Glutamate transporters: Critical components of glutamatergic transmission.

Glutamate is the major excitatory neurotransmitter in the vertebrate central nervous system. Once released, it binds to specific membrane receptors and transporters activating a wide variety of signal transduction cascades, as well as its removal from the synaptic cleft in order to avoid its extracellular accumulation and the overstimulation of extra-synaptic receptors that might result in neuronal death through a process known as excitotoxicity. Although neurodegenerative diseases are heterogenous in clinical phenotypes and genetic etiologies, a fundamental mechanism involved in neuronal degeneration is excitotoxicity. Glutamate homeostasis is critical for brain physiology and Glutamate transporters are key players in maintaining low extracellular Glutamate levels. Therefore, the characterization of Glutamate transporters has been an active area of glutamatergic research for the last 40 years. Transporter activity its regulated at different levels: transcriptional and translational control, transporter protein trafficking and membrane mobility, and through extensive post-translational modifications. The elucidation of these mechanisms has emerged as an important piece to shape our current understanding of glutamate actions in the nervous system.

GLAST Activity is Modified by Acute Manganese Exposure in Bergmann Glial Cells.

Glutamate is the major excitatory amino acid neurotransmitter in the vertebrate brain. It exerts its actions through the activation of specific plasma membrane receptors expressed in neurons and glial cells. Overactivation of glutamate receptors results in neuronal death, known as excitotoxicity. A family of sodium-dependent glutamate transporters enriched in glial cells are responsible of the vast majority of the removal of this amino acid form the synaptic cleft. Therefore, a precise and exquisite regulation of these proteins is required not only for a proper glutamatergic transmission but also for the prevention of an excitotoxic insult. Manganese is a trace element essential as a cofactor for several enzymatic systems, although in high concentrations is involved in the disruption of brain glutamate homeostasis. The molecular mechanisms associated to manganese neurotoxicity have been focused on mitochondrial function, although energy depletion severely compromises the glutamate uptake process. In this context, in this contribution we analyze the effect of manganese exposure in glial glutamate transporters function. To this end, we used the well-established model of chick cerebellar Bergmann glia cultures. A time and dose dependent modulation of [3H]-D-aspartate uptake was found. An increase in the transporter catalytic efficiency, most probably linked to a discrete increase in the affinity of the transporter was detected upon manganese exposure. Interestingly, glucose uptake was reduced by this metal. These results favor the notion of a direct effect of manganese on glial cells, this in turn alters their coupling with neurons and might lead to changes in glutamatergic transmission.

Thrombin induces Ca2+-dependent glutamate release from RPE cells mediated by PLC/PKC and reverse Na+/Ca2+ exchange.

Purpose: We analyzed the molecular mechanisms leading to glutamate release from rat primary cultures of RPE cells, under isosmotic conditions. Thrombin has been shown to stimulate glutamate release from astrocytes and retinal glia; however, the effect of thrombin on glutamate release from RPE cells has not been examined. Our previous work showed that upon the alteration of the blood-retina barrier, the serine protease thrombin could contribute to the transformation, proliferation, and migration of RPE cells. In this condition, elevated extracellular glutamate causes neuronal loss in many retinal disorders, including glaucoma, ischemia, diabetic retinopathy, and inherited photoreceptor degeneration. Methods: Primary cultures of rat RPE cells were preloaded with 1 µCi/ml 3H-glutamate in Krebs Ringer Bicarbonate (KRB) buffer for 30 min at 37 °C. Cells were rinsed and super-perfused with 1 ml/min KRB for 15 min. Stable release was reached at the 7th minute, and on the 8th minute, fresh KRB containing stimuli was added. Results: This study showed for the first time that thrombin promotes specific, dose-dependent glutamate release from RPE cells, induced by the activation of protease-activated receptor 1 (PAR-1). This effect was found to depend on the Ca2+ increase mediated by the phospholipase C-ß (PLC-ß) and protein kinase C (PKC) pathways, as well as by the reverse activity of the Na+/Ca2+ exchanger. Conclusions: Given the intimate contact of the RPE with the photoreceptor outer segments, diffusion of RPE-released glutamate could contribute to the excitotoxic death of retinal neurons, and the development of thrombin-induced eye pathologies.

