Characterization of glutamate transporter function in the tiger salamander retina

Glutamate transporters in the tiger salamander retina were studied by autoradiographic and intracellular recording techniques. When the retina was incubated with 15 microM L-[3H]glutamate, photoreceptors and Muller cells were labeled, indicating that these cells had high-affinity glutamate uptake transporters. A much higher dose of glutamate than kainate was required in the bath to produce the same membrane depolarization in horizontal cells (HCs), and the time course of glutamate-induced depolarization was much slower than that of the kainate-induced depolarization. Since glutamate is a substrate of glutamate transporters whereas kainate is not, we attribute these differences to the buffering of extracellular glutamate by glutamate transporters in the retina. D-aspartate (D-asp) increased the efficacy of bath-applied glutamate. Dihydrokainate (DHKA) exerted little effect on glutamate efficacy when applied alone, but it increased glutamate efficacy in the presence of D-asp. These results are consistent with the notion that glutamate transporters in Muller cells are D-asp sensitive and those in photoreceptors are DHKA and D-asp sensitive. Application of DHKA (1-2 mM) did not affect the dark membrane potential or the light responses in rods and cones, but it depolarized the HC dark membrane potential and reduced the HC peak and tail light responses. Our results suggest that DHKA-sensitive glutamate transporters in photoreceptors regulate glutamate levels in rod and cone synaptic clefts. They modulate dark membrane potential and the relative rod cone inputs in retinal HCs.     

Identification of functional domains of the human glutamate transporters EAAT1 and EAAT2

Glutamate transporters serve the important function of mediating removal of glutamate released at excitatory synapses and maintaining extracellular concentrations below excitotoxic levels. Excitatory amino acid transporter subtypes EAAT1 and EAAT2 have a high degree of sequence homology and similar predicted topology and yet display a number of functional differences. Several recombinant chimeric transporters were generated to identify domains that contribute to functional differences between EAAT1 and EAAT2. Wild-type transporters and chimeric transporters were expressed in Xenopus laevis oocytes, and electrogenic transport was studied under voltage clamp conditions. The differential sensitivity of EAAT1 and EAAT2 to transport blockers, kainate, threo-3-methylglutamate, and (2S, 4R)-4-methylglutamate as well as L-serine-O-sulfate transport and chloride permeability were employed to characterize chimeric transporters. One particular region, transmembrane domains 9 and 10, plays an important role in defining these functional differences. The intracellular carboxyl-terminal region may also play a minor role in conferring an effect on chloride permeability. This study provides important insight into the identification of functional domains that determine differences among glutamate transporter subtypes.     

Peak mitral inflow velocity predicts mitral regurgitation severity

Glutamate transport is a primary mechanism for the synaptic inactivation of glutamate. Excitatory amino acid transporter 4 (EAAT4) is a novel glutamate transporter with properties of a ligand-gated chloride channel that was recently cloned from human brain. Here we report the cloning of rat EAAT4 (rEAAT4) cDNA from rat cerebellum. The nucleotide sequence of rEAAT4 was 88% identical to the human sequence, and the predicted peptide was 89% identical to the human protein. The transport activity encoded by rEAAT4 has high affinity for L-glutamate. In Xenopus laevis oocytes expressing rEAAT4, L-glutamate and other transporter substrates elicited a current predominantly carried by chloride ions. Like human EAAT4, the rEAAT4 mRNA was largely restricted to cerebellar Purkinje cells; the rEAAT4 protein was localized to Purkinje cell somas and dendrites.     

Heart rate and mortality]

1. Glutamate is the predominant excitatory neurotransmitter in the brain, but it is also a potent neurotoxin. Following release of glutamate from presynaptic vesicles into the synapse and activation of a variety of ionotropic and metabotropic glutamate receptors, glutamate is removed from the synapse. This is achieved through active uptake of glutamate by transporters located pre- and also post-synaptically or, alternatively, glutamate can diffuse out of the synapse and be taken up by transporters located on the cell surface of glial cells. 2. Complementary DNA encoding a number of glutamate transporters have recently been cloned and form a family of structurally related membrane proteins with a high degree of amino acid sequence conservation. Expression of the cloned glutamate transporters in various cell types has aided in the characterization of the functional properties of the different transporter subtypes. 3. Glutamate transport is coupled to sodium, potassium and pH gradients across the cell membrane creating an electrogenic process. This allows transport to be measured using electrophysiological techniques, which has greatly aided in understanding some of the basic mechanisms of the transport process and has also allowed a detailed understanding of the molecular pharmacology of the different transporter subtypes. 4. In the present review I shall discuss some of the recent advances in understanding the molecular basis for glutamate transporter function and then highlight some of the unanswered questions concerning the physiological roles of these proteins and suggest possible strategies for pharmacological manipulation of transporters for the treatment of neurological disorders.     

