Glutamate Transporter Subtypes Research Articles

Glutamate transporter activation enhances nicotine antinociception and attenuates nicotine analgesic tolerance

Analgesic tolerance is partially mediated by enhanced glutamatergic transmission in the CNS. β-lactam antibiotics, through glutamate transporter subtype 1 (GLT-1) activation, reduce extracellular glutamate levels and attenuate tolerance to morphine analgesia in rats. Similar to opioids, nicotine has potent analgesic properties that are subject to tolerance. The purpose of this study was to evaluate the effects of ceftriaxone, a β-lactam antibiotic and GLT-1 activator on nicotine antinociception and its tolerance. Rats were pretreated for 5 days with ceftriaxone (200 mg/kg, intraperitoneally) before evaluating their analgesic response to nicotine (1.0 or 2.5 mg/kg, subcutaneously) for seven consecutive days using the tail-flick assay. Ceftriaxone-treated rats displayed an enhanced antinociceptive response to nicotine and unlike saline-injected controls, did not develop tolerance to nicotine's analgesic effects. These results suggest that GLT-1 transporter activation enhances and preserves nicotine antinociception and identify β-lactam antibiotics as potential complementary therapeutic agents for the treatment of chronic pain.

Connexin-deficiency affects expression levels of glial glutamate transporters within the cerebrum

The glial glutamate transporter subtypes, GLT-1/EAAT-2 and GLAST/EAAT-1 clear the bulk of extracellular glutamate and are severely dysregulated in various acute and chronic brain diseases. Despite the previous identification of several extracellular factors modulating glial glutamate transporter expression, our knowledge of the regulatory network controlling glial glutamate transport in health and disease still remains incomplete. In studies with cultured cortical astrocytes, we previously obtained evidence that glial glutamate transporter expression is also affected by gap junctions/connexins. To assess whether gap junctions would likewise control the in vivo expression of glial glutamate transporters, we have now assessed their expression levels in brains of conditional Cx43 knockout mice, total Cx30 knockouts, as well as Cx43/Cx30 double knockouts. We found that either knocking out Cx30, Cx43, or both increases GLT-1/EAAT-2 protein levels in the cerebral cortex to a similar extent. By contrast, GLAST/EAAT-1 protein levels maximally increased in cerebral cortices of Cx30/Cx43 double knockouts, implying that gap junctions differentially affect the expression of GLT-1/EAAT-2 and GLAST/EAAT-1. Quantitative PCR analysis further revealed that increases in glial glutamate transporter expression are brought about by transcriptional and translational/posttranslational processes. Moreover, GLT-1/EAAT-2- and GLAST/EAAT-1 protein levels remained unchanged in the hippocampi of Cx43/Cx30 double knockouts when compared to Cx43fl/fl controls, indicating brain region-specific effects of gap junctions on glial glutamate transport. Since astrocytic gap junction coupling is affected in various forms of brain injuries, our findings point to gap junctions/connexins as important regulators of glial glutamate turnover in the diseased cerebral cortex.

Beta-lactam antibiotic decreases acquisition of and motivation to respond for cocaine, but not sweet food, in C57Bl/6 mice

No medication is approved to treat cocaine addiction, but mounting evidence suggests that glutamate-directed approaches may reduce cocaine dependence and relapse. We tested the hypotheses that the glutamate transporter subtype 1 activator, ceftriaxone, disrupts acquisition of cocaine self-administration, motivation to self-administer cocaine, and conditioned place preference in mice. Repeated ceftriaxone (200 mg/kg) reduced the ability of mice to acquire cocaine and the motivation to self-administer cocaine after successful acquisition without affecting acquisition of or motivation for sweet food. Repeated ceftriaxone had no effect on cocaine-conditioned place preference. These results suggest that a β-lactam antibiotic reduces the direct reinforcing strength of cocaine without producing nonspecific deficits in conditioned learning processes.

