Removal of excitatory amino acids from the extracellular fluid is essential for synaptic transmission and for avoiding excitotoxicity. The removal is accomplished by glutamate transporters located in the plasma membranes of both neurons and astroglia. The uptake system consists of several different transporter proteins that are carefully regulated, indicating more refined functions than simple transmitter inactivation. Here we show by chemical cross-linking, followed by electrophoresis and immunoblotting, that three rat brain glutamate transporter proteins (GLAST, GLT and EAAC) form homomultimers. The multimers exist not only in intact brain membranes but also after solubilization and after reconstitution in liposomes. Increasing the crosslinker concentration increased the immunoreactivity of the bands corresponding to trimers at the expense of the dimer and monomer bands. However, the immunoreactivities of the dimer bands did not disappear, indicating a mixture of dimers and trimers. GLT and GLAST do not complex with each other, but as demonstrated by double labeling post-embedding electron microscopic immunocytochemistry, they co-exist side by side in the same astrocytic cell membranes. The oligomers are held together noncovalently in vivo. In vitro, oxidation induces formation of covalent bonds (presumably -S-S-) between the subunits of the oligomers leading to the appearance of oligomer bands on SDS-polyacrylamide gel electrophoresis. Immunoprecipitation experiments suggest that GLT is the quantitatively dominant glutamate transporter in the brain. Radiation inactivation analysis gives a molecular target size of the functional complex corresponding to oligomeric structure. We postulate that the glutamate transporters operate as homomultimeric complexes.

Antibodies to an excitatory amino acid transporter (EAAT4) label a glycoprotein of ϳ65 kDa strongly in the cerebellum and weakly in the forebrain. Cross-linking of cerebellar proteins with bis(sulfosuccinimidyl) suberate before solubilization causes dimer bands of EAAT4 and both dimer and trimer bands of the other glutamate transporters GLAST (EAAT1) and GLT (EAAT2) to appear on immunoblots. In contrast to GLAST, GLT, and EAAC (EAAT3), EAAT4 is unevenly distributed in the cerebellar molecular layer, being strongly expressed in parasagittal zones. It is located in cerebellar Purkinje cells, and the highest concentrations are seen in ones expressing high levels of zebrin II (aldolase C). The labeling of Purkinje cell spines and thin dendrites is stronger than that of large diameter dendrites and cell bodies. EAAT4 is present at low concentrations in the synaptic membrane, but is highly enriched in the parts of the dendritic and spine membranes facing astrocytes (which express GLAST and GLT) compared with parts facing neuronal membranes, suggesting a functional relationship with glial glutamate transporters. The presence of EAAT4 in intracellular cisterns and multivesicular organelles may reflect turnover of transporter in the plasma membrane. The total Purkinje cell spine surface and the EAAT4 concentration were found to be 1.1 m 2 /cm 3 and 0.2 mg/cm 3 , respectively, in the molecular layer, corresponding to 1800 molecules/m 2 . The juxtasynaptic location of EAAT4 may explain electrophysiological observations predicting the presence of a neuronal glutamate transporter near the release site at a Purkinje cell spine synapse. EAAT4 may function as a combined transporter and inhibitory glutamate receptor. Key words: neurotransmitter transport; neurons; glutamate uptake; antipeptide antibodies; immunocytochemistry; cerebellumThe extracellular concentration of the excitatory transmitter glutamate is kept low by transporter proteins located in the plasma membranes. These transporters (for review, see Danbolt, 1994;Robinson and Dowd, 1997) are essential for securing a high signal-to-noise ratio in synaptic transmission and for preventing harmf ul receptor overstimulation. The complexity of the uptake system suggests that its f unctions are more refined than simple transmitter removal. Five different glutamate (excitatory amino acid) transporters have been cloned so far: GL AST (EAAT1) (Storck et al., 1992;Tanaka et al., 1993), GLT (EAAT2) (Pines et al., 1992), EAAC (EAAT3) (Kanai and Hediger, 1992), EAAT4 (Fairman et al., 1995), and EAAT5 (Arriza et al., 1997). The proteins GLT and GL AST have only been demonstrated in astrocytes in the brain Lev y et al., 1993;Chaudhry et al., 1995;Ginsberg et al., 1995;Lehre et al., 1995;Schmitt et al., 1996Schmitt et al., , 1997. EAAC is neuronal and probably predominantly postsynaptic (Rothstein et al., 1994). EAAT4 is a neuronal postsynaptic glutamate transporter in Purkinje cell spines (Yamada et al., 1996;Nagao et al., 1997). The expression of the transporters is highly differenti...

