Multiple 5'-splice variants of the rat glutamate transporter-1

In most brain areas, uptake of extracellular glutamate predominantly occurs through the glutamate transporter subtype, glutamate transporter-1 (GLT-1), which is enriched in astroglia. Here, we report the identification of five splice variants of the 5'-leader sequence of rat GLT-1 which contain varying numbers of upstream open reading frames and encode putative GLT-1 proteins with two distinct N-terminal modifications. We further demonstrate that the identified rat 5'-GLT-1 splice variants are expressed in a brain region-specific manner. Our findings point to potential influences of RNA splicing on glial glutamate transport in the intact and injured rat brain.     

Distribution of two splice variants of the glutamate transporter GLT-1 in rat brain and pituitary

We have performed immunocytochemistry on rat brains using a highly specific antiserum directed against the originally described form of the glutamate transporter GLT-1 (referred to hereafter as GLT-1alpha), and another against a C-terminal splice variant of this protein, GLT-1B. Both forms of GLT-1 were abundant in rat brain, especially in regions such as the hippocampus and cerebral cortex, and macroscopic examination of sections suggested that both forms were generally regionally coexistent. However, disparities were evident; GLT-1alpha was present in the intermediate lobe of the pituitary gland, whereas GLT-1B was absent. Similar marked disparities were also noted in the external capsule, where GLT1A labeling was abundant but GLT-1B was only occasionally encountered. Conversely, GLT-1B was more extensively distributed, relative to GLT-1alpha, in areas such as the deep cerebellar nuclei. In most regions, such as the olfactory bulbs, both splice variants were present but differences were evident in their distribution. In cerebral cortex, patches were evident where GLT-1B was absent, whereas no such patches were evident for GLT-1alpha. At high resolution, other discrepancies were evident; double-labeling of areas such as hippocampus indicated that the two splice variants may either be differentially expressed by closely apposed glial elements or that the two splice variants may be differentially targeted to distinct membrane domains of individual glial cells.     

Early loss of the glutamate transporter splice-variant GLT-1v in rat cerebral cortex following lateral fluid-percussion injury

Glutamate transporter proteins are essential for the control of interstitial glutamate levels, with an impairment of their function or levels being a major potential contributor to excitotoxicity. We have investigated the effects of lateral fluid percussion on the levels of the glutamate transporter proteins GLT-1alpha, its splice variant GLT-1v, GLAST, and EAAC1 in the rat in order to evaluate their pathogenetic role in this model of traumatic brain injury (TBI). Immunoblot analysis revealed neuronal loss in the cerebral cortex was accompanied by a 54% decrease in GLT-1v 6 h following the insult which progressed to an 83% loss of the transporter after 24 h. No changes in GLT-1alpha, GLAST, or EAAC1 were observed in this brain region at either time point. GLT-1v content was also decreased by 55% and 68% in the hippocampus and thalamus, respectively, at 6 h post-injury, but recovered fully after 24 h in both brain regions. In contrast, levels of GLT-1alpha were increased in the hippocampus at 6 h and 24 h post-TBI. These alterations in transporter protein content were also confirmed using immunohistochemical methods. Our results show for the first time a pattern of early, dynamic changes in the levels of GLT-1 transporter splice variants in different brain regions in this trauma model. In addition, correlation of GLT-1v levels with both neuronal cell loss and alpha-internexin content in the injured cortex suggests that loss of this novel glutamate transporter may be a key factor in determining cerebral vulnerability following this type of brain injury.     

Glial glutamate transporter expression patterns in brains from multiple mammalian species