A brief comparison of the pathophysiology of inflammatory versus neuropathic pain

The causes of inflammatory pain and neuropathic pain are fundamentally different. There are, however, common mechanisms underlying the generation of each pain state. We will discuss some specific elements observed in both tissue and nerve injury pain states and consider the hypothesis that these two states actually demonstrate a convergence over time. The increased pain sensation following tissue and nerve injury results from several mechanisms, including altered ion channel expression in dorsal root ganglion neurons, enhanced dorsal horn glutamate release from primary afferents, enhanced glutamate receptor function in second-order neurons, disinhibition in the dorsal horn and glia cell activation. The role of specific subtypes of receptors, ion channels and glutamate transporters is revealed at peripheral and central sites. Importantly over time, a number of changes, in the dorsal root ganglion and in dorsal horn observed after tissue injury resemble changes observed after nerve injury. Recognition of mechanisms common to both inflammatory pain and neuropathic pain might shed light on the understanding of the transition from acute pain to persistent pain.

Nuclear Factor- B Contributes to Neuron-Dependent Induction of Glutamate Transporter-1 Expression in Astrocytes

The glutamate transporter-1 [GLT-1 (excitatory amino acid transporter 2)] subtype of glutamate transporter ensures crisp excitatory signaling and limits excitotoxicity in the CNS. Astrocytic expression of GLT-1 is regulated during development, by neuronal activity, and in neurodegenerative diseases. Although neurons activate astrocytic expression of GLT-1, the mechanisms involved have not been identified. In the present study, astrocytes from transgenic mice that express enhanced green fluorescent protein (eGFP) under the control of a bacterial artificial chromosome (BAC) containing a very large region of DNA surrounding the GLT-1 gene (BAC GLT-1 eGFP mice) were used to assess the role of nuclear factor-κB (NF-κB) in neuron-dependent activation of the GLT-1 promoter. We provide evidence that neurons activate NF-κB signaling in astrocytes. Transduction of astrocytes from the BAC GLT-1 eGFP mice with dominant-negative inhibitors of NF-κB signaling completely blocked neuron-dependent activation of a NF-κB reporter construct and attenuated induction of eGFP. Exogenous expression of p65 and/or p50 NF-κB subunits induced expression of eGFP or GLT-1 and increased GLT-1-mediated transport activity. Using wild-type and mutant GLT-1 promoter reporter constructs, we found that NF-κB sites at -583 or -251 relative to the transcription start site were required for neuron-dependent reporter activation. Electrophoretic mobility shift and supershift assays reveal that p65 and p50 interact with these same sites ex vivo. Finally, chromatin immunoprecipitation showed that p65 and p50 interact with these sites in adult cortex, but not in kidney (a tissue that expresses no detectable GLT-1). Together, these studies strongly suggest that NF-κB contributes to neuron-dependent regulation of astrocytic GLT-1 transcription.

The Discovery of Slowness: Low-Capacity Transport and Slow Anion Channel Gating by the Glutamate Transporter EAAT5

Excitatory amino acid transporters (EAATs) control the glutamate concentration in the synaptic cleft by glial and neuronal glutamate uptake. Uphill glutamate transport is achieved by the co-/countertransport of Na+ and other ions down their concentration gradients. Glutamate transporters also display an anion conductance that is activated by the binding of Na+ and glutamate but is not thermodynamically coupled to the transport process. Of the five known glutamate transporter subtypes, the retina-specific subtype EAAT5 has the largest conductance relative to glutamate uptake activity. Our results suggest that EAAT5 behaves as a slow-gated anion channel with little glutamate transport activity. At steady state, EAAT5 was activated by glutamate, with a Km= 61 ± 11 μM. Binding of Na+ to the empty transporter is associated with a Km = 229 ± 37 mM, and binding to the glutamate-bound form is associated with a Km = 76 ± 40 mM. Using laser-pulse photolysis of caged glutamate, we determined the pre-steady-state kinetics of the glutamate-induced anion current of EAAT5. This was characterized by two exponential components with time constants of 30 ± 1 ms and 200 ± 15 ms, which is an order of magnitude slower than those observed in other glutamate transporters. A voltage-jump analysis of the anion currents indicates that the slow activation behavior is caused by two slow, rate-limiting steps in the transport cycle, Na+ binding to the empty transporter, and translocation of the fully loaded transporter. We propose a kinetic transport scheme that includes these two slow steps and can account for the experimentally observed data. Overall, our results suggest that EAAT5 may not act as a classical high-capacity glutamate transporter in the retina; rather, it may function as a slow-gated glutamate receptor and/or glutamate buffering system.