Alterations in the Ha-ras-1 and the p53 pathway genes in the progression of N-methyl-N-nitrosourea-induced rat mammary tumors

Glutamate is the most prominent excitatory neurotransmitter in the retina and brain. It has become clear that the physiology of many glial cells, including retinal Müller cells, is modified by a host of neurotransmitters, including glutamate. The experiments presented here demonstrate that Müller cells isolated from the tiger salamander retina have metabotropic glutamate receptors that, when activated, lead to the release of calcium ions (Ca2+) from intracellular stores. The Ca2+-sensitive fluorescent dye, Fura-2, and video imaging microscopy were used to monitor changes in cytosolic calcium ion concentration ([Ca2+]i) evoked by glutamate (30-50 microM), (1S,3R)-ACPD (50-200 microM), quisqualate (10-50 microM), and L-AP4 (5-100 microM). Bath application of each of these metabotropic receptor agonists in the absence of extracellular Ca2+ resulted in an increase in [Ca2+]i that often began in the distal end of the cell and occurred later in the endfoot. This wavelike increase in [Ca2+]i is reminiscent of the Ca2+ waves evoked in these cells by other Ca2+ releasing agents such as ryanodine and caffeine. Extracellular application ofATP also evoked increases in [Ca2+] in Müller cells. The presence on Müller cells of receptors for retinal neurotransmitters, such as glutamate and ATP, demonstrates that these glial cells can respond to changes in the retinal extracellular environment and hence neuronal activity. Since Müller cells span almost all layers of the retina, they are likely to be exposed to most retinal neurotransmitters. The Ca2+ waves evoked in Müller cells by neurotransmitters could represent a form of signaling from the outer retinal layers to the inner ones.     

New beta-hydroxyaspartate derivatives are competitive blockers for the bovine glutamate/aspartate transporter

Four subtypes of excitatory amino acid transporters (EAAT1-4) have been identified in the mammalian brain. A number of pharmacological agents have been developed to study their intrinsic properties and function. Up to now, blockers were available only for EAAT2, whereas all the inhibitors of glutamate uptake active on the other subtypes were proved to be substrates of the transporters. We synthesized five new derivatives of DL-threo-beta-hydroxyaspartic acid, a well known general substrate of EAATs, and investigated their potential blocking activity on the cloned bovine EAAT1 expressed in the Xenopus oocyte system, by using radiotracer and voltage-clamp techniques. Two of our derivatives proved to be substrates for bovine EAAT1, with reduced electrogenicity compared with their parent compound, and an affinity of 40 and 64 microM. The last three derivatives displayed a blocking activity on bovine EAAT1. The affinity of DL-threo-beta-benzoyloxyaspartate and DL-threo-beta-(1-naphthoyl)oxyaspartate was determined by Schild analysis as 17.2 and 52.1 microM, respectively. These blockers should help in the better understanding of the key intrinsic properties of EAAT1. Moreover, they appear as good candidates for a general blocking activity on EAATs.     

Macroscopic and microscopic properties of a cloned glutamate transporter/chloride channel

The behavior of a Cl- channel associated with a glutamate transporter was studied using intracellular and patch recording techniques in Xenopus oocytes injected with human EAAT1 cRNA. Channels could be activated by application of glutamate to either face of excised membrane patches. The channel exhibited strong selectivity for amphipathic anions and had a minimum pore diameter of approximately 5A. Glutamate flux exhibited a much greater temperature dependence than Cl- flux. Stationary and nonstationary noise analysis was consistent with a sub-femtosiemen Cl- conductance and a maximum channel Po < 1. The glutamate binding rate was similar to estimates for receptor binding. After glutamate binding, channels activated rapidly followed by a relaxation phase. Differences in the macroscopic kinetics of channels activated by concentration jumps of L-glutamate or D-aspartate were correlated with differences in uptake kinetics, indicating a close correspondence of channel gating to state transitions in the transporter cycle.     