It is generally assumed that rodent brains can be used as representative models of neurochemical function in other species, such as humans. We have compared the distributions of the predominant glial glutamate transporters in rodents, rabbits, cats, pigs, monkeys, and humans. We identify similarities but also significant differences between species. GLT-1v, which is abundantly expressed by rodent astrocytes, is expressed only in a rare subset of astrocytes of cats and humans, and appears to be absent from brains of rabbits and monkeys. Conversely, in the pig brain GLT-1v is expressed only by oligodendrocytes. GLAST and GLT-1alpha expression differed significantly between species; while rodents and rabbits exhibited uniform expression patterns in cortex, higher species, including cats, pigs, monkeys, and humans, exhibited heterogeneities in cortical and hippocampal expression. Patches devoid of labeling intermingling with patches of strong labeling were evident in areas such as temporal cortex and frontal cortex. In addition, we noted that in human motor cortex, there were inconsistencies in labeling for the C-terminal of GLT-1alpha and common domains of GLT-1, suggesting that the C-terminal region may be missing or that an unidentified splicing is present in many human astrocytes. Collectively our data suggest that assumptions as to the roles of glutamate transporters in any species may need to be tested empirically.     

Loss of glial glutamate transporters and induction of neuronal expression of GLT-1B in the hypoxic neonatal pig brain

The homeostasis of glutamate is critical to normal brain function; deficiencies in the regulation of extracellular glutamate are thought to be a major determinant of damage in hypoxic brains. Extracellular levels of glutamate are regulated mainly by plasmalemmal glutamate transporters. We have evaluated the distribution of the glutamate transporter GLAST and two splice variants of GLT-1 in the hypoxic neonatal pig brain using this as model of neonatal humans. In response to severe hypoxic insults, we observe a rapid loss of two glial glutamate transporters from specific brain regions, such as the CA1 region of the hippocampus, but not the dentate gyrus. The spatial distribution of loss accords with patterns of damage in these brains. Conversely, we demonstrate that hypoxia evokes the expression of a splice variant of GLT-1 in neurons. We suggest that this expression may be induced in response to elevated extracellular glutamate around these neurons, and that this splice variant may represent a useful marker for direct quantification of the extent of likely neuronal damage in hypoxic brains.     

Astrocytes express specific variants of CaM KII delta and gamma, but not alpha and beta, that determine their cellular localizations

Multiple isoforms of type II Ca(2+)-calmodulin-dependent kinase (CaM KII) are composed of two major neuron-specific subunits, designated alpha and beta, and two less well-characterized subunits that are also expressed in non-neuronal tissues, designated delta and gamma. Regulated expression of these 4 gene products, and several variants produced by alternative splicing, shows temporal and regional specificity and influences intracellular targeting. We used immunoblotting and RT-PCR to analyze subunit and variant expression and distribution in cultured cerebellar astrocytes and neurons, and whole cerebellar cortex from rodent brain. The data indicate that: (i) astrocytes express a single splice variant of delta, namely delta(2); (ii) like neurons, astrocytes express two forms of CaM KII gamma; gamma(B) and gamma(A); (iii) these CaM KII variants are enriched in the supernate fraction in astrocytes, and the particulate fraction in neurons; (iv) unlike neurons, astrocytes do not express detectable levels of alpha or beta subunits or their respective splice variants. The results indicate that neurons and astrocytes express distinct CaM KII subunits and variants that localize to distinct subcellular compartments and, by inference, exert distinct cellular functions.     

Astrocytic glutamate transport regulates a Drosophila CNS synapse that lacks astrocyte ensheathment

Anatomical, molecular, and physiological interactions between astrocytes and neuronal synapses regulate information processing in the brain. The fruit fly Drosophila melanogaster has become a valuable experimental system for genetic manipulation of the nervous system and has enormous potential for elucidating mechanisms that mediate neuron–glia interactions. Here, we show the first electrophysiological recordings from Drosophila astrocytes and characterize their spatial and physiological relationship with particular synapses. Astrocyte intrinsic properties were found to be strongly analogous to those of vertebrate astrocytes, including a passive current‐voltage relationship, low membrane resistance, high capacitance, and dye‐coupling to local astrocytes. Responses to optogenetic stimulation of glutamatergic premotor neurons were correlated directly with anatomy using serial electron microscopy reconstructions of homologous identified neurons and surrounding astrocytic processes. Robust bidirectional communication was present: neuronal activation triggered astrocytic glutamate transport via excitatory amino acid transporter 1 (Eaat1), and blocking Eaat1 extended glutamatergic interneuron‐evoked inhibitory postsynaptic currents in motor neurons. The neuronal synapses were always located within 1 μm of an astrocytic process, but none were ensheathed by those processes. Thus, fly astrocytes can modulate fast synaptic transmission via neurotransmitter transport within these anatomical parameters. J. Comp. Neurol. 524:1979–1998, 2016. © 2016 Wiley Periodicals, Inc.     