Glutamate transporter subtype 1 (GLT-1) activator ceftriaxone attenuates amphetamine-induced hyperactivity and behavioral sensitization in rats

BackgroundThe β-lactam antibiotic and glutamate transporter subtype 1 (GLT-1) activator ceftriaxone prevents relapse to cocaine-seeking and inhibits morphine-induced physical dependence and tolerance in rats, but its efficacy against amphetamine-induced behaviors is unknown. MethodsHere, we tested the hypothesis that ceftriaxone (200mg/kg, i.p.) inhibits hyperactivity produced by acute amphetamine administration (2mg/kg, i.p.) and sensitization of hyperactivity induced by repeated amphetamine exposure (2mg/kg, i.p.). For acute experiments, rats treated with ceftriaxone for 5 days were injected with amphetamine or saline on day 6. ResultsAmphetamine elicited less ambulatory and stereotypical activity in ceftriaxone-treated rats than in ceftriaxone-naïve rats. For chronic experiments, rats injected with ceftriaxone or saline for 8 days were also injected with amphetamine or saline on days 6–8 and then challenged with amphetamine 5 days later. Amphetamine produced greater ambulatory and stereotypical activity in amphetamine-pretreated rats than in rats previously naïve to amphetamine. Amphetamine challenge produced less ambulatory and stereotypical activity in rats pretreated with a combination of ceftriaxone (200mg/kg) and amphetamine than in rats pretreated with only amphetamine. ConclusionThe present demonstration that ceftriaxone attenuates amphetamine-induced hyperactivity and behavioral sensitization suggests its documented efficacy against adverse cocaine and morphine effects extends to amphetamine.

Regulation of glutamate transporter GLT-1 by MAGI-1

The sodium-dependent glutamate transporter, glutamate transporter subtype 1 (GLT-1) is one of the main glutamate transporters in the brain. GLT-1 contains a COOH-terminal sequence similar to one in an isoform of Slo1 K(+) channel protein previously shown to bind MAGI-1 (membrane-associated guanylate kinase with inverted orientation protein-1). MAGI-1 is a scaffold protein which allows the formation of complexes between certain transmembrane proteins, actin-binding proteins, and other regulatory proteins. The glutathione S-transferase pull-down assay demonstrated that MAGI-1 was a binding partner of GLT-1. The interaction between MAGI-1 and GLT-1 was confirmed by co-immunoprecipitation. Immunofluorescence of MAGI-1 and GLT-1 demonstrated that the distribution of MAGI-1 and GLT-1 overlapped in astrocytes. Co-expression of MAGI-1 with GLT-1 in C6 Glioma cells resulted in a significant reduction in the surface expression of GLT-1, as assessed by cell-surface biotinylation. On the other hand, partial knockdown of endogenous MAGI-1 expression by small interfering RNA in differentiated cultured astrocytes increased glutamate uptake and the surface expression of endogenous GLT-1. Knockdown of MAGI-1 increased dihydrokainate-sensitive, Na(+) -dependent glutamate uptake, indicating that MAGI-1 regulates GLT-1 mediated glutamate uptake. These data suggest that MAGI-1 regulates surface expression of GLT-1 and the level of glutamate in the hippocampus.

Insulin increases glutamate transporter GLT1 in cultured astrocytes

The astroglial cell-specific glutamate transporter subtype 2 (excitatory amino acid transporter 2, GLT1) plays an important role in excitotoxicity that develops after damage to the central nervous system (CNS) is incurred. Both the protein kinase C signaling pathway and the epidermal growth factor (EGF) pathway have been suggested to participate in the modulation of GLT1, but the modulatory mechanisms of GLT1 expression are not fully understood. In the present study, we aimed to evaluate the effects of insulin on GLT1 expression. We found that short-term stimulation of insulin led to the upregulation of both total and surface expressions of GLT1. Akt phosphorylation increased after insulin treatment, and triciribine, the inhibitor of Akt phosphorylation, significantly inhibited the effects of insulin. We also found that the upregulation of GLT1 expression correlated with increased kappa B motif-binding phosphoprotein (KBBP) and GLT1 mRNA levels. Our results suggest that insulin may modulate the expression of astrocytic GLT1, which might play a role in reactive astrocytes after CNS injuries.

