Functions of glutamate transporters in cerebellar Purkinje cell synapses

Glutamate transporters play a critical role in the maintenance of low extracellular concentrations of glutamate, which prevents the overactivation of post‐synaptic glutamate receptors. Four distinct glutamate transporters, GLAST/EAAT1, GLT‐1/EAAT2, EAAC1/EAAT3 and EAAT4, are distributed in the molecular layer of the cerebellum, especially near glutamatergic synapses in Purkinje cells (PCs). This review summarizes the current knowledge about the differential roles of these transporters at excitatory synapses of PCs. Data come predominantly from electrophysiological experiments in mutant mice that are deficient in each of these transporter genes. GLAST expressed in Bergmann glia contributes to the clearing of the majority of glutamate that floods out of the synaptic cleft immediately after transmitter release from the climbing fibre (CF) and parallel fibre (PF) terminals. It is indispensable to maintain a one‐to‐one relationship in synaptic transmission at the CF synapses by preventing transcellular glutamate spillover. GLT‐1 plays a similar but minor role in the uptake of glutamate as GLAST. Although the loss of neither GLAST nor GLT‐1 affects cerebellar morphology, the deletion of both GLAST and GLT‐1 genes causes the death of the mutant animal and hinders the folium formation of the cerebellum. EAAT4 removes the low concentrations of glutamate that escape from uptake by glial transporters, preventing the transmitter from spilling over into neighbouring synapses. It also regulates the activation of metabotropic glutamate receptor 1 (mGluR1) in perisynaptic regions at PF synapses, which in turn affects mGluR1‐mediated events including slow EPSCs and long‐term depression. No change in synaptic function is detected in mice that are deficient in EAAC1.     

Glutamate transporters GLAST and EAAT4 regulate postischemic Purkinje cell death: an in vivo study using a cardiac arrest model in mice lacking GLAST or EAAT4

Cerebellar Purkinje cells represent a group of neurons highly vulnerable to ischemia. Excitotoxicity is thought to be an important pathophysiological mechanism in Purkinje cell death following ischemia. The glutamate transporter is the only mechanism for the removal of glutamate from the extracellular fluid in the brain. Therefore, glutamate transporters are believed to play a critical role in protecting Purkinje cells from ischemia-induced damage. Two distinct glutamate transporters, GLAST and EAAT4, are expressed most abundantly in the cerebellar cortex. GLAST is expressed in Bergmann glia, whereas EAAT4 is concentrated in the perisynaptic regions of Purkinje cell spines. However, the in vivo functional significance of these glial and neuronal glutamate transporters in postischemic Purkinje cell death is largely unknown. To clarify the role of these glutamate transporters in the protection of Purkinje cells after global brain ischemia, we evaluated Purkinje cell loss after cardiac arrest in mice lacking GLAST or EAAT4. We found that Purkinje cells with low EAAT4 expression were selectively lost after cardiac arrest in GLAST mutant mice. This result demonstrates that GLAST plays a role in preventing excitotoxic cerebellar damage after ischemia in concert with EAAT4.     

Roles of glial glutamate transporters in shaping EPSCs at the climbing fiber-Purkinje cell synapses

Glial glutamate transporters, GLAST and GLT-1, are co-localized in processes of Bergmann glia (BG) wrapping excitatory synapses on Purkinje cells (PCs). Although GLAST is expressed six-fold more abundantly than GLT-1, no change is detected in the kinetics of climbing fiber (CF)-mediated excitatory postsynaptic currents (CF-EPSCs) in PCs in GLAST(-/-) mice compared to the wild-type mice (WT). Here we aimed to clarify the mechanism(s) underlying this unexpected finding using a selective GLT-1 blocker, dihydrokainate (DHK), and a novel antagonist of glial glutamate transporter, (2S,3S)-3-[3-(4-methoxybenzoylamino)benzyloxy]aspartate (PMB-TBOA). In the presence of cyclothiazide (CTZ), which attenuates the desensitization of AMPA receptors, DHK prolonged the decay time constant (tau(w)) of CF-EPSCs in WT, indicating that GLT-1 plays a partial role in the removal of glutamate. The application of 100 nM PMB-TBOA, which inhibited CF-mediated transporter currents in BG by approximately 80%, caused no change in tau(w) in WT in the absence of CTZ, whereas it prolonged tau(w) in the presence of CTZ. This prolonged value of tau(w) was similar to that in GLAST(-/-) mice in the presence of CTZ. These results indicate that glial glutamate transporters can apparently retain the fast decay kinetics of CF-EPSCs if a small proportion ( approximately 20%) of functional transporters is preserved.     

