D-aspartate concentrations (0.05-1.0 mM), strongly suggesting that astrocytes have "low-affinity" uptake sites for excitatory amino acid. The PMB-TBOA insensitive D-aspartate uptake in astrocytes was sodium dependent and inhibited by succinate and to certain extent by homocysteate, but not by cystine or DIDS. We suggest that excitatory amino acid is transported into astrocytes in a "low-affinity" fashion by sodium/dicarboxylate transporters.

Previous studies have shown that a compound purified from the spider Parawixia bistriata venom stimulates the activity of glial glutamate transporters and can protect retinal tissue from ischemic damage. To understand the mechanism by which this compound enhances transport, we examined its effects on the functional properties of glutamate transporters after solubilization and reconstitution in liposomes and in transfected COS-7 cells. Here, we demonstrate in both systems that Parawixin1 promotes a direct and selective enhancement of glutamate influx by the EAAT2 transporter subtype through a mechanism that does not alter the apparent affinities for the cosubstrates glutamate or sodium. In liposomes, we observed maximal enhancement by Parawixin1 when extracellular sodium and intracellular potassium concentrations are within physiological ranges. Moreover, the compound does not enhance the reverse transport of glutamate under ionic conditions that favor efflux, when extracellular potassium is elevated and the sodium gradient is reduced, nor does it alter the exchange of glutamate in the absence of internal potassium. These observations suggest that Parawixin1 facilitates the reorientation of the potassium-bound transporter, the rate-limiting step in the transport cycle, a conclusion further supported by experiments showing that Parawixin1 does not stimulate uptake by an EAAT2 transport mutant (E405D) defective in the potassium-dependent reorientation step. Thus, Parawixin1 enhances transport through a novel mechanism targeting a step in the transport cycle distinct from substrate influx or efflux and provides a basis for the design of new drugs that act allosterically on transporters to increase glutamate clearance.

Temporal lobe epilepsy (TLE) with hippocampal sclerosis is associated with high extracellular glutamate levels, which could trigger seizures. Down-regulation of glial glutamate transporters GLAST (EAAT1) and GLT-1 (EAAT2) in sclerotic hippocampi may account for such increases. Their distribution was compared immunohistochemically in non-sclerotic and sclerotic hippocampi and localized only in astrocytes, with weaker immunoreactivity for both transporters in areas associated with pronounced neuronal loss, especially in CA1, but no decrease or even an increase in areas with less neuronal loss, like CA2 and the subiculum in the sclerotic group. Such compensatory changes in immunoreactivity may account for the lack of differences between the groups in immunoblot studies as blots show the average concentrations in the samples. These data suggest that differences in glial glutamate transporter distribution between the two groups of hippocampi may be an insufficient explanation for the high levels of extracellular glutamate in sclerotic seizure foci observed through in vivo dialysis studies.

Antibodies have been in widespread use for more than three decades as invaluable tools for the specific detection of proteins or other molecules in biological samples. In spite of such a long experience, the field of immunocytochemistry is still troubled by spurious results due to insufficient specificity of antibodies or procedures used. The importance of keeping a high standard is increasing because massive sequencing of entire genomes leads to the identification of numerous new proteins. All the identified proteins and their variants will have to be localized precisely and quantitatively at high resolution throughout the development of all species. Consequently, antibody generation and immunocytochemical investigations will be done on a large scale. It will be economically important to secure an optimal balance between the risk of publishing erroneous data (which are expensive to correct) and the costs of specificity testing. Because proofs of specificity are never absolute, but rather represent failures to detect crossreactivity, there is no limit to the number of control experiments that can be performed. The aims of the present paper are to increase the awareness of the difficulties in proving the specificity of immunocytochemical labeling and to initiate a discussion on optimized standards. The main points are: (1) antibodies should be described properly, (2) the labeling obtained with an antibody to a single epitope needs additional verification and (3) the investigators should be required to outline in detail how they arrive at the conclusion that the immunocytochemical labeling is specific.