Chronological changes of N-methyl-D-aspartate receptors and excitatory amino acid carrier 1 immunoreactivities in CA1 area and subiculum after transient forebrain ischemia

We investigated changes of immunoreactivities of N-methyl-D-aspartate receptor (NR) and of excitatory amino acid carrier 1 (EAAC-1), the neuronal glutamate transporter, in the vulnerable CA1 area and the less vulnerable subiculum of the gerbil hippocampus at various times following transient forebrain ischemia. At 30 min after ischemia-reperfusion, the intensity of NR immunoreactivity increased markedly in neurons of CA1 and subiculum, particularly NR2A/B, while EAAC-1 immunoreactivity was reduced in CA1. At 3 hr after reperfusion, the density of NR1 immunoreactivity markedly decreased in CA1. In contrast EAAC-1 immunoreactivity increased in CA1 and in the subiculum. At 12 hr after reperfusion, the decrease of NR1 immunoreactivity was not detected whereas EAAC-1 immunoreactivities in the CA1 area were intensified. In the subiculum, both NR subunits immunoreactivities decreased significantly, in contrast to the maintenance of EAAC-1 immunoreactivity. At 24 hr after reperfusion, both NR2A/B and EAAC-1 immunoreactivities decreased markedly in CA1 and subiculum. We tentatively suggest that the increase of NR immunoreactivity in CA1 at early times after ischemia-reperfusion may increase the delayed neuronal death, and that the increase or maintenance of EAAC-1 immunoreactivity at early times after ischemia-reperfusion may be an important factor in survival of neurons.     

GDNF pre-treatment aggravates neuronal cell loss in oxygen-glucose deprived hippocampal slice cultures: a possible effect of glutamate transporter up-regulation

Besides its neurotrophic and neuroprotective effects on dopaminergic neurons and spinal motoneurons, glial cell line-derived neurotrophic factor (GDNF) has potent neuroprotective effects in cerebral ischemia. The protective effect has so far been related to reduced activation of N-methyl-D-aspartate receptors (NMDAr). This study tested the effects of GDNF on glutamate transporter expression, with the hypothesis that modulation of glutamate transporter activity would affect the outcome of cerebral ischemia. Organotypic hippocampal slice cultures, derived from 1-week-old rats, were treated with 100 ng/ml GDNF for either 2 or 5 days, followed by Western blot analysis of NMDAr subunit 1 (NR1) and two glutamate transporter subtypes, GLAST and GLT-1. After 5-day exposure to GDNF, expression of GLAST and GLT-1 was up-regulated to 169 and 181% of control values, respectively, whereas NR1 was down-regulated to 64% of control. However, despite these changes that potentially would support neuronal resistance to excitotoxicity, the long-term treatment with GDNF was found to aggravate the neuronal damage induced by oxygen-glucose deprivation (OGD). The increased cell death, assessed by propidium iodide (PI) uptake, occurred not only among the most susceptible CA1 pyramidal cells, but also in CA3 and fascia dentata. Given that glutamate transporters are able to release glutamate by reversed action during energy failure, it is suggested that the observed increase in OGD-induced cell death in the GDNF-pretreated cultures was caused by the build-up of excitotoxic concentrations of extracellular glutamate released through the glutamate transporters, which were up-regulated by GDNF. Although the extent and consequences of glutamate release via reversal of GLAST and GLT-1 transporters seem to vary in different energy failure models, the present findings should be taken into account in clinical trials of GDNF.     