Neuronal high-affinity glutamate transport in the rat central nervous system

EAAC1 is a neuronal and epithelial high affinity glutamate transporter previously cloned from rabbit intestine. Here we report the isolation of EAAC 1 from rat brain* and its expression in the central nervous system based on in situ hybridization. Strong signals were detected in brain, spinal cord and retina. Expression of EAAC1 was particularly strong in pyramidal cells of the cerebral cortex, pyramidal cells of the hippocampus, mitral cells of the olfactory bulb, various thalamic nuclei and cells of certain retinal layers. EAAC1 was also expressed in non-glutamatergic neurons such as GABAergic cerebellar Purkinje cells and alpha-motor neurons of the spinal cord. We propose that EAAC1 is not only involved in the sequestration of glutamate at glutamatergic synapses and in protecting neurons from glutamate excitotoxicity, but also in the cellular metabolism involving glutamate.     

Reducing vancomycin use utilizing a computer guideline: results of a randomized controlled trial

Normal development and hypoxic-ischemic changes of glutamate-aspartate transporters (GLAST) and excitatory amino acid transporter type 4 (EAAT4) were demonstrated in the human cerebellum. GLAST-immunoreactive Bergmann's glia and EAAT4-positive Purkinje cells showed a specific distribution and localization, and developed with age in the molecular and Purkinje cell layers. The dendrites and cell bodies of Purkinje cells, which showed EAAT4 immunoreactivity, were ensheathed by GLAST processes. In neonatal hypoxic-ischemic encephalopathy (HIE), GLAST immunoreactivity decreased in the molecular layer and increased in the inner granule cell layer at an early stage, and markedly increased in the Purkinje and inner granule cell layers at a late stage. EAAT4 immunoreactivity decreased with post-ischemic changes of Purkinje cells. GLAST reactivity changed more rapidly than EAAT4 in cases of HIE. These changes of GLAST and EAAT4 may be closely related to the vulnerability of Purkinje cells in hypoxia-ischemia. The glutamate transporter of Bergmann glia may play a more important role in the regulation of the extracellular glutamate concentration in hypoxia and/or ischemia.     

The number of glutamate transporter subtype molecules at glutamatergic synapses: chemical and stereological quantification in young adult rat brain

The role of transporters in shaping the glutamate concentration in the extracellular space after synaptic release is controversial because of their slow cycling and because diffusion alone gives a rapid removal. The transporter densities have been measured electrophysiologically, but these data are from immature brains and do not give precise information on the concentrations of the individual transporter subtypes. Here we show by quantitative immunoblotting that the numbers of the astroglial glutamate transporters GLAST (EAAT1) and GLT (EAAT2) are 3200 and 12,000 per micrometer3 tissue in the stratum radiatum of adult rat hippocampus (CA1) and 18,000 and 2800 in the cerebellar molecular layer, respectively. The total astroglial cell surface is 1.4 and 3.8 m2/cm3 in the two regions, respectively, implying average densities of GLAST and GLT molecules in the membranes around 2300 and 8500 micrometer-2 in the former and 4700 and 740 micrometer-2 in the latter region. The total concentration of glial glutamate transporters in both regions corresponds to three to five times the estimated number of glutamate molecules in one synaptic vesicle from each of all glutamatergic synapses. However, the role of glial glutamate transporters in limiting synaptic spillover is likely to vary between the two regions because of differences in the distribution of astroglia. Synapses are completely ensheathed and separated from each other by astroglia in the cerebellar molecular layer. In contrast, synapses in hippocampus (stratum radiatum) are only contacted by astroglia and are often found side by side without intervening glial processes.     