Agathisflavone, a flavonoid derived from Poincianella pyramidalis (Tul.), enhances neuronal population and protects against glutamate excitotoxicity.

Flavonoids are bioactive compounds that are known to be neuroprotective against glutamate-mediated excitotoxicity, one of the major causes of neurodegeneration. The mechanisms underlying these effects are unresolved, but recent evidence indicates flavonoids may modulate estrogen signaling, which can delay the onset and ameliorate the severity of neurodegenerative disorders. Furthermore, the roles played by glial cells in the neuroprotective effects of flavonoids are poorly understood. The aim of this study was to investigate the effects of the flavonoid agathisflavone (FAB) in primary neuron-glial co-cultures from postnatal rat cerebral cortex. Compared to controls, treatment with FAB significantly increased the number of neuronal progenitors and mature neurons, without increasing astrocytes or microglia. These pro-neuronal effects of FAB were suppressed by antagonists of estrogen receptors (ERα and ERß). In addition, treatment with FAB significantly reduced cell death induced by glutamate and this was associated with reduced expression levels of pro-inflammatory (M1) microglial cytokines, including TNFα, IL1ß and IL6, which are associated with neurotoxicity, and increased expression of IL10 and Arginase 1, which are associated with anti-inflammatory (M2) neuroprotective microglia. We also observed that FAB increased neuroprotective trophic factors, such as BDNF, NGF, NT4 and GDNF. The neuroprotective effects of FAB were also associated with increased expression of glutamate regulatory proteins in astrocytes, namely glutamine synthetase (GS) and Excitatory Amino Acid Transporter 1 (EAAT1). These findings indicate that FAB acting via estrogen signaling stimulates production of neurons in vitro and enhances the neuroprotective properties of microglia and astrocytes to significantly ameliorate glutamate-mediated neurotoxicity.

Cortical Bilateral Adaptations in Rats Submitted to Focal Cerebral Ischemia: Emphasis on Glial Metabolism.

This study was performed to evaluate the bilateral effects of focal permanent ischemia (FPI) on glial metabolism in the cerebral cortex. Two and 9 days after FPI induction, we analyze [18F]FDG metabolism by micro-PET, astrocyte morphology and reactivity by immunohistochemistry, cytokines and trophic factors by ELISA, glutamate transporters by RT-PCR, monocarboxylate transporters (MCTs) by western blot, and substrate uptake and oxidation by ex vivo slices model. The FPI was induced surgically by thermocoagulation of the blood in the pial vessels of the motor and sensorimotor cortices in adult (90 days old) male Wistar rats. Neurochemical analyses were performed separately on both ipsilateral and contralateral cortical hemispheres. In both cortical hemispheres, we observed an increase in tumor necrosis factor alpha (TNF-α), interleukin-1ß (IL-1ß), and glutamate transporter 1 (GLT-1) mRNA levels; lactate oxidation; and glutamate uptake and a decrease in brain-derived neurotrophic factor (BDNF) after 2 days of FPI. Nine days after FPI, we observed an increase in TNF-α levels and a decrease in BDNF, GLT-1, and glutamate aspartate transporter (GLAST) mRNA levels in both hemispheres. Additionally, most of the unilateral alterations were found only in the ipsilateral hemisphere and persisted until 9 days post-FPI. They include diminished in vivo glucose uptake and GLAST expression, followed by increased glial fibrillary acidic protein (GFAP) gray values, astrocyte reactivity, and glutamate oxidation. Astrocytes presented signs of long-lasting reactivity, showing a radial morphology. In the intact hemisphere, there was a decrease in MCT2 levels, which did not persist. Our study shows the bilateralism of glial modifications following FPI, highlighting the role of energy metabolism adaptations on brain recovery post-ischemia.

7-Fluoro-1,3-diphenylisoquinoline-1-amine reverses the reduction in self-care behavior induced by maternal separation stress in rats by modulating glutamatergic/GABAergic systems.