Contribution of glutamate transporter GLT-1 to removal of synaptically released glutamate at climbing fiber-Purkinje cell synapses

Rapid removal of synaptically released glutamate from the extracellular space ensures a high signal-to-noise ratio in excitatory neurotransmission. In the cerebellum, glial glutamate transporters, GLAST and GLT-1, are co-localized in the processes of Bergmann glia wrapping excitatory synapses on Purkinje cells (PCs). Although GLAST is expressed six-fold more abundantly than GLT-1, the decay kinetics of climbing fiber-mediated excitatory postsynaptic currents (CF-EPSCs) in PCs in GLAST(−/−) mice are not different from those in wild-type (WT) mice. This raises a possibility that GLT-1 plays a significant role in clearing glutamate at CF-PC synapses despite its smaller amount of expression. Here, we studied the functions of GLT-1 and GLAST in the clearance of glutamate using GLAST(−/−) mice and GLT-1(−/−) mice. In the presence of cyclothiazide (CTZ) that attenuates the desensitization of AMPA receptors, the decay time constant of CF-EPSCs (τ_w) in GLT-1(−/−) mice was slower than that in WT mice. However, the degree of this prolongation of τ_w was less prominent compared to that in GLAST(−/−) mice. The values of τ_w in GLT-1(−/−) mice and GLAST(−/−) mice were comparable to those estimated in WT mice in the presence of a potent blocker of glial glutamate transporters (2S,3S)-3-[3-(4-methoxybenzoylamino)benzyloxy]aspartate (PMB-TBOA) at 10 and 100 nM, which reduced the amplitudes of glutamate transporter currents elicited by CF stimulation in Bergmann glia to ∼81 and ∼28%, respectively. We conclude that GLT-1 plays a minor role compared to GLAST in clearing synaptically released glutamate at CF-PC synapses.     

Interleukin-1 attenuates normal tension glaucoma-like retinal degeneration in EAAC1-deficient mice

Glaucoma, one of the leading causes of irreversible blindness, is characterized by progressive degeneration of retinal ganglion cells (RGCs) and optic nerves. Although glaucoma is often associated with elevated intraocular pressure, recent studies have shown a relatively high prevalence of normal tension glaucoma (NTG) in glaucoma patient populations. In the mammalian retina, glutamate/aspartate transporter (GLAST) is localized to Müller glial cells, whereas excitatory amino acid carrier 1 (EAAC1) is expressed in neural cells, including RGCs. Since the loss of GLAST or EAAC1 leads to retinal degeneration similar to that seen in NTG, we examined the effects of interleukin-1 (IL-1) on RGC death in GLAST- and EAAC1-deficient mice. IL-1 promoted increased glutamate uptake in Müller cells by suppressing intracellular Na⁺ accumulation, which is necessary to counteract Na⁺-glutamate cotransport. The observed trends for the glutamate uptake increase in the wild-type (WT), GLAST- and EAAC1-deficient mice were similar; however, the baseline glutamate uptake and intracellular Na⁺ concentration in the GLAST-deficient mice were significantly lower than those in the wild-type mice. Consistently, pretreatment with IL-1 exhibited no beneficial effects on glutamate-induced RGC degeneration in the GLAST-deficient mice. In contrast, IL-1 significantly increased glutamate uptake by Müller cells and the number of surviving RGCs in the wild-type and EAAC1-deficient mice. Our findings suggest that the use of IL-1 for enhancing the function of glutamate transporters may be useful for neuroprotection in retinal degenerative disorders including NTG.     

