Abnormal expressions of glutamate transporters and metabotropic glutamate receptor 1 in the spontaneously epileptic rat hippocampus

Excessive glutamatergic neurotransmission is considered an underlying factor of epilepsy. The modulation of the synaptic activity occurs both by the removal of glutamate from the synaptic cleft and by excitatory amino acid transporters (EAATs) and by modulation of glutamate receptors.The spontaneously epileptic rat (SER), a double mutant (zi/zi, tm/tm), exhibits both tonic convulsions and absence-like seizures from the age of 8 weeks. However, there are no reports that can elucidate the effects of EAATs and metabotropic glutamate receptors (mGluRs) in SER. The present study was undertaken to detect EAATs (GLAST, GLT-1 and EAAC-1) and Group I metabotropic glutamate receptors (mGluR1) in SER hippocampus from both the level of mRNA and protein in SERs hippocampus compared with control Wistar rats. In this study, the glutamate concentration in SERs hippocampus was increased compared with that of control rats by high performance liquid chromatography; the mRNA expressions of GLAST and mGluR1 in SERs hippocampus were significantly lower than those in control rats hippocampus, whereas an abundant increase in mRNA for GLT-1 was observed by RT-PCR; EAAC-1 and mGluR1 protein in SERs and control rats were localized widely in the hippocampus including CA1, CA3 and dentate gyrus regions by immunohistochemistry; the number of GLAST and mGluR1-positive cells in the hippocampus of SERs were less than those in control rats, especially for CA3 and DG region; the protein expression of GLT-1 was up-regulated, but the protein expressions of GLAST and mGluR1 were down-regulated in SER hippocampus by western blot. Our data show that epileptogenesis in SER are associated with regulations of glutamate transporters and mGluR1, which might be potential targets for therapy in genetic epilepsy.     

Sequential changes in glutamate transporter protein levels during Fe(3+)-induced epileptogenesis

Severe head injury in humans causes recurrent seizures; this form of epilepsy appears to correlate with occurrence of parenchymal hemorrhage. Injection of ferric cations, one component of hemoglobin, into rat amygdala, causes lipid peroxidation, and recurrent spontaneous seizures. We wondered whether regulation of extracellular glutamate might be perturbed as a mechanism of chronic epileptogenesis, therefore levels of glutamate transporter proteins GLT-1, GLAST and EAAC-1 were measured in ipsilateral and contralateral hippocampi removed from rats having spontaneous iron-induced limbic seizures. The neuronal transporter EAAC-1 was elevated bilaterally up to 30 days following the microinjection that initiated seizures. The neuronal transporter EAAC-1 was elevated bilaterally up to 30 days following the microinjection that initiated seizures. The glial transporter GLT-1 increased 5 and 15 days after iron injection on the side contralateral to the injection then returned to basal levels 30 days after the lesion. GLAST also showed an initial increase but at 15 and 30 days after injection, when experimental animals were experiencing spontaneous limbic behavioral seizures, this protein was down-regulated. The results suggest that iron-induced epileptogenesis involves alteration in glial glutamate transport that may lead to enhanced excitation within the hippocampus.     

Region- and age-specific changes in glutamate transport in the AβPP23 mouse model for Alzheimer's disease

Using 8- and 18-month-old AβPP23 mice, we investigated the involvement of high-affinity glutamate transporters (GLAST, GLT-1, EAAC1), vesicular glutamate transporters (VGLUT1-3) and xCT, the specific subunit of system x(c)?, in Alzheimer's disease (AD) pathogenesis. Transporter expression was studied in cortical and hippocampal tissue and linked to extracellular glutamate and glutamate reuptake activity as measured using in vivo microdialysis. In 8-month-old animals, we could not observe plaque formation or gliosis. Yet, in hippocampus as well as cortex GLAST and GLT-1 expression was decreased. Whereas in cortex this was accompanied by upregulated VGLUT1 expression, extracellular glutamate concentrations were decreased. Surprisingly, inhibiting glutamate reuptake with TBOA revealed increased glutamate reuptake activity in cortex of AβPP23 mice, despite decreased GLAST and GLT-1 expression, and resulted in status epilepticus in all AβPP23 mice, contrary to wildtype littermates. In hippocampus of 8-month-old AβPP23 mice, we observed increased EAAC1 expression besides the decrease in GLAST and GLT-1. Yet, glutamate reuptake activity was drastically decreased according to the decreased GLAST and GLT-1 expression. In 18-month-old AβPP23 mice, plaque formation and gliosis in cortex and hippocampus were accompanied by decreased GLT-1 expression. We also showed, for the first time, increased cortical expression of VGLUT3 and xCT together with a strong tendency towards increased cortical extracellular glutamate levels. VGLUT2 expression remained unaltered in all conditions. The present findings support the hypothesis that alterations in transport of glutamate, and more particular via GLT-1, may be involved in AD pathogenesis.     