7-Fluoro-1,3-diphenylisoquinoline-1-amine (FDPI) is a promising isoquinoline that elicits an antidepressant-like action in rodents. In this study, an animal model of stress induced by maternal separation was used to investigate the effects of FDPI in Wistar rats of 30 and 90 days of age. It was investigated the effects of maternal separation in the self-care behavior and the contribution of glutamatergic and gamma-aminobutyric acid (GABA)ergic systems in the FDPI action. Male Wistar rats were separated from their mothers for 3 h/day from postnatal day (PND) 1-10. The rats were treated at different ages (PND-30 and PND-90) with FDPI (5 mg/kg, intragastrically/7 days) and performed the splash test. Maternal separation reduced total grooming time in the splash test, an index of motivational and self-care behavior, and FDPI treatment was effective in reversing this behavior in rats at both ages. The neurochemical parameters were differently affected, dependent on the age of rats, by maternal separation and FDPI. Maternal separation increased the GABA uptake and the excitatory amino acid transporter 1 levels in the prefrontal cortices of rats at PND-30 and FDPI was effective against these alterations. At PND-90, maternal separation decreased the glutamate uptake and increased the GABA uptake and the N-methyl-D-aspartate (NMDA) receptor 2B levels in the prefrontal cortices of rats. FDPI reversed the neurochemical alterations caused by maternal separation in the prefrontal cortices of rats at PND-90. The results of this study demonstrated that FDPI reversed the reduction in self-care behavior induced by maternal separation stress in rats by modulating the glutamatergic/GABAergic systems in rats.

Reliable Genetic Labeling of Adult-Born Dentate Granule Cells Using Ascl1 CreERT2 and Glast CreERT2 Murine Lines.

Newly generated dentate granule cells (GCs) are relevant for input discrimination in the adult hippocampus. Yet, their precise contribution to information processing remains unclear. To address this question, it is essential to develop approaches to precisely label entire cohorts of adult-born GCs. In this work, we used genetically modified mice to allow conditional expression of tdTomato (Tom) in adult-born GCs and characterized their development and functional integration. Ascl1(CreERT2);CAG(floxStopTom) and Glast(CreERT2);CAG(floxStopTom) mice resulted in indelible expression of Tom in adult neural stem cells and their lineage upon tamoxifen induction. Whole-cell recordings were performed to measure intrinsic excitability, firing behavior, and afferent excitatory connectivity. Developing GCs were also staged by the expression of early and late neuronal markers. The slow development of adult-born GCs characterized here is consistent with previous reports using retroviral approaches that have revealed that a mature phenotype is typically achieved after 6-8 weeks. Our findings demonstrate that Ascl1(CreERT2) and Glast(CreERT2) mouse lines enable simple and reliable labeling of adult-born GC lineages within restricted time windows. Therefore, these mice greatly facilitate tagging new neurons and manipulating their activity, required for understanding adult neurogenesis in the context of network remodeling, learning, and behavior. SIGNIFICANCE STATEMENT: Our study shows that Ascl1(CreERT2) and Glast(CreERT2) mice lines can be used to label large cohorts of adult-born dentate granule cells with excellent time resolution. Neurons labeled in this manner display developmental and functional profiles that are in full agreement with previous findings using thymidine analogs and retroviral labeling, thus providing an alternative approach to tackle fundamental questions on circuit remodeling. Because of the massive neuronal targeting and the simplicity of this method, genetic labeling will contribute to expand research on adult neurogenesis.

Methylphenidate Increases Glutamate Uptake in Bergmann Glial Cells.

Glutamate, the main excitatory transmitter in the vertebrate brain, exerts its actions through the activation of specific membrane receptors present in neurons and glial cells. Over-stimulation of glutamate receptors results in neuronal death, phenomena known as excitotoxicity. A family of glutamate uptake systems, mainly expressed in glial cells, removes the amino acid from the synaptic cleft preventing an excessive glutamatergic stimulation and thus neuronal damage. Autism spectrum disorders comprise a group of syndromes characterized by impaired social interactions and anxiety. One or the most common drugs prescribed to treat these disorders is Methylphenidate, known to increase dopamine extracellular levels, although it is not clear if its sedative effects are related to a plausible regulation of the glutamatergic tone via the regulation of the glial glutamate uptake systems. To gain insight into this possibility, we used the well-established model system of cultured chick cerebellum Bergmann glia cells. A time and dose-dependent increase in the activity and protein levels of glutamate transporters was detected upon Methylphenidate exposure. Interestingly, this increase is the result of an augmentation of both the synthesis as well as the insertion of these protein complexes in the plasma membrane. These results favour the notion that glial cells are Methylphenidate targets, and that by these means could regulate dopamine turnover.