Involvement of group I metabotropic glutamate receptors and glutamate transporters in the slow excitatory synaptic transmission in the spinal cord dorsal horn

Our experiments demonstrate a novel role for group I metabotropic glutamate receptor (mGluR) subtypes 1 and 5 in generating a long-lasting synaptic excitation in the substantia gelatinosa (SG) and deep dorsal horn (DH) neurons of the rat spinal cord. In the present study we have investigated a slow excitatory postsynaptic current (EPSC), elicited by a brief high intensity (at Adelta/C fiber strength) and high frequency (20 or 100 Hz) stimulation of primary afferent fibers (PAFs) using whole-cell patch-clamp recordings from neurons located in the DH (laminae II-V) in spinal cord slices of young rats and wild-type and gene-targeted mice lacking mGluR1 subtype. The results shown here suggest that the activation of both mGluR1 and mGluR5 along with NK1 receptors, may be involved in the generation of the slow EPSC in the spinal cord DH. Inhibition of glial and neuronal glutamate transporters by dl-threo-beta-benzyloxyaspartate (TBOA) enhanced the group I mGluR-dependent slow EPSC about eightfold. Therefore, we conclude, that glutamate transporters strongly influence the group I mGluR activation by PAFs possibly at sensory synapses in the DH. Overall these data indicate that stimulus trains can generate a sustained and widespread glutamate signal that can further elicit prolonged EPSCs predominantly mediated by the group I mGluRs. These slow excitatory synaptic currents may have important functional implications for DH cell firing and synaptic plasticity of sensory transmission, including nociception.     

The glutamate/neutral amino acid transporter family SLC1: molecular, physiological and pharmacological aspects

The solute carrier family 1 (SLC1) includes five high-affinity glutamate transporters, EAAC1, GLT-1, GLAST, EAAT4 and EAAT5 (SLC1A1, SLC1A2, SLC1A3, SLC1A6, and SLC1A7, respectively) as well as the two neutral amino acid transporters, ASCT1 and ASCT2 (SLC1A4 and ALC1A5, respectively). Although each of these transporters have similar predicted structures, they exhibit distinct functional properties which are variations of a common transport mechanism. The high-affinity glutamate transporters mediate transport of l-Glu, l-Asp and d-Asp, accompanied by the cotransport of 3 Na(+) and 1 H(+), and the countertransport of 1 K(+), whereas ASC transporters mediate Na(+)-dependent exchange of small neutral amino acids such as Ala, Ser, Cys and Thr. The unique coupling of the glutamate transporters allows uphill transport of glutamate into cells against a concentration gradient. This feature plays a crucial role in protecting neurons against glutamate excitotoxicity in the central nervous system. During pathological conditions, such as brain ischemia (e.g. after a stroke), however, glutamate exit can occur due to "reversed glutamate transport", which is caused by a reversal of the electrochemical gradients of the coupling ions. Selective inhibition of the neuronal glutamate transporter EAAC1 (SLC1A1) may be of therapeutic interest to block glutamate release from neurons during ischemia. On the other hand, upregulation of the glial glutamate transporter GLT1 (SLC1A2) may help protect motor neurons in patients with amyotrophic lateral sclerosis (ALS), since loss of function of GLT1 has been associated with the pathogenesis of certain forms of ALS.     

Patterned expression of Purkinje cell glutamate transporters controls synaptic plasticity

Glutamate transporters are responsible for clearing synaptically released glutamate from the extracellular space. If expressed at high enough densities, transporters can prevent activation of extrasynaptic receptors by rapidly lowering glutamate concentrations to insignificant levels. We find that synaptic activation of metabotropic glutamate receptors expressed by Purkinje cells is prevented in regions of rat cerebellum where the density of the glutamate transporter EAAT4 is high. The consequences of metabotropic receptor stimulation, including activation of a depolarizing conductance, cannabinoid-mediated presynaptic inhibition and long-term depression, are also limited in Purkinje cells expressing high levels of EAAT4. We conclude that neuronal uptake sites must be overwhelmed by glutamate to activate perisynaptic metabotropic glutamate receptors. Regional differences in glutamate transporter expression affect the degree of metabotropic glutamate receptor activation and therefore regulate synaptic plasticity.     