Expression of the human excitatory amino acid transporter 2 and metabotropic glutamate receptors 3 and 5 in the prefrontal cortex from normal individuals and patients with schizophrenia

A disturbance of glutamatergic transmission has been suggested to contribute to the development of schizophrenic pathophysiology based primarily on the ability of glutamate receptor antagonists to induce schizophrenic-like symptoms, and recent studies suggesting reduced glutamatergic function in the prefrontal cortex (PFC) of individuals with a diagnosis of schizophrenia. In order to investigate this hypothesis further, the expression of several 'glutamatergic' markers, the metabotropic glutamate receptors (mGluRs; mGluR3, 5) and the human excitatory amino acid transporter (EAAT2) were compared in the PFC of normal individuals and schizophrenics. The present results showed that glial cells in the pyramidal layers of the PFC from schizophrenics had decreased EAAT2 mRNA content relative to controls in Brodmann areas 9 and 10. The cellular levels of expression of the two mGluR signals investigated (mGluR3, and 5) were not significantly changed relative to controls except for an increase in the neuronal mGluR5 in the pyramidal cell layers of area 11. Comparing the ratio of cellular mGluR expression to that of EAAT2, the mGluR/EAAT2 ratio showed that schizophrenics had a significantly increased mGluR/EAAT2 ratios in the pyramidal cell layers of all three PFC regions examined. The glutamate content of consecutive sections analyzed by high pressure liquid chromatography (HPLC), although decreased in schizophrenics did not reach significance and did not correlate with either EAAT2 or mGluR mRNA content. These results are discussed in the light of current results on the neurochemistry and pharmacology of schizophrenia.     

Arachidonic acid activates a proton current in the rat glutamate transporter EAAT4

The excitatory amino acid transporter EAAT4 is expressed predominantly in Purkinje neurons in the rat cerebellum (1-3), and it participates in postsynaptic reuptake of glutamate released at the climbing fiber synapse (4). Transporter-mediated currents in Purkinje neurons are increased more than 3-fold by arachidonic acid, a second messenger that is liberated following depolarization-induced Ca2+ activation of phospholipase A2 (5). In this study we demonstrate that application of arachidonic acid to oocytes expressing rat EAAT4 increased glutamate-induced currents to a similar extent. However, arachidonic acid did not cause an increase in the rate of glutamate transport or in the chloride current associated with glutamate transport but rather activated a proton-selective conductance. These data reveal a novel action of arachidonate on a glutamate transporter and suggest a mechanism by which synaptic activity may decrease intracellular pH in neurons where this transporter is localized.     

Cloning and localization of RPE65 mRNA in salamander cone photoreceptor cells1

RPE65 is a potential retinoid-processing protein expressed in the retinal pigment epithelium. Mutations in the RPE65 gene have been shown to cause certain inherited retinal dystrophies. Previous studies have shown that salamander cone photoreceptor cells have a unique retinoid processing mechanism which is distinct from that of rods. To determine whether RPE65 is expressed in photoreceptors, the RPE65 cDNA was cloned from a salamander retinal cDNA library. The deduced protein consists of 533 amino acids and is 85% identical to human and bovine RPE65. The RPE65 mRNA was detected in all of the single cone cells isolated from the salamander retina, as well as in the retinal pigment epithelium by RT-PCR, but not in the isolated rods. The RT-PCR products have been confirmed to be RPE65 by DNA sequencing. The results indicate that this potential retinoid processing protein is expressed in the cone photoreceptor cells but not in rods. Therefore, this protein may contribute to the unique retinoid processing capabilities in salamander cones.     

Extra-junctional localization of glutamate transporter EAAT4 at excitatory Purkinje cell synapses

We used silver-enhanced immunogold electron microscopy to reveal synaptic localization of the glutamate transporter EAAT4 in mouse cerebellar Purkinje cells (PCs). Gold-silver particles representing the EAAT4 were densely localized on extra-junctional membrane, but not on junctional membrane of PC spines in contact with parallel fiber or climbing fiber terminals. No particle accumulations were observed at inhibitory synapses formed on cell body and dendritic shafts of PCs. Therefore, the EAAT4 is selectively targeted to the extra-junctional site of excitatory PC synapses. The finding suggests that the EAAT4 transports glutamate or its related amino acids from outside the synaptic cleft, which would facilitate glutamate diffusion from the synaptic cleft to the extrasynaptic space and restrict glutamate spillover to adjacent synapses.     