Transient focal cerebral ischemia down-regulates glutamate transporters GLT-1 and EAAC1 expression in rat brain

Transient focal cerebral ischemia leads to extensive excitotoxic neuronal damage in rat cerebral cortex. Efficient reuptake of the released glutamate is essential for preventing glutamate receptor over-stimulation and neuronal death. Present study evaluated the expression of the glial (GLT-1 and GLAST) and neuronal (EAAC1) subtypes of glutamate transporters after transient middle cerebral artery occlusion (MCAO) induced focal cerebral ischemia in rats. Between 24h to 72h of reperfusion after transient MCAO, GLT-1 and EAAC1 protein levels decreased significantly (by 36% to 56%, p < 0.05) in the ipsilateral cortex compared with the contralateral cortex or sham control. GLT-1 and EAAC1 mRNA expression also decreased in the ipsilateral cortex of ischemic rats at both 24h and 72h of reperfusion, compared with the contralateral cortex or sham control. Glutamate transporter down-regulation may disrupt the normal clearance of the synaptically-released glutamate and may contribute to the ischemic neuronal death.     

Alterations in Glutamate Transporter Protein Levels in Kindling-Induced Epilepsy

Abstract: There is increasing evidence that levels of glutamate are elevated in certain brain regions immediately prior to and during induction and propagation of seizures. Modulation of high-affinity glutamate uptake is a potential mechanism responsible for the elevated levels observed with seizures. To date, three distinct Na⁺-dependent glutamate transporters have been cloned from rat and rabbit: GLT-1, GLAST, and EAAC-1. We performed a series of experiments to determine whether levels of these transporters are altered in amygdala-kindled rats. Levels of GLT-1, GLAST, and EAAC-1 were examined in three brain regions (hippocampus, piriform cortex/amygdala, and limbic forebrain) by quantitative immunoblotting using subtype-specific antibodies. GLAST protein was down-regulated in the piriform cortex/amygdala region of kindled rats as early as 24 h after one stage 3 seizure and persisting through multiple stage 5 seizures. In contrast, kindling induced an increase in EAAC-1 levels in piriform cortex/amygdala and hippocampus once the animals had reached the stage 5 level. No changes in GLT-1 were observed in any region examined. Changes in transporter levels could contribute to the changes in glutamate levels seen with kindling.     

Impairment of Neuronal Glutamate Uptake and Modulation of the Glutamate Transporter GLT-1 Induced by Retinal Ischemia

Excitotoxicity has been implicated in the retinal neuronal loss in several ocular pathologies including glaucoma. Dysfunction of Excitatory Amino Acid Transporters is often a key component of the cascade leading to excitotoxic cell death. In the retina, glutamate transport is mainly operated by the glial glutamate transporter GLAST and the neuronal transporter GLT-1. In this study we evaluated the expression of GLAST and GLT-1 in a rat model of acute glaucoma based on the transient increase of intraocular pressure (IOP) and characterized by high glutamate levels during the reperfusion that follows the ischemic event associated with raised IOP. No changes were reported in GLAST expression while, at neuronal level, a reduction of glutamate uptake and of transporter reversal-mediated glutamate release was observed in isolated retinal synaptosomes. This was accompanied by modulation of GLT-1 expression leading to the reduction of the canonical 65 kDa form and upregulation of a GLT-1-related 38 kDa protein. These results support a role for neuronal transporters in glutamate accumulation observed in the retina following an ischemic event and suggest the presence of a GLT-1 neuronal new alternative splice variant, induced in response to the detrimental stimulus.     

Regional Deafferentiation Down-Regulates Subtypes of Glutamate Transporter Proteins

Abstract: Low extracellular glutamate content is maintained primarily by high-affinity sodium-dependent glutamate transport. Three glutamate transporter proteins have been cloned: GLT-1 and GLAST are astroglial, whereas EAAC1 is neuronal. The effects of axotomy on glutamate transporter expression was evaluated in adult rats following unilateral fimbria-fornix and corticostriatal lesions. The hippocampus and striatum were collected at 3, 7, 14, and 30 days postlesion. Homogenates were immunoblotted using antibodies directed against GLT-1, GLAST, EAAC1, and glial fibrillary acidic protein and assayed for glutamate transport by d -[³H]aspartate binding. GLT-1 immunoreactivity was decreased within the ipsilateral hippocampus and striatum at 14 days postlesion. GLAST immunoreactivity was decreased within the ipsilateral hippocampus and striatum at 7 and 14 days postlesion. No alterations in EAAC1 immunoreactivity were observed. d -[³H]Aspartate binding was decreased at 14 days postlesion within the ipsilateral hippocampus and at 7 and 14 days postlesion within the ipsilateral striatum. By 30 days postlesion, glutamate transporters and d -[³H]aspartate binding returned to control levels. This study demonstrates the down-regulation of primarily glial, and not neuronal, glutamate transporters following regional disconnection.     