MRNA for the EAAC1 subtype of glutamate transporter is present in neuronal dendrites in vitro and dramatically increases in vivo after a seizure

The neuronal Na+-dependent glutamate transporter, excitatory amino acid carrier 1 (EAAC1, also called EAAT3), has been implicated in the control of synaptic spillover of glutamate, synaptic plasticity, and the import of cysteine for neuronal synthesis of glutathione. EAAC1 protein is observed in both perisynaptic regions of the synapse and in neuronal cell bodies. Although amino acid residues in the carboxyl terminal tail have been implicated in the dendritic targeting of EAAC1 protein, it is not known if mRNA for EAAC1 may also be targeted to dendrites. Sorting of mRNA to specific cellular domains provides a mechanism by which signals can rapidly increase translation in a local environment; this form of regulated translation has been linked to diverse biological phenomena ranging from establishment of polarity during embryogenesis to synapse development and synaptic plasticity. In the present study, EAAC1 mRNA sequences were amplified from dendritic samples that were mechanically harvested from low-density hippocampal neuronal cultures. In parallel analyses, mRNA for histone deacetylase 2 (HDAC-2) and glial fibrillary acidic protein (GFAP) was not detected, suggesting that these samples are not contaminated with cell body or glial mRNAs. EAAC1 mRNA also co-localized with Map2a (a marker of dendrites) but not Tau1 (a marker of axons) in hippocampal neuronal cultures by in situ hybridization. In control rats, EAAC1 mRNA was observed in soma and proximal dendrites of hippocampal pyramidal neurons. Following pilocarpine- or kainate-induced seizures, EAAC1 mRNA was present in CA1 pyramidal cell dendrites up to 200μm from the soma. These studies provide the first evidence that EAAC1 mRNA localizes to dendrites and suggest that dendritic targeting of EAAC1 mRNA is increased by seizure activity and may be regulated by neuronal activity/depolarization.

Sequences of the non-coding RNA, NTAB, are contained within the 3'-UTR of human and rat EAAT2/GLT-1 transcripts and act as transcriptional enhancers.

In the CNS, extracellular glutamate is predominantly cleared by astroglial cells through the high-affinity glutamate transporter subtype, EAAT2/GLT-1. Expression of EAAT2/GLT-1 is perturbed in various acute and chronic brain diseases eventually allowing for the onset of neurotoxic extracellular glutamate concentrations and subsequent excitotoxic neuronal cell death. The idea that glutamate-induced brain damage could be prevented by restoring glutamate homeostasis in the injured brain, spurred considerable interest in identifying the mechanisms controlling EAAT2/GLT-1 expression. Since to date most of this study was done with rat astrocytes, an emerging issue is to whether these findings would also apply to humans. While so far it is known that the promoter region of the EAAT2/GLT-1 gene is strikingly similar in rat and man, little information is available on the function of the EAAT2/GLT-1 3'-UTR in the control of EAAT2/GLT-1 expression in general as well as across both species. We now report on the presence of a homologous sequence within the 3'-UTR of the human and rat EAAT2/GLT-1 gene which we identified as a partial sequence of the putative non-coding RNA, Ntab. We further demonstrate that fragments of Ntab act as enhancers of EAAT2/GLT-1 transcription. Finally, we unravel that partial Ntab sequences are selectively present in the vicinity of the EAAT2/GLT-1 gene in several other mammalians, implying a conserved function of this sequence in the vertebrate CNS.

β-Lactam antibiotic produces a sustained reduction in extracellular glutamate in the nucleus accumbens of rats

We investigated the short- and long-term effects of ceftriaxone on glutamate transporter subtype 1 (GLT-1) transporter activity and extracellular glutamate in the rat nucleus accumbens. Repeated ceftriaxone administration (50, 100 or 200 mg/kg, i.p.) produced a dose-dependent reduction in glutamate levels that persisted for 20 days following discontinuation of drug exposure. The ceftriaxone effect was prevented by the GLT-1 transporter inhibitor dihydrokainate (1 μM, intra-accumbal). These results suggest that β-lactam antibiotics produce an enduring reduction in glutamatergic transmission in the brain reward center.