胶质细胞谷氨酸转运体在大鼠点燃效应中的作用研究

目的 研究点燃形成过程中和点燃后大鼠海马中氨酸天门氨酸转运体（GLAST）和谷氨酸转运体1（GLT－1）的变化,进一步探讨癫痫的形成机制。方法 将78只雄性成年Wistar大鼠随机分为对照组（I组）和戊四氮（PTZ）组（Ⅱ组）。Ⅱ组腹腔注射阈下剂量的PTZ（335mg/kg）,每日1次,直到达到点燃标准;I组腹腔注射等量生理盐水。采有RT－PCR方法检测海马区GLAST和GLT－1mRNA的表达。结果 PTZ组点燃后,GLASTmRNA的表达下降,60天时恢复至对照组;与对照组比较,PTZ组GLT－1mRNA的表达,在给药后15天时开始上升,点燃后0小时和48小时时显著升高,此后呈下降趋势。60天时,两组比较无明显差异。结论 海马区胶质细胞谷氨酸转运体的下降可能与癫痫敏感性的形成有关。     

Transient striatal GLT-1 blockade increases EAAC1 expression, glutamate reuptake, and decreases tyrosine hydroxylase phosphorylation at ser(19)

Three glutamate transporters, GLT-1, GLAST, and EAAC1, are expressed in striatum. GLT-1 and, to a lesser extent, GLAST are thought to play a primary role in glutamate reuptake and mitigate excitoxicity. Progressive tyrosine hydroxylase (TH) loss seen in Parkinson's disease (PD) is associated with increased extracellular glutamate. Glutamate receptor antagonists reduce nigrostriatal loss in PD models. These observations suggest that excess synaptic glutamate contributes to nigrostriatal neuron loss seen in PD. Decreased GLT-1 expression occurs in neurodegenerative disease and PD models, suggesting decreased GLT-1-mediated glutamate reuptake contributes to excitotoxicity. To determine how transient GLT-1 blockade affects glutamate reuptake dynamics and a Ca(2+)-dependent process in nigrostriatal terminals, ser(19) phosphorylation of TH, the GLT-1 inhibitor dihydrokainic acid (DHK) was delivered unilaterally to striatum in vivo and glutamate reuptake was quantified ex vivo in crude synaptosomes 3h later. Ca(2+)-influx is associated with excitotoxic conditions. The phosphorylation of TH at ser(19) is Ca(2+)-dependent, and a change resulting from GLT-1 blockade may signify the potential for excitotoxicity to nigrostriatal neurons. Synaptosomes from DHK infused striatum had a 43% increase in glutamate reuptake in conjunction with decreased ser(19) TH phosphorylation. Using a novel GLAST inhibitor and DHK, we determined that the GLAST-mediated component of increased glutamate reuptake increased 3-fold with no change in GLAST or GLT-1 protein expression. However, GLT-1 blockade increased EAAC1 protein expression ~20%. Taken together, these results suggest that GLT-1 blockade produces a transient increase in GLAST-mediated reuptake and EAAC1 expression coupled with reduced ser(19) TH phosphorylation. These responses could represent an endogenous defense against excitotoxicity to the nigrostriatal pathway.     

GLT-1 glutamate transporter levels are unchanged in mice expressing G93A human mutant SOD1.