High expression of GLT-1 in hippocampal CA3 and dentate gyrus subfields contributes to their inherent resistance to ischemia in rats

It is well known that neurons in the CA3 and dentate gyrus (DG) subfields of the hippocampus are resistant to short period of ischemia which is usually lethal to pyramidal neurons in hippocampal CA1 subfield. The present study was undertaken to clarify whether the inherent higher resistance of neurons in CA3 and DG to ischemia is associated with glial glutamate transporter-1 (GLT-1) in rats. Western blot analysis and immunohistochemistry assay showed that the basal expressions of GLT-1 in both CA3 and DG were much higher than that in CA1 subfield. Mild global brain ischemia for 8 min induced delayed death of almost all CA1 pyramidal neurons and marked GLT-1 down-regulation in the CA1 subfield, but it was not lethal to the neurons in either CA3 or DG and induced GLT-1 up-regulation and astrocyte activation showed normal soma and aplenty slender processes in the both areas. When the global brain ischemia was prolonged to 25 min, neuronal death was clearly observed in CA3 and DG accompanied with down-regulation of GLT-1 expression and abnormal astrocytes represented with hypertrophic somas, but shortened processes. After down-regulating of GLT-1 expression and function by its antisense oligodeoxynucleotides or inhibiting GLT-1 function by dihydrokainate, an inhibitor of GLT-1, the mild global brain ischemia for 8 min, which usually was not lethal to CA3 and DG neurons, induced the neuronal death in CA3 and DG subfields. Taken together, the higher expression of GLT-1 in the CA3 and DG contributes to their inherent resistance to ischemia.     

The GLT-1 and GLAST glutamate transporters are expressed on morphologically distinct astrocytes and regulated by neuronal activity in primary hippocampal cocultures

The GLT-1 and GLAST astroglial transporters are the glutamate transporters mainly involved in maintaining physiological extracellular glutamate concentrations. Defects in neurotransmitter glutamate transport may represent an important component of glutamate-induced neurodegenerative disorders (such as amyotrophic lateral sclerosis) and CNS insults (ischemia and epilepsy). We characterized the protein expression of GLT-1 and GLAST in primary astrocyte-neuron cocultures derived from rat hippocampal tissues during neuron differentiation/maturation. GLT-1 and GLAST are expressed by morphologically distinct glial fibrillary acidic protein-positive astrocytes, and their expression correlates with the status of neuron differentiation/maturation and activity. Up-regulation of the transporters paralleled the content of the synaptophysin synaptic vesicle marker p38, and down-regulation was a consequence of glutamate-induced neuronal death or the reduction of synaptic activity. Finally, soluble factors in neuronal-conditioned media prevented the down-regulation of the GLT-1 and GLAST proteins. Although other mechanisms may participate in regulating GLT-1 and GLAST in the CNS, our data indicate that soluble factors dependent on neuronal activity play a major regulating role in hippocampal cocultures.     

Traumatic Brain Injury Down-Regulates Glial Glutamate Transporter (GLT-1 and GLAST) Proteins in Rat Brain