The relative distribution of the excitatory amino acid transporter 2 (EAAT2) between synaptic terminals and astroglia, and the importance of EAAT2 for the uptake into terminals is still unresolved. Here we have used antibodies to glutaraldehyde-fixed D-aspartate to identify electron microscopically the sites of D-aspartate accumulation in hippocampal slices. About 3/4 of all terminals in the stratum radiatum CA1 accumulated D-aspartate-immunoreactivity by an active dihydrokainate-sensitive mechanism which was absent in EAAT2 glutamate transporter knockout mice. These terminals were responsible for more than half of all D-aspartate uptake of external substrate in the slices. This is unexpected as EAAT2-immunoreactivity observed in intact brain tissue is mainly associated with astroglia. However, when examining synaptosomes and slice preparations where the extracellular space is larger than in perfusion fixed tissue, it was confirmed that most EAAT2 is in astroglia (about 80%). Neither D-aspartate uptake nor EAAT2 protein was detected in dendritic spines. About 6% of the EAAT2-immunoreactivity was detected in the plasma membrane of synaptic terminals (both within and outside of the synaptic cleft). Most of the remaining immunoreactivity (8%) was found in axons where it was distributed in a plasma membrane surface area several times larger than that of astroglia. This explains why the densities of neuronal EAAT2 are low despite high levels of mRNA in CA3 pyramidal cell bodies, but not why EAAT2 in terminals account for more than half of the uptake of exogenous substrate by hippocampal slice preparations. This and the relative amount of terminal versus glial uptake in the intact brain remain to be discovered. NIH Public Access Author ManuscriptNeuroscience. Author manuscript; available in PMC 2009 November 11. Published in final edited form as:Neuroscience. Glutamate uptake into glia and neurons is essential for controlling the excitatory action of glutamate (for reviews see: Danbolt, 2001;Beart and O'Shea, 2007). It has been much debated whether the uptake activity of glutamatergic nerve terminals represents a major proportion of the total brain tissue uptake activity. The prevailing view is that most brain glutamate uptake is performed by astroglia, because (a) most of the glutamate uptake in forebrain tissue slices and synaptosome preparations is both dihydrokainate sensitive and dependent on the excitatory amino acid transporter (EAAT) 2 (GLT1; slc1a2) gene and protein, and (b) the highest numbers of dihydrokainate sensitive EAAT2 glutamate transporters are found in glial cells (for review see: Danbolt, 2001). This view, however, does not take account of a substantial amount of data showing a significant uptake into glutamatergic nerve terminals (e.g. Beart, 1976; StormMathisen, 1977;Gundersen et al., 1993Gundersen et al., , 1996Suchak et al., 2003;Xu et al., 2003;Waagepetersen et al., 2005; for a detailed discussion about the existence of nerve terminal glutamate up-take, see section 4.2 in Danbolt, 20...

The extracellular concentration of the excitatory neurotransmitter glutamate is kept low by the action of glutamate transporters in the plasma membranes of both neurons and glial cells. These transporters may play important roles, not only in the adult brain, but also in the developing brain, as glutamate is thought to modulate the formation and elimination of synapses as well as neuronal migration, proliferation and apoptosis. Here we demonstrate the developmental changes in the expression of two glutamate transporters, GLAST and GLT, by quantitative immunoblotting and by light and electron microscopic immunocytochemistry. At birth, GLT is not detectable, but GLAST is present at significant concentrations both in the forebrain and in the cerebellum. GLT is first detected in the forebrain and cerebellum in the second and third week, respectively. Both transporters reach adult levels by postnatal week 5. The development of the total glutamate uptake activity in the forebrain, as determined by solubilization and reconstitution of the transporters in liposomes, parallels that of GLT, in agreement with the observation that GLT is the predominant transporter in the adult brain. The regional distributions of both GLAST and GLT in the tissue are similar in young and adult rats. Only GLAST is detectable in the external germinal layer of the cerebellar cortex. Electron microscopical investigation demonstrated GLAST and GLT exclusively in glial cells in young as well as in adult animals.

We studied the early and late effects of L-trans-pyrrolidine-2,4-dicarboxylate (PDC), a competitive inhibitor of glutamate uptake with low affinity for glutamate receptors, in co-cultures of rat cortical neurons and glia expressing spontaneous excitatory amino acid (EAA) neurotransmission. At 100 or 200 microM, PDC induced different patterns of electrical changes: 100 microM prolonged tetrodotoxin-sensitive excitation triggered by synaptic glutamate release; 200 microM produced sustained, tetrodotoxin-insensitive and EAA-mediated neuronal depolarization, overwhelming synaptic activity. At 200 microM, but not at 100 microM, PDC caused rapid elevation of the glutamate concentration ([Glu]o) in the culture medium, resulting in NMDA receptor-mediated excitotoxic death of neurons 24 h later. The increase in [Glu]o was largely insensitive to tetrodotoxin, independent of extracellular Ca2+, and present also in astrocyte-pure cultures. By the use of glutamate transporters functionally reconstituted in liposomes, we showed directly that PDC activates carrier-mediated release of glutamate via heteroexchange. Glutamate release and delayed neurotoxicity in our cultures were suppressed if PDC was applied in a Na(+)-free medium containing Li+. However, replacement of Na+ with choline instead of Li+ did not result in an identical effect, suggesting that Li+ does not act simply as an external Na+ substitute. In conclusion, our data indicate that alteration of glutamate transport by PDC has excitotoxic consequences and that active release of glutamate rather than just uptake inhibition is responsible for the generation of neuronal injury.