Outside the community of biomedical scientists, glutamate is probably best known as “monosodium glutamate” or “MSG” which is the sodium salt of glutamic acid and a white crystalline solid used as a flavor or taste enhancer in food (food additive number E620). This, however, is not the reason for the enormous scientific interest in glutamate. The main motivation for the ongoing worldwide research on glutamate is that glutamate is the major excitatory transmitter in the brain. Like other signaling substances, the signaling effect of glutamate is not dependent on the chemical nature of glutamate, but on how cells are programmed to respond when exposed to it. Because the glutamate receptor proteins are expressed on the surface of the cells in such a way that they can only be activated from the outside, it follows that glutamate exerts its neurotransmitter function from the extracellular fluid. Consequently, control of receptor activation is achieved by releasing glutamate to the extracellular fluid and then removing glutamate from it. Because there are no enzymes extracellularly that can degrade glutamate, low extracellular concentrations require cellular uptake. This uptake is catalyzed by a family of transporter proteins located at the cell surface of both astrocytes and neurons (e.g. Danbolt 2001; Grewer and Rauen 2005; Tzingounis and Wadiche 2007; Vandenberg and Ryan 2013). Because glutamate is the major mediator of excitatory signals as well as of nervous system plasticity, including cell elimination, it follows that glutamate should be present at the right concentrations in the right places at the right time. It further follows that cells should have the correct sensitivity to glutamate and have energy enough to withstand normal stimulation, and that glutamate should be removed with the appropriate rates from the right locations. Both too much glutamate and too little glutamate are harmful. Excessive activation of glutamate receptors may excite nerve cells to their death in a process now referred to as “excitotoxicity”. This toxicity was initially perceived as a paradox like “Dr. Jekyll and Mr. Hyde”, but it is now clear that glutamate is toxic, not in spite of its importance, but because of it. As outlined before (Danbolt 2001), the intensity of glutamatergic stimulation that a given cell can tolerate, depends on several factors. As long as one variable is not extreme, it will be the combination of several factors that will determine the outcome. It took a long time to realize that glutamate is a neurotransmitter in part because of its abundance in brain tissue and in part because it is at the crossroad of multiple metabolic pathways (e.g. Erecinska and Silver 1990; Broman et al. 2000; McKenna 2007; Hertz 2013). There is 5–15 mmol glutamate per kg brain tissue, depending on the region, more than that of any other amino acid (Schousboe 1981). So although it was noted early on that glutamate plays a central metabolic role in the brain (Krebs 1935), that brain cells have a very high glutamate uptake activity (Stern et al. 1949) and that glutamate has an excitatory effect (Hayashi 1954; Curtis et al. 1959, 1960), the transmitter role was not realized until the early 1980s (for review see Fonnum 1984). In fact, glutamate metabolism is complex and compartmentalized (Berl et al. 1961, 1962; Van den Berg and Garfinkel 1971; Balcar and Johnston 1975). The important role of glutamate uptake in the control of the excitatory action of glutamate was recognized (Logan and Snyder 1971, 1972; Wofsey et al. 1971; Balcar and Johnston 1972). This became a hot research topic. A number of different glutamate and aspartate analogues were synthesized, and heterogeneity within glutamate uptake was uncovered suggesting more than one uptake mechanism (Ferkany and Coyle 1986; Robinson et al. 1991, 1993; Fletcher and Johnston 1991; Balcar and Li 1992; Rauen et al. 1992). Similarly, several families of glutamate receptor proteins were identified with molecular cloning (for review see Niswender and Conn 2010; Traynelis et al. 2010; Nicoletti et al. 2011). The receptors were classified as N-methyl-d-aspartate (NMDA) receptors (Gonda 2012; Bonaccorso et al. 2011; Santangelo et al. 2012), AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors (Rogawski 2013), kainate receptors (Lerma and Marques 2013) and metabotropic receptors (Gregory et al. 2013). Most, if not all, cells in the nervous system express at least one type of glutamate receptor (Steinhauser and Gallo 1996; Vernadakis 1996; Forsythe and Barnes-Davies 1997; Wenthold and Roche 1998; Petralia et al. 1999; Conti et al. 1999; Shelton and McCarthy 1999; Bergles et al. 2000). The locations and functional properties of each type are beyond the scope of this review. Medicinal chemists continued to synthesize new compounds and it is now possible to differentiate pretty well between the various receptors and transporters. Considering the relatively large