Nandrolone-induced aggressive behavior is associated with alterations in extracellular glutamate homeostasis in mice.

Nandrolone decanoate (ND), an anabolic androgenic steroid (AAS), induces an aggressive phenotype by mechanisms involving glutamate-induced N-methyl-d-aspartate receptor (NMDAr) hyperexcitability. The astrocytic glutamate transporters remove excessive glutamate surrounding the synapse. However, the impact of supraphysiological doses of ND on glutamate transporters activity remains elusive. We investigated whether ND-induced aggressive behavior is interconnected with GLT-1 activity, glutamate levels and abnormal NMDAr responses. Two-month-old untreated male mice (CF1, n=20) were tested for baseline aggressive behavior in the resident-intruder test. Another group of mice (n=188) was injected with ND (15mg/kg) or vehicle for 4, 11 and 19days (short-, mid- and long-term endpoints, respectively) and was evaluated in the resident-intruder test. Each endpoint was assessed for GLT-1 expression and glutamate uptake activity in the frontoparietal cortex and hippocampal tissues. Only the long-term ND endpoint significantly decreased the latency to first attack and increased the number of attacks, which was associated with decreased GLT-1 expression and glutamate uptake activity in both brain areas. These alterations may affect extracellular glutamate levels and receptor excitability. Resident males were assessed for hippocampal glutamate levels via microdialysis both prior to, and following, the introduction of intruders. Long-term ND mice displayed significant increases in the microdialysate glutamate levels only after exposure to intruders. A single intraperitoneal dose of the NMDAr antagonists, memantine or MK-801, shortly before the intruder test decreased aggressive behavior. In summary, long-term ND-induced aggressive behavior is associated with decreased extracellular glutamate clearance and NMDAr hyperexcitability, emphasizing the role of this receptor in mediating aggression mechanisms.

An acute glutamate exposure induces long-term down regulation of GLAST/EAAT1 uptake activity in cultured Bergmann glia cells.

Glutamate, the major excitatory neurotransmitter in the vertebrate brain, is a potent neurotoxin therefore its extracellular levels have to be tightly regulated by means of sodium-dependent glutamate uptake systems of the slc1A family. The glial glutamate/aspartate transporter (GLAST/EAAT1) and the glutamate transporter 1 carry most of the uptake activity in cerebellum and in the forebrain, respectively. In the cerebellar cortex, GLAST is profusely expressed in Bergmann glia cells, which completely enwrap the parallel fiber-Purkinje cells synapses. Glutamate exposure in these cells, down regulates the activity as well as the expression levels of this transporter. In order to characterize the persistence of a single glutamate exposure, we followed the [(3)H]-D-aspartate uptake activity as a function of time after the removal of the glutamatergic stimulus. We were able to demonstrate that a single 30 min exposure to glutamate reduces the uptake activity for up to 3 h. This effect is dose-dependent and it is not reproduced neither by ionotropic nor metabotropic glutamate receptors agonists. In contrast, transporter specific ligands such as D-aspartate or L-(-)-threo-3-Hydroxyaspartic acid fully reproduce the glutamate effect. Equilibrium binding experiments revealed a decrease in [(3)H]-D-aspartate Bmax without a significant change in affinity, clearly suggesting that a reduction in the availability of plasma membrane glutamate transporters is the molecular basis of this effect. Interestingly, neither Glast mRNA nor its protein levels were significantly reduced upon the single glutamate exposure. Taken together, these results favor the notion of a transporter-mediated tight control of the uptake process.

Congenital hypothyroidism alters the oxidative status, enzyme activities and morphological parameters in the hippocampus of developing rats.

