Contribution of a glial glutamate transporter to GABA synthesis in the retina

Neuronal glutamate transporters have been shown to play a role in GABA synthesis by enhancing glutamate uptake. In the present study, we have examined whether a glial glutamate transporter, GLAST, has a role in GABA synthesis in the mammalian retina. We found that the retinal GABA level was about two-fold higher in the GLAST-/- mouse retina compared to that in the wild type. Endogenous glutamate level was also increased about 2-fold in the mutant. Therefore, loss of GLAST results in a higher retinal GABA level, probably due to increased availability of its precursor, glutamate. An increase in GABAergic activity can be expected to affect trigger features such as directional selective response of neurons in the GLAST-/- mouse retina.     

Characterization of glycine transport in cultured Müller glial cells from the retina.

Rapid termination of the synaptic action of glutamate (Glu) and glycine (Gly) is achieved by uptake into the presynaptic terminal and glial cells. In the vertebrate CNS, Gly acts both as an inhibitory neurotransmitter and as a Glu modulator or coagonist at postsynaptic N-methyl-D-aspartate (NMDA) receptors. We have previously described NMDA receptors in Müller cells of chick retina coupled to the phosphoinositide cascade, the entry of calcium, and the activation of protein kinase C (PKC; López-Colomé et al. Glia 9:127-135, 1993). A colocalization of Gly transporters and NMDA receptors has been reported in brain tissue (Smith et al. Neuron 8:927-936, 1992); since the concentration of Gly could participate in the modulation of Glu excitatory transmission in the vertical pathways of the retina, transport of Gly in monolayer cultures of Müller cells was studied. Gly transport was found pH-sensitive with an optimum at pH 7.4. Kinetic analysis of the saturation curve for Gly within a concentration range of 0.01-2 mM, revealed two components of transport: a low-affinity system with Km = 1.7 mM, Vmax = 30 nmol/10 min/mg protein, and a high-affinity one with a Km = 27 microM, Vmax = 3 nmol/10 min/mg protein. Both systems were Na+ -dependent; the high-affinity system proved also dependent on external Cl- and was inhibited by sarcosine, characteristic of GLYT1 transporters. The inhibition of low-affinity uptake by 2-(methylamino)isobutyric acid (MeAIB) and 2-aminoisobutyric acid (AIB) suggests the presence of transport system A in Müller cells. The process is energy-requiring, since Gly transport was decreased by metabolic inhibitors. Data obtained are in keeping with a modulatory role for Müller glia on excitatory transmission in the retina.     

Functional glutamate transport in rodent optic nerve axons and glia

Recent findings suggest that synaptic-type glutamate signaling operates between axons and their supporting glial cells. Glutamate reuptake will be a necessary component of such a system. Evidence for glutamate-mediated damage of oligodendroglia somata and processes in white matter suggests that glutamate regulation in white matter structures is also of clinical importance. The expression of glutamate transporters was examined in postnatal Day 14-17 (P14-17) mouse and in mature mouse and rat optic nerve using immuno-histochemistry and immuno-electron microscopy. EAAC1 was the major glutamate transporter detected in oligodendroglia cell membranes in both developing and mature optic nerve, while GLT-1 was the most heavily expressed transporter in the membranes of astrocytes. Both EAAC1 and GLAST were also seen in adult astrocytes, but there was little membrane expression of either at P14-17. GLAST, EAAC1, and GLT-1 were expressed in P14-17 axons with marked GLT-1 expression in the axolemma, while in mature axons EAAC1 was abundant at the node of Ranvier. Functional glutamate transport was probed in P14-17 mouse optic nerve revealing Na+-dependent, TBOA-blockable uptake of D-aspartate in astrocytes, axons, and oligodendrocytes. The data show that in addition to oligodendroglia and astrocytes, axons represent a potential source for extracellular glutamate in white matter during ischaemic conditions, and have the capacity for Na(+)-dependent glutamate uptake. The findings support the possibility of functional synaptic-type glutamate release from central axons, an event that will require axonal glutamate reuptake.     