Nocistatin excites rostral agranular insular cortex-periaqueductal gray projection neurons by enhancing transient receptor potential cation conductance via Gαq/11-PLC-protein kinase C pathway

Rostral agranular insular cortex (RAIC) projects to periaqueductal gray (PAG) and inhibits spinal nociceptive transmission by activating PAG-rostral ventromedial medulla (RVM) descending antinociceptive circuitry. Despite being generated from the same precursor prepronociceptin, nocistatin (NST) and nociceptin/orphanin FQ (N/OFQ) produce supraspinal analgesic and hyperalgesic effects, respectively. Prepronociceptin is highly expressed in the RAIC. In the present study, we hypothesized that NST and N/OFQ modulate spinal pain transmission by regulating the activity of RAIC neurons projecting to ventrolateral PAG (RAIC–PAG). This hypothesis was tested by investigating electrophysiological effects of N/OFQ and NST on RAIC–PAG projection neurons in brain slice. Retrogradely labeled RAIC–PAG projection neurons are layer V pyramidal cells and express mRNA of vesicular glutamate transporter subtype 1, a marker for glutamatergic neurons. N/OFQ hyperpolarized 25% of RAIC–PAG pyramidal neurons by enhancing inwardly rectifying potassium conductance via pertussis toxin-sensitive Gαi/o. In contrast, NST depolarized 33% of RAIC–PAG glutamatergic neurons by causing the opening of canonical transient receptor potential (TRPC) cation channels through Gαq/11-phospholipase C-protein kinase C pathway. There were two separate populations of RAIC–PAG pyramidal neurons, one responding to NST and the other one to N/OFQ. Our results suggest that Gαq/11-coupled NST receptor mediates NST excitation of RAIC–PAG glutamatergic neurons, which is expected to cause the supraspinal analgesia by enhancing the activity of RAIC–PAG–RVM antinociceptive pathway. Opposite effects of NST and N/OFQ on supraspinal pain regulation are likely to result from their opposing effects on RAIC–PAG pyramidal neurons.

Astrocyte processes exhibit confined mGluR5 diffusion to maintain high mGluR5 density and high sensitivity to mGluR agonist

Around excitatory synapses in cerebellar Purkinje cells (PCs), GLAST and EAAT4 are expressed as predominant glial and neuronal glutamate transporters, respectively. EAAC1, another subtype of neuronal glutamate transporter, is also expressed in PCs. EAAT4 is co-localized with metabotropic glutamate receptors (mGluRs) at perisynaptic sites in excitatory synapses in PCs, and this neuronal transporter was reported to be involved in the regulation of mGluR activation induced by the stimulation of parallel fibers (PFs). However, it remains to be elucidated whether only EAAT4 is specifically involved in mGluR activation among the glutamate transporters expressed near excitatory synapses in PCs. Here we examined mGluR-mediated excitatory postsynaptic currents (mGluR-EPSCs) evoked by PF stimulation in cerebellar slices of mice deficient in EAAT4, EAAC1, or GLAST. PF-evoked mGluR-EPSCs showed larger amplitude and faster rising kinetics in EAAT4-deficient mice than in the wild-type mice. In contrast, there was no significant difference in either the amplitude or the rising kinetics of mGluR-EPSCs in GLAST- or EAAC1-deficient mice compared to wild-type mice. We conclude that EAAT4 is most closely involved in mGluR activation in PCs among the glutamate transporters.

Beta-lactam antibiotic reduces morphine analgesic tolerance in rats through GLT-1 transporter activation

Glutamate transporter subtype 1 (GLT-1) activation is a promising – and understudied – approach for managing aspects of morphine tolerance caused by increased glutamatergic transmission. Identification of beta-lactam antibiotics as pharmaceuticals which activate GLT-1 transporters prompted us to hypothesize that repeated beta-lactam antibiotic (ceftriaxone) administration blocks development of tolerance to morphine antinociception through GLT-1 activation. Here, we injected rats with morphine (10 mg/kg, s.c.) twice daily for 7 days to induce tolerance and used the hot-plate assay to determine antinociception on days 1, 4 and 7 of repeated morphine administration. Ceftriaxone and a selective GLT-1 transporter inhibitor dihydrokainate (DHK) were co-administered with morphine to determine if GLT-1 activation mediated the ceftriaxone effect. Tolerance was present on days 4 and 7 of repeated morphine administration. Ceftriaxone (50, 100 or 200 mg/kg, i.p.) administration dose-dependently blocked development of morphine tolerance. DHK (10 mg/kg, s.c.), administered 15 min before each morphine injection, prevented inhibition of morphine tolerance by ceftriaxone (200 mg/kg, i.p.). These results identify an interaction between ceftriaxone and morphine in opioid-tolerant rats and suggest beta-lactam antibiotics preserve analgesic efficacy during chronic morphine exposure.

