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 levels of excitatory amino acids are kept low by the action of the glutamate transporters. Glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) are the most abundant subtypes and are essential for the functioning of the mammalian CNS, but the contribution of the EAAC1 subtype in the clearance of synaptic glutamate has remained controversial, because the density of this transporter in different tissues has not been determined. We used purified EAAC1 protein as a standard during immunoblotting to measure the concentration of EAAC1 in different CNS regions. The highest EAAC1 levels were found in the young adult rat hippocampus. Here, the concentration of EAAC1 was ϳ0.013 mg/g tissue (ϳ130 molecules m Ϫ3 ), 100 times lower than that of GLT-1. Unlike GLT-1 expression, which increases in parallel with circuit formation, only minor changes in the concentration of EAAC1 were observed from E18 to adulthood. In hippocampal slices, photolysis of MNI-D-aspartate (4-methoxy-7-nitroindolinyl-D-aspartate) failed to elicit EAAC1-mediated transporter currents in CA1 pyramidal neurons, and D-aspartate uptake was not detected electron microscopically in spines. Using EAAC1 knock-out mice as negative controls to establish antibody specificity, we show that these relatively small amounts of EAAC1 protein are widely distributed in somata and dendrites of all hippocampal neurons. These findings raise new questions about how so few transporters can influence the activation of NMDA receptors at excitatory synapses.

Hippocampal GABA and glutamate transporter IR differ in TLE patients compared with autopsies. These data support the hypothesis that excitatory and inhibitory neurotransmission and seizure susceptibility could be altered by neuronal and glial transporters in TLE patients.

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Abstract-Specific antibodies are essential tools for identifying individual proteins in biological samples. While generation of antibodies is often straightforward, determination of the antibody specificity is not. Here we illustrate this by describing the production and characterization of antibodies to excitatory amino acid transporter 3 (EAAT3). We synthesized 13 peptides corresponding to parts of the EAAT3 sequence and immunized 6 sheep and 30 rabbits. All sera were affinity purified against the relevant immobilized peptide. Antibodies to the peptides were obtained in almost all cases. Immunoblotting with tissue extracts from wild type and EAAT3 knockout animals revealed that most of the antibodies did not recognize the native EAAT3 protein, and that some recognized other proteins. Several immunization protocols were tried, but strong reactions with EAAT3 were only seen with antibodies to the C-terminal peptides. In contrast, good antibodies were obtained to several parts of EAAT2. EAAT3 was only detected in neurons. However, rabbits immunized with an EAAT3-peptide corresponding to residues 479 -498 produced antibodies that labeled axoplasm and microtubules therein particularly strongly. On blots, these antibodies recognized both EAAT3 and a slightly smaller, but far more abundant protein that turned out to be tubulin. The antibodies were fractionated on columns with immobilized tubulin. One fraction contained antibodies apparently specific for EAAT3 while another fraction contained antibodies recognizing both EAAT3 and tubulin despite the lack of primary sequence identity between the two proteins. Addition of free peptide to the incubation solution blocked immunostaining of both EAAT3 and tubulin. Conclusions: Not all antibodies to synthetic peptides recognize the native protein. The peptide sequence is more important than immunization protocol. The specificity of an antibody is hard to predict because crossreactivity can be specific and to unrelated molecules. The antigen preabsorption test is of little value in testing the specificity of affinity purified antibodies.

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