The amino terminus of the glial glutamate transporter GLT-1 interacts with the LIM protein Ajuba

We have identified a cytoplasmic LIM protein, Ajuba, which interacts with the amino terminus of GLT-1, the most abundant plasma membrane glutamate transporter in the brain. Ajuba has a cytoplasmic location when expressed alone in COS cells, but translocates to colocalize with GLT-1 at the plasma membrane when GLT-1 is coexpressed. Ajuba is expressed in cerebellum, cortex, hippocampus, and retina and also in organs outside the CNS. Ajuba is found with GLT-1 in astrocytes, cerebellar Bergmann glia and retinal neurons, and antibodies to Ajuba coimmunoprecipitate GLT-1 from brain. For GLT-1 expressed in COS cells, coexpression of Ajuba did not affect the transporter's K(m) or V(max) for glutamate. Since Ajuba is known to activate MAP kinase enzymes, and its homologue Zyxin binds to cytoskeletal proteins, we propose that Ajuba is a scaffolding protein allowing GLT-1 to regulate intracellular signaling or interact with the cytoskeleton.     

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.     

The role of glutamate transporter-1a in the induction of brain ischemic tolerance in rats

This study was undertaken to determine the role of glutamate transporter-1a (GLT-1a), one of the splice variants of glutamate transporter-1, in the induction of brain ischemic tolerance by cerebral ischemic preconditioning (CIP). We used a rat global cerebral ischemic model and assessed changes by neuropathological evaluation, Western blot analysis, immunohistochemistry, real-time PCR, in vivo brain microdialysis, and high performance liquid chromatography. We found that CIP induced a significant upregulation of GLT-1a expression in the CA1 hippocampus in a time course corresponding to that of neuroprotection of CIP against brain ischemia. Severe brain ischemia for 8 min induced delayed downregulation of GLT-1a, an obvious increase in glutamate concentration and delayed neuronal death of the pyramidal neurons in the CA1 hippocampus. When the animals were pretreated with CIP before the severe ischemia, the above changes normally induced by the severe ischemia were effectively prevented. Importantly, such a preventive effect of CIP on these changes was significantly inhibited by intracerebroventricular administration of GLT-1a antisense oligodeoxynucleotides, which have been proven to specifically inhibit the expression of GLT-1a protein and mRNA, and had no effect on the expression of GLT-1b. In addition, the concentration of aspartate was also elevated after severe brain ischemic insult. However, CIP had no effect on the elevated aspartate concentrations. These results indicate that GLT-1a participated in the brain ischemic tolerance induced by CIP in rats.     

GRIP1 in GABAergic synapses

The glutamate receptor-interacting protein GRIP1 is present in glutamatergic synapses and interacts with the GluR2/3/4c subunits of the AMPA receptors. This interaction plays important roles in trafficking, synaptic targeting, and recycling of AMPA receptors as well as in the plasticity of glutamatergic synapses. Although GRIP1 has been shown to be present at GABAergic synapses in cultured neurons, the use of EM (electron microscopy) immunocytochemistry in the intact brain has failed to convincingly reveal the presence of GRIP1 in GABAergic synapses. Therefore, most studies on GRIP1 have focused on glutamatergic synapses. By using mild tissue fixation and embedding in EM, we show that in the intact brain the 7-PDZ domain GRIP1a/b is present not only in glutamatergic synapses but also in GABAergic synapses. In GABAergic synapses GRIP1a/b localizes both at the presynaptic terminals and postsynaptically, being frequently localized on the synaptic membranes or the synaptic junctional complex. Considerably higher density of GRIP1a/b is found in the presynaptic GABAergic terminals than in the glutamatergic terminals, while the density of GRIP1a/b in the postsynaptic complex is similar in both types of synapses. The results also show that the 7-PDZ and the shorter 4-PDZ domain splice forms of GRIP1 (GRIP1c 4-7) frequently colocalize with each other in individual GABAergic and glutamatergic synapses. The results suggest that GRIP1 splice forms might play important roles in brain GABAergic synapses.     