Functional analysis of glutamate transporters in excitatory synaptic transmission of GLAST1 and GLAST1/EAAC1 deficient mice

The high affinity, Na(+)-dependent, electrogenic glial L-glutamate transporters GLAST1 and GLT1, and two neuronal EAAC1 and EAAT4, regulate the neurotransmitter concentration in excitatory synapses of the central nervous system. We dissected the function of the individual transporters in the monogenic null allelic mouse lines, glast1(-/-) and eaac1(-/-), and the derived double mutant glast(-/-)eaac1(-/-). Unexpectedly, the biochemical analysis and the behavioral phenotypes of these null allelic mouse lines were inconspicuous. Inhibition studies of the Na(+)-dependent glutamate transport by plasma membrane vesicles and by isolated astrocytes of wt and glast1(-/-) mouse brains indicated the pivotal compensatory role of GLT1 in the absence particularly of GLAST1 and GLAST1 and EAAC1 mutant mice. In electrophysiological studies, the decay rate of excitatory postsynaptic currents (EPSCs) of Purkinje cells (PC) after selective activation of parallel and climbing fibers proved to be similar in wt and eaac1(-/-), but was significantly prolonged in glast1(-/-) PCs. Bath application of the glutamate uptake blocker SYM2081 prolonged EPSC decay profiles in both wt and double mutant glast1(-/-)eaac1(-/-) PCs by 286% and 229%, respectively, indicating a prominent role of compensatory glutamate transport in shaping glast1(-/-)eaac1(-/-) EPSCs.     

Forebrain structures involved in ovulation and release of serum LH and FSH in proestrous rats

The specific sodium-dependent binding of [3H]glutamate to membranes of the rat striatum was examined and a comparison made with high affinity glutamate uptake. In the presence of sodium, [3H]glutamate binding was saturable and of high affinity. No binding could be detected in the absence of sodium. Removal of the cortical afferents to the striatum resulted in a parallel decrease in Na+-dependent glutamate binding and in high-affinity glutamate uptake. After the injection of the neurotoxin kainic acid into the striatum, the density of Na+-dependent glutamate binding sites was reduced by 40%, while high-affinity uptake showed no significant decrease. Drugs which inhibit high-affinity uptake were also effective at inhibiting Na+-dependent binding. The results suggest that about half the Na+-dependent glutamate binding sites in the striatum represent high-affinity uptake sites on the corticostriatal terminals. The remainder of the binding sites are located on striatal neurons and may also be uptake sites.     

Sodium signals in cerebellar Purkinje neurons and Bergmann glial cells evoked by glutamatergic synaptic transmission

Glial cells express specific high-affinity transporters for glutamate that play a central role in glutamate clearance at excitatory synapses in the brain. These transporters are electrogenic and are mainly energized by the electrochemical gradient for sodium. In the present study, we combined somatic whole-cell patch-clamp recordings with quantitative Na+ imaging in fine cellular branches of cerebellar Bergmann glial cells and in dendrites of Purkinje neurons to analyze intracellular Na+ signals close to activated synapses. We demonstrate that pressure application of glutamate and glutamate agonists causes local Na+ signals in the mM range. Furthermore, we analyzed the pharmacological profile, as well as the time course and spatial distribution of Na+ signals following short synaptic burst stimulation of parallel or climbing fibers. While parallel fibers stimulation resulted in local sodium transients that were largest in processes close to the stimulation pipette, climbing fibers stimulation elicited global sodium transients throughout the entire cell. Glial sodium signals amounted to several mM, were mainly caused by sodium influx following inward transport of glutamate and persisted for tens of seconds. Sodium transients in dendrites of Purkinje neurons, in contrast, were mainly caused by activation of AMPA receptors and had much faster kinetics. By reducing the driving force for sodium-dependent glutamate uptake, intracellular sodium accumulation in glial cells upon repetitive activity might provide a negative feedback mechanism, promoting the diffusion of glutamate and the activation of extrasynaptic glutamate receptors at active synapses in the cerebellum.     