Congenital hypothyroidism is associated with delay in cell migration and proliferation in brain tissue, impairment of synapse formation, misregulation of neurotransmitters, hypomyelination and mental retardation. However, the mechanisms underlying the neuropsychological deficits observed in congenital hypothyroidism are not completely understood. In the present study we proposed a mechanism by which hypothyroidism leads to hippocampal neurotoxicity. Congenital hypothyroidism induces c-Jun-N-terminal kinase (JNK) pathway activation leading to hyperphosphorylation of the glial fibrillary acidic protein (GFAP), vimentin and neurofilament subunits from hippocampal astrocytes and neurons, respectively. Moreover, hyperphosphorylation of the cytoskeletal proteins was not reversed by T3 and poorly reversed by T4. In addition, congenital hypothyroidism is associated with downregulation of astrocyte glutamate transporters (GLAST and GLT-1) leading to decreased glutamate uptake and subsequent influx of Ca(2+) through N-methyl-D-aspartate (NMDA) receptors. The Na(+)-coupled (14)C-α-methyl-amino-isobutyric acid ((14)C-MeAIB) accumulation into hippocampal cells also might cause an increase in the intracellular Ca(2+) concentration by opening voltage-dependent calcium channels (VDCC). The excessive influx of Ca(2+) through NMDA receptors and VDCCs might lead to an overload of Ca(2+) within the cells, which set off glutamate excitotoxicity and oxidative stress. The inhibited acetylcholinesterase (AChE) activity might also induce Ca(2+) influx. The inhibited glucose-6-phosphate dehydrogenase (G6PD) and gamma-glutamyl transferase (GGT) activities, associated with altered glutamate and neutral amino acids uptake could somehow affect the GSH turnover, the antioxidant defense system, as well as the glutamate-glutamine cycle. Reduced levels of S100B and glial fibrillary acidic protein (GFAP) take part of the hypothyroid condition, suggesting a compromised astroglial/neuronal neurometabolic coupling which is probably related to the neurotoxic damage in hypothyroid brain.

Radial glia-like cells persist in the adult rat brain.

During development, radial glia cells contribute to neuronal migration and neurogenesis, and differentiate into astrocytes by the end of the developmental period. Recently, it was demonstrated that during development, radial glia cells, in addition to their role in migration, also give rise to neuroblasts. Furthermore, radial glial cells remain in the adult brain as adult neural stem cells (NSC) in the subventricular zone (SVZ) around the lateral ventricles (LVs), and generate new neurons continuously throughout adulthood. In this study, we used immunohistochemical and morphological methods to investigate the presence of radial glia-like cells around the LVs during the postnatal development period until adulthood in rats. In all ages of rats studied, we identified cells with morphological and immunocytochemical features that are similar to the radial glia cells found in the embryonic brain. Similarly to the radial glia, these cells express nestin and vimentin, and have a radial morphology, extending perpendicularly as processes from the ventricle wall. These cells also express GFAP, GLAST, and Pax6, and proliferate. In the brains of adult rats, we identified cells with relatively long processes (up to 600 mum) in close apposition with migrating neuroblasts. Our results showed that the radial glia-like cells present in the adult rat brain share several morphological and functional characteristics with the embryonic radial glia. We suggest that the embryonic radial glia cells located around the LV walls do not complete their transformation into astrocytes, but rather persist in adulthood.

Insulin-dependent regulation of GLAST/EAAT1 in Bergmann glial cells.

Glutamate is the major excitatory neurotransmitter in the central nervous system. Ionotropic and metabotropic glutamate receptors are present in neurons and glial cells and are involved in gene expression regulation. A family of sodium-dependent glutamate transporters carries out the removal of the neurotransmitter from the synaptic cleft. In the cerebellum, the bulk of glutamate transport is mediated through the excitatory amino acids transporter 1 (EAAT1/GLAST) expressed in Bergmann glial cells. Proper transporter function is critical for glutamate cycling and glucose turnover, as well as prevention of excitotoxic insult to Purkinje cells. In order to gain insight into the regulatory signals that modify this uptake activity, we investigated the effects of insulin exposure. Using the well-defined chick cerebellar Bergmann glial cell culture model, we observed a time and dose-dependent decrease in [(3)H]-d-aspartate uptake. As expected, this effect is mimicked by the tyrosine phosphatase inhibitor sodium orthovanadate, suggesting a receptor-mediated effect. Equilibrium [(3)H]-d-aspartate binding experiments as well as a reverse transcriptase/polymerase chain reaction strategy demonstrated that the decrease in the uptake activity is related to reduced numbers of transporter molecules in the plasma membrane. Accordingly, the transcriptional activity of the chick glast promoter diminished upon insulin treatment. The present findings suggest the involvement of insulin in neuronal/glial coupling in the cerebellum.