A decrease in expression of the glutamate transporter GLT-1 is thought to be responsible for the increase in extracellular glutamate observed in patients with amyotrophic lateral sclerosis (ALS) and in a transgenic mouse model of ALS. We examined protein levels of the glutamate transporters GLT-1, GLAST and EAAC1 in the G93A (SOD1) transgenic mouse model of ALS. GLT-1 was detected in two bands (72 and 150 kD). Semi-quantitative analysis of Western blots showed that GLT-1 levels in sensorimotor cortex, brain stem, and cervical and lumbar spinal cord of G93A mice did not differ significantly from controls, either at end stage or at 60- or 90-days old. Nevertheless, other differences were found in GLT-1 at end stage. The percentage of total GLT-1 in the 150 kD band increased significantly (p<0.05) in the spinal cord and was elevated in the brain stem and cortex. Furthermore, brain stem and spinal cord GLT-1 from G93A mice showed retarded mobility on gels compared to controls (M(r) approximately equal to 77.3+/-2.3 and 164.3+/-3.1 vs. 72.2+/-2.4 and 153.6+/-4.7, respectively). GLAST and EAAC1 were unchanged in both amount and mobility. These results show that a loss of GLT-1 protein is not necessary for ALS-like neurodegeneration in G93A mice. However, the changes in GLT-1 mobility and distribution indicate that GLT-1 is altered in mice with the SOD1 mutation.     

Molecular regulation of glutamate and GABA transporter proteins by clobazam during epileptogenesis in Fe(+++)-induced epileptic rats

To assess the molecular effects of the antiepileptic drug clobazam (CLB, 1,5-benzodiazepine), a benzodiazepine effective in the management of epilepsy, we performed a series of experiments using rats with chronic, spontaneous recurrent seizures induced by amygdalar injection of FeCl(3). Experimental animals were treated for 14 days with CLB. We then measured the expression of glutamate and GABA transporter proteins and evaluated the changes that occurred in these proteins using both experimental and control animals. CLB treatment was associated with an increase in the production of GLT-1 in the contra-lateral hippocampus of animals receiving amygdalar FeCl(3) and CLB treatment. CLB treatment up-regulated the GABA transporter GAT3 in the contra-lateral hippocampus of animals with chronic, recurrent seizures. In contrast, CLB had no effect on the expression of EAAC1 and GAT1 in the hippocampus or the cortex in control animal groups. Chronic epileptogenesis may be associated with down-regulation of the production of glial excitatory amino acid transporters, GLAST and GLT-1, proteins that cause increase in the basal extracellular concentrations of glutamate. Elevated GABA transporter expression results in increased reverse transport of GABA to the extracellular space during periods of excitation. In addition to allosteric activation of GABA(A) receptors, this study suggests that CLB might exhibit its antiepileptic action by increasing GLT-1 expression and GAT3 in the hippocampus of rats with chronic seizures.     

Effect of zonisamide on molecular regulation of glutamate and GABA transporter proteins during epileptogenesis in rats with hippocampal seizures

Epileptiform discharges and behavioral seizures may be the consequences of excess excitation associated with the neurotransmitter glutamate, or from inadequate inhibitory effects associated with gamma-aminobutyric acid (GABA). Synaptic effects of these neurotransmitters are terminated by the action of transporter proteins that remove amino acids from the synaptic cleft. Excitation initiated by the synaptic release of glutamate is attenuated by the action of glial transporters glutamate-aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1), and the neuronal transporter excitatory amino-acid carrier-1 (EAAC-1). GABA is removed from synaptic regions by the action of the transporters proteins GABA transporter-1 (GAT-1) and GABA transporter-3 (GAT-3). In this experiment, albino rats with chronic, spontaneous recurrent seizures induced by the amygdalar injection of FeCl3 were treated for 14 days with zonisamide (ZNS) (40 mg/kg, i.p.). Control animals underwent saline injection into the same amygdalar regions. Treatment control for both groups of intracerebrally injected animals was i.p. injection of equal volumes of saline. Western blotting was used to measure the quantity of glutamate and GABA transporters in hippocampus and frontal cortex. ZNS caused increase in the quantity of EAAC-1 protein in hippocampus and cortex and down regulation of the GABA transporter GAT-1. These changes occurred in both experimental and ZNS treated control animals. These data show that the molecular effect of ZNS, with up-regulation of EAAC-1 and decreased production of GABA transporters, should result in increased tissue and synaptic concentrations of GABA. Although many antiepileptic drugs have effects on ion channels when measured in vitro our study suggests that additional mechanisms of action may be operant. Molecular effects on regulation of transporter proteins may aid in understanding epileptogenesis and inform investigators about future design and development of drugs to treat epilepsy.     

The density of EAAC1 (EAAT3) glutamate transporters expressed by neurons in the mammalian CNS

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.     