number of proteins with ability to bind glutamate, it may seem strange that it is possible to find compounds that can distinguish between them. The reason is the high flexibility of the glutamate molecule which permits several conformations that are only minimally less favorable energetically at body temperature than the lowest energy conformation (Bridges et al. 1991). This implies that glutamate can take many shapes and explains, in part, why the various glutamate binding proteins (transporters, receptors, enzymes) can have quite different binding sites and still be able to bind glutamate. A large number of compounds are now available, and there are a number of excellent reviews on the topic (e.g. Bräuner-Osborne et al. 1997; Jensen and Bräuner-Osborne 2004; Shigeri et al. 2004; Ritzen et al. 2005; Thompson et al. 2005; Bridges and Esslinger 2005; Shimamoto 2008; Bridges et al. 2012a, b; Gregory et al. 2013; Gonda 2012; Bonaccorso et al. 2011). Early attempt to localize glutamate uptake sites were done using autoradiography in combination with tissue slices or synaptosome preparations (e.g. Minchin and Beart 1975; McLennan 1976; Beart 1976; Storm-Mathisen 1981; Storm-Mathisen and Wold 1981). To obtain higher resolution, thinner sections were needed. By using dry mount autoradiography (Young and Kuhar 1979; Danbolt et al. 1993) in combination with “sodium-dependent binding” of excitatory amino acids the uptake sites (for references see Danbolt 1994), higher resolution seemed to be within reach. However, heteroexchange complicated the interpretations as the amount of retained radioactively labeled ligand was dependent on both the number of transporter molecules and by the amount of endogenous dicarboxylic amino acid trapped within the membranes (Danbolt and Storm-Mathisen 1986a, b; Danbolt 1994). From the early days of glutamate research, it was believed that glutamate is taken up by glutamatergic nerve terminals (Fonnum 1984), but the finding that glial glutamate transporters are down-regulated after glutamatergic denervation (Levy et al. 1995), weakened the evidence (for a discussion, see sect. 4.2 in Danbolt 2001). By incubating tissue slices in d-aspartate and fixing the slices, it was possible to detect fixed d-aspartate with antibodies. With this technique, uptake in both astrocytes and nerve terminals was demonstrated at the electron microscopic level (Gundersen et al. 1993). d-aspartate is often used instead of l-glutamate as a probe for glutamate uptake because it is slowly metabolized in brain tissue (Davies and Johnston 1976). After the protein sequences of the transporters were known, synthetic peptides could be used to generate antibodies to the transporters themselves (Danbolt et al. 1998) rather than to the substrates. This led to an explosion in the use of antibodies to transporters, but, unfortunately, not all investigators validated their antibodies and procedures well enough (for detailed discussion see Holmseth et al. 2005, 2006, 2012a). The most difficult part is to obtain good negative controls. Antibodies may react with seemingly unrelated proteins (Holmseth et al. 2005; Zhou et al. 2014). In fact, antibody binding can always be achieved (see for instance Fig. 3 in: Holmseth et al. 2005). This is just a question of adjusting the assay conditions. Without a good negative control (e.g. tissue from knockout mice processed in parallel with tissue from wild-type mice), it is not possible to prove that the binding is to the antigen of interest. Therefore, antibody binding does not in itself prove that a given antigen is present. In this context it should be noted that the so called pre-adsorption test can easily give a false impression of specificity (Holmseth et al. 2012a). Whenever possible, it is a good idea to use additional methods, including in situ hybridization and Western blotting in combination with immunocytochemistry. TaqMan Real Time PCR is an excellent method for getting a first approximation of expression levels (e.g. Lehre et al. 2011; Zhou et al. 2012a). Another approach is to search available transcriptome and proteome datasets. For instance, proteome data from rat proximal tubules (http://dir.nhlbi.nih.gov/papers/lkem/pttr/) confirms the presence of EAAT3, but does not confirm expression of any of the other EAATs. Similarly, EAAT2 is in liver, but the other EAATs were not detected (http://141.61.102.16/), and neither the EAATs nor the VGLUTs were detected by proteome analysis of mouse pancreas (Zhou et al. 2014). Together, these data cast doubt over a large number of immunocytochemistry reports. The reason is obvious. Labeling with antibodies can always be obtained, and without good negative controls, it is not possible to tell if the labeling represents the antigen of interest or artifacts (see Holmseth et al. 2012a). Further, rapid post mortem proteolysis represents and additional challenge when studying human samples (Beckstrøm et al. 