Microsomal electron transfer in higher plants: cloning and heterologous expression of NADH-cytochrome b5 reductase from Arabidopsis

The mechanisms underlying the conversion of prolonged glutamate release from ribbon synapses in bipolar cells to sustained and transient excitatory postsynaptic responses in identified retinal amacrine cells were studied in tiger salamander (Ambystoma tigrinum) retina. A retina slice preparation with whole cell patch recording techniques under voltage- and current-clamp conditions was used to assay the electrical properties of bipolar and amacrine cells. Amacrine cells were categorized into two basic forms: (i) transient amacrine cells that respond to a step of light with a burst of spikes only at the transitions of the step; and (ii) sustained amacrine cells that respond with continuous spiking during the entire light step. The two cell types each had a characteristic morphology: transient amacrine cells possessed wide dendritic fields (chi = 375 microns), while sustained cells had much more narrowly confined dendritic fields (chi = 85 microns). Whole cell voltage-gated currents of the transient and sustained cell types were not significantly different. Both cell types had spikes that were sensitive to tetradotoxin (TTX, 0.3 microM) with voltage deflections of up to 100 mV. Light-evoked excitatory synaptic currents relaxed rapidly in transient neurons (tau 1/2 = 100 ms) and more slowly in sustained neurons (tau 1/2 = 1.2 s). EPSCs in both cells reversed near 0 mV. Rapid application of glutamate or kainate elicited rapidly desensitizing ionic currents (tau 1/2 = 85 ms) followed by a slowly desensitizing component. Cyclothiazide, a drug that eliminates rapid desensitization, lengthened the time course of the glutamate gated current from tau 1/2 = 85 ms to about 3 s, and the relaxation kinetics of the glutamatergic EPSC from tau 1/2 = 85 ms to about 1.0 s. These data suggest that a key determinant in forming transient versus sustained responses in amacrine cells of vertebrate retina is the differences in their excitatory, glutamatergic synaptic inputs, and that rapid desensitization of glutamate receptors plays a role in converting the presynaptic signal associated with sustained glutamate release into a postsynaptic, transient signal at the ribbon synapse.     

Ability of retinal Müller glial cells to protect neurons against excitotoxicity in vitro depends upon maturation and neuron-glial interactions

Glutamate is the most abundant excitatory amino acid in the central nervous system. It has also been described as a potent toxin when present in high concentrations because excessive stimulation of its receptors leads to neuronal death. Glial influence on neuronal survival has already been shown in the central nervous system, but the mechanisms underlying glial neuroprotection are only partly known. When cells isolated from newborn rat retina were maintained in culture as enriched neuronal populations, 80% of the cells were destroyed by application of excitotoxic concentrations of glutamate. Massive neuronal death was also observed in newborn retinal cultures containing large numbers of glia, or when neurons were seeded onto feeder layers of purified cells prepared from immature (postnatal 8 day) rat retina. When newborn retinal neurons were seeded onto feeder layers of purified glial cells prepared from adult retinas, application of excitotoxic amino acids no longer led to neuronal death. Furthermore, neuronal death was not observed in mixed neuron/glial cultures prepared from adult retina. However, in all cases (newborn and adult) application of kainate led to amacrine cell-specific death. Activity of glutamine synthetase, a key glial enzyme involved in glutamate detoxification, was assayed in these cultures in the presence or absence of exogenous glutamate. Whereas pure glial cultures alone (from young or adult retina) showed low activity that was not stimulated by glutamate addition, mixed or co-cultured neurons and adult glia exhibited up to threefold higher levels of activity following glutamate treatment. These data indicate that two conditions must be satisfied to observe glial neuroprotection: maturation of glutamine synthetase expression, and neuron-glial signalling through glutamate-elicited responses.     