The excitatory amino acid carrier 1 (EAAC1) in the rat nucleus of the solitary tract: subcellular localization suggests no major role in glutamate clearance

The excitatory amino acid carrier 1 (EAAC1) is a sodium-dependent glutamate transporter widely found in the mammalian brain and mainly localized in the somatodendritic compartment of neurons. The present study was performed to determine whether EAAC1 is present in the rat nucleus of the solitary tract (NST, a sensory brainstem nucleus involved in visceroception) and to document its subcellular localization. Using fluorescent immunolabeling, peroxidase immunostaining and quantitative immunogold labeling, we showed that both intracellular and plasma membrane-associated pools of EAAC1 transporters existed in dendrites of NST neurons. Although plasma membrane-associated transporters were more concentrated in the vicinity of synapses, no labeling was found at the axon–dendrite interface, suggesting that EAAC1 was not (or barely) expressed in this portion of dendritic membrane. Using computer simulation, we next showed that the ability of EAAC1 to efficiently take up synaptically released glutamate was very low outside the axon–dendrite interface. These data suggest that EAAC1 transporters present on NST dendrites may play a minor role if any in glutamate clearance.     

Neuronal glutathione deficiency and age-dependent neurodegeneration in the EAAC1 deficient mouse

Uptake of the neurotransmitter glutamate is effected primarily by transporters expressed on astrocytes, and downregulation of these transporters leads to seizures and neuronal death. Neurons also express a glutamate transporter, termed excitatory amino acid carrier-1 (EAAC1), but the physiological function of this transporter remains uncertain. Here we report that genetically EAAC1-null (Slc1a1(-/-)) mice have reduced neuronal glutathione levels and, with aging, develop brain atrophy and behavioral changes. EAAC1 can also rapidly transport cysteine, an obligate precursor for neuronal glutathione synthesis. Neurons in the hippocampal slices of EAAC1(-/-) mice were found to have reduced glutathione content, increased oxidant levels and increased susceptibility to oxidant injury. These changes were reversed by treating the EAAC1(-/-) mice with N-acetylcysteine, a membrane-permeable cysteine precursor. These findings suggest that EAAC1 is the primary route for neuronal cysteine uptake and that EAAC1 deficiency thereby leads to impaired neuronal glutathione metabolism, oxidative stress and age-dependent neurodegeneration.     

The Role of Glial Glutamate Transporters in Maintaining the Independent Operation of Juvenile Mouse Cerebellar Parallel Fibre Synapses

There is controversy over the extent to which glutamate released at one synapse can escape from the synaptic cleft and affect receptors at other synapses nearby, thereby compromising the synapse-specificity of information transmission. Here we show that the glial glutamate transporters GLAST and GLT-1 limit the activation of Purkinje cell AMPA receptors produced by glutamate diffusion between parallel fibre synapses in the cerebellar cortex of juvenile mice. For a single stimulus to the cerebellar molecular layer of wild-type mice, increasing the number of activated parallel fibres prolonged the parallel fibre EPSC, demonstrating an interaction between different synapses. Knocking out GLAST, or blocking GLT-1 in the absence of GLAST, prolonged the EPSC when many parallel fibres were stimulated but not when few were stimulated. When spatially separated parallel fibres were activated by granular layer stimulation, the EPSC prolongation produced by stimulating more fibres or reducing glutamate transport was greatly reduced. Thus, GLAST and GLT-1 curtail the EPSC produced by a single stimulus only when many nearby fibres are simultaneously activated. However when trains of stimuli were applied, even to a small number of parallel fibres, knocking out GLAST or blocking GLT-1 in the absence of GLAST greatly prolonged and enhanced the AMPA receptor-mediated current. These results show that glial cell glutamate transporters allow neighbouring synapses to operate more independently, and control the postsynaptic response to high frequency bursts of action potentials.     

Expression and distribution of ‘high affinity’ glutamate transporters GLT1, GLAST,EAAC1 and of GCPII in the rat peripheral nervous system