Transporter reversal as a mechanism of glutamate release from the ischemic rat cerebral cortex: studies with DL-threo-beta-benzyloxyaspartate

Elevated levels of the excitotoxic amino acids, glutamate and aspartate, have been implicated in the pathogenesis of neuronal injury and death induced by cerebral ischemia. This study evaluated the contribution of reversed high-affinity, Na(+)-dependent, glutamate transport to the ischemia-evoked release of glutamate and aspartate using DL-threo-beta-benzyloxyaspartate (DL-TBOA), a newly developed competitive, non-transported blocker of the EAAT 1-3 transporters. Changes in the extracellular levels of these and other amino acids, and of glucose and lactate in cerebral cortical superfusates during four-vessel occlusion-elicited global cerebral ischemia were examined using a cortical window technique. Basal and ischemia-evoked amino acid, glucose and lactate efflux were compared in control versus DL-TBOA (100 microM; applied topically for 35 min prior to ischemia) animals. Twenty minutes of ischemia caused large increases in aspartate, glutamate, GABA and taurine effluxes into cortical superfusates, with non-significant effects on the efflux of glycine, glutamine, alanine and serine. Application of DL-TBOA caused a 2-fold increase in basal, preischemic, extracellular glutamate levels, but did not affect those of the other compounds. In the presence of DL-TBOA, ischemia-evoked release of aspartate, glutamate, taurine and glutamine was significantly reduced; that of the other amino acids was not affected. The ischemia-evoked declines in glucose were significantly attenuated, and lactate release was enhanced above that in control animals. The amino acid data are interpreted as indicating that aspartate and glutamate releases were reduced as a consequence of DL-TBOA inhibition of reversed transport by high-affinity, Na-dependent carriers, predominantly involving the glial EAAT 2 transporter. The reduction in ischemia-evoked taurine release is interpreted as being due to a decrease in cell swelling prior to and during the initial phase of ischemia due to reduced entry of the Na(+), and other ions, associated with a decreased glutamate uptake. Glucose-sparing and availability for lactate formation would also result from a reduced glutamate/Na(+) uptake. These results indicate that reversed transport, primarily from glial cells by the EAAT 2 carrier, is responsible for a substantial (42 and 56%) portion of the ischemia-evoked increase in extracellular glutamate and aspartate levels, respectively. As a potent, competitive, non-transported blocker of high-affinity, Na(+)-dependent, glutamate transporters, DL-TBOA promises to be a valuable new compound for the study of glutamatergic mechanisms.     

Synaptosomal plasma membrane transport of excitatory sulphur amino acid transmitter candidates: kinetic characterisation and analysis of carrier specificity.

The transport kinetics of the excitatory sulphur-containing amino acid (SAA) transmitter candidates, L-cysteine sulphinate (L-CSA), L-cysteate (L-CA), L-homocysteine sulphinate (L-HCSA), and L-homocysteate (L-HCA), together with their plasma membrane carrier specificity, was studied in cerebrocortical synaptosome fractions by a sensitive high performance liquid chromatographic assay. A high affinity uptake system could be demonstrated for L-CSA (Km = 57 +/- 6 microM; Vmax = 1.2 +/- 0.1 nmol/min/mg protein) and L-CA (Km = 23 +/- 3 microM; Vmax = 3.6 +/- 0.1 nmol/min/mg protein), whereas L-HCSA (Km = 502 +/- 152 microM; Vmax = 6.1 +/- 1.3 nmol/min/mg protein) and L-HCA (Km = 1550 +/- 169 microM; Vmax = 10.3 +/- 1.1 nmol/min/mg protein) exhibited much lower affinity as transport substrates. In all cases, only a single, saturable Na(+)-dependent component of uptake could be identified, co-existing with a non-saturable, Na(+)-independent influx component. Plasma membrane carrier specificity of the SAAs was established following comparison with other high-affinity neurotransmitter systems. High-affinity L-CSA and L-CA transport and low-affinity L-HCSA and L-HCA transport demonstrate strong positive correlations in inhibition profiles when compared against each other or individually against the high-affinity transport of L-[3H]glutamate, L-[3H]aspartate, or D-[3H]aspartate. Moreover, the transport systems for the excitatory SAAs exhibited a negative correlation when compared in inhibition profiles with the high affinity transport of both [3H] gamma-aminobutyric acid (GABA) and [3H]taurine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glutamate transporters and retinal excitotoxicity