Abstract: Excess activation of NMDA receptors is felt to participate in secondary neuronal damage after traumatic brain injury (TBI). Increased extracellular glutamate is active in this process and may result from either increased release or decreased reuptake. The two high-affinity sodium-dependent glial transporters [glutamate transporter 1 (GLT-1) and glutamate aspartate transporter (GLAST)] mediate the bulk of glutamate transport. We studied the protein levels of GLT-1 and GLAST in the brains of rats after controlled cortical impact-induced TBI. With use of subtype-specific antibodies, GLT-1 and GLAST proteins were quantitated by immunoblotting in the ipsilateral and contralateral cortex at 2, 6, 24, 72, and 168 h after the injury. Sham-operated rats served as control. TBI resulted in a significant decrease in GLT-1 (by 20–45%; p < 0.05) and GLAST (by 30–50%; p < 0.05) protein levels between 6 and 72 h after the injury. d -[³H]Aspartate binding also decreased significantly (by 30–50%; p < 0.05) between 6 and 72 h after the injury. Decreased glial glutamate transporter function may contribute to the increased extracellular glutamate that may mediate the excitotoxic neuronal damage after TBI. This is a first report showing altered levels of glutamate transporter proteins after TBI.     

The Rates of Postmortem Proteolysis of Glutamate Transporters Differ Dramatically between Cells and between Transporter Subtypes

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.     

Transgenic SOD1 G93A mice develop reduced GLT-1 in spinal cord without alterations in cerebrospinal fluid glutamate levels

Glutamate-induced excitotoxicity is suggested to play a central role in the development of amyotrophic lateral sclerosis (ALS), although it is still unclear whether it represents a primary cause in the cascade leading to motor neurone death. We used western blotting, immunocytochemistry and in situ hybridization to examine the expression of GLT-1 in transgenic mice carrying a mutated (G93A) human copper-zinc superoxide dismutase (TgSOD1 G93A), which closely mimic the features of ALS. We observed a progressive decrease in the immunoreactivity of the glial glutamate transporter (GLT-1) in the ventral, but not in the dorsal, horn of lumbar spinal cord. This effect was specifically found in 14- and 18-week-old mice that had motor function impairment, motor neurone loss and reactive astrocytosis. No changes in GLT-1 were observed at 8 weeks of age, before the appearance of clinical symptoms. Decreases in GLT-1 were accompanied by increased glial fibrillary acidic protein (GFAP) levels and no change in the levels of GLAST, another glial glutamate transporter. The glutamate concentration in the cerebrospinal fluid (CSF) of TgSOD1 G93A mice was not modified at any of the time points examined, compared with age-matched controls. These findings indicate that the loss of GLT-1 protein in ALS mice selectively occurs in the areas affected by neurodegeneration and reactive astrocytosis and it is not associated with increases of glutamate levels in CSF. The lack of changes in GLT-1 at the presymptomatic stage suggests that glial glutamate transporter reduction is not a primary event leading to motor neurone loss.     

Fimbria-Fornix Transections Selectively Down-Regulate Subtypes of Glutamate Transporter and Glutamate Receptor Proteins in Septum and Hippocampus

Abstract: The effects of CNS axotomy on glutamate transporter and glutamate receptor expression were evaluated in adult rats following unilateral fimbria-fornix transections. The septum and hippocampus were collected at 3, 7, 14, and 30 days postlesion. Homogenates were immunoblotted by using antibodies directed against glutamate transporters (GLT-1, GLAST, and EAAC1) and glutamate receptors (GluR1, GluR2/3, GluR6/7, and NMDAR1), and they were assayed for glutamate transport by d -[³H]aspartate binding. GLT-1 was decreased at 7 and 14 days postlesion within the ipsilateral septum and at 7 days postlesion in the hippocampus. GLAST was decreased within the ipsilateral septum and hippocampus at 7 and 14 days postlesion. No postlesion alterations in EAAC1 immunoreactivity were observed. d -[³H]Aspartate binding was decreased at 7, 14, and 30 days postlesion within the ipsilateral septum and 14 days postlesion in the hippocampus. GluR2/3 expression was down-regulated at 30 days postlesion within the ipsilateral septum, whereas GluR1, GluR6/7, and NMDAR1 immunoreactivity was unchanged. In addition, no alterations in glutamate receptor expression were detected within hippocampal homogenates. This study demonstrates a selective down-regulation of primarily glial, and not neuronal, glutamate transporters and a delayed, subtype-specific down-regulation of septal GluR2/3 receptor expression after regional deafferentation within the CNS.     