l ‐Glutamate is one of the major excitatory neurotransmitters in the mammalian central nervous system, but recently it has been shown to have a role also in the transduction of sensory input at the periphery, and in particular in the nociceptive pathway. An excess of glutamate is implicated in cases of peripheral neuropathies as well. Conventional therapeutic approaches for treating these diseases have focused on blocking glutamate receptors with small molecules or on reducing its synthesis of the receptors through the inhibition of glutamate carboxypeptidase II (GCPII), the enzyme that generates glutamate. In vivo studies have demonstrated that the pharmacological inhibition of GCPII can either prevent or treat the peripheral nerve changes in both BB/Wor and chemically induced diabetes in rats. In this study, we characterized the expression and distribution of glutamate transporters GLT1, GLAST, EAAC1 and of the enzyme GCPII in the peripheral nervous system of female Wistar rats. Immunoblotting results demonstrated that all glutamate transporters and GCPII are present in dorsal root ganglia (DRG) and the sciatic nerve. Immunofluorescence localization studies revealed that both DRG and sciatic nerves were immunopositive for all glutamate transporters and for GCPII. In DRG, satellite cells were positive for GLT1 and GCPII, whereas sensory neurons were positive for EAAC1. GLAST was localized in both neurons and satellite cells. In the sciatic nerve, GLT1 and GCPII were expressed in the cytoplasm of Schwann cells, whereas GLAST and EAAC1 stained the myelin layer. Our results give for the first time a complete characterization of the glutamate transporter system in the peripheral nervous system. Therefore, they are important both for understanding glutamatergic signalling in the PNS and for establishing new strategies to treat peripheral neuropathies.     

Glutamate transporter dysfunction associated with nerve injury-induced pain in mice

Dysfunction at glutamatergic synapses has been proposed as a mechanism in the development of neuropathic pain. Here we sought to determine whether peripheral nerve injury-induced neuropathic pain results in functional changes to primary afferent synapses. Signs of neuropathic pain as well as an induction of glial fibrillary acidic protein in immunostained spinal cord sections 4 days after partial ligation of the sciatic nerve indicated the induction of neuropathic pain. We found that following nerve injury, no discernable change to kinetics of dl-α-amino-3-hydroxy-5-methylisoxazole-propionic acid (AMPA) or N-methyl-d-aspartate receptor (NMDAR)-mediated evoked excitatory postsynaptic currents (eEPSCs) could be observed in dorsal horn (lamina I/II) neurons compared with those of na?ve mice. However, we did find that nerve injury was accompanied by slowed decay of the early phase of eEPSCs in the presence of glutamate transporter inhibition by the competitive nontransportable inhibitor dl-threo-β-benzyloxyaspartic acid (TBOA). Concomitantly, expression patterns for the two major glutamate transporters in the spinal cord, excitatory amino acid transporters (EAAT) 1 and EAAT2, were found to be reduced at this time (4 days postinjury). We then sought to directly determine whether nerve injury results in glutamate spillover to NMDARs at dorsal horn synapses. By employing the use-dependent NMDAR blocker (±)MK-801 to block subsynaptic receptors, we found that although TBOA-induced spillover to extrasynaptic receptors trended to increased activation of these receptors after nerve injury, this was not significant compared with na?ve mice. Together, these results suggest the development of neuropathic pain involves subtle changes to glutamate transporter expression and function that could contribute to neuropathic pain during excessive synaptic activity.     

Subcellular localization of the glutamate transporters GLAST and GLT at the neuromuscular junction in rodents

The vertebrate neuromuscular junction (NMJ) is known to be a cholinergic synapse at which acetylcholine (ACh) is released from the presynaptic terminal to act on postsynaptic nicotinic ACh receptors. There is now growing evidence that glutamate, which is the main excitatory transmitter in the CNS and at invertebrate NMJs, may have a signaling function together with ACh also at the vertebrate NMJ. In the CNS, the extracellular concentration of glutamate is kept at a subtoxic level by Na(+)-driven high-affinity glutamate transporters located in plasma membranes of astrocytes and neurons. The glutamate transporters are also pivotal for shaping glutamate receptor responses at synapses. In order to throw further light on the potential role of glutamate as a cotransmitter at the NMJ we used high-resolution immunocytochemical methods to investigate the localization of the plasma membrane glutamate transporters GLAST (glutamate aspartate transporter) and GLT (glutamate transporter 1) in rat and mice NMJ regions. Confocal laser-scanning immunocytochemistry showed that GLT is restricted to the NMJ in rat and mouse skeletal muscle. Lack of labeling signal in knock-out mice confirmed that the immunoreactivity observed at the NMJ was specific for GLT. GLAST was also localized at the NMJ in rat but not detected in mouse NMJ (while abundant in mouse brain). Post-embedding electron microscopic immunocytochemistry and quantitative analyses in rat showed that GLAST and GLT are enriched in the junctional folds of the postsynaptic membrane at the NMJ. GLT was relatively higher in the slow-twitch muscle soleus than in the fast-twitch muscle extensor digitorum longus, whereas GLAST was relatively higher in extensor digitorum longus than in soleus. The findings show--together with previous demonstration of vesicular glutamate, a vesicular glutamate transporter and glutamate receptors--that mammalian NMJs contain the machinery required for synaptic release and action of glutamate. This indicates a signaling role for glutamate at the normal NMJ and provides a basis for the ability of denervated muscle to be reinnervated by glutamatergic axons from the CNS.     