Glutamate appears to play a major role in several degenerative retinal disorders. However, exogenous glutamate is only weakly toxic to the retina when glutamate transporters on Müller glial cells are operational. In an ex vivo rat retinal preparation, we previously found that exogenous glutamate causes Müller cell swelling but does not trigger excitotoxic neurodegeneration unless very high concentrations that overwhelm the capacity of glutamate transporters are administered. To determine the role of glutamate transporters in Müller cell swelling and glutamate-mediated retinal degeneration, we examined the effects of DL-threo-beta-benzyloxyaspartate (TBOA), an agent that blocks glutamate transport but that unlike most available transport inhibitors is neither a substrate for transport nor a glutamate receptor agonist. We found that TBOA triggered severe retinal neurodegeneration attenuated by ionotropic glutamate receptor antagonists. TBOA-induced neuronal damage was also diminished by riluzole, an agent that inhibits endogenous glutamate release. In the presence of riluzole, to inhibit glutamate release plus TBOA to block glutamate uptake, the addition of low concentrations of exogenous glutamate triggered severe excitotoxic neuronal damage without inducing Müller cell swelling. We conclude that TBOA-sensitive glutamate transporters play an important role in regulating the neurodegenerative effects of glutamate in the rat retina.     

Characteristics of excitatory amino acid uptake in cultures from neurons and glia from the retina

3H-D-Aspartate uptake was biochemically characterized in cultures from chick retina enriched in glial (Müller) cells or neurons during progressive days in vitro (DIV). In the neuronal cultures a high-affinity, Na(+)-dependent system was found with Km = 8-13 microM and pharmacological characteristics in agreement with those of reuptake systems in other regions of the CNS. The uptake system in glial cells showed a lower affinity, with Km = 100-135 microM. In both cases, uptake was temperature and energy dependent. A sharp increase in the Vmax of uptake was observed in both neuronal and glial cultures at 5 DIV, at which time morphologically mature synapses have been shown to be present in retinal cultures. A parallel increase in the pharmacological specificity of the uptake system in neuronal cultures was observed, with a rise in the efficiency of D-Asp, L-Asp, L-Glu, and DL-asp- beta-hydroxamate for inhibiting 3H-D-Aspartate uptake. Results suggest the possibility of reuptake participating in the regulation of extracellular glutamate concentration during development.     

Transcriptional control in myelinating glia: flavors and spices

Rapid removal of glutamate from the extracellular space is required for the survival and normal function of neurons. Although glutamate transporters are expressed by all CNS cell types, astrocytes are the cell type primarily responsible for glutamate uptake. Astrocyte glutamate uptake also plays a role in regulating the activity of glutamatergic synapses. Lastly, release of glutamate from astrocytes, via transporter reversal and other routes, can contribute to glutamate receptor activation. This review examines the mechanisms of astrocyte glutamate uptake and release, with particular focus on high-affinity Na(+)-dependent transporters. Transporter regulation, energetics, and physiological roles are discussed.     