1999; Tessler et al. 1999; Li et al. 2012). Also note that water soluble proteins present in the samples may inhibit binding of transporters to the blotting membranes (Zhou et al. 2012b). Thus, strong upregulation of other proteins should be considered a potential source of error when estimating transporter levels by immunoblotting. Electron microscopy in combination with pre-embedding immunocytochemistry without detergents on unfrozen tissue is ideal for identification of labeled cell types, but is not ideal for subcellular distribution as the peroxidase reaction product diffuses some distance before precipitating. (Depending on the strength of the reaction, the reaction product may diffuse a couple of hundred nanometers.) In contrast, post-embedding immunogold is better for collecting semi-quantitative data and gives better intracellular resolution, but when cell membranes are labeled and cells are close to each other as they typically are in the brain, then immunogold cannot tell which membrane labeling belongs to (for description of these methods, see Danbolt et al. 1998; Amiry-Moghaddam and Ottersen 2013). Another problem with post-embedding immunogold is that there must be a sufficient number of target molecules in the plane of the section. This is case for EAAT1, EAAT2 and EAAT4. These proteins are present at very high concentrations (Dehnes et al. 1998; Lehre and Danbolt 1998) making them ideal targets for immunogold investigations. This explains, in part, why our early localization studies were so successful (Chaudhry et al. 1995; Dehnes et al. 1998). In contrast, EAAT3 is expressed at lower levels resulting in too few molecules per micrometer plasma membrane length to distinguish real labeling from background noise (Holmseth et al. 2012b). It should be recalled that the tissue sections used for electron microscopy are thin (40–60 nm) and thereby only slightly thicker than the outer diameter of synaptic vesicles (40 nm), and only two/three times thicker than the width of the synaptic cleft (20 nm). The antibodies do not penetrate well into the sections. To maximize labeling, the section may be mounted so that they can be labeled on both sides. Thus, the sensitivity of the post-embedding immunogold technique is limited by the number of proteins in the exact section plane. Another challenge follows from the vulnerability of the sections and thereby also the labeling. These sections are easily damaged during processing. Consequently, there is variability and this leads to another challenge: avoiding sampling error. This challenge comes in addition to those mentioned above (specificity, proteolysis, etc.). It is also important to consider if any detected proteins are expressed at physiologically relevant levels. The number of molecules needed to accomplish a given task depends on what that task is. This consideration is particularly relevant for neurotransmitter transporters because the transport process is fairly slow. The cycling time of EAAT2 and EAAT3 are in the order of 30 glutamate molecules per second at Vmax (Otis and Jahr 1998; Otis and Kavanaugh 2000; Bergles et al. 2002; Grewer and Rauen 2005) and EAAT5 is even slower (Gameiro et al. 2011). The cycling time of the GABA transporters appear to be comparable to those of the EAATs (Mager et al. 1993; Sacher et al. 2002; Karakossian et al. 2005; Gonzales et al. 2007). This means that the number of transporters must be high. There is a rapid extracellular turnover of glutamate (Jabaudon et al. 1999), and despite this, the resting levels of extracellular glutamate in normal brains are low, possibly as low as 25 nm (Herman and Jahr 2007). Because the km-values are about 1,000 times higher (Danbolt 2001), maintenance of such low extracellular levels implies a vast excess of transporter proteins (Bergles and Jahr 1997; Dehnes et al. 1998; Lehre and Danbolt 1998; Otis and Kavanaugh 2000). A large number of papers on transporter distributions have been published, and it is not easy to navigate in the literature as many of the statements are corrected in later publications. Figure 1 is a schematic illustration of the distributions of EAAT1, EAAT2 and EAAT3 in the forebrain.  A schematic illustration of glutamate transporter distributions around synapses close to a blood vessel in the hippocampus. Four glutamatergic nerve terminals (T) are shown forming synapses onto dendritic spines (S). Astrocyte branches are indicated (G). Note that astrocytes have very high densities (Lehre et al. 1995; Ginsberg et al. 1995; Lehre and Danbolt 1998) of both EAAT2 (red dots) and EAAT1 (blue dots). The highest densities of EAAT1 and EAAT2 are in the astrocyte membranes facing neuropil, while the membranes facing the endothelium have low levels. Also note that glutamate transporters have not been detected in the endothelium. EAAT1 is selective for astrocytes (Lehre et al. 1995; Ginsberg et al. 1995), while EAAT2 is predominantly expressed in astrocytes (Danbolt et al. 1992), but there is also some (about 10 %) in hippocampal nerve terminals (Furness et al. 2008). EAAT3 (green dots) is selective for neurons, but is expressed at levels two orders of magnitude lower than EAAT2 and is targeted to dendrites and cell bodies (Holmseth et al. 2012b). Also note that the endfeet may actually overlap with no gaps in between them (Mathiisen et al. 2010) (Copyright: Neurotransporter AS; Reproduced with permission) EAAT1 (GLAST; slc1a3) is selectively expressed in astrocytes throughout the CNS (Lehre et al. 1995). This conclusion is supported both by in situ hybridization and immunocytochemistry (e.g. Ginsberg et al. 1995; Rothstein et al. 1995 Schmitt et al. 1997; Berger and Hediger 1998, 2000) and appears to be valid for all parts of the central nervous system including the regions where EAAT1 is the predominant transporter (Lehre et al. 1995; Lehre and Danbolt 1998; Takatsuru et al. 2007; Takayasu et al. 2009): the retina (Rauen et al. 1996; Lehre et al. 1997; Rauen et al. 1998; Rauen 2000; Rauen and Wiessner 2000), the inner ear (Furness and Lehre 1997; Takumi et al. 1997), and the circumventricular organs (Berger and Hediger 2000). Thus, there is no disagreement here. Other statements can be found in the literature, but these have been corrected by the authors themselves. After having determined the cell types expressing EAAT1 (Lehre et al. 1995), immunogold was performed to obtain additional information (Chaudhry et al. 1995). This revealed that EAAT1 is preferentially targeted to the plasma membranes, and that plasma membranes facing neuropil have higher densities than those facing cell bodies, pia mater and endothelium (Fig. 1). Mice lacking EAAT1 (Watase et al. 1998) develop normally, but show symptoms of insufficient glutamate uptake in regions where EAAT1 is the major glutamate transporter (Watase et al. 1998; Hakuba et al. 2000; Harada et al. 1998). The EAAT1 knockout mice also display poor nesting behavior; abnormal sociability, reduced alcohol intake and reward (Watase et al. 1998; Stoffel et al. 2004; Karlsson et al. 2009, 2012). Lack of GLAST does not lead to spontaneous seizures like those seen in connection with EAAT2-deficiency (Tanaka et al. 1997), but GLAST deficiency increases seizure duration and severity (Watanabe et al. 1999). EAAT1 mutations in humans are linked to episodic ataxia (Bröer and Palacin 2011; Jen et al. 2005; de Vries et al. 2009). EAAT2 (GLT-1; slc1a2) was the first glutamate transporter to be localized immunocytochemically. In the mature and normal brain it is predominantly expressed in astrocytes (Danbolt et al. 1992; Levy et al. 1993; Rothstein et al. 1994; Lehre et al. 1995). There is no disagreement here either, and this conclusion is supported both by later immunocytochemistry (e.g. Schmitt et al. 1996; Kugler and Schmitt 2003; Berger et al. 2005; Holmseth et al. 2009) and in situ hybridization (Torp et al. 1994, 1997; Berger and Hediger 2000, 2001) as well as by data obtained with EAAT2 eGFP BAC reporter mice (de Vivo et al. 2010a). EAAT2 is the only one of the EAAT-type of glutamate transporters that is required for survival under non-challenging conditions (Tanaka et al. 1997; Danbolt 2001). This is in agreement with biochemical data showing that the EAAT2 protein represents about 1 % of the total forebrain protein and that it is about four times more abundant than EAAT1 in the hippocampus and six times less abundant than EAAT1 in the cerebellum (Lehre and Danbolt 1998). Based on immunoadsorption of transport activity EAAT2 was shown to account for 95 % of the total glutamate uptake activity in young adult forebrain tissue (Danbolt et al. 1992; Haugeto et al. 1996). This conclusion was confirmed by deletion of the EAAT2 gene in mice (Tanaka et al. 1997; Voutsinos-Porche et al. 2003; Matsugami et al. 2006; Kiryk et al. 2008; Holmseth et al. 2012b) as well as by electrophysiological recordings of glutamate transporter currents (Otis and Kavanaugh 2000). The discussion about EAAT2 distribution concerns expression in neurons. Having said that, there is consensus that EAAT2 is expressed in cultured neurons from hippocampus and neocortex; in particular if these are cultured in the absence of astrocytes (Mennerick et al. 1998; Wang et al. 1998; Plachez et al. 2000) in agreement with observations that EAAT2 is transiently localized on growing axons of the mouse spinal cord before establishing astrocytic expression (Yamada et al. 1998). There is also consensus that EAAT2 is present in neurons in the normal and mature mammalian retina (Rauen et al. 1996, 1999; Rauen and Kanner 1994; Euler and Wassle 1995; Rauen 2000). The controversy is related to expression of EAAT2 in neurons in the normal and mature brain (cerebrum and cerebellum). All studies, however, agree that there is EAAT2 mRNA in CA3 hippocampal neurons (Torp et al. 1994, 1997; Berger and Hediger 2000, 2001; de Vivo et al. 2010a) and that their