Comparative structural and functional analysis of the GLT‐1/EAAT‐2 promoter from man and rat

In the vertebrate CNS, glutamate transport predominantly occurs through the glutamate transporter subtype, GLT-1/EAAT-2, which prevails in astrocytes. GLT-1/EAAT-2 expression is impaired in many acute and chronic brain diseases, leading to increases in extracellular glutamate and subsequent excitotoxic neuronal cell death. An obvious therapeutical approach to prevent glutamate-induced brain damage would be targeting GLT-1/EAAT-2 expression. Since so far, insights into the mechanisms modulating GLT-1/EAAT-2 expression mostly originated from work with rat astrocytes, we now sought to determine whether this modulatory network would also apply to humans. To this end, we have cloned the previously unknown rat GLT-1/EAAT-2 promoter and compared it to the human promoter sequence. In reporter assays, the cloned 2.7-kb region immediately flanking the 5'-end of the rat GLT-1/EAAT-2 gene allowed for similar increases in constitutive gene expression as the human promoter sequence. Sequence analysis demonstrated the presence of highly conserved regions on the rat and human GLT-1/EAAT-2 promoters, which turned out to be likewise essential for constitutive GLT-1/EAAT-2 expression, stimulation of gene transcription by EGF, TGFalpha, and PACAP as well as inhibition of gene transcription by TNFalpha. Intriguingly, endothelin-1 which inhibits endogenous GLT-1/EAAT-2 expression, promoted activity of both rat and human reporter constructs, indicating the existence of (an) inhibitory mechanism(s) not operational in the reporter gene assay. Our findings establish close similarities in the regulation of GLT-1/EAAT-2 expression in rat and man and, hence, validate rat astrocytes as an assay system for studying the molecular mechanisms affecting glutamate homeostasis in the healthy and diseased human brain.

Icilin-induced wet-dog shakes in rats are dependent on NMDA receptor activation and nitric oxide production

Icilin is a cold channel agonist that produces vigorous wet-dog shaking in rats. The shaking is accompanied by an increase in the level of extracellular glutamate in the brain. Hence, we hypothesized that icilin-induced wet-dog shakes are dependent on increased glutamatergic transmission and nitric oxide (NO) production. Rats injected with icilin (0.5, 1, 2.5, 5 mg/kg, i.p.) displayed a dose-related increase in wet-dog shakes. Pretreatment with LY 235959 (1, 2 mg/kg, i.p.), a NMDA receptor antagonist, or L-NAME (50 mg/kg, i.p.), a NO synthase (NOS) inhibitor, attenuated icilin-induced wet-dog shakes. The shaking was also reduced by intracerebroventricular L-NAME (1 mg/rat, i.c.v.) administration, indicating that the stimulant effect of icilin is dependent on central NO production. Pretreatment with 6,7-dinitroquinoxaline-2,3(1H,4H)-dione (DNQX) (10, 20 mg/kg, i.p.), an AMPA receptor antagonist, or ceftriaxone (200 mg/kg, i.p. for 5 days), a beta-lactam antibiotic and glutamate transporter subtype 1 (GLT-1) activator, did not alter the incidence of icilin-induced shaking. The present data reveal that icilin produces behavioral stimulation by a mechanism requiring NMDA receptor activation and nitric oxide production and suggest that glutamate and NO signaling play important roles in cold channel pharmacology.