Effects of chronic restraint stress and estradiol replacement on glutamate release and uptake in the spinal cord from ovariectomized female rats.

Glutamate is an excitatory neurotransmitter involved in neuronal plasticity and neurotoxicity. Chronic stress produces several physiological changes on the spinal cord, many of them presenting sex-specific differences, which probably involve glutamatergic system alterations. The aim of the present study was to verify possible effects of exposure to chronic restraint stress and 17beta-estradiol replacement on [3H]-glutamate release and uptake in spinal cord synaptosomes of ovariectomized (OVX) rats. Female rats were subjected to OVX, and half of the animals received estradiol replacement. Animals were subdivided in controls and chronically stressed. Restraint stress or estradiol had no effect on [3H]-glutamate release. The chronic restraint stress promoted a decrease and 17beta-estradiol induced an increase on [3H]-glutamate uptake, but the uptake observed in the restraint stress +17beta-estradiol group was similar to control. Furthermore, 17beta-estradiol treatment caused a significant increase in the immunocontent of the three glutamate transporters present in spinal cord. Restraint stress had no effect on the expression of these transporters, but prevented the 17beta-estradiol effect. We suggest that changes in the glutamatergic system are likely to take part in the mechanisms involved in spinal cord plasticity following repeated stress exposure, and that 17beta-estradiol levels may affect chronic stress effects in this structure.

Evidence that glutaric acid reduces glutamate uptake by cerebral cortex of infant rats.

The role of excitotoxicity in the cerebral damage of glutaryl-CoA dehydrogenase deficiency (GDD) is under intense debate. We therefore investigated the in vitro effect of glutaric (GA) and 3-hydroxyglutaric (3-OHGA) acids, which accumulate in GDD, on [(3)H]glutamate uptake by slices and synaptosomal preparations from cerebral cortex and striatum of rats aged 7, 15 and 30 days. Glutamate uptake was significantly decreased by high concentrations of GA in cortical slices of 7-day-old rats, but not in cerebral cortex from 15- and 30-day-old rats and in striatum from all studied ages. Furthermore, this effect was not due to cellular death and was prevented by N-acetylcysteine preadministration, suggesting the involvement of oxidative damage. In contrast, glutamate uptake by brain slices was not affected by 3-OHGA exposure. Immunoblot analysis revealed that GLAST transporters were more abundant in the cerebral cortex compared to the striatum of 7-day-old rats. Moreover, the simultaneous addition of GA and dihydrokainate (DHK), a specific inhibitor of GLT1, resulted in a significantly higher inhibition of [(3)H]glutamate uptake by cortical slices of 7-day-old rats than that induced by the sole presence of DHK. We also observed that both GA and 3-OHGA exposure did not alter the incorporation of glutamate into synaptosomal preparations from cerebral cortex and striatum of rats aged 7, 15 and 30 days. Finally, GA in vivo administration did not alter glutamate uptake into cortical slices from 7-day-old rats. Our findings may explain at least in part why cortical neurons are more vulnerable to damage at birth as evidenced by the frontotemporal cortical atrophy observed in newborns affected by GDD.

The effects of estradiol on estrogen receptor and glutamate transporter expression in organotypic hippocampal cultures exposed to oxygen--glucose deprivation.

The molecular basis of estrogen-mediated neuroprotection against brain ischemia remains unclear. In the present study, we investigated changes in expression of estrogen receptors (ERs) alpha and beta and excitatory amino acid transporters (EAAT) 1 and 2 in rat organotypic hippocampal slice cultures treated with estradiol and subsequently exposed to oxygen--glucose deprivation (OGD). Pretreatment with 17beta-estradiol (10 nM) for 7 days protected the CA1 area of hippocampus against OGD (60 min), reducing cellular injury by 46% compared to the vehicle control group. Levels of ERalpha protein were significantly reduced by 20% after OGD in both vehicle- and estradiol-treated cultures, whereas ERbeta was significantly up-regulated by 25% in the estradiol-treated cultures. In contrast, EAAT1 and EAAT2 levels were unchanged in response to estradiol treatment in this model of OGD. These findings suggest that estrogen-induced neuroprotection against ischemia might involve regulation of ERbeta and, consequently, of the genes influenced by this receptor.