Selective down-regulation of the astrocyte glutamate transporters GLT-1 and GLAST within the medial thalamus in experimental Wernicke's encephalopathy

Although earlier studies on thiamine deficiency have reported increases in extracellular glutamate concentration in the thalamus, a vulnerable region of the brain in this disorder, the mechanism by which this occurs has remained unresolved. Treatment with pyrithiamine, a central thiamine antagonist, resulted in a 71 and 55% decrease in protein levels of the astrocyte glutamate transporters GLT-1 and GLAST, respectively, by immunoblotting in the medial thalamus of day 14 symptomatic rats at loss of righting reflexes. These changes occurred prior to the onset of convulsions and pannecrosis. Loss of both GLT-1 and GLAST transporter sites was also confirmed in this region of the thalamus at the symptomatic stage using immunohistochemical methods. In contrast, no change in either transporter protein was detected in the non-vulnerable frontal parietal cortex. These effects are selective; protein levels of the astrocyte GABA transporter GAT-3 were unaffected in the medial thalamus. In addition, astrocyte-specific glial fibrillary acidic protein (GFAP) content was unchanged in this brain region, suggesting that astrocytes are spared in this disorder. Loss of GLT-1 or GLAST protein was not observed on day 12 of treatment, indicating that down-regulation of these transporters occurs within 48 h prior to loss of righting reflexes. Finally, GLT-1 content was positively correlated with levels of the neurofilament protein alpha-internexin, suggesting that early neuronal drop-out may contribute to the down-regulation of this glutamate transporter and subsequent pannecrosis. A selective, focal loss of GLT-1 and GLAST transporter proteins provides a rational explanation for the increase in interstitial glutamate levels, and may play a major role in the selective vulnerability of thalamic structures to thiamine deficiency-induced cell death.     

Intermittent Hypobaric Hypoxia Preconditioning Induced Brain Ischemic Tolerance by Up-Regulating Glial Glutamate Transporter-1 in Rats

Several studies showed that the up-regulation of glial glutamate transporter-1 (GLT-1) participates in the acquisition of brain ischemic tolerance induced by cerebral ischemic preconditioning or ceftriaxone pretreatment in rats. To explore whether GLT-1 plays a role in the acquisition of brain ischemic tolerance induced by intermittent hypobaric hypoxia (IH) preconditioning (mimicking 5,000?m high-altitude, 6?h per day, once daily for 28?days), immunohistochemistry and western blot were used to observe the changes in the expression of GLT-1 protein in hippocampal CA1 subfield during the induction of brain ischemic tolerance by IH preconditioning, and the effect of dihydrokainate (DHK), an inhibitor of GLT-1, on the acquisition of brain ischemic tolerance in rats. The basal expression of GLT-1 protein in hippocampal CA1 subfield was significantly up-regulated by IH preconditioning, and at the same time astrocytes were activated by IH preconditioning, which appeared normal soma and aplenty slender processes. The GLT-1 expression was decreased at 7?days after 8-min global brain ischemia. When the rats were pretreated with the IH preconditioning before the global brain ischemia, the down-regulation of GLT-1 protein was prevented clearly. Neuropathological evaluation by thionin staining showed that 200?nmol DHK blocked the protective role of IH preconditioning against delayed neuronal death induced normally by 8-min global brain ischemia. Taken together, the up-regulation of GLT-1 protein participates in the acquisition of brain ischemic tolerance induced by IH preconditioning in rats.     