Neuronal activity regulates glutamate transporter dynamics in developing astrocytes

Glutamate transporters (GluTs) maintain a low ambient level of glutamate in the central nervous system (CNS) and shape the activation of glutamate receptors at synapses. Nevertheless, the mechanisms that regulate the trafficking and localization of transporters near sites of glutamate release are poorly understood. Here, we examined the subcellular distribution and dynamic remodeling of the predominant GluT GLT-1 (excitatory amino acid transporter 2, EAAT2) in developing hippocampal astrocytes. Immunolabeling revealed that endogenous GLT-1 is concentrated into discrete clusters along branches of developing astrocytes that were apposed preferentially to synapsin-1 positive synapses. Green fluorescent protein (GFP)-GLT-1 fusion proteins expressed in astrocytes also formed distinct clusters that lined the edges of astrocyte processes, as well as the tips of filopodia and spine-like structures. Time-lapse three-dimensional confocal imaging in tissue slices revealed that GFP-GLT-1 clusters were dynamically remodeled on a timescale of minutes. Some transporter clusters moved within developing astrocyte branches as filopodia extended and retracted, while others maintained stable positions at the tips of spine-like structures. Blockade of neuronal activity with tetrodotoxin reduced both the density and perisynaptic localization of GLT-1 clusters. Conversely, enhancement of neuronal activity increased the size of GLT-1 clusters and their proximity to synapses. Together, these findings indicate that neuronal activity influences both the organization of GluTs in developing astrocyte membranes and their position relative to synapses.     

Modulation of GABAergic transmission by endogenous glutamate in the rat supraoptic nucleus

The presence of group III metabotropic glutamate receptors on GABAergic terminals in the supraoptic nucleus suggests that the level of glutamate in the extracellular space may regulate synaptic strength at inhibitory synapses. To test this hypothesis we examined the consequences of increasing ambient glutamate on GABA-mediated synaptic activity in supraoptic neurons. The concentration of the excitatory amino acid in the extracellular space was increased pharmacologically by blocking glutamate transporters. Inhibition of the astrocyte-specific GLT-1 glutamate transporter led to a reversible decrease in evoked inhibitory postsynaptic current amplitude. This modulation had a presynaptic origin as revealed by analysis of paired-pulse ratio and miniature inhibitory currents. Furthermore, blocking group III metabotropic glutamate receptors with the specific antagonist MAP4 prevented the depression of GABAergic transmission induced by glutamate transporter blockade. Thus, presynaptic metabotropic glutamate receptors located on inhibitory terminals in the supraoptic nucleus appear to sense changes in ambient glutamate and modify GABA release accordingly. However, it seems that such changes need to reach a certain magnitude because the discrete deficit in glutamate clearance which occurs in the supraoptic nucleus of lactating rats is not sufficient to modulate GABA-mediated transmission. These results suggest that ambient glutamate contributes to the modulation of synaptic efficacy not only at glutamatergic synapses but also at inhibitory GABAergic synapses.     