Excitatory amino acid-stimulated uptake of 22Na+ in primary astrocyte cultures

In this study we have found that L-glutamic acid, as well as being taken up by a Na+-dependent mechanism, will stimulate the uptake of 22Na+ by primary astrocyte cultures from rat brain in the presence of ouabain. By simultaneously measuring the uptake of 22Na+ and L-3H-glutamate a stoichiometry of 2-3 Na+ per glutamate was measured, implying electrogenic uptake. Increasing the medium K+ concentration to depolarize the cells inhibited L-3H-glutamate uptake, while calculations of the energetics of the observed L-3H-glutamate accumulation also supported an electrogenic mechanism of at least 2 Na+:1 glutamate. In contrast, kinetic analysis of the Na+ dependence of L-3H-glutamate uptake indicated a stoichiometry of Na+ to glutamate of 1:1, but further analysis showed that the stoichiometry cannot be resolved by purely kinetic studies. Studies with glutamate analogs, however, showed that kainic acid was a very effective stimulant of 22Na+ uptake, but 3H-kainic acid showed no Na+ -dependent uptake. Furthermore, while L-3H-glutamate uptake was very sensitive to lowered temperatures, glutamate-stimulated 22Na+ uptake was relatively insensitive. These results indicate that glutamate-stimulated uptake of 22Na+ in primary astrocytes cultures cannot be explained solely by cotransport of Na+ with glutamate, and they suggest that direct kainic acid-type receptor induced stimulation of Na+ uptake also occurs. Since both receptor and uptake effects involve transport of Na+, accurate measurements of the Na+ :glutamate stoichiometry for uptake can only be done using completely specific inhibitors of these 2 systems.     

Glial transporters for glutamate, glycine and GABA I. Glutamate transporters

The termination of chemical neurotransmission in the CNS involves the rapid removal of neurotransmitter from synapses by specific transport systems. Such mechanism operates for the three major amino acid neurotransmitters glutamate, gamma-aminobutyric acid (GABA) and glycine. To date, five different high-affinity Na(+)-dependent glutamate (Glu) transporters have been cloned: GLT1, GLAST, EAAC1, EAAT4 and EAAT5. The first two are expressed mainly by glial cells, and seem to be the predominant Glu transporters in the brain. A major function of Glu uptake in the nervous system is to prevent extracellular Glu concentrations from raising to neurotoxic levels in which glial transporters seem to play a critical role in protecting neurons from glutamate-induced excitotoxicity. Under particular conditions, glial GluTs have been shown to release Glu by reversal of activity, in a Ca(2+)--and energy-independent fashion. Furthermore, an activity of these transporters as ion channels or transducing units coupled to G-proteins has recently been reported. The localization, stoichiometry, and regulation of glial GluTs are outlined, as well as their possible contributions to nervous system diseases as ALS, AD and ischemic damage.     

gamma-Aminobutyric acid (GABA) uptake by Xenopus oocytes injected with rat brain mRNA

A major pathway for inactivation of biogenic amine and amino acid neurotransmitters is by uptake into neurons or glia cells through Na+-dependent, high-affinity uptake systems. Here, we show that Xenopus oocytes, microinjected with poly(A)+ RNA from developing rat brain, express a Na+-dependent GABA uptake system, which is similar in its properties to the high-affinity GABA uptake system, present in rat brain. These results suggest that the oocyte expression system may be useful in the isolation of mRNAs and subsequent cloning of the genes encoding the polypeptides involved in GABA transport in the nervous system.     

PSA-NCAM expression in the piriform cortex of the adult rat. Modulation by NMDA receptor antagonist administration

Uptake of biocytin and biotin was investigated in cultured transformed variants of neuronal (NB2a neuroblastoma) and glial (C6 astrocytoma) CNS cells. NB2a cells took up both compounds but biocytin was transported more efficiently than biotin in the nanomolar concentration range. In NB2a cells a single transport mechanism was found for biocytin with different kinetic parameters in the presence of high extracellular Na+ (Km 0.4 microM, Vmax 20 pmol/min/mg), K+ (Km 1.7 microM, Vmax 32 pmol/min/mg), or choline+ (Km 0.1 microM, Vmax 5 pmol/min/mg). Two transport systems (Km1 17 microM, Vmax1 53 pmol/min/mg; Km2 314 microM, Vmax2 360 pmol/min/mg) were identified for biotin with only system 1 being Na+-dependent. Biocytin uptake was competitively inhibited by excess biotin but not vice versa. Inhibition studies with structural analogs indicated different specificities for biotin and biocytin uptake. Biocytin uptake into C6 cells was hardly detectable whereas biotin was taken up by diffusion (kD 0.6 microl/min/mg) and a single saturable mechanism (Km 70 microM, Vmax 119 pmol/min/mg) at high extracellular Na+. High extracellular K+ enhanced biotin diffusion into C6 cells. Inhibition studies with structural analogs revealed a less specific biotin uptake mechanism in C6 than in NB2a cells. Biocytin normalized deficient biotin-dependent propionyl-CoA carboxylase activity within 4 h in biotin-deficient NB2a cells whereas in C6 cells reactivation was <20% thereby confirming that biocytin is only poorly transported into C6 cells. Specific biocytin uptake into NB2a cells is to our knowledge the first demonstration of a carrier-mediated transport mechanism for this compound. Neuronal biocytin uptake might contribute to the pathogenesis of biotinidase deficiency where biocytin is present in elevated levels.     