axon-terminals express the protein, at least in the CA1 (Chen et al. 2004; Furness et al. 2008; Melone et al. 2009, 2011). Further, all of the glutamate uptake activity in glutamatergic terminals in CA1 is due to EAAT2 (Furness et al. 2008). The remaining controversy concerns (a) the expression of EAAT2 in axon-terminals in other parts of the brain, and (b) the physiological importance of the uptake into terminals. Why was about half of all d-aspartate taken up by hippocampus slices found in axon-terminals when terminals only contain around 10 % of the EAAT2 protein (Furness et al. 2008)? This disproportionally large uptake cannot simply be disregarded as an in vitro artifact due to a higher rate of heteroexchange than net uptake (Zhou et al. 2013), but it might still be an artifact because the possibility has not been ruled out that astrocytes release glutamate via anion channels or similar. Preliminary data from selective deletion of EAAT2 in axon-terminals indicate disturbances in synaptic transmission (Sun et al. 2012), and thereby may suggest that EAAT2 in terminals is functionally relevant. However, further studies are required before definite conclusions can be made. In contrast to EAAT1, there is very little EAAT2 in mice and rats at birth and in the first postnatal week (Ullensvang et al. 1997; Furuta et al. 1997). This explains why EAAT2-knockout mice are inconspicuous at birth. But at 3 weeks, when the EAAT2-levels in wild-type mice have increased to 50 % of adult levels, the EAAT2-deficient mice can readily be identified because they are hyperactive, epileptic and smaller than their wild-type littermates. They have increased extracellular glutamate levels (Mitani and Tanaka 2003; Takasaki et al. 2008), and about half of them die from spontaneous seizures before they reach 4 weeks of age (Tanaka et al. 1997). The heterozygote EAAT2 knockout mice (±) have only half the EAAT2-concentrations as wild-type mice, but do not show any apparent morphological brain abnormalities (Kiryk et al. 2008), but are more vulnerable to traumatic spinal cord injury (Lepore et al. 2011). EAAT3 (EAAC1; slc1a1) has been particular hard to localize. Nevertheless, the first studies were basically correct (Kanai and Hediger 1992; Rothstein et al. 1994). EAAT3 is a neuronal transporter, and is not expressed in glial cells (Holmseth et al. 2012b; Shashidharan et al. 1997). It appears to be expressed in the majority if not all neurons throughout the CNS, but has a unique sorting motif (Cheng et al. 2002) selectively targeting it to somata and dendrites avoiding axon terminals (Holmseth et al. 2012b; Shashidharan et al. 1997). The highest levels of EAAT3 in the brain are found in the hippocampus and neocortex, but the total tissue content in young adult rat brains is about 100 times lower than that of EAAT2 (Holmseth et al. 2012b). It is also expressed in the kidney and in the ileum. In agreement, mice lacking EAAT3 (Peghini et al. 1997) develop dicarboxylic aminoaciduria, but do not show signs of neurodegeneration at young age and do not have epilepsy (Peghini et al. 1997; Aoyama et al. 2006; Berman et al. 2011). Humans lacking EAAT3 develop dicarboxylic aminoaciduria (Bailey et al. 2011) and EAAT3 polymorphisms are associated with obsessive–compulsive disorders (Brandl et al. 2012; Walitza et al. 2010). EAAT4 (slc1a6) is predominantly found in the cerebellar Purkinje cells (Fairman et al. 1995; Dehnes et al. 1998) where it is targeted to the dendrites, the spines in particular (Dehnes et al. 1998), but there is also some EAAT4 in a subset of forebrain neurons (Dehnes et al. 1998; Massie et al. 2008; de Vivo et al. 2010b) and in vestibular hair cells and calyx endings (Dalet et al. 2012). EAAT4 knockout mice are viable and appear normal (Huang et al. 2004) albeit with some alteration of receptor activation (Nikkuni et al. 2007). EAAT5 (slc1a7) is preferentially expressed in the retina, while the levels in the brain are low (Arriza et al. 1997; Eliasof et al. 1998). EAAT5 is also expressed in vestibular hair cells and calyx endings (Dalet et al. 2012). There is more than one isoform in the retina due to variable splicing (Eliasof et al. 1998). As explained above, EAAT4 and EAAT5 are not very efficient as transporters, but are efficient chloride channels suggesting that they may be more important as inhibitory glutamate receptors than as transporters. Some investigators have tried to determine the exact cellular and subcellular localization of EAAT5, but the validity of these studies is hard to judge at present because nobody has as yet made an EAAT5 knockout mouse that could serve as negative control for validation of the immunolabeling. We have previously shown how important this control is and also how inadequate the so called pre-adsorption test is (Holmseth et al. 2012a). So, validated information on EAAT5 distribution remains to be provided.
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