Synaptically Evoked Glutamate Transporter Currents in Spinal Dorsal Horn Astrocytes

BackgroundRemoving and sequestering synaptically released glutamate from the extracellular space is carried out by specific plasma membrane transporters that are primarily located in astrocytes. Glial glutamate transporter function can be monitored by recording the currents that are produced by co-transportation of Na+ ions with the uptake of glutamate. The goal of this study was to characterize glutamate transporter function in astrocytes of the spinal cord dorsal horn in real time by recording synaptically evoked glutamate transporter currents.ResultsWhole-cell patch clamp recordings were obtained from astrocytes in the spinal substantia gelatinosa (SG) area in spinal slices of young adult rats. Glutamate transporter currents were evoked in these cells by electrical stimulation at the spinal dorsal root entry zone in the presence of bicuculline, strychnine, DNQX and D-AP5. Transporter currents were abolished when synaptic transmission was blocked by TTX or Cd2+. Pharmacological studies identified two subtypes of glutamate transporters in spinal astrocytes, GLAST and GLT-1. Glutamate transporter currents were graded with stimulus intensity, reaching peak responses at 4 to 5 times activation threshold, but were reduced following low-frequency (0.1 – 1 Hz) repetitive stimulation.ConclusionThese results suggest that glutamate transporters of spinal astrocytes could be activated by synaptic activation, and recording glutamate transporter currents may provide a means of examining the real time physiological responses of glial cells in spinal sensory processing, sensitization, hyperalgesia and chronic pain.

Endothelin-1 reverses the histone deacetylase inhibitor-induced increase in glial glutamate transporter transcription without affecting histone acetylation levels

Astrocytes play a crucial role in maintaining glutamate homeostasis in the intact central nervous system. This function is profoundly impaired in the acutely and chronically diseased brain as evidenced by the decreased expression of the glial glutamate transporters, GLT-1/EAAT-2 and/or GLAST/EAAT-1, and a subsequent increase in extracellular glutamate concentrations which in turn induce excitotoxic neuronal cell death. We recently provided evidence that these disease-related disturbances of glial glutamate transport might be mediated in part by endothelins. This family of peptides not only massively reduces basal expression of GLT-1/EAAT-2 and GLAST/EAAT-1 in cultured astrocytes, but also completely overrides the exogenously induced expression of both glutamate transporter subtypes. The observed potency of this effect now prompted us to investigate whether endothelins inhibit glial glutamate transporter expression through an epigenetic, protein acetylation-dependent mechanism. We found that treating cultured astrocytes with the histone deacetylase inhibitor, trichostatin A, promotes transcription of both the GLT-1 and GLAST genes. These stimulatory influences were associated with an overall increase in acetylation of histones H3 and H4. The additional presence of endothelin-1 completely prevented the trichostatin A-induced increases in GLT-1- and GLAST expression without profoundly altering H3 and H4 acetylation levels. We conclude that endothelins inhibit glial glutamate transporter expression through an acetylation/deacetylation-independent process. We further suggest that modulating the activity of histone deacetylases represent an additional mechanism by which disease-associated disturbances of glial glutamate transporter expression are mediated.

The expression of glutamate transporters in chest compression-induced audiogenic epilepsy: a comparative study

Background: This study was conducted to compare the expression of three glutamate transporter subtypes (GLAST, GLT-1 and EAAC1) in rats undergoing chest compression-induced global cerebral ischemia in the presence and absence of cerebral ischemia-related epilepsy.Material and methods: A reliable rat model of global cerebral ischemia-related epilepsy was established. The rats were divided into the following groups: sham surgery group (Group S), global cerebral ischemia without epilepsy (Group I) and global cerebral ischemia with epilepsy (Group E). The latter two groups were further divided into four subgroups based on time (24 hours, 72 hours, 5 days and 7 days) after 8 minutes of chest compression. Electroencephalographic recordings were obtained in all rats. Hippocampal tissue samples were prepared, and the expression of GLAST, GLT-1 and EAAC1 in the hippocampal CA1 region and the motor cortex area was studied using immunohistochemical methods.Results: Seizure developed in 32 (64%) of 50 rats. Compared with that in group I, the expression of GLT-1 in the hippocampal CA1 region and the motor cortex area in group E was down-regulated, and EAAC1 was up-regulated in those regions.Conclusion: Altering the expression of GLT-1 and EAAC1 through some means might lead them to be potential targets for therapy in cerebral ischemia-related epilepsy.