Glial glutamate transporter GLT-1 down-regulation precedes delayed neuronal death in gerbil hippocampus following transient global cerebral ischemia

Glial (GLT-1 and GLAST) and neuronal (EAAC1) high-affinity transporters mediate the sodium dependent glutamate reuptake in mammalian brain. Their dysfunction leads to neuronal damage by allowing glutamate to remain in the synaptic cleft for a longer duration. The purpose of the present study is to understand their contribution to the ischemic delayed neuronal death seen in gerbil hippocampus following transient global cerebral ischemia. The protein levels of these three transporters were studied by immunoblotting as a function of reperfusion time (6 h to 7 days) following a 10 min occlusion of bilateral common carotid arteries in gerbils. In the vulnerable hippocampus, there was a significant decrease in the protein levels of GLT-1 (by 36-46%, P < 0.05; between 1 and 3 days of reperfusion) and EAAC1 (by 42-68%, P < 0.05; between 1 and 7 days of reperfusion). Histopathological evaluation showed no neuronal loss up to 2 days of reperfusion but an extensive neuronal loss (by approximately 84%, P < 0.01) at 7 days of reperfusion in the hippocampal CA1 region. The time frame of GLT-1 dysfunction (1-3 days of reperfusion) precedes the initiation of delayed neuronal death (2-3 days of reperfusion). This suggests GLT-1 dysfunction as a contributing factor for the hippocampal neuronal death following transient global cerebral ischemia. Furthermore, decreased EAAC1 levels may contribute to GABAergic dysfunction and excitatory/inhibitory imbalance following transient global ischemia.     

In vivo evidence that truncated trkB.T1 participates in nociception

Background

Clearance of synaptically released glutamate, and hence termination of glutamatergic neurotransmission, is carried out by glutamate transporters, most especially glutamate transporter-1 (GLT-1) and the glutamate-aspartate transporter (GLAST) that are located in astrocytes. It is becoming increasingly well appreciated that changes in the function and expression of GLT-1 and GLAST occur under different physiological and pathological conditions. Here we investigated the plasticity in expression of GLT-1 and GLAST in the spinal dorsal horn using immunohistochemistry following partial sciatic nerve ligation (PSNL) in rats.

Results

Animals were confirmed to develop hypersensitivity to mechanical stimulation by 7 days following PSNL. Baseline expression of GLT-1 and GLAST in naive animals was only observed in astrocytes and not in either microglia or neurons. Microglia and astrocytes showed evidence of reactivity to the nerve injury when assessed at 7 and 14 days following PSNL evidenced by increased expression of OX-42 and GFAP, respectively. In contrast, the total level of GLT-1 and GLAST protein decreased at both 7 and 14 days after PSNL. Importantly, the cellular location of GLT-1 and GLAST was also altered in response to nerve injury. Whereas activated astrocytes showed a marked decrease in expression of GLT-1 and GLAST, activated microglia showed de novo expression of GLT-1 and GLAST at 7 days after PSNL and this was maintained through day 14. Neurons showed no expression of GLT-1 or GLAST at any time point.

Conclusion

These results indicate that the expression of glutamate transporters in astrocytes and microglia are differentially regulated following nerve injury.     

Microglial self-defence mediated through GLT-1 and glutathione

Glutamate is stored in synaptic vesicles in presynaptic neurons. It is released into the synaptic cleft to provide signalling to postsynaptic neurons. Normally, the astroglial glutamate transporters GLT-1 and GLAST take up glutamate to mediate a high signal-to-noise ratio in the synaptic signalling, and also to prevent excitotoxic effects by glutamate. In astrocytes, glutamate is transformed into glutamine, which is safely transported back to neurons. However, in pathological conditions, such as an ischemia or virus infection, astroglial transporters are down-regulated which could lead to excitotoxicity. Lately, it was shown that even microglia can express glutamate transporters during pathological events. Microglia have two systems for glutamate transport: GLT-1 for transport into the cells and the x_c⁻ system for transport out of the cells. We here review results from our work and others, which demonstrate that microglia in culture express GLT-1, but not GLAST, and transport glutamate from the extracellular space. We also show that TNF-α can induce increased microglial GLT-1 expression, possibly associating the expression with inflammatory systems. Furthermore, glutamate taken up through GLT-1 may be used for direct incorporation into glutathione and to fuel the intracellular glutamate pool to allow cystine uptake through the x_c⁻ system. This can lead to a defence against oxidative stress and have an antiviral function.