Transport-mediated release of endogenous glutamate in the vertebrate retina

In the present study we measured calcium-dependent, vesicular glutamate release, and calcium-independent, transport-mediated glutamate release patterns in the vertebrate retina to better understand the sources of elevated glutamate in neural tissue under ischemic conditions. A potassium concentration of 40 mM, which mimics the extracellular potassium concentration in the central nervous system during ischemia, was applied to the bathing medium of a retinal slice prepared from zebrafish. High external potassium evoked release of endogenous glutamate that was measured using a glutamate-specific fluorometric assay applied to the bath. The slice was visualized under 668 nm light using Normarski optics and fluorescent images were captured using a cooled charge-coupled device (CCD) camera. Following the elevation of external potassium to 40 mM several bands of glutamate fluorescence, reflecting the spatial distribution of glutamate release, were observed. A calcium-dependent cloud of glutamate was observed in the inner plexiform layer, that was antagonized by bath-applied nifedipine. A relatively dense glutamate cloud (1-10 microM) was observed over the ganglion cell layer, which was blocked by dihydrokainate, a glutamate transport antagonist. In contrast, nifedipine, an inhibitor of calcium-dependent neurotransmitter release in the retina, failed to block the cloud of released glutamate in the ganglion cell layer. These data suggest that under pathological conditions in the eye where glutamate levels are elevated surrounding retinal ganglion cells, such as observed in some forms of glaucoma, a possible source of the elevated glutamate is through a glutamate transporter operating in a reversed direction. A likely candidate for mediating this reversed transport of glutamate is the retinal Muller cell.     

Synaptic inputs mediating bipolar cell responses in the tiger salamander retina

Postsynaptic receptors in bipolar cells were studied by focal application of glutamate and GABA to the outer and inner plexiform layers (OPL and IPL) under visual guidance in living retinal slices of the tiger salamander. Two different types of conductance change could be elicited in bipolar cells by applying glutamate to the OPL. In off-center cells, which had axon telodendria ramifying in the distal 55% of the IPL, glutamate elicited a conductance increase associated with a reversal potential near -5 mV. In on-center cells, which had telodendria stratified in the proximal 45% of the IPL, glutamate caused a conductance decrease associated with a reversal potential near -11 mV. These observations suggest that glutamate gates relatively nonspecific cation channels at synapses between photoreceptors and bipolar cell dendrites. Application of glutamate to the IPL elicited no conductance change in Co2+ Ringer's solution, but in normal Ringer's it generated a conductance increase associated with a reversal potential near the chloride equilibrium potential (ECl). These findings are consistent with the notion that glutamate receptors exist in GABAergic and/or glycinergic amacrine cells, and that glutamate in the IPL depolarizes these cells, causing GABA and/or glycine release and the opening of chloride channels in bipolar cell axon terminals. In Co2+ Ringer's, application of GABA at the OPL elicited no conductance changes in bipolar cells, suggesting that GABA receptors do not exist on bipolar cell dendrites. Applied at the IPL, GABA elicited large conductance increases associated with a reversal potential near ECl. Implications of these results for the functional circuitry of the tiger salamander retina are discussed.     

Stoichiometry of the glial glutamate transporter GLT-1 expressed inducibly in a Chinese hamster ovary cell line selected for low endogenous Na+-dependent glutamate uptake

Glutamate transport across the plasma membrane of neurons and glia is powered by the transmembrane electrochemical gradients for sodium, potassium, and pH, but there is controversy over the number of Na+ cotransported with glutamate. The stoichiometry of glutamate transporters is important because it determines a lower limit to the extracellular glutamate concentration, [glu]o, in both normal and pathological conditions. We used whole-cell clamping to study the stoichiometry of the glial transporter GLT-1, the most abundant glutamate transporter in the brain, expressed under control of the Tet-On system in a Chinese hamster ovary (CHO) cell line selected for low endogenous glutamate transport. After the induction of GLT-1 expression with doxycycline, glutamate evoked a Na+-dependent inward current with the voltage dependence and pharmacology of GLT-1 and acidified the cell cytoplasm. Raising [K+]o around cells clamped with electrodes containing sodium and glutamate evoked an outward reversed uptake current. These responses were reduced by the specific GLT-1 blocker dihydrokainate (DHK). DHK evoked an outward current with NO3-, but not with Cl-, as the main intracellular anion, suggesting that the anion conductance of the transporter is active even without external glutamate but generates little current in the absence of highly permeable anions like NO3-. Measuring the reversal potential of the transporter current in various ionic conditions suggested that the transport of one glutamate anion is coupled to the cotransport of three Na+ and one H+ and to the countertransport of one K+. This suggests that in ischemia, when [K+]o rises to 60 mM, the reversal of glutamate transporters will raise [glu]o to >50 microM.