Synaptic and nonsynaptic localization of protocadherin‐γC5 in the rat brain

It has been proposed that γ‐protocadherins (Pcdh‐γs) are involved in the establishment of specific patterns of neuronal connectivity. Contrary to the other Pcdh‐γs, which are expressed in the embryo, Pcdh‐γC5 is expressed postnatally in the brain, coinciding with the peak of synaptogenesis. We have developed an antibody specific for Pcdh‐γC5 to study the expression and localization of Pcdh‐γC5 in brain. Pcdh‐γC5 is highly expressed in the olfactory bulb, corpus striatum, dentate gyrus, CA1 region of the hippocampus, layers I and II of the cerebral cortex, and molecular layer of the cerebellum. Pcdh‐γC5 is expressed in both neurons and astrocytes. In hippocampal neuronal cultures, and in the absence of astrocytes, a significant percentage of synapses, more GABAergic than glutamatergic, have associated Pcdh‐γC5 clusters. Some GABAergic axons show Pcdh‐γC5 in the majority of their synapses. Nevertheless, many Pcdh‐γC5 clusters are not associated with synapses. In the brain, significant numbers of Pcdh‐γC5 clusters are located at contact points between neurons and astrocytes. Electron microscopic immunocytochemistry of the rat brain shows that 1) Pcdh‐γC5 is present in some GABAergic and glutamatergic synapses both pre‐ and postsynaptically; 2) Pcdh‐γC5 is also extrasynaptically localized in membranes and in cytoplasmic organelles of neurons and astrocytes; and 3) Pcdh‐γC5 is also localized in perisynaptic astrocyte processes. The results support the notions that 1) Pcdh‐γC5 plays a role in synaptic specificity and/or synaptic maturation and 2) Pcdh‐γC5 is involved in neuron–neuron synaptic interactions and in neuron–astrocyte interactions, including perisynaptic neuron–astrocyte interactions. J. Comp. Neurol. 518:3439–3463, 2010. © 2010 Wiley‐Liss, Inc.     

Synaptic localization of GLT-1a in the rat somatic sensory cortex

GLT-1a, the major glutamate transporter, plays an important role in both physiological and pathological conditions. Uncertainty regarding its localization in the cerebral cortex prompted us to re-examine its cellular and subcellular localization in the rat somatic sensory cortex. GLT-1a detection was sensitive to fixation; in optimal conditions approximately 25% of GLT-1a+ profiles were axon terminals. GLT-1a/VGLUT1 double-labeling and pre-embedding electron microscopy studies showed that approximately 50% of GLT-1a+ profiles were in the vicinity of asymmetric synapses. Using pre-embedding electron microscopy, we found that approximately 70% of GLT-1a located in the vicinity of asymmetric synapses was astrocytic and approximately 30% was neuronal. Post-embedding immunogold studies showed that the density of gold particles coding for GLT-1a was much higher in astrocytic processes than in axon terminals, and that in the latter they were never at the active zone. In both astrocytic processes and axon terminals most gold particles were localized in a membrane region extending for about 250 nm from active zone margin, with a peak at 140 nm for astrocytic processes and at 80 for axon terminals. We conclude that, although GLT-1a is expressed by both astrocytes and axon terminals, astrocytic GLT-1a predominates at asymmetric synapses, and that the perisynaptic localization of GLT-1a in cortex is well-suited to modulate Glu concentrations at the cleft and also to restrict Glu spillover.     

Mechanisms of substrate transport-induced clustering of a glial glutamate transporter GLT-1 in astroglial-neuronal cultures

Glutamate uptake by the Na(+)-dependent glutamate transporter GLT-1, which is predominantly expressed in astrocytes, is crucial for regulating glutamate concentration at the synaptic cleft and achieving proper excitatory neurotransmission. A body of evidence suggests that GLT-1 constitutively traffics between the plasma membrane and endosomes via an endocytosis/recycling pathway, and forms a cluster. Here, we report substrate transport via GLT-1-induced formation of GLT-1 cluster accompanied by intracellular trafficking in rat astroglial-neuronal cultures. We constructed a recombinant adenovirus expressing enhanced green fluorescence protein (EGFP)-tagged GLT-1. Adenoviral infection resulted in the expression of functional GLT-1-EGFP preferentially in astrocytes, partly as clusters. Treatment with glutamate, but not N-methyl-D-aspartate, dramatically increased the number of GLT-1 clusters within 1 h. The estimated EC(50) value of glutamate was 240 microm. In addition, glutamate decreased the cell surface expression and increased the intracellular expression of GLT-1. The GLT-1 clusters were found in early and recycling endosomes and partly in lysosomes, and were inhibited by blockade of endocytotic pathways. Ionotropic and metabotropic glutamate receptor antagonists had no effect on glutamate-induced GLT-1 clustering. The non-transportable glutamate uptake inhibitors (2S,3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate and dihydrokainate, as well as Na(+)-free conditions, prevented the glutamate-induced GLT-1 clustering, whereas the competitive substrates, aspartate and L-trans-pyrrolidine-2,4-dicarboxylate, induced GLT-1 clustering. Furthermore, the Na(+)/K(+)-ATPase inhibitor, ouabain, and the Na(+) ionophores, gramicidin and monensin, produced GLT-1 clustering. Modulators of intracellular Ca(2+)signaling or membrane depolarization had no effect on GLT-1 clustering. Taken together, these results suggest that Na(+) influx associated with GLT-1 substrate transport triggers the formation of GLT-1 clusters accompanied by intracellular trafficking via endocytotic pathways in astrocytes.     