Comparative analysis of glutamate transporter expression in rat brain using differential double in situ hybridization

 This study compares the mRNA expression pattern for the three glutamate transporters EAAC1, GLT1 and GLAST in rat brain, using a sensitive non-radioactive in situ hybridization technique. The results confirm the predominantly neuronal localization of EAAC1 mRNA, the astroglial and ependymal localization of GLAST mRNA and the astroglial and neuronal localization of GLT1 mRNA. Further, we demonstrate, using a novel differential double hybridization protocol, that the presence of GLT1 mRNA in neurons is more widespread than previously thought, and that it encompasses the majority of neurons in the neocortex, neurons in the external plexiform layer in the olfactory bulb, neurons in dorsal and ventral parts of the anterior olfactory nucleus, the majority of neurons in the anteromedial thalamic nuclei, the CA3 pyramidal neurons in the hippocampus and neurons in the inferior olive. In addition, we demonstrate marked variations in the expression levels of GLT1 and GLAST mRNAs in different brain areas, suggesting that their mRNA levels are regulated by different mechanisms. Finally, for EAAC1 we demonstrate also a widespread distribution and a marked heterogeneity in the expression levels. EAAC1 is strongly expressed by a heretofore unrecognized group of cells in white matter tracts such as the corpus callosum, fimbria-fornix or anterior commissure. Also, strong EAAC1 expression is present in groups of scattered cells in grey matter areas of much of the forebrain and the cerebellum. These results provide more detailed information about the precise cellular localization of these three glutamate transporters and their regulation at the mRNA level. Accepted: 29 January 1998     

Mammalian ion-coupled solute transporters.

Active transport of solutes into and out of cells proceeds via specialized transporters that utilize diverse energy-coupling mechanisms. Ion-coupled transporters link uphill solute transport to downhill electrochemical ion gradients. In mammals, these transporters are coupled to the co-transport of H+, Na+, Cl- and/or to the countertransport of K+ or OH-. By contrast, ATP-dependent transporters are directly energized by the hydrolysis of ATP. The development of expression cloning approaches to select cDNA clones solely based on their capacity to induce transport function in Xenopus oocytes has led to the cloning of several ion-coupled transporter cDNAs and revealed new insights into structural designs, energy-coupling mechanisms and physiological relevance of the transporter proteins. Different types of mammalian ion-coupled transporters are illustrated by discussing transporters isolated in our own laboratory such as the Na+/glucose co-transporters SGLT1 and SGLT2, the H(+)-coupled oligopeptide transporters PepT1 and PepT2, and the Na(+)- and K(+)-dependent neuronal and epithelial high affinity glutamate transporter EAAC1. Most mammalian ion-coupled organic solute transporters studied so far can be grouped into the following transporter families: (1) the predominantly Na(+)-coupled transporter family which includes the Na+/glucose co-transporters SGLT1, SGLT2, SGLT3 (SAAT-pSGLT2) and the inositol transporter SMIT, (2) the Na(+)- and Cl(-)-coupled transporter family which includes the neurotransmitter transporters of gamma-amino-butyric acid (GABA), serotonin, dopamine, norepinephrine, glycine and proline as well as transporters of beta-amino acids, (3) the Na(+)- and K(+)-dependent glutamate/neurotransmitter family which includes the high affinity glutamate transporters EAAC1, GLT-1, GLAST, EAAT4 and the neutral amino acid transporters ASCT1 and SATT1 reminiscent of system ASC and (4) the H(+)-coupled oligopeptide transporter family which includes the intestinal H(+)-dependent oligopeptide transporter PepT1.