GABA uptake by purified avian Müller glia cells in culture

GABA is the main inhibitory aminoacid transmitter present in neurons and glial cells. Its uptake is carried out by specific high-affinity Na(+)/Cl (-) dependent transporters (GATs). It has been reported in the past that, in the avian retina, [(3)H]GABA appears to be exclusively accumulated by horizontal and amacrine cells in the inner nuclear layer, and also by ganglion cells. Purified chick Müller glia cultures were able to take up [(3)H]GABA in a Na(+) and Cl(+) dependent way. Increasing GABA concentration increases GABA uptake by these cells, reaching half-maximal transport efficiency (EC50) around 0.3 mM. [(3)H]GABA uptake by Müller glia neuronal-free cultures was not mediated by neuronal transporters since it was not blocked by NNC-711, but was inhibited by beta-alanine, a specific glial transporter inhibitor. Chick Müller glia in culture express both GAT-1 and GAT-3 GABA transporters. Although mixed neuron-glial dense cultures released GABA upon glutamate, high K[(+) or veratridine stimulation, Müller glial cells did not release [(3)H]GABA upon treatment with these agents, suggesting that different from neurons, transporter mediated GABA release is not a common mechanism operating in these cells. The data also suggest that Müller cells take up GABA unidirectionally, which may constitute an important mechanism of inactivating GABA activity mediated by neurons.     

Glial transporters for glutamate, glycine, and GABA: II. GABA transporters

The termination of chemical neurotransmission in the central nervous system (CNS) involves the rapid removal of neurotransmitter from synapses. This is fulfilled by specific transport systems in neurons and glia, including those for gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the brain. Glial cells express the cloned Na(+)/Cl(-)-dependent, high-affinity GABA transporters (GATs) GAT1, GAT2, and GAT3, as well as the low-affinity transporter BGT1. In situ hybridization and immunocytochemistry have revealed that each transporter shows distinct regional distribution in the brain and the retina. The neuronal vs. glial localization of the different transporters is not clear-cut, and variations according to species, neighboring excitatory synapses, and developmental stage have been reported. The localization, stoichiometry, and regulation of glial GATs are outlined, and the participation of these structures in development, osmoregulation, and neuroprotection are discussed. A decrease in GABAergic neurotransmission has been implicated in the pathophysiology of several CNS disorders, particularly in epilepsy. Since drugs which selectively inhibit glial but not neuronal GABA uptake exert anticonvulsant activity, clearly the establishment of the molecular mechanisms controlling GATs in glial cells will be an aid in the chemical treatment of several CNS-related diseases.     

Glutamate uptake in mammalian retinal glia is voltage- and potassium-dependent

Glutamate evokes an inward membrane current in glial cells from the rabbit retina, by activating high-affinity glutamate uptake. Uptake is strongly inhibited by depolarization. It is also inhibited by removing extracellular sodium or intracellular potassium and by raising the extracellular potassium concentration, suggesting that the uptake carrier transports sodium ions into and potassium ions out of the cell. The voltage- and potassium-dependence of glutamate uptake may have clinical implications: during anoxia, when [K+]0 rises, uptake will be inhibited and the extracellular glutamate concentration may then rise to neurotoxic levels.