Induction of astrocyte differentiation by propentofylline increases glutamate transporter expression in vitro: heterogeneity of the quiescent phenotype

Reactive astrocytes display decreased glutamate transporters, such as GLT-1, and as a result synaptic glutamate clearance is impaired. In addition, these activated astrocytes are immunocompetent and release algesic mediators that can sensitize neurons in the spinal cord. Currently, we evaluated the effect of propentofylline (PPF), an experimental antiallodynic agent, on the phenotype and glutamate transporter expression of astrocytes. Primary astrocyte cultures, which represent an activated phenotype with a polygonal morphology and low GLT-1 expression, were treated for 3 or 7 days with 10, 100, or 1,000 microM PPF or dibutyryl-cAMP (db-cAMP), a known inducer of GLT-1 expression. PPF dose-dependently induced astrocytes to display a mature phenotype, with elongated processes and a stellate shape, as well as increased GLT-1 and GLAST immunoreactivity, similar to that seen with db-cAMP. Real time RT-PCR and Western blot analysis clearly demonstrated that PPF caused a potent dose-dependent induction of GLT-1 and GLAST mRNA and protein in these astrocytes. Importantly, the observed increase in glutamate transporters was found to have a functional effect, with significantly enhanced glutamate uptake in astrocytes treated with 100 or 1,000 microM PPF that was sensitive to dihydrokainate inhibition, suggesting it is GLT-1 mediated. Finally, the effect of PPF on lipopolysaccharide-induced chemokine release was investigated. Interestingly, PPF was able to dampen both MCP-1 (CCL2) and MIP-2 (CXCL2) release from astrocytes while db-cAMP significantly enhanced this chemokine expression. These findings suggest that PPF is capable of differentiating astrocytes to a homeostatic, mature phenotype, competent for glutamate clearance and distinct from that induced by db-cAMP.     

The upregulation of glial glutamate transporter-1 participates in the induction of brain ischemic tolerance in rats.

Glial glutamate transporter-1 (GLT-1) plays an essential role in removing glutamate from the extracellular space and maintaining the glutamate below neurotoxic level in the brain. To explore whether GLT-1 plays a role in the acquisition of brain ischemic tolerance (BIT) induced by cerebral ischemic preconditioning (CIP), the present study was undertaken to observe in vivo changes in the expression of GLT-1 and glial fibrillary acidic protein (GFAP) in the CA1 hippocampus during the induction of BIT, and the effect of dihydrokainate (DHK), an inhibitor of GLT-1, on the acquisition of BIT in rats. Immunohistochemistry for GFAP showed that the processes of astrocytes were prolonged after a CIP 2 days before the lethal ischemic insult, which could protect pyramidal neurons in the CA1 hippocampus against delayed neuronal death induced normally by lethal ischemic insult. The prolonged processes extended into the area between the pyramidal neurons and tightly surrounded them. These changes made the pyramidal layer look like a 'shape grid'. Simultaneously, the prolonged and extended processes showed a great deal of GLT-1. Western blotting analysis showed significant upregulation of GLT-1 expression after the CIP, especially when it was administered 2 days before the subsequent lethal ischemic insult. Neuropathological evaluation by thionin staining showed that DHK dose-dependently blocked the protective role of CIP against delayed neuronal death induced normally by lethal brain ischemia. It might be concluded that the surrounding of pyramidal neurons by astrocytes and upregulation of GLT-1 induced by CIP played an important role in the acquisition of the BIT induced by CIP.