Early expression of glutamate transporter proteins in ramified microglia after controlled cortical impact injury in the rat

Traumatic brain injury is followed by increased extracellular glutamate concentration. Uptake of glutamate is mainly mediated by the glial glutamate transporters GLAST and GLT-1. Extent and distribution of GLAST and GLT-1 were studied in a rat model of controlled cortical impact injury (CCII). Western Blot analysis revealed lowest levels of GLAST and GLT-1 with a decrease by 40%-54% and 42%-49% between 24 and 72 h posttrauma. By 8 h after CCII, CSF glutamate levels were increased (10.5 microM vs. 2.56 microM in controls; P < 0.001), reaching maximum values by 48 h. A significant increase in de novo GLAST and GLT-1 expressing ramified microglia was observed within 4 h, reached a stable level by 48 h, and remained high up to 72 h after CCII. Furthermore, ramified microglia de novo expressed the neuronal glutamate transporter EAAC1 after CCII. Following CCII, GLAST/GLT-1 and GFAP coexpressing astrocytes were immediately reduced, reaching minimum levels within 8 h. This reduction of expression could be either due to protein downregulation or loss of astrocytes. At 72 h, a marked population of GLAST- and GLT-1-positive reactive astrocytes appeared. These results support the hypothesis that reduced astrocytic GLAST and GLT-1 protein levels following CCII contribute to evolving secondary injury. Microglia are capable of de novo expressing glutamate transporter proteins, indicating that the expression of glial and neuronal glutamate transporters is not restricted to a specific glial or neuronal lineage. Ramified microglia may play an important compensatory role in the early regulation of extracellular glutamate after CCII.     

Sequential changes in glutamate transporter mRNA levels during Fe(3+)-induced epileptogenesis

Severe head injury in humans can cause recurrent seizures; this form of epilepsy appears to correlate with the occurrence of parenchymal hemorrhage. The injection of ferric cations, one component of hemoglobin, into rat amygdala, causes lipid peroxidation, and recurrent spontaneous seizures. We wondered whether the regulation of glutamate might be perturbed as a result of severe head injury, which might then act as a mechanism of chronic epileptogenesis. Levels of glutamate transporter glutamate-aspartate transporter (GLAST), glutamate transporter-1 (GLT-1), and excitatory amino-acid carrier (EAAC-1) mRNA were measured in ipsilateral and contralateral hippocampi and cerebral cortex removed from rats at 60 min, 24 h, and 5, 15 and 30 days after FeCl(3) injection into the amygdaloid body. While the neuronal transporter EAAC-1 mRNA was elevated bilaterally for up to 30 days following the microinjection that initiated seizures, GLT-1 mRNA, derived from glial cells, returned to basal levels. At 15 and 30 days after injection, however, when the experimental animals were experiencing spontaneous limbic behavioral seizures, GLAST mRNA was down-regulated. Epileptogenesis may correlate with the impairment of glial glutamate transport, leading to an excitation and imbalance of transmitter influences within the hippocampi and cerebral cortex.     

Requirement of appropriate glutamate concentrations in the synaptic cleft for hippocampal LTP induction

Although glutamate transporters maintain low extracellular levels of the excitatory neurotransmitter glutamate in the nervous system, little is known about their roles in synaptic plasticity. Here, using knockout mice lacking GLT-1, that is the most abundant glial subtype of glutamate transporters, we showed that long-term potentiation (LTP) induced by tetanic stimulation in mutant mice was impaired in the hippocampal CA1 region. When tetanic stimulation was applied in the presence of low concentrations of an N-methyl-D-aspartate (NMDA) receptor antagonist, the impairment was overcome. Consistent with these results, the increased glutamate in the synaptic cleft of mutant mice preferentially activated NMDA receptors. Furthermore, analyses of mutant mice revealed that the magnitude of NMDA receptor-dependent transient synaptic potentiation during low-frequency stimulation depended on the concentration of glutamate in the synaptic cleft. These findings suggest that GLT-1 plays critical roles in LTP induction, as well as in short-term potentiation, through regulation of extracellular levels of glutamate, which enables appropriate NMDA receptor activation.     

Altered expression of the glutamate transporter EAAC1 in neurons and immature oligodendrocytes after transient forebrain ischemia.

Glutamate uptake is reduced during ischemia because of perturbations of ionic gradients across neuronal and glial membranes. Using immunohistochemical and Western blot analyses, the authors examined the expression of the glutamate transporters EAAC1, GLAST, and GLT-1 in the rat hippocampus and cerebral cortex 8 hours and 1 to 28 days after transient forebrain ischemia. Densitometric analysis of immunoblots of CA1 homogenates showed a moderate increase in EAAC1 protein levels early after the insult. Consistently, it was observed that EAAC1 immunostaining in CA1 pyramidal neurons was more intense after 8 hours and 1 day of reperfusion and reduced at later postischemia stages. A similar transient increase of EAAC1 immunolabeling was detected in layer V pyramidal neurons of the cerebral cortex. In addition, the authors observed that EAAC1 also was located in oligodendroglial progenitor cells in subcortical white matter. The number of EAAC1-labeled cells in this region was increased after 3 and 28 days of reperfusion. Finally, changes in GLAST and GLT-1 expression were not observed in the CA1 region after ischemia using immunohistochemical study or immunoblotting. Enhanced expression of EAAC1 may be an adaptive response to increased levels of extracellular glutamate during ischemia.     

Expression and functional role of mGluR3 and mGluR5 in human astrocytes and glioma cells: opposite regulation of glutamate transporter proteins

We examined the regulation of glutamate transporter protein expression after stimulation with selective metabotropic glutamate receptor (mGluR) agonists in cultured human glial cells. mGluR3 and mGluR5 are expressed in human astrocytes and in human glioma cells in vivo as well as in vitro, as shown by either RT-PCR or western blot analysis. The selective group I agonist (S)-3,5-dihydroxyphenylglycine produced a significant down-regulation of both GLAST and GLT-1 protein expression in astrocytes cultured in the presence of growth factors. This condition mimics the morphology of reactive glial cells in vivo including an increased expression of mGluR5 protein (observed in pathological conditions). In contrast, (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine, a selective agonist of group II metabotropic glutamate receptors, positively modulates the expression of GLAST and GLT-1 proteins. A similar opposite effect of (S)-3,5-dihydroxyphenylglycine and (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine was observed for the expression of EAAT3 protein in U373 glioblastoma cell line. Selective group I and II antagonists prevented these effects. Pharmacological inhibition of mitogen-activated protein kinase and phosphatidylinositol-3-K pathways reduces the induction of GLT-1 observed in response to the group II metabotropic glutamate receptor agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine. Thus, mGluR3 and mGluR5 can critically and differentially modulate the expression of glutamate transporters and may represent interesting pharmacological targets to regulate the extracellular levels of glutamate in pathological conditions.     

CNS region-specific regulation of glial glutamate transporter expression

The neuronal cell death associated with certain neurodegenerative disorders as well as acute brain injuries is in part due to the reduced expression of glial glutamate transporters and the subsequent accumulation of toxic extracellular glutamate concentrations. Extracellular factors previously found to potently stimulate the expression of the glial glutamate transporters, GLT-1/EAAT2 and GLAST/EAAT1, in astroglial cultures of rat cerebral hemispheres are PACAP, TGF alpha, and EGF. In the present study, we sought to determine whether similar stimulatory influences apply for astroglia from other areas of the central nervous system (CNS). Immunoblot and real-time RT-PCR analysis of striatal astroglial cultures maintained for 72 h with PACAP, TGF alpha, or EGF revealed a prominent increase in GLT-1 and GLAST expression. In apparent contrast, all factors completely failed to affect GLT-1 and GLAST expression in astroglial cultures from the cerebellum, mesencephalon, and spinal cord between 36 h and 7 days. This failure was not due to the absence of functional recognition or transduction machineries for the extracellular factors as suggested by the additional observations that cerebellar, mesencephalic and spinal cord glia were capable of responding to stimulation with PACAP, TGF alpha, or EGF for 10 min with activation of CREB. Moreover, dibutyryl cyclic AMP (dbcAMP) potently promoted GLT-1 and/or GLAST expression in mesencephalic, cerebellar and spinal cord glia, further indicating that extracellular factors regulate glial glutamate transporter expression throughout the CNS. Together these findings identify PACAP, TGF alpha and EGF as potent regulators of glutamate transporter expression in striatal glia. In addition, these findings provide evidence for a CNS region-specific regulation of glial glutamate transport.